DEPARTMENT OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES

LOWER SALINITY DECREASES CARDIAC OUTPUT BUT MAINTAINS GUT BLOOD FLOW IN THE MARINE SHORTHORN SCULPIN (MYOXOCEPHALUS SCORPIUS) -REDUCED COSTS OF OSMOREGULATION

Erika Sundell

Degree project for Master of Science (120 hec) with a major in BiologyBIO727, Physiology and cell biology, 60 hecSecond cycle Semester/year: Autumn 2018/Spring 2019Supervisor: Erik Sandblom, department of biological and environmental sciencesExaminer: Michael Axelsson, department of biological and environmental sciences

Table of content

ABSTRACT.............................................................................................................................................................2

SAMMANFATTNING (ABSTRACT)..................................................................................................................3

INTRODUCTION...................................................................................................................................................4

OSMOREGULATION BY FRESHWATER AND SEAWATER TELEOSTS.........................................................................4

ENERGETIC DEMANDS ACROSS SALINITIES...........................................................................................................5

IMPORTANCE OF THE CARDIOVASCULAR SYSTEM IN RESPONSE TO WATER SALINITY..........................................6

AIMS AND HYPOTHESES........................................................................................................................................7

MATERIAL AND METHODS..............................................................................................................................7

EXPERIMENTAL ANIMALS AND HOLDING CONDITIONS.........................................................................................7

EXPERIMENTAL PROTOCOLS.................................................................................................................................7

Experiment 1: Energy expenditure in seawater and 15 ppt salinity...............................................................8

Experiment 2: Blood flow and metabolic rate during acclimation to 15 ppt salinity.....................................8

Surgical Instrumentations..............................................................................................................................................8

Experimental protocol...................................................................................................................................................9

RESPIROMETRY SET UP.......................................................................................................................................10

DATA ACQUISITION AND ANALYSIS....................................................................................................................10

Statistical analysis.........................................................................................................................................12

RESULTS..............................................................................................................................................................13

EFFECTS OF ACUTE EXPOSURE TO 15 PPT ON METABOLISM AND BLOOD COMPOSITION....................................13

EFFECTS OF ACCLIMATION TO LOWER SALINITY ON BLOOD FLOW AND METABOLISM......................................15

DISCUSSION........................................................................................................................................................18

LOWER WATER SALINITY DECREASES METABOLIC RATE AND CARDIAC OUTPUT...............................................18

Gut blood flow is maintained in across salinities.........................................................................................19

Shorthorn sculpin are weak regulators of plasma osmolality and ion composition.....................................20

A role of altered arteriovenous branchial blood flow in reduced salinity?..................................................20

CONCLUSIONS AND FUTURE PERSPECTIVES........................................................................................................21

ACKNOWLEDGEMENTS..................................................................................................................................22

REFERENCES......................................................................................................................................................23

APPENDIX I...........................................................................................................................................................1

ABSTRACT

Ongoing climate change is predicted to exacerbate future salinity variations in marine environments, which can impose challenges for fish. Decreased rates of water loss and salt load when salinity decreases in hyperosmotic environments may require osmoregulatory adjustments to maintain internal homeostasis. Freshwater-acclimated euryhaline teleost rainbow trout upregulates both gut blood flow and cardiac output in response to increased water salinity. Yet, the cardiovascular and metabolic responses to reduced salinity in marine euryhaline teleosts are presently unknown. The aim of this study was, therefore, to assess the effects of reduced seawater salinity on cardiorespiratory function in the euryhaline marine teleost shorthorn sculpin (Myoxocephalus scorpius). Uninstrumented sculpins were placed in respirometers to measure standard metabolic rate (SMR) over 24 hours in seawater (33 ppt), followed by 96 hours in 15 ppt. In another group of sculpins acclimated to seawater or 15 ppt, flow probes were placed around the celiacomesenteric artery and the ventral aorta before placing the fish in respirometers for simultaneous in vivo measurements of gut blood flow, cardiac output, heart rate and routine metabolic rate (RMR). Uninstrumented sculpin displayed lowered SMR when exposed to 15 ppt salinity compared to seawater. Similarly, RMR was reduced in instrumented sculpins acclimated to 15 ppt, which was accompanied with reduced cardiac output and stroke volume, whereas gut blood flow was maintained across salinities. The reduced metabolic rate at the lower salinity indicates lower cost of osmoregulation in 15 compared to 33 ppt salinity, which is consistent with the reduced cardiac output. As gut blood flow was maintained while cardiac output was reduced at 15 ppt, the most probable cause for the lower metabolic rate is decreased costs of osmoregulation at the gills. This may involve less shunting of blood to the osmoregulatory arteriovenous pathway, which is consistent with the overall decrease in cardiac output.

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SAMMANFATTNING (ABSTRACT)

De pågående klimatförändringarna förutspås skapa förändring i vattensalthalten, vilket kan vara utmanande för akvatiska organismer. För att bibehålla osmotisk homeostas krävs att fiskar kan ändra sin osmoreglering som svar på ändringar i omgivningens salthalt. Bland annat kan fiskar ändra regleringen av det kardiovaskulära systemet för att möta nya förhållanden. Regnbågen, som är en euryhalin sötvattensfisk, ökar både hjärtminutvolym och blodflöde till magtarmkanalen när salthalten ökar. Troligen för att kunna öka det jon-kopplade vattenupptaget över tarmen, som är nödvändigt i en hyperosmotisk miljö. Effekterna på det kardiovaskulära systemet av minskad salthalt hos euryhalina teleoster är dock okända. Syftet med denna studie var att undersöka effekten av minskad salthalt på kardiorespiratoriska funktioner hos den marina fiskarten rötsimpa (Myoxocephalus scorpius). Rötsimpor placerades i respirometrar för att mäta syreförbrukning, som mått på fiskarnas standardmetabolism. Mätningarna gjordes under 24 timmar i saltvatten (33 ppt) och därefter över 96 timmar i 15 ppt. En annan grupp av rötsimpor korttids-acklimerades till antingen 33 eller 15 ppt. Dessa fiskar instrumenterades med flödesprober runt ventralaortan och den celiakomesenteriska artären. Fiskarna placerades i respirometrar för simultana in vivo mätningar av blodflöde till magtarmkanalen, hjärtminutvolym, hjärtfrekvens och rutinmetabolism. Såväl de oinstrumenterade som de instrumenterade rötsimporna visade minskad metabolism i den lägre salthalten. Även en minskad hjärtminutvolym på grund av en minskning i hjärtats slagvolym uppmättes. Blodflödet till magtarmkanalen förändrades däremot inte. Den minskade syreförbrukningen i lägre salthalt tyder på att fisken har lägre energikostnader för osmoreglering i 15 jämfört med 33 ppt. Då blodflödet till magtarmkanalen inte påverkades av förändringar i salthalt är en trolig förklaring till minskad metabolism en lägre energikostnad för osmoreglering i gälarna. Detta leder troligen till en shuntning av blodet i gälarna från det osmoreglerande, arterio-venösa systemet och till en övergripande minskning i gälblodflöde, vilket stämmer överens med den minskade hjärtminutvolymen uppmätt i den lägre salthalten.

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INTRODUCTION

Climate predictions foresee a future with greater salinity variability in the coastal areas of the world (Elliot et al., 2015). For example, potential reductions in salinity are expected in estuaries and coastal areas due to freshwater (FW) run off and increased precipitation (Meier et al., 2012), whereas the salinity of the water is likely to increase in more arid regions due to reduced precipitation and increased evaporation (Ruby and Ahilan, 2018). Additionally, longer periods of both drought and precipitation acting on the same area may greatly exacerbate the local variations in salinity (Jeppesen et al., 2015). Changes in environmental salinity can be challenging for teleost fishes as they need to maintain plasma osmolality relatively constant ( 300 mOsm kg-1; Marshall and Grosell, 2006). Teleosts are the largest group of fishes and inhabit water salinities between <0.1 to over 2400 mOsm kg-1 (Edwards and Marshall, 2013). Some teleost species are stenohaline and tolerate only small fluctuations in salinity, whereas others are euryhaline and can regulate ion and plasma levels across a broad range of salinities (Kultz, 2015). Euryhaline teleost therefore possesses a phenotypic plasticity for osmoregulation, which allows them to manage migrations across salinities and to withstand stationary salinity fluctuations within their natural habitats.

