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Effects of acute salinity and temperature change on Pacific hagfish, Eptatretus stoutii; implications… Hastey, John Pinkerton 2011

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EFFECTS OF ACUTE SALINITY AND TEMPERATURE CHANGE ON PACIFIC HAGFISH, EPTATRETUS STOUTII; IMPLICATIONS FOR BYCATCH POST RELEASE SURVIVAL by John Pinkerton Hastey B.Sc.(Hons)., Acadia University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (ANIMAL SCIENCE)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2011 © John Pinkerton Hastey, 2011  Abstract Hagfish captured as bycatch in a commercial fishery may be exposed to a range of elevated temperatures and reduced salinities when brought to the surface but it is not known how this will affect post-release survival. In this study, hagfish were exposed to all combinations of four salinities (33, 28, 23, and 18 g/l) and three temperatures (7, 16 and 25˚C) and sampled following 1, 3 or 6 h exposure to investigate sub-lethal affects on plasma osmolality, [Na+], [Cl-], [Mg2+], and glucose, hematocrit, mean cell haemoglobin concentration , muscle water content , as well as behavioural responses and survival. An additional group of hagfish were sampled after 48 h of recovery (33 g/l at 7˚C) following 3 h exposure to all salinity/temperature combinations to investigate latent effects of exposures. In general, during exposure to salinity and temperature combinations, plasma osmolality, [Na+] and [Cl-] decreased as: i) the salinity of exposure was reduced, ii) the duration of exposure was increased or iii) the temperature to which hagfish were exposed was increased. Plasma osmolality did not equilibrate with environmental osmolality within 6 h and appeared to approach an asymptote at 60% equilibration. Behavioural effects were observed during exposure to reduced salinity conditions at all temperatures and included full body contractions, reduced slime production, reduced swimming ability, extended body posture. During exposure to 25˚C, hagfish were often unresponsiveness to touch. Following 48 h of recovery from 3 h exposure to all salinity/temperature combinations most of the parameters measured were restored, with the exception of plasma glucose levels which remained elevated indicating latent stress. Following recovery from exposure to 23 and 18 g/l at 25˚C, morbidity levels of 14 and 100% were observed. Thus, hagfish captured and immediately brought to the surface and released, without exposure to extreme salinity or temperatures, may not physiologically be negatively affected by a ii  catch and release fishery however, diminished survival should be expected if exposure salinity approaches 18 g/l or water temperature approaches 25˚C. Future research should address the implications of observed behaviours on release survival of hagfish in a natural setting.  iii  Table of Contents Abstract .................................................................................................................... ii Table of Contents ................................................................................................... iv List of Tables .......................................................................................................... vi List of Figures ........................................................................................................ vii List of Abbreviations ............................................................................................. ix Acknowledgements................................................................................................. xi Chapter 1: Introduction and Thesis Objectives ....................................................1 Introduction .............................................................................................................1 The Commercial Fishery ........................................................................................2 Catch and Release Mortality ...................................................................................3 Environmental Stress ..............................................................................................4 Ion and Water Balance in the Marine Environment ...............................................5 Thermal Stress ........................................................................................................8 Thesis Objectives ..................................................................................................11 Chapter 2: Effects of Acute Exposure to a Range of Salinity and Temperatures on Pacific hagfish (Eptatretus stoutii) ..........................................12 Synopsis ................................................................................................................12 Introduction ...........................................................................................................13 Materials and Methods..........................................................................................15 Results ...................................................................................................................19 Discussion .............................................................................................................21 Chapter 3: Conclusion ...........................................................................................42 Catch and Release Mortality .................................................................................42 Study Limitations ..................................................................................................44 Hagfish Husbandry ...............................................................................................45 Future Direction ....................................................................................................46 References ...............................................................................................................49 iv  Appendix .................................................................................................................56 Appendix A ...........................................................................................................56  v  List of Tables  Table 2.1 Linear regression results for the relationship between seawater and hagfish plasma osmolality at 1, 3 or 6 h in hagfish following exposure to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C. Data is plotted in Figure 2.6……….……………………...……………..38  Table 2.2. Behaviour displayed by hagfish when exposed to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C for 1, 3 or 6 h. The minimum exposure time that a behaviour was observed is indicated within ( ), the number referring to hours. Each behavioural response listed was the dominant behaviour(s) displayed by the entire experimental group (n=7). Behaviours are listed in the order they appeared. FBC full body contractions, RSP reduced slime production, NSP no slime produced, EBP extended body posture, RSA reduced swimming ability, UN unresponsive to touch. ……………………………………………...………………….………..39  Table 2.3. Plasma glucose and hematological measurements in hagfish exposed to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C for up to 6 h. No significant differences were found between 1, 3 and 6 h exposures within a given salinity and temperature, so data for all times were pooled to yield a single value. Values represent mean ± s.e.m.. A significant difference from the control (33 g/l at 7˚C) is indicated by “ * ” and was determined using a 2way ANOVA (p<0.05). Sample size was n=21 for each salinity condition unless otherwise indicated by (n). …………………………………...…………..………………………….……..40  Table 2.4. Plasma glucose, ion, muscle water, hematological and morbidity measurements in hagfish exposed to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C for 3 h and followed by a 48 h recovery before sampling. Values represent mean ± s.e.m.. A significant difference from the control (33 g/l at 7˚C) is indicated by “ * “ and was determined using 2-way ANOVA (p<0.05, n=7)…………………………………………………………………….…….41  vi  List of Figures Figure 2.1. Plasma osmolality (mOsm/l) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05)………………………………………………………………………………………….29  Figure 2.2. Plasma [Na+] (mM) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05)………………………………………………………………………………….………30  Figure 2.3. Plasma [Cl-] (mM) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05)……………………………………………………………..………………………...….31  Figure 2.4. Plasma [Mg2+] (mM) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05)……………………………………………………………………….…………….……32  Figure 2.5. Muscle water content (%) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature (p<0.05)…………….......33 vii  Figure 2.6. Relationship between seawater and hagfish plasma osmolality at 1 ( ), 3 ( ) or 6 ( ) h following exposure to a range of salinities (33, 28, 23 or 18g/l) at 7(a), 16(b) or 25(c) ˚C. A 6 h exposure was not completed at 25˚C. The dotted line represents the line of unity for reference. See Table 2.1 for the slope and R2 values of the regression lines……………….……34  Figure 2.7. Plasma osmolality (mOsm/l) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) for 3 h at 7 (a), 16 (b) and 25 (c)˚C and allowed to recover for 48 h at 33 g/l and 7˚C. Values reported at 3 h are presented for reference and are from Figure 2.1. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 3 h exposure value within a given salinity treatment (p<0.05)…………………………………………………………………………….…………….35  Figure 2.8. Hematocrit (%) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) for 3 h at 7 (a), 16 (b) and 25 (c)˚C and allowed to recover for 48 h at 33 g/l and 7˚C. Values reported at 3 h are presented for reference and are were sampled previous to this trial following a 3 h exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. (p<0.05)…………………………………………………………………………………………..36  Figure 2.9. MCHC (g/dl) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) for 3 h at 7 (a), 16 (b) and 25 (c)˚C and allowed to recover for 48 h at 33 g/l and 7˚C. Values reported at 3 h are presented for reference and are were sampled previous to this trial following a 3 h exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 3 h exposure value within a given salinity treatment (p<0.05)……………………………………………………………………………..