Eptatretus Stoutii and Myxine Glutinosa) Protects from Biting Predators

Home , Hagfish, Myxine glutinosa, Pacific hagfish

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

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.

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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.

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

RESULTS ……………………………………………………………………………...……. p. 32

DISCUSSION ……………………………………………………………….…………...... p. 39

LITERATURE CITED …………………………………………………….………….…….. p. 49

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

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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

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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). With the fibres running in left and right handed helices, the animal can bend and move more easily than if

the fibres ran lengthwise and circumferentially. Many invertebrates use hydrostatic skeletons for terrestrial locomotion, burrowing, and even swimming (Chapman, 1958). Hydrostats are also found in plants, worms, echinoderms, arthropods and vertebrates (Neville, 1993). Caecilians, a type of limbless amphibian, use a hydrostatic skeleton enclosed by crossed-helical tendons to improve locomotion (O’Reilly et al., 1997). In the hagfish, the notochord provides the body with

75% of its flexural stiffness (the resistance while undergoing bending) and 80% of its flexural damping (the decrease in the amplitude of the oscillation) (Long et al., 2002). Scales and vertebrae can contribute to the flexural stiffness of other fishes (Symmons, 1979; Pearson, 1981;

Long et al., 1996), but hagfishes possess neither.

Hagfish skin

The scaleless skin of hagfish is made up of an epidermis, dermis, and hypodermis

(Andrew and Hickman, 1974). One of the roles of the dermis it to provide tensile strength to the skin. The tensile properties of hagfish skin have been shown to be similar to those of other elongated fish (Clark et al., 2016). The dermis of the hagfish is composed of collagen fibrils that cross each other at angles between 90o and 110o (Welsch et al., 1998). These fibres running in left and right handed helices allow the hagfish to bend easily (Chapman, 1958). Hagfish skin is anisotropic with the longitudinal stiffness roughly twice the circumferential stiffness (Clark et al., 2016).

Hagfish have slime glands located ventral-laterally along both sides of their body (Fig.

1). These glands release both mucus and slime threads when the hagfish is provoked (Downing et al., 1981; Ferry, 1941). The majority of the resulting slime is sea-water that is trapped between

Figure 1: Slime glands on the ventral-lateral portion of the hagfish body. They are found on both sides of the hagfish. Modified from Herr et al. (2014).

the slime threads (Fudge et al., 2005). This slime is secreted as a defence against predators

(Fudge et al., 2005) and to discourage competition for food (Collins et al., 1999; Tamburri and

Barry, 1999; Davies et al., 2006).

In 2008, multiple baited remote underwater stereo-video (BRUV) units were positioned in 67 locations, 97 – 1162 m deep, along New Zealand’s Northern coast where hagfish are found

(Zintzen et al., 2011). Hagfish were recorded being attacked by a catshark (Cephaloscyllium isabellum), spiny dogfishes (Squalus griffini and Cirrhigaleus australis), a kitefin shark

(Dalatias licha), conger eels (Bassanago bulbiceps), a scorpionfish (Heliocolenus sp.), wreckfish

(Polyprion americanus) and raftfish (Hyperoglyphe Antarctica) (Zintzen et al., 2011). The

BRUVs recorded 12 videos of hagfish being attacked, with 14 different instances where slime deterred a potential predator (Zintzen et al., 2011). In all cases but one, the hagfish appear to swim away with no obvious signs of injury from the initial attack. In the instance of the attack by a kitefin shark, Dalatias licha, a single puncture wound is observed. The lack of damage observed was surprising as hagfish are scaleless and, in teleosts, scales have been shown to increase the protective qualities of the epidermis by up to 10 times when compared to skin without scales (Zhu et al., 2013; Vernerey et al., 2014). These observations raise the possibility that hagfish skin is more puncture resistant than other fish skin. Measurements of the force to puncture for hagfish skin and 19 other fish species (18 teleosts and 1 elasmobranch) showed that hagfish skin is similar in puncture resistance to other fishes (Stiles et al., unpublished; Fig. 2).

Unlike hagfish, the other fishes used for this comparison possess scales, and thus, the puncture resistance of hagfish skin is impressive. However, puncture resistance does not explain the lack of injuries that hagfishes sustain in bites from predators such as sharks. These observations

Hexagrammos stelleri Citharichthys sordidus Myoxocephalus polyacanthocephalus 4 Ophiodon elongatus Embiotoca lateralis Apodichthys flavidus Aulorhynchus flavidus 3 Blepsias cirrhosus Eopsetta jordani Gadus chalcogrammus E. stoutii Gasterosteus aculeatus 2 LoadPunctureat (N) Isopsetta isolepis Lepidopsetta bilineata Liparis florae Lumpenus sagitta 1 Lyopsetta exalis Psettichthys melanostictus Squalus suckleyi

0 Eptatretus stoutii 0 0.2 0.4 0.6 0.8 1 Oncorhynchus mykiss Thickness (mm)

Figure 2: The force required to puncture Pacific hagfish (Eptatretus stoutii), teleost and elasmobranch skin as a function of skin thickness. Hagfish skin was not found to be more puncture resistant compared to the other fishes (Stiles et al., unpublished).

prompted me to consider the possibility that hagfishes possess other biomechanical adaptations that allow them to survive the initial attacks from biting predators.

Skin attachment and the subcutaneous sinus

In most fishes, skin is firmly attached to the underlying musculature. In contrast, in hagfishes, there are minimal connections between the skin and muscle (Fig 3). The hagfish’s body is only attached to the skin at the mid-dorsal line and at the slime glands (i.e. the skin is loose) (Cole, 1907; Cole, 1926; Vogel and Gemballa, 2000). In the Atlantic hagfish (Myxine glutinosa) a few thin connections of connective tissue fibres are also seen just above the slime glands (personal observations) (Fig. 4). Some authors have suggested that in fish such as sharks and eels, taut skin can act as an exotendon (Wainwright et al., 1978; Hebrank, 1980). This external tendon helps with movements, such as swimming, by stiffening and transferring energy from the muscle down the body (Westneat et al., 1993; Szewciw and Barthelat, 2016). Other fish species such as Norfolk spot (Leiostomus xanthurus) and skipjack tuna (Katsuwonus pelamis) do not use their skin as exotendons (Hebrank and Hebrank, 1986). The detached skin of hagfish also does not act like an exotendon (Clark et al., 2016) nor does it aid much with swimming (Vogel and Gemballa, 2000).