Osmoregulation by freshwater and seawater teleostsDepending on the salinity of the surrounding water, different osmoregulatory strategies are required by fish to keep plasma and ion levels constant (Figure 1). FW generally has a volume loading effect in which fish tend to lose ions and gain water, whereas seawater (SW) has a volume depleting effect where fish tend to lose water and gain ions (Edwards and Marshall, 2013). To counteract hypervolemia in FW, fish produce and excrete large volumes of dilute urine through high glomerular filtration rates and renal reabsorption of monovalent ions (Greenwell et al., 2003). FW living fish also have an active uptake of ions from the surrounding water across specialized cells in the gills and from the diet across the intestine (Evans, 1993; Marshall and Smith, 1930).

In the gills of FW fish, Na+ is taken up by Na+/H+ exchange, Na+ channels driven by proton pumps and via Na+/Cl- co-transport (Evans, 2008). Cl- is also transported into the cells via Cl-/HCO3

- exchange (Evans, 2011; Kumai and Perry, 2010). On the contrary, fish in SW conserve water by excreting low volumes of urine and counteract salt load by an active excretion of excess ions across the gills and in the kidney (Evans et al., 2005; Grosell, 2011). Monovalent ions (e.g., Na+ and Cl-) are mainly excreted across the gills, whereas divalent ions (e.g., SO4

2- and Mg2+) are primarily excreted by the kidney (Greenwell et al., 2003; Marshall and Grosell, 2006). Moreover, marine fish actively drink large amounts of SW to restore fluid volumes (Bath and Eddy, 1979; Evans and Claiborne, 2009). Imbibed SW is rapidly desalinized in the esophagus, which is important to reduce the osmolality of the water that enters the intestinal lumen to allow for intestinal absorption of ions and water through solute-linked water absorption mechanisms (Grosell, 2010; Kirsch and Meister, 1982; Laverty and Skadhauge, 2012). In these mechanisms, basolateral Na+/K+-ATPases in the intestinal epithelium creates a Na+ gradient that drives apical Na+/Cl- and Na+/K+/2Cl- co-transporters to transport ions from the intestinal lumen across the intestinal epithelium. In turn, this creates an osmotic pressure that drives the inward directed flow of water into the blood (Grosell, 2013; Whittamore, 2012). Anion exchangers in the apical part of the intestinal epithelium also absorbs Cl- in exchange for HCO3

-. In the intestinal lumen, HCO3-

reacts with Ca2+ to form CaCO3 precipitates. This precipitation reduces the osmolality of the luminal fluid even further and thus, contributes to the intestinal water absorption (Grosell, 2011; Wilson et al., 2002).

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Figure 1. Fish osmoregulatory challenges and processes in freshwater (FW; 0 ppt salinity) and seawater (SW; 33 ppt salinity). The figure shows a schematic drawing of a river that enters the ocean via an estuary. In FW, fish tend to gain water and lose ions. This volume loading is counteracted by the active absorption of ions and by excreting high volumes of dilute urine. In SW, fish tend to lose water and gain ions and avoid hypovolemia by drinking SW and excreting ions.

Energetic demands across salinitiesThe maintenance of osmotic homeostasis across salinities is energetically costly for fish as ions are being transported against their concentration gradients (Evans and Claiborne, 2009). Since less active transport is required for osmoregulation in water near iso-osmotic to the fish plasma, an iso-osmotic external environment has been suggested to be less energetically demanding than a hyper- or hypoosmotic environment (Ern et al., 2014; Morgan and Iwama, 1998). On the other hand, water salinities to which fishes are adapted to have also been suggested to be the least energetically costly medium as alterations in physiology, morphology and behavior may be required in salinities outside of this range, potentially increasing the energetic costs (Laverty and Skadhauge, 2012; Lee et al., 1996). With that said, it may be more energetically costly to live in fluctuating salinities or salinities that deviates from the preferred range of the fish, even if that means iso-osmotic waters (Kultz, 2015). To date, research results are conflicting regarding which water osmolality that is the least energetically costly from an osmoregulatory perspective (Ern et al., 2014). In fact, the most energetically beneficial medium appears to vary both within and among species and can depend on acclimation state or the stage within the life cycle (Brijs et al., 2015, 2016; Nordlie, 1978; Toepfer, and Barton, 1992). However, changes in environmental salinity is indeed a potential stressor for most fishes and can increase the energetic demands of fish as they need to compensate for osmotic gradients to avoid altered osmolality of internal fluids (Kultz, 2015).

The minimum amount of energy used by unfed fish to cover basic functions is called the standard metabolic rate (SMR), whereas the maximum metabolic rate represents the metabolic rate at maximum activity levels, or for inactive species after activities such as feeding (Fry, 1971). In between the SMR and maximum metabolic rate is routine metabolic rate (RMR), which

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Low urine excretion

Passive loss of water

Active excretion of ions, passive loss of water & gain of salts

Drinking SW

Path of waterPath of ionsPassive processesActive processes

High urine excretion

Passive gain of water

Active uptake of ions, passive gain of water & loss of

ions

Dietary ions

Brackish water

SW

FW

refers to the metabolic rate during normal and spontaneous activity (Steffensen, 2005). The metabolic rate in fishes has commonly been estimated from measurements of oxygen consumption rate (Cech, 1990). The theoretical basis behind this is that aerobic metabolism depends on oxygen, thus, reflecting the metabolic rate when sufficient oxygen is available. However, limitations to this estimation exist as anaerobic metabolism is not taken into consideration, resulting in a possible underestimation of the true metabolic rate (Nelson, 2016). In discussions of energy expenditure across salinities, the measured variable is often oxygen uptake, but is assumed to reflect the metabolic rate and hence the energetic demands of the fish (Chabot et al., 2016). Therefore, the rate of oxygen uptake by the fish will be referred to as oxygen consumption rate, which will be used synonymously with metabolic rate throughout this thesis.

Importance of the cardiovascular system in response to water salinityRecent studies have shown that in order for teleost fish to withstand fluctuations in environmental salinity, the cardiovascular system plays an important role in perfusing osmoregulatory organs with sufficient amounts of blood for osmoregulatory functions (Brijs et al., 2015, 2016, 2017; Sundell et al., 2018). A suite of cardiovascular adjustments has been observed in FW-acclimated rainbow trout (Oncorhynchus mykiss), both when acutely exposed and after long-term acclimation to SW. For example, gut blood flow increases at least two-fold in SW compared to FW (Brijs et al., 2015, 2016). This elevation in gut blood flow is believed to reflect an increased metabolic demand of the gut tissues (e.g., due to increased activity of Na+/K+-ATPases) and serve to increase the convection of absorbed ions and water in the gut. Increases in gut blood flow can either be caused by an increased driving pressure (i.e., dorsal aortic blood pressure), a reduced gastrointestinal vascular resistance, or a combination of both (Sandblom and Gräns, 2017). In trout long-term acclimated to SW, both the systemic vascular resistance and the dorsal aortic blood pressure is reduced and consequently, increased dorsal aortic blood pressure cannot explain the elevated gut blood flow in SW (Sundell et al., 2018). Therefore, it is likely that the elevated gut blood flow is caused, at least partly, by a reduced gut vascular resistance, which is consistent with the reduced systemic vascular resistance. Moreover, to partly compensate for the decreased blood pressure in SW, rainbow trout also exhibit an increased cardiac output that is mediated by an increased stroke volume (Brijs et al., 2015, 2016; Maxime et al., 1991; Sundell et al., 2018).

To my knowledge, the response in cardiac output and gut blood flow to water salinity have been studied almost exclusively in the euryhaline teleost rainbow trout (Brijs et al., 2015, 2016, 2017; Maxime et al., 1991; Sundell et al., 2018). The anadromous strain of rainbow trout (i.e., steelhead trout) naturally migrates between FW and SW whereas most farmed rainbow trout have been farmed in FW for generations (Quinn and Myers, 2004). Still, farmed rainbow trout possesses pronounced euryhalinity and can be acclimated to full strength SW (Brijs et al., 2016). Rainbow trout has therefore served as a model organism for exploring the various cardiovascular adjustments that takes place in both acute SW transition and after long-term acclimation to SW in FW euryhaline teleosts (Brijs et al., 2016; Maxime et al., 1991; Sundell et al., 2018).

