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Of saline and sea lice : hydromineral challenges and osmoregulatory strategies associated with early… Sackville, Michael 2010

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Of Saline and Sea Lice: Hydromineral Challenges and Osmoregulatory Strategies Associated with Early Ocean Entry of Juvenile Pink Salmon, Oncorhynchus gorbuscha by Michael Sackville B.Sc., McGill University, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2010 © Michael Sackville, 2010  Abstract  Pink salmon (Oncorynchus gorbuscha) enter seawater (SW) following gravel emergence at a body mass of 0.2 g. Two hydromineral challenges associated with this remarkable early ocean entry were investigated: (1) initial exposure to a hyper-osmotic environment and (2) sea louse (Lepeophtheirus salmonis) parasitism. To survive SW, pink salmon were hypothesized to develop hypo-osmoregulatory abilities as larval alevins prior to natural SW entry as post-larval fry. To test this, alevins and fry were transferred from freshwater (FW) in darkness to SW under a simulated natural photoperiod (SNP). Ionoregulatory status was assessed at 0, 1 and 5 days post-transfer. Alevins showed no evidence of hypo-osmoregulation, marked by a loss of water balance, a 35% increase in body [Cl-], and no change in gill Na+/K+-ATPase (NKA) activity. Conversely, fry maintained water balance and increased gill NKA activity by 50%. Fry gill NKA activity also increased by 50% following exposure to SNP in FW, providing the first evidence of photoperiod-triggered smoltification for pink salmon. A 15% increase in fry body [Na+] was observed as well, perhaps representing a novel mechanism for maintaining water balance during ocean entry. Physical damage to the host epidermis is a primary proximal effect of louse infection. Such damage may exacerbate existing hydromineral flux in SW. To test this, ionoregulatory status was measured in pink salmon of varying size with and without attached-stage lice. In laboratory-infected fish (~1 wk SW; 0.2-0.4 g), body [Na+] increased by 12% when infected with 1 chalimus IV louse, and by 23% with 2-3 chalimus III lice. Mortality was 6%. In wild-infected fish (~4-12 wks SW; 0.5-1.5 g), body [Na+] did not differ from controls. Combining data sets revealed a “no effect” fish size threshold of 0.5 g for 1 chalimus IV louse. This threshold is partly due to increasing hypo-osmoregulatory ability.  ii  Pink salmon thus appear to possess a novel hypo-osmoregulatory strategy where ion balance is sacrificed to maintain water balance prior to maximum ion excretion capacity. Outmigrating fish are particularly vulnerable to sea louse parasitism at this time, and as such, BC fish farms have relocated to minimize interactions during this critical period.  iii  Table of Contents Abstract .............................................................................................................................. ii Table of Contents.............................................................................................................. iv List of Tables ..................................................................................................................... vi List of Figures .................................................................................................................. vii Acknowledgements ......................................................................................................... viii Co-authorship Statement .................................................................................................. x Chapter 1: Introduction and Thesis Goals...................................................................... 1 1.1 Osmoregulatory demands of anadromy................................................................... 2 1.2 Evolution of anadromy and smoltification .............................................................. 4 1.3 Pink salmon ocean entry.......................................................................................... 5 1.4 Hydromineral challenge 1: the hyper-osmotic environment ................................... 6 1.5 Chapter 2 objectives ................................................................................................ 7 1.6 Hydromineral challenge 2: sea louse parasitism ..................................................... 8 1.7 Sea louse biology..................................................................................................... 8 1.8 Increased louse susceptibility of juvenile pink salmon ........................................... 9 1.9 Louse-induced performance reductions may be linked to ionic homeostasis ....... 11 1.10 Chapter 3 objectives ............................................................................................ 12 1.11 References............................................................................................................ 13 Chapter 2: Water Trumps Ion Balance in Early Marine Survival of Juvenile Pink Salmon (Oncorhynchus gorbuscha)............................................................................................. 22 2.1 Introduction ........................................................................................................... 22 2.2 Materials and methods........................................................................................... 24 2.3 Results ................................................................................................................... 27 2.4 Discussion.............................................................................................................. 29 2.5 References ............................................................................................................. 41 Chapter 3: Pink Salmon (Oncorhynchus gorbuscha) Hypo-osmoregulatory Development Plays a Key Role in Sea Louse (Lepeophtheirus salmonis) Tolerance ....................... 45 3.1 Introduction ........................................................................................................... 45 3.2 Materials and methods........................................................................................... 47 3.3 Results ................................................................................................................... 52 3.4 Discussion.............................................................................................................. 55 3.5 References ............................................................................................................. 66 Chapter 4: General Discussion ....................................................................................... 71 4.1 Chapter 2 summary: the hyper-osmotic environment ........................................... 71 4.2 A new form of anadromy?..................................................................................... 71 4.3 Evolutionary implications...................................................................................... 72 4.4 Future basic research directions............................................................................. 73 4.5 Chapter 3 summary: sea louse parasitism.............................................................. 74 iv  4.6 Applied contributions ............................................................................................ 74 4.7 Future applied research directions ......................................................................... 74 4.8 References ............................................................................................................. 76  v  List of Tables Table 2.1 Gill mRNA expression of Na+/K+-ATPase 1a & 1b, Ubiquitin, and Elongation Factor 1 in alevins held in FW and transferred to SW+SNP........................ 39 Table 2.2 Gill mRNA expression of Na+/K+-ATPase 1a & 1b, Ubiquitin, and Elongation Factor 1 in fry held in FW and transferred to SW+SNP and FW+SNP ....... 40 Table 3.1 Fish mass and area of quantified sub-epidermal tissue exposure in laboratory-infected river-caught pink salmon ................................................................... 64 Table 3.2 Fish mass and area of quantified sub-epidermal tissue exposure in naturally-infected ocean-caught pink salmon.................................................................... 65  vi  List of Figures Figure 2.1 Wet mass, dry mass, body [Na+], body [Cl-] and gill Na+/K+-ATPase activity of pink salmon alevins held in FW and transferred to SW+SNP ......................... 34 Figure 2.2 Gill Na+/K+-ATPase 1a & 1b mRNA expression as function of total RNA and as an 1a/b ratio in pink salmon alevins held in FW and transferred to SW+SNP............................................................................................................................ 35 Figure 2.3 Drinking rates of pink salmon alevins and fry transferred to SW+SNP ........ 36 Figure 2.4 Wet mass, dry mass, body [Na+], body [Cl-] and gill Na+/K+-ATPase activity of fry held in FW and transferred to SW+SNP .................................................... 37 Figure 2.5 Gill Na+/K+-ATPase 1a & 1b mRNA expression as function of total RNA and as an 1a/b ratio in fry held in FW and transferred to SW+SNP...................... 38 Figure 3.1 Body [Na+] and gill Na+/K+-ATPase activity of laboratory-infected rivercaught pink salmon ............................................................................................................ 60 Figure 3.2 Body [Na+] and gill Na+/K+-ATPase activity of naturally-infected oceancaught pink salmon ............................................................................................................ 61 Figure 3.3 Body [Na+] and gill Na+/K+-ATPase activity of river-caught pink salmon following mechanical epidermal abrasion ......................................................................... 62 Figure 3.4 Combined raw data for body [Na+] and gill Na+/K+-ATPase activity of river and ocean-caught pink salmon infected with one chalimus IV louse ....................... 63  vii  Acknowledgments  My time with UBC’s department of zoology was a truly inspirational and serendipitous introduction to the world of science. I would like to express my sincerest gratitude to those that not only made my arrival to the department a reality, but one of the most positive and influential experiences of my life to date. Foremost, I wish to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for rejecting my application for master’s funding. This initially devastating event serendipitously restricted my academic pursuits to those PI’s bold enough to supervise an unfunded and unproven student. As luck would have it, I was graciously taken in by Anthony P. Farrell, Colin J. Brauner and UBC’s comparative physiology group. I suppose this sequence of events could alternatively be owed to my sub-par undergraduate performance, but thanking oneself seems slightly inappropriate for an acknowledgements section. To Big Tone, I will forever be grateful for providing that first opportunity to prove myself. His willingness to meet and chat with any eager mind is a truly admirable quality and almost entirely responsible for my arrival to UBC. As a supervisor and mentor, he continually impressed with his ability to squeeze the very best out of his students, relentlessly challenging the intellect and drive of all those around him. I thank Tony for pushing me to my limits and coaxing out a confidence I never knew existed. I’m also a huge fan of his signature analogy-style explanations, which double quite well as popular xmas skit material. To Colin, I am grateful for being a constant reminder that science is meant to be enjoyed. CJ’s living proof that being a great scientist is as much about building friendships and sharing new experiences as anything. I thank him for providing the freedom to pursue my own interests  viii  and make my own mistakes, for encouraging creative thought, and most importantly, fostering a love for science. In Steve Tang and Laura Nendick, I found a brother and sister. I thank them for a lifetime of memories in the Broughton –most of which were positive! They both possess a rare combination of intelligence, honesty and work ethic that will undoubtedly be met by success. My parents are the most positive, encouraging, inspirational and supportive people in my life. I can’t even begin to describe how important and amazing they are. I will, however, express my gratitude for their decision to fund the final six months of my time at UBC. Their generosity kept several ill-conceived money-making schemes grounded and prevented the lab bench from becoming my new bed. Finally, I would like to thank the department of zoology. My experience here can best be described as being adopted by an eccentric family of well-rounded, over-achieving, academic socialites. I’ve learned so much from faculty and students, and forged friendships and memories that will surely last a lifetime. The sense of camaraderie is what truly sets this place a notch above the rest, and I feel extremely privileged to have been part of such a special community.  ix  Co-Authorship Statement  Chapters 2 and 3 are co-authored as indicated by chapter footnotes. Research questions, experimental design, experimentation, data analyses and manuscript preparation were conducted by Michael Sackville under the co-supervision of Drs. Colin J. Brauner and Anthony P. Farrell. Laura Nendick and Stephen Tang assisted greatly with fish collection, care and experimentation for chapter 2.  x  Chapter 1: Introduction and Thesis Goals  The importance of Pacific salmon to the ecological, cultural and economic integrity of their surroundings has long been established. As keystone species of marine, freshwater and terrestrial ecosystems, they support multiple food webs across multiple habitats and serve as unique links in nutrient cycling between very distinct and often isolated systems (Quinn, 2005). Their highly productive populations have also significantly contributed to the economic development, success and well-being of coastal peoples for thousands of years, having sustained countless commercial, recreational and subsistence fisheries (Augerot and Foley, 2005; Quinn, 2005). As a result, these iconic fishes are intricately woven into the cultural fabric of virtually every coastal society within their distribution (Augerot and Foley, 2005). The extent of these impacts stems largely from a truly remarkable life history strategy. As anadromous fishes, salmon spawn in freshwater, move to the ocean for a period of rapid growth, then return to natal streams to reproduce and die. Transition to more productive marine waters supports the rapid growth of large numbers of individuals by providing access to an abundance of space and food otherwise unavailable in most temperate freshwater systems (Gross et al., 1988; McDowall, 2008; McDowall, 1997). By facilitating this high productivity, successful transition to the marine environment is a major reason why these fishes have such dramatic effects on their environs, and without it, their impacts would be far less profound. This thesis explores two hydromineral challenges associated with ocean entry that pink salmon (Oncorhynchus gorbuscha) must overcome to achieve their high-impact status: (1) the hyper-osmotic environment and (2) sea louse (Lepeophtheirus salmonis) parasitism. The strategies adopted to meet these challenges are investigated in pink salmon for reasons that are both basic and applied. The following is a brief thesis outline:  1  Chapter 2: The hyper-osmotic environment, basic research Pink salmon represent the most extreme and perhaps derived form of salmonid anadromy, yet remain largely unstudied. Elucidating the mechanisms underlying their transition to marine waters may reveal a novel strategy and potentially provide insight into the evolution of salmonid anadromy. The physiological basis for ocean entry in pink salmon will serve as the focus of chapter 2.  