In combination with its minimal connections, hagfish skin is not taut against the underlying muscle. This unattached, flaccid design allows the body to form knots to facilitate feeding on large carrion or when removing slime from their bodies (Clark et al., 2016) and also to manoeuver through tight spaces (Freedman and Fudge, 2017). This lack of pre-strain in the hagfish may confer protection to the skin by increasing the force and extension needed for it to

a) Pacific hagfish (Eptatretus stoutii) b) Sea lamprey (Petromyzon marinus)

Figure 3: Light microscopy images of a) Pacific hagfish (Eptatretus stoutii) showing minimal connections between musculature and skin compared to the attached musculature of b) the sea lamprey (Petromyzon marinus). The subcutaneous sinus contains up to 30% of the blood volume of the hagfish and is flaccid.

Figure 4: Thin connective fibres (white arrow) between the skin and muscle of the Atlantic hagfish (Myxine glutinosa) above the slime glands.

be punctured. In humans, the force needed for an object to penetrate the skin is affected by skin tension and the material properties of the underlying material, such as muscle or bone (Knight,

1975). For example, skin stretched tight, such as over the ribs, is easier to puncture than areas not in tension and/or not underpinned by rigid tissue such as bone or cartilage. If these findings are transferable to hagfish skin, the slack skin may provide protection to the internal muscles and viscera by allowing them to move out of the way of a penetrating tooth.

In addition to their loose skin, hagfishes have a large, subcutaneous sinus (Fig.3). This sinus runs the length of their body and contains up to 30% of their blood volume (Forster et al.,

1989). Although the blood volume within the subcutaneous sinus is known, the total volume it can hold has never been quantified. The function of the subcutaneous sinus as a defence mechanism for the hagfish is discussed within this thesis. Its large size combined with the lack of connections between the skin and muscles could be enough to allow the internal musculature and viscera to move out of the way of a penetrating tooth. The moving muscle and viscera may not protect the skin but perhaps allow internal structures to avoid lethal damage even when the skin is punctured.

Hagfish musculature

The parietal muscle of the hagfish extends downward from the mid-dorsal line, reaching its greatest thickness at the ventral surface of the notochord (Cole, 1907). These muscles are important for swimming (Cole, 1907). The oblique muscle covers the ventral portion of the parietal muscle, separating the parietal muscle from the slime glands (Cole, 1907; Fig. 5). The function of the oblique muscle is not well described, but it has been suggested to be involved in

Figure 5: The location of the parietal muscle and oblique muscle in a cross section of a hagfish. Modified from Cole (1907).

slime release (Cole, 1907) although there is no direct evidence for this claim. The paucity of connections between the skin and musculature likely confers a greater range of lateral motion of the muscle and viscera within the skin.

Defensive secretions

Animals employ diverse strategies to protect themselves against predators. These range from avoidance techniques, such as camouflage, burrowing, and mimicry, to anatomical adaptations such as quills and claws or limb autotomy (Skelhorn et al., 2010; Stankowich, 2011;

Nunez et al., 2014; Caro et al., 2016; Lin et al., 2017). In many cases, animals defend themselves by secreting chemicals. These secretions can be poisons, alarm signals, sensory deterrents, wound protectants, or physical deterrents. For example, octopuses, cuttlefish, and some slugs can produce ink that blocks the vision of attacking predators and allows for escape (Derby el al.,

2013). The ink of sea hares (Aplysia californica) has been shown to act as an alarm signal, affect predator feeding responses, and/or cause sensory deprivation (Derby, 2007; Nusnbaum et al.,

2012; Love-Chezem et al., 2013). The larvae of the six-spotted neolema (Neolema sexpunctata) and the three-lined potato beetle (Lema trilinea) both secrete substances into fecal matter that they then use as chemical shields against predatory ants (Morton and Vencl, 1997). The mucus that the orange-banded Arion (Arion fasciatus) and grey garden slug (Deroceras reticulatum) secrete has been shown to deter predatory beetles (Pakarinen, 1993). Parrotfish (Scarus croicensis) envelop themselves in a mucosal cocoon at night to potentially ward off predators

(Winn and Bardach, 1959). This mucus cocoon has also been shown to have antibiotic effects against several pathogenic tropical fish bacteria (Videler et al., 1999). Hawaiian wrasses,

Labroides phthirophagus, are also known to form a gelatinous cocoon around themselves for

protection while resting (Jakowska, 1963), and the southern bottletail squid, Sepiadarium austrinum, can secrete a large amount of slime, which is suspected to work as a chemical defense

(Caruana et al., 2016). The soapfish, Rypticus saponaceus, also releases slime that is suspected to repel predators (Randall, 1967) via a toxic polypeptide called ryptisin (Maretzki and Del

Castillo, 1967; Goyco et al., 1973). Even when a released substance does not harm an animal, previous exposure can cause a learned aversion response to the secretion. For example, kit foxes

(Vulpes macrotis), after being subjected to the blood-squirting of the Texas Horned Lizard

(Phrynosoma cornutum) chose other prey items in subsequent trials (Sherbrooke and

Middendorf, 2004). It has long been known that hagfishes release slime exudate as a means of defence when they are physically attacked or stressed (Ferry, 1941; Downing et al., 1981). The subsequent physical distress of the predator, caused by the clogged gills, may deter it from attacking another hagfish in the future.

Hagfish slime defense

Hagfishes have been found in the stomach contents of air-breathing marine animals such as toothed whales (Smith and Read, 1992; Schiavini et al., 1997; Gannon et al., 1998), harbor porpoises (Phocoena phocoena; Borjesson et al., 2003) and Pacific Harbour seals (Phoca vitulina ricbardsi; Orr et al., 2004). They have also been found in the stomachs of birds such as

Magellanic penguins (Spheniscus magellanicus; Scolaro et al., 1999), the Imperial Shag

(Phalacrocorax atricps; Michalik et al., 2010) and Laysan albatrosses (Phoebastria immutabilis;

Pitman et al., 2004). Hagfish are common prey of Atlantic white-sided dolphins

(Lagenorhynchus acutus) off the coast of New England, but it is not clear if they are actively hunting them or feeding on hagfish discarded by fishermen or disturbed by bottom otter trawls

(Craddock et al., 2009). In contrast, there are relatively few documented instances of hagfish predation by teleosts, chondrichthyans, or other gill breathing animals (Martini, 1998; Table 1).

