Atlantic salmon (Salmo salar - Göteborgs universitet · Atlantic salmon is an anadramous salmonid, which means its life cycle involves both freshwater (FW) and seawater (SW). They

Kirth Lumingkit

Degree project for Bachelor of Science inBiology

BIO602 Biology: Degree project 15 hecSpring 2014

Department of Biological and Environmental SciencesUniversity of Gothenburg

Examiner: Catharina OlssonDepartment of Biological and Environmental Sciences

University of Gothenburg

Supervisor: Henrik Sundh and Linda Hasselberg-Frank Department of Biological and Environmental Sciences

University

Atlantic salmon (Salmo salar) osmoregulation in sea water Effects of sexual maturation

Insert reference for image on front page here

The picture on the front page was taken from a poster by Henrik Sundh (University of Gothenburg, Sweden) and shows a smolt in SW.

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Table of Contents 1. Abstract………………………………………………………………………………………………………………………..….5

1.1. Abstract in English………………………………………………………………………………………………………5

1.2. Sammanfattning på svenska……………………………………………………………………………………….5

2. Introduction……………………………………………………………………………………………………….…………….6

2.1. Atlantic salmon aquaculture………………………………………………………………………………….…...6

2.2. The life cycle of anadromous salmonid………………………………………………………………….…...6

2.3. Osmoregulation……………………………………………………………………………………………………...….7

2.4. The sodium potassium ATPase……………………………………………………………………………….…..7

2.5. Intestinal osmoregulation and fluid absorption of Atlantic salmon in SW……………………7

2.6. Gill osmoregulation and Na+/K+-ATPase……………………………….…………………………….….…..8

2.7. Aim of study………………………………………………………………….…………………………………………...8

3. Materials & Methods …………………………………………………………………………………………………...…8

3.1. Animal and tissue sampling……………………………………………………………………………………..…8

3.2. Western Blot……………………………………………………………………………………………….…………….9

3.2.1. Membrane protein preparation…………………………………………..……………………….….9

3.2.2. Gel electrophoresis…………………………………………………………………………….…………...9

3.2.3. Membrane transfer……………………………………………………………………..…………....…..9

3.2.4. Immunoblotting……………………………………………………….…………………..…………….….10

3.2.5. Detection……………………………………………………………………………………….…..….………10

4. Results………………………………………………................................................................................11

5. Discussion…………………………………………………………………………………………………………………..…..11

5.1. Problems with Western Blot…………………….………………………………………………………….……12

5.2. Effects of sexual maturation and osmoregulatory problems in SW.………….…………..…..12

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5.3. Intestinal Na+/K+-ATPase and isoforms in SW……………………………………………………………13

5.4. Fluid absorption in the intestine of Atlantic salmon………………………………..…..….……….13

5.5. Intestinal epithelium and tight junctions of Atlantic salmon in SW……………….…………..14

5.6. Further studies………………..……………………………………………………………………………………..…14

6. Conclusion.......................................................…………………………………………………………………14

7. Acknowledgement…………………………...................................................................................15

8. References………………………………………………………………………………………………………………………15

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1. Abstract

1.1. Abstract in English

Atlantic salmon is an important species in aquaculture. Its lifecycle involves both freshwater (FW) and seawater (SW). They lay their eggs in FW, where it hatches and the juvenile spend the first years. During the process of smoltification in spring that involves changes in behavior, morphology and physiology of Atlantic salmon, the salmon population initiates migration to the ocean, where they live for years before they return to FW to reproduce. In FW, a passive gain of water and loss of ions is counteracted by producing copious dilute urine and actively taking up ions across the gills. In SW, a passive gain of ions and loss of water by osmosis is counteracted by drinking water, absorbing water across the epithelium, and secreting excess ions across the gills and through the kidney. In SW, fluid absorption is coupled to an uptake of monovalent ions driven by the intestinal Na+/K+-ATPase (NKA). Previous studies have shown that sexually mature Atlantic salmon in SW may have increased energetic cost for fluid absorption in SW. This is hypothesized to be linked to a higher intestinal NKA protein expression. Western blot was used to quantify the relative expression of NKA in proximal and distal intestine of mature Atlantic salmon by using immature Atlantic salmon as a control. The molecular mass of NKA is approximately 100 kD that was verified by gel electrophoresis. The abundance of NKA in the intestine of Atlantic salmon couldn’t be obtained due to error in the procedure of western blot. From these results we concluded that it could be due to reduced quality of antibodies. The procedure may have not been followed correctly or the detection reagent did not work. Whether intestinal NKA expression is increased or not in sexually matures Atlantic salmon remains to be investigated by Western blot. 1.2. Sammanfattning på Svenska.

