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The ontogenetic ecology of the signal crayfish, Pacifastacus leniusculus, in a small temperate stream Bondar, Carin Anne 2007

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THE ONTOGENETIC ECOLOGY OF THE SIGNAL CRAYFISH, PACIFASTACUSLENIUSCULUS, IN A SMALL TEMPERATE STREAM. By CARIN ANNE BONDAR B.Sc. Simon Fraser University, 1999 M.Sc. The University of Victoria, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Forestry)  THE UNIVERSITY OF BRITISH COLUMBIA March 2007 © Carin Anne Bondar, 2007  Abstract Organisms that undergo ontogenetic niche shifts, changes in behavior or resource use with respect to developmental stage, can have disparate ecological roles through development. This thesis examines the ontogenetic ecology of the signal crayfish, Pacifastacus leniusculus (Dana) using field and laboratory based experimentation as well as a detailed population study. I initially researched the roles o f ontogenetic stage and density o f crayfish, and how this can affect the intensity o f their roles within a community. A n enclosure experiment was conducted i n a small stream (Spring Creek) in M a l c o l m Knapp Research Forest. Both juvenile and adult crayfish were found to have major effects on the surrounding community; however, these effects were independent o f ontogenetic stage. Gut and stable isotope analyses were employed to determine the primary food sources o f the crayfish from this study in an attempt to establish the mechanism behind their effects on the stream community. Aspects o f the diet were further investigated in the laboratory by examining growth-rate o f both juveniles and adults that were provided with leaves, wood or invertebrates. From the latter results, another experimental study was designed in order to examine the effects o f predators (adult crayfish or adult cutthroat trout (Oncorhynchus clarki (Richardson)) on the feeding behavior of juvenile crayfish, as their natural diets contained a large proportion of energetically poor food sources. Based on the fact that both predators were found to have negative effects on feeding behavior juvenile crayfish, a field experiment was undertaken to explore the ontogenetic relationships between signal crayfish and cutthroat trout. I found a reciprocal negative effect o f adults o f one species on juveniles o f the other, as well as a negative effect of juvenile crayfish on juvenile fish. In order to contextualize the work accomplished in the first part of this thesis, I undertook a population mark-recapture study which provided information on crayfish density in Spring Creek, as well as ontogenetic aspects of survival, movement and microhabitat choice. Overall, the work presented in this  thesis represents a substantial contribution to our knowledge o f the ontogenetic ecology of the signal crayfish.  TABLE OF CONTENTS Abstract  ii  List o f tables  vii  List o f figures  viii  Acknowledgements  xi  Dedication  xii  Co-authorship statement  xiii  CHAPTER 1 Title page  1  Introduction and literature review  2  Chapter 2 objectives and hypotheses  7  Chapter 3 objectives and hypotheses  8  Chapter 4 objectives and hypotheses  8  Chapter 5 objectives and hypotheses  9  Chapter 6 objectives and hypotheses  10  References  11  CHAPTER 2 Title page  15  Introduction  16  Methods  18  Results  21  Discussion  23  References  36  iv  CHAPTER 3 Title page  41  Introduction  42  Methods  44  Results  50  Discussion  52  References  60  CHAPTER 4 Title page  63  Introduction  64  Methods  66  Results  68  Discussion  70  References  74  CHAPTER 5 Title page  76  Introduction  77  Methods  8  0  Results  8  4  Discussion  8  7  References  99  CHAPTER 6 Title page  102  Introduction  103  Methods  105 v  Results  109  Discussion  112  References  124  CHAPTER 7 Title page  128  Thesis chapter summary and integration  129  Directions for future research  134  Conclusion  135  References  137  vi  LIST OF TABLES Table 2.1 Probability values from A N C O V A s testing the effects o f crayfish biomass (covariate) and ontogenetic stage on the abundant members o f the leaf pack community. Statistically significant differences (p <0.05) are marked by asterisks 30 Table 2.2 Probability values from A N O V A s testing the effects o f treatment type and block on the abundant members o f the leaf-pack insect community. Statistically significant (a = 0.0045) results are marked with asterisks 30 Table 2.3 Probability values from A N C O V A s testing the effects o f crayfish biomass and ontogenetic stage on the abundant members o f the cobble bottom (Surber sample) community. Statistically significant differences (a = 0.0045) are marked by asterisks 31 Table 2.4 Probability values from A N O V A s testing the effects o f treatment type and block on the abundant members o f the cobble bottom (Surber sample) insect community. Statistically significant (a = 0.0045) results are marked with asterisks 31 Table 5.1 A N O V A on the members o f the leaf pack communities showing the significant treatment effects. A l l invertebrates shown were significantly negatively affected by the treatments relative to controls  93  Table 6.1 Top ten models predicted by program M A R X for both site 2 (a) and site 3 (b). M o d e l notation follows standard notation outlined in mark manual (2006). A ' . ' indicates a lack of time dependence. S = survival, p = probability o f capture, t = time, len = O C L length, len2 = O C L length squared 118 Table 6.2. The parameter estimates, standard errors and confidence intervals for the best models predicted by M A R X for site 2 (a) and site 3 (b). Parameter notation follows standard notation outlined in M A R X manual (Cooch and White 2006). S = survival, Gamma = emigration parameter (fixed in my models) p = probability o f capture, N = population size 119 Table 6.3. The most parsimonious models for the pooled data for sites 2 and 3 are shown in (a). The best model S(len)p(t,site) shows that length is important for survival at both sites, regardless of time, and that probability o f capture varies by both time and site, (b): Parameter estimates, standard errors and confidence intervals generated for the most parsimonious model shown in (a). M o d e l notation follows standard notation outlined in M A R X manual (Cooch and White 2006). A ' . ' indicates a lack o f time dependence. S = survival, Gamma = emigration parameter (fixed in my models), p = probability o f capture, N = population size, t = time, len = O C L length, len2 = O C L length squared 120  vii  LIST OF FIGURES Figure 2.1. L e a f pack dry weight for each o f the treatments at the end o f the experiment. The only significant difference here was between the 1 adult and 3 adult treatments (p = 0.0002). The abbreviated treatment labels are: 4j = 4 juvenile, 8j = 8 juvenile, 12j = 12 juvenile, l a = 1 adult, 2a = 2 adult, 3a = 3 adult, c = control 32 Figure 2.2. Total invertebrate abundance (both the 500 u.m-1 mm (small) fraction and the > l m m (large) fraction) for the common members o f the leaf pack community. Results from all crayfish treatments, as well as the control are shown along the x axis for each invertebrate. Graphs (a)(e) show the abundance of: Zapada, shredders, Orthocladiinae, Acari, and Tipulidae, respectively. In all cases the control was significantly different from all other treatments. Labeling follows the same conventions as Figure 2.1 33 Figure 2.3. Abundance o f Chironomini (a) and Tanypodinae (b) in the leaf packs. These data are shown by size fraction, large (>1 mm) and small (500 um - 1 mm). For each o f these organisms, the large size fraction produced a significant result but the small fraction did not. In each case, the large fraction showed all crayfish treatments to be significantly different from the control. Labeling follows the same conventions as Figure 2.1 34 Figure 2.4. Abundance o f the common invertebrates o f the cobble bottom community (Surber samples). These data are shown by size fraction, large (>1 mm), and small (500 um-1 mm). In panels (a), (c) and (d) there was a significant negative effect o f crayfish biomass for the large size fraction, but no corresponding effect in the small size fraction. Panel (b) shows that the small nemourid stoneflies were affected both by crayfish biomass and ontogenetic stage. In no cases were there significant 'control vs. a l l ' contrasts, as seen with the leaf pack communities. Labeling follows the same conventions as Figure 2.1 35 Figure 3.1. Gut contents o f adult, juvenile, and young-of-the-year Pacifastacus leniusculus from within the enclosures as well as caught directly from Spring Creek, (a) adults from within the enclosures, (b) juveniles ( 2 Y A ) from within the enclosures, (c) adults caught directly from Spring Creek, (d) juveniles ( 2 Y A ) caught directly from Spring Creek, (e) Y O Y young-of-theyear caught directly from Spring Creek. Percentages shown are averages based on n = 12 for graph (a) n = 12 for graph (b) and n = 5 for graphs (c) (d) and (e) 57 Figure 3.2. Stable isotope signatures o f carbon and nitrogen for three ontogenetic stages of crayfish (Pacifastacus leniusculus) as well as their possible dietary components. Points shown are means, and errors are standard deviations. Sample sizes range from n = 5 for each crayfish, to n = 2 (based on ground samples of 50 chironomids, 20 mayflies, 10 leaves, and 10 pieces o f woody debris). B i o f i l m samples (n = 2) are based on dried biofilm scrapings from 15 leaves or 15 pieces o f woody debris. A l l samples were collected from Spring Creek 58 Figure 3.3. Overall growth o f both juvenile and adult crayfish (Pacifastacus leniusculus) raised on three different diets in a laboratory setting. Crayfish were housed in 20 L aquaria (one crayfish per aquarium). Food types (conditioned leaves, conditioned woody debris, or invertebrates (Chironomidae (blood worms)), were provided in excess for a period of three months. Mean overall weight change is shown for each food type for both adult (four replicates  for each food type) and juvenile (six replicates for each food type) crayfish. Error bars denote standard error 59 Figure 4.1. Time to first food choice by young-of-the-year Pacifastacus leniusculus alone and in the presence of various other members o f the stream community. In all cases the first food choice was a chironomid. A statistically significant difference in the time to choice was detected between the treatments containing adult crayfish and all other treatments ( A N O V A post-hoc contrast, p = 0.0001 for both adult female and male crayfish vs. all other treatments). Numbers of replicates for each trial were n = 19 for juvenile crayfish alone, and n = 14 for each o f the other trials with added individuals. Error bars indicate standard error 73 Figure 5.1. The difference (final - initial) of Y O Y fish biomass for each treatment over the 6 week experiment. The collective biomass was used because individual Y O Y fish could not be effectively marked. Error bars indicate 1 standard error 94 Figure 5.2. Crayfish mass change as a % o f original mass. Adult crayfish were unaffected by treatment. Error bars indicate 1 standard error 94 Figure 5.3. Collective leaf mass remaining by treatment (4 leaf packs per enclosure, starting mass o f 4 g each). Adult crayfish treatments were significantly different from all other treatments (p values for least squared means post hoc comparisons < 0.001). Y O Y crayfish treatments had a significantly higher leaf mass remaining than all adult crayfish treatments (p values for post hoc comparisons < 0.001). Error bars indicate + 1 standard error 95 Figure 5.4. Total leaf pack invertebrates per gram o f leaf mass (Numbers/g). (a) Chironomini (b) Tanytarsini (c) Orthocladiinae (d) Tanypodinae (e) Tipulidae (f) Dipteran Pupae (g) Rhyacophilidae (h) Zapada. Error bars indicate +1 standard error. Treatments are abbreviated as follows: A D C = adult crayfish, Y O Y F = Y O Y Fish, A D F = Adult fish, Y O Y C = Y O Y Crayfish. 96 Figure 5.5. Fractioned organic and inorganic matter in the leaf packs per gram o f leaf mass, (a) C P O M (b) F P O M (c) fine inorganic sediment. Error bars indicate +1 standard error. Treatments are abbreviated using the same convention as for Figure 5.4 97 Figure 5.6. The interactions between Y O Y crayfish and adult fish, exhibited by the disproportionate decrease of numbers o f (a) Chironomini (b) Tanypodinae (c) Baetis (d) Orthocladiinae in the Y O Y C + A D F treatments. Error bars indicate +1 standard error. Treatments are abbreviated using the same convention as for Figure 5.4  98  Figure 5.7. Stage-specific interactions between crayfish and fish with respect to weight change during the experiment. Solid arrows indicate negative effects, and the dashed arrow indicates a positive effect. The thickness o f the arrows indicates the magnitude o f effects, based on the % growth ( Y O Y Crayfish) or % biomass change ( Y O Y fish) 98 Figure 6.1. Size distribution o f all crayfish captured at sites 2 and 3. Crayfish are divided into size classes of 1 m m O C L 121 Figure 6.2. Survival rate as a function o f length for all crayfish at sites 1 and 2. This estimates is based on the model S(len)p(t), the most parsimonious model, for the combined data at sites 2 and ix  3. The solid line indicates the estimated survival probability, and the dashed lines represent the 95% confidence intervals 122 Figure 6.3. Cumulative movement o f re-captured individuals per study site. Columns labeled ' W refer to re-captures within primary sessions and columns labeled ' B ' refer to re-captures between primary sessions. Error bars denote one standard error. A n asterisk between 2 columns indicates that the 2 columns are significantly different 122 Figure 6.4. Microhabitat use of juvenile and adult crayfish, (a) Juvenile crayfish microhabitat distribution for sites 2 and 3 combined, (b) Adult crayfish microhabitat distribution for sites 2 and 3 combined. Numbers on the graphs correspond to the following microhabitat types: 1: under a rock near the stream bank, 2: under a rock mid-stream (pool), 3: under a rock mid-stream (riffle), 4: debris filled area near the stream bank, 5: debris filled area in middle o f stream (pool), 6: uncovered on streambed (pool), 7: uncovered on stream bed close to the stream bank, 8: uncovered on streambed (riffle). Categories 7 and 8 are so rare that they do not appear in the figures 123 Figure 6.5. Adult crayfish microhabitat use at site 3. (a) shows the results for primary session 1, (b) for primary session 2 and (c) for primary session 3. Numbers on the graphs correspond to the same microhabitat types as outlined i n Figure 6.4. Categories 7 and 8 are so rare that they do not appear in the figures 123  x  ACKNOWLEDGEMENTS There are so many people that have helped in the completion of this thesis in diverse ways. I would like to acknowledge the amazing dedication of Kate Bottriell to this project, through the 2 summers that we worked together. Thanks Kate, for never letting me down. I would not have accomplished nearly as much as I did without your help. I would also like to thank others who provided assistance in the field: Katie Zeron, Lee-Ann Hamilton, Nancy Hofer, Tatiana Lee and Megan Harrison, those who provided much needed assistance in the lab: Leanne Baker, Nancy Hofer, Pina Viola, Katie Zeron and Christine Englehardt, and assistance with statistical analysis: Carolyn Huston and Simon Bonner and Carl Schwarz. Thanks to members of the StARR lab for their support over the years: Suzie Lavallee, Laurie Marczak, Trent Hoover, Maggie Branton, Alana Hilton and Ed Quilty. I would also like to thank members of my committee for their encouragement and helpful comments on manuscripts and presentations: Diane Srivastava, Jordan Rosenfeld, Bill Neill and Scott Hinch. My supervisor, John Richardson, has been a great supporter of mine through the 5 years I have worked on this thesis, and 1 would like to gratefully acknowledge his dedication to helping me with my work, and his continuous belief in my abilities.  xi  DEDICATION  This thesis would not exist without the unconditional support o f those around me. I dedicate this work to the people who I love the most and who love me the most: to my dear father and brother, who I lost along the way, I am incredibly proud to have been the object o f your admiration and pride. To my amazing mother, who inspires me every day o f my life to be strong, and who would stop at nothing to consider my needs ahead o f her own. Lastly, to my husband lan, for his never-ending patience and support, and for being the true love o f my life. For all their inspiration and their utter dedication to me, I dedicate this to them.  xii  CO-AUTHORSHIP STATEMENT Chapter 2: I was primarily responsible for identification and design o f the research, performing the research, analyzing the data and preparation o f the manuscript. M y coauthor, John Richardson, provided feedback on the manuscript, advice on experimental design and statistical analysis, and financial assistance during the field season. Chapter 3: I was primarily responsible for identification and design o f the research, performing the research, analyzing the data and preparation o f the manuscript. Kate Bottriell provided field and laboratory assistance, and assumed maintenance duties o f the experiments when I was unable to do them (I was away at a conference for 1 week during the summer. Katie Zeron accomplished the gut content analyses for the crayfish caught outside o f the enclosures. I instructed M s . Zeron as to the dissection technique and helped her learn to identify various components within the guts. She then completed several dissections on her own. John Richardson provided advice on experimental design and statistical analysis, feedback to previous versions o f the manuscript, as well as financial assistance (to myself, Kate and Katie) during the field season. Chapter 4: I was primarily responsible for identification and design o f the research, performing the research, analyzing the data and preparation o f the manuscript. Katie Zeron provided assistance with the initial set of experiments involving crayfish alone and with adult crayfish and fish. Katie then completed the experiments involving Y O Y fish with the help o f another summer N S E R C student. John Richardson provided feedback on previous versions o f the manuscript.  xiii  Chapter 5: I was primarily responsible for identification and design of the research, performing the research, analyzing the data and preparation of the manuscript. John Richardson provided advice on statistical analysis and feedback on earlier versions of the manuscript, and financial assistance to my field and laboratory assistants. Chapter 6: I was primarily responsible for identification and design of the research, performing the research, analyzing the data and preparation of the manuscript. John Richardson provided feedback on previous versions of the manuscript, and financial assistance to me during the write up of this manuscript.  xiv  Chapter 1 Introductory Chapter  Introduction and Literature Review  In order to assess the total impact of an organism on its environment, one must consider the possibility that the organism may have different ecological roles through its development. Most fish, amphibians, and invertebrates undergo distinct ontogenetic shifts, changes in resource or habitat use related to size or developmental stage (Olson 1996, Hjelm et al. 2000), which may render different life history stages o f the same organism functionally different contributors to the ecosystem. Species that develop over a large size range (in some cases up to 5 orders o f magnitude) w i l l have a wider use o f resources, and more potential to vary ontogenetically i n niche requirements than species that change little in size through development (Polis 1984, Bystrom and Garcia-Berthou 1999). According to Polis (1984) traditional models o f niche breadth of various species neglect the complete array o f ontogenetic interactions. He asserts that age-specific differences in resource use effectively increase the niche width of a species. With the addition o f the 'age structure component', the total niche width (w ) becomes a combination o f the within-phenotype 2  component ( V ) , the between-phenotype component (Vb), and the age-structure component (V ). w  a  He suggests that i n many cases the niche width o f one species is as wide as that found between species, and that i n this case different ontogenetic stages o f the same species may be referred to as 'ecological species'. The age structure component o f niche width may have considerable importance in community dynamics. Indeed, Erkisson (2002) and Piet et al. (1999) state that the most commonly used indices o f niche breadth make the incorrect assumption that all members o f a population are ecologically equivalent. The latter authors suggest that while traditional estimates of niche breadth and niche overlap based on such indices may be adequate for describing bird and mammal populations, they do not hold for populations o f fish, amphibians and invertebrates. In a similar conclusion to that o f Polis (1984), it was suggested (Piet et al. 1999) that two 2  components o f niche breadth may be recognized for organisms that undergo ontogenetic niche shifts: the within-ontogenetic stage component, and the between-ontogenetic stage component. Incorporating this information into niche breadth should enhance traditional models. Ontogenetic resource shifts can vastly complicate species interactions and have important consequences for community dynamics (Ebenman 1988, Taylor et al. 2001). There are opportunities for indirect effects in response to predation (Werner and G i l l i a m 1984) from different ontogenetic stages. The response o f a community to a top predator w i l l depend on the foraging capacity of different size classes o f the predator on different members o f the community (Whalstrom et al. 2000). Overlooking the complete array o f such interactions can severely limit the predictions and generalizations that can be made, as well as the predictive capacity for how a population w i l l recover after an environmental disturbance (Wootton 1994, Piet et al. 1999). The majority o f examples o f ontogenetic niche shifts come from studies on freshwater aquatic communities with an emphasis on planktivory and piscivory in fish (e.g. Werner and H a l l 1988, Mittelbach and Persson 1998, Persson et al. 1999, Claessen et al. 2002, Persson and Bronmark 2002, Rezsu and Specziar 2006). However, this phenomenon is also well documented in terrestrial systems (Polis 1984 and references therein, Polis 1988, Adams 1996, Dopman et al. 2002, Brito 2004). Ontogenetic changes in the ecological role o f an organism may be discrete (i.e. metamorphosing insects) and obvious, with different ontogenetic stages occupying completely different environments (Wilbur 1980) or continuous, with smaller differences between ontogenetic stages or with all stages inhabiting the same environment (e.g. Polis 1981, Livingston 1982, Werner 1988). Although there is vast potential for diverse direct and indirect effects o f predation from size-structured populations, two commonly documented phenomena are competitive juvenile bottlenecks and mixed competition/predation interactions (Werner and G i l l a m 1984, Mittelbach e t a l . 1988). Both apply to discrete and continuous shifts. 3  The juvenile bottlenecking phenomenon occurs when juveniles of a species experience intense interspecific competition, resulting in limited recruitment to subsequent ontogenetic stages, despite the fact that older stages are not resource limited (Persson et al. 1988, Piet et al. 1999). Persson et al. (1987, 1988) and several other researchers (e.g. Hammer 1985, Bystrom and Garcia-Berthou 1999) have studied the complex interactions between the piscivorous perch {Perca fluviatilis)  and the omnivorous roach (Rutilus rutilus) in field and laboratory studies.  Although the perch is piscivorous in its adult stage, juveniles must compete with roach for zooplankton in order to attain a large enough size to become piscivores. The roach are far more efficient competitors for the zooplankton, resulting in a bottleneck for the juvenile perch. Such dynamics can have complex indirect implications for the surrounding prey communities. Mixed competition and predation occurs when an adult of one species preys on a second species, but juveniles of the first species compete for resources with the second (Wilbur 1988). The perch/roach example may also be applied to a mixed competition/predation scenario, because while the roach is a superior competitor to juvenile perch for zooplankton, roach are susceptible to predation from adult perch (Persson et al. 1988). This pattern of competition and predation has been widely documented (e.g. Werner et al. 1983, Polis 1988, Cross and Stiven 1999). Cannibalism may be considered an important characteristic of size-structured population dynamics (Sillett and Foster 2000, Taylor et al. 2001), and may contribute to combined juvenile bottlenecking and competition/predation interactions. Cannibalism is a phenomenon that changes with ontogeny (assuming that larger individuals are generally more cannibalistic towards smaller ones (Mittelbach and Persson 1998)). If the organisms are severely cannibalistic, then adults would have the opportunity to limit juvenile recruitment, resulting in severe alterations to population dynamics (Claessen et al. 2002). In addition, if adults prey on juveniles as well as on shared food resources, there is rich potential for a mixed 4  competition/predation interaction (e.g. Whalstrom et al. 2000). The latter authors showed that extensive cannibalism o f adult perch (Perca fluviatilis)  on juvenile perch resulted in indirect  positive effects on the zooplankton community, since the juveniles were superior competitors for this resource. These authors suggest that the size dependent nature o f trophic interactions, including cannibalism, is extremely important in deciphering the direct and indirect effects o f predators on prey communities.  Ontogenetic Niche Shifts in Crayfish: There is considerable evidence from studies o f food webs based on either primary production or detritus that crayfish of diverse genera undergo ontogenetic niche shifts with respect to dietary preference, and to a lesser extent, spatial preference. Juveniles have been described as primarily carnivorous (e.g. Abrahamsson 1966, Creed 1994, Parkyn et al. 1997, Whitledge and Rabeni 1997, Guan and Wiles 1998), whereas adults are generally considered to be omnivorous (e.g. Lodge et al. 1994, Whitledge and Rabeni 1997, Schofield et al. 2001). Although there is not a considerable amount o f evidence, some studies suggest that juveniles tend to be located in shallow, low velocity areas o f streams and rivers (Creed 1994, Englund and Krupa 2000, G e l w i c k 2000, DiStefano et al. 2003), whereas adults are generally found in deep pools o f streams and rivers (Butler and Stein 1985, Creed 1994). The ontogenetic shifts in diet have been attributed to increased need for protein-based material of juveniles for growth (Momot 1995), and the inability o f adult crayfish to obtain small fast-moving invertebrates as prey (Abrahamsson 1966). However, Parkyn et al. (1997) and others (e.g. Ilheu and Bernardo 1993) have shown that adults do not necessarily reduce their consumption o f aquatic invertebrates once the caloric requirements for growth decrease, and that adults are indeed capable o f obtaining aquatic insect prey. Further studies on the subject may provide insight as to the mechanisms behind the ontogenetic shifts. 5  Although there is widespread agreement and documentation about the existence o f the dietary ontogenetic shifts in several crayfish genera, detailed studies on the ontogenetic ecology of crayfish are lacking. In addition, apart from some work published in the 1960s and 1970s (Mason 1963, 1970, 1974 and 1975), there has been almost no work published on the ecology o f Pacifasticus  leniusculus (Dana) (commonly referred to as the signal crayfish) in its native  environment, even though it is the only crayfish endemic to north-western North America. The vast majority o f research done on P . leniusculus has been done in environments where this species is introduced. The objective of this thesis is to address the magnitude and consequences o f differences in the ecology o f several ontogenetic stages of P. leniusculus i n its native British Columbia. The genus Pacifastacus refers to all Astacinae crayfish native to North America, west of the Rocky Mountains (Bott 1950). The natural range o f P. leniusculus extends from the southern part o f British Columbia (Hamr 1998) to the northern part o f California (Elser 1994) and east to parts o f Utah and Montana (Johnson 1986, Sheldon 1989) (cited from Bondar et al. 2005, p4). There are accounts o f maturation of P. leniusculus individuals occurring as early as the first year o f life (Miller 1960, Kirjavainen and Westman 1995, Soderback 1995 (males)); however, most studies report sexual maturity at age 2+ (Abrahamsson 1971, Shimitzu and Goldman 1983, M c G r i f f 1983, Reynolds et al. 1992, Soderback 1995 [females], Lewis and Horton 1997) or 3+ (Abrahamsson and Goldman 1970, Flint 1975, Kirjavaienen and Westman 1995). Size at maturity also varies considerably, from 60mm to 90mm total length (Miller 1960, Abrahamsson 1971, Mason 1975, M c G r i f f 1983, Hogger 1986, Kirjavainen and Westman 1999). Sexually mature females may be discerned through visibly white cement glands on their ventral side, whereas maturity in males may be detected through the presence o f white sperm in the gonads (Abrahamsson 1971). In addition, at maturity the abdomen o f female crayfish becomes  6  wider, creating a protective cavity for eggs (Goellner 1943), and the chellipeds of the males start to exhibit allometric growth (Mason 1975). (cited from Bondar et al. 2005, p6). The existence o f well-documented ontogenetic niche shifts in environments (in feeding preference) to which the signal crayfish has been introduced (e.g. Guan and Wiles 1998) provides the possibility that this organism may be playing several ecological roles through its development in its native environment as well. I therefore hypothesize that juvenile and adult crayfish have distinctive ecological roles i n the stream community. Juveniles are hypothesized to be carnivorous predators, while adults are hypothesized to be detritivores, making the overall niche width o f this species quite large. The various chapters o f this thesis have been designed in order to support or refute the claim that adult and juvenile P. leniusculus are 'ecological species' (i.e. Polis 1984). Below I outline the progression of the thesis and contents o f each chapter.  