Investigation of Physiological and Behavioral Alarm Responses in Larval White Sturgeon (Acipenser Transmontanus)

Home , Hemigrammus erythrozonus, Reedfish

Investigation of physiological and behavioral alarm responses in larval white sturgeon (Acipenser transmontanus)

Junho Eom

B.Sc., Kangwon National University, 2003

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology)

THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)

May 2016

White sturgeon populations in North America have dramatically decreased because of the commercial demand for caviar in the past and anthropogenic activities in the present. To conserve white sturgeon, recovery planning is required to ensure that the fish are self- sustaining through natural reproduction. However, little is known about many important aspects of white sturgeon biology, including their ability to detect and respond to alarm cues, the goal of this thesis. My results show that larval white sturgeon possess epidermal club cells, which in other fish are known to contain alarm cues. I investigated the effect of exposure to whole body extracts (WBE) of conspecifics (which contain the epidermal cells) on olfaction, through the use of electro-olfactogram (EOG), and investigated the effect of WBE exposure on behavioural responses and whole body cortisol levels in ~20 day post hatch white sturgeon larvae. The fish larvae showed alarm behaviors, such as avoidance, dashing, and freezing when exposed to WBE. In WBE treatment, the fish also increased whole body cortisol levels that are known as an indicator of stress. This thesis not only provides information on fundamental aspects of white sturgeon biology, but also may help fisheries management to understand how alarm substances are perceived in hatchery-reared naïve white sturgeon larvae.

ii Preface

This thesis is an original intellectual product of the author, J. Eom. The animal research reported in Chapter 2 was approved by the University of British Columbia

Animal Research Ethics Board (A15-0266) and was in accordance with the guideline of the Canadian Council on Animal Care.

The section on “2.3.5 Whole body steroid extraction for cortisol analysis” was conducted by Satbir Rai.

iii Table of contents Abstract ...... ii Preface...... iii Table of contents ...... iv List of tables ...... vi List of figures ...... vii Acknowledgements ...... ix Chapter 1: General introduction...... 1 1.1 White sturgeon in North America ...... 1 1.1.1 Description ...... 1 1.1.2 Distribution ...... 1 1.1.3 Life history ...... 2 1.1.4 Threats and limiting factors ...... 2 1.1.5 Research efforts to conserve white sturgeon ...... 3 1.2 Alarm pheromones in fish ...... 4 1.2.1 Sensory systems and alarm substances ...... 4 1.2.2 Alarm pheromones ...... 6 1.2.3 Origin of alarm substances...... 8 1.2.4 Predator-prey relationships and alarm responses ...... 9 1.3 Stress and alarm behaviors ...... 10 1.3.1 Definition of stress ...... 10 1.3.2 Stress response in fish ...... 10 1.3.3 Potential use of alarm pheromones in fisheries management ...... 11 1.4 Thesis objectives and hypotheses ...... 12

Chapter 2: Characterization of alarm responses in white sturgeon larvae ...... 15 2.1 Introduction ...... 15 2.2 Materials and methods ...... 16 2.2.1 Experimental animals and maintenance ...... 16 2.2.2 Histology of white sturgeon larvae ...... 17 2.2.3 Whole Body Extract preparation ...... 18

iv 2.2.4 Electrophysiological examination of olfactory responses to EOG stimuli ...... 18 2.2.5 Behavior assays to whole body extract ...... 21 2.2.5.1 Two-choice maze assay ...... 21 2.2.5.2 Petri dish assay ...... 22 2.2.6 Whole body steroid extraction for cortisol analysis ...... 23 2.2.7 Data analyses ...... 24 2.3 Results ...... 25 2.3.1 Histology of white sturgeon larvae ...... 25 2.3.2 Electrophysiological examination of olfactory responses to EOG stimuli ...... 25 2.3.3 Two-choice maze assay ...... 27 2.3.4 Petri dish assay ...... 27 2.3.5 Whole body steroid extraction for cortisol analysis ...... 27 2.4 Discussion ...... 28 2.4.1 Identification of club cells ...... 28 2.4.2 Electrophysiological examination of olfactory responses to EOG stimuli ...... 29 2.4.3 Avoidance behavior of larval white sturgeon ...... 30 2.4.4 Behavioral responses to alarm cues ...... 31 2.4.5 Physiological effects of alarm cues on stress responses ...... 33 2.4.6 Conspecific alarm cues in fisheries management ...... 34

Chapter 3: Conclusions ...... 49 3.1 Thesis summary ...... 49 3.2 Thesis objectives and hypotheses ...... 49 3.3 Research limitations and future directions ...... 51 References ...... 52

v List of tables

Table 1: List of representative examples of alarm cues in other species and their responses…………………………………………………………………………..…..36

vi List of figures

Figure 1: Electro-olfactogram (EOG) of early life stages of white sturgeon. In anesthetized fish (tricaine methanesulfonate, 0.0375 mg/ml, final concentration), olfactory sensitivity was examined by delivering chemical stimuli. Olfactory response magnitudes were amplified and visualized for analysis……………………………38

Figure 2: Avoidance behavior was observed in two-choice maze assay. After acclimation, A) avoidance behavior was tested with respect to freshwater (FW) or alarm pheromone contained whole body extract (WBE). In order to drain waste efficiently, B) overall system was tilted at 10 degrees. For video analysis, fish behavior was recorded…………………………………………………….39

Figure 3: Histology sections of early life stages of white sturgeon. Sliced epidermal club cells (ECC) and mucous cells (MC) in the epidermal layer (E) were magnified in A) 100X and B) 200X. The dermal layer (D) lies between epidermal layer (E) and abdominal cavity (AC). Scale bars represent 1mm.40

Figure 4: The spatial distribution (in the back, tail, head, and abdomen) of epidermal club cells in 20 dph white sturgeon larvae (n=5) consisting of A) size of club cells (p=0.0023) and B) distance to dermis, and C) number of club cells. A symbol of asterisk indicates statistically significant differences……………………………..41

Figure 5: A) Electro-olfactogram (EOG) responses and B) their response magnitudes in larval white sturgeon (n=3) exposed to 10-4 M of amino acids and bile acids. Of the 20 amino acids and 9 bile acids, fish larvae responded to 6 amino acids: L-lysine (K), L-arginine (R), L-Glutamic (E), L-histidine (H), L-cysteine (C), and L- aspartic (D), and 3 bile acids: taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDA), and taurocholic acid (TCA) that are shown here. A symbol of asterisk indicates statistically significant differences (One-way ANOVA, p<0.0001)………..42

Figure 6: Electro-olfactogram (EOG) in white sturgeon larvae (n=3) exposed to serially diluted whole body extract (WBE). Between 0.026 mg/ml and 26 mg/ml diluted WBE, the fish larvae significantly increased olfactory sensitivity (one-way ANOVA, p<0.0001). In addition, initial threshold of olfactory sensitivity occurred between 0.026 mg/ml and 0.26 mg/ml (Unpaired student’s t-test, p=0.0122)……………………………………………………….43

vii Figure 7: Percent of time larval white sturgeon spent in the two channel maze assay, with a choice of either freshwater (FW) or diluted whole body extract (WBE). Fish larvae avoided the channel supplied with 0.065 mg/ml WBE, and spent more time in the FW channel (Unpaired student t-test, p<0.0001, n=6). However, avoidance to the WBE treated channel was not observed when the WBE concentration was less than 0.052 mg/ml. Due to time spent in starting area where fish was released, sum of time spent percent in the two channel maze assay showed below 100%...... 44

Figure 8: Movement of white sturgeon larvae in the petri dish assay in response to freshwater (FW) (n=5) or whole body extract (WBE, 2.6 mg/ml final concentration) addition (n=5) as quantified by the Ethovision animal tracking software. Unlike FW treatment fish larvae that occupied entire petri dish, WBE treatment fish larvae gradually decreased activity levels and occupied areas distant from the center of the petri dish where WBE was applied…………...45

Figure 9: Alarm responses of white sturgeon larvae in the petric dish assay in fish exposed to freshwater (FW) (n=5) or diluted whole body extract (WBE, 2.6 mg/ml final concentration) (n=5). Using the Ethovision video tracking software, the movement patterns and activity levels were analyzed and the values were calculated using the equation of “(either FW or WBE treatment value – acclimation value) / acclimation value x 100 (%).” In general, WBE treatment fish larvae decreased activity levels compare to FW treatment fish (two-way ANOVA, P=0.0003), especially mean turn angle (Unpaired student’s t-test, p=0.0018) and total movement duration (p=0.0020). A symbol of asterisk indicates statistically significant differences………………………………………………………46

Figure 10: C-start body curls over 5 minutes in white sturgeon larvae in response to a predators approach in the petri dish assay with freshwater (FW) (n=5) or whole body extract (WBE, 2.6 mg/ml final concentration) (n=5). A symbol of asterisk indicates statistically significant differences (Unpaired student’s t-test, p<0.0001)……………………………………………………….47

Figure 11: Whole body cortisol levels in larval white sturgeon exposed for 1 h to freshwater (FW) (n=11) or hole body extract (WBE, 2.6 mg/ml final concentration) (n=10). A symbol of asterisk indicates statistically significant differences (Unpaired student’s t-test, p<0.0001)………………………………………………………..48

viii Acknowledgements

I would like to thank Dr. David Close and Dr. Sang-Seon Yun, who have supported me and enabled me to accomplish the white sturgeon larvae project. I also thank Dr. Colin Brauner and Dr. Steve McAdam, who encouraged me to finish this project properly.