Atlantlax är en viktig fiskart i vattenbruk. Dess livscykel involverar både sötvatten och saltvatten. De lägger sina ägg i sötvatten, där det kläcks och de små fiskarna tillbringar de första åren. Under ”smoltifieringen” på våren som involverar förändringar i beteende, morfologi och fysiologi i fisken, så påbörjar atlantlax vandringen till havet, där de lever i flera år innan de återvänder till sötvatten för att reproducera. I sötvatten, är den passiva vinsten på vatten och förlust av joner motverkas genom att producera mängder utspädd urin och aktivt ta upp joner över gälarna. I saltvatten, är den passiva vinsten på joner och förlust av vatten motverkas genom att dricka vatten, absorbera vatten i hela epitelet, och utsöndra överskott joner över gälarna och genom njuren. I saltvatten, är vätskeabsorption kopplad till upptag av monovalenta joner som drivs av Na+/K+-ATPas (NKA) i tarmen. Tidigare studier har visat att könsmogna atlantlax i saltvatten kan ha en ökad energi kostnad för vatten absorption i saltvatten. Det är en hypotes att det skulle vara kopplat till ökat NKA uttryck i tarmen. Western blot användes för att kvantifiera den relativa expressionen av NKA i fram- och bak-tarm av mogna atlantlax genom att använda omogna atlantlax som kontroll. Massan av NKA är ungefär 100 kD som verifierats med gelelektrofores. Mängden NKA i tarmen av mogna atlantlax kunde inte erhållas på grund av fel i utförandet av Western blot. Från detta resultat har vi dragit slutsatsen att det kan vara på grund av försämrad kvalitet av antikroppar. Utförandet har inte följts korrekt eller att detektionsreagenset inte har fungerat. Huruvida tarm uttrycket ökar eller inte i mogna atlantlax står att utredas av Western blot.

Keywords: osmoregulation, Anadramous salmonid, intestine, Western-Blot, Na+/K+-ATPase. 5


2. Introduction

2.1. Atlantic salmon aquaculture

Today, aquaculture is one of the fastest growing industries that produce food. Atlantic salmon is an important species in aquaculture and it’s one of the most commonly farmed salmon. In commercial salmon farming, sexual maturation results into reduced growth rate (Figure 1).

Recently, early maturation has been hypothesized to cause osmoregulatory problems. This could lead to economical loses for the fish farming industry since it might lead to death of the fish if they are kept in SW for a longer period of time after maturation. In order to develop aquaculture in a sustainable way, maintaining good health and welfare of the fish is of major importance.

2.2. The life cycle of anadromous salmonid

Atlantic salmon is an anadramous salmonid, which means its life cycle involves both freshwater (FW) and seawater (SW). They lay their eggs in fresh water, where the eggs hatches and the juvenile spend the first years often in a small territory within a single stream (McCormick, 2013). In fresh water there are fewer predators that eat their eggs and fry, thereby increasing their chances of survival (Stefansson et al., 2008). While still in fresh water the juvenile Atlantic salmon, now called parr has reach a critical size threshold initiates the process of smolfication, during the spring. Smoltification is a morphological, behavioral and physiological pre-adaptation of Atlantic salmon for a life in SW, while the fish is still in FW (Sundell & Sundh, 2012). The parr become silvery and streamlined, lose territoriality and the tendency to move towards a current of water, begin schooling and have increased salinity preference (McCormick et al., 1998). Smoltification is initiated by environmental cues such as photoperiod and temperature through their impact on the neuroendocrine system, which results in the release of developmental hormones such as cortisol, IGF-I and thyroid hormones (McCormick et al., 1998). At the physiological level, these hormones govern the development of hypoosmoregulatory capability in SW. During the development migration is initiated. Their journey begins by downstream migration in the river, through estuaries, and hundreds or thousands kilometers to the ocean, where they feed. The synchronous migration of smolts occurs over 3-6 weeks period in most populations. After 1-4 years the adult salmon usually return to their river of origin to reproduce (McCormick., 2013).