Chapter 3: Sea louse parasitism, applied research Recent pink salmon population declines have been attributed by some to sea lice of fish farm origin; however, the physiological effects of parasitism have yet to be determined. Chapter 3 aims to bring concrete measures of sub-lethal impact to this controversial issue, ideally promoting pink salmon conservation and sustainable industry practice.  The remainder of this introduction will further contextualize and explain thesis research drivers while listing specific research objectives.  1.1 OSMOREGULATORY DEMANDS OF ANADROMY Anadromy has its obvious benefits with respect to species abundance and distribution, but it is not without obstacles. As osmoregulators, salmon homeostatically maintain very specific concentrations of water and ions within their extracellular fluid largely independent of the external environment (Evans, 2008; Krogh, 1939; Smith, 1932). Hydromineral balance within this internal milieu is essential to maintaining the electrochemical gradients required for nearly every cellular process imaginable; therefore, any disruption could have devastating effects at the cellular, tissue and even organismal level. Because the gill epithelium is permeable to both water and ions, fish must actively defend this balance against passive fluxes. In freshwater, where 2  osmolarity is 1/300th of plasma, teleosts passively gain water and lose ions to the external medium, thus requiring active ion uptake at the gills and excretion of excess water as dilute urine. In seawater, where osmolarity is roughly three times that of plasma, the scenario is reversed; fish combat passive ion gain and water loss with active ion excretion at the gills and water uptake at the gut. These two strategies, termed hyper- and hypo-osmoregulation respectively, demand very different and highly specialized machinery at the gills, gut and kidney (for reviews see Evans, 2008; Evans et al., 2005; Krogh, 1939; Smith, 1930). Anadromy and its many benefits require fish to switch between these opposing strategies, which is arguably the greatest physiological challenge associated with ocean entry. The failure to do so results in a rapid loss of hydromineral balance and, ultimately, death (Boeuf and Harache, 1982). A successful switch to hypo-osmoregulation requires extensive physiological change at the osmoregulatory organs (for reviews see Boeuf, 1993; Folmar and Dickhoff, 1980; Hoar, 1988; McCormick and Saunders, 1987). In the intestine, specialized epithelial cells develop to create a favourable osmotic gradient that draws water into the extracellular fluid (ECF) paracellularly from the lumen (Grosell, 2006; Grosell et al., 2009; Grosell et al., 2007). The cells secrete bicarbonate (HCO3-) into the intestinal fluid which binds to calcium (Ca2+) and precipitates out of solution, while sodium (Na+) and chloride (Cl-) are actively transported transcellularly into the ECF. This process is driven largely by Na+/K+-ATPase (NKA) on the basolateral membrane and by Na+, K+, Cl--cotransporter (NKCC) and Cl-/HCO3- anion exchanger on the apical side. Water does move into the ECF as a result, but an excess amount of Na+ and Cl- accumulates as well. To maintain ion balance, specialized mitochondria-rich cells (MRC’s) situated on the interlamellar regions of the gill epithelium excrete Cl- transcellularly while Na+ leaves paracellularly down a local electrochemical gradient (Evans, 1993; Evans et al., 2005; Keys and Willmer, 1932; Potts, 1984). This process is driven largely by NKA and NKCC on the basolateral membrane and by an apical Cl- channel. Divalent ions are primarily secreted 3  by the kidney, which also reduces urine production to conserve water in the hyper-osmotic environment (Smith, 1930). However, the associated changes to renal physiology are relatively understudied.  1.2 EVOLUTION OF ANADROMY AND SMOLTIFICATION Depending on the nature and timing of the osmoregulatory switch, salmonids can generally be classified under one of two main types of anadromy; facultative or predictive. Facultative anadromy is common among more ancestral salmonids such as brook charr (S. fontinalis) of the genus Salvelinus (McCormick, 2009), and the switch to hypo-osmoregulation is triggered by direct exposure to seawater (McCormick and Naiman, 1984a; McCormick and Naiman, 1984b; McCormick et al., 1985). These fishes typically spend extended periods of time in brackish water while undergoing the necessary transformations for marine life (~3 weeks; McCormick and Naiman, 1984b), which results in considerable lag time between the onset of downstream migration and ocean entry. Conversely, predictive anadromy is common among more derived species such as members of the genera Oncorhynchus and Salmo, and the switch to hypo-osmoregulation is triggered by changes in photoperiod in anticipation of ocean entry (McCormick, 2009). This preparatory metamorphosis, which also includes a host of behavioural and morphological changes tailored to marine life, is termed smoltification (Boeuf, 1993; Folmar and Dickhoff, 1980; Hoar, 1976; Hoar, 1988; McCormick, 1994; McCormick and Saunders, 1987). Smoltification facilitates the transition to marine waters by initiating the upregulation of hypo-osmoregulatory mechanisms almost two months prior to seaward migration (Dickhoff et al., 1985; McCormick et al., 1987; Nilsen et al., 2007; Stefansson et al., 2007). Controlled experiments demonstrate that preparatory increases in gill NKA activity and mRNA expression of the enzyme’s 1 subunit seawater isoform greatly improve seawater adaptability, evidenced 4  by increased growth, survival and a reduction in both the duration and magnitude of ionic disturbances following seawater exposure (Boeuf and Harache, 1982; Boeuf and Harache, 1984; Brauer, 1982; Folmar et al., 1982; McCormick et al., 1987; Nielsen et al., 1999; Nilsen et al., 2007; Nilsen et al., 2003; Otto, 1971). Preparatory smolt changes run to completion within a matter of days to weeks of initial seawater entry, resulting in a significantly reduced acclimation period for this more derived form of anadromy. Interestingly, transition to the marine environment within the predictively anadromous salmonids may occur earlier in development and at smaller fish sizes in those more derived species (Clarke, 1982; Hoar, 1976; Hoar, 1988; McCormick, 2009; McCormick and Naiman, 1984b; McKay et al., 1996; Oakley and Phillips, 1999; Quinn and Myers, 2004; Rounsefell, 1958). Consequently, there may be an evolutionary trend toward increasingly early ocean entry, from facultative to predictive anadromy, and from large body size to small. This earlier access to more productive marine waters may also be a successful strategy, as those more derived salmonids that enter the ocean earliest are generally the most abundant and widely distributed (Augerot and Foley, 2005; Quinn, 2005). Although predation is often considered the dominant selective pressure dictating size at ocean entry (Hargreaves and LeBrasseur, 1986; Holtby et al., 1990; Saloniemi et al., 2004), osmoregulatory challenges likely play a significant role as well (Clarke et al., 1989; McCormick and Naiman, 1984b). Investigating the strategies used to overcome the hydromineral challenges accompanying increasingly early ocean entry may thus provide insight into the evolution of salmonid anadromy. Similarly, physiological adaptations key to the productivity and associated high-impact status of these fishes may be revealed.  1.3 PINK SALMON OCEAN ENTRY Pink salmon (Oncorhynchus gorbuscha) are an especially interesting salmonid in which to study ocean entry because they represent the most extreme, abundant, and perhaps derived 5  form of salmonid anadromy (Clarke, 1982; Hoar, 1988; McCormick, 1994; McCormick, 2009; Quinn and Myers, 2004; Rounsefell, 1958). Pink fry migrate seaward directly following gravel emergence at a size of 0.2 g (Grant et al., 2009; Heard, 1991), while most other anadromous salmonids do so after at least one year in freshwater at sizes between 2-30 g (Clarke, 1982; Rounsefell, 1958). Even chum and those runs of chinook and sockeye that migrate seaward in the same year of emergence are twice as large as pink fry and spend weeks to months in freshwater prior to ocean entry (Clarke, 1982; Hoar, 1988; McCormick and Naiman, 1984b; Quinn and Myers, 2004). Thus, pink salmon possess the smallest body size and shortest freshwater residency of any salmonid to regularly undergo this transition. As further described below, this exceptionally early ocean entry renders the hydromineral challenges explored in this thesis particularly daunting. Understanding how pink salmon overcome these challenges is therefore interesting from physiological, ecological and evolutionary perspectives alike.  1.4 HYDROMINERAL CHALLENGE 1: THE HYPER-OSMOTIC ENVIRONMENT A major challenge associated with the early ocean entry of pink salmon is the demand for a sufficiently developed seawater tolerance upon yolk sac absorption and gravel emergence. This scenario is particularly daunting because of the high body surface area to volume ratio facing small pink fry and the relative lack of freshwater preparatory time commonly afforded to other salmonids. The near absence of post-emergent preparatory time has spurred speculation that pink salmon may employ a novel strategy in which they develop hypo-osmoregulatory abilities as larval alevins in the redd -perhaps without the usual photoperiod cue (Hoar, 1988; McCormick, 2009; Sullivan et al., 1983; Weisbart, 1968). Despite this exceptional life history, smoltification and the development of seawater tolerance in pink salmon remains largely unstudied. Previous work has shown that although larval pink alevins fail to survive seawater exposure prior to yolk sac absorption, they do exhibit 6  signs of plasma ion regulation and possess a higher LT50 than other oncorhynchids, surviving almost three times as long as coho, chinook and sockeye alevins (Weisbart, 1968). While these results suggest larval hypo-osmoregulatory development, extra-branchial MRC’s as seen on the yolk sac of chum alevins (Kaneko et al., 1995) could explain this effect. Further complicating matters is that fry experience a doubling of whole body Na+ and Cl- content that coincides with naturally timed seawater entry (Grant et al., 2009). This elevation in whole body ion levels persists until gill NKA activity peaks two months later, which is far longer than the mere hours to days reported for other salmonids (O. keta, O. mykiss, O. kisutch, S. salar; Black, 1951; Leray et al., 1981; Miles and Smith, 1968; Prunet and Boeuf, 1985) and may indicate an underdeveloped hypo-osmoregulatory capacity. Furthermore, the majority of the observed increase in gill NKA activity occurs after seawater entry (Grant et al., 2009; Honma, 1982), suggesting that compensatory rather than preparatory changes may occur. Evidently, when and how seawater tolerance and hypo-osmoregulatory ability of juvenile pink salmon develops remains unclear.  1.5 CHAPTER 2 OBJECTIVES Chapter 2 thus has two objectives. 1) To determine the extent to which pink salmon develop adult-like hypo-osmoregulatory abilities as larval alevins, and 2) identify the physiological differences between alevins and post-larval fry critical to seawater survival. Studying the physiological mechanisms underlying early ocean entry in what might be the most derived oncorhynchid may provide valuable insight into the evolution of salmonid anadromy.  Chapter 2 Hypothesis Pink salmon have adult-like hypo-osmoregulatory abilities as larval alevins.  7  Chapter 2 Predictions 1. Larval alevins increase gill NKA activity, 1b isoform expression and drinking rate following seawater exposure. 2. Post-larval fry exhibit increases in gill NKA activity, 1b isoform expression and drinking rate equal to or greater than those exhibited by larval alevins following seawater exposure. 3. Hydromineral balance is better maintained in post-larval fry than larval alevins.  1.6 HYDROMINERAL CHALLENGE 2: SEA LOUSE PARASITISM Unfortunately for juvenile pink salmon, the hydromineral challenge that accompanies such an early transition to the marine environment can be exacerbated by other factors in the ocean. The ectoparasitic salmon louse, Lepeophtheirus salmonis, is one such factor that has become of particular concern for management and conservation groups as of late. The effects of louse infection on the ionoregulatory status of post-emergent pink salmon will serve as the focus for chapter 3 of this thesis.  1.7 SEA LOUSE BIOLOGY Endemic to the Pacific salmon of British Columbia’s coastal waters (Kabata, 1988), L. salmonis is a naturally occurring marine ectoparasite also commonly associated with Atlantic salmon in open net-pen fish farms (Orr, 2007). Lice first encounter their hosts as planktonic copepodids, drifting freely in the water column while surviving on limited internal energy reserves (Pike and Wadsworth, 1999; Tully, 1992; Wootten et al., 1982). Upon infection, copepodids anchor to the fish’s surface with a chitinous filament and begin feeding on host mucus, epidermal tissue and blood (Brandal et al., 1976; Kabata, 1974). This on-host feeding fuels louse development through four attached stages (termed chalimus 1-4) and three unattached motile stages (termed pre-adult 1-2 & adult) to sexual maturity (Johnson and Albright, 1991b). 8  Growth from a 1mm copepodid to a 14 mm adult female is highly temperature dependent, ranging anywhere from four to twelve weeks (Johnson and Albright, 1991a; Tully, 1992). Beyond the physical damage imparted by mouthparts while feeding, lice secrete proteases that suppress host immune function and aid tissue digestion (Fast et al., 2003; Fast et al., 2007a; Fast et al., 2007b; Fast et al., 2004; Fast et al., 2006; Firth et al., 2000; Wagner et al., 2008). As shown in 200 g Atlantic salmon and 90 g sea trout, this surface feeding can lead to significant hydromineral disruption (up to 60% increase in plasma electrolytes; Bjorn and Finstad, 1997; Bowers et al., 2000; Grimnes and Jakobsen, 1996; Nolan et al., 1999; Wootten et al., 1982). This disruption is thought to result from either epidermal lesions that damage the external osmotic barrier or from a stress-related increase in the osmo-respiratory compromise (Gonzalez and McDonald, 1994; Gonzalez and McDonald, 1992; Nolan et al., 1999; Randall et al., 1972; Wendelaar Bonga, 1997). The osmo-respiratory compromise is the trade-off between respiration and passive hydromineral flux across the gills. High permeability of the gill epithelium better facilitates the gas exchange required for respiration, but unfortunately also permits undesirable passive flux of water and ions. Furthermore, this trade-off increases with elevated respiration following a generalized stress response (Gonzalez and McDonald, 1992; Wendelaar Bonga, 1997). Regardless of origin, hydromineral imbalance is a major concern as it can lead to a myriad of associated complications; reduced swim performance (Brauner et al., 1994; Brauner et al., 1992), stunted growth (Brauer, 1982; Folmar et al., 1982; McCormick et al., 1987) and death (Boeuf and Harache, 1982) have all been linked to hydromineral disruption.  