Satbir Rai performed radioimmunoassay (RIA) analysis for whole body cortisol measurements. The animal care was performed by Catherine Cheung. Adam Goulding assisted me in designing the two-choice maze experiment. Dr. Anthony G. Phillips in the

Department of Psychology at the University of British Columbia (UBC), kindly shared the Ethovision software for the video tracking analyses of fish behavior. The microscopic imaging of club cells was done at the UBC BioImaging Centre with partial assistance from Kevin Hodgson.

This project was funded by BC Ministry of Environment and the Department of

Fisheries and Oceans.

ix Chapter 1: General introduction

White sturgeon are a conservation concern in North America due to population declines caused by commercial demand for caviar in the past and anthropogenic activities in the present. Although the species overall is not endangered some populations are legally listed as endangered in both Canada and US. Recovery planning is recommended to ensure that those white sturgeon populations are self-sustaining through natural reproduction. However, there continue to be important biological uncertainties for this species.

1.1 White sturgeon in North America

1.1.1 Description

The white sturgeon, Acipenser transmontanus, is the largest freshwater fish found in North America and comes from an ancient lineage of ray-finned fish (Scott and

Crossman 1973). The overall body of the adult white sturgeon is more rounded than that of other sturgeon species. The white sturgeon has a large and bluntly rounded head that includes small eyes, a relatively large olfactory system, and four conspicuous barbels anterior to the mouth. The ventral surface possesses a toothless mouth that siphons gape- fitted prey items. Most of the skeleton is cartilaginous except for the superficial bones of the skull, jaw, and pectoral girdle (Scott and Crossman 1973).

1.1.2 Distribution

After Lord (1866) reported the existence of white sturgeon, this species has been recorded in North America. Specifically in Canada, the white sturgeon is distributed in

1 the Fraser River system, including the Harrison, Lower Pitt, and Stellako rivers, Fraser and Stuart lakes, the Taku River, and the Columbia River watershed including the mainstream Columbia River, Arrow Lakes Reservoir, as well as Duncan Reservoir,

Kootenay Lake and Kootenay River (COSEWIC 2003, COSEWIC 2013, McPhail 2007,

Smith et al. 2002).

1.1.3 Life history

The semi-anadromous mature white sturgeon migrate into upstream river areas for spawning in May and June. Spawning occurs over cobbled and graveled bottoms in swift currents when water temperatures are around 14 C (Paragamian and Wakkinen 2002).

After spawning, the eggs adhere to the bottom of the river over cobbled and graveled substrates, which provide protection from predators (Parsley and Beckman 1994). The hatched larvae are initially nourished by the yolk; after 12 days at 17.5 C or 16 days at

13.5 C, the larvae have absorbed the yolk (Boucher et al. 2014), emerge from the substrate for feeding, and drift downstream during the night. White sturgeon consume benthic living organisms, including invertebrates, insect larvae, mollusks, isopods, and relatively small fish (Carl et al. 1967, McPhail 2007).

1.1.4 Threats and limiting factors

Although white sturgeon are listed as “Least Concern”, some populations, such as the Kootenay River, remain in the threatened categories (IUCN 2004). In general, sturgeon species have suffered from habitat fragmentation between dams (Prince 2001,

Shmigirilov et al. 2006), habitat and spawning site losses (Billard and Lecointre 2001,

2 Rosenau and Angelo 2005), and recruitment failure (Partridge 1983, McAdam 2015).

Moreover, by-catch in commercial salmon fisheries and catch-and-release recreational activities in North America are thought to increase fish mortality (COSEWIC 2013,

Fisheries and Oceans Canada 2013). Regarding recruitment failure, the fine substratum that builds up in regulated rivers is known to result in a high mortality rate for larval sturgeon (McAdam 2011, McAdam 2012) because their inability to hide in the fine sediment leads to high predation in aquatic environments.

1.1.5 Research efforts to conserve white sturgeon

In North America, white sturgeon are a valuable fish for recreational and guided angling, aboriginal fisheries, scientific inquiry, aquaculture, and ecosystem education.

However, this valuable fish has been in decline. To protect white sturgeon in decline, federal legislation has been triggered. For example, under the Federal Species at Risk Act

(SARA), white sturgeon in the Kootenay, Nechako, Columbia, and Upper Fraser rivers are listed as a Schedule 1 endangered species (Minister of Justice 2002). Notably white sturgeon populations in the middle and lower Fraser River are not listed under SARA.

Provincially, the Fraser and Stuart rivers are the only two rivers containing white sturgeon that have been designated “protected rivers” by the British Columbia Fish

Protection Act (Fisheries and Oceans Canada 2013). In such a small population size, one general problem is limited genetic diversity which may limit population recovery.

Recovery planning for an overall increase in white sturgeon populations is required for multiple reasons, with the goal of restarting self-sustaining populations through natural reproduction (Duke et al. 1999, Fisheries and Oceans Canada 2013).

3 In general, a recovery plan is designed to lead to the survival of a species at risk.

To fulfill the recovery strategies of endangered species including white sturgeon, it is essential to incorporate environmental considerations into the development of public policies and recovery strategies (Billard and Lecointre 2001). The following knowledge gaps need to be addressed to develop a recovery plan and national guidelines that incorporate environmental considerations: the basis for recruitment failure in dam-caused geographical fragmentation, development of methods for fish culture for stocking programs, clarification of existing threats, and biological needs in each life cycle of the fish (Fisheries and Oceans Canada 2013).

Hatchery programs are required due to the presence of long-term recruitment failure (COSEWIC 2013), however, hatcheries lead to concerns regarding cost effective and feasible white sturgeon recovery plan (Boucher et al. 2014, Irelands et al. 2002). For example, low survival rates from predation remain a concern for hatchery-reared predator naïve fish including white sturgeon (Suboski and Templeton 1989). Life-skills training with alarm substances mediated by the olfactory system along with visual cues to potential predators may make it possible for prey fish to learn to recognize actual predators and thus may increase survival rates in early life stages, especially in hatchery- reared fish (Sloychuk and Chivers 2016). The potential for life skills training is dependent upon the underlying biology to detect and respond to appropriate substances, and one outcome of this thesis is the ability to evaluate that potential.

4 1.2 Alarm pheromones in fish

1.2.1 Sensory systems and alarm substances

Fish use sensory cues such as visual, olfactory, and tactile in predator-prey interactions. The visual cues are essential for recognizing potential predators. For example, Chivers and Smith (1994) claimed that the association between instinctive alarm behavior and alarm substances mediated by the olfactory system along with visual cues to potential predators may make it possible for prey fish to learn to recognize actual predators. Specifically, in laboratory experiments, after 2 days of training combining visual cues of potential predators with the alarm pheromones of skin damaged conspecific homogenates, naïve fathead minnows (Pimephales promelas) showed significant alarm responses to a natural predator (pike) and to a non-piscivorous exotic

(goldfish). However, visual cues are greatly limited in opaque underwater environments; therefore, benthic or nocturnal fish including sturgeon species rely on the olfactory system to sense alarm substances, including the alarm pheromones released by damaged conspecifics (Abrahams and Kattenfeld 1997, Liley 1982).

In opaque underwater environments where the visual cues are greatly limited, the olfactory cues that are detected by olfactory organs are critical in predator-prey interactions. The olfactory organs in fish are generally situated in paired snouts. Each olfactory organ contains one olfactory chamber that is divided by a nasal bridge. An olfactory rosette covered by olfactory epithelium is situated on the floor of the olfactory chamber. The olfactory epithelium contains different types of chemoreceptors which are directly connected to the olfactory bulb in the brain. Eventually, fish behaviors are

5 influenced by olfactory sensitivity to specific chemical substances including alarm pheromones (Zeiske et al. 1992).

Fish are also known to use their flow-sensitive lateral line system for detecting their environment. The lateral line system is a sensory organ detecting movement and vibration in the surrounding water and is found mainly in fish (McHenry et al. 2009). In predator-prey interactions, fish are known to use lateral line system and increase undulatory swimming that is triggered by the C-start body curls with respect to a predator’s approach (Stewart et al. 2010). In the lateral line system of prey fish, the sensory hair cells, which include mechanoreceptors, allow the fish to sense the flow from a predator (Dijkgraaf 1963). Stewart et al. (2010), who also examined the function of mechanosensory receptors in the lateral line system, found that fish failed to avoid the predator’s attack after an experimental ablation of the hair cells. In general, the sensory- motor system provides an additional predator detection mechanism when available in any circumstance where visual cues are more likely to be limited. Moreover, the sensory- motor system responds faster than the visual system because of the different latency time in response to a predator’s approach (Stewart et al. 2010). However, further studies are required to understand whether the prey fish generate the sensory-motor response specifically in response to a predator or as a way to avoid any object in aquatic environments.