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Figure 1. Problems with commercial fish farming. Sexual maturation results in reduced growth rate. Illustration is borrowed from (Taranger et al., 2010).


2.3. Osmoregulation

FW and SW are two different osmotic environments for the fish. In FW the Atlantic salmon is hyperosmotic compared to the external medium. Due to osmotic forces, water passively enters the fish over all epithelia, while ions are lost to the environment. As a consequence, fish in FW have low drinking rates and there is no need to absorb more water. In FW, active absorption of ions from their food and retaining ions in the kidney (Grosell., 2011) along with actively taking up sodium and chloride across the gills is crucial (Sundell & Sundh., 2012). These activities in combination with excretion of the excess amounts of water through production of copious dilute urine by the kidney and urinary bladder are important (McCormick et al., 2009). In SW the Atlantic salmon is hypoosmotic compared to the external medium. Consequently, due to the osmotic forces over all epithelia the fish is constantly losing water while gaining excess amounts of ions. To solve these problems, the fish have to increase their drinking rate and the absorption of water through a coupled uptake of NaCl in the intestine, where NaCl are transported against its concentration gradient (Grosell., 2011). The active uptake of NaCl along with its diffusion into the fish leads to surplus of these ions, which are excreted across the gills (Grosell., 2011). In two of the most important osmoregulatory tissues, intestine and gills, fluid absorption and excretion of excess ions respectively, is energy demanding task, which requires transport of NaCl against their electrochemical gradient. This energy is provided by the sodium potassium ATPase.

2.4. The sodium potassium ATPase

Na+/K+-ATPase (NKA) is an antiporter enzyme located in the cellmembrane of all animal cells. It’s composed of three important subunits, which is α, β and ɣ. The α-subunit is the binding sites for ATP, Na+, K+ and the inhibitor ouabain, which makes it the catalytic component of the enzyme (McCormick et al., 2009). The β-subunit is a glycosylated polypeptide that helps to fold and position the protein into the basolateral membrane of intestine (McCormick et al., 2009). The ɣ-subunit works to modulate the NKA enzyme by changing their affinity for Na+ and K+ for the function of different cell types (McCormick et al., 2009). Na+/K+-ATPase pumps three Na+ ions out for every two K+ ions pumped in (Stefansson et al., 2008), which cost hydrolyzation of one ATP to produce ADP.

2.5 Intestinal osmoregulation and fluid absorption of Atlantic salmon in SW In order for fluid absorption to occur the osmolality of the ingested sea water must decrease. Further, a coupling compartment must be created in the fish with an osmolality exceeding the luminal salinity for fluid to be transported from the intestinal lumen to the blood. In SW, fluid absorption is coupled to an uptake of monovalent ions driven by the intestinal Na+/K+-ATPase, located in the lateral membrane of the intestinal epithelial cells. It’s important that the tight junctions decrease the permeability of ions across the epithelium to build up the osmotic gradient in the lateral intracellular space (LIS) (Sundell & Sundh., 2012). Consequently, the osmolality in the LIS

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is elevated to approximately 650 mOsm (Grosell., 2011). Simultaneously, electrochemical gradient for Na+ entry into the cell builds up by the NKA is utilized by Na-K-Cl cotransporter 2 (NKCC2). NKCC2 that is located on the apical membrane of enterocytes works by actively transporting luminal K+, Na+ and Cl- into the cell, and in part reduces the osmolality of sea water in the gut lumen that has come from drinking of the fish. The following transport of ions from the lumen to the blood leads to a reduced osmolality in the lumen. As soon as the osmolality in the lumen is lower than the osmolality in the LIS, fluid will diffuse from lumen to the blood side. The diffusion of water from lumen to the blood circulation occurs via paracellular/transcellular pathway (Sundell & Sundh., 2012). The exact pathway by which water follows is not well established (Madsen et al., 2011). The paracellular route involves the diffusion of water between enterocytes through tight junctions. Transcellular water flux may involve three different pathways including passive diffusion across the lipid bilayers, co-transport with ions and nutrients, and the diffusion through aquaporins in the apical as well as lateral membranes (Madsen et al., 2011).