1.8 INCREASED LOUSE SUSCEPTIBILITY OF JUVENILE PINK SALMON Adult pink salmon are believed to be well equipped to cope with the effects of sea louse infection in the wild (Beamish et al., 2005), but the case for out-migrating juveniles is far from clear. Foremost, pink salmon enter the ocean as small as 0.2 g and are thus three orders of 9  magnitude smaller than their adult counterparts upon first encountering lice (Grant et al., 2009; Heard, 1991; Jones and Hargreaves, 2007). Furthermore, ocean entry by these fish appears truly precocious, as hypo-osmoregulatory, immune and epidermal systems all still develop over the first few months at sea. Gill NKA activity peaks 2 months post-entry (Grant et al., 2009), immunocompetence is reached at approximately 2.5 months post-entry (Johnson et al., 1982), while scale formation remains incomplete until almost 3 months post-entry (Jones et al., 2008). Based on the aforementioned proximal effects of L. salmonis parasitism, the development of these physiological systems undoubtedly plays a critical role in successfully resisting and/or tolerating louse infection. Thus, juvenile pink salmon are likely particularly vulnerable to sea louse parasitism during this precocious out-migration. With the exception of stickleback, G. aculeatus (Jones et al., 2006), most natural host populations are fortunately situated offshore during this critical period in pink salmon development (Gottesfeld et al., 2009; Quinn and Myers, 2004). This migratory allopatry may naturally restrict parasite exposure to more developed pink fry. However, open net-pen salmon farms often house adult S. salar in coastal waters year-round. These cultured fish can potentially serve as a nearshore parasite source that exposes out-migrating pink fry to unnaturally high and/or premature levels of L. salmonis infection (Orr, 2007). In fact, sea lice of fish farm origin have recently been implicated in pink salmon population declines and extinction predictions in British Columbia’s Broughton Archipelago (Krkosek et al., 2007a; Krkosek et al., 2007b; Krkosek et al., 2006a; Krkosek et al., 2005; Krkosek et al., 2009; Morton and Routledge, 2005; Morton et al., 2004; Morton et al., 2005). Although the correlational data and mathematical models presented by these studies are compelling, major questions regarding parasite transmission and infection impacts on host physiology remain unknown. The resulting uncertainty has led to considerable debate in both scientific and public fora alike (Beamish et al., 2006; Beamish et al., 2007; Brooks, 2005; Brooks and Jones, 2008; Brooks and Stucchi, 2006; 10  Butterworth et al., 2008a; Butterworth et al., 2008b; Dill et al., 2009; Krkosek et al., 2008a; Krkosek et al., 2008b; Krkosek et al., 2006b; Morton and Routledge, 2008; Riddell et al., 2008). Two major unknowns are of particular interest: (1) the relative contribution of fish farms to local parasite populations and (2) the level of parasitism required to sub-lethally impair juvenile pink salmon. Chapter 3 of this thesis will address the latter.  1.9 LOUSE-INDUCED PERFORMANCE REDUCTIONS MAY BE LINKED TO IONIC HOMEOSTASIS To date, only one study has examined the sublethal effects of louse infection on outmigrating juvenile pink salmon between 0.2 and 3 g in body mass (Nendick et al. 2010). Over this range, the effect of 1 motile louse per fish was size-related, significantly reducing swimming ability in fish below, but not beyond, a body mass of 0.7 g. Interestingly, this body mass threshold corresponds with the completion of pink salmon hypo-osmoregulatory development (Grant et al., 2009), indicating that performance impacts are perhaps linked to hypoosmoregulatory ability. Further supporting this notion is the correlation between similar reductions in swimming ability and elevated plasma ions in 8-20 g O. kisutch following SW entry (Brauner et al., 1994; Brauner et al., 1992; Randall and Brauner, 1991) and 600 g S. salar infected with 80 lice/fish (Wagner and McKinley, 2004; Wagner et al., 2003; Wagner et al., 2004). Moreover, 200 g infected S. salar have also been shown to upregulate gill NKA activity by as much as 60%, likely as an energetically costly attempt to restore hydromineral balance (Nolan et al., 1999). Based on these studies, the proximal effects of louse infection and the precocious nature of juvenile pink salmon ocean entry, we hypothesize that louse-induced performance reductions are related to ionoregulatory status.  11  1.10 CHAPTER 3 OBJECTIVES The ionic disruptions and ionoregulatory compensations associated with sea louse infection were measured in post-emergent pink salmon in field and laboratory settings. These data were subsequently used to determine a fine-scale threshold fish body mass for sub-lethal louse effects. These findings will hopefully promote more informed aquaculture management in British Columbia, ideally minimizing the interaction between open net-pen farms and wild pink salmon below the identified threshold.  Chapter 3 Hypothesis Louse infection impairs ionic homeostasis. Chapter 3 Predictions 1. Infected fish will increase gill NKA activity to compensate for the ionic disruption associated with increasing louse loads. 2. 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P. and Myers, K. W. (2004). Anadromy and the marine migrations of Pacific salmon and trout: Rounsefell revisited. Reviews in Fish Biology and Fisheries 14, 421. Randall, D. J., Baumgarten, D. and Malyusz, M. (1972). The relationship between gas and ion transfer across the gills of fishes. Comparative Biochemistry and Physiology Part A: Physiology 41, 629-637. Randall, D. J. and Brauner, C. J. (1991). Effects of environmental factors on exercise in fish. The Journal of Experiment Biology 160, 113-126. Riddell, B. E., Beamish, R. J., Richards, L. J. and Candy, J. R. (2008). Comment on "Declining wild salmon populations in relation to parasites from farm salmon". Science 322, 1790b-. Rounsefell, G. A. (1958). Anadromy in North American salmonidae. US Fish and Wildlife Fishery Bulletin 58, 171-185. Saloniemi, I., Jokikokko, E., Kallio-Nyberg, I., Jutila, E. and Pasanen, P. (2004). Survival of reared and wild Atlantic salmon smolts: size matters more in bad years. ICES Journal of Marine Science 61, 782-787. Smith, H. W. (1930). The abosrption and excretion of water and salts by marine teleosts. Am J Physiol 93, 480-505. Smith, H. W. (1932). Water Regulation and Its Evolution in the Fishes. The Quarterly Review of Biology 7, 1. Stefansson, S. O., Nilsen, T. O., Ebbesson, L. O. E., Wargelius, A., Madsen, S. S., Björnsson, B. T. and McCormick, S. D. (2007). Molecular mechanisms of continuous light inhibition of Atlantic salmon parr-smolt transformation. Aquaculture 273, 235-245. Sullivan, C. V., Brewer, S. D., Johnston, G. P. and Dickhoff, W. W. (1983). Plasma thyroid hormone concentrations and gill Na+,K+-ATPase activities in post-emergent pink salmon. Transactions of the American Fisheries Society 112, 825-829. Tully, O. (1992). Predicting infestation parameters and impacts of caligid copepods in wild and cultured fish populations. Invertebrate Reproduction and Development 22, 91-102. Wagner, G. N., Fast, M. D. and Johnson, S. C. (2008). 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Aspects of the biology of the parasitic copepods Lepeophtheirus salmonis and Caligus elongatus on farmed salmonids, and their treatment. Proceedings of the Royal Society, Edinburgh 81B, 185-197.  21  Chapter 2: Water Trumps Ion Balance in Early Marine Survival of Juvenile Pink Salmon (Oncorhynchus gorbuscha)1 2.1 INTRODUCTION Smoltification is a complex suite of behavioural, morphological and physiological changes that prepares anadromous salmonids for the transition from freshwater (FW) to the marine environment (for reviews see Boeuf, 1993; Folmar and Dickhoff, 1980; Hoar, 1976; Hoar, 1988; McCormick, 1994; McCormick and Saunders, 1987). Arguably the most dramatic change associated with this metamorphosis is the acquisition of seawater (SW) tolerance, in which fish switch from a hyper- to hypo-osmoregulatory strategy. To facilitate this remarkable shift, extensive remodelling of the major osmoregulatory organs is triggered by changes in photoperiod up to 2 months prior to seaward migration (Dickhoff et al., 1985; McCormick et al., 1987; Nilsen et al., 2007; Stefansson et al., 2007). The resulting preparation, which includes increases in gill Na+/K+-ATPase (NKA) activity and mRNA expression of the enzyme’s 1 subunit SW isoform, has been shown to vastly improve SW adaptability (Boeuf and Harache, 1982; Boeuf and Harache, 1984; Brauer, 1982; Folmar et al., 1982; McCormick et al., 1987; Nielsen et al., 1999; Nilsen et al., 2007; Nilsen et al., 2003; Otto, 1971). Pink salmon are an especially interesting animal system in which to study smoltification because they possess the smallest body size and shortest FW residency of any salmonid upon ocean entry. Pink fry migrate seaward directly following gravel emergence at a size of 0.2 g (Grant et al., 2009; Heard, 1991), while most other salmonids do so after at least one year in FW at sizes between 2-30 g (Clarke, 1982; Hoar, 1988; Quinn and Myers, 2004; Rounsefell, 1958). Even chum and those runs of chinook and sockeye that migrate seaward in the same year of  1  A version of this chapter will be submitted for publication. Sackville, M., Farrell, A.P. and Brauner, C.J. (2010) Water trumps ion balance in early marine survival of juvenile pink salmon, Oncorhynchus gorbuscha.  22  emergence are at least twice as large and spend weeks to months in FW prior to ocean entry (Clarke, 1982; Hoar, 1988; Quinn and Myers, 2004; Rounsefell, 1958). Seaward migration is thus particularly challenging to pink fry because of the high body surface area to volume ratio and relative lack of post-larval FW preparatory time. The near absence of preparatory time has spurred speculation of a novel strategy where hypo-osmoregulatory development begins at larval stages in the redd -perhaps without the usual photoperiod cue (Hoar, 1988; McCormick, 2009; Sullivan et al., 1983; Weisbart, 1968). Despite this exceptional life history, smoltification and the development of seawater tolerance in pink salmon remains largely unstudied. Previous work has shown that although larval pink alevins fail to survive seawater exposure prior to yolk sac absorption, they do exhibit signs of plasma ion regulation and possess a higher LT50 than other oncorhynchids (Weisbart, 1968). These results suggest larval hypo-osmoregulatory development, but extra-branchial MRC’s as seen on the yolk sac of chum alevins (Kaneko et al., 1995) could explain this effect. Further confounding interpretation is that fry experience a doubling of whole body Na+ and Clcontent that coincides with naturally timed SW entry (Grant et al., 2009). This elevation in whole body ion levels persists until gill NKA activity peaks two months later, which is well beyond the hours to days reported for other salmonids (O. keta, O. mykiss, O. kisutch, S. salar; Black, 1951; Leray et al., 1981; Miles and Smith, 1968; Prunet and Boeuf, 1985) and may indicate an underdeveloped hypo-osmoregulatory capacity. Furthermore, the majority of the increase in gill NKA activity occurs after seawater entry (Grant et al., 2009; Honma, 1982), suggesting compensatory rather than preparatory changes. Evidently, when and how SW tolerance and hypo-osmoregulatory ability of juvenile pink salmon develops remains unclear. The objective of this study was thus two-fold. 1) To determine if pink salmon possess adult-like hypo-osmoregulatory abilities as larval alevins, and 2) identify the physiological differences between alevins and post-larval fry critical to SW survival. To address these issues, I 23  measured ionoregulatory status of larval alevins and post-larval fry before and after 5 days of SW exposure.  2.2 MATERIALS AND METHODS Experimental Animals Pink salmon alevins aged 500 accumulated thermal units (ATU’s) were transported from Seymour River Hatchery to the University of British Columbia on February 28th 2008. Fish were separated and held in two 90 L static charcoal-filtered glass aquaria in dechlorinated Vancouver city tap water ([Na+], 0.17 mM; [Cl-], 0.21 mM; hardness, 30mg L-1 as CaCO3; pH 5.8-6.4) and maintained at a temperature of 3.5oC in total darkness to mimic gravel rearing conditions. Fish were treated according to University of British Columbia animal care protocol #A07-0055.  Transfer Protocol At 540 and 600 ATU’s, fish were randomly transferred to new 90 L static charcoalfiltered glass aquaria filled with either freshwater (FW; 3.5oC; composition listed above) or 100% seawater (SW; 3.5oC; 32.0 g L-1, prepared in dechlorinated Vancouver city tap water with Instant Ocean sea salt mix); transfer tanks were maintained on a simulated natural photoperiod (SNP) of 12L:12D to mimic gravel emergence. A total of 38 fish (N=10 for wet/dry mass and total body [Na+] and [Cl-]; N=10 for gill Na+/K+-ATPase activity; N=10 for drinking rates; N=8 for gill Na+/K+-ATPase  subunit mRNA expression) were sampled from the respective FW holding tanks pre-transfer (pre-transfer FW), and from FW and SW treatment tanks under SNP (FW+SNP, SW+SNP) at 1 and 5 days post-transfer. Fish aged 540 ATU’s, referred to as alevins, possess an externally visible yolk sac and do not naturally emerge from gravel at this stage in development to enter SW (M. Casselman; personal communication, Seymour River Hatchery). Fish aged 600 ATU’s, referred to as fry, no 24  longer possess an externally visible yolk sac and do naturally emerge from gravel at this stage in development to enter SW. Due to a limited number of fish, the FW+SNP treatment was foregone during alevin transfers. Additionally, drinking rates were only measured in SW+SNP treatment groups, as they were expected to be very low or absent in FW.  Body Mass and Total Body [Na+] and [Cl-] Fish were anaesthetized with a lethal dose of buffered tricaine methanesulfonate (0.8 g L1  MS-222; Syndel Laboratories, Vancouver, BC, Canada), rinsed in de-ionized water, blotted dry  and weighed. Fish were dried in pre-weighed 15 mL polystyrene tubes at 65oC until no further reduction in mass was observed. Dried fish were digested in 2 mL of 1 M nitric acid for 3 days at 65oC and the supernatant subsequently analyzed for Na+ and Cl- content. Supernatant [Na+] was measured using flame atomic absorption spectroscopy (Spectra AA-220FS; Varian, Mulgrave, VC, Australia) and standardized to wet and dry body mass for total body [Na+]. Supernatant [Cl-] was measured using the colorimetric mercuric thiocyanate method (Zall et al., 1956) and standardized to wet mass for total body [Cl-].  Gill Na+/K+-ATPase Activity Whole gills were removed and stored at -80 oC following anaesthetization as described above. A modified version of the method outlined by McCormick (1993) was used to determine NKA activity. This method couples ouabain-sensitive ATP hydrolysis to the oxidation of NADH by way of pyruvate kinase and lactate dehydrogenase. Briefly, whole frozen gills were homogenized on ice in SEI buffer (250 mM sucrose, 10 mM EDTA, 50 mM imidazole; pH 7.3), centrifuged at 5000 g for 1 min and the supernatant removed. Supernatant ATPase activity was measured spectrophotometrically in the presence and absence of ouabain (1 mM), and activity was taken as the difference between these conditions. Protein concentration was measured using 25  the bicinchoninic acid method (Sigma-Aldrich) and bovine serum albumin standards. NKA activity is reported as mol ADP mg-1 protein h-1. All samples were run in triplicate within 2 h of homogenization.  Drinking Rates Ten fish were placed in static polyethylene chambers containing 60 mL of aerated SW. To minimize disturbance, trials were completed in darkness and fish were permitted a 1 h chamber acclimation period. At trial onset, 90 Ci of [3H]polyethylene glycol (PEG-4000; American Radiolabeled Chemicals Inc., St. Louis, MO, USA) was added to the system and 5 mL of water sampled. Following 4 h of exposure, fish were lethally anaesthetized as described above and an additional 5 mL water sample was taken. Fish were rinsed thrice in de-ionized water to remove residual PEG, blotted dry and weighed. Fish were digested with 2 mL of 10% perchloric acid for 48 h at 65oC, then homogenized, vortexed and left to settle. 1 mL aliquots of clear supernatant and trial water samples were analyzed for radioactivity using a liquid scintillation counter (LSC-2000; Beckman-Coulter Inc., Fullerton, CA, USA). Water uptake was determined by dividing total counts per fish by the specific activity (Ci/ml H2O) of reference water samples. Drinking rates were calculated by expressing total water uptake relative to fish mass and [3H]polyethylene glycol exposure time. Drinking rates are reported as mL H2O kg-1 h-1.  