……………37  Figure A1. Critical thermal limit of hagfish when acclimated to 4, 8 and 14˚C before exposure to increasing temperature conditions of 0.45˚C/min. The thermal limit was reached when hagfish were no longer responsiveness to touch. Values are mean ± s.e.m. (n=6). Letters that differ indicate statically significant differences among acclimation groups (p<0.05)…………………57  viii  List of Abbreviations ANOVA  analysis of variance  ATPase  adenosine triphosphatase  ˚C  degree Celsius  cm  centimeter  Cl-  chloride  [ ]  concentration  EBP  extended body posture  FBC  full body contractions  g  gram  g/dl  gram per deciliter  g/l  gram per liter  Hb  haemoglobin  Hct  hematocrit  h  hour  K+  potassium  l  liter  Mg2+  magnesium  MCHC  mean corpuscular haemoglobin concentration  m  meter  mM  millimolar  mOsm/l  milliosmoles  Na+  sodium  NSP  no slime produced  ix  %  percent  RBC  red blood cell  RSP  reduced slime production  RSA  reduced swimming ability  s.e.m.  standard error of the mean  UN  unresponsive to touch  x  Acknowledgements  To Scott. Thank you for providing me with an opportunity to complete a Masters project at UBC. I could never have imagined the diversity of presented opportunities, as well as the range of people I would encounter on this journey. This has truly been a voyage from which I have gained not only research experiences, but more importantly life experiences. You enabled opportunities, which allowed me to prove myself in situations where success was seemly impossible. It has built a confidence within me that will last my lifetime, and for that I am grateful. Colin, being a welcomed part of your lab broadened my UBC community and introduced me to the wonders of physiology. Your support throughout this thesis has been invaluable and a true inspiration for me to see this through to the end. Thank you! Thank you to Janice, Leo and Carl at CAER who were always ready to help all along the way. A special thanks to William Wong for always being ready to lend a hand. I have a strong respect for your organization, dedication and work ethic. Thanks to Aimee and all the Hastey’s for their continued love and support, especially little Rope, who sacrificed two weeks of his summer to come out west and work for his big bro.  xi  Chapter 1: Introduction and Thesis Objectives Introduction Pacific hagfish Eptatretus stoutii (Lockington 1878) are primitive, jawless, benthic marine craniates belonging to the class Agnatha (Martini et al., 1997). Pacific hagfish are found in areas of both soft bottom and rocky substrate at depths of 18-1000m (Leask and Beamish, 1999; Martini, 1998) where temperature does not rise above 10˚C and the salinity is quite stable between 31-33 g/l (McInerny and Evans, 1970). Hagfish are scavengers of the deep, feeding on moribund animals or benthic invertebrates and likely play a significant role in nutrient cycling (Lesser et al., 1997; Martini et al., 1997). They are osmoconformers (Cholette et al., 1970; McFarland and Munz, 1958; Morris, 1965) and thus have a limited tolerance to low salinity (Martini, 1998; Strahan, 1962; Worthington, 1905) and may be susceptible to rapid temperature changes because they live in a stable temperature environment (Gustafson, 1935; Palmgren, 1927; Robertson, 1963). Commercial fisheries exists for hagfish, however, there is a size limit below which they must be released back into the environment. Current fishery practices may result in hagfish being exposed to high temperature and low salinity surface waters and it is not known what the implications of these conditions are for subsequent release of the bycatch (Grant, 2006; Martini, 1998). While there has been a great deal of interest in some aspects of the basic biology and physiology of hagfish to evolutionary physiologists, largely because of its phylogenetic position, there is insufficient knowledge on how relatively short duration exposure to changes in salinity and temperature will affect the physiology and ultimate survival of hagfish. The objective of this thesis was to investigate the effects of acute exposure to a change in environmental parameters 1  on the physiological status of hagfish as well as assessing their recovery ability, post exposure, from experimental conditions. The following is a brief discussion of the commercial fishery for hagfish and the interaction of ion and water balance, thermal stress and the potential effects on the release survival of hagfish, post capture in a commercial fishery.  The Commercial Fishery Recently, an international market has developed for hagfish products and as a result, there is a renewed interest in development of a fishery on the west coast of Canada (Leask and Beamish, 1999). Currently there is an experimental fishery for Atlantic hagfish (Myxine glutinosa) on the east coast of Canada (Grant, 2006) and both the east and west coast of the USA (Brass, 1993). The largest portion of the market is driven by Korea’s demand for hagfish skin as a fine leather product, and its flesh as a food product (Brass, 1993). Despite the development of a commercial fishery for hagfish, significant knowledge gaps exist in their general biology and ecology. These gaps include maximum age, age at maturity, growth rate and spawning behaviour, all of which are required to establish a sustainable fishery. Capturing hagfish in a fishery involves setting a long line of baited traps on the seafloor with a bottom time of 12 to 24 h. Typically, the fishery targets depths of 100-150 m where the summer bottom temperatures off the west coast of Vancouver Island are 7-8˚C and salinity 31-33 g/l, compared to surface water temperatures of 16-18˚C and air temperatures ranging from 1530˚C (Davis and Olla, 2001; Olla et al., 1998). On deck the traps are emptied and hagfish are size sorted with under sized individuals being released, descending back through the thermocline to the seafloor.  2  The experimental fishery in British Columbia 1988-92 was initially conducted using cylindrical Korean style traps with an inverted cone entrance at one end (Leask and Beamish, 1999). All traps require 3cm escape holes in an attempt to reduce the catch rate of undersized individuals but the effectiveness of this protocol has yet to be quantified. During this experimental fishery, the average capture weight was 110 g and mean length from 30.3 cm to 48.3 cm (Leask and Beamish, 1999). The minimum size of hagfish acceptable for the market is 30 cm, equal to the mean capture length of the B.C. experimental fishery. This creates a potential situation where a significant portion of the haul could be undersized individuals (< 30cm) that would be discarded as bycatch at the time of capture. The bycatch of undersized hagfish on the east coast can reach 20% of the total catch which is released, however, nothing is known about their subsequent survival (Grant, 2006).  Catch and Release Mortality Exposure to low salinity and elevated temperature of surface water during capture and subsequent release of undersized hagfish was thought to go beyond their range of tolerance, resulting in high mortality (Adam and Strahan, 1963; Martini, 1998) but data directly supporting this theory is very limited (Gustafson, 1935; McFarland and Munz, 1958; McFarland and Munz, 1965; Palmgren, 1927; Strahan, 1962). The probability of survival following catch and release in a commercial fishery is dependent on the interaction of stressors, environmental and biological (Rummer, 2007). Davis (2002) has stressed the importance of conducting controlled laboratory experiments to understand the principals controlling discard mortality to be correlated with behavioural and physiological measures to create mortality assays. Bycatch stressors include but are not limited 3  to, capture and gear type, light conditions, temperature fluctuations, anoxia, sea conditions, air exposure and handling. Survival of fish captured in a commercial fishery are reduced as the number of acting stressors increases (Davis, 2002; Olla et al., 1998). Predation post capture is an important factor when looking at release mortality but difficult to quantify. The stressors associated with capture often lead to behavioural modifications that reduce predator avoidance capabilities (Davis and Parker, 2004; Olla et al., 1995). Hagfish captured in a commercial fishery are exposed to a range of changing environmental conditions including temperature and salinity, as they are hauled to the surface where they are exposed to elevated air temperature and handling stress, a scenario shown to produce high mortality in other fisheries (Davis and Olla, 2001; Davis et al., 2001; Olla et al., 1998). The characterization of a hagfish response to environmental variables and stress is an important aspect in managing catch and release mortality, promoting a more sustainable fishery.  Environmental Stress Stress can be defined as a change in the environment which upsets the normal physiological balance, potentially negatively impacting an individual’s performance (Barton et al., 2002). When an individual is exposed to environmental stress, a primary, and if the stressors continue, a secondary response is initiated. A primary stress response involves neuroendocrine pathways where a secondary stress response involves a change or disruption of physiological processes requiring compensation, presumably at an increased energetic cost (Barton et al., 2002). If stressful conditions persist, sub-lethal and in extreme cases, lethal responses are observed unless a sustainable balance can be achieved between energy required for physiological 4  compensation and routine metabolic demands. The parameters related to stress investigated in this thesis were the degree of physiological change in hagfish resulting from acute exposure to the environmental stressors of reduced salinity and elevated temperature, in isolation and in combination.  Ion and Water Balance in the Marine Environment Marine fish constantly face water stress due to the high salt content of seawater requiring strategies to actively or passively cope with water and ion balance between the extracellular compartment and the external environment. A delicate balance exists between the extracellular and intracellular compartment, important to all cellular functions (Somero and Yancey, 1997). The osmoregulatory strategy of a fish largely determines its ability to tolerate a change in external osmolyte concentration. The most common strategy among marine fish is osmoregulation, where extracellular osmolality is maintained within a narrow range, largely independent from the external environment. Teleosts manage a balance between passive ion gain and water loss through active water uptake across the gut, and ion excretion at the gills. Active processes in the gut transcellularly take up Na+ and Cl- into the extracellular fluid, establishing an osmotic gradient drawing water paracellularly into the extracellular fluid (Grosell, 2006). The excess solutes in the blood accumulated at the gut are removed at the gills to maintain ion balance. Cl- is actively excreted transcellulary by Cl- channels in mitochondria-rich cells on the apical membrane of gills and driven by Na+/K+ ATPase and Na+,K+, 2Cl- cotransporter. Na+ is removed paracellularly by a transepithelial membrane potential across the gill epithelium (Evans et al.,  5  2005). Although, there is a cost to osmoregulation, the benefit is that an animal can inhabit a range of environmental salinities while maintaining a relatively stable internal environment. In contrast to osmoregulators, osmoconformers have a blood osmolality that is isosmotic with the external environment. Any change in the external salinity will cause a resulting shift in the osmolality of the extracellular compartment. Hagfish and elasmobranchs are both osmoconformers, however, their strategy of osmolyte management differs greatly. Elasmobranchs replace a portion of blood Na+ and Cl- with urea, an organic metabolic byproduct. Urea is normally a perturbing solute that disrupts macromolecule function, the effects of which are offset by also elevating methylamines, a group of counteracting organic osmolytes such as trimethylamine oxide (TMAO), glycine betaine and carcosine (Yancey et al., 1982). Elasmobranchs rely on a similar process for Na+ and Cl- excretion as described above for osmoregulators but the excretory organ, known as the rectal glad, is located in the intestine (Evans et al., 2005). Thus, in elasmobranchs, there must be a cost associated with ionoregulation, but because they are isoosmotic, they are in equilibrium with their environment in terms of water balance. In contrast to sharks, hagfish rely on inorganic osmolytes, predominantly Na+ and Cl- as solutes in the extracellular fluid (Robertson, 1976; Robertson, 1986). Hagfish lack the ability to regulate plasma [Na+] and [Cl-] and consequently levels are only slightly different than seawater (McFarland and Munz, 1965; Sardella et al., 2009; Smith, 1932). Although hagfish are osmoconformers, they do appear to have the ability to regulate plasma [Mg2+ ], [Ca2+ ] , [SO42+ ] different from the external environment, but the specific mechanisms are unknown. The divalent cations are likely regulated due to effects on nervous function and muscle contraction when elevated in concentration but the extent to which these cations can be regulated is unknown.  