Thesis Chapter 2: The effects o f ontogenetic stage and density on the ecological role o f the freshwater crayfish (Pacifastacus leniusculus) in a stream within its native range.  Objectives: To determine whether ontogenetic stage or density o f crayfish within in-stream enclosures had effects on the surrounding community in two microhabitat types. Hypotheses: I hypothesized that juvenile and adult crayfish would have different ecological roles in the detritus-based stream communities where they occur, and that the effect o f increased crayfish density (biomass) would alter these effects. Based on the ontogenetic niche shifts described above I predicted a decrease in the abundance and diversity o f the invertebrate community in response to juvenile crayfish, and a diminished leaf-pack dry weight due to consumption by adult crayfish. These effects were predicted to intensify with increasing crayfish density to a point, after which crayfish would be limited by intraspecific aggression, as crayfish, and P. leniusculus in particular, have cannibalistic tendencies. The effects of crayfish 7  within the enclosures were expected to be more substantial in the leaf-pack microhabitat than the cobble bottom microhabitat.  Thesis Chapter 3 : Does trophic position of the omnivorous signal crayfish (Pacifastacus leniusculus) i n a stream food web vary with life history stage or density? Objectives: 1) To determine the mechanism (direct or indirect) of the effects described in Chapter 2 of crayfish on the stream ecosystem through determination of the major food sources contributing to the diet of both juveniles and adults from within the enclosures; 2) To determine whether crayfish density had an effect on the diet; 3) To compare the diet o f crayfish from within the enclosures to the diet of crayfish caught in the natural environment; 4) To utilize different food sources in a laboratory-based growth experiment to determine which food sources permit crayfish to experience the most growth. Hypotheses: I predicted a preponderance of carnivory in juveniles at low density, followed by increased ingestion of lower quality food resources and lower levels of gut-fullness at higher densities. For adults I predicted that detritivory would be prevalent at low density, and that there would be an increase in the incidence of cannibalism by the largest individuals at high densities. In addition, I predicted a decrease in gut-fullness of subordinate (smaller) adults at high densities resulting from a restriction of movement or inhibition by the largest individuals.  Thesis Chapter 4 : Risk-sensitive foraging by juvenile signal crayfish (Pacifastacus leniusculus)  Objectives: The components of the diet of juvenile P. leniusculus determined from the research in chapter 3 showed there to be a large reliance on detrital material. This chapter attempts to clarify reasons for inclusion of such a low quality food source. M y objective was to use fieldbased choice experiments to assess the food selections made by P. leniusculus juveniles in the 8  presence o f different predators (cutthroat trout) and conspecifics (adult or juvenile crayfish), in order to determine i f these organisms had any effect on feeding behavior. Hypotheses: Due to the aggressive, cannibalistic nature o f this species I expected there to be a strong negative impact o f the presence o f adult crayfish (e.g. no food choice, attempting to seek refuge instead o f feeding) on the feeding behavior of juveniles. Similarly, as adult cutthroat trout have been shown to actively prey on juvenile P. leniusculus, I expected to see a strong negative influence o f their presence. The presence o f conspecific juvenile crayfish was expected to alter the time to a decision, but not the overall choice, and the presence of Y O Y (young-ofthe-year) cutthroat trout was not expected to alter the behavior o f the juvenile crayfish, as there is little size difference between these two organisms.  Thesis Chapter 5: Stage-specific interactions between dominant consumers within a small stream ecosystem: direct and indirect consequences.  Objectives: In chapter 4 juvenile crayfish were shown to alter their feeding behavior in the presence o f adult cutthroat trout, therefore, this experiment was designed to: 1) assess the interactions between adult fish and crayfish, the effects o f adults of each species on Y O Y o f the other species, and finally, the interactions between Y O Y o f both species; and 2) determine the effects o f these interactions both directly (in terms o f individual mass change during the experiment), and indirectly (in terms o f invertebrate community composition, and accumulation of organic and inorganic matter measured between treatments). Hypotheses: I predicted that interactions between adults o f one and Y O Y o f the other would result in negative effects on the Y O Y due to the threat o f predation. Interactions between Y O Y crayfish and fish and adult crayfish and fish were hypothesized to be o f a competitive nature, as  9  these organisms have been observed co-habiting in similar small-stream habitats. These results were predicted to resemble a mixed competition-predation scenario, as outlined above.  Thesis Chapter 6: Survival, movement and microhabitat choice o f signal crayfish in a small temperate stream: does individual size matter?  Objectives: This chapter provides age-specific information to substantiate the research in the previous chapters by clarifying aspects o f the density and size-based microhabitat preferences o f crayfish in a small stream. M y objective was to investigate aspects of Pacifastacus leniusculus populations in a small stream ecosystem in north-western North America, including overall population size and size structure, microhabitat uses and movement patterns of both juvenile and adult crayfish. Hypotheses:  I hypothesized that crayfish populations in this small stream would be large, with a  greater number of juveniles than adults. I predicted that juvenile and adult crayfish would display microhabitat preferences, as they are most often found in pools or in areas with a large amount o f debris. I predicted that, based on the aggressive nature o f adult P. leniusculus, juvenile crayfish would move around more than adults in order to avoid contact.  10  References Abrahamsson, S. A . 1966. Dynamics o f an isolated population o f the crayfish, Astacus astacus Linne. Oikos 17:96-107. Adams, R. A . 1996. Size-specific resource use in juvenile little brown bats, Myotis lucifugus (Chiroptera: Vespertilionidae): is there an ontogenetic shift? Canadian Journal o f Zoology 74:1204-1210. Brito, J.C. 2004. Feeding ecology o f Vipera Jatastei in northern Portugal: Ontogenetic shifts, prey size and seasonal variations Herpetological Journal 14(1): 13-19. Butler, M . J., and R. A . Stein. 1985. A n analysis o f the mechanisms governing species replacements in crayfish. Oecologia 66:168-177. Bystrom, P., and E . Garcia-Berthou. 1999. Density dependent growth and size specific competitive interactions in young fish. Oikos 86:217-232. Claessen, D . , C . V a n Oss, A . M . de Roos, and L . Persson. 2002. The impact o f size-dependent predation on population dynamics and individual life history. Ecology 83:1660-1675. Creed, R. P. 1994. Direct and indirect effects o f crayfish grazing i n a stream community. Ecology 75:2091-2103. Cross, R. E . , and A . E . Stiven. 1999. Size-dependent interactions in salt marsh fish (Fundulus heteroclitus Linnaeus) and shrimp (Palaemonetes pugio Holthuis). Journal o f Experimental Marine B i o l o g y and Ecology 242:179-199. DiStefano, R . J . , J.J. Decoskke, T . M Vanguilder and L . S . Barnes. 1993 Macrohabitat partitioning among three crayfish species in two Missouri Streams, U . S . A . Crustaceana 76(2): 343-362. Dopman, E . B . , G . A . Sword, and D . M . Hillis. 2002. The importance o f the ontogenetic niche in resource-associated divergence: evidence from a generalist grasshopper. Evolution 56:731-740. Ebenman, B . 1988. Dynamics o f Age- and Size-Structured Populations: Intraspecific Competition. Pages 127-139 in B . Ebenman and L . Persson, editors. Size-structured Populations: Ecology and Evolution. Springer Verlag, N e w Y o r k . Eriksson, O. 2002. Ontogenetic niche shifts and their implications for recruitment in three clonal V a c c i n i u m shrubs: Vaccinium myrtillus, Vaccinium vitis-idaea, and Vaccinium oxycoccos. Canadian Journal of Botany 80:635-641. Gelwick, F . P. 2000. Grazer identity changes the spatial distribution o f cascading trophic effects in stream pools. Oecologia 125:573-583. Guan, R., and P. R. Wiles. 1998. Feeding ecology o f the signal crayfish Pacifastacus in a British L o w l a n d River. Aquatic L i v i n g Resources 9:265-272.  leniusculus  Hammer, C . 1985. Feeding Behavior o f roach (Rutilus-rutilus) larvae and the fry o f perch (Perca 11  fluviatilis) in Lake Lankau. Archiv Fur Hydrobiologie 103(1): 63-74. Hjelm, J., L . Persson, and B . Christensen. 2000. Growth, morphological variation and ontogenetic niche shifts in perch (Perca fluviatilis) in relation to resource availability. Oecologia 122:190-199. Ilheu, M . , and J. M . Bernardo. 1993. Experimental evaluation o f food preference o f red swamp crawfish, Procambarus clarkii: vegetal versus animal. Freshwater Crayfish 9:359-364. Livingston, R. J. 1982. Trophic organization o f fishes in a coastal seagrass system. Marine Ecology Progress Series 7:1-12. Lodge, D . M . , M . W . Kershner, and J. E . A l o i . 1994. Effects o f an omnivorous crayfish (Orconectes rusticus) on a freshwater littoral rood web. Ecology 75:1265-1281. Mason, J. C . 1963. Life history and production o f the crayfish, Pacifastacus leniusculus trowbridgii (Stimson), in a small woodland stream. M . S c . Oregon State University, Oregon. Mason, J. C . 1970. Copulatory behavior o f the crayfish, Pacifastacus American Midland Naturalist 84:463-473.  leniusculus (Stimson). The  Mason, J. C. 1974. Aquaculture potential o f the freshwater crayfish, (Pacifastacus). during 1970. Fisheries Research Board o f Canada. Technical Report 440.  I. Studies  Mason, J. C . 1975. Crayfish production in a small woodland stream. Freshwater Crayfish 2:449 479. Mittelbach, G . G . , C. W . Osenberg, and M . A . Leibold. 1988. Trophic relations and ontogenetic niche shifts in aquatic ecosystems, in B . Ebenman and A . Persson, editors. Size structured populations: ecology and evolution. Springer Verlag, N e w York. Mittelbach, G . G . , and L . Persson. 1998. The ontogeny o f piscivory and its ecological consequences. Canadian Journal of Fisheries and Aquatic Sciences 55:1454-1465. Momot, W . T. 1995. Redefining the role o f crayfish in aquatic ecosystems. Review o f Fisheries Science 3:33-63. Olson, M . H . 1996. Ontogenetic niche shifts in largemouth bass: variabililty and consequences for first-year growth. Ecology 77:179-190. Parkyn, S. M . , C. F. Rabeni, and R. J. Collier. 1997. Effects of crayfish (Paranephrops planifrons: Parastacadae) on in-stream processes and benthic faunas: a density manipulation experiment. N e w Zealand Journal of Marine and Freshwater Research 31:685-692. Persson, L . 1987. Effects o f habitat and season on competitive interactions between roach (Rutilus rutilus) and perch (Perca fluviatilis). Oecologia 73:170-177. Persson, L . 1988. Asymmetries in competitive and predatory interactions in fish populations. Pages 203-218 in B . Ebenman and A . Persson, editors. Size structured populations: ecology and 12  evolution. Springer Verlag, N e w Y o r k . Persson, L . , K . Leonardsson, A . M . de Roos, M . Gyllenberg, and B . Christensen. 1998. Ontogenetic scaling o f foraging rates and the dynamics o f a size-structured consumer-resource model. Theoretical Population Biology 54:270-293. Persson, L . , P. Bystrom, E . Whalstrom, J. Andersson, and J. Hjelm. 1999. Interactions among size-structured populations in a whole lake experiment: size and scale dependent processes. Oikos 87:139-156. Persson, A . , and C . Bronmark. 2002. Foraging capacities and effects of competitive release on ontogenetic diet shift in bream, Abramis brama. Oikos 97:271-281. Piet, G . J., J. S. Pet, W . A . H . P. Guruge, J. Vijverberg, and W . L . T. V a n Densen. 1999. Resource partitioning along three niche dimensions in a size-structured tropical fish assemblage. Canadian Journal o f Fisheries and Aquatic Sciences 56:1241-1254. Polis, G . 1981. The evolution and dynamics o f interspecific predation. Annual Review o f Ecology and Systematics 12:225-251. Polis, G . A . 1984. A g e structure component o f niche width and intraspecific resource partitioning: can age groups function as ecological species? American Naturalist 123:541-564. Polis, G . 1988. Exploitation competition and the evolution o f interference, cannibalism, and intraguild predation in age/size structured populations. Pages 185-202 in B . Ebenman and L . Persson, editors. Size structured populations: ecology and evolution. Springer Verlag, N e w York. Rezsu, E . and A . Specziar. 2006. Ontogenetic diet profiles and size-dependent diet partitioning of ruffe Gynmocephalus cernuus, perch Perca fluviatilis and pumpkinseed Lepomis gibbosus in Lake Balaton. Ecology o f Freshwater Fish 15(3): 339-349. Schofield, K . A . , C . M . Pringle, J. L . Meyer, and A . B . Sutherland. 2001. The importance o f crayfish in the breakdown o f rhododendron leaf litter. Freshwater Biology 46:1191-1204. Sillett, K . B . , and S. A . Foster. 2000. Ontogenetic niche shifts in two populations of juvenile threespine stickleback, Gasterosteus aculeatus, that differ in pelvic spine morphology. Oikos 91:468-476. Taylor, R. C , J. C . Trexler, and W . F. Loftus. 2001. Separating the effects o f intra-and interspecific age-structured interactions in an experimental fish assemblage. Oecologia 127:143152. Werner, E . E . , and J. F. Gilliam. 1984. The ontogenetic niche and species interactions in sizestructured populations. Annual Review o f Ecology and Systematics 15:393-425. Werner, E . E . 1988. Size, scaling and the evolution o f complex life cycles. Pages 60-81 in B . Ebenman and L . Persson, editors. Size structured populations: Ecology and evolution. Springer Verlag, N e w York. 13  Werner, E . E . , and J. D . H a l l . 1988. Ontogenetic habitat shifts i n bluegill: the foraging ratepredation risk trade-off. Ecology 69:1352-1366. Werner, E . E . , J. F. Gillam, J. D . H a l l , and G . G . Mittelbach. 1983. A n experimental test of the effects o f predation risk on habitat use in fish. Ecology 64:1540-1548. Whalstrom, E . , L . Persson, S. Diehl, and P. Bystrom. 2000. Size-dependent foraging efficiency, cannibalism and zooplankton community structure. Oecologia 123:138-148. Whitledge, G . W . , and C . F. Rabeni. 1997. Energy sources and ecological role o f crayfishes in an Ozark stream: insights from stable isotopes and gut analysis. Canadian Journal of Fisheries and Aquatic Sciences 54:2555-2563. Wilbur, H . M . 1980. Complex life cycles. Annual Review o f Ecology and Systematics 11:67-93. Wilbur, H . M . 1988. Interactions between growing predators and growing prey. Pages 157-171 in B . Ebenman and A . Persson, editors. Size structured populations: ecology and evolution. Springer Verlag, N e w York. Wootton, J. T. 1994. Putting the pieces together: testing the independence o f interactions among organisms. Ecology 75:1544-1551.  14  'Chapter 2 T h e effects o f o n t o g e n e t i c stage a n d d e n s i t y o n the e c o l o g i c a l r o l e o f the freshwater crayfish  (Pacifastacus leniusculus) i n a s t r e a m w i t h i n its n a t i v e r a n g e .  A version o f this chapter has been submitted for publication. Bondar, C A . and J.S. Richardson. The effects o f ontogenetic stage and density on the ecological role of the freshwater crayfish (Pacifastacus leniusculus) in a stream within its native range. Oecologia. ]  15  Introduction Ecologists are becoming increasingly aware of the complexities involved in determining the role o f a certain species within an ecosystem, as this role is determined by several factors other than its mere presence in an area. For example, several organisms exhibit ontogenetic niche shifts, which correspond to different ecological roles through development (Olson 1996, Hjelm, et al. 2000). Behavioral shifts during development result in ecological differences between ontogenetic stages that are not simply a result o f higher biomass o f larger individuals. Ontogenetic niche shifts can result in increased complexity o f species interactions and have important consequences for community dynamics (Ebenman 1988, Taylor et al. 2001). In addition to the increased diversity in an organisms' ecological role resulting from behavioral differences through ontogeny, the density o f an organism in a specific area can have major impacts on the surrounding community as well. A n organism may be found in higher densities in certain microhabitat types, resulting in context-dependent effects on the surrounding community at localized scales. This is especially true in heterogeneous environments, such as lotic and lentic freshwater environments, where the potential for diverse microhabitat structure exists. Indeed, the heterogeneous nature of stream ecosystems requires a thorough investigation into the spatial component o f an organisms' ecology (Mcintosh et al. 2004). There is considerable evidence from studies in freshwater systems that crayfish o f many genera undergo ontogenetic niche shifts with respect to dietary preference. Juveniles are primarily carnivorous (e.g. Abrahamsson 1966, Parkyn et al. 1997, Whitledge and Rabeni 1997, Guan and Wiles 1998), whereas adults are omnivorous (e.g. Lodge et al.1994, Whitledge and Rabeni 1997, Creed and Reed 2004, Usio and Townsend 2004). The need for a protein-rich diet for small crayfish has often been cited as a primary reason behind the ontogenetic niche shifts (Momot 1995, Whitledge and Rabeni 1997), while the diminished ability o f large crayfish to obtain small, fast-moving invertebrates as prey has also been identified as an important factor 16  (Abrahamsson 1966, Nystrom et al. 1999). However, Parkyn et al. (2001) have shown that adults are indeed capable o f obtaining aquatic invertebrate prey, and that these food sources make up a substantial part o f their diet. Others (e.g. Nystrom et al. 1999, Stenroth and Nystrom 2003) suggest that the ingestion o f detritus of vascular leaves by crayfish provides the energy required for maintenance, whereas energy for growth is provided by protein-rich sources. However, the biofilm that grows on detritus is suggested to be easily digestible and highly nutritious (Whitledge and Rabeni 1997, Hollows et al. 2002). While the ecological role o f sexually mature crayfish has been extensively studied, the role of juveniles is not as clearly characterized, l n stream systems, adults have been shown to have a substantial role as both predators (Momot 1995, Parkyn et al. 1997, Whitledge and Rabeni 1997, Usio 2000, Usio and Townsend 2004) and processors o f vascular plant detritus (Huryn and Wallace 1987, Parkyn et al. 1997, Whitledge and Rabeni 1997, Schofield et al. 2001, Hollows, Townsend and Collier 2002, Creed and Reed 2004), leading several researchers to proclaim their significant role in stream ecosystems. A s little is known about the ecology o f juvenile crayfish (but see Parkyn et al. 2001), adult and juvenile members o f one crayfish species could be playing distinctive roles in the stream ecosystem. The ecology o f large crayfish has often been studied using single individuals in enclosures not accounting for the ecological impacts o f crayfish density or microhabitat use (although see Creed and Reed 2004, DiStefano et al. 2003 for exceptions). However, Pacifastacus  leniusculus (Dana) is often patchily distributed i n streams, with areas of high  density (usually areas o f low flow and high organic matter buildup) and areas o f low density (fast-moving riffles with little debris buildup) ( C . A . Bondar, personal observation), allowing for great disparity between the ecological effects o f crayfish in these different microhabitat types. I hypothesized that juvenile and adult crayfish would have different ecological roles in the detritus-based stream communities where they occur, and that the effect o f increased crayfish 17  density (biomass) would alter these effects. I was interested in how crayfish ontogeny and density (and the interaction between these two factors) would affect their community. Based on the ontogenetic niche shifts described above I predicted a decrease in the abundance and diversity o f the invertebrate community in response to juvenile crayfish, and a diminished leafpack dry weight due to consumption by adult crayfish. These effects were predicted to intensify with increasing crayfish density to a point, after which crayfish would be limited by intraspecific aggression. The effects were expected to be more substantial in the leaf-pack microhabitat than the cobble bottom microhabitat. I used a controlled enclosure experiment to test my hypotheses using P. leniusculus, the only crayfish native to north-western North America.  Methods Study organism and study site: Pacifastacus leniusculus, the only crayfish native to British Columbia, was used i n these experiments. Its natural range extends from northern California to southern British Columbia (Bondar et al. 2005a); however; most o f what is known about the ecology o f this organism is as an introduced species. This is because it was widely introduced to many parts of Europe and A s i a in the mid to late 1900s following the loss o f native European and Asian species from the crayfish plague (Abrahamsson and Goldman 1970, Svardson 1995). This experiment took place in the University of British Columbia's M a l c o l m Knapp Research Forest, located in south-western British Columbia, in the Coastal Western Hemlock biogeoclimatic zone. Spring Creek, a second order stream (mean width 2m, mean depth 45cm) (see descriptions in Richardson 1992; Reece and Richardson 2000) within the research forest, was the location o f my experiment. The riparian vegetation surrounding Spring Creek consists primarily o f red alder (Alnus rubra) with a smaller representation o f vine maple (Acer  18  circinatum); while the dominant forest cover is largely Douglas-fir (Pseudotsuga menziesii) and western hemlock (Tsuga  heterophyUd).  Experiment: I conducted a randomized, complete block experiment that contrasted effects o f crayfish ontogenetic stage and density on leaf pack weight, and community abundance and composition of both leaf packs and mixed cobbles on the bottom (based on Surber samples). Enclosures (length 90 cm X width 90 cm X height 50 cm) were constructed using 1.25 cm diameter P V C pipe and plastic hardware cloth (mesh size 1 cm ), and dug into the stream bed to a depth o f 30 2  cm. T o create microhabitat structure within the enclosures, four 5 g leaf packs were placed in each (at the corners), and allowed to condition for 10 days prior to the start o f the experiment. Senescent leaves o f red alder (Alnus rubra) were collected from the riparian area o f Spring Creek in the previous fall and air-dried in the laboratory. After weighing, leaves were re-wetted and bound with wire-based garden ties and affixed to the inside o f the enclosures. Crayfish treatment densities were based on the range of natural field densities ( C . A . Bondar, personal observation, see data i n chapter 6). Adult treatments had one, two or three individuals (average occipital carapace length 32.5 mm) per enclosure, with average total  1  biomass 23.8, 44.4 and 72.0 g, respectively. Juvenile treatments consisted o f four, eight or 12 individuals (average occipital carapace length 18.5 mm), with average total biomass per enclosure 19.4, 30.0 and 49.1 g, respectively. A control treatment consisted o f enclosures with no crayfish. A l l treatments were replicated in four complete blocks (one o f each treatment per block), spaced out along Spring Creek. One replicate o f the one adult crayfish treatment was lost during the experiment. The enclosure experiment was carried out on a homogeneous reach o f the stream (approximately 100 m in length), with a depth o f approximately 30 cm and a cobble and gravel substrate. 19  Once crayfish were inside, tops of enclosures were covered with galvanized steel hardware cloth (mesh size 1 cm ) to allow for maximum exposure to light while preventing the 2  crayfish from escaping or predators from entering. The experiment ran for 6 weeks, from early June to mid-July 2002, at which time the stream temperature was between 8 and 12 °C and stream flow was between 0.16 - 0.005m /s. The sides and tops o f the enclosures were brushed 3  twice per week to prevent debris buildup.  Sampling: A t the end o f the experiment, leaf packs and crayfish were removed. Surber samples (250 um mesh, 2 minute sampling time, one sample per enclosure) were taken from the center o f each enclosure to account for the effects o f crayfish on the stream bed community. Leaves were thoroughly washed o f any debris and invertebrates, air dried for 72 h and weighed. A l l debris and invertebrates removed from the leaves were washed into a large basin that was subsequently drained through a sieve with 63 urn mesh. The invertebrates from the leaf packs and Surber samples were stored i n 70% ethanol for subsequent analysis. Invertebrates were identified to genus under a dissecting microscope, and enumerated in two size categories. These categories w i l l subsequently be referred to as large (>1 mm) or small (0.5 m m to 1 mm).  Data Analysis: I used two analyses o f the data, one to examine the effects o f biomass (a continuous variable) within age classes ( A N C O V A ) , and another to test all seven treatments separately ( A N O V A ) . The control treatment was included only i n the second ( A N O V A ) analysis since there was no crayfish biomass in these enclosures (hence no covariate). Leaf pack dry weight and invertebrate communities were analyzed as a blocked A N C O V A , with crayfish ontogenetic stage as the main treatment effect (juvenile vs. adult) and biomass as the covariate (excluding the 20  control treatment). A Simpson's index o f diversity was calculated to assess effects of crayfish on species diversity. I used a two-way A N O V A for block design (four blocks) to compare all seven treatments including the control, for which there was no crayfish biomass. In each case ( A N C O V A leaf pack, A N C O V A cobble bottom, A N O V A leaf pack, A N O V A cobble bottom) a non-sequential Bonferonni correction for the P values was performed to account for the large number of tests. Pre-planned, orthogonal contrasts were carried out on all analyses (see tables for descriptions). Throughout the results section I w i l l refer to each test ( A N O V A vs. A N C O V A ) as it is used. Analyses were carried out using the M I X E D procedure with the maximum likelihood method ( S A S version 8e) with a = 0.0045 (the Bonferonni corrected value).  Results Crayfish effects on leaf pack decomposition: There was a significant negative effect o f crayfish biomass on leaf pack dry weight ( A N C O V A : biomass as covariate, p < 0.0001, Figure 2.1); however, this effect was not dependent on ontogenetic stage (p = 0.1318) and there was no stage*biomass interaction (p = 0.9747). It is important to note that the slope o f the crayfish biomass: leaf dry weight relationship is -0.06 for both adult and juvenile crayfish. This indicates that there is not a proportional decrease in leaf dry weight to increase in crayfish weight (95% confidence intervals around the slope -0.0226 to -0.097 do not overlap with a proportional slope of-1). The effects o f treatments on leaf pack breakdown rates were also significant, as final leaf pack dry weight differed significantly between the high adult and the low adult biomass (density) treatments ( A N O V A , pre-planned orthogonal contrast: high adult vs. low adult: p = 0.0002, Figure 2.1). The mean leaf pack dry weight o f the control treatment was not significantly heavier than all crayfish treatments ( A N O V A , pre-planned orthogonal contrast: control vs. all: p  21  > 0.05). There was a significant block effect observed for both the A N C O V A (p = 0.0003) and the A N O V A (p < 0.0001).  