Gill mRNA Total RNA was extracted from whole gills using the Invitrogen TRIzol Reagent according to the manufacturer’s instructions, and concentrations subsequently determined spectrophotometrically. First-strand cDNA was reverse transcribed according to Scott et al. (2004). Gene expression was assessed using quantitative real-time PCR (qRT-PCR) on an ABI Prism 7000 sequence analysis  26  system (Applied Biosystems). PCR reactions contained 1 l of cDNA, 4 pmol of each primer and Universal SYBR green master mix (Applied Biosystems) in a total volume of 21 l. Statistical Analyses Data are expressed as means ± s.e.m. A one-way analysis of variance (ANOVA) was used to analyze alevin data, and a two-way ANOVA was used for fry. The Holm-Sidak post-hoc test was subsequently applied for pairwise comparisons when effects were found to be significant. All data passed tests for normality and homogeneity of variance. All statistical analyses were conducted with Sigmastat (version 3.0, Systat Software Inc., San Jose, CA, USA), and a significance level of P<0.05 was used throughout.  2.3 RESULTS Alevin SW+SNP Transfers Alevin wet body mass was significantly reduced by 8% and 15% at 1 and 5 days postSW+SNP exposure, respectively (P<0.05, Fig. 2.1A). No significant change in alevin dry body mass was observed post-transfer (Fig. 2.1B), indicating that reductions in wet mass were due to water loss. Total body [Na+] of alevins expressed as a function of wet mass increased significantly by 15% relative to the pre-transfer FW control at 5 days post-SW+SNP exposure (P<0.05, Fig. 2.1C); however, total body [Na+] did not change when expressed as a function of dry mass (Fig. 2.1D). Total body [Cl-] expressed as a function of wet mass increased by 35% relative to the pretransfer FW control at 1 day post-SW+SNP exposure and remained elevated at 50 mol g wet mass-1 for trial duration (P<0.05, Fig. 2.1E). Gill NKA activity of alevins did not change significantly following SW+SNP exposure (Fig. 2.1F), but significant differences in 1 subunit mRNA expression were observed. The 1a/1b ratio was almost completely inversed relative to the pre-transfer FW value by day 5, 27  decreasing significantly from 1.91 to 0.54 (Fig. 2.2A). When expressed relative to total RNA, this relationship appears to be driven by a significant 60% decrease in 1a (Fig. 2.2A), as 1b did not change significantly over trial duration (Fig. 2.2C). A similar pattern in 1a &b expression was observed when expressed relative to the control genes EF-1 and Ub (Table 2.1), but a significant increase in 1b also appears to be driving this relationship. It should be noted that trends expressed relative to EF-1 and Ub were affected by significant decreases in both of these control genes (Table 2.1). Alevin drinking rates were measured as approximately 1.75 mL kg-1 h-1 at both 1 and 5 days post-SW+SNP transfer (Fig. 2.3).  Fry FW+SNP & SW+SNP Transfers No significant changes in wet or dry mass were observed in fry relative to the pretransfer FW values following FW+SNP and SW+SNP exposure (Fig. 2.4A,B), indicating that water balance was maintained. Total body [Na+] in fry expressed as a function of wet body mass increased significantly by 11% and 35% relative to the pre-transfer FW control at 1 and 5 days post-SW+SNP exposure, respectively (P<0.05, Fig. 2.4C). This increase exceeds that reported for alevins by more than two-fold. Additionally, values for fry 5 days post-SW+SNP exposure were significantly greater than time-matched FW+SNP values by roughly 20% (P<0.05, Fig. 2.4C). Total body [Na+] in fry expressed as a function of dry body mass also increased significantly relative to the pre-transfer FW control at 5 days post-SW+SNP exposure, with SW+SNP values significantly exceeding FW+SNP by roughly 15% (P<0.05, Fig. 2.4D). This result contrasts the lack of change observed in alevins. However, total body [Cl-] expressed as a function of wet mass in SW+SNP fry exhibited a pattern closely resembling that observed in alevins, increasing significantly above  28  pre-transfer FW and time-matched FW+SNP values to a plateau of approximately 50 mol g wet mass-1 for trial duration (P<0.05, Fig. 2.4E). While alevins failed to upregulate NKA activity following SW transfer, fry exhibited significant parallel increases relative to the pre-transfer value following both SW+SNP and FW+SNP exposure at 1 and 5 days post-transfer (P<0.05, Fig. 2.4F). The gill NKA 1a/1b mRNA ratio expressed relative to total RNA also exhibited parallel changes following FW+SNP and SW+SNP exposure (Fig. 2.5A), matching the trend observed in SW+SNP alevins. The 1.49 pre-transfer 1a/1b ratio decreased significantly to 0.54 by day 5 in FW+SNP and even further to 0.22 by day 5 in SW+SNP (P<0.05, Fig 2.5A). As in alevins, this relationship appears to be driven largely by significant decreases in 1a (Fig. 2.5B), as 1b did not change significantly over trial duration. When expressed relative to the control genes EF-1 and Ub (Table 2.2), similar patterns in the 1a/1b ratio were observed, but as with the alevin trials, a significant increase in 1b also appears to be driving this relationship. However, as also seen in the alevin trials, the trends expressed relative to EF-1 and Ub are affected by significant decreases in both of these control genes (Table 2.2). Fry drinking rates were measured as approximately 1.75 mL kg-1 h-1 at 1 and 5 days postSW+SNP transfer and are not significantly different from those reported for alevins (Fig. 2.3).  2.4 DISCUSSION The current study does not support the hypothesis that pink salmon possess adult-like hypo-osmoregulatory abilities as larvae. This point is clearly illustrated by a failure of larval alevins to maintain ion and water balance following SW exposure and a lack of upregulation in gill NKA activity. Furthermore, the key difference between larval alevins and post-larval fry critical to SW survival appears to be the maintenance of water balance, for which fry seemingly 29  sacrifice Na+ balance in doing so. We also provide the first evidence to suggest that photoperiod may trigger smolt-like increases in gill NKA activity of pink fry.  Do Larval Alevins Develop Adult-Like Hypo-Osmoregulatory Ability? Larval alevins show no evidence of adult-like hypo-osmoregulatory ability following SW exposure. Gill NKA activity does not increase as seen in post-larval stages (Honma, 1982, Grant et al. 2009), and levels remain low and comparable to those previously reported for FW presmolt salmonids (Madsen et al., 2009; Richards et al., 2003). A 35% increase in body [Cl-] persists 5 days post-SW entry, indicating a failure to maintain ion balance [the closely related chum regains [Cl-] balance 24 h post-transfer (Black 1951)], and the measures of wet and dry body mass clearly indicate a failure to maintain water balance. The measured drinking rate of 1.75 ml kg-1 h-1 for alevins transferred to SW+SNP equals that reported for fry and exceeds that reported for many salmonids in FW (0.1 for S. salar, 0.25 for O. mykiss; Fuentes and Eddy, 1997). However, alevins still dehydrate, suggesting that water uptake may not be occurring. Several points further support this interpretation. First, the method employed to determine drinking rate estimates water ingested into the gastrointestinal tract and not that absorbed across the gut wall, thus it is not a true measure of water uptake. Second, the primary source of Na+ uptake in SW salmonids is by active transport at the gut to facilitate water absorption (Grosell, 2006; Kirschner et al., 1974; Smith, 1930), yet [Na+] does not increase relative to dry mass. Finally, the lack of increase in gill NKA activity suggests that other changes typical of SW acclimation (i.e. intestinal remodelling for water uptake) may not take place, as they often co-occur (Collie and Bern, 1982; Loretz et al., 1982). Thus, the reported drinking rates may represent an ineffective behavioural response to the SW+SNP transfer.  30  Keys to Post-Larval Fry SW Survival In contrast to alevins, and as expected, fry appear to hypo-osmoregulate at the whole animal level following SW exposure. A significant increase in gill NKA activity accompanied by a significant decrease in the 1a/1b mRNA expression ratio indicates the induction of compensatory measures, while a significant increase in body [Na+] coupled with a lack of change in wet and dry mass suggests that water balance is maintained by drinking. Maintaining water balance thus appears to be the key difference between alevins and fry critical to SW survival; however, fry unexpectedly experience a greater whole body ionic disturbance than alevins following SW transfer. Anadromous salmonids are typically assessed as being prepared for the marine environment by the degree of ionic disturbance sustained at the plasma level following SW entry (24 h seawater challenge; Blackburn and Clarke, 1987; Clarke, 1982; Clarke and Blackburn, 1977). On a whole body level, water content may be a more suitable evaluation for pink salmon than a traditional ionic assessment as “prepared” fry experience an ionic perturbation three fold greater than “unprepared” alevins. Water balance therefore seems more critical to early marine survival, as pink fry actively sacrifice Na+ balance (nearly a two-fold increase; Grant et al. 2009) by drinking to maintain hydration. The prioritization of water balance does not imply that ion balance is unimportant to fry SW survival. Whole body levels still remain well below those of ambient SW, and the upregulation of gill NKA activity indicates an increased capacity for ion excretion. Although ion balance is not restored until gill NKA activity peaks eight weeks later (Grant et al. 2009), the degree of ion excretion afforded may be just as important to performance and survival if ion levels are kept below a certain critical threshold. This may explain the observed similarity between whole body [Cl-] of alevins and fry despite the presumed increase in intestinal uptake by the latter. 31  Photoperiod May Trigger Smoltification in Pink Salmon Upon controlled emergence from darkness to a natural photoperiod, changes in gill NKA activity and 1 isoform mRNA expression in FW fry parallel those of fry transferred directly to SW. These results demonstrate for the first time that photoperiod may trigger preparatory changes in pink salmon typical of smoltification. This differs from the current belief that ontogeny largely dictates the acquisition of salinity tolerance in pink salmon, which is supported by previously reported increases in gill NKA activity of FW fry following a surge in plasma thyroxine and yolk absorption (Sullivan et al., 1983). Although the present study lacks a suitable control (FW dark) to conclusively dissociate developmentally timed change from that triggered by environmental cues, the likelihood of coincidence between controlled emergence and ontogenetic change is low enough to merit speculation. The observed drinking rates and changes in 1 mRNA expression reported for alevins following SW+SNP exposure also support the notion of smolt-like environmental cue sensitivity, as they match those observed in FW/SW+SNP fry and occur well before the proposed ontogenetic switch at yolk absorption. Pink salmon are thus clearly capable of responding to environmental cues at transcriptional and behavioural levels prior to yolk absorption, but fail to mount an effective change in whole animal performance. Instead of an ontogenetic switch for hypo-osmoregulatory development, we propose that yolk absorption may mark a point in pink development where fry become capable of fully translating environmental cues into a preparatory smolt response. This proposed smolt competence could be linked to endocrine development and certainly warrants further investigation. If our hypothesis is confirmed, future work should also determine whether pinks respond to a specific photoperiod or if light exposure is sufficient to cue the appropriate changes.  32  Summary The current study sheds new light on the acquisition of SW tolerance in pink salmon. Contrary to long-held pre-conceptions, it was shown that pinks do not possess adult-like hypoosmoregulatory abilities as larvae and that post-larval preparatory change might be triggered by a smolt-like photoperiod cue rather than ontogenetic change. Furthermore, maintaining water balance appears to be the key difference between alevins and fry critical to SW survival. Fry actively incur large ion loads in SW to maintain hydration prior to the completion of hypoosmoregulatory development, which appears to be a novel strategy among salmonids. The ability to tolerate large, sustained ionic loads at the whole body level may be a key adaptation underlying this most extreme and perhaps derived form of salmonid anadromy.  33  0.22  LD 0:24  LD 12:12  0.056  A  LD 0:24 LD 12:12  B  0.20  0.052  a  Dry Mass (g)  Wet Mass (g)  0.21  0.19 b  0.18  0.048  0.044 b  0.17 0.16  0.040 LD 0:24  LD 12:12  LD 0:24  LD 12:12  D  LD 0:24  LD 12:12  F  280  +  60  50 b ab  40  320  C Total Body [Na ] -1 (mol.g dry mass )  +  Total Body [Na ] . -1 (mol g wet mass )  70  240  200  a  160  6 LD 0:24  LD 12:12  E Gill Na /K -ATPase Activity . -1. -1 mols ADP mg protein hr )  -  Total Body [Cl ] . -1 (mol g wet mass )  60  50  b  4  +  +  b  5  40 a  3  2 30  0 0  1  2  3  4  Days Post-seawater Transfer  5  0  1  2  3  4  5  Days Post-seawater Transfer  Figure 2.1. Wet mass (A), dry mass (B), body [Na+] as function of wet and dry mass (C, D), body [Cl-] as a function of wet mass (E) and gill Na+/K+-ATPase activity (F) of pink salmon alevins. Open circles represent alevins reared in freshwater under total darkness (pre-transfer FW), and closed circles represent alevins simultaneously exposed to seawater and a simulated natural photoperiod (SW+SNP). Values are means ± s.e.m. (n = 10); letters indicate significance (P<0.05).  34  2.0  LD 0:24  LD 12:12  A  a  1.5 b  1.0  +  +  Gill Na /K -ATPase 1a expression (relative to 1b )  2.5  c  0.5  0.0 LD 0:24  B  LD 12:12  2.0  1.5 a  a  1.0  +  +  Gill Na /K -ATPase 1a expression (relative to total RNA)  2.5  b  0.5  0.0 LD 0:24  C  LD 12:12  2.0  1.5  1.0  +  +  Gill Na /K -ATPase 1b expression (relative to total RNA)  2.5  0.5  0.0 0  1  2  3  4  5  Days Post-seawater Transfer  Figure 2.2. Gill Na+/K+-ATPase 1a mRNA expression relative to 1b (A) and total RNA (B), and 1b mRNA expression relative to total RNA (C) of pink salmon alevins. Open circles represent alevins reared in freshwater under total darkness (pre-transfer FW), and closed circles represent alevins simultaneously exposed to seawater and a simulated natural photoperiod (SW+SNP). Values are means ± s.e.m. (n = 6-10); letters indicate significance (P<0.05).  35  Drinking Rate (ml.kg-1.hr-1)  3  LD 0:24  LD 12:12  2  1  0 0  1  2  3  4  5  Days Post-seawater Transfer  Figure 2.3. Drinking rates of pink salmon alevins (open circles) and fry (closed circles) following simultaneous exposure to seawater and a simulated natural photoperiod (SW+SNP). Values are means ± s.e.m. (n = 10). No significant differences were found between groups.  36  0.056  0.22 LD 0:24  LD 12:12  A  B  LD 0:24  LD 12:12  LD 0:24  LD 12:12  LD 0:24  LD 12:12  0.21  Dry Mass (g)  Wet Mass (g)  0.052 0.20 0.19 0.18  0.048  0.044 0.17  0.040  0.16  70  LD 0:24  LD 12:12  320  C  * +  Total Body [Na ] -1 (mol.g dry mass )  280  +  60 +  +  50  40  +  +  +  Total Body [Na ] . -1 (mol g wet mass )  D  *  240  200  160  6  -  Total Body [Cl ] . -1 (mol g wet mass )  + +  50  40  5 +  * *  F  +  60  Gill Na+/K+-ATPase Activity . -1. -1 mols ADP mg protein hr )  E  +  LD 12:12  +  LD 0:24  4  3  2  30  0  0  1  2  3  Days Post-transfer  4  5  0  1  2  3  4  5  Days Post-transfer  Figure 2.4. Wet mass (A), dry mass (B), body [Na+] as function of wet and dry mass (C, D), body [Cl-] as a function of wet mass (E) and gill Na+/K+-ATPase activity (F) of pink salmon fry. Open circles at day “0” represent fry reared in freshwater under total darkness (pre-transfer FW), open circles at day “1” & “5” represent fry exposed to a simulated natural photoperiod while still in freshwater (FW+SNP), and closed circles represent fry simultaneously exposed to both seawater and a simulated natural photoperiod (SW+SNP). Values are means ± s.e.m. (n = 10). “+” indicates a significant difference from the pre-transfer FW value; asterisks indicate significant differences between time-matched FW+SNP and SW+SNP values (P<0.05).  