1.2.2 Alarm pheromones

In fish, an alarm pheromone is defined as a chemical mixture released from damaged individuals (von Frisch 1938). In fish, release of this pheromone can trigger

6 alarm behavior in neighboring conspecifics to avoid insecure situations, especially predators (Brown and Dreier 2002, Brown and Godin 1999, Chivers and Smith 1994).

Since von Frisch (1938) observed alarm responses in the European minnow (Phoxinus phoxinus), a large number of studies have reported alarm behaviors in various fish species in response to skin-extracted chemical substances known to contain alarm pheromones (Table 1). Alarm pheromones also elicit more complex responses including attraction of secondary predators and the potential interruption of initial predation (Lima and Dill 1989).

Alarm pheromones are thought to be species-specific (Brown et al. 2000, Smith

1979, Hazlett 1990). For example, conspecific skin extract but not swordtail

(Xiphophorus helleri) skin extract elicited alarm behaviors in fathead minnows

(Pimephales promelas) (Brown et al. 2000). In fathead minnows, Pfeiffer et al. (1985) characterized hypoxanthine-3-N-oxide as the alarm pheromone in the skin extract.

Similarly, hypoxanthine-3-N-oxide is thought to be the active alarm ingredient of ostariophysan because it triggers species-specific fright responses, such as fast swimming followed by avoidance or shelter-seeking behavior (Argentini 1976, Pfeiffer et al. 1985).

The skin extract is thought to trigger the fright responses, however, little is known about chemical structures which trigger the fright responses in fish (Pfeiffer et al. 1986).

Brown et al. (2000) claimed that general types of alarm pheromones include nitrogen-oxide chemical compounds that are similar in structure based upon the observation that fathead minnows displayed alarm behaviors in response to pyridine-N- oxide and hypoxanthing-3-N-oxide which are structurally similar. However, fish did not show alarm behaviors to structurally similar molecules, such as guanine, hypoxanthine,

7 xanthine, 4(3H)-pyrimidine, and pyridine. In addition, zebrafish showed slow swimming without darting in extracts with a high molecular weight (~30 kD by protein standard) while darting increased in extracts with a low molecular weight fraction (~1 kD)

(Mathuru et al. 2012). These prior findings suggest that fish such as white sturgeon might sense a range of compounds as alarm pheromones rather than just a single-structure molecule.

1.2.3 Origin of alarm substances

In addition to helping us understand alarm behaviors, knowing the origin of alarm pheromones may improve our understanding of the mechanism of alarm responses in fish. In their studies of the superorder Ostariophysi, especially minnow species,

Pfeiffer (1960) and Smith (1979) hypothesized that large numbers of specialized “club cells” situated in the epidermis release hypoxanthine 3-N-oxide or a similar nitrogen- oxide functional group (Brown et al. 2000) as alarm pheromones into the aquatic environment after mechanical skin damage. Club cells are situated in the surface layers of the epidermis and their contents are among the first to be released to the environment following a predator’s attack. Consequently, club cells are thought to represent the origin of alarm pheromones in fish (Pfeiffer 1960). To date, abundant data support hypothesis that skin extraction triggers conspecifics’ alarm behavior because of the large number of epidermal club cells (Barreto et al. 2010, Mittal and Munshi 1969). For example, immature goldfish (Carassius auratus) are lured by conspecifics’ holding water but are repelled by adding conspecific skin extract (Chivers and Smith 1994). Moreover, lake sturgeon (Acipenser fulvescens) larvae and juveniles significantly increase their alarm

8 activity levels after exposure to conspecific whole-body extract (WBE) (Wishingrad et al.

2014).

During breeding season, decreases in the number of club cells as a result of hormonal changes are also reported. Such decreases are known to reduce the release of alarm substances from abrasive spawning behavior (Smith 1976). To understand whether the hormonal changes affect fright responses in fish, however, interactions between decreasing epidermal club cells during breeding season and changing alarm activity levels to skin extract need to be investigated.

1.2.4 Predator-prey relationships and alarm responses

In predators, it is obvious that locomotion is essential to catching prey during the stages of detection, identification, approach, and subjugation (Endler 1986). However, the prey also uses locomotion changes in anti-predator defense mechanisms to avoid predation. For example, freezing behavior is known as an anti-predator defense behavior because it minimizes the probability of detection by decreasing the signal-to-noise ratio which predators perceive (Endler 1986). Mimicry is also a defense behavior in prey fish as mimicking something similar in the background delays identification (Feder and

Lauder 1986). With respect to the predator’s approach and subjugation, dashing movements with C-start body curls for rapid direction changes have also been described as a means to escape from predators (Stewart 2010). Although an individual prey fish will be consumed if the predator’s attack is successful, the anti-predator defense mechanisms also work to alert neighboring conspecifics through the alarm pheromones released from the damaged individual prey fish.

9 1.3 Stress and alarm behaviors

1.3.1 Definition of stress

One definition of stress is that it is the state of an organism with respect to an environmental or other factor which extends the adaptive responses of the organism beyond the normal range or which disturbs the normal functioning to such an extent that, in either case, the chances of survival are significantly reduced (Brett 1958). The stress response in an organism includes the integration of all levels of responses, including neuro-endocrine responses, physiological responses, and behavioral responses (Mazeaud et al. 1977, Wedemeyer and McLeay 1981). Thus, the measurement of one level of response is insufficient for understanding the full extent of the stress response.

1.3.2 Stress response in fish

Stress stimulates the central nervous system of prey fish initially and affects acute body processes, including physiological changes. In fish, an increase in plasma cortisol levels through the hypothalamo-pituitary-interrenal (HPI) axis is widely accepted as a primary component of the stress response (Alsop and Vijayan 2008, Bonga 1977).

Cortisol is a steroid hormone belonging to a class of glucocorticoids produced by the adrenal gland or interrenal tissues in fish. The primary function of cortisol is to redistribute energy to enhance rapid physical movement, such as avoiding or fighting, in response to stressful circumstances (Kloas et al. 1997).

Stress levels in fish are known to increase in response to various stressors, such as predator’s chasing, water depth reduction, and net confinement (Lankford et al. 2005).

Fleeing from a predator’s chasing, in particular, is known as a stress response in prey fish

10 because prey fish perish if they fail to escape from predators (Feder and Lauder 1986).

For example, the plasma cortisol levels in hatchery-reared juvenile green sturgeon (age

1+, 2.6 – 2.9 kg live weight) significantly increased, from ~3 ng/ml to 18.8 ng/ml, after 5 min of chasing (Lankford et al. 2005). In this chasing experiment, the elevated cortisol levels gradually returned to resting levels within 3 hrs.

1.3.3 Potential use of alarm pheromones in fisheries management

Hatchery-reared fish are routinely stocked in the wild to restore or maintain stable populations, especially with salmonids (Brown and Smith 1998). After releasing hatchery-reared fish into the wild, a dramatic increase in mortality levels of the newly stocked individuals by predation is a major problem (Suboski and Templeton 1989). The mortality levels of hatchery-reared fish are thought to be increased by their failure to recognize predators (Berejikian 1995). Several attempts have been made to decrease mortality levels from predation and increase survival rates in hatchery-reared fish by allowing naïve fry to interact with electric current stimulus paired with a model predator

(Thompson 1966) or a live predator (Kanid’hev et al. 1970). However, the reduction in mortality was not significant in these studies.

Instead of electric current stimulus or a live predator, some researchers have suggested life-skill training using conspecific alarm cues with predator visual cues and/or predator chemical cues (Magurran 1989, Chivers et al. 1995, Suboski et al. 1990) to train fish to avoid potential predators. For example, predator naïve fish show lack of alarm responses from a novel predator. However, fish elicited alarm responses after treating with alarm pheromones and predator visual cues (Suboski et al. 1990).

11 This life-skill training may make it possible for prey fish to recognize actual predators in all of their life stages (Chivers and Smith 1994, Suboski et al. 1990). For example, Chivers and Smith (1994) suggested the possibility of life-skill training in conservation biology, especially in hatchery-reared fish which would not normally be exposed to predators (Chivers and Smith 1994). However, further research is required into the possibility of life-skills training in hatchery-reared fish, especially white sturgeon, which is the focus of this study.

1.4 Thesis objectives and hypotheses

The general objective of this thesis is to understand physiological and behavioral alarm responses in the early life stages of white sturgeon. To understand the physiological and behavioral alarm responses in white sturgeon, I first examined for the presence of club cells using histology of whole body epidermis. Then I characterized the electrophysiology of olfactory sensitivity, and conducted behavioral assays in response to a conspecific alarm substance, and finally investigated the endocrinological stress response to whole body extracts of conspecifics.

Objective 1: Club cell identification in the epidermis of white sturgeon larvae.