Microhabitat dependent effects o f crayfish: A : L e a f Pack Community Overall, crayfish had significant negative effects on total invertebrate abundance on the leaf packs as compared to those o f the control ( A N O V A , Figure 2.2, Table 2.2). The predominant taxa on the leaf packs were the nemourid stonefly Zapada, orthoclad chironomids, acarid mites, Tipulidae, and members o f the Chironomini and Tanypodinae. A l l other taxa were excluded from the individual analyses because of low numbers. Overall, the A N O V A showed that crayfish significantly reduced abundance o f Zapada spp., total number o f shredders, Orthocladiinae, A c a r i and Tipulidae on the leaf packs (Figure 2.2, Table 2.2) as compared to control treatments ( A N O V A , pre-planned orthogonal contrast: control vs. all, Table 2.2). The impacts o f crayfish on the number o f total shredders, acarid mites, tipulidae, Zapada, and Orthocladiinae were not dependent on either biomass or ontogenetic stage ( A N C O V A , Table 1). In addition, there were no significant interactions between ontogenetic stage and biomass. These results show that while the presence o f crayfish had a large negative effect on the abundance of these taxa on the leaf packs, the same effect was realized for 72 g o f adult or 20 g of juvenile. A size-related effect was observed for Chironomini and Tanypodinae. Large individuals were affected by the presence o f crayfish as compared to the controls, while the smaller ones were not ( A N O V A , Figure 2.3, Table 2.2). The effects of the crayfish on larger Chironomini and Tanypodinae were independent o f both crayfish biomass and ontogenetic stage ( A N C O V A , Table 1). Although crayfish negatively affected total invertebrate abundance on the leaf packs compared to the control, a Simpson's index o f diversity showed that crayfish did not affect species diversity ( A N O V A , Table 2.2). Neither total invertebrate abundance nor species 22  diversity was significantly affected by either crayfish biomass or ontogenetic stage ( A N C O V A , Table 2.1).  B : Cobble bottom community The effects o f crayfish on the cobble bottom community were much less pronounced than those on the leaf pack community, as there were no effects o f crayfish that were significantly different from control treatments ( A N O V A , pre-planned orthogonal contrasts: control vs. all, all p values > 0.05, Table 2.4). Overall, there were no effects o f crayfish presence, biomass or ontogenetic stage on any taxa or total invertebrate abundance in the cobble substrates ( A N C O V A Table 2.3, A N O V A , Table 2.4). The predominant taxa in the cobble samples were chironomids, nemourid stoneflies (Zapada spp.), and baetid and leptophlebiid mayflies. Chironomini and Tanypodinae, two chironomid taxa that showed a size-specific response in the leaf-pack communities, were not affected by crayfish as members o f the cobble bottom community.  Discussion Crayfish effects on leaf pack decomposition: Adult crayfish have been described as having major effects on the rate o f leaf decomposition in other donor-controlled stream ecosystems (Creed and Reed 2004, Parkyn et al. 1997, Whitledge and Rabeni 1997, Parkyn et al. 2001, Schofield et al. 2001, Hollows et al. 2002, Huryn and Wallace 1987). However, Usio and Townsend (2004) found that the rate o f leaf decomposition was similar i n stream channels with and without crayfish, which they attributed to the compensatory grazing and shredding capabilities o f the invertebrate community. A s the shredding invertebrate guild was so much more abundant in my control enclosures, it is likely that a similar form of functional compensation was occurring. However, trends for both adult 23  and juvenile crayfish illustrate that increased crayfish biomass had a negative impact on leaf pack dry weight, indicating that higher crayfish biomass (regardless o f ontogenetic stage) negatively influenced leaf biomass in areas o f leaf debris buildup. Although I would expect to see a higher rate o f loss o f leaves in high-density crayfish areas, intraspecific crayfish interference in these areas likely results in a non-proportional decrease in leaf biomass to crayfish biomass (i.e. the rate o f leaf litter consumption by crayfish decreases with an increased crayfish density).  Crayfish effects on the invertebrate community: Research on the direct and indirect effects o f crayfish in food webs o f several streams suggests that their role as predators on several invertebrate taxa may be substantial (Momot 1995, Parkyn et al. 1997, Whitledge and Rabeni 1997, Usio 2000, Stenroth and Nystrbm 2003, Creed and Reed 2004). For example, studies on the N e w Zealand crayfishes planifrons and P. zealandicus  Paranephrops  (Parkyn et al. 2001 and Hollows et al. 2002 respectively) showed  that these species directly prey on gastropods, cased caddisflies, chironomids, oligochaetes, and ephemeropterans.  Similarly, Whitledge and Rabeni (1997) found that preferred prey types o f the  crayfishes Orconectes luteus and O. punctimanus  were chironomids, ephemeropterans and  trichopterans. These results were based on data from gut contents, showing direct consumption of these organisms by the crayfish. Other research has re-iterated the importance o f crayfish to stream invertebrate communities by demonstrating a decreased density o f certain leaf-pack dwelling invertebrates subsequent to crayfish exposure (e.g. Usio 2000, Usio and Townsend 2004). This provides evidence that crayfish presence has effects on the stream community; however it does not substantiate whether such effects are direct or indirect since these latter studies did not provide evidence o f direct predation. The results presented here do not provide insight as to whether the effects o f crayfish are due to direct predation of invertebrates or to 24  indirect effects o f crayfish presence. However, I found (Bondar et al. 2005b) that the gut content and stable isotope analyses o f crayfish from this study show that the crayfish were not directly preying on invertebrates. M y results therefore indicate that the effects o f crayfish on the leaf pack microhabitat invertebrate community are largely indirect. Indirect effects of crayfish are likely to be the result o f either bioturbation and sediment removal from the leaf packs, or a predatory chemical cue released by the crayfish. Sediment removal by crayfish can have a large effect on stream communities (e.g. Parkyn et al. 1997, Creed and Reed 2004, Usio and Townsend 2004, Zhang et al. 2004), and Pringle et al. (1993) showed that the bioturbatory effects o f large decapod crustaceans (aytid shrimp) have profound effects on the composition o f invertebrate communities o f tropical streams. Several studies have demonstrated the capacity for crayfish to indirectly affect stream invertebrate groups such as snails (e.g. Alexander and Covich 1991, Covich et al. 1994, Hoverman et al. 2005), amphibians (e.g. Saenz et al. 2003), and fish (e.g. Kusch and Chivers 2004) through chemical cues. Crayfish may similarly induce emigration o f invertebrates from areas o f low or high crayfish density. The ability o f stream invertebrates to sense chemical cues from predators has been demonstrated for several taxa (e.g. Soluk and Collins 1988, Dahl 1998). Mcintosh, Peckarsky and Taylor (2002) showed that there may be a critical threshold of such cues for the mayfly Baetis bicaudatus from brook trout (Salvelinus fontinalis),  above which drift  takes place. I suspect that there may be a similar threshold o f chemical cues exuded from the crayfish that resulted in the drift o f invertebrates from my enclosures. This would be specifically apparent in the leaf pack microhabitat areas where the crayfish appear to spend most o f their time. Indeed, it has been recently demonstrated that mayfly nymphs exhibit several anti-predator behaviors in the presence of chemical cues from Orconectes rusticus (Richmond and Lasenby 2006). Investigation of invertebrate responses to crayfish cues i n the surrounding environment, and whether there is a critical threshold for such responses is an area that merits future study. 25  The finding that the negative impacts o f crayfish on large Chironomini and Tanypodinae numbers were not seen for small individuals has been documented previously for other chironomid taxa (e.g. Creed and Reed 2004, Usio and Townsend 2004). The response of different instars to chemical or bioturbatory disturbances o f crayfish could be vastly different, which may explain this result. Indeed, A l l a n (1978) found that early instars or small taxa are less constrained in their habitat choices and drift periodicity than large individuals, perhaps making them less prone to drift in response to the presence o f environmental perturbations. Samples taken from the enclosure cobble substrates showed that the crayfish did not affect overall invertebrate number or species diversity in those areas. This result is contrary to literature that predicts an increase in collector invertebrates facilitated by the leaf-shredding activities o f the crayfish (the shredder-collector facilitation hypothesis, see Heard and Richardson 1995). In addition, Creed and Reed (2004) noticed that heptageniid mayfly numbers were higher in treatments with crayfish, which was hypothesized to be due to reduction of sediments by crayfish, something I also did not observe. A predator may affect the community in some habitat types within a system, and not others (Gibson et al. 2004). This may be an explanation for why the same types o f organisms were greatly reduced in leaf pack samples but not at all affected in cobble samples. I hypothesize that since the effects o f crayfish on the leaf packs were so much more pronounced, the crayfish did not spend much time foraging in the cobble bottom areas o f the enclosures, and instead spent the majority of time i n the debris-filled areas. A s mentioned above, crayfish were variably distributed within Spring Creek, most often being found in high densities i n areas with substantial organic matter buildup. Results o f mark-recapture studies confirm the long-term residence o f crayfish in such areas (see chapter 6), and this is precisely the microhabitat within the enclosures where they were found to be having the most profound ecological effects. It is becoming increasingly evident that ecological studies should incorporate spatial heterogeneity 26  into studies o f organism abundance in stream systems as opposed to assessing mean overall abundance when investigating ecological impacts.  N o effect o f crayfish biomass: The effects o f crayfish in leaf-pack microhabitats were entirely independent o f crayfish biomass. This is contrary to the results of other researchers who have found that the magnitude of crayfish effects on the invertebrate community increased with increased crayfish biomass (e.g. Parkyn et al 1997). This could be due to the highly aggressive nature of P. leniusculus as opposed to other crayfish species (including P. planifrons, the subject of Parkyn et al.'s (1997) study). Pacifastacus leniusculus is frequently described as aggressive and cannibalistic (Nakata and Goshima 2002, Pockl and Pekny 2002, Westman and Savolainen 2001, Tierney et al. 2000), characteristics also noted during this study. Indeed, Stenroth and Nystrom (2003) found only minor differences in their enclosure study between two densities of P. leniusculus.  I noticed in  several cases during this study that four to five weeks into its duration, one o f the adult crayfish was either severely injured or killed by another, resulting in a large weight gain o f one individual and weight loss in another. These observations are similar to those o f Corkum and Cronin (2004) who found that i n experimental treatments with high food availability, feeding o f Orconectespropinquus  was limited due to mutual interference.  A s discussed above, another possibility for the lack o f effects o f increased crayfish biomass may be that the effects on the leaf-pack invertebrates were indirect, and mediated by a chemical cue exuded from the crayfish. If the concentration of chemical signals required to elicit a response from leaf-pack dwelling invertebrates was at a threshold met at my low crayfish biomass treatments, it would make sense that the effects seen at all densities would be the same. A similar argument could be made for a threshold o f disturbance that would cause leaf-pack dwelling invertebrates to drift. If even a low level o f disturbance o f the leaf packs causes 27  emigration o f the invertebrates, this could also explain the lack o f effects o f increased crayfish biomass. Further research on the feeding behavior o f P. leniusculus in its native environment, as w e l l as on the effects o f certain invertebrate taxa to chemical and physical disturbances elicited by crayfish is necessary in order to understand the mechanism behind the lack o f increased biomass effect of crayfish on the leaf-dwelling invertebrates.  N o effect of crayfish ontogenetic stage: The results o f this study indicate that the ontogenetic stage o f the crayfish did not play a role in the effects produced by them. A l l treatments with crayfish showed the same effects on the leaf-pack invertebrate community (regardless of ontogenetic stage), indicating that adult and juvenile crayfish are playing similar ecological roles. It is possible that the juveniles that I used in this experiment (mean O C L o f 18.5 mm, corresponding to a 2-year-old crayfish) had already undergone an ontogenetic shift, and were functionally the same as adults in terms o f their ecological niche. However, Parkyn et al. (2001) demonstrate that the ontogenetic diet shift in Paranephrops planifrons occurs at an O C L size of 20.0 m m . They based this conclusion on gut content analysis data o f crayfish smaller and larger than this size. In addition, Guan and Wiles (1998) reported that P. leniusculus juveniles with a carapace length o f > 20 m m were far more carnivorous than adults with a carapace length o f 33 to 45 m m , and these were the same sizes used i n my study. I maintain that the juvenile crayfish used in this study were sufficiently small that growth should still have been more o f a priority than for adults. Hence a need for an increased amount o f protein-rich food for the frequently-molting juveniles should make them more substantial predators than adults, which molt only once or twice per year (Shimizu and Goldman 1983). Future work should examine the ecology o f young-of-the-year juveniles to determine whether an ontogenetic shift in diet occurs earlier in the life cycle. Specifically, a  28  comparison of the ecology o f young-of-the-year and adult P. leniusculus in its native habitat would address the question o f whether an ontogenetic difference in their ecology exists. Most research done on the ecology of P. leniusculus in lotic environments (e.g. Guan and Wiles 1998, Stenroth and Nystrom 2003, Bubb et al. 2004) has been undertaken in parts o f the world where it is introduced. For example, Stenroth and Nystrom (2003) showed P. leniusculus to be a substantial predator on several invertebrate taxa in a Swedish stream, having the largest effects on predatory invertebrates and Ephemeroptera, and no significant effects on abundance o f Diptera and Plecoptera. Interestingly, my results were just the opposite in that the largest effects of the crayfish were on the abundances o f Diptera and Plecoptera, and I found no impact on the abundance of Ephemeroptera. This illustrates that the ecology o f an organism in an environment where it is introduced may differ markedly from its ecology within its native range. Comparative research on the ecology o f P. leniusculus as an introduced and native species could provide insight into the contrasting roles it plays on different sides o f the world. Overall, it is important to examine the roles that ontogeny and microhabitat density play in the ecology o f organisms. Great disparities have been observed in the ecology of different ontogenetic stages (e.g. Polis 1988, Mittelbach and Persson 1998, Persson and Bronmark 2002), making inclusion of these stages in ecological studies imperative. M y results do not follow a pattern o f changes in ecology through ontogeny despite research that provides a clear expectation for such changes. In addition, my results show a difference in the microhabitat effects o f crayfish, although these effects appear to be independent o f crayfish density. Closer examination o f the mechanisms by which organisms affect their surroundings may reveal that the indirect effects of an organisms' presence (e.g. chemical or physical cues, intraspecific aggression) may have similar results at several densities or ontogenetic stages of that organism.  29  Insect Type  Effect of Biomass (D.F. 1,13)  Effect of Stage (Juvenile vs. Adult) (D.F. 2,13)  Block Effect (D.F. 3,13)  Total Shredders Total Zapada Total Orthocladiinae Total Acari Total Tipulidae 1 mm Chironomini  0.5280 0.7666 0.2358 0.0291 0.0421 0.3470 0.9070 0.2853 0.8402 0.4154  0.9901 0.8450 0.5085 0.0698 0.4693 0.8648 0.5166 0.8323 0.6897 0.5891  0.0643 0.0004* 0.1081 0.0046* 0.0072 0.0025* 0.4164 0.0786 0.0468 0.0747  0.4513  0.8519  0.0694  500 um Chironomini  1 m m Tanypodinae 500 urn Tanypodinae  Total Number o f Invertebrates Species Diversity  Table 2.1. Probability values from A N C O V A s testing the effects o f crayfish biomass (covariate) and ontogenetic stage on the abundant members o f the leaf pack community. Statistically significant differences (p <0.0045) are marked by asterisks.  Insect T y p e  Effect of Treatment (D.F, = 6,17)  Effect of Block (D.F. = 3,17)  Total Shredders Total Zapada  0.0015* 0.0211 0.0009* 0.0003* 0.0024* 0.0527 0.2455 0.0034* 0.2050  0.1734 0.0112 0.4552 0.1390 0.0757 0.4360 0.1873 0.6537 0.2432  Control Control Control Control Control Control None Control None  0.0010*  0.1636  Control vs. A l l : O . 0 0 0 1  0.0767  0.0053  none  Total Orthocladiinae  Total Acari Total Tipulidae 1 m m Chironomini 500 pm Chironomini  1 m m Tanypodinae 500 urn Tanypodinae  Total Number o f Invertebrates Species Diversity  Significant Contrasts  vs. A l l : 0.0001 vs. A l l : 0.0011 vs. A l l : 0.0001 vs. A l l : O . 0 0 0 1 vs. A l l : 0.0001 v s . A l l : 0.0026 vs. A l l : 0.0001  Table 2.2. Probability values from A N O V A s testing the effects o f treatment type and block on the abundant members o f the leaf-pack insect community. Statistically significant (a = 0.0045) results are marked with asterisks.  30  Insect Type  Effect of Biomass (D.F. 1,13)  Effect of Stage (Juvenile vs. Adult) (D.F. 2,13)  Block Effect (D.F. 3,13)  500 pm Zapada spp. 1 m m Zapada spp. 500 pm Orthocladiinae 1 m m Orthocladiinae 500 pm Baetis 1 m m Baetis 500 pm Paraleptophlebia 1 m m Paraleptophlebia 500 pm Chironomini 1 m m Chironomini 500 pm Tanypodinae 1 m m Tanypodinae Total Number o f Invertebrates  0.0410 0.416 0.6731 0.0308 0.2497 0.0108 0.2319 0.0195 0.4401 0.1380 0.1723 0.1402 0.1346  0.0174 0.2067 0.6907 0.8260 0.7679 0.0574 0.1935 0.1555 0.2521 0.3777 0.5202 0.4663 0.1397  0.0214 0.1237 0.0676 0.0028* 0.2410 0.008 0.0707 0.0886 0.0483 0.0020* 0.0145 0.0888 0.0570  Table 2.3. Probability values from A N C O V A s testing the effects o f crayfish biomass and ontogenetic stage on the abundant members o f the cobble bottom (Surber sample) community. Statistically significant differences (a = 0.0045) are marked by asterisks.  Insect Type  Effect of Treatment (D.F. 6,17)  Effect of Block (D.F. 3,17)  Significant Contrasts  500pm Zapada spp. 1mm Zapada spp.  0.0455 0.0524 0.6186 0.0974 0.1033 0.0934 0.3535 0.0177 0.7523 0.0629 0.4769 0.3378 0.4005  0.1261 0.0849 0.0300 0.0028 0.2522 0.0250 0.1519 0.0107 0.0671 0.0001* 0.0974 0.0753 0.1059  None None None None None None None None None None None None None  500u.m Orthocladiinae  1mm Orthocladiinae 500pm Baetis 1mm Baetis 500um Paraleptophlebia 1mm Paraleptophlebia  500pm Chironomini 1mm Chironomini 500pm Tanypodinae 1mm Tanypodinae Total Number of Invertebrates  Table 2.4. Probability values from A N O V A s testing the effects o f treatment type and block on the abundant members o f the cobble bottom (Surber sample) insect community. Statistically significant (a = 0.0045) results are marked with asterisks.  31  8  4j  8j  12j  1a  2a  3a  Figure 2.1. L e a f pack dry weight for each o f the treatments at the end o f the experiment. The only significant difference here was between the 1 adult and 3 adult treatments (p = 0.0002). The abbreviated treatment labels are: 4j = 4 juvenile, 8j = 8 juvenile, 12j = 12 juvenile, l a = 1 adult, 2a = 2 adult, 3a = 3 adult, c = control.  32  (a)  (b)  A 4i  8i  12i  1a  2a  3a  c  (d)  4j  8j  12j  1a  4j  8j  12j  1a  2a  3a  16 14  §10  4j  8j  12j  1a  2a  3a  c  4i  8i  12i  1a  2a  3a  c  2a  3a  c  Figure 2.2. Total invertebrate abundance (both the 500 um-1 m m (small) fraction and the > l m m (large) fraction) for the common members o f the leaf pack community. Results from all crayfish treatments, as well as the control are shown along the x axis for each invertebrate. Graphs (a)-(e) show the abundance of: Zapada, shredders, Orthocladiinae, A c a r i , and Tipulidae, respectively. In all cases the control was significantly different from all other treatments. Labeling follows the same conventions as Figure 2.1.  33  4j  8j  12j  1a  2a  3a  Figure 2.3. Abundance o f Chironomini (a) and Tanypodinae (b) in the leaf packs. These data are shown by size fraction, large (>1 mm) and small (500 um - 1 mm). For each o f these organisms, the large size fraction produced a significant result but the small fraction did not. In each case, the large fraction showed all crayfish treatments to be significantly different from the control. Labeling follows the same conventions as Figure 2.1.  34  (a)  (b) Large Zapada Small Zapada  (c)  Figure 2.4. Abundance o f the common invertebrates o f the cobble bottom community (Surber samples). 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Annual Review o f Ecology and Systematics 15: 393-425. Westman, K . and R. Savolainen, R. 2001. Long term study o f competition between two cooccurring crayfish species, the native Astacus astacus L . and the introduced Pacifastacus leniusculus Dana, in a Finnish lake. Bulletin Francais de la Peche et de la Pisciulture 361: 613627. Whalstrom, E . , L . Persson, S. Diehl, S. and P. Bystrom. 2000. Size-dependent foraging efficiency, cannibalism and zooplankton community structure. Oecologia 123: 138-148.  39  Whitledge, G . W . and C F . Rabeni. 1997. Energy sources and ecological role of crayfishes in an Ozark stream: insights from stable isotopes and gut analysis. Canadian Journal o f Fisheries and Aquatic Sciences 54: 2555-2563. Zhang, Y . X . , J.S. Richardson and J . N . Negishi. 2004. Detritus processing, ecosystem engineering and benthic diversity: a test o f predator-omnivore interference. Journal of A n i m a l Ecology 73: 756-766.  40  'Chapter 3 D o e s t r o p h i c p o s i t i o n o f the o m n i v o r o u s s i g n a l c r a y f i s h  {Pacifastacus leniusculus)  i n a s t r e a m f o o d w e b v a r y w i t h l i f e h i s t o r y stage o r d e n s i t y ?  A version o f this chapter has been published. Bondar, C . A . , K . Bottriell, K . Zeron and J.S. Richardson. 2005. Does trophic position o f the omnivorous signal crayfish (Pacifastacus leniusculus) in a stream food web vary with life history stage or density? Canadian Journal o f Fisheries and Aquatic Sciences 62: 2632-2639. ]  41  Introduction Omnivory has been recognized as an important aspect o f both aquatic and terrestrial food webs (Polis and Strong 1996, Pringle and Hamazaki 1998). In contrast to specialist consumers, omnivores may have large direct and indirect impacts on several trophic levels, and consequently affect ecosystems in ways that are difficult to predict. Omnivores can have diets that include plants, animals and detritus, and therefore w i l l simultaneously have negative influences on both numbers of prey and habitat availability for prey (Dorn and Wojdak 2004). To complicate things further, the diet of an omnivore may not remain consistent either through its life or within specific life history stages. Comprehension o f the factors that drive food selection behavior o f omnivores is central to understanding their potential roles in the ecosystem (Singer and Bernays 2003). Here I focus on two factors that could potentially affect the food choices made by omnivores, the ontogenetic stage o f individuals and the density o f their populations. Omnivorous organisms can undergo shifts with respect to food preference at certain times within their life cycles. Such shifts, prevalent i n organisms such as fish, amphibians and invertebrates, may render different life history stages o f the same organism ecologically different contributors to the ecosystem (Polis 1984, Olson 1996, Hjelm et al. 2000). Life history omnivory (Pimm and Rice 1987) is prevalent in the aquatic environment (Tavares-Cromar and Williams 1996) and generates the potential for organisms to have disparate effects on their surroundings at different life history stages. Stream-dwelling crayfish are described as having an ontogenetic shift in their food preference from juveniles to adults. Juvenile crayfish are primarily carnivorous (e.g. Abrahamsson 1966 (Astacus astacus), France 1996 (Orconectes rusticus), Guan and Wiles 1998 (Pacifastacus leniusculus)), whereas adults are omnivorous (e.g. Lodge et al. 1994 (Orconectes rusticus), Whitledge and Rabeni 1997 (Orconectes luteus and O. punctimanus),  Schofield et al. 2001 (Cambarus bartonii)).  Evidence for this shift comes from  work done on dietary (gut) analysis in crayfish o f several genera (e.g. Paranephrops (Parkyn 42  2001), Procambarus  (Correia 2003), and Pacifastacus (Guan and Wiles 1998)), and provides the  possibility that there may be a strong ontogenetic component to the effects o f crayfish on the stream community. In addition to the complicating factor o f ontogeny, omnivores may alter their diet through density dependent processes i f resources are limiting or i f there is strong intraspecific aggression. Svanback and Persson (2004) have shown that individual dietary specialization among populations o f perch (Perca fluviatilis)  fluctuates with the density o f the population and is  related to the degree of intraspecific conflict. Individuals, as well as the population, were prone to consume more generalized diets at higher densities, which concurs with research showing that increased levels of competition w i l l result in sub-optimal resources becoming more valuable (Bolnick 2001). A t present, little is understood about the variation in resource use within a species driven by density dependent processes (Svanback and Persson 2004). Stream-dwelling crayfish are likely subject to density dependence with respect to dietary choices, as they are known to achieve high densities in certain microhabitat types. Both juvenile and adult Pacifastacus  leniusculus are found in areas o f stream with low flow and high  allochthonous detritus buildup (personal observation), and are both intra- and interspecifically aggressive (Guan and Wiles 1998, Nakata and Goshima 2003, Pockl and Pekny 2002). One may postulate that diet w i l l be altered in some individuals in these areas o f high density due to increased rates of aggression. Such behavior-based dietary changes, coupled to ontogenetic changes, lead to a complex array o f potential crayfish impacts on stream ecosystems. The purpose o f this study was to assess the dietary choices of juvenile and adult P. leniusculus at three densities i n experimental enclosures. I predicted a preponderance o f caraivory in juveniles at low density, followed by increased ingestion o f lower quality food resources and lower levels o f gut-fullness at higher densities. For adults I predicted that detritivory would be prevalent at low density, and that there would be an increase in the 43  incidence o f cannibalism by the largest individuals at high densities. In addition, I predicted a decrease in gut-fullness o f subordinate (smaller) adults at high densities resulting from a restriction o f movement or inhibition by the largest individuals. Subsequent to the dietary analysis experiment, I undertook growth experiments in the laboratory to determine the effects on growth o f the food choices made in the field, thus the potential fitness consequences of these choices.  Methods Study organism: Pacifastacus  leniusculus, the only crayfish native to British Columbia, was used in these  experiments. The natural range of P. leniusculus extends from the southern part o f British Columbia (Hamr 1998) to the northern part o f California (Elser et al. 1994) and east to parts o f Utah and Montana (Johnson 1986, Sheldon 1989). This organism was widely introduced to many parts o f Europe and Asia in the mid to late 1900s to compensate for the loss o f native European and A s i a n species caused by the crayfish plague (Abrahamsson and Goldman 1970, Svardson 1995). Apart from a small number of studies done in the 1970's i n British Columbia (e.g. Mason 1975), most of what is known about the ecology of this organism is as an introduced species, including descriptions of the ontogenetic niche shifts in diet (e.g. Guan and Wiles 1998).  Field diet study: This experiment took place in Spring Creek (see descriptions in Richardson 1992, Reece and Richardson 2000, Negishi and Richardson 2003), a small stream located in the University o f British Columbia's M a l c o l m Knapp Research Forest. This forest is located in southwestern British Columbia ( 4 9 ° 1 8 ' 4 0 " N , 1 2 2 ° 3 2 ' 4 0 " W ) in the Coastal Western Hemlock biogeoclimatic zone. The riparian vegetation surrounding Spring Creek consists primarily o f red alder (Alnus 44  rubra) with a smaller representation of vine maple (Acer circinatum), while the dominant forest cover is largely Douglas-fir (Pseudotsuga menziesii) and western hemlock (Tsuga  heterophylla).  