37  LD 0:24  LD 12:12  A  2.0  1.5  1.0  *  +  0.5  +  +  +  +  Gill Na /K -ATPase 1a expression (relative to 1b )  2.5  0.0 LD 0:24  B  LD 12:12  2.0  1.5 *  + 0.5 *  +  +  +  1.0  +  Gill Na /K -ATPase 1a expression (relative to total RNA)  2.5  0.0 LD 0:24  LD 12:12  C  2.0  1.5  1.0  +  +  Gill Na /K -ATPase 1b expression (relative to total RNA)  2.5  0.5  0.0 0  1  2  3  4  5  Days Post-transfer  Figure 2.5. Gill Na+/K+-ATPase 1a mRNA expression relative to 1b (A) and total RNA (B), and 1b mRNA expression relative to total RNA (C) of pink salmon fry. Open circles at day “0” represent fry reared in freshwater under total darkness (pre-transfer FW), open circles at day “1” & “5” represent fry exposed to a simulated natural photoperiod while still in freshwater (FW+SNP), and closed circles represent fry simultaneously exposed to both seawater and a simulated natural photoperiod (SW+SNP). Values are means ± s.e.m. (n = 6-10). “+” indicates a significant difference from the pre-transfer FW value; asterisks indicate significant differences between time-matched FW+SNP and SW+SNP values (P<0.05).  38  Table 2.1. Expression of Na+/K+-ATPase 1a & 1b mRNA relative to EF-1 & Ub in pink salmon alevin gills pre- and post-SW+SNP exposure. Data are means + s.e.m.; letters indicate significance.  Treatment  1a/EF-1  1b/EF-1  EF-1/total RNA  1a/Ub  1b/Ub  Ub/total RNA  Pre-transfer FW  1.30±0.10 a  0.76±0.10 a  1.04±0.06 a  1.59±0.17 a  0.94±0.14 a  0.89±0.08 a  SW+SNP day 1  1.12±0.07 a  1.01±0.13 a  1.09±0.15 a  1.30±0.14 a,b  1.13±0.13 a  0.93±0.07 a  SW+SNP day 5  0.74±0.07 b  1.52±0.19 b  0.71±0.06 b  0.90±0.11 b  1.82±0.21 b  0.61±0.07 b  39  Table 2.2. Expression of Na+/K+-ATPase 1a & 1b mRNA relative to EF-1 & Ub in pink salmon fry gills pre- and post-FW+SNP & SW+SNP exposure. Data are means + s.e.m.; “t” indicates significance from pre-transfer FW values; asterisks indicate significance between timematched FW+SNP and SW+SNP values.  Treatment  1a/EF-1  1b/EF-1  EF-1/total RNA  1a/Ub  1b/Ub  Ub/total RNA  Pre-transfer FW  1.36±0.06  1.04±0.13  0.97±0.04  1.59±0.14  1.13±0.08  0.86±0.04  FW+SNP day 1  1.35±0.08  0.91±0.07  1.17±0.14  1.56±0.12  1.07±0.14  1.00±0.08  SW+SNP day 1  1.02±0.05 t*  1.14±0.11  0.94±0.12  1.21±0.10  1.35±0.16  0.86±0.16  FW+SNP day 5  1.34±0.25  3.08±0.54 t  0.49±0.10 t  2.27±0.55 t  4.79±0.72 t  0.29±0.03 t  SW+SNP day 5  0.72±0.07 t*  3.53±0.33 t  0.31±0.02 t  0.83±0.13 t*  4.40±0.75 t  0.34±0.10 t  40  2.5 REFERENCES Black, V. S. (1951). Changes in body chloride, density, and water content of chum (Oncorhynchus keta) and coho (O. kisutch) salmon fry when transferred from fresh water to sea water. Journal of the Fisheries Research Board of Canada 8, 164-177. Blackburn, J. and Clarke, W. C. (1987). Revised procedure for the 24 hour seawater challenge test to measure seawater adaptability of juvenile salmonids. Canadian Technical Report of Fisheries and Aquatic Sciences 1515, 1-35. Boeuf, G. (1993). Salmonid smolting: A pre-adaptation to the oceanic evironment. In Fish Ecophysiology, eds. J. C. Rankin and F. B. Jensen), pp. 105-135. London: Chapman Press. Boeuf, G. and Harache, Y. (1982). Criteria for adaptation of salmonids to high salinity seawater in France. Aquaculture 28, 163. Boeuf, G. and Harache, Y. (1984). Adaptation osmotique a l'eau de mer de differentes especes (Salmo trutta, Salmo gairdneri, Salvelinus fontinalis) et hybride (Salmo trutta ♀ × Salvelinus fontinalis ♂) de salmonides. Aquaculture 40, 343-358. Brauer, E. P. (1982). The photoperiod control of coho salmon smoltification. Aquaculture 28, 105-111. Clarke, W. C. (1982). Evaluation of the seawater challenge test as an index of marine survival. Aquaculture 28, 177-183. Clarke, W. C. and Blackburn, J. (1977). A seawater challenge test to measure smolting of juvenile salmon. Canadian Fisheries and Marine Service Technical Report 705, 11. Collie, N. L. and Bern, H. A. (1982). Changes in intestinal fluid transport associated with smoltification and seawater adaptation in coho salmon, Oncorhynchus kisutch (Walbaum). Journal of Fish Biology 21, 337-348. Dickhoff, W. W., Sullivan, C. V. and Mahnken, C. V. W. (1985). Thyroid hormones and gill ATPase during smoltification of Atlantic salmon (Salmo salar). Aquaculture 45, 376. Folmar, L. C. and Dickhoff, W. W. (1980). The parr-smolt transformation (smoltification) and seawater adaptation in salmonids: A review of selected literature. Aquaculture 21, 1-37. Folmar, L. C., Dickhoff, W. W., Mahnken, C. V. W. and Waknitz, F. W. (1982). Stunting and parr-reversion during smoltification of coho salmon (Oncorhynchus kisutch). Aquaculture 28, 91-104. Fuentes, J. and Eddy, F. B. (1997). Drinking in Atlantic salmon presmolts and smolts in response to growth hormone and salinity. Comparative Biochemistry and Physiology Part A: Physiology 117, 487-491. Grant, A., Gardner, M., Nendick, L., Sackville, M., Farrell, A. P. and Brauner, C. J. (2009). Growth and ionoregulatory ontogeny of wild and hatchery-raised juvenile pink salmon (Oncorhynchus gorbuscha). Canadian Journal of Zoology 87, 221-228. 41  Grosell, M. (2006). Intestinal anion exchange in marine fish osmoregulation. The Journal of Experiment Biology 209, 2813-2827. Heard, W. R. (1991). Life history of pink salmon (Oncorhynchus gorbuscha). In Pacific Salmon Life Histories, eds. C. Groot and L. Margolis), pp. 119-230. Vancouver: UBC Press. Hoar, W. S. (1976). Smolt transformation: evolution, behavior, and physiology. Journal of the Fisheries Research Board of Canada 33, 1234–1252. Hoar, W. S. (1988). The Physiology of Smolting Salmonids. In Fish Physiology, vol. 11 (B) eds. W. S. Hoar and D. J. Randall), pp. 275-343: Academic Press. Honma, M. (1982). Studies on the sea water adaptation of pink salmon (Oncorhynchus gorbuscha) in the early stages. Scientific Reports of the Hokkaido Fish Hatchery (Japan) 37, 2332. Kaneko, T., Hasegawa, S., Takagi, Y., Tagawa, M. and Hirano, T. (1995). Hypoosmoregulatory ability of eyed-stage embryos of chum salmon. Marine Biology 122, 165. Kirschner, L. B., Greenwald, L. and Sanders, M. (1974). On the mechanism of sodium extrusion across the irrigated gill of sea water-adapted rainbow trout (Salmo gairdneri). Journal of General Physiology 64, 148-165. Leray, C., Colin, D. A. and Florentz, A. (1981). Time course of osmotic adaptation and gill energetics of rainbow trout (Salmo gairdneri) following abrupt changes in external salinity. Journal of Comparative Physiology 144, 175-181. Loretz, C. A., Collie, N. L., Richman Iii, N. H. and Bern, H. A. (1982). Osmoregulatory changes accompanying smoltification in coho salmon. Aquaculture 28, 67-74. Madsen, S. S., Kiilerich, P. and Tipsmark, C. K. (2009). Multiplicity of expression of Na+,K+ATPase -subunit isoforms in the gill of Atlantic salmon (Salmo salar): cellular localisation and absolute quantification in response to salinity change. The Journal of Experiment Biology 212, 78-88. McCormick, S. (1993). Methods for nonlethal gill biopsy and measurement of Na+,K+-ATPase activity. Canadian Journal of Fisheries and Aquatic Sciences 50, 656-658. McCormick, S. D. (1994). Ontogeny and evolution of salinity tolerance in anadromous salmonids: Hormones and heterochrony. Estuaries and Coasts 17, 26-33. McCormick, S. D. (2009). Evolution of the hormonal control of animal performance: Insights from the seaward migration of salmon. Integrative and Comparative Biology 49, 408-422. McCormick, S. D. and Saunders, R. L. (1987). Preparatory physiological adaptations for marine life of salmonids: osmoregulation, growth and metabolism. Transactions of the American Fisheries Society 1, 211–229. McCormick, S. D., Saunders, R. L., Henderson, E. B. and Harmon, P. R. (1987). Photoperiod control of parr–smolt transformation in Atlantic salmon (Salmo salar): changes in 42  salinity tolerance, gill Na+,K+-ATPase activity and plasma thyroid hormones. Canadian Journal of Fisheries and Aquatic Sciences 44, 1462-1468. Miles, H. M. and Smith, L. S. (1968). Ionic regulation in migrating juvenile coho salmon. Oncorhynchus kisutch. Comparative Biochemistry and Physiology 26, 381-398. Nielsen, C., Madsen, S. S. and Björnsson, B. T. (1999). Changes in branchial and intestinal osmoregulatory mechanisms and growth hormone levels during smolting in hatchery-reared and wild brown trout. Journal of Fish Biology 54, 799-818. Nilsen, T. O., Ebbesson, L. O. E., Madsen, S. S., McCormick, S. D., Andersson, E., Bjornsson, B. T., Prunet, P. and Stefansson, S. O. (2007). Differential expression of gill Na+,K+-ATPase - and -subunits, Na+,K+,2Cl- cotransporter and CFTR anion channel in juvenile anadromous and landlocked Atlantic salmon Salmo salar. The Journal of Experiment Biology 210, 2885-2896. Nilsen, T. O., Ebbesson, L. O. E. and Stefansson, S. O. (2003). Smolting in anadromous and landlocked strains of Atlantic salmon (Salmo salar). Aquaculture 222, 71-82. Otto, R. G. (1971). Effects of salinity on the survival and growth of pre-smolt coho salmon (Oncorhychus kisutch). Journal of the Fisheries Research Board of Canada 28, 343-349. Prunet, P. and Boeuf, G. (1985). Plasma prolactin level during transfer of rainbow trout (Salmo gairdneri) and atlantic salmon (Salmo salar) from fresh water to sea water. Aquaculture 45, 167176. Quinn, T. P. and Myers, K. W. (2004). Anadromy and the marine migrations of Pacific salmon and trout: Rounsefell revisited. Reviews in Fish Biology and Fisheries 14, 421. Richards, J. G., Semple, J. W., Bystriansky, J. S. and Schulte, P. M. (2003). Na+/K+-ATPase -isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer. The Journal of Experimental Biology 206, 4475-4486. Rounsefell, G. A. (1958). Anadromy in North American salmonidae. US Fish and Wildlife Fishery Bulletin 58, 171-185. Scott, G. R., Richards, J. G., Forbush, B., Isenring, P. and Schulte, P. M. (2004). Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. American Journal of Physiology -Cell Physiology 287, C300-309. Smith, H. W. (1930). The abosrption and excretion of water and salts by marine teleosts. American Journal of Physiology 93, 480-505. Stefansson, S. O., Nilsen, T. O., Ebbesson, L. O. E., Wargelius, A., Madsen, S. S., Björnsson, B. T. and McCormick, S. D. (2007). Molecular mechanisms of continuous light inhibition of Atlantic salmon parr-smolt transformation. Aquaculture 273, 235-245. Sullivan, C. V., Brewer, S. D., Johnston, G. P. and Dickhoff, W. W. (1983). Plasma thyroid hormone concentrations and gill Na+,K+-ATPase activities in post-emergent pink salmon. Transactions of the American Fisheries Society 112, 825-829. 43  Weisbart, M. (1968). Osmotic and ionic regulation in embryos, alevins, and fry of the five species of Pacific salmon. Canadian Journal of Zoology 46, 385-397. Zall, D. M., Fisher, D. and Garner, M. G. (1956). Photometric determination of chlorides in water. Analytical Chemistry 28, 1665-1668.  44  Chapter 3: Pink Salmon (Oncorhynchus gorbuscha) HypoOsmoregulatory Development Plays a Key Role in Sea Louse (Lepeophtheirus salmonis) Tolerance2 3.1 INTRODUCTION The ectoparasitic salmon louse Lepeophtheirus salmonis has been recently implicated in pink salmon (Oncorhynchus gorbuscha) population declines and extinction predictions in British Columbia’s Broughton Archipelago (Krkosek et al., 2007a; Krkosek et al., 2007b; Krkosek et al., 2006; Krkosek et al., 2005; Krkosek et al., 2009; Morton and Routledge, 2005; Morton et al., 2004; Morton et al., 2005). Although these studies are compelling, major questions regarding parasite transmission and infection impacts on host physiology remain unknown. The uncertainty has led to considerable debate as to whether sea lice, specifically of farm origin, are negatively impacting local pink salmon populations (Beamish et al., 2006; Beamish et al., 2007; Brooks, 2005; Brooks and Jones, 2008; Brooks and Stucchi, 2006; Butterworth et al., 2008a; Butterworth et al., 2008b; Dill et al., 2009; Krkosek et al., 2008a; Krkosek et al., 2008b; Morton and Routledge, 2008; Riddell et al., 2008). A critical knowledge gap which the present study aims to address is the level of louse infection required to sub-lethally impair performance of outmigrating juvenile pink salmon weighing between 0.2 and 3 g. Lice feed on host mucous, epidermal tissue and blood (Brandal et al., 1976; Kabata, 1974) as they develop through four attached stages (termed chalimus 1-4) and three motile stages (termed pre-adult 1-2 & adult) on the fish’s exterior surface (Brooks, 2005; Johnson and Albright, 1991; Tully, 1992). As shown in 200 g Atlantic salmon and 90 g sea trout, this surface feeding can lead to significant increases in plasma electrolytes (up to 60%; Bjorn and Finstad, 2  A version of this chapter has been submitted for publication. Sackville, M., Tang, S., Nendick, L., Brauner, C.J. and Farrell, A.P. (2010) Pink salmon (Oncorhynchus gorbuscha) hypo-osmoregulatory development plays a key role in tolerating Lepeophtheirus salmonis infection.  45  1997; Bowers et al., 2000; Grimnes and Jakobsen, 1996; Nolan et al., 1999; Wootten et al., 1982). This hydromineral disruption is thought to result either directly from epidermal lesions that damage the external osmotic barrier or indirectly from a stress-related increase in the osmorespiratory compromise (Gonzalez and McDonald, 1994; Gonzalez and McDonald, 1992; Wendelaar Bonga, 1997). Regardless of origin, hydromineral imbalance is a major concern as it can lead to stunted growth (Brauer, 1982; Folmar et al., 1982; McCormick et al., 1987), reduced swim performance (Brauner et al., 1994; Brauner et al., 1992), and even death (Boeuf and Harache, 1982). While adult pink salmon are likely well equipped to cope with parasitism in the wild (Beamish et al., 2005), out-migrating juveniles may be less prepared. Foremost, fry enter seawater as small as 0.2 g (Heard, 1991) and are thus three orders of magnitude smaller than adult pinks when first exposed to lice (Jones and Hargreaves, 2007). Additionally, epidermal (Jones et al., 2008a), immune (Johnson et al., 1982) and hypo-osmoregulatory (Grant et al., 2009) systems all still develop during the first few months at sea. Since these physiological characteristics are presumably critical to L. salmonis infection resistance and tolerance, juvenile pink salmon are likely particularly vulnerable to parasitism during this precocious out-migration. Currently, only one study has quantified the sublethal effects of infection on outmigrating, 0.2-3 g pink salmon. One motile louse was shown to significantly reduce swimming performance in fry below, but not beyond, a body mass of 0.7 g (Nendick et al. 2010). Interestingly, this body mass threshold corresponds with the completion of pink salmon hypoosmoregulatory development (0.7-1.0 g; Grant et al., 2009), suggesting the observed effects might be linked to hypo-osmoregulatory ability. Similar reductions in swimming performance have also been correlated with elevated plasma ions in 8-20 g O. kisutch following SW entry (Brauner et al., 1994; Brauner et al., 1992; Randall and Brauner, 1991) and 600 g Salmo salar infected with 80 lice per fish (Wagner et al., 2003; Wagner et al., 2004). Furthermore, 200 g 46  infected S. salar have been shown to upregulate gill NKA activity by as much as 60% (Nolan et al., 1999), likely as an attempt to restore hydromineral balance. Based on these findings, we hypothesized that louse-induced performance reductions are ionoregulatory in origin. In a controlled laboratory setting and the wild, we investigated the ionic disruptions and ion regulatory compensations associated with sea louse-infection in post-emergent pink salmon for the first time. These data were used to determine a fine-scale threshold infection level for sublethal louse effects.  