Epidermal club cells are thought to release alarm pheromones after a predator’s attack, which subsequently trigger alarm responses in neighboring conspecifics (Pfeiffer

1960, Smith 1979). The first component of characterizing the alarm response in white sturgeon was to investigate whether club cells exist in the surface layer of the epidermis of white sturgeon larvae.

12 Null hypothesis: White sturgeon larvae do not contain club cells.

Alternative hypothesis: White sturgeon larvae contain club cells.

Objective 2: Electrophysiological examination of olfactory responses to EOG stimuli.

White sturgeon are a benthic animal in aquatic environments. Because visual signals are greatly limited in opaque and dim-lighted benthic areas, white sturgeon larvae may rely heavily on non-visual sensory systems, mostly the olfactory system, to avoid dangerous situations including potential predators. Therefore, investigation of olfactory sensitivity with respect to potential alarm substances is an essential process to establishing a functional alarm response.

Null hypothesis: White sturgeon larvae do not sense chemical substances through their olfactory system.

Alternative hypothesis: White sturgeon larvae sense chemical substances through their olfactory system.

Objective 3: Investigation of alarm behaviors in the presence of whole body extract

(WBE) as a potential alarm pheromone.

After a predator’s attack, it is known that the prey fish release alarm substances from damaged epidermal club cells that trigger alarm responses in neighboring conspecifics. Although the attacked fish is damaged and may perish, neighboring conspecifics may survive because of their responses to the conspecific alarm signals.

Therefore, WBE that contains damaged epidermal club cells may trigger alarm behaviors in white sturgeon larvae.

13 Null hypothesis: WBE does not trigger alarm behaviors in white sturgeon larvae.

Alternative hypothesis: WBE triggers alarm behaviors in white sturgeon larvae.

Objective 4: Determine whether exposure to whole body extracts elevated whole body cortisol levels.

Because prey fish perish if they fail to escape from predators in predator-prey relationships, fleeing from a predator is known as a stress response. It is well known that stress hormone levels, especially the plasma cortisol levels, increase in the stressed fish.

Therefore, cortisol levels in white sturgeon larvae may increase in response to WBE treatment as part of stress response.

Null hypothesis: Cortisol levels in white sturgeon larvae do not increase in response to

WBE treatment.

Alternative hypothesis: Cortisol levels in white sturgeon larvae increase in response to

WBE treatment as part of the stress response.

14 Chapter 2: Characterization of alarm responses in white sturgeon larvae

2.1 Introduction

Since von Frisch (1938) observed alarm responses in the European minnow

(Phoxinus phoxinus), a large number of researchers have reported alarm behaviors in various fish species related to skin-extracted chemical substances known as alarm pheromones. Skin extracts that contain alarm substances, especially the epidermal club cells have been shown to trigger alarm behavior in neighboring conspecifics.

Behavioural responses to skin extracts include avoidance and dashing movements with

C-start body curls and freezing or shelter seeking (Brown and Dreier 2002, Brown and

Godin 1999, Chivers and Smith 1994).

Although several studies have dealt with the susceptibility of sturgeon species to predators (Gadomski and Parsley 2005, McAdam 2011), relatively little is known about sturgeon alarm pheromones. Only a few studies have reported responses to alarm cues in the different life phases of sturgeon (Hintz et al. 2013, Wishingrad et al. 2014). For example, Wishingrad et al. (2014) investigated fright responses in larval and juvenile lake sturgeon and concluded that fish increase activity levels when exposed to conspecific skin extract. In lake sturgeon, larvae exhibit a more developed fright response than juveniles, which is thought to be associated with reduced predation risks in juvenile stages (Wishingrad 2014). Predator avoidance was also examined in shovelnose sturgeon.

After a shovelnose sturgeon was bitten by predators, the sturgeon spent more time in the predator-free area than in the predator-occupied area (Hintz et al. 2013). Therefore, Hintz et al. (2013) concluded that the alarm response might be a learned behavior instead of an innate behavior in fish.

15 In the present study, physiological and behavioral patterns of alarm responses in larval white sturgeon were examined. First, specialized club cells situated in the surface layers of the epidermis that are known to release an alarm substances upon rupture were identified as described elsewhere (Pfeiffer 1960, Smith 1979). Then olfactory responses to alarm pheromones contained in the epidermal club cells using whole body extract

(WBE) were studied. Electrophysiological responses of olfactory organs to WBE were also studied based on the understanding that alarm behavior in fish are likely elicited by the transfer of sensory signals from olfactory receptor neurons to the olfactory bulb

(Hamdani and Doving 2007). Two behavior assays, a two-choice maze and a petri dish assay, were also used to investigate alarm behaviors in response to WBE, known as a potential alarm pheromone. Finally, stress responses of larval white sturgeon upon exposure to WBE were examined by measuring whole body cortisol levels. The findings not only demonstrate the early development of olfactory sensitivity that provides the alarm behavior response to conspecific alarm pheromones, but also suggest the possibility of using alarm pheromone treatments to train predator naïve hatchery fish as part of recovery programs for endangered populations of white sturgeon.

2.2 Materials and methods

2.2.1 Experimental animals and maintenance

All procedures were approved by the University of British Columbia Animal

Research Ethics Board and were in accordance with the guidelines of the Canadian

Council on Animal Care. The dechlorinated Vancouver city freshwater (pH 7.0, 14 C) was used during experiments.

16 White sturgeon embryos (6 days post fertilization) were shipped to the Vancouver

International Airport by airfreight from the Columbia Sturgeon Hatchery in Fort Steele,

British Columbia, Canada. The embryos were hatched in 38 L glass aquaria provided with aeration. After hatching, fish larvae were maintained in gravel substratum (~2 cm diameter) tanks (30 x 15 x 20.25 cm) filled with dechlorinated Vancouver city freshwater. The fish larvae were maintained at 15 C, and were fed with the brine shrimp

(Artemia International LLC, Fairview, TX, USA) starting at 12 days post hatch (dph). To maintain water quality in the tanks, half of water was replaced every day.

2.2.2 Histology of white sturgeon larvae

Prior to fixation, 20 dph fish larvae (n=6) were euthanized in MS-222 (0.0375 mg/ml, final concentration). Larval fish were then initially fixed in 4% para- formaldehyde in phosphate buffered saline (PBS) for 24 hours, and then transferred to a

30% sucrose solution until the fish larvae sank to the bottom of container at 4 C. After a few days of fixation, fish samples were embedded in paraffin after dehydrating through a graded series of ethanol and xylene respectively, and were sagittal sectioned (6 m thicknesses) using a rotary microtome. Deparaffinization and rehydration were conducted on a slide warmer, and slides were stained using a periodic acid-Schriff (PAS) staining method (Smith 1976). The sectioning and staining procedures were undertaken by Wax-it

Histology Services (Vancouver, BC, Canada).

The sagittal histology sections were visualized using a Zeiss AxioPlan 2 upright microscope connected to QImaging 12 bit cooled Charge-Coupled Device (CCD) camera and saved by QCapture software in the UBC BioImaging Centre. The entire fish

17 histology images were then divided by head, dorsal, ventral, and tail regions. The divided images were analyzed by image analysis software (ImageJ, Ver. 1.48, National Institute of Mental Health, Bethesda, MD, USA).

ImageJ is a public domain Java image-processing program written by National

Institutes of Health (NIH). Prior to club cell analyses, the scale bar imbedded TIFF histology image was opened in the ImageJ. The scale bar was used for setting scale by converting unit from pixels to international system of units. After setting the scale, I measured average number, surface area, and distance to dermis of club cells per 1 mm2

(n=10) in each designated body region. As depicted in figure 3C, the club cells were counted and measured from 5 sampling boxes (1mm x 1 mm) which were randomly designated in the middle of head, dorsal, tail, and ventral area.

2.2.3 Whole Body Extract preparation

To prepare whole body extract (WBE), donor larval fish (between 13-15dph, 25.9 g wet weight in total combined) were euthanized using a blow to body technique (Hugie and Smith 1986). The euthanized fish were then snap-frozen using liquid nitrogen and the frozen fish were ground using a mortar and pestle. To the ground tissue, 100 ml of distilled water was added and mixed thoroughly to prepare the 0.259 g/ml WBE stock solution. The WBE was then filtered (Whatman, Little Chalfont, Buckinghamshire, UK) and frozen in aliquots until use and was diluted with freshwater to achieve the alarm substances concentrations necessary in each experiment and stored at 4 C until next use.

18 2.2.4 Electrophysiological examination of olfactory responses to stimuli

Electro-olfactogram (EOG) was employed to examine olfactory sensitivity with respect to chemical stimulus (Figure 1). At first, screening EOG with amino acids and bile acids was conducted to assess the presence or absence of a response. Because amino acids and bile acids are known as food substances and sex pheromones in fish (Lemly and Smith 1985, 1987), these chemical substances are commonly used for analysis of fish olfactory sensitivity. Therefore, these chemicals are great candidates to examine the presence or absence of smell in fish EOG. After that, the EOG response to dose- dependent increases in WBE was measured to quantify olfactory sensitivity to WBE.