With anthropogenic impacts on the environment, the salinity in coastal areas and estuaries are predicted to change and to fluctuate even more in the future (Gillanders et al., 2011; Meier et al., 2012). In a changing world, the trait of euryhalinity may therefore be of added importance for the survival and success of teleost fishes and may be particularly beneficial for marine species inhabiting coastal waters and estuaries (Smyth and Elliot, 2016). However, in order to understand the possible benefits and associated constrains of euryhalinity in marine teleost fishes compared with fishes that have a narrower salinity tolerance range, there is a need for improved knowledge of the cardiovascular adjustments and the metabolic costs that emanates when salinity decreases. It is possible that marine euryhaline teleosts exhibit reverse cardiovascular adjustments compared to FW euryhaline teleost like the rainbow trout. These adjustments could include a lowering of both cardiac output and gut blood flow as drinking rates and intestinal absorption of ions and water are reduced when salinity decreases. Moreover, although there are no conclusive trends of

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how changing environmental salinity affects the metabolism in teleost fishes (Ern et al., 2014), long-term exposure to reduced salinity in a marine teleost would possibly lower the metabolic costs due to an external environment that is closer to isosmotic to the plasma of the fish. On the other hand, altered physiological, morphological and behavioral functions in response to decreased water salinity could also increase the metabolic cost for marine teleost fishes. The genus Myoxocephalus contain species native to both FW, SW and brackish waters, and so fishes in this genus have evolved mechanisms to cope with a wide range of salinities (Nelson et al., 2016). One species in this genus is the Shorthorn sculpin (Myoxocephalus scorpius), which is a marine teleost species with a wide latitudinal distribution, ranging from the Arctic south to Florida and Portugal (www.iobis.org). The shorthorn sculpin is a benthic species that typically inhabit rocky shore bottoms, but have also been encountered in shallow waters in the littoral zone and in estuaries, which can be subjected to great salinity fluctuations (Bone and More, 2008; Coad and Reist. 2004). These habitats suggest euryhalinity in the shorthorn sculpin, which is supported by a reported low salinity tolerance down to 150 mM Na+ l-1 (Foster, 1969) and an ability to increase water excretion by the kidney (Oikari, 1978a, 1978b). Collectively, since the shorthorn sculpin is a territorial sit-and wait predator that is frequently encountered in areas of fluctuating salinities, it has the potential to serve as an excellent species for studying the effect of reduced salinity on cardiovascular function and metabolism in a marine euryhaline teleost.

Aims and hypothesesThe aim of this study was to examine the role of the cardiovascular system and the energetic cost of osmoregulation in the Shorthorn sculpin (Myoxocephalus scorpius) across water salinities. To do this cardiac output, gastrointestinal blood flow and oxygen consumption rate was measured in shorthorn sculpin short-term acclimated to 15 and 33 ppt salinity, and oxygen consumption rate was also measured in shorthorn sculpin acutely transitioned from 33 to 15 ppt salinity. I hypothesize that cardiac output and gastrointestinal blood flow are reduced in 15 ppt compared to seawater as drinking and intestinal water absorption are predicted to decline when the surrounding water salinity decreases. Moreover, I hypothesize that near iso-osmotic conditions (i.e., 15ppt) will result in reduced oxygen consumption rate as less energetic costs due to active transport is predicted in 15 ppt compared to full strength SW.

MATERIAL AND METHODS

Experimental animals and holding conditionsShorthorn sculpin (Myoxocephalus Scorpius, Linneaus, 1758) of both sexes were caught by a fisherman outside of the Swedish west coast (Gullmarsfjorden, Grundsund, Sweden). The fish were kept on a 12h:12h light:dark photoperiod in a tank receiving recirculating aerated SW (33 ppt salinity) at 10°C. The holding tank contained half clay pots and gravel that provided a suitable environment for the bottom-dwelling sculpins. To ensure habituation to the new environment, fish were kept in the holding tank for a minimum of four weeks before experiments were performed. The sculpins were fed once a week with whole herring and fasted for six days prior to experimentation. All animal handling and experimentation was performed in accordance with ethical permit 165-2015 from the ethical committee in Gothenburg.

Experimental protocolsTo assess how blood flow and metabolic rate were affected by reduced salinity, two different experiments were performed. In the first experiment (experiment 1), the effect of lowered water salinity (15 ppt salinity) on standard metabolic rate in uninstrumented fish was examined

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continuously over 5 days, followed by analysis of blood composition. The second experiment (experiment 2), compared routine metabolic rate and blood flow in shorthorn sculpin that were chronically acclimated to SW (33ppt) or short-term acclimated (4-9 days) to 15 ppt salinity. The respective lengths and masses of the fish were 20.33.3 cm and 127.657.4 g in experiment 1, and 25.44.5 cm and 223.014.1 g in experiment 2.

Experiment 1: Energy expenditure in seawater and 15 ppt salinityShorthorn sculpin were transferred from the holding tank to four identical PVC respirometers. The respirometer sides were opaque and covered with black plastic to avoid visual disturbance of the fish. The mass:volume ratio between the mass of the fish and the volume of the respirometer was 1:26, which is within the recommended range (Clark et al., 2013). All fish were allowed 35 hours in SW to recover from the stress of being transferred from the holding tank to the respirometers before the protocol was started.

The experiment consisted of two treatment groups: a treatment group that received recirculating aerated SW at 10°C for 24 hours before being exposed to 15 ppt salinity for 96 hours and a control group that received recirculating aerated SW (33 ppt salinity) at 10°C throughout the entire protocol duration. To dilute the SW, the SW inflow was turned off and FW was added to the tank. The inflow of FW was turned off with regular intervals to allow the salinity in the tank to stabilize. Once the salinity of the water within the tank had stabilized at 15 ppt salinity, the FW inflow was turned off and a closed recirculating system was maintained for the remaining duration of the protocol. The water quality of the closed circulation system was monitored regularly to ensure good levels of the nitrogenous compounds’ nitrite, nitrate and ammonia. Ammonia ranged between 0-0.08, nitrite between 0.2-0.5 and nitrate between 6.2-7.8 mg l-1, which were all within the recommended ranges (Handy and Poxton, 1993). The temperature of the water within the tank was maintained at 10°C by recirculating the water in the tank through the heat exchanger, which used SW from the main system for temperature control. Lowering of the salinity took approximately 3 hours. To control for the potential effect of time spent in the respirometers, the control group was kept in SW throughout the experimental protocol (i.e., 120 hours), but exposed to a mimicked salinity decrease after 24 hours. This was done by turning on and off the SW inflow with regular intervals over 3 hours, similar to what was done to lower the salinity for the treatment group. Throughout the experimental protocols, 25-minute respirometry cycles were run. In these cycles, the flush pump was turned on for 10 minutes, flushing the respirometers with fully oxygenated water and subsequently turned off for 15 minutes to allow for measurements of oxygen consumption rate from the change in oxygen content of the water (see below for details).

When the protocol was finished, the fish were killed with a sharp blow to the head and a caudal blood sample was taken from each fish using heparinized 1 ml syringes and 23-gauge needles. A subset of blood was transferred to micro haematocrit capillary tubes (BrandTM, Wertheim, Germany) to measure hematocrit and a subset of blood was added to Microcuvettes (HemoCue 201+, Ängelholm, Sweden) to measure hemoglobin. Once blood was added to the microcuvettes, they were left for 10 minutes to allow for completion of all reactions (Clark et al., 2008). The remaining blood was centrifuged for five minutes at 3000 rpm to separate the red blood cells from the plasma, which was collected and transferred to 1.5 ml Eppendorf tubes that were frozen at -18°C until further analysis of plasma osmolality and plasma concentration of Na+, K+ Cl- and Ca2+.

Experiment 2: Blood flow and metabolic rate during acclimation to 15 ppt salinity Before any surgical procedures were performed, shorthorn sculpin were either kept in their acclimation salinity (33 ppt salinity) or acclimated to 15 ppt salinity for a minimum of 4 days (acclimation period ranged between 4-9 days).