The enclosure experiment was carried out within a large run reach o f the stream (approximately 100 m), with a depth of approximately 30 cm and a cobble/gravel substrate. The experiment took place from early June to mid-July 2002, at which time the stream temperature was between 8 °C and 12 °C. Enclosures ( l m x l m x 6 0 cm) were constructed with 1.25cm diameter P V C (polyvinyl chloride) pipe and plastic hardware cloth (mesh size 1 cm ), and dug into the stream bed to a 2  depth o f 30cm. Once the enclosures were embedded within the stream bed, they were re-filled with substrate to a depth that matched the level on the outside. This allowed for a full representation of food items larger than the mesh size to be included within the enclosures. Small items would also have been added in this manner, and would additionally have been able to drift into the enclosures throughout the experiment. To standardize the leaf-litter content within each enclosure, four 5g leaf packs (Alnus rubra) were added, and allowed to condition for ten days prior to the start o f the experiment. Senescent leaves had been collected from the riparian area o f Spring Creek in the previous fall and air-dried in the laboratory. After weighing, leaves were re-wetted and bound with wire-based garden ties and affixed to the inside of the enclosures. Crayfish treatment densities were in the same range as natural field densities: 1, 2 or 3 adults (average occipital carapace length [ O C L ] o f 32.5 mm) per enclosure or 4, 8 or 12 juveniles (average O C L 18.5mm, corresponding to 2-year-old sexually immature juveniles (Mason 1975)) per enclosure. A l l six treatments were replicated in four complete blocks. Once all crayfish were placed into the enclosures, they were covered with galvanized steel hardware cloth (mesh size 1 cm ) to allow for maximum exposure to light while preventing the crayfish from escaping or predators from entering. The experiment ran for a total o f 6 weeks, during 45  which time the sides and tops o f the enclosures were brushed bi-weekly to prevent debris buildup.  Data Collection: After six weeks all crayfish were collected and immediately euthanized in carbonated water. They were frozen upon return to the laboratory to prevent further digestion o f food items. In addition, five adult crayfish and five juvenile crayfish were collected from Spring Creek (approximately 100 m downstream o f the enclosures) and immediately euthanized and frozen in the same manner. These specimens were collected for gut analysis in order to assess potential effects o f the enclosures on crayfish diet. I also collected five Y O Y (young-of-the-year, O C L average 11.6mm) crayfish to assess how their gut contents compared to those o f the 2-year-old juveniles used in the experiment.  Gut content analysis The foreguts o f all crayfish from each treatment (apart from eight juvenile and twelve juvenile treatments, where five specimens from each enclosure (n = 20) were used) were dissected out and analyzed for fullness and content. The contents of the foregut were used for this analysis as they are more easily identifiable than the contents of the hindgut, which have been subject to further mechanical and chemical digestion. The methods of Parkyn et al. (2001) were used. The foreguts were dissected out o f the crayfish, and all contents were then flushed through a 500um sieve and preserved in ethanol until subsequent analysis. A l l particles greater than 500um (0.5mm) were counted and identified, allowing for more accuracy in calculating percentages o f each component within the diet. The diets o f the additional crayfish caught from Spring Creek were analyzed in the same similar manner. To avoid pseudoreplication from within the enclosures, gut-content proportion data were averaged per enclosure, then arcsine square root transformed and assessed for statistical 46  differences between different densities o f each ontogenetic stage (e.g. to test for differences between the 4, 8, and 12 juvenile treatment or the 1, 2, and 3 adult treatment) using the M A N O V A (multiple analysis o f variance) procedure in S A S version 8e (SAS Institute Inc., Cary, North Carolina). Apart from one food type for juvenile crayfish, no statistically significant differences were found between densities for either juveniles or adults (see results section). Data from all density treatments for each ontogenetic stage were therefore pooled, resulting in a total of n = 12 for juvenile guts (based on a total of 56 individuals dissected), and n = 14 adult guts (based on a total of 24 individuals dissected). I then performed a M A N O V A in order to compare the diets of juvenile versus adult crayfish. A second M A N O V A was performed in order to compare the diets of crayfish (both adult and juvenile) from the experiment with those caught directly from Spring Creek (n = 5 for the juvenile and n = 5 for the adult crayfish caught directly from Spring creek). The proportions o f food types within the guts were the response measures, although the lowest proportion was left out o f the analysis to avoid sum = 1. Gut fullness measures were assessed for statistical differences between different densities o f each ontogenetic stage o f crayfish from within the enclosures using the G L M procedure i n S A S version 8e.  Stable isotope analysis The use o f stable isotope analysis in corroboration with gut content analysis provides for a more robust approximation o f the diet, as it takes into account the food types that are assimilated into crayfish tissue, thereby eliminating overestimation o f some diet items that have a long latency period in the gut. A s there were no significant differences in the gut contents between juveniles in different density treatments and adults in different density treatments (see results section), five individuals o f each age category were selected at random for analysis o f stable isotopes o f carbon and nitrogen, in addition to the five Y O Y juveniles caught directly from Spring Creek. Frozen tissue from the tail muscle was used for this analysis. Conditioned  47  leaf litter and woody debris were collected from Spring Creek, well rinsed, and immediately frozen. The biofilm layer from 15 leaves and 15 chunks o f woody debris were removed using a soft-bristled toothbrush and distilled water. The biofilm solutions were centrifuged and freezedried prior to homogenization with a mortar and pestle. Ten leaves and ten pieces o f woody debris were dried at 60 °C for 72 hours and homogenized in the same manner. Chironomids (n = 50), and heptageniid (n = 20) and leptophlebiid (n = 20) mayflies were collected from Spring Creek, and the guts of the mayflies were dissected out before drying all organisms in a 60 °C oven for 24 hours. Samples were then ground prior to stable isotope analysis. For the biofilm samples, leaves, wood, and invertebrates other than crayfish, two samples o f the homogenized ground tissues were run for stable isotope analysis (n = 2). A s these homogenized samples were not independent, they reveal subsampling error rather than the true individual-individual variation shown by the crayfish data. A l l samples were analyzed using a Finnigan Deltapluss mass spectrometer for measuring isotope ratios o f carbon and nitrogen according to standard methods.  Laboratory feeding experiment: A feeding experiment was set up based on the results o f the gut-content analysis described above. Thirty 20-liter aquaria were filled with water from Spring Creek (sieved at 63 pm), and attached to a system o f aeration. Adult and juvenile crayfish were caught from Spring Creek and brought directly to the lab for the experiment. Each aquarium contained one crayfish in order to preclude any effects o f competition between crayfish. Treatment (food) types were based on the predominant food types found in the gut content analysis, plus invertebrates: conditioned leaf-litter, conditioned woody debris, and a mixture o f live and dead invertebrates (Chironomidae (blood worms)). Leaves o f red alder (Alnus rubra) had been collected from the vicinity o f Spring Creek the previous autumn, and air-  48  dried. Bundles of these leaves were placed in the creek at least 10 days prior to being used in the experiment to allow for conditioning. Crayfish were given two leaves per day (or more as needed). Woody debris was collected from the field as well, and broken into 1 cm x 1 cm pieces to allow for crayfish handling. Crayfish were given at least four pieces daily to ensure a surplus of food. All leaves and wood were thoroughly washed prior to being placed in aquaria to remove any invertebrates or other debris. Invertebrates were provided to the crayfish twice daily; however, the aquaria were closely monitored to ensure that there were always enough to eat while not allowing for a buildup that might have altered aquarium pH, bacteria or oxygen levels. For the juvenile crayfish there were a total of six replicates for each food source, and for adults there were four replicates for each food source. The experiment was carried out from early May until the end of July 2003, at a constant water temperature of 16 °C. Aquaria were cleaned bi-weekly to remove debris and feces, and half of the water was replaced with freshly filtered water from Spring Creek. At each bi-weekly cleaning session, each crayfish was weighed and its O C L measured to assess growth.  Data analysis: Total weight gain and total change in O C L for adults and juveniles for each food source was analyzed separately using the G L M (general linear model) procedure in SAS version 8e (SAS Institude Inc., Cary, North Carolina). The ratio of O C L change to weight gain was also assessed in the same manner. For significant A N O V A results, post-hoc least-squared means (LS means) were calculated to determine which treatments differed from each other.  49  Results Field diet study:  Gut content analysis A total of seven categories were discernable from the foregut contents. These were: deciduous leaves, wood, macrophytes, conifer needles, invertebrates, crayfish parts/molts, and other (unidentifiable organic matter). Juvenile crayfish exhibited a difference in the amount o f macrophytes in the diet between different density treatments (F 2,9) = 8.13, p = 0.0096); however, (  this difference was not a result o f increased or decreased density, as the highest levels o f macrophytic material were found in the 8 juvenile treatment. Levels o f macrophytes in the diet of juveniles in the low density (4) treatment were the lowest, and those in the diet of juveniles in the high density (12) treatment were in the mid-range. A s this difference in diet did not reflect a trend o f altered diet with increased density, and there were no other significant differences in dietary composition between any of the density treatments for juveniles or adults, all juvenile data were pooled together (n = 12) and all adult data were pooled together (n = 12) prior to the comparison between juvenile and adult diets. In addition, there were no differences in gut fullness for adults or juveniles in any o f the different treatments. Juveniles had significantly more leaves and needles in their diets ( M A N O V A : F i,22) = 12.31, p = 0.002 and F i,22) = 13.10 p (  (  = 0.001 respectively) (Figure 3.1a,b) than adults. Juvenile guts also had a significantly more insects than adult guts ( M A N O V A : F(  1>22  ) = 17.59, p = 0.0004); however, for both adults and  juveniles insects were among the smallest components of the diet (0.6% and 1.6% respectively). Adult guts contained more crayfish/molts than the juvenile guts ( M A N O V A : F 22) = 18.98, p = (1;  0.0003); however, the incidence o f cannibalism did not increase with an increase in adult crayfish density, with one incident occurring in the 2 adult treatment (reduced to 1 individual), and 1 incident occurring in the 3 adult treatment (reduced to 1 individual).  50  There were no significant differences in dietary composition between individuals (either juvenile or adult) from within the enclosures and those caught directly from Spring Creek (Figure 3.1 c,d), with the exception o f the proportion o f insects found in the guts ( M A N O V A : F(i,3i) = 7.04, p = 0.01). There were fewer insects found in the guts o f the crayfish caught directly from Spring Creek. Gut content analysis o f the Y O Y juveniles revealed that these individuals had a similar diet to all other crayfish assessed in this study. Woody debris was by far the most represented item in the diet o f the crayfish, followed by leaves, macrophytes and needles (Figure 3.1 e). Insects were the least represented item i n the diet, comprising around 0.5% of the gut contents.  Stable isotope analysis Stable isotopes of carbon and nitrogen did not vary to a great extent with crayfish ontogenetic stage (Figure 3.2). Adult crayfish were slightly more depleted in nitrogen than juvenile or Y O Y crayfish; however, this difference was < 1.0 %o and therefore not large enough to represent a complete change in trophic level (Parkyn et al. 2001). Adult and Y O Y crayfish were very similar in their carbon signatures, while juvenile crayfish were slightly more depleted. Crayfish were not found to be more nitrogen enriched than chironomids or heptageniid mayflies, indicating that they are on the same trophic level as these herbivorous grazers. Crayfish were more nitrogen enriched than leptophlebiid mayflies and all detrital samples. For both leaves and wood, the nitrogen and carbon signals varied between the biofilm scrapings and the entire fragments. Leaf biofilm was considerably more nitrogen enriched than the actual leaves (approximately 4 %o), while wood biofilm was approximately 2 %o more enriched than wood. In both cases this represents a change in trophic level (Peterson and Fry 1987).  51  Laboratory feeding experiment: Overall growth on the invertebrate diet was significantly higher for both adults 4.87, p = 0.03) and juveniles  (F(2,i5)  (F 2,9) = (  = 22.66, p < 0.0001) compared to a diet o f leaves or wood  (Figure 3.3). This same trend was evident for O C L increase. There was no significant difference between weight gain for either adults or juveniles being fed leaves or wood ( L S means post-hoc test p = 0.3 for adults, and p = 0.51 for juveniles), while significant differences between invertebrates and detrital sources were observed for both adults (p = 0.07 for invertebrates vs. leaves, p = 0.01 for invertebrates vs. wood) and juveniles (p < 0.0001 for invertebrates vs. leaves, p < 0.0001 for invertebrates vs. wood). In addition, there were no differences in the length gained per gram o f weight gained ( O C L change/weight change) for any of the treatments.  Discussion Results from my gut content and stable isotope analyses do not support the ontogenetic niche shifts for crayfish diet described by other authors (e.g. Abrahamsson 1966, Lorman and Magnuson 1978, France 1996), despite the fact that a clear growth advantage for juvenile crayfish would be realized from incorporating animal prey into their diets. In fact, my results show that regardless o f whether or not they were in enclosures, juvenile P. leniusculus consumed food types that were the opposite of those purported to be o f most nutritional value to them. In all cases, detrital matter was the largest component o f the diet for both adults and juveniles. It was also the greatest component o f the diet o f the Y O Y crayfish I assessed. The results o f Guan and Wiles (1998) and Stenroth and Nystrom (2003) show P. lenisuculus to be a substantial predator on chironomids, simuliids, and ephemeropterans in lentic systems; however, my results did not lead to the same conclusion. It may be important to note that the studies o f both Guan and Wiles (1998) and Stenroth and Nystrom (2003) were done on P. leniusculus as an introduced 52  species in Europe. Apart from one study (Mason 1975) on the feeding ecology of P. leniusculus in a small woodland stream in British Columbia, little is known about the ecology o f these organisms in their native North American habitats. In addition, my results do not corroborate those o f Mason (1975), who found a greater proportion o f insects in the diets o f both juvenile and adult crayfish. Although juveniles have a physiological need for larger amounts o f protein (Momot 1995, Paglianti and Gherardi 2004), it does not seem as though juvenile P. leniusculus in my system were getting substantial input o f protein through animal sources in their diet. Whitledge and Rabeni (1997) determined the assimilation efficiencies o f several food sources for the crayfish O. luteus and O. punctimanus,  and found that chironomids were much more efficiently  assimilated (92%) than vascular plant detritus (14%). This indicates that a small portion o f the diet may be composed o f protein-based material to maintain an adequate growth rate. Despite the fact that the size and nature o f insect matter may mean a shorter assimilation time in the crayfish guts, my sample size o f 70 crayfish should have yielded some individuals who had recently processed insect matter i f indeed they were doing so to a great extent in this system. The assimilation efficiency results o f Whitledge and Rabeni (1997) described above are corroborated by research showing that the stable isotope signature o f crayfish reflects a diet o f protein-based matter, while gut content studies show that adults ingest a substantial amount o f vascular detritus (Parkyn et al. 2001, Hollows et al. 2002). However, my stable isotope results disagree with this conclusion as well. I would expect values o f carbon and nitrogen to be substantially more enriched (as found by Parkyn et al. (2001) and Hollows et al. (2002)) i f crayfish were predators in my system. I instead found that all ontogenetic stages were at the same trophic level as chironomids, despite the fact that chironomids have been hypothesized to provide the major protein-based food source for several crayfish genera including Pacifastacus (Guan and Wiles 1998, Hollows et al. 2002, Stenroth and Nystrom 2003). 53  M y results show crayfish to be approximately 2 - 3.5%o more enriched than the biofilms on the wood and leaves, putting them one trophic level above these food sources (Peterson and Fry 1987), and indicating their role as detritivores. The carbon signatures o f all crayfish most closely match those o f wood and wood biofilm, agreeing with my gut content analyses and emphasizing the probable importance o f these food types. However, interpretation o f the dual stable isotope plot must be exercised with caution, as crayfish clearly do not assimilate insect and detrital food sources with the same efficiency (see discussion above), which violates a critical assumption of the use o f stable isotopes to verify trophic position (Gannes et al. 1997). Though the results of the isotope plot must be interpreted with caution, my gut-content analysis re-affirms that all stages of P. leniusculus in its native habitat are utilizing detritus and detrital biofilms to a great extent in their diets. Whitledge and Rabeni (1997) suggest that the easilydigested biofilms may contribute significantly to crayfish production given the large amount o f detritus ingested by crayfish. Indeed, i f more detritus was available to crayfish at a lower foraging cost, the energy intake may be higher on this resource despite the lower digestion efficiency. Several others suggest that the fungal/bacterial biofilm on allochthonous detritus is easily digestible and highly nutritious (e.g. M c C l a i n et al. 1992, Hollows et al. 2002). In addition, some researchers disagree that animal material is more efficiently assimilated by crayfish. Gherardi et al. (2004) showed that the assimilation efficiency o f vascular detritus by Austropotamobiuspallipes Procambarus  was 81%, agreeing with the results o f Ilheu and Bernardo (1993) for  clarkii (73%). The former authors attribute this efficiency to the cellulolytic  activity revealed in the hindgut o f A.  pallipes.  The results o f my growth experiment showed no significant difference in the growth rate for either juveniles or adults fed leaves or wood. A steady ingestion o f biofilm from either o f these substrates could have provided a large portion of the energy required for growth, making the overall rates o f weight-gain for these two treatments indistinguishable from one another. 54  While the detrital biofilm may indeed be a nutritious food source, it was clear that there were major fitness consequences (in terms of growth rate) for those crayfish raised on detritus versus those fed invertebrates. Both juvenile and adult P. leniusculus grew significantly more when fed on a diet o f invertebrates, similar to the results o f Paglianti and Gherardi (2004) for A pallipes and P. clarkii raised on animal matter or detritus. The question remains as to why I did not see any substantial ingestion o f invertebrates by either adult or juvenile crayfish. M y only indication that P. leniusculus may attain some nutrition from animal sources in the field was the occurrence o f cannibalism in the two and three adult treatments. Cannibalism by adult P. leniusculus was also documented by Guan and Wiles (1998). Large amounts o f cover and habitat heterogeneity in the areas with higher crayfish density are clearly important features for adult crayfish survival. Despite the fact that crayfish and crayfish molt parts were found in the guts o f several adult specimens, the stable isotope results do not reflect crayfish tissue as a major source o f nutrition. This may indicate that the majority o f food items in this category were molts (as opposed to actual crayfish tissue), ingested primarily for their calcium content. In addition, there was no indication that the occurrence o f cannibalism was higher in the three adult treatment as compared to the two adult treatment, as a single incidence o f cannibalism was observed in both cases. It is possible that the occurrence of cannibalism in this species may be a by-product o f several interacting factors, including habitat heterogeneity, resource availability and timing of molts (which leaves newly molted crayfish in a highly vulnerable state). Other than the single incidences o f cannibalism in the two and three adult treatments, density o f either juvenile or adult crayfish in the enclosures did not affect either gut fullness or gut contents. In addition, the smaller adults within the multiple adult treatments did not exhibit a difference in dietary composition or gut fullness than the larger adults in these treatments. This is not surprising given that all crayfish (except those preying on other crayfish) were exhibiting a 55  large reliance on lower-quality food. Populations o f P. leniusculus in Spring Creek are generally found in areas containing large amounts of conditioned allochthonous debris, so it is not surprising that this constitutes a major part o f the diet. However, it is surprising that crayfish did not choose food sources (such as chironomids or other invertebrates) that would result in a higher growth rate. Overall, this research has shown that the omnivorous crayfish Pacifastacus  leniusculus  does not appear to undergo ontogenetic niche shifts in dietary preference that are commonly described for freshwater crayfish in its native North American habitat. Although there were some ontogenetic differences in the amounts o f specific types o f detritus ingested, all ontogenetic stages o f crayfish appear to be primarily detritivorous, despite large protein requirements for fast-growing juveniles, and a clear demonstration that an invertebrate-based diet would result in higher growth rate. In addition, insects were clearly able to colonize the enclosures (e.g. up to 700 individuals per enclosure have been counted, Bondar and Richardson, unpublished data) and were therefore available as food sources to the crayfish. Density o f crayfish did not appear to change the nature o f the choices selected by most crayfish (e.g. larger ingestion o f lower quality food at higher densities), indicating that presence of conspecifics was not responsible for the ingestion of vascular detritus over invertebrate food sources. These results show that neither ontogeny nor density have major effects on the ecological roles of crayfish in this stream community. Indeed, there were few differences in the insect communities within the enclosures that were dependent on either crayfish ontogenetic stage or density (Bondar and Richardson, unpublished data). Although these omnivores can potentially switch their diets to include food types from several trophic levels, there appear to be constraints preventing them from doing so to a great extent. Further work should focus on the mechanisms behind the selection of low-quality food in both juvenile and adult P. leniusculus in its native stream habitat. 56  Leaves 10.7%  Leaves 35.5% 0.5%  1.4%  Wood 45%  Macro. Vinsects 1.6%  Other 2.7%  /  .12% /  Insects 1.6°/? Needles 4% (c) Wood 35%  Leaves 17%  Needles  Wood 59%  Leaves 14.7%  Other 2.4% Crayfish & molts 2% Needles 7.4% Insects 0.7%  Figure 3.1. Gut contents of adult, juvenile, and young-of-the-year Pacifastacus leniusculus from within the enclosures as well as caught directly from Spring Creek, (a) adults from within the enclosures, (b) juveniles ( 2 Y A ) from within the enclosures, (c) adults caught directly from Spring Creek, (d) juveniles ( 2 Y A ) caught directly from Spring Creek, (e) Y O Y young-of-theyear caught directly from Spring Creek. Percentages shown are averages based on n = 12 for graph (a) n = 12 for graph (b) and n = 5 for graphs (c) (d) and (e).  57  6  YOY crayfish  Juvenile crayfish T Hii < « k' iI  4  Heptageniidae (Ephemeroptera)  T1  *  *1  Chironomidae  i• i  Adult crayfish  1  Biofilm LEAF HtH Leptophlebiidae (Ephemeroptera) m  Biofilm WOOD -2  1—*H  Leaves  -31  -30  -29  W  Wood  -28  5 C 1 3  -27  -26  -25  -24  (%o)  Figure 3.2. Stable isotope signatures o f carbon and nitrogen for three ontogenetic stages of crayfish (Pacifastacus leniusculus) as well as their possible dietary components. Points shown are means, and errors are standard deviations. Sample sizes range from n = 5 for each crayfish, to n = 2 (based on ground samples o f 50 chironomids, 20 mayflies, 10 leaves, and 10 pieces of woody debris). Biofilm samples (n = 2) are based on dried biofilm scrapings from 15 leaves or 15 pieces o f woody debris. A l l samples were collected from Spring Creek.  58  6  Wood  Leaves  Invertebrates  Diet Figure 3.3. Overall growth of both juvenile and adult crayfish (Pacifastacus leniusculus) raised on three different diets in a laboratory setting. Crayfish were housed in 20 L aquaria (one crayfish per aquarium). Food types (conditioned leaves, conditioned woody debris, or invertebrates (Chironomidae (blood worms)), were provided in excess for a period of three months. Mean overall weight change is shown for each food type for both adult (four replicates for each food type) and juvenile (six replicates for each food type) crayfish. Error bars denote standard error.  59  References  Abrahamsson, S . A . 1966. Dynamics of an isolated population o f the crayfish, Astacus astacus Linne. Oikos 17:96-107. Abrahamsson, S.A., and C R . Goldman. 1970. Distribution, density and production o f the crayfish Pacifastacus leniusculus (Dana) in Lake Tahoe, California-Nevada. Oikos 21:83-91. Bolnick, D . I. 2001. Intraspecific competition favors niche width expansion in Drosophila melanogaster. Nature 410:463-466. Bott, R. 1950. Die flusskrebse Europas (Decapoda, Astacidae). Adhandlungen der Senckenbergischen Naturforschenden Gessellschaft. 483:1-36. Correia, A . M . 2003. Food choice by the introduced crayfish Procambarus Zoologici Fennici 40:517-528.  clarkii. Annales  Dorn, N . J . , and J . M . Wojdak. 2004. The role o f omnivorous crayfish in littoral communities. Oecologia 140:150-159. Elser, J. J., C . Junge and C R . Goldman. 1994. Population structure and ecological effects o f the crayfish Pacifastacus leniusculus in Castle Lake, California. Great Basin Naturalist 54:162-169. France, R. 1996. Ontogenetic shift in crayfish 13C as a measure o f land-water ecotonal coupling. Oecologia 107:239-242. Gannes, L . Z . , D . M . O ' B r i e n , and C . Martinez del R i o , C . 1997. Stable isotopes i n A n i m a l ecology: assumptions, caveats and a call for more laboratory experiments. Ecology 78: 12711276. Gherardi, F., P. Acquistapace and G . Santini. 2004. Food selection in freshwater omnivores: a case study o f crayfish Austropotamobius pallipes. A r c h i v fur Hydrobiologie 159:357-376. Guan, R., and P.R. Wiles. 1998. Feeding ecology o f the signal crayfish Pacifastacus in a British lowland river. Aquaculture 169:177-193.  leniusculus  Hamr, P. 1998. Conservation Status o f Canadian Freshwater Crayfishes. W o r l d Wildlife Fund Canada and the Canadian Nature Federation, Toronto, Ontario. Hjelm, J., L . Persson and B . Christensen. 2000. Growth, morphological variation and ontogenetic niche shifts in perch (Perca fluviatilis) in relation to resource availability. Oecologia 122:190-199. Hollows, J.W., Townsend, C . R . , and Collier, K . J . 2002. Diet of the crayfish Paranephrops zealandicus in bush and pasture streams: insights from stable isotopes and stomach analysis. N e w Zealand Journal o f Marine and Freshwater Research 36:129-142. Ilheu, M . and J . M . Bernardo. 1993. Experimental evaluation o f food preference o f red swamp crawfish, Procambarus clarkii: vegetal versus animal. Freshwater Crayfish 9:359-364. 60  Johnson, J.E. 1986. Inventory o f Utah crayfish with notes on current distribution. Great Basin Naturalist 46:625-631. Lodge, D . M . , M . W . Kershner and J.E. A l o o i . 1994. Effects o f an omnivorous crayfish (Orconectes rusticus) on a freshwater littoral rood web. Ecology 75:1265-1281. Lorman, J.G. and J.J. Magnuson. 1978. Role o f crayfishes in aquatic ecosystems. Fisheries 3:810. Mason, J.C. 1975. Crayfish production in a small woodland stream. Freshwater Crayfish 2:449479. M c C l a i n , W.R., W . H . N e i l and D . M . I . Gatlin. 1992. Nutrient profiles o f green and decomposed rice-forages and their utilization by juvenile crayfish (Procambarus clarkii). Aquaculture 101:251-265. Momot, W . T . 1995. Redefining the role o f crayfish in aquatic ecosystems. Review o f Fisheries Science 3:33-63. Nakata, K . , and S. Goshima. 