Prior to the EOG measurements, sensory pipettes were prepared with borosilicate glass capillaries (1B150F-4, World Precision Instruments, Sarasota, FL, USA). The borosilicate glass capillaries were pulled by the dual-stage glass micropipette puller (PC-

10, Narishige, East Meadow, NY, USA) to make a sharp tip-end (between 150 to 200

µm). The sharp tip-end glass capillaries were filled with 8% gelatin (Sigma) melted in

0.8% NaCl at 120 C and stored in 0.8% NaCl until use at 4 C. The gelatin filled fine tip-end glass capillaries were inserted into silver chloride (Ag/AgCl) microelectrodes

(World Precision Instruments, Sarasota, FL, USA) filled with 3M potassium chloride and used as sensory pipettes in EOG.

After preparing sensory pipettes, fish larvae were anesthetized in 3 mg/ml of tricaine methanesulfonate (MS-222, Western Chemicals, Ferndale, WA, USA) which was buffered with NaHCO3 for 5 seconds and placed on the vibration-free EOG table. Under a dissection microscope (SMZ645, Nikon, Mississauga, ON, Canada), nostrils skin was gently removed to expose the olfactory epithelium for direct contact with the recording

19 sensory pipette while attaching the other sensory pipette on skin near the nasal cavity as reference pipette. Background EOG signals were monitored while flowing freshwater over the fish olfactory epithelium to set the control baseline. After a quick MS-222 treatment, the anesthetization was lasted about 10-15 min during EOGs.

During the EOG recordings, either freshwater or chemical substances were applied to the olfactory epithelium by a gravity-fed flowing delivery system. The overall delivery system was controlled by a dual tubing 2-way pinch valve (100PD3MP12-02S,

Biochem Fluidics, Berlin, CT, USA) and electronic switching timer (Model 655, GRA

Lab, Centerville, OH, USA). The 2-way pinch valve is designed to open and close flexible silicone tubing (1/16 ID x 1/8 OD inches) to achieve controlled fluid flow. In the

2-way pinch valve, freshwater flow was 12.9 ml/min through normally open flow path.

Due to the electronic switching timer controlling system, the FW flow path closed while the other flow path opened to flow chemical stimulus for 2 seconds (0.43 ml) in the olfactory epithelium. To monitor the desensitization of olfactory responses, 10-4M of L- arginine was used as internal standard in EOGs. Following addition of the chemical substances, the electrical output was magnified by an amplifier (ML132, ADInstruments,

Colorado Springs, CO, USA) and recorded by a digitizer (PowerLab 4/35,

ADInstruments). Finally, action potentials were visualized and analyzed using analysis software (LabChart Ver 7.0, ADInstruments). In the analysis software, a cut-off frequency using a 50 Hz low-pass filter was applied which eliminated electrical noise in

EOG chamber.

In the early life stages of white sturgeon EOG experiments, chemicals including amino acids, bile acids, and conspecific WBE were tested. These compounds, with the

20 exception of WBE, were purchased from Sigma-Aldrich (St. Louis, MO, USA) which consisted of 20 amino acids: L-alanine (A), L-arginine (R), L-asparagine (N), L-aspartic

(D), L-cysteine (C), L-glutamic (E), L-glutamine (Q), glycine (G), L-histidine (H), L- isoleucine (I), L-leucine (L), L-lysine (K), L-methionine (M), L-phenylalanine (F), L- proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), and L-valine

(V); 7 bile acids: glycocholic acid (GCA), taurochenodeoxycholic acid (TCDA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA).

2.2.5 Behavior assays to whole body extract

With respect to WBE, two different behavior assays were conducted: i) a two- choice maze assay (Body Weight (BW): 53.63  1.01 mg, Body Length (BL): 21.42 

0.42 mm, n=12) and ii) a petri dish assay (BW: 54.59  1.93 mg, BL: 23.20  0.44 mm, n=10) were conducted in 23-25 dph white sturgeon larvae during August, 2014.

2.2.5.1 Two-choice maze assay

The two-choice maze assay was designed to investigate avoidance behaviors in white sturgeon larvae (n=6) (Gerlach et al. 2007) (Figure 2). The two-choice maze system consists of a flowing system and a visualization system. To minimize unknown light effects, nocturnal fish larvae were tested after sunset.

In the flowing system, a two-channel clear PVC tubing system was placed in each lane divided by partition. In each lane, one of the two-channels was continuously supplied with freshwater at 4 ml/sec while the peristaltic pump (Masterflex, Vernon Hills,

21 IL, USA) controlled the rest of the channel to supply freshwater and diluted WBE (2.59 mg/ml) at a rate of 3.6 ml/min, 4.8 ml/min, and 6 ml/min to result in a final concentration of 0.0381 mg/ml, 0.052 mg/ml, and 0.065 mg/ml, respectively in the two-choice maze system. Freshwater or WBE were randomly applied either left or right lane to prevent fish preference for specific lane. To contain fish larvae within the system, fine plastic mesh was placed between drainage and partition. The plastic mesh was fixed temporarily on the plexiglass by transparent tape for convenient washing after each experiment.

In the visualization system, a web-camera (C310, Logitech, Newark, CA, USA) was placed on top of the two-choice maze, and recorded the entire process using the visualizing software (Photo Booth, Ver. 7.0, Apple Inc., Cupertino, CA, USA). In recorded video files, the total time that individual fish spent in either the freshwater or the

WBE lane was calculated.

Fish were released and acclimatized (5 min) between the plastic mesh and the partition in two-choice maze system. After acclimatization, the time an individual fish larvae spent in either freshwater or diluted WBE , was counted using recorded video files.

2.2.5.2 Petri dish assay

The petri dish assay was conducted to examine the patterns of alarm behaviors in early life stages of white sturgeon. Prior to the assay, a glass petri dish (diameter 10 cm) was prepared by filling it with 99 ml of freshwater. Then, an individual fish was released into the glass petri dish and acclimatized for 5 min before either 1 ml of freshwater (n=5) or 1 ml of WBE (2.595 mg/ml final concentration, n=5) was added in the center of peridish. A top-mounted surveillance camera (ADV1-84500, Night Owl) recorded all fish

22 movements over the duration of exposure (5 min). The recorded fish movement was automatically analyzed by the video tracking software (Ethovision 3.0, Noldus,

Wageningen, The Netherlands). For the video tracking analysis, a glass petri dish was placed on a white paper background so that the dark bodied larvae were more easily visualized.

2.2.6 Whole body steroid extraction for cortisol analysis

For cortisol analysis, fish larvae were acclimatized for an hour in a darkened one litre chamber filled with 990 ml of freshwater at 15 C. Fish were treated by adding either

10 ml of freshwater (n=11) or 10 ml of WBE (final concentration 2.6 mg/ml, n=10).

After an hour treatment, fish were euthanized and then snap-frozen using liquid nitrogen.

The snap-frozen samples were stored at -80 C until whole body cortisol analysis was performed. To extract cortisol in each fish, one ml of 90% methanol was added to each

Eppendorf tube containing an individual larval fish and metal beads, and homogenization was performed using a Precellys 24 homogenizer (Bertin Technologies, Toulouse,

France) by applying a 3 x 11 sec procedure. The tubes were centrifuged at 20,000g for 5 minutes and the supernatant was decanted to a separate tube. This procedure was repeated to produce a total of 2 ml of 90% methanol containing the extract. Before cortisol measurement, the methanol extracts were vacuum dried using a CentriVap concentrator

(Labconco, Kansas City, MO, USA) connected with a VirTis Benchtop Freeze Dryer (SP

Industries, New York, USA). To measure cortisol levels in individual fish, radioimmunoassay (RIA) was performed as in Scott et al, (1980). Briefly, using culture tubes (10 mm x 75 mm, Fisher, Ottawa, Canada), nine standard tubes were made ranging

23 from 1.95-500 pg of standard cortisol steroid in 100 μl assay buffer (0.05 M NaPO4, pH

7.4 containing 0.2% BSA, 137 mM NaCl, 0.40 mM EDTA, and 0.77 mM sodium azide).

Blank, total and maximum tubes received 100 μl of assay buffer. Samples re-suspended in 100 μl of assay buffer were transferred to assay tubes with radiolabel-buffer solution with 5000 dpm. All tubes (except blank tubes) received 100 μl of this antibody-label- buffer solution. Tubes were incubated overnight at 4 °C. The next day tubes were placed on ice and 500 μl of 0 °C charcoal solution (50 mM sodium phosphate, pH 7.4, 0.1% gelatin, 1.0% dextran-coated charcoal) was added to all tubes except that total tubes received 500 μl assay buffer). After 15 min, the tubes were centrifuged at 1,000 x g, 4 °C for 12 min, decanted into 7 ml scintillation vials (Fisher) and mixed with 5 ml scintillation cocktail (RPI Corp., Mount Prospect, IL, USA). DPM were counted with an

LS-6500 scintillation counter (Beckman Coulter, Mississauga, Ontario, Canada). The data were processed using preformatted Excel program to determine cortisol concentrations in each sample.