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Surgical InstrumentationsIndividual shorthorn sculpin were anesthetized with MS-222 (Tricaine methanesulfonate; Scanvacc, Hvam, Norway) until ventilation ceased. Depending on the treatment group, fish were either anaesthetized in SW containing MS-222 (100 mg L-1), or in water with a salinity of 15 ppt containing MS-222 (100 mg L-1) buffered with NaHCO3 (200 mg L-1; Farrell et al., 2013). Weight and length were obtained before placing the fish on water-soaked foam on a surgical table. The gills were irrigated with aerated recirculating SW at 10°C containing anesthetic throughout the surgery. The SW only contained MS-222 (50 mg L-1), whereas the 15 ppt water contained MS-222 (50 mg L-1) buffered with NaHCO3 (100 mg L-1; Seth and Axelsson, 2009).

To measure gut blood flow, the abdominal wall was opened by a ventral incision posterior to the pectoral fin (Gräns et al., 2013). The celiacomesenteric artery, which branches off the dorsal aorta and further divides into several vessels that perfuses the gut, was localized by moving the liver, spleen, intestine, stomach and gonads using cotton-free compresses. (Seth and Axelsson, 2009). Once the vessel was localized, it was dissected free of surrounding tissues by blunt dissection using forceps (Figure 2A; Gräns et al., 2013). Care was taken not to damage any surrounding nerves or vessels. A 1.5 PRS Transonic flow probe (Transonic systems, Inc, Ithaca, NY, USA) calibrated at 10°C was placed around the vessel. In some cases, the celiacomesenteric artery was difficult to access before it divided into two vessels (i.e., bifurcated). On these occasions, the flow probe was placed around both vessel branches resulting from the bifurcation, as close to the bifurcation point as possible (2A). Appropriate positioning of the probe (i.e., aligning the direction of the vessel) was verified before attaching the probe lead to the abdominal wall and closing the wound using silk sutures (4.0; Figure 2B). Finally, the probe lead was attached dorsally to the skin.

The operculum and the gills were then carefully lifted to expose the opercular cavity. The ventral aorta was dissected free using sharp and blunt dissection without damaging nearby nerves or vessels (Figure 2C; Brijs et al., 2014). To avoid damaging the pericardium, the vessel was exposed close to the afferent branchial arteries. A 2.5 PSL Transonic flow probe (Transonic systems, Inc, Ithaca, NY, USA) calibrated at 10°C was placed around the vessel (Figure 2D). The probe was aligned in accordance with the direction of the vessel and a silk suture was attached as close to the probe head as possible to keep the probe in place. The probe lead was then attached with several silk sutures to the skin and finally dorsally together with the lead from the celiacomesenteric artery probe. After surgery the fish were placed in SW or in water with the salinity of 15 ppt and transferred to the experimental set up.

Experimental protocolSurgically instrumented fish were placed in two identical custom-made polyethylene respirometers (PlastKapTek Sverige, Sweden) equipped with an opening at the top to exit flow probe leads. To avoid contact between the fish and the stress of being exposed to the surroundings, a wall shielded the two respirometers and black plastic covered each respirometer. As recommended by Clark et al., (2013), the mass:volume ratio of the fish and the respirometers was 1:27 in this experiment, which is within the desirable range of 1:20-1:100. The tank contained aerated SW or water with the salinity of 15 ppt at 10°C depending on the treatment group. Similar to experiment 1, the control group received recirculating aerated SW whereas the treatment group was enclosed within a closed recirculation system. The temperature in the closed circulation was maintained by recirculating the water in the tank through the heat exchanger and the water levels of nitrogenous compounds were monitored carefully to ensure good water quality. Fish were allowed 24 hours of recovery from surgery, after which metabolic rate, cardiac output, gut blood flow and heart rate were measured during 24 hours in SW or in 15 ppt salinity depending on the treatment group. When the protocol was finished, the fish were killed with a sharp blow to the head.

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Respirometry set upIn experiment 1 (uninstrumented sculpins) four 3.1 L respirometers submersed into a 160 L tank were used (Figure 3A) and in experiment 2 (instrumented sculpins) two 6.1 L respirometers submersed into a 180 L tank were used (Figure 3B). Each respirometer was connected to a common automated flush pump and an individual recirculation pump (Eihem, Germany) through Tygon tubing (Clark et al., 2013). To ensure sufficient mixing of the water within the respirometers, the outflow and inflow for the recirculation pump was connected diagonal and an overflow pipe was placed above the water level diagonal from the flush pump inflow. An oxygen optode that constantly recorded the partial pressure of oxygen in the respirometer water was placed in the outflow of the recirculation loop. The flush pump was turned on for 10 minutes, flushing the respirometers with fully aerated water, and then turned off for 15 minutes such that the rate of oxygen removal from the water by the fish could be obtained. To account for any bacterial oxygen consumption within the respirometers, measurements of oxygen consumption rate was obtained from empty respirometers for a minimum of 2 hours after each protocol (Rosewarne et al., 2016; Svendsen et al., 2016).

Data acquisition and analysisFlow probes were connected to a Transonic flow meter (Transonic systems, Inc, Ithaca, NY, USA). The partial pressure of oxygen in the respirometer water was measured using a fiberoptic FireSting O2 system (PyroScience, Aachen, Germany). Changes in water oxygen content recorded by the FireSting optodes were logged by Pyro oxygen logger software (v. 3.213, PyroScience, Aachen, Germany). Each optode was calibrated at 0 and 100% oxygen saturation before each protocol by placing the optode together with a FireSting temperature sensor in sodium sulphite saturated water (0% oxygen saturation) followed by fully oxygen saturated water (100% oxygen saturation).

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A 16SP PowerLab unit connected to a computer containing the data acquisition software LabChart pro (7.3.2, ADIinstruments) was used to record signals from the Pyro oxygen loggers and the Transonic flow meters. Heart rate was calculated in LabChart pro based on the pulsatile cardiac output flow recordings. Metabolic rate was calculated according to Equation 1:

Equation (1). M O2=⌊ (V r−V f )∗ΔCwO 2 ⌋

( Δt∗M f )

where MO2 is the oxygen consumption rate, Vr is the volume of the respirometer, Vf is the volume of the fish (assumed to equal the body mass (Mf ) of the fish), ΔCwO2 is the change in oxygen concentration in the water, which is dependent on salinity and temperature, and Δt is the time period during which ΔCwO2 was measured (Clark et al., 2013). During the respirometry cycles, the slopes used to calculate ΔCwO2 were generated during the 15 minutes closed periods. However, as it takes time for a new equilibrium to stabilize after turning off the flush pump, the slopes were only calculated from the last 10 minutes of the closed respirometer period (Svendsen et al., 2016). Background respiration measurements generated a minimum of 5 slopes which were used to calculate the mean slope for background respiration. The mean background respiration slope was then subtracted from the fish oxygen consumption rate slopes to correct for background respiration. To assess whether oxygen could leak into the respirometers, leak tests were performed on all respirometers before any experiments were initiated. This was done by adding oxygen deficient water to closed respirometers, reducing the oxygen content of the water to 80-90% oxygen saturation, and subsequently measuring the oxygen content in the water over a minimum of 12 hours (Svendsen et al., 2016).

The SMR in experiment 1 was calculated based 12-hour periods between 07:00-19:00 each day when the light was turned on and between 19:00-07:00 each night when the light was turned off. In total, 26-29 metabolic rate measurements were obtained from each 12-hour period. To

12

assess the presence of outliers, these slopes were used to calculate a mean value for metabolic rate over the whole period. If any of the individual metabolic rate data deviated more than two standard deviations from the mean metabolic rate they were considered as outliers and removed from the data set. After potential outliers had been removed, the lowest 10% of the metabolic rate data (most commonly comprising three slopes) were used to calculate a mean value that represented the SMR for each fish at each 12-hour period (Clark et al., 2013). In experiment 2, cardiorespiratory and metabolic variables were most stable between 24- and 40-hours post-surgery, and so mean values for each fish were calculated over a 1-3 hour period within this time interval. This representative period was visually determined when blood flows and metabolic rate were low and stable. The metabolic rate measurements received from experiment 2 is defined as the RMR since the metabolic rate was obtained from a relatively short time interval (1-3 hours), and possibly involved effects of spontaneous activity (Steffensen, 1989). Linear slopes representing the decline in oxygen content of the water was used to calculate a mean value for RMR for each fish individually. If metabolic rate data resulting from these slopes deviated more than two standard deviations from the calculated mean, they were considered as outliers and removed from the data set (Clark et al., 2013). The RMR for individual fish were based on 4-6 metabolic rate measurements.