6  Generally, cell volume is regulated by manipulating the concentrations of intracellular organic osmolytes in response to a change in osmolality of the extracellular fluid. Intracellular organic osmolytes such as amino acids tend to be compatible with biochemical processes at a wide range of concentrations in contrast to inorganic osmolytes which, above normal intracellular concentration, have a perturbing effect on the functional properties of proteins (Somero and Yancey, 1997). Hagfish make use of amino acids to regulate intracellular osmolality (Cholette and Gagnon, 1973; Cholette et al., 1970). As the extracellular osmotic gradient becomes hyper or hypo-osmotic relative to the intracellular milieu, concentrations of amino acids, predominantly proline, are modified to compensate to some degree. Amino acids account for up to 46% of the cellular osmolytes, the remainder being of inorganic composition (Brodal and Fange, 1963; Cholette et al., 1970). Even with some ability to regulate intracellular osmolyte levels, muscle water content is directly influenced by changes in extracellular osmolality indicating that intracellular water balance is incomplete (Cholette and Gagnon, 1973; Sardella et al., 2009). Given that hagfish are osmoconformers, it is likely that their salinity tolerance is low and is a limiting factor determining species distribution (Adam and Strahan, 1963; Gustafson, 1935; Martini, 1998) but surprisingly few studies have been conducted to investigate their salinity tolerance. When M. glutinosa is exposed to salinities below 20 g/l, individuals will struggle violently and become morbid within 5 h (Gustafson, 1935) but they can be maintained at 29 g/l for months (Adam and Strahan, 1963; Gustafson, 1935). E. stoutii survives exposure to 27 g/l seawater for 7 days but cannot survive salinity at 20-25 g/l (McFarland and Munz, 1965) while E. atami experiences 100% mortality during a 30 h exposure to 27 g/l salinity (Strahan, 1963). Hagfish likely do not experience dramatic fluctuations in their natural environment because they 7  live in the stable full strength salinity of the ocean floor but exposure to surface waters that vary in salinity is likely to occur during their capture in a fishery. There is limited data to infer the effect of short duration exposure to low salinities on subsequent survival in hagfish which is required for managing the hagfish fishery.  Thermal Stress Temperature affects almost every aspect of cellular and whole animal function, and consequently species have evolved a myriad of physiological and behavioural strategies to deal with temperature enabling organism to inhabit virtually all thermal niches on the planet. There are high physiological costs associated with changes in internal temperatures and consequently, temperature is one of the dominant controlling factors limiting species distribution and how they interact with the environment (Fry and Randall, 1971; Hochachka and Somero, 2002). Physiologically, temperature influences chemical interactions affecting biochemical reactions important for the proper functioning of organisms, specifically fish for this discussion. Beginning with the cardiovascular system, a change of an ectotherms internal temperature can lead to large changes in heart and ventilation rates, reflective of the new metabolic demands imposed by the given temperature modification (Huey and Kingsolver, 1993). During heating, metabolic rate typically doubles or triples for every 10˚C change in temperature, referred to as the Q10 effect. The enhancement of biochemical processes at elevated temperature is due to the increase in kinetic energy of the molecules, increasing the likelihood of exceeding the activation energy required for a chemical reaction to proceed (Huey and Kingsolver, 1993). The increase in metabolic rate during thermal stress also leads to an increased oxygen demand and production of metabolic wastes, increasing the demand on the cardiovascular system. 8  At some point, an increase in temperature has negative effects. Proteins and enzymes can lose functionality during thermal stress due to the physical effects of temperature on protein structure. To counteract the denaturing or malfunctioning of proteins and enzymes, cells produce a heat shock protein (Hsp) which binds to and corrects denatured proteins, potentially returning some functionality. The hydrophobic effect, ionic interactions, hydrogen bonds and van der Waals interaction are other protein stabilizing forces to maintain function during thermal stress (Hochachka and Somero, 2002) all of which are associated with some metabolic cost. The membrane is the most sensitive component of a cell to thermal stress due to its lipid composition and complex role as a physical barrier as well as being the site of cellular regulatory and transmission processes (Hochachka and Somero, 2002). Cellular signaling through synaptic transmission is the most sensitive process of the cell membrane to thermal stress. The temperature induced change to membrane lipids causes a disruption of the membrane based ion flux and the fabrication of signal molecules leading to a breakdown in cellular signaling known as synaptic block (Hochachka and Somero, 2002). Cossins et al., (1977) demonstrated the breakdown of the synaptic pathway in goldfish following acute exposure to high temperatures which caused behaviour modifications related to synaptic block. Cold adapted species of Antarctic fishes cannot survive direct transfers to temperatures of 4˚C and when exposed to 10˚C the fish lose equilibrium, swim rapidly in short bursts and become uncoordinated (Hofmann et al., 2000; Somero and DeVires, 1967). Heat death is caused in Antarctic fishes first by the rapid release of the neurotransmitter acetylcholine, followed by the breakdown of an enzyme responsible for degrading acetylcholine and preventing neural function (Baldwin, 1971). The break down in synapse transmission during elevated temperature exposure may be involved in setting thermal tolerances limits for species (Bowler and Manning, 1994). 9  Thermal stress can also upset the water ion balance by modifying gill permeability and transporter function. When exposed directly to an upper thermal limit, fish experience a water and ion flux greater than would be experienced if the fish were exposed to their acclimation temperature (Motais and Isaia, 1973). An important driver for this process during high thermal stress is directly related to metabolism, bringing a greater amount of blood into contact with the gills. Experiments demonstrating temperature dependent diffusion and transport with Goldfish (Carassius auratus) and flounder (Platichthys flesus) found passive Na+ flux was less temperature dependent with a Q10=2 than the active Na+/K+ transport with a Q10=3-6 (Crawshaw, 1979; Maetz and Evans, 1972). The temperature driven imbalance between passive flux and active transport of solutes can disrupt the internal osmotic balance, further impacting intracellular processes and increasing the effects of thermal stress. In their natural habitat, Pacific hagfish are a homeothermic ectotherm, not documented at locations where temperature rises above 10˚C (McInerny and Evans, 1970). They live in a consistent thermal environment and have no control over internal temperatures. What is known of hagfish temperature tolerance is based on observations of holding conditions in captivity (Adam and Strahan, 1963; Gustafson, 1935; Palmgren, 1927; Worthington, 1905). M. glutinosa found in the eastern Atlantic, has be maintained for extended periods at 0-4˚C (Martini, 1998) and has been held at 10˚C (Gustafson, 1935) while tolerating brief exposure to 15˚C (Palmgren, 1927). The only known migratory population of hagfish, E. burgeri, found in Koajiro Bay, Japan has an inshore migration when water temperatures fall below 21˚C (Fernholm, 1974). E. stoutii was reported to tolerate brief exposures to 30˚C when previously acclimated to 22˚C (Worthington, 1905). Relating to the depth of habitat and associated temperatures, Eptatretus have a wider range of tolerance to elevated temperature than Myxine species when held in 10  aquaria (Ota and Kuratani, 2006). The range of temperatures shown to support hagfish in a lab have only accounted for short term survival following acclimation and the latent effects of increased metabolic demand and perturbing effects of thermal stress on protein function following direct transfers has not been considered.  Thesis Objectives Little is known about the ultimate survival of the bycatch of undersized hagfish generated by the commercial fishery but salinity and temperature are likely two of the most important variables to investigate. The objectives of my thesis are: 1) To assess the physiological and behavioural effects on Pacific hagfish of an acute exposure to a combination of salinity and temperatures relevant to their catch and release in a fishery. Hagfish were directly transferred to salinities of 33, 28, 23 or 18 g/l at temperatures of 7, 16 or 25˚C, and then terminally sampled at1, 3 or 6 h. Plasma osmolality, ions (Na+, Cl-, Mg2+), glucose, hematocrit, haemoglobin and muscle water content were measured to quantify the effect. 2) To assess the extent of recovery within 48 h following a 3 h exposure to all combinations of salinity and temperature mentioned in objective 1, providing insight into the potential for delayed release mortality. 3) To discuss the findings as they pertain to the catch and release mortality of hagfish in a commercial fishery.  11  Chapter 2: Effects of Acute Exposure to a Range of Salinity and Temperatures on Pacific hagfish (Eptatretus stoutii) Synopsis In this study, hagfish were exposed to all combinations of four salinities (33, 28, 23, and 18 g/l) and three temperatures (7, 16 and 25˚C) and sampled following 1, 3 or 6 h exposure to investigate sub-lethal affects on plasma osmolality, [Na+], [Cl-], [Mg2+], and glucose, hematocrit, mean cell haemogloin concentration , muscle water content, as well as behavioural responses and survival. An additional group of hagfish were sampled after 48 h of recovery (33 g/l and 7˚C) from a 3 h exposure to all combinations of salinities and temperatures to investigate latent effects of exposures. Significant physiological effects were detected following a 1 h exposure to reduced salinity (≤ 28 g/l) and temperature combinations with increasing effects as exposure time was increased, salinity was reduced and temperature was increased. Following 48 h of recovery from 3 h exposure to all salinity/temperature combinations most of the parameters measured were restored, with the exception of plasma glucose levels which remained elevated indicating latent stress. Following recovery from exposure to 23 and 18 g/l at 25˚C, morbidity levels of 14 and 100% were observed. Hagfish captured and quickly brought to the surface and immediately released, without large deviations from acclimation salinity and temperature, may not be negatively affected by a catch and release fishery however, induced behavioural changes such as reduced slime production and swimming ability may lead to reduced survival. Low survival is likely if hagfish are exposed to salinity approaching 18 g/l or water temperature approaching 25˚C. These conditions were shown to cause latent stress and/or morbidity.  