2.6. Gill osmoregulation and Na+/K+-ATPase

To maintain stable plasma osmolality in SW secretion of surplus ions is crucial. Ion secretion is primarily carried out by chloride cells in the gill filament. Na+/K+-ATPase located in the basolateral membrane provides low Na+ levels and a negative charge within the chloride cell by secreting Na+ ions, which exits the gill by paracellular pathway (McCormick., 2013). The Na+/K+/2Cl- cotransporter (NKCC) that is also located in the basolateral membrane utilizes the low Na+ concentration to transport Cl- ions into the chloride cells (McCormick., 2013). Chloride ions then leave the cell on a “downhill” electrical gradient through the cystic fibrosis transmembrane regulator (CFTR) channel, located on the apical membrane (McCormick., 2013). Depending on the environment of the fish, whether its FW or SW, different isoforms of NKA are important.

2.7 Aim of study

Previous studies have revealed that the intestinal permeability towards Na+ increases in sexually mature Atlantic salmon in SW (Sundh et al., unpublished). Consequences of leaky epithelia are it become harder to maintain a high osmolality in LIS. It’s hypothesized that more NKA are needed to compensate for the leaky epithelial barrier. Therefore, the aim of the study was to investigate if NKA is induced to higher level in the intestine of sexually mature Atlantic salmon compared to immature Atlantic salmon, which we use as a control by using the Western blot methodology.

3. Materials & Methods

3.1. Animal and tissue sampling

Two year old Atlantic salmon post-smolts were kept under simulated natural light and temperature conditions in four indoor tanks containing SW during the period of September to January at the Matre research station, Institute of Marine Research, Norway. The tanks contained both mature and immature fish. Normally, mature fish do not eat and food was therefore withheld from all fish in

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order to avoid feed as a confounding factor. The experiment had been approved by the Norwegian Ethical Committee and the animals were treated according to the Norwegian national legislation for laboratory animals. In January 10, fish were anesthetized in metomidate (12,5 mg/L) and killed by a sharp blow to the head. The body cavity was opened laterally and mesenteries and adipose tissues were carefully removed. The intestine, from the last pyloric caeca to the anus, was removed and opened longitudinally, and divided to a proximal and distal part. The mucosa from the intestinal segments (n=4) were scraped of using two glass-slides. Then the tissue were frozen at -80°C in a freezer and kept until analyses.

3.2. Western Blot

3.2.1. Membrane protein preparation

The focus of this study is NKA that is a membrane-bound enzyme. To do the experiment we prepared the membrane-fraction from the tissue. The tissue samples were transferred to each homogenization tube. This was followed by adding 1 ml of homogenization buffer (0.25 M sucrose in 0.25 M Tris and 0.01 M MgCl2, pH 7.4) with protease inhibitors cocktail tablets (Roche Diagnostic GmbH, Mannheim, Germany) into the tubes. Tissues were homogenized using a glass-glass homogenizer. It was done to mechanically disrupt the cells of the tissue. Tubes with the homogenate were centrifuged using Eppendorf centrifuge 5415R in 2000 g for 20 min at 4°C. The supernatant contains proteins while the pellet constitutes part of the tissue that has not been homogenized. Supernatant was ultracentrifuge using Beckman Coulter Optima LE-80K Ultracentrifuge in 50000 g for 30 min at 4°C. Now, the pellet contains membrane-proteins. The pellet was resuspended in resuspension buffer (25 mM Tris, 10 mM MgCl2, pH 7.4) with protease inhibitors cocktail tablets (Roche Diagnostic GmbH, Mannheim, Germany). Protein concentration was measured with BCATm Protein Assay Kit (Pierce, Rockford, USA) at A540, using bovine serum albumin as standard.