Mean values for cardiac output, gut blood flow and heart rate were obtained during the same period as the metabolic rate in experiment 2. Thus, mean values of cardiac output, gut blood flow and heart rate were calculated as the means during 1-3 hours. Stroke volume was calculated from the cardiac output and heart rate of each individual using Equation 2. The percentage of cardiac output directed to the gut was calculated by equation 3. The oxygen extraction by the tissues was estimated using equation 4.

Equation (2). Stroke volume=Cardiac outputHeart rate

Equation (3). % blood flow shunted ¿the gut=Gut blood flowCardiac output

Equation (4). Oxygen extractionby tissues=M O2

Cardiac output

Blood filled capillary tubes were centrifuged in a hematocrit centrifuge (Hematocrit 210, Hettich, Tuttlingen, Germany) at 10000 rpm for five minutes before the hematocrit (% red blood cells) was read using a Hawksley hematocrit reader (Hawskey, Sussex, England). Microcuvettes were placed in a haemoglobin analyser (HemoCue 201+, Ängelholm, Sweden) that detects the hemoglobin content of the blood in mg l-1. Blood osmolality was analyzed using a freezing point osmometer (micro osmometer 3300, Advanced instruments, Norwood, USA) and sodium, potassium, chloride and calcium concentrations were determined using an electrolyte analyser (ISE comfort Electrolyte Analyzer, Convergent technologies, Cölbe, Germany). All blood analyses were performed in duplicates.

Statistical analysisAll data are reported as means SEM. Prior to statistical comparisons of treatment groups, data were tested for assumptions of normality (Shapiro-Wilk test) and homogeneity of variances (Levene´s test). The respirometry data from experiment 1 in uninstrumented shorthorn sculpin were assessed using a two-way mixed-model ANOVA. SMR was included as the dependent variable and treatment group and time as independent variables. When statistical interactions 13

were found, main effects of the independent variables were tested. One-way ANOVAS were performed to examine differences in SMR between treatment groups and one-way repeated measures ANOVAS, corrected for multiple comparisons using Bonferroni correction, were performed to assess differences within groups. To analyze differences in blood variables between treatment groups, independent sample t-tests were used. Welch t-tests were performed for analyses of group differences in chloride concentrations and plasma osmolality since the assumption of homogeneity of variances were violated. To compare the effect of salinity on cardiorespiratory variables between treatment groups in experiment 2 (surgically instrumented sculpins), independent sample t-tests containing one dependent factor were conducted. However, the assumption of homogeneity of variances were violated when analyzing the difference between treatment groups in stroke volume and in the proportion of cardiac output shunted to the gut. On these occasions, Welch t-tests containing one dependent variable were used. All statistical analyses were performed in SPSS statistics (v.24, IBM Corporation, US). Statistical significance was accepted at P < 0.05.

RESULTS

Table 1. Morphological characteristics of Shorthorn sculpin (Myoxocephalus scorpius).

Experiment 1 Experiment 2

33 ppt 15 ppt 33 ppt 15 ppt

Body mass (g) 138.

011.3

102.913.1 246.621.4 199.416

Length (cm) 21.85.4 20.07.4 26.06.8 24.75.4

The table show the body mass and length of shorthorn sculpin in seawater (33 ppt) or 15 ppt salinity (15 ppt) in experiment 1 and 2, respectively. Data are presented as means SEM.

Shorthorn sculpin generally remained calm and still in the respirometers and no obvious behavioral differences were observed between treatment groups. Moreover, there were no differences in body mass or length between treatment groups in the respective experiments (table 1).

Effects of acute exposure to 15 ppt on metabolism and blood compositionIn sculpins exposed to 15 ppt, the SMR decreased significantly 12 hours after the reduction of water salinity (272 mg O2 h-1 kg-1) compared to the first 12 hours in SW (373 mg O2 h-1 kg-1; Figure 4). This lower SMR in 15 ppt was maintained for the remaining part of the protocol. Further, a significant interaction on SMR was found between treatment group and time in uninstrumented sculpins (F3.951, 51.364=2.992, P=0.027), suggesting that the two treatment groups responded differently in SMR over time. Indeed, the SMR was significantly reduced in shorthorn sculpin exposed to 15 ppt salinity compared to sculpins kept in SW at all 12-hour periods after the decrease in salinity, whereas there were no differences in SMR between groups in full strength SW (Figure 4). Yet, a small significant time effect was also found in the control sculpins continuously exposed to SW (F9,54=2.094, P=0.046). However, when comparing each 12-hour period in the control group with each other, there were no significant differences between 12-

14

hour periods found. This indicates that the time effect observed within the control group was likely too weak to be picked up during a multiple comparisons test.

Both hemoglobin (Figure 5A) and hematocrit (Figure 5B) content were similar between treatment groups in un-instrumented sculpins (t14=0.285, P=0.780 and t14=0.616, P=0.548 for hematocrit and hemoglobin respectively). This resulted in a similar mean corpuscular hemoglobin concentration between the treatment groups (Figure 5C; t14=1.143, P=0.272). The hemoglobin content was 457 mg l-1 in SW-acclimated sculpin and 589 mg l-1 in the 15 ppt-acclimating group. The hematocrit was 152 % red blood cells in SW and 173 % red blood cells in 15 ppt.

Shorthorn sculpin exposed to 15 ppt salinity displayed a significantly lower plasma osmolality (3446 mOsm kg-1) compared to sculpin kept in 33 ppt salinity (4043 mOsm kg-1; Figure 6A; t8.205=2.963, P=0.018). The lower plasma osmolality at 15 ppt was also reflected by lower plasma ion concentrations of chloride (Figure 6B; t11.471=5.458, P<0.001), sodium (Figure 6C; t14=5.581, P<0.001), and calcium (Figure 6D; t14=2.199, P=0.045). There were no differences in plasma potassium concentrations between treatment groups, although a tendency for increased potassium levels in sculpin exposed to 15 ppt was indicated (Figure 6E; t14=1.932; P=0.074).

15

BA

Time (h)

33 ppt 15 pptTreatment*Time:F3.951, 51.364=2.992, P=0.027

Figure 4. Standard metabolic rate (SMR) in uninstrumented shorthorn sculpin (Myoxocephalus scorpius) during exposure to 15 ppt salinity. A control group was kept in seawater (SW; 33 ppt) for 120 hours (open circles) and a treatment group was exposed to 15 ppt salinity (15 ppt) for 96 hours after 24 hours in SW (closed squares). Red hatched line represents a decrease in salinity for the treatment group or a mimicked salinity decrease for the control group. SMR data are based on 12-hour periods and were obtained between 19:00-07:00 each night (shadowed stripes) and 07:00-19:00 each day (white stripes). Data are presented as means SEM (n= 7 SW, 8 15 ppt). Asterisk (*) denote significant differences between groups at each time period, different lower-case letters (control group) and capitals (treatment group) denote significant differences between 12-hour periods within groups. A significant interaction between treatment group and time is denoted in the white box. Statistical significance was accepted at P<0.05.

* *******

BBBBBBBABABA

aaaaaaaaaa

Effects of acclimation to lower salinity on blood flow and metabolismCardiac output was 232 ml min-1 kg-1 in sculpin chronically acclimated to SW, while sculpin short term acclimated to 15 ppt had a significantly lower cardiac output at 191 ml min-1 kg-1

(Figure 7A). Thus, acclimation to lower salinity reduced cardiac output by 4 ml min-1 kg-1

(t18=2.278, P=0.035). There was no difference in heart rate between the treatment groups (t18=1.523, P=0.145; Figure 7B), although stroke volume was significantly lower in fish short-term acclimated to 15 ppt (t13.196=2.529, P=0.025). The effect size of water salinity on stroke volume was 0.23 ml beat-1 kg-1; Figure 7C). Similar to the uninstrumented fish, instrumented shorthorn sculpin displayed a significantly lower RMR (39.4 mg O2 h-1 kg-1) after short-term acclimation to 15 ppt compared to chronic acclimation to SW (47.7 mg O2 h-1 kg-1; t18=3.772, P=0.002; Figure 7D). As both RMR and cardiac output were reduced in fish short-term acclimated to 15 ppt, there were no differences in the oxygen extraction by tissues between the two treatment groups (Figure 7E).