3.2 MATERIALS AND METHODS Research Facility All experiments were conducted at an autonomous field laboratory in the Broughton Archipelago, British Columbia, Canada between March and July of 2008. Constructed on a floathouse adjacent to Doctor Islets in Knight’s Inlet, the laboratory location provided easy access to juvenile pink salmon throughout the entirety of their near-shore migration (transport time from collection areas never exceeded 4 h). Laboratory holding tanks were equipped with flow-through aerated seawater (SW) drawn from depth at 30 m to ensure stable temperature (7.0-8.5 oC) and salinity (32-34 ppt), and the facility’s translucent canopy permitted a natural photoperiod throughout study duration. All fish were treated in accordance with the University of British Columbia Animal Care Committee and the Canadian Council on Animal Care.  Series 1: River-caught (RC) Fish, Laboratory Infection Experimental Animals River-caught (RC) fish: Approximately 2,000 post-emergent pink fry were collected with rotary screw-traps from the Glendale River during their down-stream migration on March 27th 2008. Fish were immediately transported to the field laboratory where they were gradually 47  exposed to full strength SW (34 ppt) over a period of 24 h. Initially at an approximate size of 25 mm and 0.2 g, pink fry were divided into four 100 L fibreglass holding tanks in which they were fed commercial trout chow twice daily (Bio-Vita starter feed; Bio-Oregon, Longview, WA). Glendale River was selected as a fish source because it is highly representative of the region’s fish, accounting for more than 35 and 85% of total Broughton pink salmon in even and odd years, respectively (Brooks and Jones, 2008). Out-migrating RC fish were used for laboratorybased infections for two reasons: (1) they had no prior exposure to sea lice; and (2) they represented the earliest and presumably most sensitive life-stage that could become infected in the wild given their precocious seawater entry. Lepeophtheirus salmonis: Approximately 300 gravid female lice were collected from adult Atlantic salmon on March 24th 2008 during a commercial harvest at Marine Harvest Canada’s Wicklow Point farm. Upon transport to the Doctor Islets laboratory, louse egg strings were incubated at 7 oC in four 4 L closed containers filled with aerated SW (34 ppt). Active nauplii were filtered out every two days with 96 m plankton mesh and placed in separate 2 L rearing chambers. Incubation water was changed every other day and nauplii monitored daily for copepodid development. Active copepodid densities were estimated by averaging counts from five 10 mL aliquots sampled from each rearing chamber. Infection was initiated when a copepodid:nauplii ratio of 3:1 was reached. Infection Protocol and Results L. salmonis infection was initiated on April 3rd 2008 after RC fish had spent 1 week in SW. A total of 585 fish was divided into subgroups of ~75 and placed into 8 separate 11 L infection chambers (modified POS plastic totes; Rubbermaid). Each chamber was filled with 3 L of static aerated SW and approximately 1,700 active copepodids, yielding a bath density of ~560 copepodids . L-1 and a copepodid:fish ratio of 23:1. Chamber dimensions were such that infection bath depth was only 5 cm, minimizing fish/louse stratification and maximizing host/parasite 48  interaction. Louse exposure was carried out in darkness for a total of 4 h, followed by a switch to flow-through SW to flush the infection bath. An additional 150 fish were subjected to an identical sham infection without lice. Approximately 48 h post-infection, all fish were lightly anaesthetized (0.05 g.L-1 MS222) and sorted according to louse load. 511 of 581 fish were successfully infected (a prevalence of ~88 %): 129 individuals (22%) were infected with 1 louse; 101 individuals (17%) with 2 lice; 99 individuals (17%) with 3 lice; 182 individuals (31%) with 4+ lice (where up to 20 lice were observed on some fish). Sampling Protocol and Constraints Following sorting, fish were placed in 10 separate 11 L holding tanks according to five categories of louse load (0, 1, 2, 3 and 4+ lice/fish; 2 replicate tanks/treatment). Each tank was equipped with flow-through SW (34 ppt, 7.0-8.5oC), held no more than 75 fish and was supplied with commercial trout chow twice daily. Fish were sampled over the ensuing 24 days as sea lice developed towards adults. For the experiments described in this study, a total of 20 fish (10 for whole body [Na+] and 10 for gill NKA activity) were sampled for four louse loads (0, 1, 2 and 3 lice/fish) at each of the four attached louse stages [chalimus 1-4; 3, 7, 15 and 24 days postinfection (DPI)]. All fish were examined by microscope at the time of sampling to determine louse number, developmental stage and surface area of epidermal tissue penetration. As reported in previous laboratory studies (Jones et al., 2008a; Jones et al., 2008b), infected pink salmon fry experienced high rates of louse loss throughout trial duration. Consequently, the number of infected fish (especially those with more than one louse) declined with time. To increase infected fish numbers, fish that were initially placed in the 4+ lice/fish group were re-sorted at 14 DPI (~chalimus 3 stage) and redistributed appropriately among the 13 lice/fish treatment tanks. A total of 99 fish were added to the sampling population (48 fish with 1 louse, 31 fish with 2 lice and 20 fish with 3 lice). Even with these additional animals, infected 49  fish numbers remained limited; hence measurements of gill NKA activity were necessarily restricted to fish with 1-2 lice of chalimus 1-3 stages, while measurements of whole body [Na+] beyond the chalimus 3 stage were limited to fish with 1 louse. All reported louse loads correspond to infection levels at the time of sampling and, as a result, louse loads for some individuals would have been higher prior to sampling. Thus, if anything, the impacts of particular louse loads are overestimated.  Series 2: Ocean-caught (OC) Fish, Wild Infection Over 10 000 wild pink salmon fry were captured by beach or purse seine during their near-shore migration through the Broughton Archipelago between April and June of 2008. These ocean-caught (OC) fish were graded directly following collection so that excess, uninfected fry could be released immediately on site and infected fry transported promptly back to the field laboratory. Fish were permitted a 24-h recovery period in the laboratory, then lightly anaesthetized (0.05 mg L-1 MS-222; Syndel) for sorting according to louse number and developmental stage. After an additional 24-h recovery period post-sort, fish were sampled for whole body [Na+] and/or gill NKA activity, and the area of epidermal tissue penetration measured as in Series 1. Fish were only sampled if a minimum of 8 infected, size-matched fish of similar louse number and developmental stage were collected at the same location on the same date. A group of 10 uninfected, size-matched fish were also sampled from the same collection as a control. This sampling regime was designed to minimize any variation attributable to fish development and environmental experience. Total louse prevalence among this large sample of fish was roughly 10%, with an approximate intensity of 1. This low infection intensity restricted analysis to fish infected with 1 attached stage louse. As with Series 1, assay priority was given to whole body [Na+] analysis.  50  Series 3: River-caught (RC) Fish, Mechanical Epidermal Abrasion To mimic mechanical damage inflicted by motile stage lice, the epidermis of uninfected RC fish was subjected to various degrees of artificial abrasion following completion of Series 1. Using even pressure with a #2 scalpel blade, lightly anaesthetized fish approximately 0.7 g and 50 mm in size were subjected to: 1) 1 mm2 surface scale removal, 2) 1 mm2 epidermal penetration, or 3) 2 mm2 epidermal penetration. The surface areas for abrasion were selected to match motile louse mouth sizes, and fish size corresponded to that of wild fish first encountering motile lice. All lesions were located posterior to the dorsal fin on the lateral line, and abrasion time never exceeded 30 s. A sham group was abraded in identical fashion with the scalpel handle to serve as a control. Following abrasion, fish were held in duplicate tanks for each of the four treatment groups. Holding tanks, water conditions and fish care were identical to those of Series 1. For whole body [Na+] and gill NKA activity, a total of 20 fish from each treatment were sampled pre-abrasion, and at 1 and 5 days post-abrasion as described below. Gill NKA activity was not measured in the 2 mm2 abrasion group due to limited fish availability.  Assays Wet & Dry Mass, Total Body [Na+] Fish were anaesthetized with a lethal dose of buffered tricaine methanesulfonate (0.8 g L1  MS-222; Syndel Laboratories, Vancouver, BC, Canada), rinsed in de-ionized water, blotted dry  and weighed for wet body mass. Individual fish were subsequently dried at 65oC to a constant dry mass and digested in 1 M nitric acid (1 mL of acid for every 0.1 g of wet tissue mass). Digest supernatant was subsequently analyzed for Na+ concentration ([Na+]) using flame atomic absorption spectroscopy (Spectra AA-220FS; Varian, Mulgrave, VC, Australia). Total body [Na+] is reported as mol Na+ g-1 wet mass. 51  Gill Na+/K+-ATPase Activity Whole gills were removed from lethally anaesthetized fish (see above) and stored at 80oC. To determine gill NKA activity, a modified version of the method outlined by McCormick (McCormick, 1993) was used. Briefly, individual whole frozen gills were homogenized on ice in SEI buffer (250 mM sucrose, 10 mM EDTA, 50 mM imidazole; pH 7.3). Homogenates were centrifuged at 5,000 g for 1 min and the supernatant placed on ice. Supernatant ATPase activity was measured spectrophotometrically in the presence and absence of ouabain (1 mM), and taken as the difference between conditions. Protein concentration was measured using the bicinchoninic acid method (Sigma-Aldrich) with bovine serum albumin standards. NKA activity is reported as mol ADP mg-1 protein h-1.  Statistical Analyses Data are expressed as means ± s.e.m. A two-way analysis of variance (ANOVA) was used throughout to determine significant differences, and the Holm-Sidak post-hoc test was subsequently applied for pairwise comparisons. All data passed tests for normality and homogeneity of variance. All statistical analyses were conducted with Sigmastat (version 3.0, Systat Software Inc., San Jose, CA, USA), and a significance level of P<0.05 was used throughout.  3.3 RESULTS Series 1: River-caught (RC) Fish, Laboratory Infection Fish Mortality, Growth and Louse-induced Damage Of the initial 438 infected fish (1-3 lice/fish), only 25 animals died during the 24-day trial. There was no mortality among control fish, and so overall louse-induced mortality was approximately 6%. Of these 25 dead fish, 17 originated from the 4+ lice/fish group and were 52  added to the 1-3 lice/fish groups following a re-sort at 14 DPI. These fish could have been initially infected with as many as 20 lice/fish. Mortality among fish originally infected with 1-3 lice/fish was therefore 2.4 %. Control fish grew significantly from 0.224 + 0.014 g to 0.358 + 0.019 g over a 24-day period, yielding a specific growth rate (SGR) of approximately 2.26 + 0.66 % body mass day-1 (Table 3.1). Growth of infected fish was similar, with SGR ranging from 1.23 to 1.86 % body mass day-1. Fish mass only differed significantly from the control group at 7 DPI in the 2 lice/fish treatment (P < 0.05; Table 3.1). Superficial, louse-inflicted epidermal damage was visible on host fish as early as 3 DPI (chalimus 1 stage), and epidermal penetration was present in all three treatments by 15 DPI (chalimus 3 stage; Table 3.1). The area of exposed subepidermal tissue increased significantly by 3-fold between 15 and 24 DPI in the 1 louse/fish treatment (P < 0.05; Table 3.1), during which time lice developed from the chalimus 3 to 4 stage. No significant differences among louse loads were found, but statistical power was low (0.187).  Total body [Na+] and Gill Na+/K+-ATPase As expected for a normal developmental trajectory in SW (see Grant et al., 2009), total body [Na+] of control fish decreased steadily and significantly from approximately 81 mol.g-1 at day 0 to 73 mol.g-1 by 24 DPI (P < 0.05; Figure 3.1A). Total body [Na+] of fish infected with 1 louse did not differ significantly from control values until 24 DPI (chalimus 4 stage, Figure 3.1 A; P < 0.05), when a 12 % increase was observed. A similar but larger and earlier significant increase in total body [Na+] occurred in fish infected with 2 and 3 lice, where a 23 % increase was observed by 15 DPI (chalimus 3 stage, Figure 3.1A; P < 0.05). Thus, all three infection loads had a threshold louse developmental stage that triggered an ionic disturbance. This threshold was reached earlier in louse development when fish were host to more than one louse. 53  Although two lice triggered a greater ionic disturbance than 1 louse, no significant differences in total body [Na+] were observed between 2 and 3 lice/fish. As also expected for a normal developmental trajectory in SW, gill NKA activity of control fish increased steadily and significantly from approximately 7 mol ADP.mg-1.h-1 at day 0 to 11mol ADP.mg-1.hr-1 by 15 DPI (Figure 3.1B; P < 0.05). In contrast, gill NKA activity of infected fish was significantly depressed as early as 3 DPI with just 1 louse (chalimus 1 stage, Figure 3.1B; P < 0.05). This approximate 40 % depression in enzyme activity was maintained independent of louse load until at least 7 DPI (chalimus 2 stage). By 15 DPI (chalimus 3 stage), gill NKA activity had recovered to control levels in all treatments. Therefore, rather than showing a compensatory increase in association with elevated ionic loads, infected fish exhibited an initial reduction in gill NKA activity.  Series 2: Ocean-caught (OC) Fish, Wild Infection As with Series 1, epidermal penetration was not visible in infected OC fish prior to the chalimus 3 stage. Epidermal damage was also only visible in the smallest size class of OC fish (0.474 + 0.043 g; Table 3.2.) Total body [Na+] and gill NKA activity of uninfected OC fish followed the same patterns observed for control fish of Series 1. As fish mass varied from ~0.5 g to ~1.5 g, total body [Na+] decreased significantly from ~ 70 mol.g-1 to ~ 50 mol.g-1 (Figure 3.2A; P < 0.05). At the same time, gill NKA activity increased significantly from ~10 mol ADP.mg-1.h-1 to ~14 mol ADP.mg-1.h-1 (Figure 3.2B; P < 0.05). In contrast to Series 1, total body [Na+] and gill NKA activity of infected OC fish did not differ significantly from size-matched uninfected OC fish (Figure 3.2; P > 0.05).  54  Series 3: River-caught (RC) Fish, Mechanical Epidermal Abrasion The area of epidermal abrasion applied to RC fish exceeded the maximum area of louseinduced abrasion by nearly 10-fold (Series 1 & 2). Despite this excessive damage, neither total body [Na+] nor gill NKA activity differed significantly from respective time-matched controls (Figure 3.3; P > 0.05). Furthermore, total body [Na+] and gill NKA activity were similar to sizematched control fish in other experiments.  