2003. Competition for shelter o f preferred sizes between the native crayfish species Cambarides japonicus and the alien crayfish species Pacifastacus leniusculus in Japan in relation to prior residence, sex difference, and body size. Journal o f Crustacean Biology 23:897-907. Negishi, J.N. and J.S. Richardson. 2003. Responses o f organic matter and macroinvertebrates to placements of boulder clusters in a small stream o f southwestern British Columbia, Canada. Canadian Journal of Fisheries and Aquatic Sciences 60:247-258. Olson, M . H . 1996. Ontogenetic niche shifts in largemouth bass: variability and consequences for first-year growth. Ecology 77:179-190. Paglianti, A . , and F. Gherardi. 2004. Combined effects o f temperature and diet on growth and survival o f young of year crayfish: a comparison between indigenous and invasive species. Journal o f Crustacean Biology 24:140-148. Parkyn, S . M . , K . J . Collier, R.J. and B . J . Hicks. 2001. N e w Zealand stream crayfish: functional omnivores but trophic predators? Freshwater Biology 46:641-652. Peterson, B . J . , and B . Fry. 1987. Stable isotopes in ecosystem studies. Annual Review of Ecology and Systematics 18:293-320. P i m m , S.L. and J.C. Rice. 1987. The dynamics o f multispecies, multi-life-stage models o f aquatic food webs. Theoretical Population Biology 32:303-325. Pockl, M . , and R. Pekny. 2002. Interaction between native and alien species of crayfish in Austria: Case studies. Bulletin Francais de la Peche et de la Pisciculture 367:763-776. Polis, G A . 1984. A g e structure component of niche width and intraspecific resource 61  partitioning: can age groups function as ecological species? American Naturalist 123:541-564. Polis, G . A . and D . R . Strong, D . R . 1996. Food web complexity and community dynamics. American Naturalist 147:813-846. Pringle, C M . , and T. Hamazaki. 1998. The role o f omnivory in a neotropical stream: separating diurnal and nocturnal effects. Ecology 79:269-280. Reece, P.F. and J.S. Richardson. 2000. Benthic macroinvertebrate assemblages o f coastal and continental streams and large rivers o f southwestern British Columbia, Canada. Hydrobiologia 439:77-89. Richardson, J.S. 1992. Food, microhabitat, or both? Macroinvertebrate use o f leaf accumulations in a montane stream. Freshwater Biology 27:169-176. Schofield, K . A . , C M . Pringle, J.L. Meyer and A . B . Sutherland. 2001. The importance o f crayfish in the breakdown o f rhododendron leaf litter. Freshwater Biology 46:1191-1204. Sheldon, A . L . 1989. Reconnaissance o f crayfish populations in western Montana. Montana Department o f Fish, Wildlife and Parks, Montana. Singer, M . S . , and E . A . Bernays. 2003. Understanding omnivory needs a behavioral perspective. Ecology 84:2532-2537. Stenroth, P. and P. Nystrom, P. 2003. Exotic crayfish in a brown water stream: effects on juvenile trout, invertebrates and algae. Freshwater Biology 48:466-475. Svanback, R. and L . Persson. 2004. Individual diet specialization, niche width and population dynamics: implications for trophic polymorphisms. Journal o f A n i m a l Ecology 73:973-982. Svardson, G . 1995. The early history o f signal crayfish introduction into Europe. Freshwater Crayfish 8:68-77. Tavares-Cromar, A . F . and D . D . Williams. 1996. The importance o f temporal resolution in food web analysis: evidence from a detritus-based stream. Ecological Monographs 66:91-113. Whitledge, G . W . and C F . Rabeni. 1997. Energy sources and ecological role o f crayfishes in an Ozark stream: insights from stable isotopes and gut analysis. Canadian Journal of Fisheries and Aquatic Sciences 54:2555-2563.  62  'Chapter 4 Risk-sensitive foraging by juvenile signal crayfish (Pacifastacus leniusculus).  A version o f this chapter has been published. Bondar, C . A . , Zeron, K . and J.S. Richardson. 2006. Risk-sensitive foraging by juvenile signal crayfish (Pacifastacus leniusculus). Canadian Journal of Zoology 84(11): 1693-1697. 1  63  Introduction Omnivory in aquatic food webs has been recognized as a widespread occurrence (Menge and Sutherland 1987, Polis and Strong 1996). However, food choices made by omnivorous organisms are difficult to predict because an understanding o f when and why different food groups are more likely to be consumed is required. Most often it is assumed that nutritional constraints uniquely determine the food mixing behavior o f omnivores; however, several other factors may ultimately play a role (Singer and Bernays 2003). Factors such as predation risk, density o f a preferred food source, biotic interactions (both intraspecific and interspecific), and microhabitat use all contribute to the dietary choices o f an omnivore, which may lead to selection of food sources that are sub-optimal in terms o f nutrition. For example, it has been well established that freshwater predatory invertebrates often incorporate algae and detritus (nutritionally poor food choices) into their diets, though the reasons for this inclusion are poorly understood (Lancaster et al. 2005). The effects o f predation on feeding behavior have traditionally been observed through an organisms' feeding rate on one specific food type (Sih 1982, G i l l i a m and Fraser 1987); however, few studies have investigated how predation risk affects the feeding behavior o f omnivores. Understanding the mechanisms contributing to food choice in omnivorous organisms is important both in terms o f the feeding ecology o f the omnivores themselves, and in terms o f the potential effects of omnivorous organisms on their surrounding communities. The dynamics o f food selection can ultimately dictate whether omnivory stabilizes population, community and food web dynamics (Singer and Bernays 2003). Stream-dwelling crayfish have been shown to be true omnivores (Pimm and Lawton 1978), having both plant and animal-based components in their diets (Whitledge and Rabeni 1997, Parkyn et al. 2001, Hollows et al. 2002). In addition to the omnivory observed within ageclasses, crayfish have also been described to exhibit 'life history omnivory' (Woodward and 64  Hildrew 2002), in that there are shifts i n the feeding behavior o f crayfish through development. Juvenile crayfish have traditionally been described as carnivorous and adults as detritivorous, and this trend has been shown for many crayfish genera (e.g. Abrahamsson 1966, Parkyn et al. 1997, Whitledge and Rabeni 1997). However, the ontogenetic shifts previously described i n the literature may not be ubiquitous to all crayfish genera. In particular, research on the North American signal crayfish, Pacifastacus  leniusculus (Dana, 1852), in its native environment has  documented that juveniles feed primarily on conditioned woody debris found in the stream bed (Bondar et al. 2005), in contrast to the primary food base o f aquatic invertebrates found by other authors (e.g. Stenroth and Nystrom 2003). While a certain amount o f nutrition is clearly available from detrital food sources via the biofilm (Whitledge and Rabeni 1997), growth rates were significantly higher when crayfish were raised on a diet o f invertebrates i n comparison to detritus (Bondar et al. 2005). Juvenile P. leniusculus are therefore making foraging decisions that are not entirely based on the nutritive value o f the food source, leading to the question about what determines the behavioral aspects o f food choice in this omnivore. There are several reasons why juvenile P. leniusculus may select food that is not optimal in terms o f its nutritional value. Members of this species are aggressive, and exhibit strong cannibalistic tendencies at several ontogenetic stages, especially by adults (Guan and Wiles 1998). Juvenile and adult P. leniusculus are found in similar stream habitats in their native environment, in areas o f low flow and high debris buildup ( C . A . Bondar unpublished data). Although crayfish in these areas can be quite dense (see chapter 6), the woody debris and other vascular detritus provide an abundance o f refugia. In addition to the threat of intraspecific cannibalism, there is a threat o f predation for juvenile crayfish from other sources. Cutthroat trout (Oncorhynchus  clarkii (Richardson, 1836)) are numerous in small streams containing  crayfish, and prey on juvenile P. leniusculus in Spring Creek ( C . A . Bondar personal 65  observation). Little is known about the relationship between juvenile crayfish and juvenile cutthroat trout; however, these organisms are known to co-exist in similar areas of the stream. I used field-based choice experiments to assess the food selections made by P. leniusculus  juveniles. Specifically I asked whether juvenile crayfish would select invertebrate  prey over woody debris when given a choice in a non-threatening environment, and if the feeding behavior of these organisms would be influenced by the presence of conspecifics (adult or juvenile crayfish) or predators (cutthroat trout). Due to the aggressive, cannibalistic nature of this species I expected there to be a strong negative impact of the presence of adult crayfish (no food choice, attempting to seek refuge instead of feeding). I used both male and female adult crayfish in this study to determine whether there was an influence of adult gender on juvenile behavior. Similarly, as adult cutthroat trout have been shown to actively prey on juvenile P. leniusculus,  I expected to see a strong negative influence of their presence. The presence of  conspecific juvenile crayfish was expected to alter the time to a decision, but not the overall choice, and the presence of YOY cutthroat trout was not expected to alter the behavior of the juvenile crayfish, as there is little size difference between these two organisms.  Methods  Experiments: The choice experiments took place in Spring Creek (see descriptions in Reece and Richardson 2000, Negishi and Richardson 2003), a second-order stream located in the University of British Columbia's Malcolm Knapp Research Forest. Young of the year (YOY) juvenile P. leniusculus  (occipital carapace length [OCL] average 11.01 mm, range 8.2 to 12.5 mm) were  hand-netted and kept in 25 L plastic containers without food for 48 hours prior to being tested. Lids were kept on the containers to minimize disturbance and predation by birds and mammals. The vessels containing the crayfish were kept in the stream to maintain cool temperatures. River 66  rocks (3-5 cm) purchased from a landscape supplier were thoroughly washed and placed into the containers with the crayfish to provide them with hiding places without providing any food. Small (5 L ) plastic vessels were used for the food choice tests. Purified water from the laboratory was brought to the stream site so that the water that was free o f any chemical signals that might influence the crayfish. The food choices added at random places to each vessel were: five pieces o f conditioned woody debris (approximately 1 cm x 1 cm), ten live chironomid larvae, and five live baetid mayfly nymphs. The invertebrates were chosen based on their occurrence in diets o f crayfish (including P. leniusculus) from other research (Guan and Wiles 1998, Hollows et al. 2002). Food sources were added to the enclosures two minutes prior to the addition o f the crayfish. Crayfish were placed in the enclosures (one per trial) within the confines of a piece o f 5.25cm P V C tubing held vertically, and were given three minutes to acclimatize prior to being released. Subsequent to the release o f the crayfish the P V C tubing was removed from the vessel. The first item eaten by the crayfish was taken to be a positive trial for that food source. Time to consumption was also recorded. If a trial progressed for 20 minutes without consumption o f a food item, it was not included. Only one trial was performed in each enclosure before rinsing and re-adding the food sources. A total of 19 trials were performed this way, each crayfish was used only once. For the treatments involving the reaction o f the juvenile crayfish to potential predators or conspecifics, a basket (13.0cm diameter) made o f 0.32cmplastic mesh was hung in the center of the vessel so that the presence o f the added fish or crayfish could be experienced by the juvenile crayfish without the surface area o f the arena being altered in any way. Mesh was used so that chemical signals could easily permeate the vessel, and so that the entire floor o f the treatment area would be visible once the basket was in place. The treatment organism was added to the basket, and the basket was suspended over the treatment area for 2 minutes prior to the addition of the food sources. Trials then proceeded as outlined above. A total o f 14 juvenile feeding trials 67  were performed for each o f the following added organisms: adult female crayfish ( O C L (occipital carapace length) < 30mm, adult male crayfish ( O C L < 30mm), conspecific juvenile crayfish ( O C L approximately 11mm), juvenile (mass approximately 0.5g) and adult (mass approximately lOg) cutthroat trout. The added organisms were caught by hand-netting in Spring Creek, except for adult cutthroat trout, which were caught in minnow traps. These organisms were held without food in minnow traps (adult crayfish and fish) or plastic vessels (juvenile fish) within the stream for 24 hours prior to the experiment. A l l treatments were conducted during daylight hours, in a shaded area o f the stream bank.  Data Analysis: Time to a choice data were analyzed in a 1-way A N O V A using the M I X E D procedure in S A S version 8e (SAS Institute, Cary, N C ) for the choice made by the juvenile crayfish for all treatments. The time to a choice for each treatment was assessed in the same manner, and treatments were compared using post-hoc contrasts.  Results For each o f the choice treatments apart from the cutthroat trout, there was one trial for which the crayfish did not make a choice within 20 minutes. Otherwise, all trials for all treatments yielded the same result: the crayfish ate the chironomid larvae. It was noted i n a few instances that the crayfish attempted to obtain a mayfly; however, they were never successful in this endeavor. Often, crayfish would climb over the wood fragments in order to reach a chironomid, or would turn them over with their chelipeds i f a chironomid had crawled underneath it. The crayfish never selected the wood as a food source in these trials. The amount o f time before a food item was consumed by the crayfish varied between treatments (F  (4>6 7 )  = 10.35, p < 0.0001) Figure 4.1). It was significantly faster when the crayfish 68  were alone or with conspecific juveniles or cutthroat trout as opposed to when they were with adult crayfish (significant contrasts for alone versus with adult female (p < 0.0001 (D.F.= 67)) or adult male (p < 0.0001 (D.F.= 67)). The time to consumption was 285% and 242% longer in the presence o f adult female and male crayfish, respectively. There was no statistically significant difference in the time it took for the juvenile crayfish to consume a food item in the presence of either female versus male adult crayfish. Although there were no statistically significant differences in the time to consumption for the alone, conspecific juvenile crayfish or cutthroat trout trials, there was a trend of increased time with any introduced organism. Time to consumption was 31%, 64% and 87% longer in the presence of Y O Y cutthroat trout, conspecific juvenile crayfish and adult cutthroat trout, respectively. Although not statistically significant, there was a trend o f increased time to consumption in the presence o f adult cutthroat trout (p = 0.10). In the trials where juvenile crayfish were alone, several unsuccessful attempts to obtain mayflies occurred. This happened with 26% o f individuals (5 o f 19 individuals), as opposed to an average o f 6% across all other trials (4 o f 70 individuals). In each o f the 5 individuals that attempted to obtain mayflies in the juvenile crayfish alone treatment, there was an average o f 3 tries per individual, while there was only one for those crayfish in the other treatments where these attempts occurred. The movement patterns o f the crayfish differed between trials. In trials with adult crayfish and adult cutthroat trout, the juvenile crayfish were still and movements were slow as opposed to when they were alone or with conspecific juveniles or Y O Y fish. Movements became especially limited, or would cease altogether when the adult crayfish or fish within the cages were active.  69  Discussion Although gut content and stable isotope analyses have demonstrated that young-of-theyear juvenile P. leniusculus in Spring Creek have a substantial portion o f woody debris (and almost no invertebrates) in their diet (Bondar et al. 2005), wood was not the food source consumed under any circumstances during this experiment. W h y are invertebrates not a larger part o f the natural diet of juvenile P. leniusculus?  The answer may come in part from the clear  inhibition exhibited by the juvenile crayfish in the presence o f adult crayfish, and to a lesser extent the large cutthroat trout. Despite the fact that the larval chironomids were always chosen, the time to consumption was much longer, suggesting a degree o f inhibition. In addition, the movement patterns o f the juvenile crayfish in the presence o f adult crayfish and fish suggested a greater degree o f caution. The threat o f cannibalism by larger conspecifics has been shown to alter foraging decisions in other organisms as well. For example, Sih (1992) documented that avoidance o f cannibalistic adults for juvenile aquatic insects (Notonecta hoffmanni) was achieved by a reduction in movement, similar to the results o f my study. If the threat o f cannibalism to juvenile crayfish was causing a restriction o f movement, the ingestion o f woody debris may be a by-product o f the increased risk. Since crayfish are omnivorous, and therefore have the capacity to feed on several food-types, the presence o f adults may shift juveniles to a diet that is composed largely o f detritus, a more easily obtained food source. The woody debris may be serve a dual function as a refuge from predators and a food source. Although chironomids and other invertebrates may also be present in areas with an abundance o f woody debris, juvenile crayfish may refrain from ingesting them i f a great deal o f movement is required to do so. The occasional ingestion o f invertebrates may occur for this reason, although stable isotope data (Bondar et al. 2005) show that generally this does not occur to a large extent.  70  Although not statistically significant, my results show a pattern o f reduced movement and inhibition o f feeding behavior o f the juvenile crayfish in presence of the large cutthroat trout, leading us to speculate that adult cutthroat trout have impacts on the ecology o f these organisms. The lack o f significance found in my study may be a result o f the fact that my experiments took place during daylight hours as opposed to at night. Indeed, Nystrom (2005) found a greater effect offish on the activity level of P. leniusculus outside o f refugia during the night. However, Soderback (1994) researched the behavior of juvenile P. leniusculus in the presence o f adult European perch and found a strong antipredator response in pool-based experiments conducted during daylight hours. The latter study found the use o f refugia in experimental pools was greater in the presence o f perch. Although I did not use refugia in this study, I speculate that larger pieces o f woody debris in Spring Creek may serve such a function for juvenile crayfish, and serve secondarily as a food source. Further work should incorporate this possibility by providing juvenile crayfish with a choice between edible and non-edible types o f refugia. Additionally, the effects o f cutthroat trout on juvenile crayfish behavior should be investigated at several times o f day and night. When juvenile crayfish were alone in the choice trials, several unsuccessful attempts to obtain the mayflies occurred. Crayfish were clearly unable to obtain the mayflies, as I did not observe any successful captures. However, they were almost always successful in capturing a chironomid on their first attempt. This shows that there may be some food sources that would be preferable to the crayfish but are too costly to obtain both in terms of energy expenditure and the potential exposure to predators through increased movement (since few attempts to obtain mayflies occurred when any other organism was present in the vessel). Several researchers have speculated that adult crayfish are detritivorous due to their inability to obtain small, fast-moving invertebrates in their large chelipeds (Abrahamsson 1966, Nystrom et al. 1999). This study shows that the same may be true of juvenile crayfish for certain fast-moving invertebrates, such 71  as the baetid mayflies used in my experimental vessels. The fact that the crayfish were always able to obtain a chironomid, even when they had to overturn wood pieces to find them, shows that this should be a likely food source for them in the absence o f predation risk in a natural setting. M y research has demonstrated that predation risk can affect the feeding behavior of omnivores, and I speculate that this may be the reason for the inclusion o f nutritionally-poor items in the diet in a natural setting. The decreased movement patterns, and the increased time to food consumption may be contributing factors to the seemingly 'poor' diet of juvenile P. leniusculus in Spring Creek. Feeding behavior o f omnivorous organisms presents ecologists with a substantial challenge. However, detailed studies on feeding behavior w i l l provide a broader insight into the circumstances that govern when a switch i n food choice takes place. This i n turn w i l l lead to a greater knowledge o f the complex web o f ecological impacts that omnivores have on their communities.  72  800  600 A  Figure 4.1. Time to first food choice by young-of-the-year Pacifastacus leniusculus alone and in the presence of various other members o f the stream community. In all cases the first food choice was a chironomid. A statistically significant difference in the time to choice was detected between the treatments containing adult crayfish and all other treatments ( A N O V A post-hoc contrast, p = 0.0001 for both adult female and male crayfish vs. all other treatments). Numbers of replicates for each trial was n = 19 for juvenile crayfish alone, and n = 14 for each of the other trials with added individuals. Error bars indicate standard error.  73  References Abrahamsson, S.A.1966. Dynamics o f an isolated population o f the crayfish, Astacus astacus Linne. Oikos, 17: 96-107. Bondar, C . A . , K . Bottriell, K . Zeron and J.S. Richardson. 2005. Does tropic position o f the omnivorous signal crayfish (Pacifastacus leniusculus) in a stream food web vary with life history stage or density? Canadian Journal o f Fisheries and Aquatic Sciences 62: 2632-2639. Gilliam, J.F. and D . F . Fraser. 1987. Habitat selection under predation hazard: test o f a model with foraging minnows. Ecology 68(6): 1856-1862. Guan, R. and P . R . Wiles. 1998. Feeding ecology o f the signal crayfish Pacifastacus in a British lowland river. Aquaculture 169: 177-193.  leniusculus  Hollows, J.W., C R . Townsend and K . J . Collier. 2002. Diet o f the crayfish Paranephrops zealandicus i n bush and pasture streams: insights from stable isotopes and stomach analysis. N e w Zealand Journal o f Marine and Freshwater Research 36: 129-142. Lancaster, J., D . C Bradley, A . Hogan and S. Waldron.2005. Intraguild omnivory i n predatory stream insects. Journal o f A n i m a l Ecology 74: 619-629. Menge, B . A . and J.P. Sutherland. 1987. Community regulation: variation in disturbance, competition, and predation in relation to environmental stress and recruitment. American Naturalist 130: 730-757. Negishi, J . N . and J.S. Richardson. 2003. Responses o f organic matter and macroinvertebrates to placements o f boulder clusters in a small stream o f southwestern British Columbia, Canada. Canadian Journal o f Fisheries and Aquatic Sciences 60: 247-258. Nystrom, P., C B r o n m a r k and W . Graneli. 1999. Influence of an exotic and a native crayfish species on a littoral benthic community. Oikos 85: 545-553. Nystrom, P. 2005. Non-lethal predator effects on the performance o f a native and an exotic crayfish species. Freshwater Biology 50: 1938-1949. Parkyn, S . M . , C F . Rabeni and K . J . Collier. 1997. Effects of crayfish (Paranephrops planifrdns: Parastacidae) on in-stream processes and benthic faunas: a density manipulation experiment. N e w Zealand Journal o f Marine and Freshwater Research 31: 685-692. Parkyn, S . M . , K . J . Collier and B . J . Hicks. 2001. N e w Zealand stream crayfish: functional omnivores but trophic predators? Freshwater Biology 46:641-652. Pimm, S.L. and J . H . Lawton. 1978. O n feeding on more than one trophic level. Nature (London), 275: 542-544. Polis, G . A . and D . R . Strong. 1996. Food web complexity and community dynamics. American Naturalist 147: 813-846.  74  Reece, P.F. and J.S. Richardson. 2000. Benthic macroinvertebrate assemblages of coastal and continental streams and large rivers o f southwestern British Columbia, Canada. Hydrobiologia,  439:77-89. Sih, A . 1982. Foraging strategies and the avoidance of predation by an aquatic insect, Notonecta hoffmanni. Ecology 63(3): 786-796. Sih, A . 1992. Prey uncertainty and the balancing o f antipredator and feeding needs. American Naturalist 139(5): 1052-1069. Singer, M . S . and E . A . Beraays. 2003. Understanding omnivory needs a behavioral perspective. Ecology 84(10): 2532-2537. Soderback B . 1994. Interactions among juveniles o f two freshwater crayfish species and a predatory fish. Oecologia 100(3):229-235. Stenroth, P. and P. Nystrom. 2003. Exotic crayfish in a brown water stream: effects on juvenile trout, invertebrates and algae. Freshwater Biology 48: 466-475. Whitledge, G . W . and C F . Rabeni. 1997. Energy sources and ecological role o f crayfishes in an Ozark stream: insights from stable isotopes and gut analysis. Canadian Journal of Fisheries and Aquatic Sciences 54: 2555-2563. Woodward, G . and A . G . Hildrew. 2002. Body-size determinants of niche overlap and intraguild predation within a complex food web. Journal o f A n i m a l Ecology 71: 1063-1074.  75  'Chapter 5 Stage-specific interactions between dominant consumers within a small stream ecosystem: direct and indirect consequences.  ' A version of this chapter has been submitted for publication. Bondar, C A . and J.S. Richardson. Stage-specific interactions between dominant consumers within a small stream ecosystem: direct and indirect consequences. Ecology.  76  Introduction Direct and indirect interactions between dominant members o f a freshwater community have important implications for the surrounding ecosystem. Negative (or positive) effects of one organism on another that result in an altered food choice can affect the direction o f ecosystem processes (Lima and D i l l 1990, Werner and Anholt 1996). This is especially important in systems with omnivorous organisms that may switch to lower quality food sources in the presence o f a predator or competitor. Further, the process o f ontogeny must be taken into account as ecologists continue to demonstrate the importance of ontogenetic stage-specificity in interspecific interactions (e.g. Rudolf 2006). There is great potential for variation in both the strength and the sign o f interactions between organisms over their lifespan, especially with organisms that vary greatly in size or ecological niche through ontogeny. If two organisms occupy a similar environment though their lifespan, the interactions between different ontogenetic stages are as important as those between adult stages, although they are rarely investigated (although see Persson et al. 1999 and Woodward and Hildrew 2002). Food web models that assume constant interaction strengths between species can therefore yield misleading results. Investigating the ontogenetic stage-specific interactions between two species is imperative when the species occupy a similar environment though several ontogenetic stages. For example, freshwater crayfish and fish are known to inhabit similar areas in small stream environments. A large number of studies have investigated the relationships between crayfish and fish (e.g. Englund 1999, Englund and Kruppa 2000, Usio and Townsend 2000, Dorn and Mittelbach 2001), as these two organisms are often dominant consumers in small stream ecosystems. In several cases, strong interactions have been documented between adults of one species and juveniles or young-of-the-year ( Y O Y ) o f the other.  