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Mean gut blood flow for SW shorthorn sculpin was 5.60.6 ml min-1 kg-1 whereas the gut blood flow in sculpin acclimating to 15 ppt was 5.10.6 ml min-1 kg-1 (Figure 8A). Yet, there were no significant differences in gut blood flow between treatment groups (t15=0.595, P=0.561; Figure 8A). However, the gut blood flow data showed a lot of variation in both treatments group and ranged from 4.0 to 8.1 ml min-1 kg-1 in SW-acclimated sculpin and from 3.1 to 8.7 ml min-1 kg-1 in sculpin acclimating to 15 ppt. Even though cardiac output was significantly reduced in 15 ppt-acclimating sculpin while gut blood flow was maintained across treatment groups there was no significant difference in the proportion of cardiac output shunted to the gut (t9.580=0.699, P=0.501; Figure 8B). The proportion of cardiac output shunted to the gut was 241% in the control group and 273 % in the treatment group.

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1.0

0.8

0.6

0.4

0.2

E

D

B

C

A

*

*

*

*

0

0

0

Figure 6. Plasma osmolality and plasma ion composition in shorthorn sculpin (Myoxocephalus scorpius) kept in full strength seawater (SW; 33 ppt; open bars) or exposed to 15 ppt salinity (15 ppt; closed bars). A) shows the plasma osmolality and B-E shows the plasma ion concentrations of chloride (Cl-1), sodium (Na+), calcium (Ca2+) and potassium (K+), respectively. Data are presented as means SEM (n= 8 SW, 8 15 ppt). Asterisk (*) denote significant differences between treatment groups. Statistical significance was accepted at P0.05.

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Figure 8. Gut blood flow in shorthorn sculpin (Myoxocephalus Scorpius) acclimating to different water salinities. A) show the gut blood flow and B) show the proportion (%) of cardiac output shunted to the gut in chronically SW acclimated sculpin (33 ppt; open bars) and sculpin short-term acclimating to 15 ppt salinity (15 ppt; closed bars). Data are presented as means SEM. No significant differences were found between treatment groups (n=8 SW, 9 15 ppt). Statistical significance was accepted at P<0.05.

BA

33 ppt 15 ppt33 ppt 15 ppt

0

33 ppt 15 ppt

D1.0

0.8

0.6

0.4

0.2

0

*

33 ppt 15 ppt

E

C*

BA*

Figure 7. Cardiorespiratory variables in shorthorn sculpin (Myoxocephalus Scorpius) acclimating to different water salinities. The figure shows cardiac output (CO; A), heart rate (HR; B), stroke volume (SV; C), routine metabolic rate (RMR; D) and the oxygen extraction by tissues (E) in fish chronically acclimated to seawater (SW; 33 ppt; open bars) and fish short-term acclimating to 15 ppt salinity (15 ppt; closed bars). Data are presented as means SEM (n= 10 SW, 10 15 ppt). Asterisk (*) denote significant difference between treatment groups. Statistical significance was accepted at P<0.05.

DISCUSSIONLower water salinity decreases metabolic rate and cardiac output

Standard metabolic rate decreased in uninstrumented sculpins exposed to 15 ppt compared to sculpins kept in SW. The decrease in SMR occurred 12 hours post-exposure to 15 ppt and was thereafter maintained over the full 96-hour period in the lower salinity. Thus, my study concludes that in the shorthorn sculpin, the energetic cost of osmoregulation is lower in 15 compared to 33 ppt water salinity. The advantage of analyzing SMR over RMR, as was done in the present study for uninstrumented sculpins, is that normal and spontaneous activity occasionally elevating the metabolic rate is avoided (Clark et al., 2013). This means that I can draw the conclusion that the decrease in SMR in 15 ppt is in fact due to the changed water salinity and not because of alterations in behavior leading to lower activity. In turn, this supports the finding of decreased RMR as an effect of short-term acclimation to 15 ppt in instrumented sculpin. The lower costs of osmoregulation in 15 ppt water salinity is likely a result of smaller osmotic gradients between the surrounding water and the internal environment of the fish (Foster, 1969). Smaller osmotic gradients should allow for reduced rates of active processes (i.e., ion excretion and water absorption) needed to maintain osmotic homeostasis (Costa, 2009). Since these active processes require energy, the resulting outcome is reduced energetic costs reflected in reduced metabolic rates.

Previous research results have been conflicting regarding the energetic cost of osmoregulation in different water salinities, with some studies showing lower costs of osmoregulation in the acclimation salinity while others show lower costs of osmoregulation in iso-osmotic waters (Ern et al., 2014). While the present study examined SMR and RMR at 15 ppt salinity, iso-osmotic water is closer to 10 ppt so an even lower cost of osmoregulation could be expected in iso-osmotic water. Marine shorthorn sculpin has been observed to survive in 10 ppt salinity, but not in salinities below that (Foster 1969), whereas a Baltic sea population of shorthorn sculpin that normally live at 6 ppt survived in 2.5 ppt salinity, but not in FW (Oikari, 1978a). In water salinities below 10 ppt, osmotic gradients increase compared to iso-osmotic conditions as the surrounding water is more dilute than the plasma of the fish. This causes water to move into and ions to move out from the fish. While the osmotic costs for osmoregulation in euryhaline marine teleost below iso-osmotic waters warrants future investigation, it is reasonable to think that the cost of osmoregulation likely increases again due to the combination of increased osmotic gradients and the need to alter the osmoregulatory strategy going from coping with water loss to water load.

Moreover, although not statistically significant, it appears as if the shorthorn sculpin exhibits daily fluctuations in SMR, which is a common phenomenon amongst fishes (Chabot et al., 2016; Fry, 1971). For the shorthorn sculpin studied here, these fluctuations appear to follow a circadian fashion, with the energetic cost being higher during night than during day. The more active fish are the more oxygen they consume. Hence, the nocturnal trends observed here are likely due to increased activity during night time (Castanheira et al., 2011; Sánchez et al., 2009). Indeed, by observing the shorthorn sculpin during holding conditions it was obvious that the sculpins preferred dark hiding places during the day when the light was turned on. While the respirometers were covered with dark plastic bags, there was still a difference in light intensity reaching the fish between day and night time.

Seawater-acclimated sculpin at 10C had a cardiac output of 232 ml min-1 kg-1, which is in accordance with previous measurements of 26±5 ml min–1 in SW at 8C (MacCormack and Driedzic 2004). In sculpins short-term acclimated to 15 ppt the cardiac output was 191 ml min-1

kg-1, which is 17.8% lower than the cardiac output in SW. This shows that marine shorthorn sculpin reduces their cardiac output in response to decreased seawater salinity. Interestingly, the RMR decreased by 17.4% in sculpins short-term acclimated to 15 ppt compared to sculpins chronically acclimated to SW, and accordingly correlates with the decrease in cardiac output. 19

This is not surprising as fishes generally respond to changes in tissue oxygen demand with changes in cardiac output (Farrell et al., 2009; Steinhausen et al., 2008). The decrease in cardiac output was primarily mediated by a decreased stroke volume. However, stroke volume was reduced by 27.6%, which is greater than the reduction in cardiac output. Although not statistically significant, a trend towards higher heart rate was observed in sculpin acclimating to 15 ppt: heart rate was 312 and 271 beats min-1 for 15 ppt and SW-acclimated sculpin, respectively. Thus, a slight elevation in heart rate in the lower salinity could explain the substantial reduction in stroke volume that served to reduce cardiac output as energetic demands declined.

As water loss is predicted to decrease when the salinity is reduced from 33 to 15 ppt, dehydration seem as an improbable cause for reduced stroke volume (Olson hand Hoagland, 2008). However, stroke volume can be regulated by altering the size of the ventricle (Farrell, 2011; Gamperl and Farrell, 2004). Thus, one possible explanation for the decreased stroke volume in 15 ppt is a morphological reduction of the ventricular mass. Indeed, an increased proportion of compact myocardium in the type II heart of rainbow trout have been demonstrated after long-term acclimation to SW compared to FW (Brijs et al., 2017). It is also likely that neurohumoral control play a role in the regulation of stroke volume and cardiac contractility in different salinities. For example, neurohumoral control via -adrenergic tonus has been shown to, at least in part, regulate the decrease in systemic vascular resistance observed in rainbow trout acclimated to SW compared to trout acclimated to FW (Sundell et al., 2018). This may contribute to the increased central venous pressure underlying increased stroke volume in SW-acclimated trout (Brijs et al., 2017) Mammals have displayed direct vasodilatory effects of hyperosmotic and hypertonic fluids in the intestine (Levine et al., 1978; Steenbergen and Bohlen, 1993). Therefore, a direct effect of altered plasma osmolality on myogenic contractions could be another explanation for the decreased stroke volume. Indeed, different osmolality of imbibed water have shown to affect the cardiac sympathetic activity and the cardiac vagal tone in humans (Brown et al., 2005).