12  Introduction Pacific Hagfish, Eptatretus stoutii (Lockington 1878) have long been studied by evolutionary biologists who have added greatly to knowledge of their physiology and biochemistry but the general biology and ecology has remained largely unstudied. The resulting knowledge gap has left fisheries managers without the basic data required to manage a fishery. The objective of this thesis was to investigate how relatively short duration exposure to changes in salinity and temperature will affect the physiology and ultimate survival of hagfish. Capturing hagfish in a fishery involves setting a long line of baited traps on the seafloor typically targeting depths of 100-150 m (Grant, 2006) where temperature does not rise above 10˚C and the salinity remains stable between 31-33 g/l (McInerny and Evans, 1970). During capture hagfish are exposed to high temperature and low salinity surface waters before being sorted on the ship deck and undersized individuals are discarded as bycatch, however, nothing is known about their subsequent survival. The probability of survival following catch and release in a commercial fishery is dependent on the interaction of stressors, environmental and biological (Rummer, 2007). A better understanding of the effects of acute exposure to a range of temperature and reduced salinity conditions will aid in the development of sustainable management plan for the commercial fishery. The little that is known about hagfish temperature tolerance is based on observations of holding conditions which have sustained hagfish in captivity (Adam and Strahan, 1963; Gustafson, 1935; Palmgren, 1927; Worthington, 1905). M. glutinosa found in the eastern Atlantic, can be maintained for extended periods at 0-4˚C (Martini, 1998) and have been held at 10˚C (Gustafson, 1935) while tolerating brief exposure to 15˚C (Palmgren, 1927). E. stoutii was 13  reported to tolerate brief exposures to 30˚C when maintained at 22˚C (Worthington, 1905) but in the wild are only documented at locations where temperature does not rise above 10˚C (McInerny and Evans, 1970). The stable temperature of hagfish habitat is thought to make them particularly vulnerable to rapid temperature changes but there is limited data to suggest what the effects may be (Gustafson, 1935; Palmgren, 1927; Robertson, 1963). The physiological implication of salinity challenges in hagfish has received some attention (Cholette et al., 1970; McFarland and Munz, 1965) but surprisingly few studies have investigate their salinity tolerance. Species E. stoutii can survive exposure to 27 g/l seawater for 7 days but does not survive sustained exposure to 20-25 g/l (McFarland and Munz, 1965) while E. atami are moribund within a 30 h exposure to 27 g/l (Strahan, 1963). M. glutinosa can be maintained for months at 29 g/l but struggle violently and become mobid when exposed for 5 h to salinities below 20 g/l (Gustafson, 1935). Due to limited data many questions still exist about the effect of short duration exposure to low salinities on subsequent survival in hagfish important for the fishery to be managed sustainability. As osmoconformers, fluctuating salinity of the external environment has a rapid physiological impact on hagfish (McFarland and Munz, 1958; McFarland and Munz, 1965). Even though hagfish are osmoconformers they have fairly constant water and ion levels due to the stable salinity and temperature regime of their environment. A change in intracellular osmolyte concentration, particularly inorganic, as well as temperature, effect every cellular process, specifically water balance and volume regulation, membrane processes, enzyme, protein and synapse function which together greatly impact their overall performance (Hochachka and Somero, 2002; Huey and Stevenson, 1979; Somero, 2004; Somero and Yancey, 1997; Yancey, 2005). 14  The objectives of this study were 1) to assess the physiological and behavioural effects on Pacific hagfish of acute exposure to a combination of salinity and temperatures relevant to their catch and release in a fishery, 2) to assess the extent of recovery within 48 h following a 3 h exposure, providing insight into the potential for delayed release mortality and 3) to discuss the findings as they pertain to the catch and release mortality of hagfish in a commercial fishery.  Materials and Methods  Experimental Animals Hagfish (48 cm ± 0.25) were captured off the west coast of Vancouver Island, BC Canada in June, 2008 and held at the West Vancouver Laboratories at the Department of Fisheries and Oceans, Centre for Aquaculture and Environmental Research in 4000 L tanks serviced with flow-through sand filtered sea water. Temperature and salinity of the flow-through seawater ranged from 32-33 g/l and 9-13˚C. Hagfish were fed dead rainbow trout once a week to satiation but feeding was withheld two weeks prior to experiments. A total of 308 hagfish were used in this experiment which was performed according to University of British Columbia animal care protocol #A08-0368.  Experimental Design Hagfish were exposed to all combinations of the following salinities and temperatures and terminally sampled at 1, 3 or 6 h (n=7). Salinities investigated were 33, 28, 23 and 18 g/l with 18 g/l representing the minimum expected surface salinity. Temperatures investigated were 7.0 ± 0.39, 15.6 ± 0.61 and 24.6˚C ± 0.49 (referred to as nominal values of 7, 16 and 25˚C from this point forward) where 25˚C was chosen as the upper limit based on previous critical thermal 15  maximum trials reported in Appendix A. Exposure times of 1, 3 and 6 h were chosen to embody the range of possible exposure times to surface conditions prior to release as bycatch. A 150L fiberglass tank was used for each salinity/temperature experimental trial. Diluted seawater was created by mixing full strength seawater (33 g/l) with fresh well water until the desired salinity was obtained. Premixed, temperature adjusted seawater was continuously supplied to the experimental tank to maintain stable conditions. Salinity was measured using a refractometer (VeeGee, STX-3, error ± 2%). Temperature was measured using an OxyProbe (OxyGuard, Handy Polaris).  Transfer Protocol Hagfish were acclimated to 7˚C ± 0.40 and 33 g/l seawater in flow through conditions, for a minimum of two weeks prior to experiments. 24 h prior to experimentations individual hagfish were transferred underwater (to prevent air exposure), into a 17 cm x 28 cm x 8 cm plastic tupperware container. Each container had a 4 cm x 17 cm hole cut on the long side and two 6 cm x 12 cm holes cut in the lid, each covered with black 1 cm diameter aquaculture screen to ensure water flow through the experimental chamber. When a container was lifted from the acclimation tank, 2.5 cm of water remained in the bottom, preventing air exposure and minimizing hagfish disturbance during transfer to the experimental tank. The majority of hagfish remained in coiled while the chamber was being transferred. Upon initiation of an experimental salinity/temperature trial, a total of 21 containers (each housing a single hagfish to be sampled at 1, 3 or 6 h (n=7) were placed into the 150 L experimental tank containing the preset salinity/temperature combination. Introduction of the containers was staggered by 3 minutes to allow for sufficient time to sample all 7 hagfish at each 16  pre-determined exposure duration. The above protocol was completed separately for each of the salinity/temperature combinations with behavioural observations (see below) made throughout the duration of exposure. The 6 h exposure to 23 and 18 g/l at 25˚C were not completed due to imminent morbidity.  Recovery Trial Recovery trials were completed using the same methods outlined above for hagfish exposed to all salinity and temperature combinations for 3 h, following which hagfish were returned to acclimation conditions (33 g/l, 7˚C) for 48 h before sampling (n=7). The 28 and 23 g/l salinity trials at 7˚C were not competed for the recovery experiments due to a restricted availability of animals.  Sampling Procedure Following exposure to a given salinity and temperature combination for the predetermined time, one container was removed from the experimental tank drained of excess water and the hagfish removed. Length was recorded and blood was withdrawn from the caudal sinus into a 1 ml heparinized syringe. The blood was expelled into three capillary tubes for measurement of hematocrit and the remaining whole blood was expelled into a 1.5 ml eppendorf tube. A 10 µl aliquot of blood was added to Drabkin’s reagent for measurement of haemoglobin concentration (Dacie and Lewis, 1968), and the remaining blood was centrifuged at 3000 rpm for 5 minutes and plasma was removed and stored at -80˚C for later analysis of plasma [Na+], [Cl-], [Mg2+] and glucose. Following blood collection, hagfish were decapitated and a 1.0 g muscle sample was removed from the dorsal epaxial muscle. The muscle was dried at 65˚C for 96 h, or until no further weight change was measured and muscle water content was calculated. 17  Blood Analyses Haemoglobin was measured spectrophotometrically, and mean corpuscular haemoglobin concentration (MCHC) was calculated as haemoglobin x 100/hematocrit. Glucose was analyzed spectrophotometrically using a Cayman Chemical assay kit (cat no.10009582). Plasma osmolality was measured using a Wescor 5500 Vapor Pressure Osmometer (Wescor, Logan, UT). Plasma [Na+] and magnesium was measured using an atomic absorption spectrophotometer (Spectra AA-220FS; Varian, Mulgrave, VC, Australia) and plasma chloride was measured using an HBI digital chloridometer.  Behaviour Observations Behaviours were recorded when displayed by the majority of individuals within a given salinity and temperature combination at a given duration of exposure. Hagfish under control conditions remained quiescent and coiled with minimal movements; behaviours observed in the wild (McInerny and Evans, 1970). Multiple behaviours were noticed for some exposure times and were recorded in the order they were observed. The most common behaviour observed was full body contractions (FBC) which was displayed every few minutes throughout a trial and consisted of sporadic body convulsions lasting anywhere from a few seconds, up to 4 minutes. The second most common behaviour was the reduction in slime production (RSP). This was a subjective measure characterized by a ≥ 50% reduction in slime production capacity when hagfish were removed from the experimental container prior to sampling. When no slime was detected the observation no slime produced (NSP) was recorded. Extended body posture (EBP) refers to the change from the coiled position to the straight and extended body posture. The reduced swimming ability (RSA) was characterized by a reduction in the sinusoidal body 18  movement when stimulated to swim by hand prodding at the time of sampling. If hagfish did not respond with movement when handled they were deemed unresponsive (UN).  Statistical Analyses The results are expressed as means ± s.e.m.. Analysis was completed using a two-way ANOVA followed by a Holm-Sidak post-hoc test when significance was found using p<0.05. All statistics were performed using Sigmaplot software (Version 11).  Results  Plasma Ions At 7˚C, within 1 h exposure to the different salinities there was a significant reduction in plasma osmolality (Fig 2.1A), [Na+] (Fig 2.2A) and [Cl-] (Fig. 2.3A) at 18 g/l relative to 33 g/l, with values at all salinities differing from one another at 3 and 6 h exposure (except 18 vs 23 g/l at 3 h for [Cl-] which did not differ). At 16˚C, this pattern was exaggerated with plasma osmolality (Fig 2.1B), [Na+] (Fig 2.2B) and [Cl-] (Fig. 2.3B) significantly different between 18 and 23g/l relative to 33g/l at 1 h, and all values differing from one another at 3 and 6 h. At 25˚C, plasma osmolality (Fig 2.1C) differed among all salinity exposures at 1, 3 and 6 h, however, due to morbidity prior to 6 h exposure, no values were obtained at 18 and 23 g/l. Similar changes were observed for plasma [Na+] (Fig 2.2C; except values at 28 vs 33 g/l at 1h and 23 vs 28 g/l at 3 h did not differ) and [Cl-] (Fig 2.3C; except values at 18 vs 23 g/l and 28 vs 33 g/l at 1 h and 18 vs 23 g/l at 3 h did not differ). At 7˚C (Figure 2.4A) and 16˚C (Figure 2.4B), there was a significant reduction in plasma [Mg2+] at 18 and 23 g/l relative to 33g/l at 3 and 6 h. At 25˚C (Figure 2.4C) there was a 19  significant reduction in plasma [Mg2+] at 3h in 18 g/l salinity relative to all other salinities. All of the hagfish from the 25˚C and 18 g/l combination were moribund after the 3 h exposure and thus 6 h samples were not obtained.  