The proportionately low number of fishes known to prey on hagfish is thought to be a result of their slime defense and the fact that in most fishes, food and respiratory water both pass through the oral cavity (Fernholm, 1981; Martini, 1998). In a laboratory setting, the slime has been shown to cause an increased resistance to water flow through the gills of the predator (Lim et al., 2006). This could thwart fish predators by threatening them with suffocation if they continued an attack. Evidence for this gill clogging effect has also been observed in the wild with various species of fishes (Zintzen et al., 2011). Slime is released when hagfishes are physically attacked or stressed (Ferry, 1941; Downing et al., 1981). It is released only by glands in the proximity of the attack and does not form a protective layer around the hagfish (Lim et al.,

2006).

Videos of hagfish being attacked in the wild show that the slime defense is only activated after the hagfish is attacked. In the case of suction feeders, such as wreckfishes (Polyprion americanus) and scorpionfishes (Helicolenus sp.), the utility of the slime is straightforward. The ingestion of the hagfish by the suction feeder induces the hagfish to release exudate from the slime glands. Exudate is pulled into the fish’s mouth and, when mixed with sea water, forms the gill-clogging slime. The hagfish is then able to escape while the predator tries to dislodge the slime. In the case of biting predators, especially those with sharp teeth, the slime’s utility is less obvious. The physical attack of the teeth on the hagfish’s body results in the release of the exudate but the initial bite has the potential to do serious damage to the hagfish. Slime release

Table 1: Recorded instances of hagfish found in the stomach contents of gill breathing animals. Species Source Broadnose sevengill sharks (Notorynchus Crespi-Abril et al., 2003 cepedianus) Greenland shark (Somniosus microephalus) Nielsen et al., 2014 North Sea whiting (Merlangius merlangus) Stafford et al., 2007 Thorny skate (Amblyraja radiata) Link and Almeida, 2000 Southern giant octopus (Octopus magnificus) Villanueva, 1993

may prevent a second attack, but that would be of little use if serious or lethal damage has already occurred.

How do hagfish survive shark bites?

A kitefin shark (Dalatias licha) biting into the middle of a hagfish was filmed in the wild

(Zintzen et al., 2011), resulting in the production of large volumes of slime, which filled the shark’s mouth and trailed out of its gills. The hagfish was then observed to swim away, with only a single obvious puncture wound (Fig. 6). Kitefin sharks have 19 small, smooth, hooked teeth in their upper jaw, and triangular, serrated teeth in their lower jaw (Fig. 7). Narrow cusp teeth, such as in the upper jaw of kitefin sharks, are usually better at puncturing prey than broad triangular teeth (Whitenack and Motta, 2010). Serrated teeth, such as those in the lower jaw of the kitefin shark, have a larger total bite force due to the friction between them and the material they are biting (Frazzetta, 1988). When sharks bite, the jaw opening phase begins when the lower jaw is depressed and the upper jaw (and cranium) is raised (Frazzetta and Prange, 1987). The upper jaw then protrudes as the mouth is closed. It is speculated that the kitefin shark bites into its prey and then spins to remove chunks of skin and flesh (Clark and Kristof, 1990). In the video presented by Zintzen et al. (2011), the slime from the hagfish causes the shark to release the hagfish before it has a chance to spin; this could explain why the hagfish was not killed, but it does not explain why the initial bite did not cause more damage.

Hypothesis and predictions

It is likely that hagfish possess other adaptations that allow them to survive attacks by biting predators as slime is not released until after the initial attack, and hagfish skin is not

Figure 6: A single puncture wound (white arrow) is seen on the side of a New Zealand hagfish (Eptatretus cirrhatus) after it has been bitten and released by a kitefin shark (Dalatias licha). The shark is seen trying to dislodge the hagfish slime from its gills. Modified from Zintzen et al. (2011).

Figure 7: The jaw of a kitefin shark, Dalatias licha, in anatomical position revealing the differences in tooth morphology between the upper and lower jaw. https://commons.wikimedia.org/wiki/File:Dalatias_licha_jaw.JPG

puncture resistant. In this thesis, I research the possibility that the flaccid, loosely attached skin in hagfish minimizes injuries from biting predators by allowing the body to move out of the way of penetrating teeth. This hypothesis makes several predictions. First, a substantial amount of fluid should be able to be injected into the subcutaneous sinus before there is an increase in pressure. This quantification of the flaccidity of the sinus would indicate if enough space is present for the internal organs and musculature to move out of the way of the penetrating shark tooth. Second, this hypothesis predicts that increasing connections between the skin and musculature will cause an increase in damage during simulated shark attacks as the internal muscle and viscera would not be able to move out of the way and the tooth would penetrate both skin and muscle rather than slipping between the two. Third, decreasing the connections between the skin and musculature and increasing the flaccidity of the skin will decrease damage, and it would provide opportunity and means for the muscle to slip out of the way of the penetrating tooth and the therefore avoid damage. And finally, this hypothesis predicts the distance a tooth can travel after making contact with the skin before generating enough stress to puncture it will increase as skin slackness is increased.

Three sets of experiments were conducted:

a) Hagfish inflation experiments

b) Shark teeth impact experiments

c) Isolated skin puncture tests

To test these predictions, I quantified flaccidity by measuring pressure as a function of fluid volume injected into the subcutaneous sinus. I tested the importance of skin looseness by manipulating both the flaccidity of the skin as well as its degree of attachment to the underlying

musculature. I simulated shark attacks using a spring-driven guillotine to drive shark teeth into freshly dead hagfish and lamprey. Here I provide evidence consistent with the hypothesis that the loose and flaccid design of the hagfish body confers protection against the initial attack of biting gill breathing predators, allowing them to subsequently use slime to clog the gills and gain release.

