The Body Design of Hagfishes (Eptatretus stoutii and Myxine glutinosa) Protects from Biting Predators by Sarah Boggett A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science In Integrative Biology Guelph, Ontario, Canada © Sarah Boggett, April, 2017 ABSTRACT The Body Design of Hagfishes (Eptatretus stoutii and Myxine glutinosa) Protects from Biting Predators Sarah Boggett Advisors: University of Guelph, 2017 Dr. D. S. Fudge Dr. P. A. Wright This thesis investigates how the body structure of hagfishes plays a role in defence against biting predators. Evidence of hagfish being attacked by biting predators show them swimming away relatively unscathed even after a violent initial attack. I hypothesized that the flaccid and loose body design of hagfishes protects them from biting predators by the minimal attachments between the skin and musculature combined with a large subcutaneous sinus allowing its internal organs to avoid damage from penetrating teeth. To test this, I quantified the flaccidity of the subcutaneous sinus, simulated shark attacks with a customized guillotine, and manipulated the adhesion and flaccidity between the skin and body of hagfishes and lamprey. Here I provide evidence consistent with my hypothesis. This ability of hagfishes to survive initial attacks from biting predators may be an essential component of a strategy that relies on defensive slime to thwart further attacks. iii ACKNOWLEGEMENTS I would like to start this off by thanking Dr. Douglas Fudge. You took a big risk is accepting a high school teacher who had been out of academia for 15 years. It is something I will always be grateful for. You have not only made me a better student but also a better teacher. There will be a whole stream of high school students coming through with a better understanding of science and the scientific method thanks to you. I would like to thank: Dr. Pat Wright, you were always there to answer questions and for support even before you became my official advisor. You are a role model to me. Thank you for always making me feel welcome and a part of your lab and for all of your advice. Dr. Matthew Vickaryous, thank you for being a part of my advisory committee and for all of your assistance and advice. My parents, Helen and John Boggett, for supporting me no matter what it is I decide to try next. You have always had my back and supported my choices. My sister, for always being there to talk to and discuss ideas with. My Aunty Sheila for your support and for providing me the opportunity to pursue other opportunities while back at school. Sarah Schorno, you were always willing to lend an ear, educate, and help out. Thank you for being a mentor to me while I learned about hagfish. And most importantly thank you for being a friend. Tessa Blanchard, Andy Turko, Lauren Gatrell, Calli Freedman, and Evan McKenzie thank you for listening, giving advice, and helping me when I had questions. Grad school is always better when you have people you can count on. Thank you Steve Wilson for building the original guillotine. Ian Moore for being able to take my ideas for the skin stretcher and make it into something that actual worked and for all of the modifications to the guillotine as my project progressed. And finally, thank you Mike Davies and Matt Cornish at the Hagen Aqualab for your care of the hagfish. iv TABLE OF CONTENTS ABSTRACT …………………………………………………………………………………. p. ii ACKNOWLEDGEMENTS …………………………………………………………………. p. iii TABLE OF CONTENTS ……………………………………………………………………. p. iv LIST OF TABLES …………………………………………………………………………... p. vi LIST OF FIGURES …………………………………………………………………………. p. vii INTRODUCTION …………………………………………………………………………... p. 1 Hagfish phylogeny …………….………………………………………..…………… p. 1 The notochord ……………………………………………………………………...... p. 1 Hagfish skin …………………………………………………………………….…… p. 2 Skin attachment and the subcutaneous sinus …………………………………………p. 6 Hagfish musculature ………………………………………………………………… p. 9 Defensive secretions ……………………………………………………………....… p. 11 Hagfish slime defence ………………………………………………………………. p. 12 How do hagfish survive shark bites? …………………………………….……..…… p. 15 Hypothesis and predictions ……………………..…………………………………… p. 15 METHODS ………………………………………….…………………………………….… p. 19 Animal care and collection ……………………………….…………………….…… p. 19 Inflation experiments …………….……………………………..…………………… p. 20 Shark teeth impact experiments ………….………………………..………...………. p. 22 Skin strain experiments …….……………………………………..………………… p. 27 Statistical analysis ……………………………………………………………..…….. p. 29 v RESULTS ……………………………………………………………………………...……. p. 32 DISCUSSION ……………………………………………………………….…………......... p. 39 LITERATURE CITED …………………………………………………….………….