PERCEPTION AND USE OF MAGNETIC FIELD INFORMATION IN NAVIGATION BEHAVIORS IN ELASMOBRANCH FISHES A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE IN ZOOLOGY, SPECIALIZING IN MARINE BIOLOGY JULY 2018 By James Murray Anderson Dissertation Committee: Kim Holland, Chairperson Timothy C. Tricas Kathleen Cole Masato Yoshizawa Patricia Couvillon Keywords: Magnetoreception, Navigation, Shark, Behavior, Magnetite, Raman Spectroscopy Dedication & Acknowledgements I dedicate this dissertation and the body of work it represents to the memory of my mother, Irene, who always believed in me, with unwavering love and support. I would like to thank my wife, Catherine and my children, Sonny, Zane, Loki and Kai (a.k.a. the four horsemen), for their own unwavering love and support, for putting up with me, and providing me with a counterbalance to the stresses and demands of graduate school. There are a host of people whom I need to thank; my advisors through my graduate career, Kim Holland and Tim Tricas, for taking me on, having faith in me, and for their help and guidance through a tricky and complicated research topic. I owe a debt of thanks to several amazing people who took an active interest in my research, gave their time and effort to help me, and ultimately provided critical input to the success of the study – so thank you Kristen Ewell and Miyoko Bellinger (U.H. Histology Core), Tina Weatherby Carvalho (B.E.M.F), Fukun Hoffman (CMB), Alexandra Gurary (Molecular and Cellular Immunology Core), Kara Yopak (UNCW). I also owe a debt of thanks to Tamrynn Clegg for her dedication and hard work in assisting with much of the study. I also extend my thanks to Shaun Collin, Gary Cowin, Marilyn Dunlap, Carl Meyer, Kelly Williams, Kelsey Maloney, Christine Ambrosino, Luisa Queiroz, Mark Royer, Danny Coffey, Melanie Hutchinson, Kevin Izumi, Kaylee Rossing, Jeff Muir, Mariana Azevedo and the many interns in the lab for their help in data collection and husbandry. I thank the members of my dissertation committee, Drs. K. Cole, M. Yoshizawa, P. Couvillon, K. Holland, and T. Tricas, for help with experimental methodology and constructive manuscript editing. Finally, I would like to thank all my friends and family for their support and help throughout my time as a graduate student. ii Abstract Three principal hypotheses prevail regarding the sensory means by which animals, including elasmobranchs, perceive and use magnetic field information. Of these three hypotheses, the iron (magnetite) based magnetoreceptor hypothesis is regarded as being the most basal, and is proposed to be conserved across taxa, from invertebrates through to high vertebrates. Magnetoreception via magnetic-electric induction and the radical pairs mechanism is considered more derived. Modern elasmobranch and teleost fish share a common ancestry. Although they evolved independently, the apparent evolutionary conservation of functional magnetite containing structures (as in teleosts and birds) might suggest such structures are also present in the elasmobranchs. Elasmobranch fishes have been both hypothesized and empirically shown to respond to changes in magnetic fields. However, empirical evidence to support orientation and navigation via magnetic field information in elasmobranch fishes is scant; sensory acuity to magnetic stimuli is undescribed, and the physical mechanisms and sensory pathways by which sharks may perceive and use magnetic information continue to be the subject of debate. The investigations detailed in the following chapters aim to examine the ability of sharks to perceive and use magnetic field information in navigation behaviors. Using conditioned behavior experiments as a proxy, these experiments confirm sharks’ ability to perceive magnetic stimuli, quantify sensory acuity, determine ability to discriminate contrasting magnetic stimuli, and provide insight into the possible mechanisms used. Morphological and physiological analyses aimed to identify critical structures required for an elasmobranch homologue to the iron/magnetite based olfactory magnetoreceptor described in teleost fish. Sharks were able to perceive magnetic field changes as low as 0.03 microtesla (µT), and could repeatedly and reliably discriminate between contrasting but similar magnetic landmarks. This demonstrates sharks could not only perceive ecologically relevant magnetic stimuli, but could learn, internalize and organize that information – a key iii component in the formation of a cognitive map, required in navigation. Sensory deprivation/impairment techniques incorporated suggested the likely use of at least two sensory mechanisms (magnetic-electric induction & a putative magnetite based magnetoreceptor). Finally, morphological and physiological investigations suggest sharks possess the critical structures required in a magnetite-based olfactory housed magnetoreceptor. iv Table of Contents Acknowledgements………………………………………………………………………………………………...ii Abstract ..