2.2.7 Data analyses

All data are represented as means  SD (n=sample size). Statistical differences between two different groups were assessed using a student’s t-test. When there were more than two groups, differences were detected using ANOVA followed by a Tukey multiple comparisons test (GraphPad Prism 6.0, La Jolla, CA, USA). The cut off for statistical significance was p<0.05.

24 2.3 Results

2.3.1 Histology of white sturgeon larvae

Club cells in surface layer of the epidermis were investigated in the white sturgeon larvae. The epidermal club cells were located along the entire fish body.

However, the average surface area of individual club cells varied. The club cells in white sturgeon larvae lacked pores but were thin-walled cells with peripheral nuclei. In general, the club cells were larger than mucous cells.

The average area of club cells and their distance to the dermis varied among body regions. For example, the average sectioned area in club cells located in the head (490.2 

247.7 m2), dorsal (631.6  155.0 m2), and tail (509.8  175.1 m2) were significantly larger than in ventral (120.3  34.0 m2) region (one way ANOVA, p=0.0023) (Figure

4A). In addition, the club cells in the dorsal (39.68  5.21 m) and tail (38.96  7.97 m) regions were significantly closer to the dermis than the cells in head (56.58  6.21 m) and ventral (73.49  10.98 m) surfaces (one way ANOVA, p<0.0001) (Figure 4B).

However, the average number of club cells was not significantly different between body regions (one way ANOVA, p=0.0875) (Figure 4C).

2.3.2 Electrophysiological examination of olfactory responses to EOG stimuli

The white sturgeon larvae (~25 dph) showed EOG responses during exposure to chemical substances. With respect to chemical stimulus (430 l/2 sec), the fish larvae generated immediate negative action-potentials that reached peak magnitude after 5 sec.

The negative excitation followed by slow recovery, requiring up to five seconds to return to baseline. EOG responses showed varied sensitivity to either the position of the

25 recording and reference pipettes or the distance of the recording pipette from the olfactory epithelium. However, the varied EOG sensitivity was minimized in white sturgeon larvae because the fish olfactory epithelium was small enough to be covered by the recording pipette.

The EOG response to a range of amino acids and bile acids was investigated in larval fish (n=3) (Figure 5). Out of 20 amino acids tested at 10-4M, fish showed significant olfactory responses (relative to freshwater EOG control (27.83  3.53 V)) to

6 amino acids: L-lysine (643.80  161.84 V), L-arginine (628.80  27.77 V), L- glutamic (621.30  43.40 V), L-histidine (553.80  71.27 V), L-cysteine (378.83 

59.12 V), and L-aspartic (475.56  19.45 V). In addition, out of 9 bile acids tested at

10-4M, fish displayed significant olfactory responses to 3 bile acids, including taurodeoxycholic acid (1114.93  394.98 V), taurochenodeoxycholic acid (856.50 

177.63 V), and taurocholic acid (698.13  57.48 V).

The EOG response to serially diluted WBE (n=3) was tested and is reported in

Figure 6. The EOG response increased as the WBE concentrations increased (one-way

ANOVA, p<0.0001). In addition, the initial EOG threshold occurred between 0.026 mg/ml (112.70  39.29 V) and 26 mg/ml (1149.13  173.13 V) (Unpaired student’s t- test, p=0.0122). The initial EOG threshold might represent the minimal WBE concentration that triggers alarm behaviors. The minimal WBE concentration ranges that trigger the initial EOG threshold, therefore, is useful in behavior assays.

26 2.3.3 Two-choice maze assay

Avoidance behavior in fish larvae was tested in a two-choice maze assay. Within the 5 min assay, fish larvae (n=6) showed avoidance behavior in 0.065 mg/ml WBE

(11.06  1.03%, Unpaired student’s t-test, p<0.0001), and spent more time in freshwater

(22.17  2.56%). However, fish did not show any avoidance behavior in 0.052 mg/ml and

0.038 mg/ml WBE (Figure 7).

2.3.4 Petri dish assay

The Petri dish assay was designed to investigate alarm behavior patterns in fish larvae (Figure 8). In the assay, the fish larvae changed behavior patterns when exposed to

WBE relative to FW. WBE treatment fish larvae significantly decreased mean body turn angle (Unpaired student’s t-test, p=0.0018) while increased total movement time

(p=0.0020) (Figure 9). There was a trend toward a decreased mean heading and mean angular velocity after WBE treatment. However, the values were not significant.

After WBE treatment, fish also increased C-start body curls (Figure 10). Unlike freshwater treatment (6.2  3.0), the fish significantly increased body curls (80  17.6) during exposure to WBE (Unpaired student’s t-test, p<0.0001).

2.3.5 Whole body steroid extraction for cortisol analysis

Whole body cortisol levels were examined as part of stress response in WBE treatment (Figure 11). Compared to freshwater treatment (130.8  30.85 ng/g body weight, n=11), the fish significantly increased average cortisol level in WBE treatment

(212.4  32.53 ng/g body weight, n=10) (Unpaired student’s t-test, p<0.0001).

27 2.4 Discussion

This study examined the physiological and behavioral alarm response in larval white sturgeon and demonstrated that white sturgeon conform to the general alarm substances model seen in other species. My study demonstrates that predator-naïve larval white sturgeon exhibit alarm behaviors, presumably sensed through olfaction, in response to conspecific WBE containing club cells which are widely considered to be the source of putative alarm cues. While fish larvae respond to alarm pheromones, increases in stress hormone levels were also observed in the one-hour WBE treatment. Including club cell- based alarm pheromones, the WBE also contains various chemical compounds such as amino acids. Therefore, characterization of active chemical structure which triggers alarm responses in WBE is essential in future research.

2.4.1 Identification of club cells

Similar to Acipenser persicus (Saadatfar et al. 2010), this research clearly shows that white sturgeon larvae contain club cells over the entire body surface layer of the epidermis. Epidermal club cells are known to contain alarm substances in other teleosts.

Hence, it is assumed that white sturgeon larvae might release alarm cues after a predator attack from the ruptured epidermal club cells triggering alarm responses in neighboring conspecifics.

While the specific alarm pheromones have not been fully characterized alarm pheromones are thought to be a single chemical compound which is species-specific

(Brown et al. 2000, Smith 1979), artificial chemical compounds that include functional nitrogen-oxide (Brown et al. 2003) or different molecular weight fractions in skin

28 extracts (Mathuru et al. 2012) are thought to represent other types of alarm pheromones in fish. Mathuru et al. (2012) in particular claimed that glycosaminoglycan (GAG) chondroitin purified from skin extract is an alarm pheromone in zebrafish since it triggers fear responses. Additionally, the GAG chondroitin was also shown to activate the medial- dorsal posterior olfactory bulb in fish brains, a region which is innervated by hair-like sensory cells located in the olfactory epithelium (Mathuru et al. 2012). Together, these findings suggest that the fright response is mediated by the central nervous system and is triggered by conspecific alarm pheromones. However, general types of alarm pheromones that trigger alarm behaviors between interspecific species require further investigation (Karplus 1987, Mathis et al. 1996, Snyder 1967).

2.4.2 Electrophysiological examination of olfactory responses to EOG stimuli

Demonstration of the olfactory capacity to detect alarm substances and the response thresholds for WBE detection establish the second component of the alarm substance model seen in other fish.

With respect to amino acids and bile acids, the presence of olfactory responses was examined in EOG experiments. In fish, amino acids are known as food substances

(Lemly and Smith 1985, 1987) while bile acids are known as sex pheromones that attract spawning-ready individuals or trigger spawning behavior during breeding season (Hara

1992, Li et al. 1995). However, in some fish, both amino and bile acids are used as alarm pheromones. For example, Tucker and Suzuki (1972) suggested that mixtures of amino acids and oligopeptides may act as alarm pheromones in white catfish (Ictalurus catus).

In addition, juvenile Persian sturgeon (Acipenser persicus) were repelled by some amino

29 acids, such as aspartic acid, tyrosine, and glutamic acid (Shamushaki et al. 2007) but attracted by others, such as alanine and glycine (Kasumyan 1999). Although EOG revealed the ability of the olfactory system in white sturgeon larvae to detect specific chemical substances, the basis for their preferences is unknown. Therefore, further behavioral assays are required to determine whether amino acids or bile acids are attractants or repellants and whether they could be used in white sturgeon management.

The EOG study revealed that in their early life stages, white sturgeon can sense a range of chemical substances. Around 20 dph the overall olfactory sensitivity of fish larvae is, however, not enough to sense these chemicals at concentrations lower than

10-7 M, a concentration that the fish might encounter in natural streams (Moor et al.

2002). These concentrations are equivalent to adult teleosts sensing a square centimeter of skin extract in 58 000 L of water (58 000 L/cm2) (Smith 1992). Whereas white sturgeon larvae elicited avoidance behavior at 0.065 mg/ml of WBE which is equivalent to ~166 L/cm2 (conversion equation from O’Shea et al. 2006). Although volumetric ratios of body size and length might affect the number of olfactory receptor neurons in the olfactory epithelium are considered, early life stages of white sturgeon seem to have less olfactory sensitivity compared to adult teleost fish.