The relationship between fish  and crayfish has the added dimension that crayfish are omnivorous consumers that have been 77  shown to alter feeding behavior in the presence o f a threatening organism (Bondar et al. 2006), which can result in a wide variety o f ecosystem-level effects. Adult crayfish have been shown to have negative effects on Y O Y fish through direct predation (Rubin and Svensson 1993, Mueller et al. 2006) and through exclusion o f Y O Y fish from sheltered areas (Rahel and Stein 1988, Griffiths et al. 2006), thereby making them more vulnerable to predation and to increased energy expenditure to remain in the currents o f the water column. Large fish have been shown to prey directly on Y O Y crayfish (Hein et al. 2006, Seiler and Turner 2004), and have even been shown to influence the size-structure o f the slowgrowing crayfish populations. The aforementioned studies suggest that there are substantial ontogenetic components to the interactions between stream-dwelling fish and crayfish. However, most previous research on this subject has focused specifically on adults o f one species and Y O Y of the other, which gives a one-sided view o f the ontogenetic interactions within each specific system. Few studies have looked at a larger spectrum o f ontogenetic interactions between fish and crayfish, including Y O Y and adult stages of each organism. The interactions between Y O Y fish and Y O Y crayfish have not been investigated and may be more complex than those across age classes. The signal crayfish, Pacifastacus leniusculus, has been the subject o f several studies investigating the interactions between fish and crayfish (e.g. Griffiths et al. 2006, Guan and Wiles 1997, Stenroth and Nystrom 2003), as it is abundant in several areas of the world. However, in all o f the aforementioned study systems P. leniusculus is an introduced organism. It has recently been demonstrated (Bondar et al. 2005) that the ecology of P. leniusculus in its native environment may be substantially different from where it has been introduced. In one o f the few studies undertaken to investigate the crayfish-fish interactions of P. leniusculus in its native environment, Zhang et al. (2004) studied the interaction o f adult P. leniusculus and adult cutthroat trout (Oncorhynchus clarkii), in terms o f the effects on growth of crayfish and fish, and 78  the invertebrate assemblages and organic matter accumulation in experimental enclosures. Although no significant interactions between the adults o f these two species were found, the potential for interactions between other ontogenetic stages of the signal crayfish and cutthroat trout in their native environment merits investigation. In this study I assessed the interactions between adult fish and crayfish (see also Zhang et al. 2004), the effects o f adults o f each species on Y O Y o f the other species, and finally, I look at interactions between Y O Y o f both species. I determined the interactions both directly (in terms of individual mass change during the experiment), and indirectly (in terms o f invertebrate community composition, and accumulation o f organic and inorganic matter measured between treatments). I predicted that interactions between adults o f one and Y O Y of the other would result in negative effects on the Y O Y due to the threat o f predation. Interactions between Y O Y crayfish and fish and adult crayfish and fish were hypothesized to be of a competitive nature, as these organisms have been observed co-habiting in similar small-stream habitats and may have similar dietary components (Dorn and Mittelbach 1999).  Ecologists are becoming increasingly aware (e.g. Mcintosh, Peckarsky and Taylor 2004) that environmental heterogeneity may render the interactions and effects of organisms on the surrounding ecosystem dependent on the context o f the microhabitat. The signal crayfish and the cutthroat trout inhabit similar microhabitat areas o f the small stream environment (personal observation), areas o f low flow near the stream bank with debris and areas o f refuge. Large cutthroat trout have been observed using refugia similar to what a crayfish would use (e.g. woody debris, large rocks), leading us to hypothesize that there would be a microhabitat-specific component to the effects o f both fish and crayfish between the leaf-pack and benthic areas o f the enclosures.  79  Methods Experiment: This experiment took place in Spring Creek a second order stream, located in the M a l c o l m Knapp Research Forest in the Coastal Western Hemlock biogeoclimatic zone o f British Columbia (49°18'40"N 122°32'40"W) (average stream width = 2m, average stream depth 45cm). I carried out a randomized block experiment with nine treatments in five blocks for six weeks during the months o f June and July 2003 when the average stream temperature was between 10°C and 12°C, and the flow level o f the stream was between 0.006 - 0.005m /s. A 3  total o f 45 enclosures (length 90 cm X width 90 cm X height 50 cm) were dug into the cobbled streambed to a depth of 30 cm. These enclosures were constructed out o f 2.63 cm P V C pipe and the bottom and all sides were covered with 0.5 cm galvanized steel hardware mesh. This size of mesh was used so that the Y O Y crayfish and cutthroat trout would not be able to escape the enclosures. Each enclosure was outfitted with two general microhabitat types. First, leaf packs were placed at each corner o f the enclosure (four leaf packs per enclosure) to provide an area of high debris accumulation and lower water flow. Leaf packs were expected to provide shelter for fish, crayfish and other invertebrates, as well as to be a food source for crayfish and other invertebrates. Senescent red alder (Alnus rubra) leaves that had been collected from the stream bank the previous fall were washed and dried in order to allow for accurate measurement. Subsequent to being weighed (5 g per leaf pack), the leaves were re-wetted and tied together using garden ties. The leaf packs were fastened directly onto the mesh o f the enclosure in each o f the four corners. Second, the central benthic area o f the enclosures was free o f leaf litter and characterized by a higher water flow. A gravel basket was placed in the center o f each enclosure to allow for a uniform sampling o f the benthos subsequent to the experiment. A 18.75 cm plastic disc was 80  used as the base for the gravel basket, and plastic hardware cloth (1 cm mesh spacing) was attached around the perimeter to create a basket with sides that were 10 cm high. Each basket was then filled with 1 cm pea gravel to 5 cm depth, and then filled with 3 to 5 cm of river rock before being dug into the center o f the enclosure to a depth ensuring that the top o f the gravel basket was flush with the stream bed. The pea gravel and river rock was purchased from a garden supply center so that each gravel basket began the experiment with no C P O M (coarse particulate organic matter), F P O M (fine particulate organic matter), fine inorganic sediment, or organisms. The enclosures were left in the stream for 1 week prior to the addition o f the treatment organisms, to allow for the gravel baskets and leaf packs to become conditioned and colonized with invertebrates. Large cutthroat trout were trapped in minnow traps baited with salmon roe. The traps were placed in large pools. Y O Y cutthroat trout and all crayfish were caught by handnetting. Each Y O Y crayfish was measured (average O C L length 11.2 mm), weighed (average mass 0.91 g) and individually marked using visible implant elastomer (Northwest Marine Technologies) prior to being placed i n the appropriate enclosure. Adult crayfish were weighed (average mass 22.9 g) and measured (average O C L 32.5 mm) and adult and Y O Y cutthroat were weighed (average masses 8.1 g and 0.45 g respectively) prior to being placed in the enclosures. The treatments were as follows: 1 adult crayfish; 1 adult crayfish + 1 adult fish; 1 adult crayfish + 6 Y O Y fish; 8 Y O Y crayfish; 8 Y O Y crayfish + 1 adult fish; 8 Y O Y crayfish + 6 Y O Y fish; 1 adult fish; 6 Y O Y fish and a control with no fish or crayfish. Treatment densities o f fish and crayfish were based on my observations o f natural densities o f the organisms in the field as well as estimates o f large cutthroat density from a previous study in Spring Creek (Boss and Richardson 2002). Subsequent to the addition o f the animals, each enclosure was sealed at the top with 0.5 cm galvanized steel hardware mesh to allow for maximum light exposure while keeping the treatment organisms in and potential predators out. 81  The experiment began in early July and ran for a total o f 6 weeks. The outsides o f the enclosures were brushed twice weekly to prevent debris build-up. After the 6 week period, the leaf packs and gravel basket samples were taken from the enclosures and preserved in formaldehyde. L e a f packs were placed directly into a formaldehyde solution; however, the gravel basket samples were thoroughly rinsed through a series o f sieves (4 cm, 5 m m , 63 pm) i n order to remove the pea gravel and river rocks while retaining all invertebrates. The remaining sample was then preserved i n formaldehyde. The fish and crayfish from within the enclosures were weighed, measured and released.  Data Processing: In the lab, leaf packs were washed thoroughly to remove coarse particulate organic matter ( C P O M ) , fine particulate organic matter ( F P O M ) , inorganic sediment and invertebrates. The leaves were dried and weighed. The invertebrates were quantified and identified to genus, and the remaining debris was fractioned into C P O M and F P O M . These samples were dried and ashed, and from the post-ashing mass the inorganic sediment was quantified. The amount o f C P O M , F P O M and inorganic sediment was standardized to the mass o f leaves remaining, i.e., g per g leaf mass. The invertebrates in the gravel basket samples were quantified and identified, and the remaining debris was fractioned as above.  Data Analysis: The change in biomass o f fish and crayfish in each o f the enclosures was calculated for each treatment as simply the collective mass o f all organisms at the end o f the experiment minus the mass o f all organisms at the start. Growth o f each individual Y O Y and adult crayfish and adult fish was assessed and.calculated as a percentage o f the original mass o f the organism. Y O Y fish were individually weighed; however, since they were not uniquely marked they could 82  not be individually assessed for growth. Due to the fact that there was differential survival o f the Y O Y fish, I also calculated an average individual size both before and after the experiment based on the surviving individuals. Differences in individual growth were assessed using a two-way A N O V A for block design, and I used the probability o f differences in the post-hoc comparison o f least square means ( S A S P R O C G L M , S A S Institute, Cary, N C ) to assess differences between specific treatments. Survival o f individuals within enclosures was calculated as the average number remaining at the end o f the experiment divided by the number at the beginning o f the experiment. Data from the leaf packs and gravel baskets were analyzed using two-way A N O V A s for block design. Probability o f differences i n the post-hoc comparison o f least square means was used to assess differences between specific treatments, and significance levels were altered using the Bonferonni correction. Significant results were further analyzed in a series o f reduced factorial models involving one ontogenetic stage o f fish and one ontogenetic stage o f crayfish. For example, for each significant result, four reduced models (e.g. one factorial would include adult crayfish, adult fish, adult fish + adult crayfish, and control, another would include adult crayfish, Y O Y fish, adult crayfish + Y O Y fish, and control) were carried out in order to assess crayfish effects, fish effects, and the interactions between fish and crayfish. The leaf pack invertebrates were assessed in two ways: first, the total number of each o f the abundant invertebrates was analyzed. Second, the total number o f each invertebrate per gram of dry leaf mass was analyzed to account for the significant differences in the amount o f leaf 'habitat' for the invertebrates. In addition to analyzing the abundant invertebrates on the leaf packs, I assessed the total overall number of invertebrates and estimated a Simpson's Index o f diversity on the entire leaf pack community.  83  Results Growth o f Fish and Crayfish: The overall biomass o f adult fish, adult crayfish and Y O Y crayfish in the enclosures did not change significantly during the experiment ( A N O V A , p > 0.05). However; the overall biomass o f Y O Y fish in the enclosures varied significantly between treatments (Figure 5.1, A N O V A , p = 0.0007,  F ,7) (2  = 24.27). Y O Y fish had a significantly lower biomass when paired  with either adult or Y O Y crayfish (Figure 5.1a), compared to when they were alone (post-hoc comparison o f least square means, p = 0.003 and p < 0.001, respectively), and the effect was significantly greater in the presence o f Y O Y crayfish than with adult crayfish (post-hoc comparison o f least square means p = 0.035). The average difference in biomass o f Y O Y fish was 94% lower in the presence o f adult crayfish and 149% lower in the presence o f Y O Y crayfish than when alone. When I accounted for the differential survival o f the Y O Y fish, 1 found a significantly lower average individual biomass o f Y O Y fish (p = 0.0512, F(2j) = 4.68 Figure 5.1b) when paired with either adult or Y O Y crayfish. There was no significant difference between treatments o f individual growth o f either adult crayfish (Figure 5.2) or adult fish during the experiment. However, individual Y O Y crayfish grew significantly more (post-hoc comparison o f least square means, p = 0.02, a 37% increase in growth) when they were in an enclosure with Y O Y fish (Figure 5.2) than in other treatments. There was also a trend o f lower individual Y O Y crayfish growth in the presence o f adult fish compared to when they were alone (a 20% decrease in growth, not statistically significant). Survivorship of all adults (fish and crayfish) was 100%. Survivorship o f Y O Y crayfish alone and with adult fish was the same (87.5%), while it declined for Y O Y crayfish with juvenile fish (75%>). Survivorship of juvenile fish (93% alone), declined in the presence o f adult crayfish (90%) and further declined in the presence of juvenile crayfish (83.3%). 84  Leaf pack dry mass: There were significant differences between treatments with respect to remaining leaf pack dry mass ( A N O V A , p < 0.0001,  F(g,30)  = 32.8). Adult crayfish treatments had significantly  lower leaf pack mass than Y O Y crayfish treatments, fish alone treatments or the control (Figure 5.3), post hoc comparisons between least squared means o f adult crayfish treatments and other treatments, p all < 0.001). W i t h respect to the adult crayfish treatments, the reduced-factorial A N O V A s showed that crayfish had a significant negative effect on remaining leaf mass, whereas fish did not (adult crayfish p < 0.0001; adult fish p = 0.153; Y O Y fish p = 0.426). There were no significant interactions on leaf pack mass between adult crayfish and either adult or Y O Y fish (adult crayfish*adult fish, p = 0.610, adult crayfish*YOY fish, p = 0.453). Y O Y crayfish treatments had significantly more leaf mass remaining than the adult crayfish treatments (Figure 5.3), all post hoc comparisons between adult and Y O Y crayfish treatments had p < 0.001). There was a significantly larger amount of leaf mass remaining in the Y O Y crayfish + Y O Y fish treatment compared to the Y O Y crayfish treatment alone (p = 0.005). In addition, the reducedfactorial showed a significant interaction between Y O Y fish and Y O Y crayfish (p = 0.001). The Y O Y crayfish alone treatment was not significantly different from the control (p = 0.068); however, significantly more leaf litter remained in the Y O Y crayfish + Y O Y fish treatment than in the control (p < 0.0001). The leaf pack dry masses were also analyzed per gram o f crayfish mass, to correct for the larger biomass o f the adult crayfish. The standardization made no difference to the relative effect sizes and their significance, as reported above. A l s o , there were no significant differences between the fish alone treatments and the control with respect to leaf pack mass remaining.  Leaf Pack Communities: 85  For the invertebrate communities on the leaf packs, the results were the same whether actual numbers o f invertebrates or numbers per leaf mass were analyzed. For this reason I w i l l present only the results corrected per gram o f leaf. The most abundant members of the leaf pack community were Chironomini, Tanytarsini, Orthocladiinae, Tanypodinae, and large (>lmm) Tipulidae, dipteran pupae, Rhyacophilidae and Zapada (Figure 5.4). For each o f these invertebrates, as well as for the total number o f invertebrates, the results o f A N O V A controlling for blocks were the same (Table 5.1): crayfish had a significant negative effect on the number of these invertebrates. There were no differences between the effects o f adult or Y O Y crayfish; however, all crayfish treatments were significantly different from the fish alone treatments and the controls (Figure 5.4, Table 5.1). The reduced model, factorial analyses showed that crayfish had significant effects on the leaf pack biota (all crayfish effect terms p < 0.01), whereas fish had no significant effects, and there were no crayfish*fish interactions. The Simpson's index showed that crayfish (either adult or Y O Y ) did not affect the species diversity ( A N O V A , p = 0.249,  F ,22) (8  = 1.4), just the total overall number o f invertebrates  ( A N O V A , p < 0.0001, F 8,22) = 39.25; reduced factorial model for crayfish effects in all (  treatments p < 0.0001).  Fractioned Matter from the L e a f Packs: The C P O M , F P O M and inorganic sediment from the leaf packs showed the same trend as for the invertebrates.  Both adult and Y O Y crayfish treatments had significant negative effects  on the amount o f fractioned matter present (Figure 5.5) i n comparison to the fish alone treatments and the control. The A V O V A was significant for all three parameters between treatments (p = 0.0001, p < 0.0001, p < 0.0001, F  (8;2  i ) = 7.66, 34.11, 13.65, for C P O M , F P O M  and inorganic sediment respectively). The reduced factorial analysis showed that crayfish  86  presence resulted i n significantly less o f the fractioned matter, whereas the fish did not, and there were no significant crayfish*fish interactions.  Gravel Basket Community: The most abundant invertebrates i n the gravel baskets were: Chironomini, Tanypodinae, Baetis, Orthocladiinae and Chloroperlidae. There were significant block effects for the first four analyses by taxon (p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0004, and 12.15, and 8.06 respectively).  F , 2) (4  2  = 21.06, 17.82,  There were no significant differences i n densities o f  invertebrates by treatment apart from Chloroperlidae (p = 0.0002,  F ,22) (8  = 6.53). This result  demonstrates that the effects o f crayfish in the gravel basket microhabitat were not as general as for the leaf pack microhabitat. The Simpson's index o f diversity showed that there were no significant treatment effects on species diversity or evenness (p = 0.177,  F 8,22) (  = 1.63).  Fractioned Matter from the Gravel Baskets: A s with the invertebrate community, there were significant block effects for the C P O M , F P O M and inorganic sediments within the gravel baskets (p = 0.0177, 0.0005, < 0.0001 respectively and  F , i) ( 4  2  = 3.8, 7.94, 23.01, respectively). There was a significant treatment effect  for the inorganic sediment (p = 0.022,  F , 2) (8  2  = 3.05); however, there was no clear pattern o f fish  or crayfish effects on any organic fraction.  Discussion I have shown that ontogenetic stage is important in determining the direction and magnitude o f interactions between the signal crayfish and cutthroat trout (Figure 5.7). The individual growth o f Y O Y crayfish, and the overall biomass change o f the Y O Y fish are two examples o f factors mediated by stage-specific interactions between species in my study. There 87  was no overall change in Y O Y crayfish biomass in the enclosures, although there were significant differences in individual growth rate. It makes sense that due to the aggressive nature of this species (e.g. Nakata and Goshima 2002, Pockl and Pekny 2002) some individuals would lose mass or perish, while others would grow, resulting in the same total biomass being allocated in different ways. Y O Y crayfish experienced a decreased individual growth rate (although not significant) in the presence o f adult fish, which was predicted, as other research has demonstrated a decrease in movement and general foraging behavior of juvenile crayfish in the presence o f large fish (Hein et al. 2006, Nystrom 2005). The fact that this result is not significant indicates that while adult cutthroat trout may have a slight negative effect on Y O Y crayfish feeding behavior, it is not as profound as results that have been presented elsewhere. More interestingly, juvenile crayfish experienced a substantial increase in growth rate in the presence o f juvenile fish. The presence o f Y O Y fish facilitated growth o f the Y O Y crayfish, as they grew significantly more than when they ( Y O Y crayfish) were alone. I have shown experimentally (Bondar et al in press) that Y O Y crayfish are unaffected in their feeding behavior by Y O Y cutthroat trout; however, I do not have any information on the feeding behavior of cutthroat trout in the presence o f crayfish. Y O Y fish experienced a decrease i n biomass whether they were paired with adult or Y O Y crayfish, indicating that in both cases they were negatively affected. However, the survival rate o f Y O Y fish was lower in the presence of Y O Y crayfish than in the presence o f adult crayfish. Despite the possibility for a lowered amount o f competition between Y O Y fish in these treatments (fewer individuals present), they gained significantly less weight than when they were alone, re-iterating that Y O Y crayfish have negative effects on Y O Y fish. The lack o f any other differences between the Y O Y crayfish + Y O Y fish treatment and all other treatments with respect to the invertebrate community or fractioned organic matter suggests that crayfish consumption o f the fish may have been the reason for the increased Y O Y crayfish biomass in 88  these treatments. A s mentioned above, survivorship o f Y O Y fish was lower in the presence of juvenile crayfish than in the presence of adult crayfish or alone. A t least one juvenile fish perished per enclosure in the Y O Y crayfish + Y O Y fish treatments, which may explain the greater leaf mass remaining in these treatments, i.e., crayfish may be less likely to feed on leaf matter when they are feeding on fish. Further investigation is required to determine whether Y O Y fish are threatened by the presence o f Y O Y crayfish, and to verify whether Y O Y crayfish prey on Y O Y fish. The dry mass o f leaves remaining after the experiment indicates that adult crayfish consume a greater amount o f leaf matter than Y O Y crayfish. Several researchers (Parkyn et al. 1997, Whitledge and Rabeni 1997, Creed and Reed 2004) have also demonstrated a decreased amount of leaf litter in the presence of adult crayfish. When corrected for the larger amount of crayfish biomass in the adult treatments, the differences remain, supporting the common assumption o f an ontogenetic change in leaf litter consumption between adult and juvenile crayfish (Lodge et al. 1994, Schofield et al 2001). This result differs from Bondar et al. (2005), who found no ontogenetic difference in the amount o f leaf-matter consumed between adult and juvenile P. leniusculus.  The latter study used 5-year-old juveniles (average O C L 18.5 mm) as  opposed to Y O Y crayfish, which indicates that i f an ontogenetic shift towards consumption o f leaf litter occurs in this species, it happens at a much earlier developmental stage than it does for other species, or for P. leniusculus in environments where it has been introduced. For example, Parkyn et al. (2001) demonstrated that the ontogenetic diet shift in Paranephrops  planifrons  occured at an O C L size o f 20.0 mm, and Guan and Wiles (1998) reported that introduced P. leniusculus juveniles with a carapace length o f > 20 m m were far more carnivorous than adults with a carapace length o f 33 to 45 mm. The decreased amount o f leaf matter remaining i n the fish alone and control treatments is likely explained through a functional compensation o f the detritivore invertebrate community  (detritivorous invertebrates consuming the leaf matter instead of the crayfish), a phenomenon also documented by Usio and Townsend (2004), as other detritivores were found to be so much more abundant in these samples. The invertebrate community on the leaf packs was strongly impacted by crayfish presence. I speculate that chemical cues from the crayfish would be a likely explanation for exhibited effects on the leaf packs for several reasons: 1) the effects on the leaf pack community were the same regardless o f whether there were 8 Y O Y crayfish or 1 adult crayfish; and 2) the crayfish effects on the community were not affected by fish presence, despite the fact that Y O Y crayfish may have been behaviorally inhibited in the adult fish treatments (smaller individual growth % during experiment), and spending less time feeding on the leaf packs i n the Y O Y fish treatments (see further discussion below). Recent research (Richmond and Lasenby 2006) has re-iterated the potential importance o f invertebrate detection o f crayfish chemical cues in stream ecosystems, showing that mayfly nymphs respond defensively to cues from the crayfish Orconectes rusticus. Although the responses o f snails to chemical cues of crayfish has received a good deal o f attention (Alexander and Covich 1991, Hoverman et al. 2005), the responses o f other stream invertebrates to chemical cues from crayfish are largely unknown and present an excellent opportunity for further study. The C P O M , F P O M and inorganic sediments left on the leaf packs provide further information on the movement patterns o f both fish and crayfish in the enclosures. Overall, the fish treatments and controls contained more sediments than any o f the crayfish treatments. Neither adult nor Y O Y fish appear to have been actively feeding i n the leaf packs, as evidenced in the high number of invertebrates present and also the high amount o f F P O M and inorganic sediments within the leaf packs i n these treatments. This is in agreement with the assertion o f Zhang et al (2004) that large cutthroat trout are not effective feeders on leaf-pack invertebrates.  90  The amount of inorganic sediment in the Y O Y crayfish + Y O Y fish treatment was close to being significantly higher than for all other juvenile crayfish treatments, indicating that Y O Y crayfish were not spending as much time foraging in the leaf packs when Y O Y fish were present. This further supports the idea that Y O Y crayfish were feeding outside the leaf packs (and perhaps on the Y O Y fish) when these two organisms were together. The data from the gravel basket samples (both the invertebrate community and the fractioned organic and inorganic material) is strongly suggestive that near bed water velocity (e.g. Effenburger et al. 2006) or some other environmental factor played a larger role in structuring their contents. For most o f the populous invertebrates, the highly significant block effects overshadowed the treatment effects. Chloroperlidae was the only invertebrate for which there were significant negative effects o f crayfish and fish. In all treatments except for the control, the number of Chloroperlidae was significantly reduced, suggesting either direct predation, or emigration (induced by crayfish or fish chemical cues). The results o f this study have lent a small amount o f support to the common assumption that adult fish have a negative effect on Y O Y crayfish. However, I have demonstrated that there are several other stage-specific interactions between these two species, and that in this system it is actually the crayfish that are having more profound negative effects on the fish. Adult crayfish had significant negative effects on growth rate Y O Y fish, and Y O Y crayfish inhibit the feeding behavior, and perhaps even prey upon Y O Y fish. These results demonstrate the complexities involved in the ontogenetic interactions between two species. This research demonstrates the need for ontogenetic stage-specific relationships to be included i n ecological studies and food-web analyses. Our ability to correctly predict species interactions requires an understanding o f the spectrum of interactions that may take place through an organisms' lifetime. Further, the environment in which such interactions take place  91  is another important aspect o f ecological studies, as organisms vary in their ability to effect change within the context o f different microhabitats.  92  Invertebrate Type  Chironomini Tanytarsini Orthocladiinae Tanypodinae Tipulidae Pupae Rhyacophilidae  Zapada Sum o f Invertebrates  (Treatment)  Treatment Probability Values  9.31 8.53 23.72 19.26 3.94 8.27 3.78 4.38 39.25  <0.0001 <0.0001 <0.0001 <0.000T 0.005 O.0001 0.006 0.003 <0.0001  F (8,22)  F(4,22)  B l o c k Effects  (Block) 3.13 3.51 6.82 6.20 0.20 1.04 7.01 4.73 3.00  0.035 0.023 0.001 0.002 0.936 0.407 0.001 0.007 0.053  Table 5.1. A N O V A on the members of the leaf pack communities showing the significant treatment effects o f crayfish. A l l invertebrates shown were significantly negatively affected by the crayfish treatments relative to controls. Bonferonni corrected a = 0.0056.  93  (b)  SJ  0.4  0)  0.0  E  b  40" 40"  Figure 5.1. a) The difference (final - initial) o f Y O Y fish biomass for each treatment over the duration o f the 6 week experiment. The collective biomass was used because individual Y O Y fish could not be effectively marked, b) The average individual weight gain o f Y O Y fish for each treatment during the experiment. Error bars in both a and b indicate 1 standard error.  Figure 5.2. Crayfish mass change as a % o f original mass. Adult crayfish were unaffected by treatment. Error bars indicate 1 standard error.  7  Figure 5.3. Collective leaf mass remaining by treatment (4 leaf packs per enclosure, starting mass o f 5 g each). Adult crayfish treatments were significantly different from all other treatments (p values for least squared means post hoc comparisons < 0.001). Y O Y crayfish treatments had a significantly higher leaf mass remaining than all adult crayfish treatments (p values for post hoc comparisons < 0.001). Error bars indicate + 1 standard error.  (a)  (c)  (b)  (d)  95  (e)  (f)  (h)  (g)  S  2-1  I  £L Figure 5.4. Total leaf pack invertebrates per gram of leaf mass (Numbers/g). (a) Chironomini (b) Tanytarsini (c) Orthocladiinae (d) Tanypodinae (e) Tipulidae (f) Dipteran Pupae (g) Rhyacophilidae (h) Zapada. Error Bars indicate + 1 standard error. Treatments are abbreviated as follows: A D C = adult crayfish, Y O Y F = Y O Y Fish, A D F = Adult fish, Y O Y C = Y O Y Crayfish.  96  Figure 5 . 5 . Fractioned organic and inorganic matter in the leaf packs per gram o f leaf mass, (a) C P O M (b) F P O M (c) fine inorganic sediment. Error bars indicate + 1 standard error. Treatments are abbreviated using the same convention as for Figure 5 . 4 .  97  (a)  (b)  Figure 5.6. The interactions between Y O Y crayfish and adult fish, exhibited by the disproportionate decrease o f numbers o f (a) Chironomini (b) Tanypodinae (c) Baetis (d) Orthocladiinae in the Y O Y C + A D F treatments. Error bars indicate +1 standard error. Treatments are abbreviated using the same convention as for Figure 5.4.  Adult Crayfish  Adult Fish  Y O Y Crayfish  Y O Y Fish  Figure 5.7. Stage-specific interactions between crayfish and fish with respect to weight change during the experiment. Solid arrows indicate negative effects, and the dashed arrow indicates a positive effect. The thickness o f the arrows indicates the magnitude o f effects, based on the % growth ( Y O Y Crayfish) or % biomass change ( Y O Y fish).  98  References Alexander, J. E . and A . P. Covich. 1991. Predation risk and avoidance behavior in two freshwater snails. Biological Bulletin 180:387-393. Bondar, C . A . , Bottriell, K . , Zeron, K . and J.S. Richardson. 2005. 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Bullfrog tadpole (Rana catesbeiana) and red swamp crayfish (Procambarus clarkia) predation on early life stages o f endangered razorback sucker (Xyrauchen texanus). The Southwestern Naturalist 51(2):258-261. Nakata, K . and S. Goshima. 2003. Competition for shelter o f preferred sizes between the native crayfish species Cambarides japonicus and the alien crayfish species Pacifastacus leniusculus in Japan in relation to prior residence, sex difference, and body size. Journal o f Crustacean Biology 23:897-907. Nystrom, P. 2005. Non-lethal predator effects on the performance o f a native and an exotic crayfish species. Freshwater Biology 50:1938-1949. Parkyn, S. M . , Collier, R. J. and B . J. Hicks. 2001. N e w Zealand stream crayfish: functional omnivores but trophic predators? Freshwater Biology 46:641-652. Parkyn, S. M . , Rabeni, C . F. and K . J. Collier. 1997. 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Predation by the noble crayfish, Astacus astacus (L.), on emerging fry of sea trout, Salmo trutta (L.). Nordic Journal o f Freshwater Research 68:100-104. Rudolf, V . H . W . 2006. The influence of size specific indirect interactions in predator prey systems. Ecology 87:362-371. Schofield, K . A . , Pringle, C . M . , Meyer, J. L . and A . B . Sutherland. 2001. The importance o f crayfish in the breakdown o f rhododendron leaf litter. Freshwater Biology 46:1191-1204. 100  Seiler, S . M . and A.M.Turner. 2004. Growth and population size o f crayfish in headwater streams: individual- and higher level consequences of acidification. Freshwater Biology 49:870881. Stenroth, P. and P. Nystrom. 2003. Exotic crayfish in a brown water stream: effects on juvenile trout, invertebrates and algae. Freshwater Biology 48:466-475. Usio, N . and C . R . Townsend. 2000. Distribution o f the N e w Zealand crayfish Paranephrops zealandicus in relation to stream physico-chemistry, predatory fish, and invertebrate prey. N e w Zealand Journal o f Marine and Freshwater Research 34:557-567. Usio, N . and C . R . Townsend. 2004.Roles o f crayfish: Consequences of predation and bioturbation for stream invertebrates. Ecology 85:807-822. Werner, E . E . and B . R . Anholt. 1996. Predator-induced behavioural indirect effects: consequences to competitive interactions i n anuran larvae. Ecology 77(1): 157-169. Whitledge, G . W . and C . F. Rabeni. 1997. Energy sources and ecological role o f crayfishes in an Ozark stream: insights from stable isotopes and gut analysis. Canadian Journal o f Fisheries and Aquatic Sciences 54:2555-2563. Woodward, G . and A . Hildrew. 2002. Body-size determinants o f niche overlap and intraguild predation within a complex food web. Journal of A n i m a l Ecology 71(6): 1063-1074. Zhang, Y . X . , Richardson, J. S. and J . N . Negishi. 2004. Detritus processing, ecosystem engineering and benthic diversity: a test o f predator-omnivore interference. Journal o f A n i m a l Ecology 73:756-766.  101  'Chapter 6 Survival, movement and microhabitat choice of signal crayfish in a small temperate stream: does individual size matter?  ' A version o f this chapter w i l l be submitted for publication. Survival, movement and microhabitat choice o f signal crayfish in a small temperate stream: does individual size matter? Freshwater Biology.  102  Introduction Freshwater invertebrates play important roles i n community and food web ecology (e.g. Spooner and Vaughn 2006). Invertebrates are often more dense and may comprise more biomass in freshwater ecosystems than vertebrates, making an understanding o f their population sizes and demographics o f great importance. In addition to overall population size, an understanding o f the size-structure and size-specific demographic rates o f a population is important to appreciate the magnitude o f the ecological role played through the life history o f the organism. This is especially important in populations that undergo ontogenetic niche shifts, i.e. changes in resource or habitat use related to size or developmental stage (Olson 1996, Hjelm et al. 2000), which may render different life history stages o f the same organism functionally different contributors to the ecosystem. Invertebrates are more likely than vertebrates to undergo ontogenetic niche shifts, as they often develop over a large size range (in some cases up to 5 orders o f magnitude), w i l l have a wider breadth o f resource use, and therefore a greater potential to vary ontogenetically in niche requirements than species that change little in size through development (Polis 1984, Bystrom and Garcia-Berthou 1999). It has been repeatedly demonstrated that crayfish are important in freshwater food webs. Crayfish have been shown to play an important role as detritivores (Parkyn et al. 1997, Whitledge and Rabeni 1997), and predators (Momot 1995), and may have profound effects on the surrounding physical habitat (Zhang et al. 2004). Although the ecological role of crayfish is well-established in streams, there is little information on overall population size, size-specific demographic rate estimates and movement metrics in these areas. Such information is needed in order to fully comprehend the extent to which crayfish effect change in these ecosystems. Stream-dwelling crayfish have been demonstrated to undergo ontogenetic niche shifts in feeding behavior (Abrahamsson 1966, Guan and Wiles 1998; but see Bondar et al. 2005) and habitat use (Butler and Stein 1985, Maher and Stein 1993, Creed 1994, Englund 1999, Flinders 103  and Magoulick 2003). Ontogenetic changes in interspecific interactions have been documented between crayfish and fish in small streams, and have the potential to influence the surrounding ecosystem. For example, large fish have been shown to prey directly on Y O Y crayfish (Hein et al. 2006, Seiler and Turner 2004), and have even been shown to influence the size-structure o f the slow-growing crayfish populations. Changes in crayfish size structure caused by large fish may in turn greatly influence the magnitude of effects that crayfish have on the surrounding ecosystem i f large and small crayfish have disparate ecological roles. In addition to the more commonly described negative effects o f adult fish on juvenile crayfish through direct predation and adult crayfish on juvenile fish through direct predation or exclusion o f small fish from favorable habitats (Rubin and Svensson 1993, Mueller et al. 2006), it has recently been shown that there may be a negative influence of juvenile signal crayfish on juvenile cutthroat trout (Oncorhynchus clarki) (Bondar and Richardson in review). A comprehensive knowledge o f the population size structure o f these organisms is therefore important to be able to reflect on the magnitude o f such interactions in the small stream ecosystem. The effects o f crayfish and other organisms in freshwater ecosystems have been shown to be specific to certain microhabitat types (Creed 1994, Mcintosh et. al 2002, Bondar et al 2005). It is therefore important to ascertain whether crayfish are always found in certain areas o f the stream, and whether this changes with ontogeny or through the season. Although crayfish have been shown to play a vital ecological role in the small stream community, there is a limited understanding o f their physical habitat use (Demers et al. 2003, DiStefano et al 2003). The effects o f crayfish on their environment w i l l depend on where and when they interact with the ecosystem. Some crayfish have been shown to occupy territories (Figler et al. 2005), while others have been shown to maintain 'ephemeral home ranges' (Robinson et al. 2000), with individuals remaining in the same place for limited periods o f time. Such phenomena have been  104  demonstrated for adult crayfish, but little is known about the movement patterns and microhabitat use of juvenile crayfish (see DiStefano et al. (2003) for an exception). The purpose of this study was to investigate aspects o f signal crayfish (Pacifastacus leniusculus Dana) populations in a small stream ecosystem in north-western North America, the native environment for these crayfish. In addition to investigating overall population size, I investigated population size-structure, age-specific survival rates, and microhabitat use and movement patterns o f both juvenile and adult crayfish in order to gain a greater understanding o f the magnitude o f the role these organisms play though ontogeny in a small stream ecosystem.  Methods Sites: I used two sites for my population study. Both sites were in Spring Creek, located in the M a l c o l m Knapp Research Forest in the Coastal Western Hemlock biogeoclimatic zone o f British Columbia (49°18'40"N 122°32'40"W). Site #2 was a 120 m reach o f stream bordered by strong riffles at the up and downstream ends, while Site #3 was 130 m , and located several k m upstream o f site 2. A culvert was located at the upstream end o f site 3, while at the downstream end the stream branched into several very small ( > 0.5 m wide) channels which rejoined approximately 1 k m downstream. I felt that both of these sites were bordered by areas that would not be hospitable to crayfish, thereby restricting (but not preventing) movements between the sites (although no crayfish were recaptured between sites). Each site (stream width approximately 1.5 m) contained a variety o f microhabitat types typical o f small stream geomorphology. Such microhabitats included pools, riffles, runs, debris-filled pools (largely woody debris and other allochthonous detritus).  Visits and capture techniques: 105  A mark-recapture study was conducted using Pollock's robust design (Pollock 1982, Kendall and Pollock 1992, Kendall and Nichols 1995, Kendall et al. 1995, 1997), combining the Cormack-Jolly-Seber (CJS) open recapture model (Cormack 1964, Jolly 1965, Seber 1965) and Huggins' closed capture estimator (Huggins 1989, 1991), in order to accurately estimate population size, capture probability, and survival rate o f the crayfish. Each stream site was visited during three primary sampling sessions through the season. The first session occurred in mid-May, the second in late June and the third in early August. Each primary sampling session consisted o f several visits to each site (5 for the first session, 4 each for the second and third sessions). A s stated above, this method allows for both closed (within session) and open (between session) estimates o f population parameters to be calculated. Each stream site was divided into 10 m sections using flagging tape on the stream bank. A total o f 20 person-minutes (two people for 10 minutes each) were spent in each 10 m section of the stream to ensure a constant sampling effort o f the entire site. The area was searched by looking under rocks or debris, and scanning the stream bed, and all crayfish were caught by hand-netting. Once a crayfish was caught, it was measured (occipital carapace length (OCL)), sexed (during primary sessions 2 and 3 only), and habitat characteristics (including the stream segment where it was found) were noted. Each crayfish was given an individual mark using Visible Implant Elastomer (VIE) (Northwestern Marine Technology, Shaw Island, Washington, U S A ) on the abdominal area when it was first captured. O n subsequent captures of the same individual the mark was noted, in addition to the O C L , sex, stream section and habitat characteristics.  Data Analysis - population parameters: Overall mark-recapture data were analyzed for a closed captures, robust design using the program M A R K (Cooch and White 2006). I constructed 'random emigration' models forcing all 106  X (emigration) parameters to be equal for my data since there were only 3 primary periods (Williams et al. 2002). I tested models that incorporated O C L (len = O C L , a measure representing age o f the organism), and O C L (len2) into both survival (S) and capture probability 2  (p), as well as models that incorporated whether either survival or capture probability varied with time (t). Each o f these models was combined with the 'Random Emigration' model explained above. I compared the statistical fit o f the competing models from the candidate set using an information theoretic approach based on Akaike information criterion, treating models with A I C c which differ by < 2 as indistinguishable statistically (Burnham and Anderson 1998). In addition, I used likelihood ratio tests between models to assess the importance o f specific covariates (e.g. length) on survival. From the best model at each site, I utilized the real function parameters to obtain estimates o f capture probabilities, survival rates and N . I plotted the best models for both sites to obtain survival estimates based on O C L . This was done by inverting the logit transform used to fit the model. Under the transform, survival probability (cp) was modeled as: log (cp/1 -<p) = p + o  pi * O C L . Post-hoc 'Program Capture' tests were performed to assess the goodness o f fit o f my selected models.  Data Analysis - movement: For each recaptured individual, I assessed overall movement as well as directional movement. This was done by calculating the numerical difference (in number o f sections) from where a crayfish was last encountered to where it was first encountered in the next visit. For example, i f a crayfish was last seen in section 2, and next seen again in section 7, it was assigned an overall movement designation of 5 (5 sections, representing a movement o f approximately 50 m). In a separate analysis, I obtained data about the directional movement o f the crayfish. A positive value was assigned to upstream movement, and a negative value to downstream 107  movement. I assessed whether there were differences in overall movement between adult and juvenile crayfish, and also whether there were differences between adults and juveniles with respect to the direction o f their movement using the general linear model approach in S A S ( S A S P R O C G L M , S A S Institute, Cary, N C ) . A l l crayfish with an O C L < 20 m m were designated as juvenile crayfish (Guan and Wiles 1998, Bondar et al. 2005), and all crayfish with an O C L > 20 m m were designated as adults. Re-capture data for movement was sub-divided into two categories: first, crayfish that were re-captured within a primary session (individually assessed for primary sessions 1-3), and second, crayfish that were re-captured between primary sessions. Movement data for those individuals caught between primary sessions was further subdivided into 3 categories: those captured in session 1 and then recaptured in session 2, those captured in session 1 and then recaptured in session 3, and those captured in session 2 and recaptured in session 3. These were assessed separately prior to pooling the data so that I could determine whether there were differences in movement patterns through the season.  Data Analysis - microhabitat use: Each individual crayfish was assessed for habitat choice each time it was captured. A generalized classification o f 8 habitat types was constructed based on where crayfish were found in the stream. These habitat types were: 1 - under a rock near the stream bank; 2 - under a rock near the middle o f the stream (pool); 3 - under a rock near the middle o f the stream (riffle); 4 - in a debris-filled area near the stream bank; 5 - in a debris-filled area near the middle o f the stream (pool); 6 - uncovered in the stream bed (pool); 7 - uncovered in the stream bed near the stream bank; and 8 - uncovered in the stream bed (riffle). I used the habitat where each individual was first captured to assess differences in habitat use between adult and juvenile crayfish ( S A S P R O C G L M , S A S Institute, Cary, N C ) . In 108  addition, I assessed whether microhabitat use varied with the season by analyzing the data between primary sessions separately. 1 used the probability o f differences in the post-hoc comparison o f least squared means to determine whether microhabitat selection was different between primary sessions for either adults or juveniles.  Results Population parameters: A total of 520 individual crayfish were caught at site 2 and 509 crayfish were caught at site 3 during the course of the 13 visits to each site. The sex ratio for all crayfish caught was close to 50:50, with 46% male and 54% female at site 2, and 49% male and 5 1 % female at site 3. O f the 520 crayfish caught at site 2, 417 were juvenile and 103 were adult. There were 332 juvenile and 177 adult crayfish caught at site 3. Figure 6.1 shows the age structure of the crayfish populations at both sites. For all models I ran in M A R K , I was restricted to 'zero emigration' models, fixing all gamma (k) parameters to zero. This is due to the fact that the estimates o f emigration provided by M A R K were extremely close to zero, leading to numerical instability in the other parameter estimates (C. Huston, S F U Department o f Statistics and Actuarial Sciences, Pers. Comm.). Once I fixed gamma to zero, I obtained suitable estimates o f all other parameters. The most parsimonious models selected by A I C c for each site incorporated a time component into probability o f capture, and a size component into the probability o f survival (Table 6.1). The best model for each site suggested that length, not time was not an important component o f survival (overall S = 0.50 for site 2, and 0.53 for site 3). In a likelihood ratio test between models incorporating length as a covariate o f survival (S(.)p(t,site) versus S(len)p(t,site), I found a significant difference between models (p > 0.0001, C h i 19.52 i), indicating a strong impact o f 2  length on survival. Probability o f capture increased at both sites through the season (Table 6.2) 109  from 5.8 to 19.5% at site 2 and 9.5% to 21.0% at site 3. Overall population estimates for each site are shown in Table 2, and were approximately 428 at site 2 and 450 at site 3 (based on the estimate from the third primary sampling session for each site). This translates to a density of 3.6 and 3.8 crayfish per linear meter for sites 2 and 3 respectively; however, my data on microhabitat use indicates that crayfish are not distributed evenly throughout the stream (see below). Since the estimates o f survival rate at both sites were both dependent only on size, I pooled the data from both sites and used site as an additional covariate in order to assess whether survival based on size would still be the strongest model, or whether the survival rates varied by site. The most parsimonious model for the pooled data showed that survival was indeed independent o f site and still dependent only on size (Table 6.3). Probability o f capture varied with time, and also varied with site. However, the probability o f survival (S) (for the pooled data set) varied only with length according to the following function: exp(2.0034-0.1031*(9CL) 1 + exp(2.0034 - 0.1031 * OCL) The graph o f survival probability vs. O C L (Figure 6.2) shows a steady decrease i n survival rate with increasing O C L . The 95% confidence intervals (calculated using the p estimates in the variance covariance matrix) for this function are also plotted on the graph (Figure 6.2).  Movement: N o statistical differences between adults and juveniles were found when I analyzed all data for movement within each primary session separately and movement between each primary session separately (p values from A N O V A s > 0.05), so these data were pooled for each site. This left a pooled set o f within primary session recaptures for sites 2 and 3 (for both juveniles and adults), as well as a pooled set o f between primary session recaptures for sites 2 and 3. A t 110  site 2 juvenile crayfish moved an average o f 2 sections (each section was, on average, 10 m long) within the primary sessions and 3.27 sections between the primary sessions (Figure 6.3). Adults moved significantly less than juveniles both within (p = 0.05, F(i g4) = 3.85) and between )  (p = 0.03, F(i,92) = 4.40) primary sessions at this site. Adults at site 2 moved an average o f 0.81 and 1.33 sections for within and between primary sessions respectively. The movement of juveniles at site 3 showed that individuals moved and average o f 1.69 and 2.27 sections within and between primary sessions. Adult movement at site 3 was higher than adult movement at site 2, with 1.5 sections and 2.96 sections for within and between primary session movement respectively (Figure 6.3). There were no significant differences with respect to ontogenetic stage at site 3, and there were no significant differences found at either site for directional movement.  Microhabitat use: I found significant differences in the habitat use between adults and juveniles at both site 2 (p < 0.0001, F ( i i 4 ) = 21.41) and site 3 (p = 0.0003, F ( , , 0 7 ) = 13.24). When adults and j5  5  juveniles from both sites were pooled together, the ontogenetic difference in habitat use remained (p = 0.0002, F i i o 2 i ) = 13.83), with the highest number of juveniles found under rocks (  !  in pools and the highest number o f adults in debris-filled pools (although the second-highest proportion of juveniles was found in debris-filled pools and the second-highest proportion o f adults was found under rocks in pools (Figure 6.4)). The habitat choices o f both juveniles and adults at site 2 did not vary seasonally, nor did the habitat choice of juveniles at site 3. However, post-hoc comparisons o f least squared means showed that adults at site 3 differed in their microhabitat use between visits 1 and 2 (p = 0.0046) and between visits 2 and 3 (p = 0.05) (Figure 6.5).  Ill  Discussion Population parameters: M y data from the mark-recapture study showed some trends that have implications for the interpretation of the ecological role o f these organisms in small streams. The estimates o f population size at both my study sites indicated that crayfish w i l l likely comprise a large proportion o f the total biomass o f the biota in these areas. However, my results also clearly show that crayfish are not evenly distributed throughout the stream. They are instead clustered in specific microhabitats (see discussion below), indicating that they are likely even more dense in certain areas. Consequently, the effects of crayfish on certain microhabitat areas of small streams have the potential to be profound. M y overall estimates o f survival (the general estimates for all sizes) are similar to those found by Jones and Coulson (2006) for a large population of Astacoides granulimanus in Madagascar. However, my results show that survival was largely independent of season, but dependent upon size. It is interesting, that in spite of the increased probability o f capture through the season, that survival remains the same. This clearly shows that size is important in determining survival in this species. Since I did not measure crayfish smaller than 10 m m O C L I cannot draw conclusions about the survival rate o f crayfish smaller than this, although I speculate that it is low at this early, vulnerable stage. M y results are also similar to size-specific survival rates found by Jones and Coulson (2006), although I found a great deal o f variation from within their 'small' and 'medium' size classes. These researchers used total carapace length ( T C L ) as an alternative to O C L ; however, using the conversion factor o f O C L = 0 . 6 3 T C L  1 0 6 6 3  (Beatty  et al. 2005) I was able to discern the  general size classes utilized by Jones and Coulson (2006) for their small (18.11-26.99mm O C L ) , medium (27.0-39.59mm O C L ) and large (> 39.6mm O C L ) crayfish (these estimates are only approximations since the conversion factor worked out by Beatty et al. (2005) was done so for 112  Cherax quinquecarinatus).  In addition, I was able to mark crayfish o f a considerably smaller  size than the former authors (10 m m minimum O C L in my study, 18.11 m m minimum O C L for their study). M y study indicates that Y O Y to 1-year-old crayfish have a better chance o f survival than those that are larger. I suggest that some aspect of intraspecific aggression is important in determining the survival o f larger crayfish in Spring Creek, in addition to an increased level o f predation from birds and mammals on larger crayfish. The greatest rate of survival o f crayfish in Spring Creek was found for small-sized crayfish (12-15 m m O C L ) , followed by a general decrease in survival for all larger size classes. Jones and Coulson (2006) found a similar decrease in survival for crayfish between their small and medium size classes, but the greatest probability o f survival was for the large (>39.6 m m O C L ) crayfish. M y largest crayfish fall into the 'medium' size category o f those observed by the latter authors, so I cannot make a similar conclusion about crayfish larger than 35 m m O C L . A s stated above, the highly aggressive nature o f P . leniusculus may result in a lower survival rate for adults, and may also result in a decreased overall size for the largest adults as compared to the largest adults found in populations o f other crayfish genera. Capture probabilities o f the crayfish at both sites consistently increased through the season, and were not at all dependent on size. This is likely because the small stream environments become even smaller during the dry period o f mid to late summer. A larger number o f organisms are therefore forced to cohabitate in a smaller amount o f space. Indeed, similar trends o f seasonal fluctuations in freshwater crustacean density have been documented by Hart (1981) and O h et al. (2003) for atyid shrimp. In both o f the aforementioned studies, higher densities o f shrimp were coincident with lower stream levels. However, I must also take into account that capture success is likely affected by the level o f the stream. Lower water levels and lower flow rates facilitate visual surveys and hand-catching of crustaceans and other organisms. 113  Although capture success may be higher at certain times o f year, it still seems likely that the density o f crayfish in preferred microhabitat areas reaches a high point in the late summer. This indicates that the effects of crayfish on the small stream ecosystem may be more prominent during the summer months when stream levels are at their lowest.  Movement: It must be noted that in this study I was assessing movement on a very limited scale, as adult crayfish have been shown to move great distances through a season (Bubb et al. 2004). However, I was primarily interested in finding out whether the crayfish in my study occupied home ranges, and whether there was an ontogenetic component to the amount o f movement within these areas. A t site 2, where there was 80.2% juveniles and 19.8% adults, juveniles were shown to be moving around significantly more than adults. Given the aggressive cannibalistic nature of this species (Nakata and Goshima 2002), it holds that adults would move around less, and perhaps be more likely to hold a home range or territory than a juvenile. However, at site 3, where there were markedly more adults present (34.8%, with a similar density o f crayfish per linear meter as site 2 (see results)) both ontogenetic stages exhibited a high degree o f movement both within and between primary sessions throughout the season. This suggests that adults may be less likely to occupy territories or home ranges when there are a greater number o f competitors around. Overall, it seems as though adult P. leniusculus in Spring Creek may have territories or home ranges when intraspecific competition is low, but juveniles do not appear to exhibit any form of territorial behavior. I did not find a difference in the overall direction o f adults or juveniles in my study, which agrees with the short-term tracking studies o f Bubb et al. (2004). However, overall Bubb et al. (2004) and Light (2003) found that adult crayfish generally migrated in a downstream direction through their study seasons. Since my study sites were limited to the two relatively 114  small patches o f the stream, I am not able to conclude whether there were overall differences in up or downstream migration.  