METHODS

Animal collection and care

Pacific hagfish (Epatretus stoutii) were obtained from the Bamfield Marine Sciences

Centre in Bamfield, British Columbia and were housed in the Hagen Aqualab at the University of Guelph in 2000-L Environmentally Controlled Aquatic Recirculating System (ECARS) tanks at 10oC and 34 ppt salinity. All housing and feeding conditions were approved by the University of Guelph Animal Care Committee (AUP #2519). Atlantic hagfish (Myxine glutinosa) were obtained and utilized at the Shoals Marine laboratory in Maine. They were caught in herring- baited traps that were set at an approximate depth of 91 m in an area 12 km southeast of

Appledore Island, Maine. Sea lamprey (Petromyzon marinus) were obtained from the Humber

River in Toronto, ON. They were euthanized by the Toronto Region Conservation Authority using a sharp blow to the head before being transported to Guelph for experiments. Mako shark

(Isurus sp) jaws were ordered from the Taiwan-Shark shop in Taiwan. The jaws were boiled in water for 1 hour and the teeth from the upper and lower jaws were removed using forceps close to the root to pry them loose. The second row of teeth was used during experimentation to ensure sharpness. Based on preliminary trials, each tooth was used for a maximum of six tests to minimize dulling effects (Corn et al. 2016). Mako shark teeth were utilized as a representative of teeth used to puncture prey.

Inflation experiments

To quantify the flaccidity of hagfish bodies, hagfish Ringer’s was injected into the subcutaneous sinus of freshly dead Atlantic and Pacific hagfish specimens. I define the slack volume as the volume of fluid that can be injected before the subcutaneous sinus is ‘full’ (Fig. 8), which I detected by monitoring the pressure as fluid was injected. To test the prediction that a substantial amount of fluid would be able to be injected into the subcutaneous sinus before there was an increase in pressure and therefore quantify the flaccidity of the hagfish body, hagfish were euthanized using an overdose of buffered MS-222 (2 mg ml−1) in seawater for 30 minutes.

The euthanized hagfish were weighed, measured (from their nostril to the tip of their tail) and their total body volume was determined using water displacement. To determine skin strain during inflation, six dots of India ink, two rows of three, were applied to the skin caudal to the gill pouches at 0.5 cm increments using a sharp toothpick. Pressure of the subcutaneous sinus was measured using a custom single column manometer made from a serological pipette attached to a t-junction with a 60-ml syringe for injection of fluid on one side, and a needle (18 gauge, 1.5 inch) inserted into the subcutaneous sinus at the caudal end.

-1 Hagfish Ringer’s solution (in mmol l : 410 NaCl, 10 KCl, 14 MgSO4, 4 urea, 20 glucose,

10 HEPES, 4 CaCl2, pH 7.8; modified from Gillis et al., 2015) was injected into the hagfish in 5 ml increments and the resultant fluid height in the manometer was measured. The internal pressure of the hagfish was determined using the equation:

pressure = density X acceleration due to gravity X height

Which can be simplified as P = pgh where p is the density of hagfish Ringer’s solution (1025 kg/m3), g is acceleration due to gravity (9.807 m/s2) and h is the measured height (in meters) of

a) b)

a )

Figure 8: Pacific hagfish (Eptatretus stoutii) with an initial mass of 58.95 g, length of 36.1 cm, and total body volume of 55.5 ml. Before a) and after b) inflation with approximately 70 ml hagfish Ringer’s solution. The maximum volume injected before leaking was 104% of the total body volume with a pressure of 646 Pa. The maximum circumferential strain was 0.20 and the maximum longitudinal strain was 0.06.

fluid in the single column manometer. For a given 5 ml increment, the volume injected into the hagfish was calculated by subtracting out the additional volume that entered the manometer column. A photo of the dots on the hagfish was taken after every 10 ml of fluid injected. The change in distance between the dots was measured using digital calipers to calculate the strain on the skin. The experiment was terminated when fluid was seen seeping out of the epidermis, slime glands, cloaca or gill pouches.

Shark teeth impact experiments

For guillotine experiments, the hagfish were anesthetized using a clove oil protocol described in Winegard and Fudge (2010), followed by decapitation. A custom spring-driven guillotine (Fig. 9) was created to investigate the effect of high speed impact of shark teeth on a variety of specimens and treatments. The guillotine consisted of a metal mouton set on high precision rails. The mouton was held up using a pin that, when removed, allowed the mouton to accelerate down, reaching a velocity of approximately 0.57 m/s when it contacted the specimen.

Mouton velocity was quantified using high speed videography. Mako shark (Isurus sp.) teeth were glued to the corner of metal plates using Clubmaker Shafting epoxy (Golfsmith

International, Austin, TX). The tooth was glued to the plate so that the tip of the tooth was parallel to the direction of mouton travel. The metal plates were created using single-edge razor blades with the sharp edge facing away from the hagfish.

Each hagfish (n = 6 for both Pacific and Atlantic hagfish) was tested with the guillotine in four states: natural state (‘natural’ treatment), with the skin glued tight to the underlying musculature (‘glued’ treatment), with glue applied to the musculature, but allowed to cure

Spring

Mouton

Shark tooth

Base

Figure 9: The customized guillotine used for quantifying the effects of shark teeth driven into a variety of specimens and treatments. Note a single shark tooth is attached to the mouton and driven down by the spring, achieving a velocity of 0.57 m/s near the base.

before the skin was replaced (‘glue-control’ treatment) and unattached tight, where the skin was detached from the hagfish muscle and then pinned to form a tight sleeve around the muscle (Fig.

10). For the glued treatment, a lateral flap of skin was created by cutting with scissors from the dorsal midline to the slime glands, caudally above the slime glands and back up to the dorsal midline. Small dots (approximately 300 µm) of Loctite 454 Prism (Henkel Corp., Rocky Hill,

CT) were applied to every second myomere and on alternate myomeres above and below the lateral midline and the skin was replaced before the glue had a chance to cure, ensuring adhesion.