2.4.3 Avoidance behavior of larval white sturgeon

The third component to characterizing the alarm substance response in white sturgeon was the successful demonstration of behavioral responses to WBE. Avoidance behavior is a common fright response associated with alarm pheromones discharged from injured conspecifics (Pfeiffer 1974, Snyder and Snyder 1970). When sensing alarm cues,

30 most fish respond with C-start body curls to direct the fish away from danger (Domenici and Blake 1997). This reaction has also been observed in the early life stages of fish such as lake sturgeon (Wishingrad et al. 2014) and zebrafish (Egan et al. 2009).

My results showed that white sturgeon larvae spent significantly more time in freshwater than in conspecific WBE in a two-choice maze assay, indicating that larval white sturgeon can recognize and avoid WBE. The avoidance behavior of the fish larvae was triggered in black covered Plexiglas under dim light, an experimental environment in which visual cues were limited. Tactile cues were also restricted because of similar amounts of freshwater flowing without predators in the two-choice maze assay.

Therefore, in their larval stage, white sturgeon might strongly rely on olfaction to avoid diluted WBE, at least at concentrations higher than 0.065 mg/ml.

2.4.4 Behavioral responses to alarm cues

In contrast to the food attraction behavior where fish crowd into areas of concentrated food odor (Kasumyan 1999), white sturgeon larvae initially increased dashing (data not shown) and significantly decreased overall activity levels during exposure to WBE. This pattern of alarm responses can be categorized as a fright response that includes avoidance and sheltering, as generalized by Pfeiffer et al. (1986). In other teleosts, avoidance behavior is triggered by an initial C-start startle response that may be used to escape predators which is also observed after alarm cue treatment (Egan et al.

2009, Stewart et al. 2013). In addition, motionless behavior, such as less circling and nibbling and circling under a shelter, are also commonly observed in fathead minnows

(McMilan and Smith 1974). However, Hintz et al. (2013) and Wishingrad et al. (2014)

31 suggested that sturgeon species’ escape behaviors and activity levels increased during alarm pheromone treatment. For example, Wishingrad et al. (2014) found that larval sturgeon elicited a higher increase in escape responses (~125% changes in activity level) than did juvenile fish (~30%). They concluded that sturgeon alarm activity levels increased in the presence of alarm cues, particularly in larval white sturgeon when the skin is still thin and more vulnerable to damage than in juveniles (Wishingrad et al.

2014). Together, movement patterns and activity levels in response to alarm cues are varied on the species or their life stages. Therefore, it is assumed that white sturgeon could also have varied movement patterns and activity levels in different life stages; further research is required to address this.

During exposure to WBE, overall activity levels of larval white sturgeon decreased while C-start body curls increased. In teleosts, an increase in C-start body curls and a reduction in activity have been observed in response to alarm pheromones

(Laurence and Smith 1989). In the case of white sturgeon larvae, however, C-start body curls occurred in conjunction with overall decreased activity levels. One possible explanation is that the white sturgeon larvae, which were trapped and stressed continuously within the boundaries of a glass petri dish, could not avoid the concentrated

WBE and their olfactory system was constantly stimulated by the WBE. Hence, the highly stimulated olfactory sensitivity might have triggered the C-start body curls.

However, olfactory fatigue is easily observed in electrophysiology experiments (Eom et al. 2009). Thus, it is also possible that the olfactory system cooperates with the mechanosensory system, such as the lateral line system, which is known to trigger C-start body curls (Stewart et al. 2010).

32 2.4.5 Physiological effects of alarm cues on stress responses

The final component in my evaluation of whether white sturgeon conform to the established alarm substance model was the demonstration that WBE exposure causes the initiation of broader stress response. The results of this study indicate that alarm cues not only elicit a series of alarm behaviors but also increase stress hormone levels in larval white sturgeon. Because fleeing from a predator’s attack is a stress response in prey fish

(Feder and Lauder 1986), examination of cortisol levels is valuable in understanding stress levels of prey fish with respect to alarm cues. In this study, whole body concentrations of cortisol levels were examined in ~15 dph white sturgeon larvae.

In general, cortisol levels in white sturgeon larvae increased significantly (212.4 

30.85 ng/ml) with WBE treatment compared to freshwater treatment control fish (130.8 

32.53 ng/ml) indicating that conspecific WBE is likely perceived as a stressor. An increase in cortisol concentration is common in acipenseridae (Bates et al. 2014,

Simontacchi et al. 2008, Zubair et al. 2012) and in other teleost (Barton and Iwama 1991,

Pickering and Pottinger 1995) under stress circumstances. For examples, Simontacchi et al. (2008) suggested resting cortisol levels of 4.30  0.65 ng/ml and 4.05  0.67 ng/ml in

10 dph and 35 dph white sturgeon larvae respectively. After 30 min of exposure to a turbulent water flow, whole body cortisol in sturgeon larvae increased to 8.42  1.54 ng/ml and 37.66  10.01 ng/ml respectively in 7 dph and 35 dph larvae respectively

(Simontacchi et al. 2008). In resting salmonids, resting whole body cortisol is 5 ng/ml of cortisol concentration in a resting status (Pickering and Pottinger 1995) with 10 to 100- fold increases after acute stress (Barton and Iwama 1991). In the current study, resting whole body cortisol levels were 120 ng/g and increased to 220 ng/g. Resting cortisol

33 levels are clearly elevated relative to other studies, and the reason for this difference is unknown. In this study, attempts were made to minimize stress in control conditions, such as keeping light intensity low and maintaining fish at appropriate water temperatures. In addition, MS-222 was used in larvae euthanasia to minimize sampling related stress. While the density of fish in the current study based upon mass was not high

(~0.5 kg/m3), density based upon numbers (10-11 fish/L) may have contributed to elevated resting cortisol levels.

Elevated plasma cortisol stimulates proteolysis by releasing free amino acids and supports enough energy for alarm responses (Milligan 1997, Moberg 1985). The alarm responses are energy-dependent behavior to avoid threats. Including salmonidae, fish are known to use protein turnover in a muscle for energy intake, however, it is slow process compare to that in tissues such as liver, kidney, and gills (Narayansingh and Eales 1975).

Therefore, tissues are thought to be a major energy supplier for energy-dependent acute alarm responses because cortisol-stimulated proteolysis is occurred by releasing free amino acids through high affinity cortisol receptors especially in liver (Henderson and

Garland 1980). In addition, Milligan (1997) claimed that cortisol synthesis inhibitors decrease tissue amino acid levels after fish exercise. Together, elevated plasma cortisol levels are positive physiological response for energy-dependent stress responses in fish.

2.4.6 Conspecific alarm cues in fisheries management

Given the importance of hatchery based augmentation for many endangered fish evaluating the biological basis of alarm substance responses provides an important foundation when considering the potential for predator conditioning. Conspecific alarm

34 cues might be applied to fisheries management including threatened white sturgeon especially to hatchery systems where fish are reared in predator-free environments

(Wisenden et al. 2004). After hatchery-reared naïve fish are released in the wild they commonly exhibit high mortality from predators (Santucci and Wahi 1993). Similarly, hatchery-reared white sturgeon larvae might suffer from predation pressures just as other similar-sized fish, even though adult white sturgeon are top predators. Increasing the survival rates of hatchery-reared naïve fish would improve the cost-effectiveness of hatchery programs (Suboski and Templeton 1989). The present study provides the basis for further evaluation of this potential in white sturgeon. For example, a logical next step is evaluation of simultaneous exposure to conspecific alarm cues and predator visual cues which have been shown to elicit alarm behaviors in hatchery-reared naïve prey fish such as fathead minnows (Chivers and Smith 1994) and brook char (Mirza and Chivers 2000,

Brown and Smith 1998). Instead of conspecific alarm cues in threatened white sturgeon, affordable amino acids or bile acids could also be candidates as repellants in fisheries management especially in hatchery systems; further behavior assays are required to gain insight into the feasibility of this approach.

35 Table 1. List of representative examples of alarm cues in other fish species and their responses.

Species References Origin Responses

Arctic charr Conspecific Increase avoidance and Lautala and Hirvonen 2008 Salvelinus alpinus skin extract freezing

Blacknose shiner Conspecific Decrease activities Wisenden et al. 2004 Notropis heterolepis skin extract Increase vertical distribution

Brook stickleback Conspecific and Friesen and Chivers 2006 Avoidance Culaea inconstans heterospecific skin extract

Brook trout Skin extract within Decrease movement and Mirza and Chivers 2000 Salvelinus fontinalis most taxonomic groups feeding

Central mudminnow Conspecific Decrease movement, Wisenden et al. 2008 Umbra limi skin extract increase time spent on the bottom

Chinook salmon Conspecific Berejikian et al. 1999 Motionless Oncorhynchus tshawytscha skin extract

Cichilids Conspecific Decrease movement and Pollock et al. 2005 Archocentrus nigrofaciatus skin extract foraging

Common bully Conspecific Increase dashing, Kristensen and Closs 2004 Gobiomorphus cotidianus skin extract decrease activity

Fathead minnow Friesen and Chivers 2006 Increase shoaling, Epidermal club cells Pimephales promelas Wisenden and Barbour 2005 freezing

Glowlight tetras Increase shoaling, Brown and Dreier 2002 tetras-fed predators Hemmigrammus erythrozonus dashing, freezing Brown and Zachar 2002 conspecific-fed predators

Golden shiner Snake predator’s feces Increase Godard et al. 1998 Notemigonus crysoleucas and urines fed golden shiners shelter seeking

Goldfish Byproducts of predators Increase shoaling and Zhao et al. 2006 Carassius auratus that fed goldfish dashing

Lake Sturgeon Conspecific whole-body Wishingrad et al. 2014 Increase activities Acipenser fulvescens extract

36 Table 1. List of representative examples of alarm cues in other fish species and their responses.

Figure 1. Electro-olfactogram (EOG) of early life stages of white sturgeon. In anesthetized fish (tricaine methanesulfonate, 0.0375 mg/ml, final concentration), olfactory sensitivity was examined by delivering chemical stimuli. Olfactory response magnitudes were amplified and visualized for analysis.