Gut blood flow is maintained in across salinitiesGut blood flow was not affected by acclimation to 15 ppt salinity but rather maintained across treatment groups. Previous studies on FW rainbow trout have shown an upregulation of gut blood flow when water salinity increases (Brijs et al., 2015, 2016, 2017), which is thought to be the result of increased drinking rates that results in increased absorption of water and ions in the gut (Brijs et al., 2015 Takie et al., 1998). In turn, this led to the hypothesis of reduced gut blood flow in marine teleost species exposed to decreased salinity. In 15 ppt, drinking rates are predicted to decline, leading to decreased absorption of ions and water in the gut (Bath and Eddy, 1979; Smith, 1930), and possibly reducing the oxygen demands, which is consistent with the reduced RMR displayed here in 15 ppt. The reason for maintaining gut blood flow similar in 15 ppt and SW is therefore presently unknown. Since the shorthorn sculpin live in marine environments that occasionally encounter salinity fluctuations, whereas the rainbow trout contain strains that naturally migrate between FW and SW during their life cycle, it is possible that the two species have evolved different mechanism to respond to altered environmental salinity (Henriksson et al., 2008; Kendall et al., 2015). Hence, a simple explanation for the maintained gut blood flow across seawater salinities examined here could be that the shorthorn sculpin does not encompass the ability to adjust gut blood flow in response to changed water salinity. To fully understand the impact that pronounced euryhalinity have on decreased water salinity, future studies could therefore investigate cardiovascular responses, including gut blood flow, in SW-acclimated rainbow trout acutely exposed and long-term acclimated to FW. Moreover, even though cardiac output was reduced in shorthorn sculpin acclimated to 15 ppt while gut blood flow was maintained, there were no differences in the relative amount of cardiac output shunted to the gut. Theoretically, when cardiac output is decreased while gut blood flow is maintained, the

20

proportion of cardiac output to the gut should increase. This inconsistency is possibly best explained by the large inter-individual variation in gut blood flow in both treatment groups.

Shorthorn sculpin are weak regulators of plasma osmolality and ion compositionThe plasma osmolality was 4043 mOsm kg-1 in SW, which is relatively high but not uncommon for marine species, and 3446 mOsm kg-1 in 15 ppt. Although a significantly lower plasma osmolality was displayed in the lower seawater salinity, sculpin kept their plasma osmolality within normal ranges for teleost fishes in both SW and 15 ppt, possibly indicating osmotically stable fish across seawater gradients (Nordlie, 2009). The decreased plasma osmolality was also reflected by decreased plasma concentrations of sodium, potassium and chloride in 15 ppt. This suggests that the shorthorn sculpin does not downregulate ion excretion sufficiently to match the lower environmental osmotic forces acting on the fish in the lower salinity. Tendencies towards increased potassium levels were also observed, which could indicate some degree of cellular osmotic stress at 15 ppt (Weed and Bowdler, 1966). Cellular osmotic stress can be energetically costly and therefore elevate the metabolic rate in fish (Kultz, 2015; Ern et al., 2014). Thus, if the sculpins exposed to 15 ppt experienced cellular osmotic stress, it is possible that the effect of lowered salinity on the SMR was underestimated compared to fish that does not experience osmotic stress. Still, no significant effects of water salinity were found on neither potassium levels nor mean corspuscular hemoglobin concentrations and so the degree of cellular stress was likely relatively minor. Further, the similar hematocrit between treatment groups in uninstrumented shorthorn sculpin indicated an ability to maintain blood volumes constant across SW gradients (Olsson, 1992). This suggests reduced rates of absorption of ions and water in the gut, as maintained absorption rates would likely result in volume loading due to less water loss to the environment in 15 compared to 33 ppt salinity.

Collectively, my data show that the shorthorn sculpin does not regulate plasma osmolality and ion composition strictly, which allows the plasma osmolality and ion composition to fluctuate with the environment within certain ranges. However, they likely have a narrow regulation of cellular osmolality and ion concentration. Indeed, shorthorn sculpin normally live in areas that are subjected to frequent salinity fluctuations, and so allowing for fluctuations in plasma osmolality and ion composition likely saves energy for these fish, which is reflected in the reduced SMR (Henriksson et al., 2008). Yet, since the shorthorn sculpin does not normally long-term acclimatize to lower salinities, there is a possibility that long-term exposure to lower salinities eventually impairs physiological functions within these fish. Thus, the energetic cost of osmoregulation may increase again if the lower salinity persists.

A role of altered arteriovenous branchial blood flow in reduced salinity?Keeping osmotic homeostasis in hyperosmotic environments involve a match between the active and passive absorption of ions and the excretion of excess ions across the gills and kidney (Evans et al., 2005). That is, when less ions are absorbed in 15 compared to 33 ppt salinity, less ions also need to be excreted across the gills. Such decreased requirement for ion excretion by the gills can be met by reduced branchial perfusion for osmoregulatory purposes. Blood flow from the heart flow through the ventral aorta to afferent branchial arteries followed by afferent filamentous arteries and afferent lamellar arterioles that direct the blood into the secondary lamellae, which are the site for gas exchange (Sandblom and Gräns, 2017). From this point, the blood can either flow in the arterio-arterial pathway through efferent lamellar arterioles, efferent filamentous arteries and efferent branchial arteries, which enters the dorsal aorta and the systemic circulation, or it can flow into the arterio-venous pathway, which is the primary site for osmoregulation in the gills (Marshall and Grosell, 2006). This latter circuit involves a shunting of blood from the arterio-arterial pathway, via arteriovenous anastomoses, to the arterio-venous pathway (Evans et al., 2005; Olsson, 2002b). The control of branchial blood flow in teleost fishes is complex and

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involves neural, hormonal and local signaling (Sundin and Nilsson, 2002). Yet, the regulation of arterio-venous blood flow has been shown to be controlled primarily by -adrenergic innervation, acting on -adrenergic receptors within arteriovenous anastomoses (Evans, 1987; Nilsson and Pettersson, 1981). Decreasing arteriovenous blood flow through increased -adrenergic tone will force more blood to flow the arterio-arterial pathway (Olsson, 2002a). However, elevations in arterio-arterial flow can be restored by reducing the total cardiac output, which is consistent with the decreased cardiac output in the present study. Collectively, reduced requirements for ion extrusion across the gills, resulting in lower arteriovenous blood flow, seem to be a probable explanation for the reduction in cardiac output. Indeed, adjustments in the gills play an important role for the ability of sculpins to cope with lower water salinity (Henriksson et al., 2008).

In the present study, the reduction of cardiac output in 15 ppt compared to SW was 4 ml min-1, which corresponds to 17.8% of the total cardiac output in SW. Routine arteriovenous blood flow is 8% of cardiac output in Atlantic cod (Gadus morhua) in SW and 7% of cardiac output in rainbow trout in FW (Ishimatsu et al., 1988; Sundin and Nilsson, 1992). As both the Atlantic cod and the rainbow trout are more active than the shorthorn sculpin, they likely demand higher blood flow through the arterio-arterial pathway, which may result in a larger proportion of the total cardiac output directed to the arteriovenous pathway in the shorthorn sculpin. In turn, this could explain the relatively large decrease in cardiac output in shorthorn sculpin acclimated to 15 ppt, if the reduction in cardiac output observed was indeed explained by reduced arteriovenous blood flow.