The skin was held in place by silk thread sutures (size 3/0) along the bottom edge of the flap close to the slime glands. This allowed up to test the prediction that increasing connections would increase the damage done to the muscle. In the glue-control treatment, the same procedure was followed but the glue was allowed to cure before the skin was replaced, thereby minimizing adhesion between the skin and glue, but maintaining the presence of the hardened glue dots.

The hagfish was placed on a Styrofoam base with wooden toothpicks used to hold it dorsal side up (Fig. 11). The hagfish was placed so that the tip of the tooth would make contact just lateral to the dorsal midline. Damage to the skin was recorded by being given a rating of 1 if the skin was punctured, and a rating of 0 if the skin was not punctured. The hagfish was removed from the guillotine and the parietal muscle was examined to assess whether it was punctured by the tooth. If the parietal muscle was punctured, it was given a rating of 1 and if it was not punctured, it was given a rating of 0.

a) b)

c) d)

Figure 10: The testing of hagfish specimens for puncture testing using a custom shark tooth guillotine. Glued and glue-control specimens were created by cutting away a flap of skin and glueing the skin down to the underlying musculature (glued) or allowing the glue to cure before the skin was replaced and sutured in place along the ventral edge of the flap (glue-control). Unattached tight specimen had their skin detached and replaced tight around the muscle. a) Natural specimen, with only the skin punctured by the tooth b) Glued specimen, with the tooth puncturing both the skin and the parietal muscle. c) Glue-control specimen, with tooth puncturing the skin but not the underlying muscle. d) Unattached tight specimen, with the tooth puncturing both the skin and the parietal muscle.

Figure 11: Hagfish holder made from wooden toothpicks to maintain proper anatomical position of the hagfish while out of water.

The lamprey (n = 6) treatments were opposite that of the hagfish as the lamprey’s natural state is with the skin tight and attached to its body. The lamprey was tested in its natural state, with its skin dissected and loose, and with its skin dissected and tight (Fig. 12). In the natural treatment, the lamprey was placed dorsal side up so the apex of the tip of the tooth would hit lateral to the dorsal midline. Damage to the skin and muscle were recorded. For the loose skin treatment, the skin was dissected away from the muscle of the lamprey using a #11 scalpel blade while maintaining the integrity of the skin in a tubular shape. If the lamprey was female, the eggs were removed to create extra space within the skin and the remaining portion was placed back into the skin tube. If the lamprey was male, a smaller area of the body was dissected away from the muscle and the remaining portion was placed into the bigger skin tube. The lamprey was then positioned so the tip of the tooth would make contact lateral to the dorsal midline and damage to the skin and muscle were recorded. In the tight treatment group, the skin was dissected away from the muscle and then held tight around the muscle using pins on the ventral side of the skin.

Skin strain experiments

For the skin strain experiments, the hagfish were anesthetized using a clove oil protocol described in Winegard and Fudge (2010), followed by decapitation. In order to test the effect of skin flaccidity on the extension and force needed to puncture the skin, a skin stretcher was made using a hollow tube of acetal Delrin rod. The tube had a lip at the top to brace the clamp used to

trap the skin. It had a rough surface to increase traction with the skin and a slot to mount it to a

model 3343 Instron Universal Testing machine (Illinois Tool Works Inc., Norwood, MA) base

once strain was applied to the skin. A snug-fitting thin hollow tube was inserted into the outer

a) b)

c) d)

Figure 12: Preparation (a) and testing (b-d) of sea lamprey (Petromyzon marinus) specimens for testing with a shark tooth guillotine. a) The skin dissected away from the muscle. b) Natural lamprey specimen with tooth penetrating both skin and underlying musculature. c) Dissected “tight” lamprey specimen, with tooth penetrating both skin and muscle. d) Dissected “loose” lamprey specimen, with tooth penetrating the skin only. See methods for details.

tube and used to generate radial strain in the skin before puncture testing (Fig. 13). Mako shark

(Isurus sp.) teeth were glued onto stainless steel plates using Clubmaker Shafting epoxy with the tip pointing in a direction parallel to the movement of the Instron crosshead. An excised piece of hagfish skin (approximately 5x5 cm) was placed on top of the outer cylinder and clamped in place. Four dots of India ink were placed on the skin, in the shape of a square. The distance between the dots was measured using digital calipers. The insert was placed into the bottom of the outer tube and placed onto a rod mounted to the Instron base. Radial strain was applied by pulling down the outer cylinder with the insert mounted in place (Fig. 14). Once the desired strain was achieved, a set screw was tightened to hold the outer tube in place. The distances between the dots were measured again and the strain was calculated by dividing the change in length by the original length between the dots. The radius of the stretched skin was marked and then measured once strain was removed. The relative radius of strained skin to unstrained skin was 0.82. Slack, i.e. loose skin, was created by raising the inner tube 0.5 cm above the outer tube before laying the skin on and clamping it in place. The inner tube was brought flush with the top of the outer tube and the skin lay slack on the apparatus. This increased the radius of skin laying within the stretcher by a factor of 1.5. Extension was defined as the distance the tooth moved relative to first skin contact with strained skin. Puncture tests were carried out using a crosshead velocity of 0.33 mm/s. Force and extension at puncture were obtained using the Instron

Universal Testing machine.

Statistical analysis

All statistical tests were two tailed t-tests, and the statistical threshold, α, was set at ≤

0.05. IBM SPSS statistics 24 was used to perform linear regression analysis, t-tests, and one-way

Figure 13: Hagfish skin stretcher made from acetal Delrin rod. From left to right: clamp, outer tube, insert. The excised skin was laid over the outer cylinder and clamped in place. The insert was used to subject the skin to radial strain before puncture testing.

a) b)

Figure 14: The skin stretcher attached to the Instron universal testing machine. a) Excised hagfish skin clamped at a strain of zero. b) A skin sample with an approximate radial strain of 0.12 before puncture testing.

ANOVAS. I tested the prediction that the extension of the shark tooth would increase as slack increased using linear regression of strain vs extension and a one-way ANOVA to test the effect of slack and pre-strain on extension. A linear regression was performed to test the effect of pre- strain on force needed to puncture the skin and a one-way ANOVA was used to test the effect of slack and strain on the force.