Figure 2. Avoidance behavior was observed in two-choice maze assay. After acclimation, A) avoidance behavior was tested with respect to freshwater (FW) or alarm pheromone contained whole body extract (WBE). In order to drain waste efficiently, B) overall system was tilted at 10 degrees. For video analysis, fish behavior was recorded.

39 A) B) AC

ECC E

C) Back Head Tail

Abdomen

Figure 3. Histology sections of early life stages of white sturgeon. Sliced epidermal club cells (ECC) and mucous cells (MC) in the epidermal layer (E) were magnified in A) 100X and B) 200X. The dermal layer (D) lies between epidermal layer (E) and abdominal cavity (AC). Scale bars represent 1mm.

Figure 4. The spatial distribution (in the back, tail, head, and abdomen) of epidermal club cells in 20 dph white sturgeon larvae (n=5) consisting of A) size of club cells (p=0.0023) and B) distance to dermis, and C) number of club cells. A symbol of asterisk indicates statistically significant differences.

Figure 5. A) Electro-olfactogram (EOG) responses and B) their response magnitudes in larval white sturgeon (n=3) exposed to 10-4 M of amino acids and bile acids. Of the 20 amino acids and 9 bile acids, fish larvae responded to 6 amino acids: L-lysine (K), L- arginine (R), L-Glutamic (E), L-histidine (H), L-cysteine (C), and L-aspartic (D), and 3 bile acids: taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDA), and taurocholic acid (TCA). A symbol of asterisk indicates statistically significant differences (One-way ANOVA, p<0.0001).

Figure 6. Electro-olfactogram (EOG) in white sturgeon larvae (n=3) exposed to serially diluted whole body extract (WBE). Between 0.026 mg/ml and 26 mg/ml diluted WBE, the fish larvae significantly increased olfactory sensitivity (one-way ANOVA, p<0.0001). In addition, initial threshold of olfactory sensitivity occurred between 0.026 mg/ml and 0.26 mg/ml (Unpaired student’s t-test, p=0.0122).

Figure 7. Percent of time larval white sturgeon spent in the two channel maze assay, with a choice of either freshwater (FW) or diluted whole body extract (WBE). Fish larvae avoided the channel supplied with 0.065 mg/ml WBE, and spent more time in the FW channel (Unpaired student t-test, p<0.0001, n=6). However, avoidance to the WBE treated channel was not observed when the WBE concentration was less than 0.052 mg/ml. Due to time spent in starting area where fish was released, sum of time spent percent in the two channel maze assay showed below 100%.

Figure 8. Representative movement of white sturgeon larvae in the petri dish assay in response to freshwater (FW) (n=5) or whole body extract (WBE, 2.6 mg/ml final concentration) addition (n=5) as quantified by the Ethovision animal tracking software. Unlike A) FW treatment fish larvae that occupied entire petri dish, B) WBE treatment fish larvae gradually decreased activity levels and occupied areas distant from the center of the petri dish where WBE was applied.

Figure 9. Alarm responses of white sturgeon larvae in the petridish assay in fish exposed to freshwater (FW) (n=5) or diluted whole body extract (WBE, 2.6 mg/ml final concentration) (n=5). Using the Ethovision video tracking software, the movement patterns and activity levels were analyzed and the values were calculated using the equation of “(either FW or WBE treatment value – acclimation value) / acclimation value x 100 (%).” In general, WBE treatment fish larvae decreased activity levels compare to FW treatment fish (two-way ANOVA, p=0.0003), especially mean turn angle (Unpaired student’s t-test, p=0.0018) and total movement duration (p=0.0020). A symbol of asterisk indicates statistically significant differences.

Figure 10. C-start body curls over 5 minutes in white sturgeon larvae in response to a predators approach in the petri dish assay with freshwater (FW) (n=5) or whole body extract (WBE, 2.6 mg/ml final concentration) (n=5). A symbol of asterisk indicates statistically significant differences (Unpaired student’s t-test, p<0.0001).

Figure 11. Whole body cortisol levels in larval white sturgeon exposed for 1 h to freshwater (FW) (n=11) or whole body extract (WBE, 2.6 mg/ml final concentration) (n=10). A symbol of asterisk indicates statistically significant differences (Unpaired student’s t-test, p<0.0001).

48 Chapter 3. Conclusion

3.1 Thesis summary

This research provides evidence that predator naïve early life stages of white sturgeon elicit alarm responses when subjected to conspecific alarm cues. Similar to other fish, sturgeon larvae contained epidermal club cells known to contain alarm substances in fish. Regarding to alarm substances, larval fish showed alarm responses such as avoidance, dashing, freezing, and C-start startle movement using olfactory systems.

Furthermore, an increase in cortisol levels was observed in larvae exposed to WBE, all of which taken together, indicates that larval white sturgeon display physiological responses to conspecific alarm pheromones that may be associated with mobilizing energy reserves to meet the demands of dealing with stressful situations.

3.2 Thesis objectives and hypotheses

Objective 1: Club cell identification in the epidermis of white sturgeon larvae.

White sturgeon larvae contain epidermal club cells. In their early life stage, white sturgeon have epidermal club cells that in other species are known to release alarm pheromones. The fish contain relatively large epidermal club cells in the dorsal-caudal region. In addition, the club cells in the dorsal-caudal are closer to the dermis than they are in the other regions.

Objective 2: Electrophysiological examination of olfactory responses to EOG stimuli.

White sturgeon larvae sense chemical substances through their olfactory system.

In their early life stage, white sturgeon use their olfactory system to sense various

49 chemical substances, such as amino acids, bile acids, and conspecific WBE. Overall, olfactory sensitivity to bile acids is higher than amino acids. With respect to WBE, the fish’s initial threshold of olfactory sensitivity increases between 0.026 mg/ml and 0.26 mg/ml.

Objective 3: Investigation of alarm behaviors in the presence of whole body extract

(WBE) as a potential alarm pheromone.

Whole body extract triggers alarm behaviors in white sturgeon larvae. White sturgeon larvae show alarm behaviors such as avoidance behavior and dashing and freezing in the presence of conspecific WBE. Unlike control fish in freshwater, WBE treatment fish in a two-choice maze assay clearly show avoidance and spend more time in freshwater. In a petri dish assay, the fish show dashing and freezing-like fatigue while the

C-start startle response increases after WBE treatment. Compared to freshwater treatment fish, which show general searching behaviors, the duration and distance of the WBE treatment fish movement decreased after 5 min and 5-8 min respectively; however, the C- start startle response increased significantly, mostly after the 5-8 min treatment.

Objective 4: Determine whether exposure to whole body extracts elevated whole body cortisol levels.

Cortisol levels in white sturgeon larvae increase in response to WBE treatment as part of the stress response. Cortisol levels in white sturgeon larvae exposed to acute stress for a one-hour WBE treatment increased 62.39% compared with freshwater treatment

50 control fish. This study clearly indicates that the WBE triggers fish alarm responses and consequently affects cortisol levels as a chemical stressor in the fish larvae. Hence, I conclude that alarm pheromones not only elicit a series of alarm behaviors but also increase stress hormone levels in larval white sturgeon.

3.3 Research limitations and future directions

In general, alarm pheromones are thought to be species-specific. In this experiment, white sturgeon larvae show alarm behaviors in response to conspecific WBE.

Characterization of white sturgeon-specific alarm pheromones in a chemical mixture of

WBE to identify the specific compounds that are eliciting the observed responses is a task for future research. From a conservation management perspective, white sturgeon- specific alarm pheromones characterized in WBE might be valuable in training hatchery- reared fish to condition them in a way to avoid predators when they are released.

The EOG responses were varied due to large growth variation in larval white sturgeon. Therefore, generalization of EOG among individuals using internal standard with amino acids was limited. This means that my thesis represent the olfactory sensitivity of specific ages post hatch larvae and are limited to generalizing the entire larval stages. Thus the EOG data represent the olfactory sensitivity of white sturgeon larvae only around 20 dph and further studies are required to understand variations in olfactory sensitivity at different larval stages.

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