Conclusions and future perspectivesThe present study examined hypo-osmoregulation in shorthorn sculpin over a gradient of SW salinities (i.e., 15 and 33 ppt salinity). In 15 ppt the SMR was reduced, likely because the energetic costs of osmoregulation decreased with the lower osmotic gradient. The reduction in SMR was consistent with a decrease in cardiac output, which could be explained by a decreased arteriovenous perfusion. While the relationships between branchial flow and environmental salinity discussed here is largely speculative, measurements of branchial arteriovenous blood flow during changes in water salinity would be an interesting topic for future studies. The fact that cardiac output decreased in response to reduced water salinity in a marine euryhaline teleost, whereas cardiac output increases in response to increased water salinity in freshwater euryhaline teleost, suggests that regulating cardiac output is a necessary physiological response to meet altered water salinity, which may be universal for euryhaline teleost fishes. Yet, this study also indicates that while cardiovascular regulations when transitioning from hyper- to hypo-osmoregulation appear necessary for the survival of the fish, alterations in cardiovascular physiology across hyper- or hypo-osmotic environments may serve energy saving purposes only. Consequently, cardiovascular responses to increasing or decreasing water salinities may not be a linear, but rather a U-shaped relationship. While the present study sheds light onto the broad osmoregulatory ability of marine euryhaline teleost to survive in environments with lower salinities, questions of the success of marine teleost exposed to even lower salinities, i.e., below iso-osmotic, remain unanswered. Moreover, to understand how marine euryhaline teleost will manage salinity fluctuations in nature, physiological responses to swimming and feeding across salinities would also be relevant for future studies.

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ACKNOWLEDGEMENTS

This thesis would not have been possible without the influence and help from a lot of talented people, which I therefore would like to express my sincerest gratitude to.

First of all, I would like to thank Erik who has served as my supervisor throughout this project. You have provided me with everything I have needed to make this happen and have had faith in both me and the sculpins through some rough times. A big congratulations to your professor’s title, you are SO worth it!

I would like to say thanks to Michael, for agreeing to be my examiner, and for very valuable input trying to come up with possible solutions for the progression of this project. Also, a big thanks for helping me print respirometry connectors with your supercool 3D printer.

Daniel, my surgeon in crime – thank you for hours of help in the surgery room. I would not have been able to do this without you. Thanks for a lot of good talks, and for always having my back. You are truly a great person, and amazingly skilled at surgical techniques. I am certain that you will succeed and generate some hardcore in vivo studies for your PhD. (P.S. – don’t forget to invite me to celebrate when the day comes)!

Andreas, thank you for always having an easy attitude on life and making me feel so welcome at Zoologen and in the ECG group. A special thanks for all the help with the respirometry and for being patient during the first part of my thesis when I constantly disturbed you with questions. Although not directly involved in my thesis work, I would also like to say thank you for having me with you for the research project in Forsmark. It was such a fun project and such a good experience for me. I would like to wish you the best of luck in Canada, I am certain that you will have a fantastic time producing some really neat research.

A big thanks to the whole ECG group. Thank you for a lot of fun activities, and for good discussions. Not least trying to resolve the mystery of the dying sculpins (we could probably write a book soon), and for very valuable input after my practice presentation, improving it like a million times. Keep up the good spirit, you are all fantastic people and co-workers. 

Jag skulle också vilja tacka min fantastiska mamma. Du är en riktig superwoman, som tar på dig så mycket och som ändå klarar av att ge ditt allt i allting du tar dig an. Jag förstår inte hur du gör det, men det inspirerar mig varje dag. Jag hoppas att jag en dag har ett jobb som jag älskar lika mycket som du gör och att jag, åtminstone lite grann, har fått din förmåga att sätta människors behov och välmående framför alltihop. För mitt masterarbete vill jag tacka för ditt stöd, för din hjälp att bygga upp mitt självförtroende och för många bra diskussioner som har breddat mitt sätt att se och tänka på fiskfysiologi. Du är bäst!

Sist, men inte minst ett stort tack till min fantastiska familj (Pappa, Mamma, Helena, Tim, Ola, Simon, Emilia, Farmor, Farfar, Vallson och Snurrson) och till mina fina vänner (Amanda, Nellsan, Soffan, Elin, Jompa, Melanie, Josefin och många fler) för att ni alltid har trott på mig och stöttat mig, inte bara under detta masterprojekt, utan under mina 5 år av studier. Ord kan inte beskriva hur tacksam och glad jag är över att ha varenda en av er i mitt liv. Oavsett vilka utmaningar och hinder som livet bringar, så vet jag att jag kommer ta mig igenom alltihop så länge jag har er vid min sida.

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APPENDIX I

Ett liv i brackvatten sparar energi.

Den mänskliga aktiviteten på jorden påverkar vårt klimat på flera olika sätt. Både avdunstningen från hav och sjöar, samt nederbörds- och avrinningsmönstret påverkas, vilket i sin tur förutspås påverka salthalten i haven, framförallt i kustnära vatten. Det är därför viktigt att förstå hur såväl fisk som andra vattenlevande djur tolererar och klarar av att svara på variationer i den omgivande salthalten. Eftersom fiskar generellt sett håller en konstant sammansättning av salter och vatten i blodet, undersöker vi forskare hur fiskar svarar på ändrad salthalt i omgivningen. Detta görs genom att studera de processer som reglerar blodets salt- och vattenbalans, något som kallas för osmoreglering, samt hur och varför dessa processer förändras i olika salthalter. I havet (salthalt 33‰) kämpar fiskar mot vattenförlust och ökning av mängden salter i blodet. Detta reglerar de genom att dricka saltvatten, ta upp vatten över tarmen och avge överskottssalter över gälarna. I sötvatten (0‰) kämpar fiskarna istället mot utspädning av blodet och förlust av salter, som de reglerar genom att avge stora volymer utspädd urin och ta upp salter över gälarna.

Att ta upp- och avge salter kräver energi. En teori är därför att det kostar mindre energi för en fisk att osmoreglera i vatten med liknande sammansättning av salter som den som är i blodet, vilket vanligen motsvarar cirka 9–10‰. Många studier har gjorts för att undersöka energikostnaden av osmoreglering, men inga entydiga bevis för den teorin har påvisats. I en studie på den marina fisken rötsimpa, har vi visat att energikostnaden för osmoreglering är lägre i havsvatten som hade spätts ut till hälften, 15‰, jämfört med i havsvatten som är 33‰ . Vi tror att den lägre energikostnaden i utsötat havsvatten är ett resultat av minskad vattenförlust, vilket minskar kostnaden för att bibehålla blodets salt- och vattenbalans.

Stora ändringar i salthalt kräver att fisken helt ändrar sin strategi för osmoreglering, vilket även inkluderar ändringar i cirkulationssystemet. Blodcirkulationen ansvarar för att transportera joner och vatten till de organ i kroppen som sköter upptag och sekretion, samt för att förse dessa vävnader med syre och även transportera bort restprodukter. Till exempel har man sett att när sötvattensfisken regnbåge flyttas från sötvatten till havsvatten ökar den sin hjärtminutvolym (den totala mängden blod som hjärtat pumpar per minut) och sitt blodflöde till magtarmkanalen, något som är nödvändigt för att tolerera och överleva i den högre salthalten. När en rötsimpa går från havsvatten till utsötat havsvatten har vi sett en minskad hjärtminutvolym medan flödet till magtarmkanalen är bibehållet i de båda salthalterna. Eftersom rötsimpans energikostnad för osmoreglering är lägre i utsötat havsvatten, tror vi att minskningen i hjärtminutvolym är ett sätt för rötsimpan att spara energi på, snarare än en nödvändig respons för rötsimpans överlevnad (som när regnbågen går från sött till salt). Då rötsimpan bibehåller flödet till magtarmkanalen i utsötat havsvatten men minskar den totala hjärtminutvolymen är det troligt att rötsimpan minskar blodflödet till någon annan del än magtarmkanalen. I utsötat havsvatten behöver gälarna förses med mindre salter och mindre syre jämfört med i havsvatten och därav tror vi att rötsimpan har lägre blodflöde till gälarna i utsötat havsvatten.

Sammanfattningsvis vet vi idag att söt- och havsvattenfisk måste kunna reglera cirkulationssystemet för att klara av stora ändringar i vattensalthalt och möta olika stora kostnader av osmoreglering. Mycket återstår dock fortfarande att studeras, så som att undersöka blodflödet över gälarna i olika salthalter. Förhoppningen är att denna kunskap, ihop med fortsatta studier, kan öka vår förståelse för hur fiskar kommer att klara framtida ändringar i vattensalthalt och bidra till ett bättre arbete i att bevara och upprätthålla fiskbestånden.