RESULTS

The average slack volume for Pacific hagfish, measured as a percent of body volume, was 46.1 ± 6.6 % and for Atlantic hagfish it was 36.1 ± 2.3%, a difference that was not statistically significant (p=0.33). Slack volume values were consistent for Atlantic hagfish, but more variable for Pacific hagfish. Pressure vs. volume curves revealed an initial rise in pressure after the slack volume was filled, followed by a plateau region in which relatively large volumes could be injected with little increase in pressure. The plateau region ended with another steep rise in pressure, which ultimately culminated in leakage and an end to the test (Fig. 15). For the

Pacific hagfish, the average final strain before leakage occurred was 0.21 ± 0.03 circumferentially and 0.050 ± 0.010 longitudinally. The maximum volume of Ringer’s that could be injected before leakage occurred, expressed as a percentage of body volume, was 120 ± 8.2 % for Pacific hagfish and 105 ± 6.3 % for the Atlantic hagfish, a difference that was not statistically significant (p=0.13).

Pacific hagfish, Atlantic hagfish and sea lamprey were tested for damage done by a Mako shark tooth mounted on a spring-driven guillotine (Table 2). Multiple trials were performed on each specimen in different locations. With the skin in its natural state, Pacific hagfish and

2500

2000

1500

1000 Pressure Pressure (Pa)

500

0 0 50 100 150 200 250 Injected Volume (% Body Volume)

Figure 15: Pressure (Pascals, Pa) in the subcutaneous sinus of two species of freshly dead hagfish as a function of injected fluid volume. For Pacific hagfish (Eptatretus stoutii) (n=13), the mean volume that could be injected without any measured increase in pressure (i.e. the slack volume), was approximately 46.1% ± 6.6 and for Atlantic hagfish (Myxine glutinosa) (n=6), it was 36.1% ±2.3.

Table 2: Comparison of puncture damage by Mako shark (Isurus sp.) teeth to the skin and parietal muscle of Atlantic and Pacific hagfish.

Species Treatment Organ Total punctures Puncture totals % Punctured with multiple averaged by trials per animal animal Natural Skin 8/8 6/6 100 (loose) Parietal 0/8 0/6 0 Pacific hagfish Muscle (Eptatretus Glued Skin 13/13 6/6 100 stoutii) (tight) Parietal 11/13 5.5/6 92 Muscle Glue-Control Skin 14/15 5.75/6 96 (loose) Parietal 1/15 1/6 17 Muscle Unattached Skin 35/36 5.83/6 97 tight Parietal 31/36 5.17 86 Muscle Natural Skin 18/18 6/6 100 (loose) Parietal 0/18 0/6 0 Muscle Glued Skin 13/13 6/6 100 Atlantic hagfish (tight) Parietal 12/13 5.5/6 92 (Myxine Muscle glutinosa) Glue-Control Skin 15/15 6/6 100 (loose) Parietal 0/15 0/6 0 Muscle Unattached Skin 35/36 5.83/6 97 tight Parietal 33/36 5.66/6 94 Muscle Natural Skin 13/13 7/7 100 (tight) Parietal 13/13 7/7 100 sea lamprey Muscle (Petromyzon Dissected Skin 7/17 2.8/7 40 marinus) (loose) Parietal 4/17 1.7/7 24 Muscle Dissected Skin 6/6 4/4 100 (tight) Parietal 6/6 4/4 100 Muscle

The experiment was performed on hagfish (Eptatretus stoutii and Myxine glutinosa): in their natural state (natural), with glue on the muscle but the skin unattached to the muscle (glue- control), the skin attached to the muscle with glue (glued), and skin unattatched but tight (unattached tight). As the lamprey (Petromyzon marinus) skin is already attached to the muscle, it was tested: with the skin dissected away from the muscle (loose), the skin dissected away and then made tight around the muscle (tight), and in its natural state (natural).

Atlantic hagfish were punctured 100% of the time, but the underlying parietal muscle was never damaged. When the skin was artificially attached to the underlying muscle using small dots of glue, the parietal muscle was damaged 92% of the time in both Pacific hagfish and Atlantic hagfish. For the glue-control trials, in which there was glue on the muscle but the skin was unattached, the skin punctured 96% of the time in the Pacific hagfish with the parietal muscle being damaged only 17% of the time. In the Atlantic hagfish glue-control treatment, the skin was punctured 100% of the time and the parietal muscle was never damaged. When the skin was fully detached and then held tight against the muscle, the skin was damaged 97% of the time in both the Pacific and Atlantic hagfishes and the parietal muscle was punctured 86% of the time in the Pacific hagfish and 94% of the time in the Atlantic hagfish. As a comparison, lamprey were tested in three states: natural, with skin dissected and loose, and with skin dissected and made tight around the muscle. In its natural state with the skin attached to the muscle, both the skin and the muscle were damaged 100% of the time. For the dissected and loose trials, the skin was punctured 40% of the time and the parietal muscle was damaged 24% of the time. When the skin was dissected away but made tight around the muscle, both the skin and the muscle were damaged 100% of the time (Table 2).

The amount of slack or strain placed on a sample of Pacific hagfish skin affected the distance the shark tooth extended before puncture occurred. Pre-strain, the amount of strain applied before testing occurred, was negatively correlated with extension (Fig. 16). The negative linear relationship between the strain and extension was statistically significant (p=0.001). The slack, determined by the radius of the skin contained within the clamp, was positively and significantly correlated with extension of the shark tooth (p<0.001; Fig. 17). In contrast, pre- strain was not a significant predictor of the force needed to puncture the skin (p=0.484; Fig. 18).

6 R² = 0.8329 5

2 Extension at Puncture (mm) Puncture at Extension 1

0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Pre - strain

Figure 16: The effect of Pacific hagfish (Eptatretus stoutii) skin pre-strain on the distance a shark tooth can travel after contact with the skin before puncture occurred. The negative linear relationship between the strain and extension is statistically significant (p=0.001).