Blood Glucose and Hematological Parameters In hagfish exposed to all combinations of salinity and temperature, there were no statistically significant differences between 1, 3 and 6 h measurements for plasma glucose, Hct, [Hb] and MCHC and consequently data for all time points were combined (Table 2.3). Of these parameters, only glucose differed significantly from control values where it was elevated during exposure to 23 g/l at 7C˚ and 18 g/l at all temperatures.  Muscle Water Content At 7˚C there was no significant effect of salinity on muscle water content (Figure 2.5). At 16˚C there was a significant increase in muscle water content at 18 g/l relative to 33 g/l after 3 h and by 6 h muscle water content differed among all salinities. At 25˚C, there were no statistically significant differences in muscle water content.  Hagfish Behaviour The most common behaviours displayed was full body contraction (FBC) and a reduction in the slime production (RSP) which were observed in all trials except the control (Table 2.2). Reduced swimming ability (RSA) and extended body posture (EBP) were observed at salinity conditions of 23 and 18 g/l at 7 and 16˚C and at all salinities at 25˚C. Hagfish were unable to produce slime (NSP) at all salinities at 25˚C and eventually became unresponsive to stimulation (UR) except at 33 g/l. 20  Recovery Trial After a 3 h exposure to all salinity/temperature combinations, hagfish were returned to the control conditions for 48 h at which time a morbidity of 14 and 100% was observed in hagfish that had been exposed to 23 and 18 g/l at 25˚C, respectively (Table 2.4). In general, most parameters measured were not significantly different from control values following 48 h recovery with the following exceptions observed at the lower salinities combined with higher temperatures. Plasma osmolality was reduced in hagfish exposed to 18 g/l at 25˚C (Figure 2.7), plasma [Na+] and [Cl-] was reduced in hagfish exposed to 23 and 18 g/l at 25˚C (Table 2.4), plasma [Mg2+] was elevated in hagfish exposed to 23 g/l at 16 and 25˚C and to 18 g/l at 7˚C and 25˚C (Table 2.4), plasma glucose was elevated at all reduced salinities at 25˚C, and at 18 g/l at 16˚C (Table 2.4), Hct was elevated in hagfish exposed to 18 g/l at all temperatures and at 23 g/l at 25˚C (Figure 2.8). Muscle water content in hagfish exposed to 18 g/l at 16˚C all returned to control levels (Table 2.4). [Hb] was reduced in hagfish exposed to 18 g/l at 7 and 16˚C and 28 g/l at 25˚C (Table 2.4). MCHC increased following exposure to control salinity at all temperatures (Figure 2.8).  Discussion As expected, significant physiological effects resulted from exposure to reduced salinity which were exacerbated by elevated temperatures. The extreme salinity and temperatures explored in this thesis resulted in latent morbidity in hagfish when allowed to recover from a 3 h exposure. There is evidence of sublethal disturbances in surviving individuals exposed to 18 g/l salinity at all temperatures and 25˚C at all salinities. Experimental conditions induced behaviours 21  likely to have immediate effects on hagfish survival in the wild which clearly need to be investigated in more detail.  Plasma Ions Hagfish are osmoconformers and so it is not surprising that plasma osmolality, [Na+] and [Cl-] are affected by a reduction in salinity, but previous studies in which this has been investigated have incorporated exposure durations of days, while this is the first to investigate the much shorter exposure durations of hours. At 7˚C, an exposure duration of as little as 1 h to 18 g/l significantly reduced plasma osmolality, [Na+] and [Cl-], while at 16 and 25˚C a significant effect was noticed within 1 h at 28, 23 and 18 g/l indicating that changes occur rapidly and are exacerbated by elevated temperature. When exposed to reduced salinity, water passively permeates the gills and skin (McFarland and Munz, 1958) increasing the blood volume and diluting plasma osmolality and ion levels in proportion to the osmolality of the environmental water. McFarland and Munz (1965) showed that E. stoutii are highly susceptible to the influx of water but resistant to the efflux of ions and thus most of the changes in plasma ion levels and osmolality in this study are likely due to the fluid shifts between the environment and the hagfish. Through unknown mechanisms, hagfish have the ability to regulate plasma [Mg2+] to levels significantly below ambient (Robertson, 1954; Robertson, 1976; Sardella et al., 2009). However, when transferred to reduced salinity at 7 and 16˚C for 1 and 3 h, hagfish plasma [Mg2+] decreased proportionately with the experimental salinity, consistent with that observed for plasma [Na+] and [Cl-] (Figure 2.1, 2.4). Following 6 h exposure to 18 g/l at 7 and 16˚C, plasma [Mg2+] was reduced by 25 and 28% respectively, while plasma osmolality, [Na+] and  22  [Cl-] was reduced by about 30%. These data indicate that within 6 h even at 18 g/l, hagfish show only a minor recovery of plasma [Mg2+] (Figure 2.1, 2.4). Higher than expected plasma [Mg2+] following transfers at 25˚C to 23 g/l could be explained by the elevated temperature accelerating Mg2+ recovery mechanisms. Sardella etal. (2009) reported no difference in plasma [Mg2+] after a 48 h exposure to 24 g/l seawater despite large reductions in plasma [Na+] and [Cl-], illustrating the ability of hagfish to regulate plasma [Mg2+].  Rate of Osmotic Shift The rate of osmotic change in hagfish is important when considering the impacts of temperature on acute exposure to low salinity. Plasma osmolality resulting from salinity exposures at 7˚C were linear over time but as the exposure temperature was increased plasma osmolality became temperature dependent. The greatest rate of change in plasma osmolality occurred within 1 h of exposure to salinity conditions resulting in an 18% change in plasma osmolality at 7˚C and a maximum change of 47% at 25˚C within that hour (Table 2.1). Interestingly the rate of change in plasma osmolality approaches an asymptote at 60% equilibration. The effect of increasing temperature on the rate of osmotic change is predominantly the consequence of an increase in the metabolic rate and the resulting increase in ventilation rate and blood flow to the gills. The rates observed during exposures to 16 and 25˚C are in the expected range when applying the accepted Q10 of 2 (Hochachka and Somero, 2002) to the increase in metabolic rate up to the point of 60% equilibration. The asymptote approached at 60% equilibration is likely a sub-lethal point beyond which hagfish experience increasing mortality.  23  Muscle Water Exposure to reduced salinity at 16 C˚ resulted in a salinity dependent change in muscle water content, consistent with the findings of other studies during longer exposures to reduced salinity (Cholette et al., 1970; McFarland and Munz, 1965; Sardella et al., 2009). It has been demonstrated that hagfish have the ability to manipulate intracellular osmolytes when exposed to hyper and hyposaline conditions but muscle water content generally remains directly proportional to the external osmotic pressure, indicating a very limited ability to regulate muscle water content (Cholette and Gagnon, 1973; Cholette et al., 1970). However, this was not observed following transfer to reduced salinities up to 18 g/l at 7˚C. There were no statistically significant increases in muscle water content following up to 6 h exposure to any salinities at 7˚C (Figure 2.5). It is possible that at 7˚C more time is required for muscle water to equilibrate with plasma osmolality, but it is also possible that at low temperatures hagfish are more capable of regulating muscle water through intracellular osmolyte manipulation than previously thought. The fact that muscle water content was actually reduced rather than increased at 23 g/l following 6 h supports the latter. Future research should explore intracellular osmolyte manipulation by hagfish during reduced salinity at temperatures ≤ 7˚C to determine whether the lack of cell volume regulation during exposure to reduced salinities has been potentially confounded by the higher experimental temperatures which have been used in past studies. This is especially important in the Pacific hagfish which are generally thought to inhabit environments ≤ 10˚C (McInerny and Evans, 1970).  24  Hagfish Behaviour Throughout trials, hagfish displayed a range of behaviours, some of which are important to consider when assessing the risk of their mortality when released as bycatch in a commercial fishery. The probability of predation increases when behaviour impairments induced by environmental conditions have altered an organisms predator avoidance (Little, 2002). Key behaviours I observed that may increase susceptibility to predation of hagfish released following exposures used in this study include a reduction of slime production ( RSP ), reduced swimming ability (RSA) , no slime produced (NSP) and unresponsiveness to touch (UN), all of which were observed to some degree here. In fact, the only experimental condition where no effects were observed was the control condition of 7 ˚C and 33 g/l salinity. A RSP was observed when hagfish were transferred to a salinity ≤ 28 g/l or under all conditions where temperature was elevated above 7˚C, NSP was observed at all salinities at 25˚C, within as little as 1 h. A RSA was displayed during lower salinity exposure at 7 and 16˚C when the muscle water content was 77% or higher and UN was observed following transfers to ≤ 28 g/l salinity at 25˚C (Table 2.2). Behaviours of RSP and NSP are particularly important to consider because slime production is one of the only predator avoidance mechanisms of hagfish (Lim et al., 2006; Martini, 1998). It was also noticed throughout the study that even under control conditions, hagfish stimulated to produce slime more than twice successively exhibited a reduction in the volume of slime produced, even to the point that no slime could be produced following continued successive sliming stimulation, which has important implications for how hagfish are handled prior to release. In general, changes in behavior occurred earlier and the magnitude of the response was more severe with an increase in temperature, and to a lesser extent a reduction in salinity. Based on these results it is recommended that hagfish should not be exposed to a salinity of 18 g/l or 25  lower at any temperature or to a temperature of 25 ˚C at any salinity based upon the severity of the behavioural responses observed. It is also recommended that a salinity of 23 g/l or lower be avoided when the water temperature is ≥16˚C. Of course, if possible a condition of 7 ˚C and 33 g/l salinity is preferred as this is the only condition tested where no noticeable behavioural effects were observed.  Recovery Trial After a 48 h recovery following 3 h exposure to all combinations of salinity and temperature, the majority of measured parameters returned to acclimation levels with the exception of glucose, [Hb] and MCHC. At 25˚C, morbidity levels of 14 (n=7) and 100% (n=7) were observed following recovery from exposure to 23 and 18 g/l, respectively (Table 2.4). An increase in plasma glucose concentrations is a standard response for organisms coping with an environmental stress (Barton et al., 2002). Significantly elevated glucose levels remained following recovery from transfers to 18 g/l at 16˚C and reduced salinities at 25˚C, indicating latent stress (Table 2.4). The elevated glucose levels suggest that hagfish post 48 h recovery were still coping with the physiological demand resulting from exposure to the experimental conditions. Elevated glucose could indicate an increase in metabolic activity required for physiological rebalancing or that acute exposure to elevated temperatures at 18 g/l salinity resulted in permanent damage, however, further studies are required to address this. Significant effects beyond those conditions causing morbidity (23 and 18 g/l, 25˚C) were observed in some blood parameters. Measures of Hct and [Hb] should be interpreted with caution due to the dynamic nature through which red blood cells (RBC) are regulated within the circulation of hagfish. Hagfish generally maintain a higher Hct in the central vascular system, 26  separate from the subcutaneous sinus (Foster and Forster, 2007; Johansen et al., 1962) however, the two values can be equal after physical activity due to skimming of RBCs from the central circulation, which complicates hematological comparisons. Plasma osmolality returned to baseline following a 48 h recovery making any resulting increase in Hct in the subcutaneous sinus related to an increase in [RBC] (Figure 2.8). E. cirrhatus has been shown to turnover blood in the subcutaneous sinus, mixing into the rest of the vascular system, every 20-24 h (Forster et al., 1989) making the remaining increase [RBC] following a 48 h recovery from exposure to reduced salinities potentially indicative of a latent effect.  