…….. p. 49 vi LIST OF TABLES Table 1: Recorded instances of hagfish found in the stomach contents of gill breathing animals ……………………………………………………………………………. p. 14 Table 2: Comparison of puncture damage to the skin and parietal muscle of Pacific hagfish (Eptatretus stoutii), Atlantic hagfish (Myxine glutinosa) and sea lamprey (Petromyzon marinus) done by mako shark teeth mounted on a spring-driven guillotine ………………………………………………………………………………….… p. 34 vii LIST OF FIGURES Figure 1: Slime glands on a hagfish ………………………………….………….…………. p. 3 Figure 2: The force of puncture for Pacific hagfish skin and skin from 18 species of fishes as a function of skin thickness……………………………………………………. p. 5 Figure 3: Cross section of a fixed Pacific hagfish (Eptatretus stoutii) showing lack of attachments between the skin and musculature and lamprey (Petromyzon marinus) with muscle skin connections……….…...………………………………….……. p. 7 Figure 4: Thin connective tissue connections between the skin and muscle of the Atlantic hagfish (Myxine glutinosa) above the slime glands………………………………. p. 8 . Figure 5: The location of the parietal muscle and oblique muscle as seen in the cross section of a hagfish ...……….……………………………………………………..p. 10 Figure 6: Kitefin shark biting into a New Zealand hagfish (Eptatretus cirrhatus)…….….... p. 16 Figure 7: The jaw of a kitefin shark (Dalatias licha).............................................................. p. 17 Figure 8: Pacific hagfish (Eptatretus stoutii) before and after inflation…………..……........ p. 21 Figure 9: The customized guillotine used for quantifying the effects of shark teeth driven into a variety of specimens and treatments……… …………………..……..……. p. 23 Figure 10: Preparation and testing of hagfish specimens using the shark tooth guillotine.… p. 25 Figure 11: Hagfish holder made from wooden toothpicks to maintain water anatomical positioning while out of water ……………………………………………...…… p. 26 Figure 12: Preparation and testing of sea lamprey (Petromyzon marinus) specimens for testing with the shark tooth guillotine………………………………………..….. p. 28 Figure 13: Hagfish skin stretcher….……………………………………….……..…............. p. 30 Figure 14: Hagfish skin stretcher attached to the Instron universal testing machine…...…... p. 31 Figure 15: Pressure in the subcutaneous sinus of two species of freshly dead hagfish as a function of injected fluid volume…………………...……………………..…….. p. 33 Figure 16: The effect of skin strain on the distance a shark tooth can travel after contact with the skin before puncture occurred………………………………………….. p. 36 Figure 17: The effect of the radius of skin within the skin stretcher extension at puncture… p. 37 viii Figure 18: The effect of pre-strain on the force needed to puncture isolated Pacific hagfish (Eptatretus stoutii) skin……………………………...…………………………... p. 38 Figure 19: The effect of the radius of skin within the skin stretcher on the force at puncture………………………………………………………………………….. p. 40 Figure 20: Typical location of damage when muscle penetration occurred……….………... p. 43 Figure 21: A Pacific hagfish (Eptatretus stoutii) in an anatomically correct position for water vs the effects of gravity out of water……..…………………………………….…… p. 44 INTRODUCTION Hagfish phylogeny Hagfishes represent one of the most basal forms of vertebrates (Janvier, 1981; Takezaki et al., 2003). They are a group of extant jawless craniates found mostly in deep marine habitats (Martini, 1998) though both the Japanese hagfish, Eptatretus burgeri, and New Zealand hagfish, Eptatretus cirrhatus, can be found in shallower waters (Fernholm, 1974). Hagfishes and lamprey belong to a monophyletic group of agnathans. Due to this close phylogenetic connection and similar morphology, the lamprey (Petromyzon marinus) was chosen as my control animal. The monophyletic grouping of hagfishes and lamprey is supported by recent studies of their microRNA (Heimberg et al., 2010) and the craniofacial development of embryos (Oisi et al., 2013). Using nucleotide and amino acid sequencing, it has been estimated that the phylogenetic split between hagfish and lamprey occurred between 470 – 390 million years ago (Kuraku and Kuratani, 2006). Hagfish and lamprey share derived characteristics such as the absence of a jaw, a large notochord, horny teeth, and pouched gills (Takezaki et al., 2003). They are both cartilaginous, scaleless and maintain their notochord into adulthood. The Notochord In contrast to elasmobranchs and teleosts, hagfish do not have vertebrae, but instead maintain their notochord into adulthood. The notochord of the hagfish acts like a hydrostat (Long, 2002). A hydrostatic skeleton can be created when there is a fluid under pressure in a closed container. The container resists tension and the fluid resists compression. Most biological hydrostatic skeletons have helically reinforcing fibres (Chapman, 1958).