Figure 17: The effect of the radius of Pacific hagfish (Eptatretus stoutii) skin within the skin stretcher on the extension of a shark tooth at puncture. The radius of the control strain (no strain or slack) was designated 1. Slack skin had a relative radius ratio of 1.5. Strained skin was marked while strained and then the radius measured with no strain on the skin. The relative ratio of the strained skin was 0.82 (df =14, f=172.947, p<0.001).

R² = 0.223 6

Force at Puncture (N) Punctureat Force 2

0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 Pre - Strain

Figure 18: The effect of strain on the force to puncture isolated Pacific hagfish (Eptatretus stoutii) skin. The level of pre-strain was not a significant predictor of puncture force (p=0.484).

The relationship between slack, the radius within the clamp, and force needed to puncture the skin was also not significant (p=0.514; Fig. 19).

DISCUSSION

The unattached, flaccid skin of hagfishes confers protection to the underlying musculature and viscera. Testing that varies the flaccidity and attachment of the skin show that in order for protection to be conferred, the both requirements must be met. As video of a kitefin shark attack and my results both show the skin of the hagfish is not puncture resistant, a different explanation was needed to explain how hagfish survive the initial bites of attacking predators. I found that hagfish employ a novel method of avoiding subcutaneous tissue damage by allowing internal structures to move out of the way of penetrating objects. In support of my hypothesis, I showed that to avoid internal damage, the hagfish skin needs to be both flaccid and have minimal connections, as these conditions allow the tooth to slide between the skin and the body musculature instead of damaging internal organs.

I have shown that flaccidity protects against internal damage from penetrating teeth. If there is no slack in the skin to create space and allow movement between the skin and body, a tooth that has penetrated the skin will continue on to penetrate the underlying muscle. However, with a flaccid body design, the extra room allows the tooth to slide along the body rather than puncturing it. The data acquired through my inflation testing are the first quantification of flaccidity in hagfishes and show that the slack volume is substantial. Although it has long been known that the subcutaneous sinus in hagfish has room to hold more blood than is normally present (Forster, 1997) both the Pacific and Atlantic hagfish were found to have enough room to accommodate over 36% of their total volume without a measurable rise in pressure. Assuming the volume enclosed by the skin does not change much, changes in the volume of food in the gut

Figure 19: The effect of the radius of Pacific hagfish (Eptatretus stoutii) skin within the skin stretcher on the force at puncture. The radius of the control strain (no strain or slack) was designated 1. Slack skin had a relative radius ratio of 1.5. Strained skin was marked while strained and then the radius measured with no strain on the skin. The relative ratio of the strained skin was 0.82. (df=14, f=0.705, p=0.514)

and the volume of the swimming musculature and viscera will affect both the absolute slack volume, as well as slack volume expressed as a percentage of total body volume. The Pacific hagfish used varied widely in when they had last eaten (1-5 weeks) and how long they had been in captivity, which increased the variability in the Pacific hagfish slack volumes. The Atlantic hagfish slack volumes were more consistent than those of the Pacific hagfish, which may have been the result of these animals being tested shortly after being caught in the wild, and having similar nutritional status (i.e. they all likely fed in the trap). In a recent publication, Freedman and Fudge (2017) showed that flaccidity in hagfishes has consequences for their ability to move through small openings. The flaccidity allowed for blood within the sinus to redistribute and delay a pressure increase in the lagging end of the hagfish thereby allowing them to move through tight spaces. My inflation data suggest that slack volume may be affected by nutritional status, although more data is needed to confirm this. The findings of Freedman and Fudge (2017) along with my own results suggests that hagfish may become trapped inside a carcass if they eat too much.

Hagfish skin has been shown to be anisotopic with the skin being stiffer longitudinally than circumferentially (Clark et al., 2016). This was supported by my findings during the inflation experiments. According to Laplace’s law, in organisms where skin is isotropic, we would expect circumferential strain to be twice that of longitudinal strain. The maximum circumferential strain of the skin of the Pacific hagfish when leakage occurred was 4.3 times that of the longitudinal strain. Having anisotropic skin could give hagfishes greater advantage when attacked by biting predators as the skin would be able to stretch more before the force of puncture is reached allowing the musculature to move out of the way before penetration occurs.

It could also allow the musculature and organs to move farther out of the way than just the subcutaneous space would allow.

During the shark teeth impact experiments with the guillotine, the parietal muscle of the hagfishes (Eptatretus stoutii and Myxine glutinosa) was never damaged by a puncturing tooth

(Table 2), but in many cases the oblique muscle did sustain damage (Fig. 20). Therefore, it is possible that damage to the oblique muscle does not hinder a hagfish’s escape from attacking predators. It is also possible that the penetration of the oblique muscle could be an artifact of the hagfish being out of water and not in typical anatomical position. Gravity acting upon the compliant tissue of the hagfish causes the dorsal portion of the body to spread laterally, forming a widened support that would not be present if the hagfish were immersed in water (Fig. 21).

Although a cradle was used to maintain a biologically relevant position, it was not possible to eliminate the effects of gravity. This may have allowed the tooth to find purchase in the oblique muscle or near the slime glands, whereas in nature this may not occur. Therefore, damage to the oblique muscle may either not hinder the hagfish’s ability to escape and/or may not be a true representation of damage done in nature.

Hagfish skin does not stop a shark tooth from entering the subcutaneous sinus but the minimal connections and flaccidity provide protection from puncture to the underlying muscles.

Guillotine testing showed that hagfish skin was almost always punctured by the shark tooth(81 out of 82 times; Table 2). These results are consistent with previous work on puncture resistance of fish skin (Stiles et al., unpublished; Fig. 2) and with the obvious puncture wound on the hagfish in the Zintzen et al. (2011) video. The blood pressure of hagfishes is considerably lower

Figure 20: A Pacific hagfish (Eptatretus stoutii) with a pin marking the typical location of damage (black arrow) when shark tooth penetration of muscle occurred. The tooth tended to puncture between the parietal and oblique muscle and exit dorsal to the slime glands. This picture shows the glue-control treatment, in which glue was placed on the muscle but was allowed to cure before the flap of skin was closed and sewn.

a) b)

Figure 21: A Pacific hagfish (Eptatretus stoutii) (viewed from above) a) held up in an anatomically correct position in water vs b) out of water where gravity causes the hagfish muscle to splay out laterally (shown by arrow).