Estimation of Threshold for Lice Effects The data sets for total body [Na+] from Series 1 & 2 were pooled for analysis across a greater size range to determine the body mass threshold for louse-induced ionoregulatory disturbance. Second order polynomial regression lines were fitted to data of total uninfected and total infected fish for statistical comparison. Total body [Na+] of fish infected with one chalimus 4 louse was significantly higher than that of uninfected fish when fish mass was below 0.5 g (Figure 3.4A), as clearly indicated by non-overlapping 95% confidence intervals. The observed difference in body [Na+] between infected and uninfected fish below this threshold body mass of 0.5 g decreased with increasing fish mass and gill NKA activity (Figure 3.4A&B).  3.4 DISCUSSION Using ecologically relevant infection levels of 1-3 attached stage lice/fish, we document some of the first sublethal physiological impacts of sea lice on post-emergent pink salmon. We conclude that beyond a threshold fish size of 0.5 g, one attached-stage louse (chalimus 1-4) no longer significantly disrupts hydromineral balance. Furthermore, we propose that this threshold is linked to pink salmon hypo-osmoregulatory development, which likely plays a key role in coping with the effects of any louse-induced damage or stress incurred. This notion is further strengthened by the maintenance of hydromineral balance in hypo-osmoregulatory advanced fish 55  (~0.7 g body mass) despite facing levels of experimenter-induced skin abrasion ten-fold that inflicted by lice in Series 1 and 2. Results from the Series 1 laboratory infection study clearly show that 1 louse at the chalimus 4 stage and 2 to 3 lice at the chalimus 3 stage are sufficient to significantly disturb total body Na+ balance in pink salmon weighing less than 0.5 g. Because louse loads decreased over time in many fish, reported infection loads for a given sample point may have been less than actual infection histories. Similarly, louse numbers for our Series 2 wild-infected OC fish also could have been greater prior to examination. Consequently, our measures of impact and the identified threshold, if anything, err on the side of caution. We propose that this size-related louse tolerance is linked to pink salmon hypoosmoregulatory development, adding to a previously suggested multi-factorial mechanism based on epidermal and immune system development (Jones et al., 2008a). A primary proximal effect of louse infection is physical damage to the host epidermis (Brandal et al., 1976; Kabata, 1974), which can potentially lead to hydromineral imbalance by disrupting the osmotic barrier and/or inducing a stress response (Wendelaar Bonga, 1997). While the immune system and epidermis combat proximal effects of parasitism (Jones et al., 2008a; Jones, 2001; Jones et al., 2008b), hypo-osmoregulatory ability enables fish to cope with the hydromineral consequences of any damage or attachment stress that might occur. The inverse relationship between total body Na+ load and gill NKA activity of infected fish supports this notion, as does the absence of any ionic loading beyond attainment of maximum gill NKA activity. The more hypo-osmoregulatory advanced fish from Series 3 even maintain Na+ balance despite levels of skin abrasion ten-fold that inflicted by lice in Series 1 and 2. Although the nature of this damage differs considerably, this result suggests the potential to tolerate louse loads beyond those explored here upon reaching this developmental threshold. It should also be noted that our findings are consistent with the proposed role of epidermal and immune system development in louse resistance (Jones 56  et al., 2008a), as the epidermal damage for a given louse load decreases with increasing fish mass. Louse-induced mortality of small, river-caught (RC) fish from the laboratory infection study was only 6% over 24 days. This does not exclude the possibility of increased lethality with further louse development, nor does it preclude the possibility that the observed sub-lethal ionic disturbances negatively impact fish performance and, ultimately, survival in the wild. An associated study found reductions in pink salmon swimming performance to occur at infection thresholds similar to those identified here for hydromineral balance (Nendick et al. 2010), indicating that the ion load may indeed impart a performance cost. Growth could also be negatively impacted, and although differences between infected and control fish were not observed here, low statistical power could be masking an effect (power = 0.112). The mechanisms through which small juvenile pink salmon cope with this louse-induced ion load remain unclear. As in hydrominerally challenged rainbow trout, other areas of the body may buffer plasma and critical tissue osmolarity (Bath and Eddy, 1979; Wood and Randall, 1973a; Wood and Randall, 1973b; Wood and Randall, 1973c), potentially coming at a cost to aerobic scope, energy stores and/or recipient buffer tissue function. Any of these impacts could explain the previously reported reductions in swimming ability (Nendick et al. 2010). Even the larger, more developed fish from Series 2 & 3 could be incurring substantial energetic costs despite maintaining ion balance following infection and abrasion. Measuring resting metabolic rate and metabolic scope would be a simple and effective way to quantify the energetic costs associated with louse infection and any resulting ionoregulatory challenge on a finer scale, thus making an excellent complement to future studies examining the effects of parasitism on fish development and survival. Originally, gill NKA activity of infected fish was predicted to increase as a compensatory response to any resulting hydromineral disturbance. This was surprisingly not observed in any 57  louse-infected or abraded fish. In fact, gill NKA activity was temporarily lower in laboratoryinfected RC fish. This reduction might be the product of a generalized stress response (Wendelaar Bonga, 1997), as stress-related elevations in cortisol have previously been paired with similar reductions in gill NKA activity of uninfected, confined chinook salmon (Strange et al., 1978). Alternatively, the observed reduction could reflect an infection bias toward weaker and less developed fish. A major limitation to studying louse impact is the inability to control which fish become infected and retain lice. As a result, infected fish may represent a subset of the population more susceptible to parasitism. Such fish may have less developed immune and epidermal systems, which may facilitate preferential louse attachment and retention (Jones et al., 2008a). If infected fish were relatively underdeveloped physiologically, hypo-osmoregulatory ability might also lag behind the population mean, thus potentially explaining the lower gill NKA activity. Sampling those fish that reject parasites or resist initial infection would help clarify this issue while providing further insight into the mechanisms underlying host susceptibility.  Summary This study provides some of the first sublethal measures of physiological impact by sea lice on juvenile pink salmon in both a controlled laboratory setting and the wild. We clearly show that hypo-osmoregulatory development is important in tolerating the effects of sea louse parasitism and identify a threshold fish mass of 0.5 g below which one attached stage louse results in sub-lethal hydromineral disruption. This size threshold is also consistent with those previously based on louse-induced swimming performance reductions (0.7 g; Nendick et al., 2010) and the development of infection resistance mechanisms (immunocompetence and epidermis; 0.5-0.7 g; Jones et al., 2008a). From these data, we recommend that management decisions be made to minimize the anthropogenic-induced interactions between sea lice and 58  juvenile pink salmon prior to the completion of hypo-osmoregulatory development; specifically, fish weighing less than 0.7-1.0 g (Grant et al., 2009). These results should also be further integrated with surrogate measures of ecological fitness before more definitive conclusions regarding fish survival can be made. Despite low levels of fish mortality, high levels of louse rejection and the limitation of sublethal hydromineral disruption to the smallest of fish, the consequences of louse infection may prove to be quite different in the wild.  59  A * *  90  +  Total Body [Na ] . -1 (mol g wet mass )  100  *  a  80  ab b  b  b  70 C1  C2  C3  C4  Gill Na+/K+-ATPase Activity . -1. -1 mols ADP mg protein hr )  0 14  B  12  b b  10 a 8  *  a  Control 1 Louse 2 Lice 3 Lice  *  6  *  4  C1  * C2  C3  0 0  5  10  15  20  25  30  Days Post-infection  Figure 3.1. Total body [Na+] (A) and gill Na+/K+-ATPase activity (B) of river-caught pink salmon artificially infected with L. salmonis copepodids (0-3 lice/fish) and held over time as indicated by days post-infection. Fish were sampled at each of the four chalimus developmental stages (C1-4). Data points represent mean ± SEM, n = 10. Asterisks indicate statistically significant differences from time-match controls; letters that differ indicate statistically significant differences within the control group (P < 0.05).  60  +  Total Body [Na ] . -1 (mol g wet mass )  80  A  70 a  60  b  c  50  B  16  14  b ab  12  +  +  Gill Na /K -ATPase Activity . -1. -1 mols ADP mg protein hr )  0  a  10  Control C1/2 C3/4  0 0.0  0.5  1.0  1.5  2.0  Wet Mass (g)  Figure 3.2. Total body [Na+] (A) and gill Na+/K+-ATPase activity (B) of ocean-caught pink salmon naturally infected with one L. salmonis chalimus. Infected data are pooled according to louse stage [chalimus 1&2 (open circles) or chalimus 3&4 (shaded circles)] and expressed relative to fish wet mass. Data points represent mean ± SEM, n = 10. Infected fish do not differ significantly from size-matched controls; letters that differ indicate statistically significant differences within the control group (P < 0.05).  61  A  60  +  Total Body [Na ] . -1 (mol g wet mass )  65  55  50 Fish Mass = 0.74 +/- 0.02 g  B  16  14  12  +  +  Gill Na /K -ATPase . -1. -1 mols ADP mg protein hr )  0  10  Control 2 De-scaled (1 mm ) 2 Lesion (1 mm ) 2 Lesion (2 mm )  0 0  1  2  3  4  5  Days Post-abrasion  Figure 3.3. Total body [Na+] (A) and gill Na+/K+-ATPase activity (B) of river-caught pink salmon following various degrees of mechanical epidermal abrasion. Mean fish mass is 0.738 g + 0.019 SEM. Data are means ± SEM, n = 10; treatment groups did not differ significantly from control (P < 0.05).  62  100  A  +  Total Body [Na ] . -1 (mol g wet mass )  90 Series 1 -Control Series 1- C4 Series 2- Control Series 2- C4  80 70 60 50 40 0  B 20 16 12  +  +  Gill Na /K -ATPase Activity . -1. -1 mols ADP mg protein hr )  24  8 4 0 0.0  0.5  1.0  1.5  2.0  Wet Mass (g)  Figure 3.4. Total body [Na+] (A) and gill Na+/K+-ATPase activity (B) of infected (shaded symbols) and uninfected (open symbols) pink salmon fry expressed as a function of wet mass. Circles represent individual river-caught fish from Series 1 infected in a laboratory setting, and triangles represent individual ocean-caught fish from Series 2 infected in the wild. All infected fish possessed a load of one stage 4 chalimus louse. Regression lines are second order polynomials based on total infected [white; y = 84.6-(41.7x)+(12.5x2); R2 = 0.87; P < 0.001; F = 140] and uninfected [grey; y =109.3–(99.0x)+(43.8x2); R2 = 0.89; P < 0.001; F = 84.5] data points. Thin lines represent 95 % confidence intervals.  63  Table 3.1. River-caught Fish, Laboratory Infection (Series 1): Fish mass and quantified area of exposed sub-epidermal tissue  DPI  Control  1 Louse  2 Lice  3 Lice  Day 0  0.224 ± 0.014 g ----  N/A  N/A  N/A  Day 3 (C1)  0.243 ± 0.009 g ----  0.224 ± 0.011 g ND  0.234 ± 0.009 g ND  0.235 ± 0.008 g ND  Day 7 (C2)  0.272 ± 0.011 g ----  0.254 ± 0.016 g ND  0.231 ± 0.015* g ND  0.249 ± 0.010 g ND  Day 14 (C3)  0.283 ± 0.010 g ----  0.313 ± 0.015 g 0.10 ± 0.05 a mm2  0.279 ± 0.019 g 0.20 ± 0.06 ab mm2  0.259 ± 0.014 g 0.15 ± 0.06 ab mm2  Day 24 (C4)  0.358 ± 0.019 g ----  0.336 ± 0.031 g 0.30 ± 0.08 b mm2  N/A  N/A  Specific Growth Rate  2.26 ± 0.66 % over 24 days  1.86 ± 1.00 % over 24 days  1.48 ± 0.86 % over 14 days  1.23 ± 0.34 % over 14 days  Mean ± SEM, n = 10. DPI refers to days post infection; C1-4 refers to chalimus developmental stages 1-4; ND = not detectable. Asterisks indicate significant differences from time-matched controls for fish mass; different letters indicate statistically significant differences from timematched controls for tissue damage (P < 0.05). Specific growth rate is calculated as % body weight/day.  64  Table 3.2. Ocean-caught (OC) Fish, Wild Infections (Series 2): Fish mass and area of subepidermal tissue exposure  Fish Mass (g)  C1/2  C3/4  0.474 ± 0.043  ND  0.16 ± 0.11 mm2  0.974 ± 0.065  ND  ND  1.409 ± 0.085  ND  N/A  Mean ± SEM, n = 10. C1-4 refers to chalimus developmental stages 1-4. ND = not detectable. All fish were infected with 1 louse. Reported damage is the total area of exposed sub-epidermal tissue.  65  3.5 REFERENCES Bath, R. N. and Eddy, F. B. (1979). Salt and water balance in rainbow trout (Salmo gairdneri) rapidly transferred from fresh water to sea water. Journal of Experiment Biology 83, 193-202. Beamish, R. J., Jones, S., Neville, C.-E., Sweeting, R., Karreman, G., Saksida, S. and Gordon, E. 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The occurrence and mechanisms of innate immunity against parasites in fish. Developmental & Comparative Immunology 25, 841-852. Jones, S. R. M., Fast, M. D. and Johnson, S. C. (2008b). Influence of reduced feed ration on Lepeophtheirus salmonis infestation and inflammatory gene expression in juvenile pink salmon (Oncorhynchus gorbuscha). Journal of Aquatic Animal Health 20, 103-109. Jones, S. R. M. and Hargreaves, N. B. (2007). The abundance and distribution of Lepeoptheirus salmonis (copepoda: caligidae) on pink (Oncorhynchus gorbuscha) and chum (O. keta) salmon in coastal British Columbia. Journal of Parasitology 93, 1324-1331. Kabata, Z. (1974). Mouth and mode of feeding of Caligidae (Copepoda), parasites of fishes, as determined by light and scanning electron microscopy. Journal of the Fisheries Research Board of Canada 31, 1583-1588. Krkosek, M., Ford, J. S., Morton, A., Lele, S. and Lewis, M. A. (2008a). Response to comment on "declining wild salmon populations in relation to parasites from farm salmon". Science 322, 1790c-. Krkosek, M., Ford, J. S., Morton, A., Lele, S. and Lewis, M. A. (2008b). Sea lice and pink salmon declines: a response to Brooks and Jones (2008). Reviews in Fisheries Science 16, 413 420. Krkosek, M., Ford, J. S., Morton, A., Lele, S., Myers, R. A. and Lewis, M. A. (2007a). Declining wild salmon populations in relation to parasites from farm salmon. Science 318, 17721775. Krkosek, M., Gottesfeld, A., Proctor, B., Rolston, D., Carr-Harris, C. and Lewis, M. A. (2007b). Effects of host migration, diversity and aquaculture on sea lice threats to Pacific salmon populations. Proceedings of the Royal Society B: Biological Sciences 274, 3141-3149. Krkosek, M., Lewis, M. A., Morton, A., Frazer, L. N. and Volpe, J. P. (2006). Epizootics of wild fish induced by farm fish. Proceedings of the National Academy of Sciences 103, 1550615510. Krkosek, M., Lewis, M. A. and Volpe, J. P. (2005). Transmission dynamics of parasitic sea lice from farm to wild salmon. Proceedings of the Royal Society B: Biological Sciences 272, 689-696. Krkosek, M., Morton, A., Volpe, J. P. and Lewis, M. A. (2009). Sea lice and salmon population dynamics: effects of exposure time for migratory fish. Proceedings of the Royal Society B: Biological Sciences 276, 2819-2828. McCormick, S. (1993). Methods for nonlethal gill biopsy and measurement of Na+,K+-ATPase activity. Canadian Journal of Fisheries and Aquatic Sciences 50, 656-658. McCormick, S. D., Saunders, R. L., Henderson, E. B. and Harmon, P. R. (1987). Photoperiod control of parr–smolt transformation in Atlantic salmon (Salmo salar): changes in salinity tolerance, gill Na+,K+-ATPase activity and plasma thyroid hormones. Canadian Journal of Fisheries and Aquatic Sciences 44, 1462-1468. 