3.2.2. Gel electrophoresis

Proteins samples were diluted up to 60 µl with ddH20, and added 20 µl Laemmli buffer (0.5 M Tris HCl, pH 6.8, glycrol, 10% SDS, 1% bromophenol blue) with addition of β-mercaptoethanol. The final concentration would be 1µg/µl in 80 µl. Then the samples were heated in a dry path at 65°C for 10 minutes. After that they were cooled down on ice. 7.5% separation gel was cast and assembled into an electrophoresis chamber. 1x electrophoresis buffer was added into the electrophoresis chamber between the gels covering the top of the wells in the gel. The rest of the electrophoresis buffer was added to the large container in the electrophoresis chamber, covering the bottom of the gel. Amersham full-range rainbow molecular weight marker (GE Healthcare, Buckinghamshire, UK) was added into the first lane of the gel. The molecular weight marker allows monitoring of protein separation during electrophoresis and verification of protein weight of the samples. 20 µl of each protein samples were loaded into the gel. Electrophoresis was performed at 150 volt during 75 min.

3.2.3. Membrane transfer

During membrane transfer the separated proteins are transferred out of the gel to a membrane in

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which the protein of interest can be detected with antibody. First, proteins were blotted onto polyvinylidene diflouride (PVDF) microporous membrane (Millipore Immobilon-P Transfer Membrane, 0.45 · m pore size) in a tris-glycine transfer buffer (25 mM Tris base, 192 mM glycine and 10% methanol) at 40 mA overnight at 4°C.

3.2.4. Immunoblotting

The PVDF membrane was removed from the cassette and put in a petri dish containing small amount of ddH2O to prevent the membrane from drying out. 25 ml Ponceau red solution was added into the petri dish for 1 minute under slow shaking using an orbital shaker. Ponceau red stains the proteins in the membrane to verify the proteins were transferred successfully. The Ponceau red solution was poured off and the PVDF membrane was washed with ddH2O until the dye was completely removed. 50 ml blocking solution (5% non-fat dried milk in TBST) was added to the petri dish and incubated for 1 hour at room temperature on an orbital shaker. The blocking solution contains proteins from milk that binds to the membrane preventing non-specific binding of the primary antibody/secondary antibody to the membrane. Next, blocking solution was poured off and the membrane was washed 2X for 1 min with 1x TBST (TBS with 0,1% Tween ® 20 ). The membrane was incubated with the NKA antibody (0,01 µg/ml) for 1 hour in room temperature under slow shaking on an orbital shaker. The primary antibody was poured off and the membrane washed 2X for 1 min with 1x TBST and further washes 6X with 1x TBST for 5 minutes. The membrane was incubated with the secondary antibody (ECLTM anti-mouse IgG) diluted to 1:50000 in TBST for 1 hour in room temperature under slow shaking on an orbital shaker. Secondary antibody binds specifically to the primary antibody on the membrane that is bound to the target protein. It’s also conjugated to horse radish peroxidase from sheep, which is used for later detection. The secondary antibodies was poured off and wash 2X for 1 min with 1x TBST and further washes 6X with 1x TBST for 5 minutes to prevent background.

3.2.5. Detection

Visualisation of proteins on the membrane was performed with the AmershamTMECLTMPrime Western Blotting Detection Reagens kit (GE Healthcare Bio-Sciences, Uppsala, Sweden) and X-ray film, developer and fix from Kodak (VWR, Sweden) according to kit instructions.

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4. Results

Figure2. Ponceau red staining of membrane proteins on PVDF membranes from Western blot. A) PVDF membrane in which 9 protein samples were transferred from a gel. Lane-1 (molecular weight ladder),- 2 (PI3), -3 (PI11), -4 (PI1), -5 (PI16), -6 (DI5), -7 (DI15), -8 (DI9), -9 (DI14). B) PVDF membrane in which 10 other protein samples were transferred from a gel. Lane 1-2 (molecular weight ladder), -3 (PI5), -4 (PI15), -5 (PI7), -6 (PI9), -7 (DI8), -8 (DI11), -9 (DI7), -10 (DI10). DI and PI on the labels refer to as distal intestine and proximal intestine respectively, which are derived from mature and immature Atlantic salmon in SW. The numbers indicates the different individuals of Atlantic salmon. The colors blue, red, green, yellow and purple of the molecular weight ladder on the PVDF membranes refers to the sizes 225 kD, 150 kD, 102 kD, 76 kD and 52 kD, respectively. The arrows on each figure indicate the relative molecular weight of the membrane proteins on PVDF membranes.