than in other fishes (Forster et al., 1991). The resting ventral aortic blood pressure of the Atlantic hagfish (Myxine glutinosa) is 1.04 kPa (Axelsson et al., 1990) and that of the New Zealand hagfish (Eptatretus cirratus) is 1.44 kPa.(Forster et al., 1988). In comparison, the ventral aortic blood pressure of the dogfish (Scyliorhinus canicula) is 5.1 kPa (Short et al., 1979) and the

Atlantic cod (Gadus morhua) is 4.9 Even with the skin punctured, the low blood pressure of hagfishes could minimize the amount of blood loss before clotting. Future studies could examine the wound healing abilities of the hagfish as skin puncture may occur regularly. Learning how the wounds heal and if the antibacterial properties of the slime (Subramanian et al., 2008) contribute to healing by possibly preventing infection and sepsis, could answer questions about how they survive even when their skin is punctured. Studying the skin of wild hagfish for evidence of puncture wounds would be an interesting addition to this research as we could then determine if hagfish are regularly attacked by biting predators and survive.

My results suggest that a hagfish’s defence against fish predators involves a two-part strategy – an ability to survive an initial attack, and an ability to deploy large volumes of gill clogging slime before the attack can be escalated or the hagfish can be ingested. The functional morphology of the large subcutaneous sinus, and minimal attachments between skin and musculature, complements the impressive defensive sliming abilities of hagfishes. Hagfishes release slime when attacked or provoked (Ferry, 1941; Downing et al., 1981). Getting hagfish slime on the gills is known to discourage fish predators from continuing a predatory attack, but it does not seem to prevent the initial attack. This is the observation that inspired my study. In its natural state, the paucity of connections between the skin and the musculature, as well as the slackness of the skin, means that predator teeth must travel further before the skin becomes taut and the force needed to penetrate the skin is achieved. Having slack in the skin does not confer a

defensive benefit in terms of the load needed to puncture the skin, but it may delay if or when puncture occurs due to the skin moving with the sharp object. Hagfish release slime exudate in less than 0.4 seconds after a predator makes contact (Zintzen et al., 2011). The loose skin may provide enough time for the exudate to be expelled, and for slime to be formed and sensed by the predator before damage can be done to the hagfish. In the case of shark predators, the musculature and internal organs are able to move out of the way of penetrating teeth.

Simultaneously, the slime causes the hagfish to be released before the subsequent head shaking of the shark that is used to break apart large prey or to subdue prey (Springer, 1961; Moss, 1972;

Moss, 1977; Frazzetta and Prange, 1987; Motta et al., 1997; Wilga and Motta, 1998). The kitefin shark, seen in the Zintzen et al. (2011) video is speculated to employ a bite and spin strategy to overcome prey (Clark and Kristof, 1990). The lack of connections also precludes avulsion injuries that would be caused by the skin being pulled away from the musculature. Avulsion injuries result in increased tissue injuries as compared to shearing injuries, where tissues slide over each other (Trott, 1988).

One limitation of my study was the use of a single tooth in the guillotine puncture tests.

An advantage of a predator having multiple teeth is that some teeth can hold the skin taut, thereby making it easier for other teeth to puncture and slice. Future studies could be done to visualize the movement of skin and internal musculature with multiple, simultaneously penetrating objects. Multiple penetrating teeth could affect or inhibit the movement of body musculature and viscera out of the way. Conversely, having multiple teeth could decrease stress on the skin as stress is inversely related to surface area. In studies on human bite marks on human skin, applied stress was greatly increased as the number of teeth was reduced (Bush et al.,

2010). Future research could show if this holds true for teeth that come to a sharp point such as

those found in sharks, as well as other animals. Teeth of various known predators of hagfish could also be used to determine if the results from this study are applicable in other situations.

Skin catching between teeth, stiffness of substrate, anatomic location, and skin tension are factors that have been shown to affect bite marks (Bush et al., 2009). Future research could focus on how the skin enfolds the teeth during biting and if fluid in the subcutaneous sinus acts as a lubricant or disperses energy thereby helping protect the musculature.

My results describe the passive properties of the hagfish body design in avoiding damage from sharp teeth. It is also of course possible and likely that hagfish engage in active strategies to minimize injury when they are attacked. These might include reflexive muscle contractions that cause the underlying musculature to shrink away from a penetrating tooth (Boggett, pers obs).

Other evasive maneuvers may also be utilized, such as rolling motions away from the penetrating objects to prevent musculature and organ damage. For the second part of the strategy, sliming, the only hagfish fossil ever found, from approximately 330 mya, does not appear to possess slime glands (Bardack, 1991). As fish mucus is used for multiple functions ranging from locomotion, feeding, and defence against environmental stresses, the subsequent evolution of defensive sliming is possible.

The loose and flaccid skin in hagfishes has previously been shown to be useful in carrying out knot-tying behaviours, to escape from predators or slime (Clark et al., 2016), and to gain purchase when scavenging on carcasses (Clark and Summers, 2007). It has also been shown to facilitate burrowing (Freedman & Fudge, 2017). Because loose skin has evolved multiple times in burrowing animals, such as naked mole rats and some caecilians (Tucker, 1981;

Herrel and Measey, 2010), it is possible that loose skin might have evolved for a similar function

in hagfish as they burrow into muddy seafloors and into carrion fall to feed. The defensive properties of the loose, flaccid skin may therefore be an example of exaptation.

Conclusion

Hagfish appear to avoid deep (subcutaneous) penetrating wounds due to their skin being only loosely adhered to the underlying musculature, and having a flaccid body morphology.

Hagfish skin itself is no more puncture resistant than that of other fish species (Stiles et al., unpublished). This unique combination of features permits the underlying body (including musculature and viscera) to be displaced out of the way of a penetrating tooth. The loose and flaccid skin also increases the time it takes to reach the force of puncture of the skin, which may minimize damage that can be done before the defensive slime can induce the predator to abandon an attack. Using an unattached, loose approach to protective equipment design may inspire the production of layered fabrics and personal protective equipment that can help prevent damage from penetrating objects, which could then be used in such fields as medicine, military, marine biology, and field research.

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