Microhabitat Preference: The microhabitat use exhibited by the crayfish in my study is o f great importance i n assessing the overall impact o f these organisms on the small stream habitat. There are clear differences between adult and juvenile crayfish in Spring Creek for use o f some microhabitat types over others. DiStefano et al (2003) also found an ontogenetic difference in microhabitat preference between 3 species o f crayfish (Orconectes luteus, O. ozarkae, and O. punctimanus). Y O Y crayfish were found to prefer vegetation patches, backwaters and shallow pools, whereas adults were found to prefer riffle habitats. In my study, the three same microhabitat types were preferred by both adults and juveniles: under rocks in pools, under rocks near the stream bank, and in debris-filled pools; however, the ratio of juvenile to adult crayfish within these habitats showed that juveniles were more likely to be in the shallower, stream bank areas, whereas adults were most often found in debris-filled pools. The fact that juvenile crayfish were most-often found in the shallower habitats agrees with the results o f several other authors (Creed 1994, Englund and Krupa 2000, Flinders and Magoulick 2003). Although these general differences between adult and Y O Y crayfish exist, it should be noted that a substantial number o f both Y O Y and adult crayfish were found in all three of the top microhabitat types. M y results are similar to those o f DiStefano et al (2003) in that my juvenile crayfish were most often found in more shallow areas; however, m y adults were primarily found in debris-filled pools and not in riffles as were those found by DiStefano et al. (2003). In addition, the opposite trend in ontogeneticspecific microhabitat preference was found for O. neglectus by Gore and Bryant (1990). These diverse results indicate that the microhabitat-specific use o f individuals w i l l depend on a plethora of biological community and environmental factors. The most important point o f my study is 115  that there are several microhabitat types where crayfish (either juvenile or adult) are likely to be found, and there are several others where they are not likely to be found. Several reasons have been postulated for why crayfish are found in specific microhabitat types (e.g. for improved food consumption (Whitledge and Rabeni 1997), for decreased exposure to predators (including intraspecific predation) (Maher and Stein 1993, Englund 2000) or competitors, and for facilitation o f growth (juvenile crayfish only) by inhabiting areas of higher temperature (Mundahl and Benton 1990). It seems as though a combination o f all of these parameters may variably act to influence the position o f crayfish in the small stream environment. The fact that exposure to predators or competitors may affect microhabitat use is evident i n my data from site 3 where there were many more adults present. It is likely that the increased competition experienced by adult crayfish at this site resulted in a constant change o f microhabitats through the season (which was also found for the movement data), whereas at site 2 (where fewer adults were present), adults did not change microhabitats through the season. This research has demonstrated that P. leniusculus is a dominant member of the small stream ecosystem in Spring Creek. This species is found in high numbers, and has a high survival rate, ensuring its presence in the ecosystem throughout the seasons. I have suggested that the effects o f this organism on the surrounding environment w i l l be most pronounced in specific microhabitat types (which also differ by ontogenetic stage), and that these effects w i l l likely intensify through the season due to an overall decrease in available environment (as indicated by an increased probability o f capture at both sites through the season). This work substantiates experimental studies on the ecology o f crayfish in small streams because it gives evidence o f the massive presence o f crayfish in these ecosystems. However, in addition to the role of this work in studies on crayfish biology, I have provided a detailed population study on a long-lived freshwater invertebrate organism. This work shows the importance o f estimating population-level rates o f freshwater invertebrates, as in some systems 116  they may comprise more biomass than vertebrates. In addition, since invertebrates are likely to show ontogenetic niche shifts through development, population studies including a description of size structure are imperative in order to fully understand the magnitude o f influence that a specific ontogenetic stage may have on its surrounding ecosystem.  117  (a) Model  AICc  AAICc  AICc Weight  {S{.len,len2}p{t}} {S{.len}p{t}} {S{t,len,len2}p{t}} {S{t,len}p{t}} {S{.len,len2}p{t,len2}} {S{.len}p{t len2}} {S{.len}p{t,len}| {S{.len}p{t,len,len2)| {S{t,len}p{t,len}} {S {.len,len2 } p {t,len,len2 } (b) Model {S{.len}p{t} {S{.len,len2}p{t} {S{.len}p{t,len}} {S{.len}p{t,len2}} {S{t,len}p{t}} {S{t,len2}p{t}} {S{t,len,len2}p{t}} {S{t,len}p{t,len}} {S{.len}p{t,len,len2}} {S{.len,len2}p{t,len,len2}}  Model  2644.63 2644.74 2645.66 2645.77 2646.04 2646.19 2646.44 2647.39 2647.56 2647.99  0.00 0.11 1.04 1.14 1.42 1.56 1.81 2.77 2.94 3.36  0.18 0.17 0.11 0.10 0.09 0.08 0.07 0.04  1.00 0.94 0.59 0.56 0.49 0.45 0.40 0.25  0.04 0.03  0.23 0.18  AICc  AAICc  AICc Weight  3160.36 3161.52 3162.14 3162.15 3162.29 3163.21 3163.47 3164.10 3164.18 3165.12  0.00 1.15 1.78 1.79 1.93 2.85 3.11 3.74 3.82 4.76  0.26 0.15 0.11 0.10 0.10 0.06 0.05 0.04 0.03 0.02  #Par  Deviance  Likelihood  Model Likelihood  1.00 0.56 0.41 0.40 0.38 0.24 0.21 0.15 0.14 0.09  6 5 7 6 7 6 6 7 7 8  2632.50 2634.65 2631.49 2633.64 2631.87 2634.06 2634.31 2633.22 2633.39 2631.77  #Par  5 6 6 6 6 6 7 7 7 8  Deviance  3150.29 3149.41 3150.03 3150.05 3150.18 3151.10 3149.33 3149.96 3150.03 3148.94  Table 6.1. Top ten models predicted by program M A R K for both site 2 (a) and site 3 (b). M o d e l notation follows standard notation outlined in mark manual (2006). A ' . ' Indicates a lack o f time dependence. S = survival, p = probability of capture, t = time, len = O C L length, len2 = O C L length squared.  118  (a)  Parameter  S Gamma" p Session 1 p Session 2 p Session 3 N Session 1 N Session 2 N Session 3  Estimate  0.50 0.00 0.06 0.07 0.20 472 748 432  Standard E r r o r  0.05 0.00 0.01 0.01 0.02 108 123 32  95% Confidence Interval Lower Upper  0.40 0.00 0.04 0.05 0.16 316 554 380  0.60 0.00 0.09 0.09 0.23 755 1043 506  Fixed  (b) Parameter S(bctwccn 1 and 2)  Gamma" p Session 1 p Session 2 p Session 3 N Session 1 N Session 2 N Session 3  Estimate  0.53 0.00 0.10 0.11 0.21 340 552 448  Standard E r r o r  0.04 0.00 0.02 0.01 0.02 53 62 29  95% Confidence Interval Lower Upper  0.44 0.00 0.07 0.09 0.18 261 450 401  0.61 0.00 0.13 0.14 0.24 472 698 516  Fixed  Table 6.2. The parameter estimates, standard errors and confidence intervals for the best models predicted by MARK for site 2 (a) and site 3 (b). Parameter notation follows standard notation outlined in MARK manual (Cooch and White 2006). S = survival, Gamma = emigration parameter (fixed in my models) p = probability of capture, N = population size.  119  (a) AICc AICc  Model  {S(len)p(t,site)| {S(len)p(t,site,len2)| {S(len)p(t,site,len)} (S(len,len2)p(t,site)} {S(len,site)p(t,site)} {S(len)p(t)| {S(len)p(t,site,len,len 2)} {S(len2)p(t,site)| {S(.)p(f)|  5841.19 5842.94 5843.17 5843.20 5843.20 5844.42 5844.66 5853.54 5860.17  A AICc  0.00 1.75 1.98 2.01 2.01 3.23 3.47 12.35 18.98  Weight  0.34 0.14 0.12 0.12 0.12 0.06 0.06 0.00 0.00  Model Likelihood  1.00 0.41 0.37 0.36 0.36 0.19 0.17 0.00 0.00  #Par  6 7 7 7 7 5 8 6 4  Deviance  5829.13 5828.86 5829.09 5829.12 5829.12 5834.38 5828.56 5841.48 5852.14  (b)  Parameter  Estimate  S(ovcrall)  0.53 0.00 0.07 0.09 0.20 778 205 885  Gamma" p Session 1 p Session 2 p Session 3 N Session 1 N Session 2 N Session 3  Standard Error 0.03 0.00 0.01 0.01 0.01 101 111 43  95% Confidence Interval Lower Upper 0.47 0.00 0.05 0.07 0.18 613 1015 809  0.59 0.00 Fixed 0.10 0.11 0.22 1018 1453 981  Table 6.3. The most parsimonious models for the pooled data for sites 2 and 3 are shown in (a). The best model S(len)p(t,site) shows that length is important for survival at both sites, regardless of time, and that probability o f capture varies by both time and site, (b): Gives the parameter estimates, standard errors and confidence intervals generated for the most parsimonious model shown in (a). M o d e l notation follows standard notation outlined in M A R K manual (Cooch and White 2006). A ' . ' Indicates a lack o f time dependence. S = survival, Gamma = emigration parameter (fixed in my models), p = probability o f capture, N = population size, t = time, len = O C L length, len2 = O C L length squared.  120  60  Site 2  q  q q q q, q o, Oj q q, qj 0 ) 0 , 0 , 0 , 0 , 0 , 0 , 0  q q q q q q , q>  Size Class (OCL)  ^  s  N" s  J  ^  v  ^ £>' rf?' r,>' r!V' rf?' r,*' rf?' rf?' riV rf*>' rf?' r»' r>' rfl-' r£>' r>'  Size Class (OCL)  Figure 6.1. Size distribution of all crayfish captured at sites 2 and 3. Crayfish are divided into size classes of 1 m m O C L .  121  1.0  ro  0.6  ro >  to  0.4  H  0.2 •  0.0 • 10  15  25 30 20 Length (OCL)  35  40  45  Figure 6.2. Survival rate as a function of length for all crayfish at sites 2 and 3. This estimates is based on the model S(len)p(t), the most parsimonious model, for the combined data at sites 2 and 3. The solid line indicates the estimated survival probability, and the dashed lines represent the 95% confidence intervals.  Figure 6.3. Cumulative movement of re-captured individuals per study site. Columns labeled ' W refer to re-captures within primary sessions and columns labeled ' B ' refer to re-captures between primary sessions. Error bars denote one standard error. An asterisk between 2 columns indicates that the 2 columns are significantly different.  122  Figure 6.4. Microhabitat use o f juvenile and adult crayfish, (a) Juvenile crayfish microhabitat distribution for sites 2 and 3 combined, (b) Adult crayfish microhabitat distribution for sites 2 and 3 combined. Numbers on the graphs correspond to the following microhabitat types: 1: under a rock near the stream bank, 2: under a rock mid-stream (pool), 3: under a rock mid-stream (riffle), 4: debris-filled area near the stream bank, 5: debris filled area in middle o f stream (pool), 6: uncovered on stream bed (pool), 7: uncovered on stream bed close to the stream bank, 8: uncovered on stream bed (riffle). Categories 7 and 8 are so rare that they do not appear in the figures.  Figure 6.5. Adult crayfish microhabitat use at site 3. (a) shows the results for primary session 1, (b) for primary session 2 and (c) for primary session 3. Numbers on the graphs correspond to the same microhabitat types as outlined in Figure 6.4. 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Ontogenetic niche shifts in largemouth bass: variability and consequences for first-year growth. Ecology 7 7 : 179-190. Parkyn, S. M . , C F . Rabeni and K . J . Collier. 1997. Effects o f crayfish (Paranephrops planifrons: Parastacadae) on in-stream processes and benthic faunas: a density manipulation experiment. N e w Zealand Journal o f Marine and Freshwater Research 3 1 : 685-692. Polis, G . A . 1984. A g e structure component o f niche width and intraspecific resource partitioning: can age groups function as ecological species? American Naturalist 1 2 3 : 541-564. Pollock, K . H . 1982. A capture-recapture design robust to unequal probability o f capture. Journal of Wildlife Management 4 6 : 752-757. Robinson C . A . , T.J. Thorn and M . C Lucas. 2000. Ranging behaviour o f a large freshwater invertebrate, the whiteclawed crayfish Austropotamobiuspallipes. Freshwater Biology 4 4 : 509521. Rubin, J. and M . Svensson. 1993. Predation by the noble crayfish, Astacus astacus (L.), on emerging fry o f sea trout, Salmo trutta (L.). Nordic Journal o f Freshwater Research 6 8 : 100-104. 126  Seber, G . A . F . 1965. A note on the multiple-recapture census. Biometrika 52: 249-259. Seiler, S . M . and A . M . Turner. 2004. Growth and population size o f crayfish in headwater streams: individual- and higher level consequences o f acidification. Freshwater Biology 49: 870881. Spooner, D . E . and C . C . Vaughn. 2006. Context-dependent effects o f freshwater mussels on stream benthic communities. Freshwater Biology 51: 1016-1024. White, G . C . and K . P . Burnham. 1999. Program M A R K : survival estimation from populations o f marked animals. Bird Study 46(Suppl): 120-138. Whitledge, G . W . and C F . Rabeni. 1997. Energy sources and ecological role of crayfishes in an Ozark stream: insights from stable isotopes and gut analysis. Canadian Journal of Fisheries and Aquatic Sciences 54: 2555-2563. Williams, B . K . , J.D. Nichols and M . J . Conroy. 2002. Analysis and management of animal populations. N e w York, Academic Press. Zhang, Y . X . , J.S. Richardson and J.N. Negishi. 2004. Detritus processing, ecosystem engineering and benthic diversity: a test o f predator-omnivore interference. Journal o f A n i m a l Ecology 73: 756-766.  127  Chapter 7 D i s c u s s i o n , Integration a n d Suggestions for Future R e s e a r c h  Thesis chapter summary and integration The research in this thesis has advanced our knowledge of the ontogenetic ecology o f the signal crayfish, Pacifastacus  leniusculus, in its native environment. This work presents the first  set o f large scale experiments done on this organism where it is not an introduced species, since the studies of Mason (1963, 1970, 1974 and 1975). In addition, my results show a disparity to some o f the conclusions o f the former author. This may be due to the large differences in our study systems and techniques. Chapter 2 provided an initial look at the ontogenetic ecology of P. leniusculus using an in-stream enclosure experiment. In addition to the investigation o f different ontogenetic stages, I incorporated the effects o f density and microhabitat into this experiment. I did this in order to assess whether the effects o f crayfish were density dependent, as might be expected from an aggressive, cannibalistic organism such as P. leniusculus (Nakata and Goshima 2003). In addition, I wanted to incorporate the microhabitat specificity o f this organisms' ecology into this study, as recent work has highlighted the importance o f spatial ecology in the heterogeneous stream environment (Mcintosh et al. 2002). In order to test whether there were microhabitatspecific effects o f the crayfish, I looked at their effects in two microhabitat types within the enclosures: leaf packs and benthic areas. These habitat types were selected based on the type of microhabitats in which I had observed P. leniusculus in the wild. Surprisingly, the results o f the enclosure experiment outlined above suggested that the effects of both juvenile and adult crayfish on the surrounding environment were the same: the leaf pack microhabitats were drastically changed in the presence o f crayfish, whereas the benthic areas o f the enclosures were not clearly affected in any way. The effects o f crayfish remained the same regardless o f both ontogenetic stage and density, leading me to speculate that the nature of these effects is that o f an indirect chemical cue. If the effects o f crayfish on the surrounding ecosystem are largely based on a chemical signal that they exude, it stands to reason that neither 129  ontogenetic stage nor density would make a difference (assuming the threshold tolerance o f stream invertebrates to a chemical cue from the crayfish is quite low). Recent research attesting to the effects of crayfish chemical cues on mayflies (Richmond and Lasenby 2006) substantiates this claim, in addition to several earlier studies on the effects o f crayfish chemical cues on gastropod ecology and development (e.g. Covich and C r o w l 1994). The determination o f whether the effects o f crayfish from this experiment were a result o f either indirect effects or direct consumption was investigated in Chapter 3, where I looked at the diet o f all crayfish from this study. Using a combination o f gut content analysis and stable isotope analysis (which are commonly used to assess trophic position in stream-dwelling crayfish (Parkyn et al. 2001, Hollows et al. 2002)), I was able to determine that the crayfish in my study were not ingesting a significant amount o f invertebrate-based material. In addition to researching crayfish from within the enclosures, I also assessed crayfish that were not involved in the enclosure study. Both the juveniles and adults I tested (from both environments) showed a large dependence on detrital products as their main food source. This is in direct contrast to other studies on P. leniusculus in introduced environments (e.g. Guan and Wiles 1998, Stenroth and Nystrom 2003), which show a substantial part o f the diet o f both juveniles and adults is composed o f invertebrate material. This led me to speculate about the nature of growth crayfish would experience when on a diet o f detritus versus invertebrates. The laboratory feeding study was designed to verify that crayfish would experience a greater increase in growth on a diet o f invertebrates as opposed to leaves and wood. Similar growth rates o f the crayfish resulted from either detrital diet (leaves vs. wood), leading me to speculate about the nutritional value o f the fungal biofilms on these food sources (i.e. Whitledge and Rabeni 1997); however, a much larger growth rate was exhibited by the crayfish that were consuming a diet o f invertebrates. This confirmed that crayfish were indeed consuming a sub-optimal diet i n the field, and led to the  130  design o f the next experiment, which assessed possible reasons for a sub-optimal diet in juvenile P. leniusculus. I developed a field-based enclosure study (chapter 4) to assess the effects of different organisms on the feeding behavior o f Y O Y (young-of-the-year) crayfish. This study addressed the issue o f food-switching in omnivorous organisms. I found that the feeding behavior of these organisms was indeed negatively affected by the presence o f both adult crayfish and adult fish (cutthroat trout, Oncorhynchus clarkii).  Although the same food source (chironomid larvae) was  ultimately chosen, the time to a choice was substantially higher in the presence o f both adult fish and adult crayfish. This result led me to conclude that Y O Y crayfish may not have an optimal diet in the field due in part to the threat o f both intra and interspecific predation. The fact that adult cutthroat trout had a negative impact on the feeding behavior o f the Y O Y crayfish introduced the idea for the 5 chapter o f the thesis, to carry out a comprehensive investigation o f th  the ontogenetic relationships between signal crayfish and cutthroat trout. The relationship between fish and crayfish has been studied by several researchers (Englund 1999, Englund and Kruppa 2000, Usio and Townsend 2000, Dora and Mittelbach 2001); however, most often the impact o f adults of one species on juveniles o f the other species is assessed. I wanted to examine the relationship o f adults o f one species on juveniles o f the other, but additionally I wanted to examine the reciprocal relationship, as well as the effects o f adults on adults and juveniles on juveniles. Another large-scale enclosure experiment was carried out in order to assess both direct (through monitoring o f individual weight gain or loss) and indirect (through monitoring o f the benthic and leaf pack communities) effects o f these pairings. I found that there was a reciprocal direct negative relationship o f adults of one species on juveniles o f the other. In each case the juvenile organisms did not experience as much weight gain as when they were alone. In addition, I found some evidence o f a negative impact o f Y O Y crayfish on Y O Y fish (a relationship that had not been studied previously); the Y O Y fish lost a 131  considerable amount o f weight when paired with Y O Y crayfish, and also experienced a higher rate o f death than when they were alone or paired with adult crayfish. Overall the research presented in this chapter outlines a need for further investigation into ontogenetic relationships among species in freshwater ecosystems. The indirect effects o f the different pairings in this experiment provided further verification o f my claim that the effects o f crayfish on the leaf pack community are largely due to indirect chemical cues. The same results were realized for any crayfish treatment, and fish did not have an effect on these results. In order to substantiate the work done i n chapters 2-5 of this thesis, it was imperative for me to investigate aspects of the crayfish populations in Spring Creek. I wanted to have a general idea about crayfish density, survival, movement patterns, and microhabitat use. I was also interested in whether any o f the above had an ontogenetic component to them. I carried out large-scale capture-mark-recapture studies at two sites in Spring Creek (chapter 6) in order to investigate these questions. A mark-recapture study was conducted using Pollock's robust design (Pollock 1982), combining the Cormack-Jolly-Seber (CJS) open recapture model (Cormack 1964, Jolly 1965, Seber 1965) and Huggins' closed capture estimator (Huggins 1989, 1991). This study included three primary capture sessions. Each primary session consisted of a series o f secondary visits (5 for session 1, 4 for sessions 2 and 3). Through the analysis o f the data from this study (using the program M A R X (Cooch and White 2006)) I concluded that there were large populations size (>400) o f crayfish found at both sites. I found that survival of crayfish was dependent on size (i.e. ontogeny), and that probability of capture was dependent on time and place (and not size). In addition, I found that there were differences in microhabitat use between adult and juvenile crayfish. A l l crayfish generally preferred still pools and woody-debris filled areas; however, juveniles tended to be found more often near the stream bank or pool edge. Despite the overall differences in microhabitat preferences found by juveniles and adults, any ontogenetic stage could be found i n the favorable 132  microhabitat types. The main point from this part o f the study is that there are areas in the small stream environment where crayfish of any size are likely to be found, and there are areas where crayfish of any size are unlikely to be found. This study also provided information on movement patterns of juvenile and adult crayfish. The results showed there to be an ontogenetic component to the movement patterns o f the crayfish. Juveniles tended to move around more than adults; however, adults moved more when a greater number o f adults were present. Adult crayfish appear to establish 'ephemeral home ranges' (Robinson et al. 2000), and do not move around too much from these areas (at least in the short term) The data from this chapter provides a framework i n which to interpret the research presented in chapters 2-5, because it provides information about the size o f crayfish populations in addition to the breakdown o f the population size structure. It shows that crayfish are a major component to the stream community, and that their effects on the ecosystem (and more specifically in habitats with low flow and high debris buildup) are likely to be substantial. A s mentioned above, this work represents the first set o f detailed studies done on P. leniusculus in its native environment for several decades. The fact my results are quite different from results obtained by other researchers studying P. leniusculus i n introduced habitats is noteworthy. Work done in Europe and A s i a (based on gut content analyses) suggests that both juvenile and adult P. leniusculus are predatory on several invertebrate groups (juveniles have been shown to be more predatory than adults), while my work suggests that this organism is primarily a detritivore during all life history stages in its native environment. Overall this shows that an organism may have a markedly different ecology i n introduced habitats (perhaps due to founder effects, biotic and/or abiotic disparities between systems). For example, the invertebrates that co-exist with P. leniusculus in its native environment should be adapted to its presence, and therefore should be less affected by its presence than invertebrates that have not 133  been exposed to it before. It w i l l be important to bring attention to this aspect of my results when assessing the appropriateness o f future introductions o f crayfish and other species to foreign ecosystems.  Directions for future research I suggest that future work in this area focus on the sensitivity o f other stream invertebrates, fish and amphibians to chemical cues from crayfish. The results of my enclosure experiments suggest strongly that this may be the way in which crayfish affect the surrounding ecosystem, but apart from several studies on snails (e.g. C r o w l and Covich 1990, Alexander and Covich 1991) and one mayfly (Richmond and Lasenby 2006), studies in this regard are scarce. In addition to work exploring the effects o f chemical cues o f crayfish on members o f the stream ecosystem, the relationship between Y O Y fish and crayfish should be further investigated. M y suggestion in chapter 5 that Y O Y crayfish potentially have a negative effect on Y O Y fish is based on the fact that Y O Y fish lost a significantly greater amount o f weight when paired with Y O Y crayfish. Further work both i n the field and in laboratory-based experiments is necessary in order to provide additional support to this claim. If this negative relationship is shown to exist in more than one scenario, it w i l l be an interesting and substantial contribution to the ontogenetic ecology o f organisms in small streams. Crayfish are long lived, and therefore must have several different ontogenetic relationships with other organisms i n the stream community. If the relationship between Y O Y crayfish and fish is indeed substantiated by future research, this allows for the possibility that Y O Y crayfish have relationships (either negative or positive) with other members o f the benthic community. The relationship of juvenile crayfish with other invertebrates of a similar size, especially large predators such as dragonfly and damselfly larvae, would be another valuable area o f study. 134  The research presented in chapter 3 showed that an equal amount o f growth occurred for both adult and juvenile crayfish whether they were fed a constant diet o f leaves or wood. This leads me to speculate about the nature o f the biofilms that accumulate on these allochthonous sources. Although neither leaves nor wood were as profitable (in terms o f growth) as invertebrates for the crayfish, it is clear that there is a general dependence o f P. leniusculus on these food types in a natural setting. Research into the composition o f the biofilms, their specific nutritional values, and how they are assimilated by crayfish would provide a substantial contribution to our understanding o f their contribution to the diet of crayfish and other invertebrates. In addition, an investigation into how the biofilms vary depending on substrate and season would also provide valuable insight into the dynamics of this food source. The differential microhabitat use o f adult and juvenile crayfish that I found in chapter 6 is another area which requires a more detailed investigation. Particularly, it would be valuable to have a quantifiable method to discern the habitat use o f crayfish in terms o f how much 'desirable' habitat is available. Future surveys should incorporate a measurement o f the occurrence o f different habitat types in order to substantiate my assertion that specific microhabitat types are more often utilized by crayfish.  Conclusion Overall, this thesis has provided a detailed look at the ontogenetic ecology o f a freshwater invertebrate. The developing field o f ontogenetic ecology w i l l benefit from this work, and the future work that w i l l follow. The areas that I have outlined above provide several different and interesting research opportunities for which the ground work has been established by the research accomplished in this thesis. In addition to these areas, I suggest that research in the area o f ontogenetic ecology should focus on invertebrates in freshwater systems. Most of the work on this topic has been done on fish communities (e.g. Werner and H a l l 1988, Bystrom and 135  Garcia-Berthou 1999, Mittelbach and Persson 1998), and my research shows that invertebrates are important candidates for such study as well. In addition to the fact that invertebrates often comprise the majority o f organismal biomass in freshwater systems, the ontogenetic niche shifts exhibited by most invertebrates make the study o f their ecology through development a necessity in order to fully comprehend freshwater food web and ecosystem dynamics.  136  References Alexander, J. E . and A . P. Covich. 1991. 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The behavioral response o f mayfly nymphs (Stenonema sp.) to chemical cues from crayfish (Orconectes rusticus). Hydrobiologia 560:335-343. Seber, G . A . F . 1965. A note on the multiple-recapture census. Biometrika 52:249-259. Stenroth, P. and P. Nystrom. 2003. Exotic crayfish in a brown water stream: effects on juvenile trout, invertebrates and algae. Freshwater Biology 48:466-475. Usio, N . and C.R. Townsend. 2000. Distribution of the N e w Zealand crayfish Paranephrops zealandicus in relation to stream physico-chemistry, predatory fish, and invertebrate prey. N e w Zealand Journal of Marine and Freshwater Research 34:557-567. Werner, E . E . and J. D . Hall. 1988. Ontogenetic habitat shifts in bluegill: the foraging rate predation risk trade-off. Ecology 69:1352-1366. Whitledge, G . W . and C . F. Rabeni. 1997. Energy sources and ecological role o f crayfishes in an Ozark stream: insights from stable isotopes and gut analysis. Canadian Journal o f Fisheries and Aquatic Sciences 54: 2555-2563.  138  

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