The separation of membrane proteins by gel electrophoresis followed by transfers of the membrane proteins from the gels to PVDF membranes by blotting were done successfully. Ponceau-red staining of membrane proteins on the PVDF membrane indicates successful membrane transfer (Fig 2A and B). The arrows on the figures indicate visible bands on PVDF membranes with the expected molecular weight of approximately 100 kD of the membrane proteins including Na+/K+-ATPase (Fig 2A and B). However, no bands of NKA on x-ray film were detected.

5. Discussion

In the current study, proteins were successfully isolated from the intestine of mature and immature Atlantic salmon. General Ponceau staining indicated proteins of the correct size. However, detection using antibodies directed towards the NKA was unsuccessful. No quantitative data on possible differences in NKA abundance between mature and immature Atlantic salmon could be obtained. Therefore the discussion is based on previous studies.

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5.1. Problems with Western Blot

Protein samples used in western blot were treated cautiously. These were put on ice at all time to prevent protein degradation, in other words the protein samples were of good quality. Also, the samples were treated equally during the different steps of the experiment to prevent variability. Gel electrophoresis and the transfer of proteins from gel to PVDF membrane has been performed correctly. The pre-stained multi-colored molecular weight marker was visible on both gel (not shown) and PVDF membrane (Fig2 A and B). Also, the staining of membrane proteins on PVDF membrane with Ponceau red indicated proteins of the correct size. The PVDF membrane used has high protein binding capacity and mechanical strength, which are good properties when doing the experiment. However, a problem occurred during exposure of the membrane bound NKA antibody to x-ray film in a darkroom. No bands of NKA could be detected when the film was being developed. Though previous test control detected NKA on x-ray film. From these results we concluded that it could be due to reduced quality of the antibodies, which doesn’t bind properly to NKA. It’s known that the α5 NKA antibody is believed to cross react will all α isoforms in Atlantic salmon (Sundh et al., unpublished). Further, during the test control different concentrations of α5 NKA antibody were tested. Both concentrations worked and the concentration 0,01 µg/ml of α5 NKA antibody was used in the experiment with the anti-mouse IgG Horseradish Peroxidase-linked whole antibody (Sundh et al., unpublished,). Other explanations could be that the experimental procedure may have not been followed correctly. In this way Western blot is a sensitive method to human errors. Membrane may also have been damaged that makes it unusable, or it was may be due to short exposure time. The exposure time 15 seconds and 5 minutes were tested, but 5 minutes were recommended by the manufacturer and was used in experiment. Also, the detection reagent may not have worked which is important for enhancement and duration of signal in the chemiluminescent reaction. Without the detections reagent NKA couldn’t be detected on x-ray film. Overall, we couldn’t quantify the expression of NKA in proximal and distal intestine of mature and immature Atlantic salmon.

5.2. Effects of sexual maturation and osmoregulatory problems in SW

It has been hypothesized that early maturation of Atlantic salmon in SW leads to osmoregulatory problems. Previous study has shown that permeability of the intestine to Na+ is increased in mature Atlantic salmon (Sundh et al., unpublished). Consequences are it become harder to maintain a high osmotic gradient in LIS. Therefore the activity of NKA must increase. This can be done by increasing the number of NKA or the activity of NKA available increases. We then tried to quantify the numbers of NKA located in the cell membrane of enterocytes. At the moment we don’t have experimental evidence that support this theory. However, previous studies have indicated increased short circuit current (SCC) suggesting increased activity of NKA (Sundh et al., unpublished). This means there is an active ongoing absorption of fluid in the intestine of mature Atlantic salmon. Another aspect of early maturation is that the fish may reverse its function from SW to FW state. Following transfer to FW most NKA are found to localize on the basal part of the enterocyte membrane with low impact on fluid absorption (Sundh et al., unpublished). In this scenario, if the mature Atlantic salmon is kept for a long period of time in SW the Na+,K+-ATPase activity wouldn’t

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keep up with the leakage of Na+ ions and/or the amount of water lost couldn’t be completely replaced. The consequences would be internal osmotic disturbance and hemoconcentration, which lead to death of fish (Gonzalez., 2012).