Recommendations for Fishery Hagfish captured and quickly brought to the surface and immediately released without large deviations from acclimation salinity and temperature may not be negatively affected by a catch and release fishery which is supported by data from recovery trials following exposures to the less extreme conditions investigated here. However, it is important to note that significant physiological effects were detected within as little as 1 h of exposure to 28 g/l at 7˚C and the measurable effects on hagfish increased dramatically as the duration of exposure to reduced salinity and elevated temperature were increased. Diminished survival is likely if hagfish are exposed to a salinity approaching18 g/l for ≥ 3 h or when water temperature approaches 25˚C, which are conditions shown to cause physiological effects as well as latent stress and/or morbidity (Table 2.4). Hagfish are unlikely to come into contact with 25˚C water temperatures, however air temperatures on ship decks in the Pacific northwest can approach 30˚C during summer seasons (Olla et al., 1998) so clearly care must be taken to ensure that holding conditions for hagfish to be released do not approach these levels. Behavioural responses were noted opportunistically in this study but are likely a key component to hagfish release survival. 27  Future research should better characterize behavioural effects and their implications for subsequent survival and associated field predation estimates. This study did not account for multiplicative stressors associated with a commercial fishery such as air exposure, handling and pressure change, all of which will only decrease the probability of survival (Wedemeyer et al., 1990) and should be considered when assessing hagfish release mortality.  28  900  7 oC  A a  a a a,b  850  a  b  800 b  b*  c*  750  d*  700  c*  650 600  Plasma Osmolality (mOsm/l)  900  d* B  850  a  a  b  b  800  a  16oC  b* c c  750 700  c* d*  c*  650 d*  600  900 850  C a  a  a  b  b  b  25oC  800 c  750 c*  700  d d  650 600 1  3  6  Exposure Time (h)  Figure 2.1. Plasma osmolality (mOsm/l) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05). 29  450  A 7 oC  a a  a a,a  400  b  b  b c* d*  350  c*  300  d*  250 B a  a  450  a  16oC  Plasma [Na+] (mM)  b b*  400  b*  c c c*  350  c* d*  300  d*  250 C 25oC  450 a a  a  400 b  350  a,b  b  b  b  c  300  c  250 1  3  6  Exposure Time (h)  Figure 2.2. Plasma [Na+] (mM) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05).  30  450  A  7oC a a  400  b b b  a  b b* c*  350  c*  c*  300 d*  250 450  B 16oC  b b  350  c c  a  b  -  Plasma [Cl ] (mM)  a  400  b* c* c*  300  d* d*  250 450  400  C  25oC  a  a a  a b*  b*  350 b b  c* c  300  250 1  3  6  Exposure Time (h)  Figure 2.3. Plasma [Cl-] (mM) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05). 31  A  14  7oC  a  a  a  a a a  12  b,b  a,b  b  b b  a  a  a,b  a  10  8 B  2+  Plasma [Mg ] (mM)  14  a  16oC  a a,a  12  b b*  b  b*  10  8 C  14  25oC  a a  a  12  a a  a a  a a  b*  10  8 1  3  6  Exposure Time (h)  Figure 2.4. Plasma [Mg2+] (mM) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 1 h exposure value within a given salinity and temperature treatment (p<0.05). 32  80  7 oC  A  78  a  76  a a a  74  a,a  a a a  a a  a  72 70 B  16oC  Muscle Water Content (%)  80 a  78  a b  76  a a a  74  a,b c a,b  a b  72  d  70 25oC  C  80 78 76  a  74  a a  a  a,a,a a  a  a  72 70 1  3  6  Exposure Time (h)  Figure 2.5. Muscle water content (%) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) at 7 (a), 16 (b) and 25 (c)˚C and sampled 1, 3 or 6 h following exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature (p<0.05).  33  A  900  7oC  800  700  600  Plasma Osmolality (mOsm/l)  500 B  900  16oC  800  700  600  500 C  900  25oC  800  700  600  500 300  400  500  600  700  800  900  1000  Water Osmolality (mOsm/l)  Figure 2.6. Relationship between seawater and hagfish plasma osmolality at 1 ( ), 3 ( ) or 6 ( ) h following exposure to a range of salinities (33, 28, 23 and 18g/l) at 7(a), 16(b) or 25(c) ˚C. A 6 h exposure was not completed at 25˚C. The dotted line represents the line of unity for reference. See Table 2.1 for the slope and R2 values of the regression lines.  34  900  A  a* a  7 oC  a a*,a*  16oC  a  850 800  b  750 700 650 600  Plasma Osmolality (mOsm/l)  900  B a  a*  850 b  800 750 700  c d  650 600 900  C  a a*,a*  a  25oC  850 800  b*  b  750 c  700 650  d  600 3  48  Exposure Time (h)  Figure 2.7. Plasma osmolality (mOsm/l) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) for 3 h at 7 (a), 16 (b) and 25 (c)˚C and allowed to recover for 48 h at 33 g/l and 7˚C. Values reported at 3 h are presented for reference and are from Figure 2.1. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 3 h exposure value within a given salinity treatment (p<0.05).  35  16  A  7o C  14 12 10 a  a  b  b  8 6 4 16  16oC  B a  Hematocrit (%)  14 12 a  10  a,b a,b b  8  b,b  6  b,c  4 16 14  C  25oC  a  a  12 10  a  b b,b  8 b b  6 4 3  48  Exposure Time (h)  Figure 2.8. Hematocrit (%) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) for 3 h at 7 (a), 16 (b) and 25 (c)˚C and allowed to recover for 48 h at 33 g/l and 7˚C. Values reported at 3 h are presented for reference and were sampled previous to this trial following a 3 h exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. (p<0.05). 36  A  7o C  40 a*  30  20 a b  a  10  0 B 16oC  MCHC (g/dl)  40  30 a,b *  20  a,b  a  b  a a a  10  c  0 C 25oC  40  30  20 b*  a  10  b,b b  a a  0 3  48  Exposure Time (h)  Figure 2.9. MCHC (g/dl) in hagfish transferred to a range of salinities (33 g/l ( ), 28 g/l ( ), 23 g/l ( ) or 18 g/l ( )) for 3 h at 7 (a), 16 (b) and 25 (c)˚C and allowed to recover for 48 h at 33 g/l and 7˚C. Values reported at 3 h are presented for reference and were sampled previous to this trial following a 3 h exposure. Values are mean ± s.e.m. (n=7). Letters that differ indicate statically significant differences within a given exposure time and temperature. “ * ” indicates a significant change relative to the 3 h exposure value within a given salinity treatment (p<0.05).  37  Table 2.1 Linear regression results for the relationship between seawater and hagfish plasma osmolality at 1, 3 or 6 h in hagfish following exposure to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C. Data is plotted in Figure 2.6. Exposure Time  7˚C  16˚C  25˚C  1h  m=0.18 R2=0.540  m=0.35 R2=0.754  m=0.47 R2=0.960  3h  m=0.29 R2=0.762  m=0.61 R2=0.931  m=0.61 R2=0.962  6h  m=0.62 R2=0.875  m=0.69 R2=0.937  -  38  Table 2.2. Behaviour displayed by hagfish when exposed to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C for 1, 3 or 6 h. The minimum exposure time that a behaviour was observed is indicated within ( ), the number referring to hours. Each behavioural response listed was the dominant behaviour(s) displayed by the entire experimental group (n=7). Behaviours are listed in the order they appeared. FBC full body contractions, RSP reduced slime production, NSP no slime produced, EBP extended body posture, RSA reduced swimming ability, UN unresponsive to touch. 33 g/l Coiled (1)  28 g/l FBC (1) RSP (3)  23 g/l FBC RSP (1)  18 g/l FBC RSP (1) EBP RSA (3)  16˚C  FBC (1) RSP (3)  FBC RSP (1)  FBC RSP (1) RSA (3)  FBC RSP (1) EBP RSA (3)  25˚C  FBC RSP NSP EBP (1)  FBC RSP NSP EBP (1) UN (6)  FBC RSP NSP EBP (1) UN (3)  FBC RSP NSP EBP (1) UN (3)  7˚C  39  Table 2.3. Plasma glucose and hematological measurements in hagfish exposed to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C for up to 6 h. No significant differences were found between 1,3 and 6 h exposures within a given salinity and temperature, so data for all times were pooled to yield a single value. Values represent mean ± s.e.m.. A significant difference from the control (33 g/l at 7˚C) is indicated by “ * ” and was determined using a 2way ANOVA (p<0.05). Sample size was n=21 for each salinity condition unless otherwise indicated by (n).  Glucose (mM)  7˚C 16˚C 25˚C  33g/l 1.96±0.42 1.51±0.26 1.45±0.14  28g/l 0.87±0.10 0.71±0.07 1.46±0.19  23g/l 1.82±0.69 1.00±0.24 1.24±0.16 (14)  18g/l 1.07±0.16 1.38±0.16 2.12±0.34 (14)  Hematocrit (%)  7˚C 16˚C 25˚C  7.2±0.3 7.4±0.3 8.4±0.5  6.6±0.2 6.7±0.4 7.9±0.4  8.0±0.3* 8.6±0.4 9.2±0.4 (14)  8.4±0.4* 9.4±0.5* 12.2±0.8* (14)  [Hb] (g/dl)  7˚C 16˚C 25˚C  1.14±0.15 0.74±0.08 0.95±0.22  1.13±0.12 0.90±0.10 0.78±0.08  1.02±0.12 0.95±0.09 1.01±0.16 (14)  0.85±0.11 1.19±0.12 1.10±0.14 (14)  MCHC (g/dl)  7˚C 16˚C 25˚C  16.1±2.0 10.4±1.3 12.2±2.3  17.4±1.8 14.1±1.9 10.5±1.2  13.7±2.0 11.5±1.2 11.5±2.3 (14)  11.0±1.6 13.0±1.2 9.5±1.4 (14)  40  Table 2.4. Plasma glucose, ion, muscle water, hematological and morbidity measurements in hagfish exposed to a range of salinities (33, 28, 23 or 18 g/l) at 7, 16 and 25˚C for 3h and followed by a 48 h recovery before sampling. Values represent mean ± s.e.m.. A significant difference from the control (33 g/l ay 7˚C) is indicated by “ * “ and was determined using 2-way ANOVA (p<0.05, n=7).  [Na+] (mM)  7˚C 16˚C 25˚C  33g/l 446.5±6.2 441.5±7.2 433.1±7.2  28g/l 430.2±5.9 430.2±7.2  23g/l 433.0±7.6 419.1±7.0*  18g/l 433.6±6.1 420.1±7.4 380.6±4.7*  [Cl-] (mM)  7˚C 16˚C 25˚C  399.1±3.5 397.0±2.9 406.0±5.8  395.4±2.7 400.7±6.5  382.0±12.1 383.6±7.1*  393.1±5.5 387.5±6.2 357.7±4.2*  [Mg2+] (mM)  7˚C 16˚C 25˚C  12.5±0.5 12.5±0.5 13.2±0.4  13.2±0.6 13.6±1.0  14.5±0.4* 15.6±0.6*  14.0±0.6* 14.0±0.5 18.1±0.7*  Glucose (mM)  7˚C 16˚C 25˚C  0.83±0.14 1.13±0.37 1.86±0.64  1.00±0.12 2.23±0.36*  0.76±0.16 3.06±0.52*  1.30±0.25 2.12±0.35* 6.38±0.84*  [Hb] (g/dl)  7˚C 16˚C 25˚C  2.00±0.24 1.41±0.25 1.07±0.23  1.19±0.15 0.65±0.12*  1.51±0.27 1.05±0.14  0.98±0.24* 0.76±0.18* 1.18±0.33  % Muscle Water  7˚C 16˚C 25˚C  73.9±0.7 72.7±0.5 74.2±0.5  73.6±0.7 73.8±0.5  72.4±0.7 73.7±0.5  73.7±0.7 71.6±0.7* 74.4±0.4  Morbidity  25˚C  1 of 7 (14%)  7 of 7(100%)  41  Chapter 3: Conclusion The overall objective of this thesis was to assess, under laboratory conditions, the effects of acute exposure to reduced salinity and elevated temperature on the physiology of hagfish to better understand potential impacts of a surface release of bycatch in a commercial fishery. The ability for hagfish to recover from experimental conditions was also explored to investigate latent effects.  