68  Morton, A. and Routledge, R. (2005). Mortality rates for juvenile pink (Oncorhynchus gorbuscha) and chum (O. keta) salmon infested with sea lice (Lepeophtheirus salmonis) in the Broughton Archipelago. Alaska Fishery Research Bulletin 11, 146–152 11, 146-152. Morton, A. and Routledge, R. (2008). Comment on Butterworth, Cubitt, and McKinley (2008). Fisheries Research 92, 345. Morton, A., Routledge, R., Peet, C. and Ladwig, A. (2004). Sea lice (Lepeophtheirus salmonis) infection rates on juvenile pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon in the nearshore marine environment of British Columbia, Canada. Canadian Journal of Fisheries & Aquatic Sciences 61, 147. Morton, A., Routledge, R. D. and Williams, R. (2005). Temporal patterns of sea louse infestation on wild pacific salmon in relation to the fallowing of Atlantic salmon farms. North American Journal of Fisheries Management 25, 811-821. Nolan, D. T., Reilly, P. and Wendelaar Bonga, S. E. (1999). Infection with low numbers of the sea louse Lepeophtheirus salmonis induces stress-related effects in post-smolt Atlantic salmon (Salmo salar). Canadian Journal of Fisheries and Aquatic Sciences 56, 947-959. Randall, D. J. and Brauner, C. J. (1991). Effects of environmental factors on exercise in fish. The Journal of Experiment Biology 160, 113-126. Riddell, B. E., Beamish, R. J., Richards, L. J. and Candy, J. R. (2008). Comment on "Declining wild salmon populations in relation to parasites from farm salmon". Science 322, 1790b-. Strange, R. J., Schreck, C. B. and Ewing, R. D. (1978). Cortisol concentrations in confined juvenile chinook salmon (Oncorhynchus tshawytscha). Transactions of the American Fisheries Society 107, 812-819. Tully, O. (1992). Predicting infestation parameters and impacts of caligid copepods in wild and cultured fish populations. Invertebrate Reproduction and Development 22, 91-102. Wagner, G. N., McKinley, R. S., Bjørn, P. A. and Finstad, B. (2003). Physiological impact of sea lice on swimming performance of Atlantic salmon. Journal of Fish Biology 62, 1000-1009. Wagner, G. N., McKinley, R. S., Bjørn, P. A. and Finstad, B. (2004). Short-term freshwater exposure benefits sea lice-infected Atlantic salmon. Journal of Fish Biology 64, 1593-1604. Wendelaar Bonga, S. E. (1997). The stress response in fish. Physiological Reviews 77, 591625. Wood, C. M. and Randall, D. J. (1973a). The influence of swimming activity on sodium balance in the rainbow trout (Salmo gairdneri). Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 82, 207-233. Wood, C. M. and Randall, D. J. (1973b). The influence of swimming activity on water balance in the rainbow trout (Salmo gairdneri). Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 82, 257-276. 69  Wood, C. M. and Randall, D. J. (1973c). Sodium balance in the rainbow trout (Salmo gairdneri) during extended exercise. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 82, 235-256. Wootten, R., Smith, J. W. and Needham, E. A. (1982). Aspects of the biology of the parasitic copepods Lepeophtheirus salmonis and Caligus elongatus on farmed salmonids, and their treatment. Proceedings of the Royal Society, Edinburgh 81B, 185-197.  70  Chapter 4: General Discussion  This thesis is a blend of basic and applied research that investigates the hydromineral challenges associated with early ocean entry in pink salmon. Chapter two furthers our understanding of the most extreme and perhaps derived form of salmonid anadromy, while chapter three applies this knowledge to promote sustainable policy development within British Columbia’s aquaculture industry.  4.1 CHAPTER 2 SUMMARY: THE HYPER-OSMOTIC ENVIRONMENT The near absence of post-emergent freshwater preparatory time afforded to pink salmon makes their remarkable early ocean entry particularly daunting. To meet this challenge, pinks were hypothesized to undergo ontogenetically driven hypo-osmoregulatory development as larval alevins in the redd (Hoar, 1988; McCormick, 2009; Weisbart, 1968). Chapter two rejects this hypothesis, as alevins neither increase gill NKA activity nor maintain hydromineral balance following seawater exposure. Furthermore, evidence suggests pink fry may undergo a photoperiod-triggered smoltification similar to that shared by other members of the genus Oncorhynchus. Active ion loading to facilitate water balance is revealed as the true novel strategy underlying this unique life history. By successfully sustaining large ionic disturbances on a whole body level, pink fry maintain water balance and thus survive ocean entry two months prior to the completion of branchial ion excretion machinery development.  4.2 A NEW FORM OF ANADROMY? Active ion loading to facilitate water balance is an essential component of what appears to be a distinct form of salmonid anadromy. Two major osmoregulatory hurdles accompany  71  increasingly early ocean entry: (1) an accelerated transition from FW to SW and (2) an increased body surface area to volume ratio (SA:V). Predictive anadromy meets the first challenge by significantly reducing the lag-time between downstream migration and ocean entry common to facultative anadromy (McCormick, 1994; McCormick, 2009). Likewise, increased hypoosmoregulatory ability can overcome the greater osmoregulatory challenge associated with a higher SA:V in smaller fish (McCormick and Naiman, 1984). However, the internal ion loading of pink salmon may further combat both constraints by masking hypo-osmoregulatory deficiency and thus eliminating the need for post-emergent FW preparatory time. Entering seawater with underdeveloped ion excretion machinery distinguishes pink salmon anadromy from the more ancestral facultative and predictive forms. We therefore propose that this unique life history be termed “precocious anadromy.”  4.3 EVOLUTIONARY IMPLICATIONS Two relationships suggest that natural selection may favour early ocean entry among salmonids. First, those more derived species generally enter the ocean earlier and at smaller sizes (Clarke, 1982; Hoar, 1988; McCormick, 2009; McCormick and Saunders, 1987; McKay et al., 1996; Oakley and Phillips, 1999). Second, those species entering the ocean earliest are also the most abundant and widely distributed (Augerot and Foley, 2005; Quinn, 2005; Quinn and Myers, 2004). Although predation is accepted as the major selective pressure dictating size at ocean entry (Hargreaves and LeBrasseur, 1986; Holtby et al., 1990; Saloniemi et al., 2004), osmoregulatory challenges are also believed to play a significant role (Clarke et al., 1989; McCormick and Naiman, 1984). Internal ion loading could thus be argued as an adaptation for what is perhaps the most derived and successful form of salmonid anadromy, as it likely incurs selective advantages beyond those provided by predictive anadromy and increased hypoosmoregulatory ability alone. For example, by facilitating earlier access to productive marine 72  waters, this trait could result in earlier rapid growth and maturation. Accelerated maturation could result in the short generation time possessed by pinks, which in turn benefits population growth and abundance. Shorter FW residency may also disrupt natal imprinting, perhaps leading to the observed high stray rate and increased distribution of pinks (Quinn, 2005). The implications are even more pronounced if this trait facilitates larval rearing in estuaries. Reduced spawning migration distance could increase fecundity by allowing adults to invest more into reproduction rather than migratory effort. Furthermore, reduced dependency on FW rearing conditions could improve the likelihood that strays encounter suitable habitat, thus increasing species dispersal. A seemingly limitless number of plausible scenarios can be generated to rationalize the benefits of internal ion tolerance to salmonid abundance and distribution.  4.4 FUTURE BASIC RESEARCH DIRECTIONS Future research should investigate the suspected photoperiod response in pink fry and determine how they tolerate such high internal ion loads. The mechanism underlying this tolerance may resemble that observed in rainbow trout, where plasma osmolarity is buffered with other tissues following osmoregulatory stress (Bath and Eddy, 1979; Eddy and Bath, 1979; Wood and Randall, 1973a; Wood and Randall, 1973b; Wood and Randall, 1973c). If pink salmon are shown to possess a similar but greater internal buffer capacity, this trait could represent a constraint on size at ocean entry. Comparisons within and across salmonid species that vary in smolt size could perhaps reveal the adaptive role, if any, of this capacity in shaping salmonid evolution and anadromy in general. On a broader scale, this trait could be expressed to different degrees by many fishes, serving as a general strategy to overcome hydromineral challenges when whole body osmoregulatory ability is insufficient.  73  4.5 CHAPTER 3 SUMMARY: SEA LOUSE PARASITISM Interestingly, the same life history facilitating early ocean entry in pink salmon appears to result in greater susceptibility to louse infection. Chapter three clearly shows an increased vulnerability to sea louse parasitism prior to the completion of hypo-osmoregulatory development, as evidenced by the inverse relationship between total body Na+ load and gill NKA activity of infected fish. Hypo-osmoregulatory developed fish even maintain Na+ balance despite levels of artificial abrasion ten-fold that inflicted by lice in our experimental trials. Although the immune system and epidermis likely combat proximal effects of infection, hypo-osmoregulatory ability clearly enables fish to cope with the hydromineral consequences of any damage or attachment stress that might occur.  4.6 APPLIED CONTRIBUTIONS Generating a concrete threshold fish mass for sublethal louse impacts is perhaps this chapter’s greatest contribution. Our identified window of sensitivity agrees with that proposed by Jones and colleagues (2008) and matches previous measurements of louse-induced performance reductions (Nendick et al., 2010). Together, these findings have directly influenced current aquaculture management in the Broughton Archipelago. Recommendations based heavily on these findings have led Marine Harvest Canada Ltd. to remove active fish farms along the pink out-migration route and initiate parasite control measures earlier in season. This chapter can therefore be deemed a success, as the ultimate goal of applied research is to positively shape industry practice.  4.7 FUTURE APPLIED RESEARCH DIRECTIONS Sustainable aquaculture strives to conserve and enrich its surrounding natural and socioeconomic environments (Frankic and Hershner, 2003). Management decisions taken to 74  ensure the conservation of local ecological integrity thus require an understanding of the complex interactions between open-net pens and their environs. Although this chapter has already positively shaped aquaculture practice in British Columbia with respect to sea louse exposure, many important questions remain. Beyond the obvious repetition of our experiments with higher parasite loads and more developed lice, the additional measurements of resting metabolic rate and aerobic scope would provide excellent estimates of the total energetic cost associated with louse infection. These numbers, integrated with measures of performance and ecological fitness, would provide a more complete understanding of infection impacts for individual fish. However, exactly how fish farms contribute to local louse populations still remains unknown. This issue must be resolved if open-net pen aquaculture is to be managed confidently in the presence of wild fish populations.  75  4.8 REFERENCES Augerot, X. and Foley, D. N. (2005). Atlas of Pacific Salmon: The First Map-Based Assessment of Salmon in the North Pacific: University of California Press. Bath, R. N. and Eddy, F. B. (1979). Salt and water balance in rainbow trout (Salmo gairdneri) rapidly transferred from fresh water to sea water. Journal of Experiment Biology 83, 193-202. Clarke, W. C. (1982). Evaluation of the seawater challenge test as an index of marine survival. Aquaculture 28, 177-183. Clarke, W. C., Shelbourn, J. E., Ogasawara, T. and Hirano, T. (1989). Effect of initial daylength on growth, seawater adaptability and plasma growth hormone levels in underyearling coho, chinook, and chum salmon. Aquaculture 82, 51. Eddy, F. B. and Bath, R. N. (1979). Ionic regulation in rainbow trout (Salmo gairdneri) adapted to fresh water and dilute sea water. The Journal of Experiment Biology 83, 181-192. Frankic, A. and Hershner, C. (2003). Sustainable aquaculture: developing the promise of aquaculture. Aquaculture International 11, 517-530. Hargreaves, N. B. and LeBrasseur, R. J. (1986). Size selectivity of coho (Oncorhynchus kisutch) preying on juvenile chum salmon (O. keta). Canadian Journal of Fisheries and Aquatic Sciences 43, 581-586. Hoar, W. S. (1988). The Physiology of Smolting Salmonids. In Fish Physiology, vol. 11 (B) eds. W. S. Hoar and D. J. Randall), pp. 275-343: Academic Press. Holtby, L. B., Andersen, B. C. and Kadowaki, R. K. (1990). Importance of smolt size and early ocean growth to interannual variability in marine survival of coho salmon (Oncorhynchus kisutch). Canadian Journal of Fisheries and Aquatic Sciences 47, 2181-2194. Jones, S., Kim, E. and Bennett, W. (2008). Early development of resistance to the salmon louse, Lepeophtheirus salmonis (Kroyer), in juvenile pink salmon, Oncorhynchus gorbuscha (Walbaum). Journal of Fish Diseases 31, 591-600. McCormick, S. D. (1994). Ontogeny and evolution of salinity tolerance in anadromous salmonids: Hormones and heterochrony. Estuaries and Coasts 17, 26-33. McCormick, S. D. (2009). Evolution of the hormonal control of animal performance: Insights from the seaward migration of salmon. Integrative and Comparative Biology 49, 408-422. McCormick, S. D. and Naiman, R. J. (1984). Osmoregulation in the brook trout, Salvelinus fontinalis, II. Effects of size, age and photoperiod on seawater survival and ionic regulation. Comparative Biochemistry and Physiology Part A: Physiology 79, 17. McCormick, S. D. and Saunders, R. L. (1987). Preparatory physiological adaptations for marine life of salmonids: osmoregulation, growth and metabolism. Transactions of the American Fisheries Society 1, 211–229.  76  McKay, S. J., Devlin, R. H. and Smith, M. J. (1996). Phylogeny of Pacific salmon and trout based on growth hormone type-2 and mitochondrial NADH dehydrogenase subunit 3 DNA sequences. Canadian Journal of Fisheries and Aquatic Sciences 53, 1165-1176. Oakley, T. H. and Phillips, R. B. (1999). Phylogeny of Salmonine Fishes Based on Growth Hormone Introns: Atlantic (Salmo) and Pacific (Oncorhynchus) Salmon Are Not Sister Taxa. Molecular Phylogenetics and Evolution 11, 381. Quinn, T. P. (2005). The Behavior and Ecology of Pacific Salmon and Trout: University of Washington Press. Quinn, T. P. and Myers, K. W. (2004). Anadromy and the marine migrations of Pacific salmon and trout: Rounsefell revisited. Reviews in Fish Biology and Fisheries 14, 421. Saloniemi, I., Jokikokko, E., Kallio-Nyberg, I., Jutila, E. and Pasanen, P. (2004). Survival of reared and wild Atlantic salmon smolts: size matters more in bad years. ICES Journal of Marine Science 61, 782-787. Weisbart, M. (1968). Osmotic and ionic regulation in embryos, alevins, and fry of the five species of Pacific salmon. Canadian Journal of Zoology 46, 385-397. Wood, C. M. and Randall, D. J. (1973a). The influence of swimming activity on sodium balance in the rainbow trout (Salmo gairdneri). Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 82, 207-233. Wood, C. M. and Randall, D. J. (1973b). The influence of swimming activity on water balance in the rainbow trout (Salmo gairdneri). Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 82, 257-276. Wood, C. M. and Randall, D. J. (1973c). Sodium balance in the rainbow trout (Salmo gairdneri) during extended exercise. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 82, 235-256.  77  

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