………………………………………………………………………………………………………………iii List of Tables………………………………………………………………………………………………………….vi List of Figures ................................................................................................................................ …vii List of Abbreviations……………………………………………………………………………………………...xi Chapter 1 - An Introduction To Navigation In Sharks & Other Animals …………………….1 1.1 General introduction ……………………………………………………........…………………………………….1 1.2 Sensory systems in navigation and homing behaviors in shark ………………………………….6 1.3 The sixth and seventh senses – Electroreception and Magnetoreception in Orientation and Navigation………………………………………………………………………………………………………… 12 1.4 Scope of the study & dissertation organization……………………………………………………….21 Chapter 2 - Magnetic Field Perception In The Sandbar Shark (Carcharhinus Plumbeus)……………………………………………………………………………………………………………… 24 2.1 Abstract…………………….......………………………………………………………………………………..…..24 2.2 Introduction ……………………………………………………………………………...………………………..25 2.3 Materials and Methods…...…………………………………………………………………………………….27 2.4 Results………………………………………………………..……………………………………………………….34 2.5 Discussion…………………………………………………………………………………………………………...45 2.6 Conclusions…………………………………………………………………………………………………………53 Chapter 3 - Magnetic Field Discrimination & Sensory Mechanisms…….………..….……56 3.1 Abstract…………………….......………………………………………………………………………………..…..56 3.2 Introduction ……………………………………………………………………………...………………………..57 3.2 Materials and Methods…...…………………………………………………………………………………….61 3.4 Results………………………………………………………..……………………………………………………….70 3.4 Discussion…………………………………………………………………………………………………………...93 Chapter 4 – Searching For An Olfactory Magnetoreceptor …….……..…………………..……102 v 4.1 Abstract…………………….......………………………………………………………………………………..……...102 4.2 Introduction ……………………………………………………………………………...……………………...103 4.3 Materials and Methods…...…………………………………………………………………………………..107 4.4 Results………………………………………………………..………………………………………………….....115 4.5 Discussion…………………………………………………………………………………………………………145 4.6 Conclusions……………………………………………………………………………………………………….155 References….………..………………………………………………………………………………...………..……158 vi List of Tables Table 1. Taxa demonstrated to perceive or respond to magnetic field changes……..14 Table 2.1 Proportion of passes over the target, per shark, across the series of ten unimpaired trials ................................................................................................................................. 37 Table 2.2 Conditioned behavioural responses of sensory-impaired sharks to presentation of stronger magnetic stimuli…………………..…………………………………………………………..38 Table 2.3 Mean proportion of passes over the target, per shark, across the series of ten impaired trials…….……………………………………………………………………………………………..41 Table 3.1 Ethogram of S. lewini experimental behaviors………………………………………63 Table 3.2 Metrics considered for analyses.…………………………………………………………..68 Table 3.3 Comparison of orientation behaviors to magnetic & non-magnetic landmarks...……………………………………………………………………………………………………….71 Table 3.4 Comparison of combined orientations vs passes by landmark type ………….………………………………………………………………………………………………………………72 Table 3.5 Comparison of orientation behaviors produced in individual sharks by landmark type…………………………………………………………………………………………………...73 Table 3.6 Orientation behaviors of individual sharks in 4-target trials ...………………78 Table 3.7 Ranked generalized linear mixed-effects models of magnetic landmark on probability of behavioral orientation to a landmark ....…………………………………………81 Table 3.8 Effects of highly influential variables in mixed model analyses ....……….….82 Table 3.9 Behavioral orientations to landmarks according to treatment groups…...87 Table 3.10 Ranked generalized linear mixed-effects models of magnetic landmark on probability of behavioral orientation to a landmark…………………………………………….89 Table 3.11 Effects of highly influential variables in mixed model analyses of two-target sensory trials……………………………………………………………………………………………………..90 Table 4.1 Signal strength and occurrence across olfactory rosette samples….……..129 Table 4.2 Cell counts and magnetic filtration yields from dissociated olfactory rosettes………………………………………………………………………………………………………...…132 vii Table 4.3 Comparison of iron oxides examined, their associated Raman bands, and laser wavelength applied………………….………………………………………………………………………153 List of Charts/Figures Figure 1.1 Parameters of the geomagnetic field that may provide organisms