Catch and Release Mortality This data suggests that hagfish physiologically have the ability to tolerate short-term exposures to the environmental conditions that may be associated with capture and release in a commercial fishery. However, diminished survival, post release is likely when considering the behavioural modifications caused by exposure to the range of temperatures and salinities used in this study. In this thesis I have demonstrated that hagfish survival following exposure to all levels of salinity at 16˚C but experienced morbidity during exposure to 25˚C at 23 and 18 g/l salinity (Table 2.4). Exposure to reduced salinity conditions at 25˚C eliminates sliming ability and leaves hagfish in a catatonic state before eventual morbidity. Hagfish are unlikely to come into contact with 25˚C water temperatures, however air temperatures on ship decks in the Pacific northwest can approach 30˚C during summer seasons (Olla et al., 1998). Behaviours displayed during exposure to the experimental conditions chosen in this study may result in increased potential for predation of hagfish that are released into the natural environment. Exposure to reduced salinity at 7˚C produced significant behavioural impairments, the occurrence of which was exacerbated by increasing temperature. Behaviours of reduced 42  ability to produce slime (RSP), reduced swimming ability (RSA) and extended body posture (EBP) could have a significant effect on the predator avoidance of hagfish as they drift back to the seafloor. Behavioural impairments resulting from catch and release activities are known to increase release mortality rates for other commercially exploited demersal species (Olla et al., 1995; Ryer, 2002; Ryer et al., 2004). Measured physiological parameters indicate that hagfish can recover within 48 h from a 3 h exposure to salinities of 18 g/l at temperatures of 7 and 16˚C. This does not account for multiplicative stressors associated with a commercial fishery such as air exposure, handling and pressure change, all of which will likely decrease the probability of survival (Wedemeyer et al., 1990). A hagfishes ability to recover from the acute effects associated with catch and release still leave open the possibility for latent effects, even after physiological parameters have returned to baseline, such as hydrodynamic instability, behavioural impairment and predator evasion, feeding , growth and reproduction just to name a few (Davis, 2002; Rummer, 2007). Taking into account the physiological unbalance, behavioural modification and observed mortality that result from acute exposure to salinity and temperature stress on hagfish there is cause for concern regarding release survival rates post capture in a commercial fishery. Hagfish were shown to physiologically rebalance post exposure within 48 h recovery but observed behaviours preceding recovery such as reduced slime production (RSP), reduced swimming ability (RSA), extended body posture (EBP) will likely reduce predator avoidance/defense in the wild, making the fact that hagfish are physiologically capable of recovering from fishing disturbances irrelevant. Displayed behaviours such as unresponsiveness (UN) and inability to produce slime (NSP) combined with the observed mortality resulting from exposure to 25˚C reinforce that hagfish should not be exposed to conditions of any salinity when temperature 43  approaches 25˚C. It is also recommended that hagfish exposure to salinities approaching 18 g/l at any temperature be avoided.  Study Limitations Data collected for this study represent the first steps toward predicting the ultimate fate of hagfish released at sea from a commercial fishery, however, if anything the results obtained here are conservative due to many other potentially interacting variables not investigated in this study. The salinity and temperature values used in this study were chosen to encompass conditions relevant to what a hagfish may be exposed to during capture in a fishery. The multiplicative stressors associated with a commercial fishery such as air exposure, handling and pressure change each have a significant role in the release of survival bycatch (Davis, 2002; Wedemeyer et al., 1990) but were not investigated here. Due to the large number of animals required to investigate the time, salinity and temperature conditions a sample size of 7 was chosen. This sample size did not allow for a threeway analysis of variance among exposure time, salinity and temperature levels due to high variability within sample groups. In retrospect a larger sample size with fewer factors being investigated would have improved the robustness of the data for use in the analysis by potentially improving statistical power among experimental treatments. This approach would not have changed the overall results of the study but would have allowed a more in-depth analysis among the physiological parameters collected. Behavioural observations were opportunistically collected and fine scale observations could have been missed as a result. Changes in hagfish behaviours resulting from environmental conditions seem to be predictable and repeatable and a study focusing exclusively on  44  environmentally manipulated behavioural modifications would greatly add to the strength of a catch and release mortality estimate.  Hagfish Husbandry Hagfish were captured off the west coast of Vancouver Island, BC Canada in June, 2008 and held at the West Vancouver Laboratories at the Department of Fisheries and Oceans, Centre for Aquaculture and Environmental Research for 1.5 years for use in this study. They were held in 4000 liter tanks serviced with flow-through sand filtered sea water. Temperature and salinity of the flow-through seawater ranged from 32-33 g/l and 9-13˚C. Hagfish were fed dead rainbow trout once a week to satiation but feeding was withheld two weeks prior to experiments. On one occasion hagfish plugged an outflow drain causing a tank to overflow in the night, spilling a portion of the hagfish out onto the concrete walkway. Once noticed the hagfish were understandably put back into the tank by an onsite security personal making the identification and separation of affected hagfish rather difficult. The longest period of time a hagfish could have spent out of the tank was 1 h but it was reported they were still quite active when reintroduced into the tank so a shorter duration spent outside of the tank is likely. The following morning all moribund hagfish were removed and the decision was made to observe the recovery of the remaining hagfish that were not physically affected by the event but were obviously stressed indicated by an elongated body position. Over the following two weeks all hagfish showing signs of stress succumbed to infection which had also spread to other initially “healthy” hagfish that had likely not exited the tank during the flooding event. During this time affected fish began showing bite marks on the skin around the anterior side of the gills and cloaca region and swelling around the cloacal vent, something not ever observed on hagfish in healthy tanks. It is recommended that hagfish continuously laying in an elongated position, have 45  swelling around the cloacal region and do not respond to handling with rapid swimming be immediately removed from the holding tank because recovery is highly unlikely. It is important to note that hagfish may be elongated and not be mortally stressed. Generally, if still healthy, hagfish found in the elongated positions will respond to probing or handling with rapid swimming behaviour as short term periodic elongation was observed post feeding and during high heat days in the summer months. A similar series of events as described above was observed in a tank of hagfish collected from the wild during August and transported to the West Vancouver facility where all hagfish eventually succumbed within weeks to some form of infection likely resulting from heat stress incurred during capture and transportation. Local holding conditions in terms of temperature and salinity differential from the wild habitat are important factors to consider when thinking about collecting hagfish, especially during summer months.  Future Direction Future research could focus on field based predictions of hagfish predation post release in a commercial fishery, determining if the risk for high predation mortality following exposure to the range of salinity and temperature used here is a valid concern. The only documented predators of E. stoutii are the Harbour seal (Phoca vitulina) while hagfish, M. glutinosa in the Atlantic ocean have a much longer list of predators which include demersal fish such as Codfish (Gadus callarias), White hake (Urophycis tenuis) and Halibut (Hippoglossus hippoglossus) as well as Harbour porpoise (Phocoena phocoena) (Martini, 1998). The list of predators of E. stoutii is sure to grow as research continues.  46  Behaviours observed in the lab may not be relevant to what actually occurs in association with a commercial fishery and is a shortcoming of this research. Now that it has been demonstrated that hagfish can survive acute exposure to 18 g/l at temperatures up to 16˚C, the greatest additions to the questions surrounding hagfish catch and release survival will come from field based investigations beginning first with the questions of predation risk. Effects of air exposure, elevated air temperatures and handling influences could also be explored. Deck conditions could significantly contribute to stress accumulated during captures due to extended sorting time required to separate the undersized hagfish to be returned as bycatch (Grant, 2006). The field based characterization of behavioural changes and the resulting effect on predator avoidance combined with the physiological observations from this thesis will provide a strong estimate of release survival of hagfish following capture in a commercial fishery. Outside of the applied question, it would also be interesting to further investigate intracellular osmolyte manipulation in hagfish at temperatures ≤ 7˚C. It was observed that muscle water content at this temperature was not significantly changed following a 6 h exposure (Figure 2.5) to salinities down to 23 g/l and exposure times were not thought to have influenced this result suggesting some form of volume regulation may have been employed. It would be a novel discovery if hagfish were found to posses the ability to regulate cellular volume of muscle tissues. It has been demonstrated that hagfish can manipulate intracellular osmolytes using amino acids when exposed to hyper and hyposalinity but not to the extent where cellular volume can be controlled independent of the extracellular osmolality (Cholette and Gagnon, 1973; Cholette et al., 1970). Cholette etal. (1970) found that intracellular inorganic osmolytes in the form of amino acids can be actively regulated to increase or decrease in concentration relative to the extracellular osmotic pressure, a necessary requirement for cellular volume regulation. Given 47  the significant influence of temperature on cellular processes, protein function, cell membrane function and permeability, it is possible that cell volume regulation in hagfish has been missed by previous mentioned studies due to the use of experimental temperatures at or above the upper limit of preferred habitat temperatures of hagfish. The ability for cell volume regulation would aid in preventing membrane damage while maintaining function and protect intracellular cellular biochemical processes in the event of osmotic stress potentially encountered by a hagfish when entering a hyposaline carcass of carrion during feeding.  48  References Adam, H. and Strahan, R. (1963). Notes on the habitat, aquarium maintenance, and experimental use of hagfishes. In: The biology of Myxine. Oslo: Universitetsforlaget, 33-42. Baldwin, J. (1971). Adaptation of enzymes to temperature: acetylcholinesterases in the central nervous system of fishes. Comparative Biochemistry and Physiology 40, 181-187. Barton, B. A., Morgan, J. D. and Vijayan, M. M. (2002). Physiological and condition-related indicators of environmental stress in fish. Bethesda, MD: American Fisheries Society, 111-148. Becker, C. and Genoway, R. (1979). Evaluation of the critical thermal maximum for determining thermal tolerance of freshwater fish. Environmental Biology of Fishes 4, 245-256. Bowler, K. and Manning, R. (1994). Membranes as critical targets in cellular heat injury and resistance adaptation. London: Portland Press, 196-223. Brass, W. H. (1993). Pacific hagfish, Eptatreus stoutii, and Black hagfish, E. deani: the Oregon fishery and port sampling observations, 1988-92. Marine Fisheries Review, 55 (4), 19-30. Cholette, C. and Gagnon, A. (1973). Isosmotic adaptation in Myxine glutinosa L.