Investigation of physiological and behavioral alarm responses in larval white sturgeon (Acipenser transmontanus) by Junho Eom B.Sc., Kangwon National University, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2016 © Junho Eom, 2016 Abstract 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