--II. Variations of the free amino acids, trimethylamine oxide and potassium of the blood and muscle cells. Comparative Biochemistry and Physiology Part A: Physiology 45, 1009-1012. Cholette, C., Gagnon, A. and Germain, P. (1970). Isosmotic adaptation in Myxine glutinosa L. -I. Variations of some parameters and role of the amino acid pool of the muscle cells. Comparative Biochemistry and Physiology 33, 333-358. Cossins, A. R., Friedlander, M. J. and Prosser, C. L. (1977). Correlations between behavioral temperature adaptations of goldfish and the viscosity and fatty acid composition of their synaptic membranes. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 120, 109-121. 49  Crawshaw, L. I. (1979). Responses to rapid temperature change in vertebrate ectotherms. American Zoologist 19, 225-237. Dacie, J. V. and Lewis, K. (1968). Practical haematology. London: Churchill, 238-242. Davis, M. W. (2002). Key principles for understanding fish bycatch discard mortality. Canadian Journal for Fisheries and Aquatic Science 59, 1834-1843. Davis, M. W. and Olla, B. L. (2001). Stress and delayed mortality induced in Pacific halibut by exposure to hooking, net towing, elevated seawater temperature and air: implications for management of bycatch. North American Journal of Fisheries Management 21, 725-732. Davis, M. W., Olla, B. L. and Schreck, C. B. (2001). Stress induced by hooking, net towing, elevated sea water temperature and air in sablefish: lack of concordance between mortality and physiological measures of stress. Journal of Fish Biology 58, 1-15. Davis, M. W. and Parker, S. J. (2004). Fish size and exposure to air: potential effects on behavioral impairment and mortality rates in discarded sablefish. North American Journal of Fisheries Management 24, 518-524. Dohn, N. and Malte, H. (1998). Volume regulation in red blood cells. In The Biology of Hagfishes, eds. J. M. Jorgensen J. P. Lomholt R. E. Weber and H. Malte). London: Chapman and Hall, 301-306. Evans, D. H., Piermarini, P. M. and Keith, P. C. (2005). The multiffunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation and excretion of nitrogenous waste. Physioogical Review 85, 97-177. Fernholm, B. (1974). Diurnal variations in the behaviour of the hagfish Eptatretus burgeri. Marine Biology 27, 351-362.  50  Fincham, D. A., Wolowyk, M. W. and Young, J. D. (1990). Characterisation of Amino Acid Transport in Red Blood Cells of a Primitive vertebrate, the Pacific Hagfish (Eptatretus Stouti). Journal of Experimental Biology 154, 355-370. Foster, J. and Forster, M. (2007). Effects of salinity manipulations on blood pressures in an osmoconforming chordate, the hagfish, Eptatretus cirrhatus. Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology 177, 31-39. Fry, F. E. J. and Randall, W. S. H. a. D. J. (1971). The effect of environmental factors on the physiology of fish. In Fish Physiology, vol. 6: Academic Press, 5-61. Grant, S. M. (2006). An exploratory fishing survey and biological resource assesment of Atlantic hagfish (Myxine glutinosa) occurring on the southwest slope of the Newfoundland Grand Bank. Journal of Northwest Atlantic Fishery Science 36, 91-110. Grosell, M. (2006). Intestinal anion exchange in marine fish osmoregulation. Journal of Experimental Biology 209, 2813-2827. Gustafson, G. (1935). On the biology of Myxine glutinosa L. Arkiv for Zoologi 28, 1-8. Hochachka, P. W. and Somero, G. N. (2002). Biochemical adaptations: mechanism and process in physiological evolution. New York: Oxford, 317-431. Hofmann, G. E., Buckley, B. A., Airaksinen, S., Keen, J. E. and Somero, G. N. (2000). Heatshock protein expression is absent in the Antarctic fish Trematomus bernacchii. Journal of Experimental Biology 203, 2331-2339. Huey, R. B. and Kingsolver, J. G. (1993). Evolution of resistance to high temperature in ectotherms. The American Naturalist 142, 21-28. Huey, R. B. and Stevenson, R. D. (1979). Integrating thermal physiology and ecology of ectotherms: a discussion of approaches. American Zoologist 19, 357-366.  51  Johansen, K., Fänge, R. and Waage Johannessen, M. (1962). Relations between blood, sinus fluid and lymph in Myxine glutinosa L. Comparative Biochemistry and Physiology 7, 23-33. Leask, K. D. and Beamish, R. J. (1999). Review of the fisheries and biology of the Pacific hagfish (Eptatreus stoutii) in British Columbia, with recommendations for biological sampling in a developmental fishery, vol. 99/205 (ed. DFO): Canadian Stock Assesment Secretariat, 6-11. Lemons, D. E. and Crawshaw, L. I. (1985). Responses to rapid temperature change in the Pacific lamprey (Lampetra tridentata). Canadian Journal of Zoology 63, 1027-1032. Lesser, M. P., Martini, F. H. and Heiser, J. B. (1997). Ecology of the hagfish, Myxine glutinosa L. in the Gulf of Maine I. Metabolic rates and energetics. Journal of Experimental Marine Biology and Ecology 208, 215-228. Lim, J., Fudge, D. S., Levy, N. and Gosline, J. M. (2006). Hagfish slime ecomechanics: testing the gill-clogging hypothesis. Journal of Experimental Biology 209, 702-710. Little, E. E. (2002). Behavioral measures of environmental stressors in fish. Bethesda, MD: American Fisheries Society, 431-472. Maetz, J. and Evans, D. H. (1972). Effects of temperature on branchial sodium-exchange and extrusion mechanisms in the seawater-adapted flounder Platichthys flesus. Journal of Experimental Biology 56, 565-585. Martini, F., Lesser, M. and Heiser, J. B. (1997). Ecology of the hagfish, Myxine glutinosa L., in the gulf of Maine: II. Potential impact on benthic communities and commercial fisheries. Journal of Experimental Marine Biology and Ecology 214, 97-103. Martini, F. H. (1998). The ecology of hagfish, In: The biology of hagfishes. London: Chapman and Hall, 57-73.  52  McFarland, W. N. and Munz, F. W. (1958). A re-examination of the osmotic properties of the Pacific hagfish, Polistotrema stouti. Biological Bulletin 114, 348-356. McFarland, W. N. and Munz, F. W. (1965). Regulation of body weight and serum composition by hagfish in various media. Comparative Biochemistry and Physiology 14, 383-397. McInerny, J. E. and Evans, D. O. (1970). Habitat characteristics of the Pacific hagfish, Polistotrema stouti. Journal of the Fisheries Research Board of Canada, 966-968. Morris, R. (1965). Studies on salt and water balance in Myxine glutinosa (L.). Journal of Experimental Biology 42, 359-371. Motais, R. and Isaia, J. (1973). Temperature-dependence of permeability to water and to sodium of the gill epithelium of the eel Anguilla anguilla. Journal of Experimental Biology 56, 587-600. Olla, B. L., Davis, M. W. and Schreck, C. B. (1995). Stress-induced impairment of predator evasion and non-predator mortality in Pacific salmon. Aquaculture Research 26, 393-411. Olla, B. L., Davis, M. W. and Schreck, C. B. (1998). Temperature magnified postcapture mortality in adult sablefish after simulated trawling. Journal of Fish Biology 53, 743-760. Ota, K. G. and Kuratani, S. (2006). The history of scientific endeavors towards understanding hagfish embryology. Zoological Science 23, 403-424. Palmgren, A. (1927). Aquarium experiments with the hagfish (Myxine glutinosa L.). Acta Zoologica 8, 135-147. Robertson, J. D. (1954). The chemical composition of the blood of some aquatic chordates, including members of the Tunicata, Cyclostomata and Osteichthyes. Journal of Experimental Biology 31, 424-442.  53  Robertson, J. D. (1963). Osmoregulation and ionic composition of cells and tissues. In The Biology of Myxine, eds. A. Brodal and R. Fange). Oslo: Universitetsforlaget, 503-515. Robertson, J. D. (1976). Chemical composition of the body fluids andmuscle of the hagfish Myxine glutinosa and the rabbit-fish Chimaera monstrosa. Journal of Zoology 178, 261-287. Robertson, J. D. (1986). The osmotic concentration of parietal muscle of the hagfish Myxine glutinosa L., including estimates of free and bound constituents. Comparative Biochemistry and Physiology Part A: Physiology 84, 751-759. Rummer, J. L. (2007). Factors affecting catch and release (CAR) mortality in fish: Insight into CAR mortality in red snapper and the influence of catastrophic decompression. Bethesda, MD: Transactions of the American Fisheries Society, 113-131. Ryer, C., H. (2002). Trawl stress and escapee vulnerability to predation in juvenile walleye pollock: is there an unobserved bycatch of behavoirally impaired escapees? Marine Ecology Progress Series 232, 269-279. Ryer, C. H., Ottmar, M. L. and Sturm, E. A. (2004). Behavioral impairment after escape from trawl codends may not be limited to fragile fish species. Fisheries Research 66, 261-273. Sardella, B., Baker, D. and Brauner, C. (2009). The effects of variable water salinity and ionic composition on the plasma status of the Pacific hagfish (Eptatretus stoutii). Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology 179, 721733. Smith, H. W. (1932). Water regulation and its evolution in the fishes. The Quarterly Review of Biology 7, 1-18. Somero, G. N. (2004). Adaptation of enzymes to temperature: searching for basic "strategies". Comparative Biochemistry and Physiology 139, 321-333.  54  Somero, G. N. and DeVires, A. L. (1967). Temperature tolerance of some Antarctic fishes. Science 156, 257-258. Somero, G. N. and Yancey, P. H. (1997). Osmolytes and cell-volume regulation:physiological and evolutionary principles. Oxford: Oxford University Press, 442-484. Strahan, R. (1962). Survival of the hagfish, Paramyxine atami Dean, in diluted sea water. Copeia 1962, 471-488. Strahan, R. (1963). The Behaviour of Myxinoids. Acta Zoologica 44, 73-85. Wedemeyer, G. A., Barton, B. A. and McLeay, D. J. (1990). Stress and acclimation. In Methods for Fish Biology, eds. C. B. Schreck and P. B. Moyle), Bethesda, Maryland: American Fisheries Society, 213-272. Welsch, U. and Potter, I. C. (1998). Dermal capillaries. In The Biology of Hagfishes, eds. J. M. Jorgensen J. P. Lomholt R. E. Weber and H. Malte), London: Chapman and Hall, 273-284. Worthington, J. (1905). Contribution to our knowledge of the Myxinoids. The American Naturalist 39, 625-639. Yancey, P. H. (2005). Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. Journal of Experimental Biology 208, 2819-2830. Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D. and Somero, G. N. (1982). Living with water stress: evolution of osmolyte systems. Science 217, 1214-1217  55  Appendix Appendix A  Critical Thermal Max Trial Preceding salinity and temperature trials a critical thermal max (CT max) was completed to provide insight into the lethal limit of hagfish when exposed to rapid temperature change following various acclimation temperatures. Hagfish were acclimated to 4, 8 and 14˚C at 33 g/l for a minimum of two weeks prior to use in CT max trials. CT max trials were performed using one hagfish at a time for a total of 6 hagfish per acclimation temperature. The experimental tank was a closed system with roughly 20 L of water constantly in circulation through to maintain homogenous temperature throughout the water column. Trials began at the acclimation temperature and increased at a rate of 0.45 ± 0.004 ˚C/min. The endpoint was determined as the time when hagfish no longer responded to stimulus and the corresponding temperature was recorded as the CT max (Becker and Genoway, 1979). Once the endpoint was reached hagfish were returned to acclimation temperatures. Within minutes of reintroduction into the acclimation tank of 4, 8 or 14˚C hagfish regained movement and eventually made a full recovery.  56  33.0  B  o  Critical Thermal Limit ( C)  32.5  32.0  B  31.5  A 31.0  30.5  30.0 4  8  14 o  Acclimation Temperature ( C)  Figure A1. Critical thermal limit of hagfish when acclimated to 4, 8 and 14˚C before exposure to increasing temperature conditions of 0.45˚C/min. The thermal limit was reached when hagfish were no longer responsiveness to touch. Values are mean ± s.e.m. (n=6). Letters that differ indicate statically significant differences among acclimation groups (p<0.05).  57  

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