5.3. Intestinal Na+/ K+- ATPase and isoforms in SW

Other studies in Atlantic salmon have shown that acclimation to SW increases the abundance of enterocyte Na+, K+- ATPase (Sundh et al., unpublished). This is apparent in both anterior and posterior intestine (Sundell et al., 2003). Na+,K+-ATPase is richly abundant in basal and lateral membranes of the columnar enterocytes (Grosell., 2011), but in greater number are present in the latter (Sundh et al., unpublished). NKA is positioned on the latter part of enterocytes to efficiently create a high osmotic gradient within LIS that drives fluid absorption in the intestine. In the current study, we investigated the abundance of intestinal NKA in Atlantic salmon during SW acclimation without considering the different isoforms of NKA. Recently, intestinal NKA isoforms such α1c, α1a and α1b has been examined with their essential roles in intestinal fluid absorption after SW transfer (Sundh et al., unpublished). These have been studied at both transcriptional and protein levels. The NKA α1c isoform is regarded as the main driving force in NaCl-coupled water transport in the intestine during SW-acclimation (Sundh et al., unpublished). Transcription of NKA α1c mRNA in both proximal and distal intestine is 1000-fold higher than any other isoform observed (Sundh et al., unpublished). At the protein level, NKA α1c is the dominating NKA isoform in the proximal intestine, but in the distal intestine NKA α1c was most abundant in the lower region of the mucosal folds (Sundh et al., unpublished). NKA- α1a and - α1b was localized to the intestinal smooth muscle layers suggesting they have a house keeping role in excitatory tissues (Sundh et al., unpublished). Overall, each NKA α isoform along with their specific distribution in the intestine of Atlantic salmon shows us that they have distinct physiological function and regulation during SW acclimation.

5.4. Fluid absorption in the intestine of Atlantic salmon

In SW fluid absorption in the intestine of Atlantic salmon is important to prevent the fish from dehydration. From previous studies the observed variation in the intestinal epithelial barrier function of mature and immature Atlantic salmon in SW tells us what their function might be between the two fishes. The decreased of intestinal barrier function in mature Atlantic salmon suggests increase water uptake via both paracellular and transcellular pathway in the intestine of Atlantic salmon (Sundell et al., 2003). This compared to immature Atlantic salmon that has been observed to have increased intestinal barrier function, which allows for building up a sufficient osmotic gradient in the LIS. It has been suggested increase water uptake only via transcellular pathway in the intestine of Atlantic salmon (Sundell et al., 2003). Further, the decreased intestinal epithelial barrier function in mature Atlantic salmon is consistent with increasing levels of cortisol in plasma of Atlantic salmon in SW, which has been observed to promote fluid absorption in proximal intestine and distal intestine via paracellular pathway (Sundell & Sundh., 2012). Cortisol is believed to down-regulate claudin-25b in tight junctions between adjacent enterocytes (Sundell & Sundh., 2012) making it more permeable. Tight junctions will be discussed later in this section. In conclusion, mature Atlantic salmon in SW has reduced intestinal epithelial barrier function that promotes fluid absorption via paracellular pathway.

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5.5. Intestinal epithelium and tight junctions of Atlantic salmon in SW

Paracellular permeability in the intestine of Atlantic salmon is determined by the sealing properties of tight junctions between adjacent enterocytes. This structure is composed of different kind of membrane proteins, including claudins and the MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain containing proteins; occludin and tricellulin. In mature Atlantic salmon the increased intestinal permeability indicates that tight junctions are down-regulated. Compared to immature Atlantic salmon intestine, which we used as a control for this study, elevation in transepithelial resistance was observed in both proximal and distal intestine, with distal intestine having 50% higher transepithelial resistance than the proximal intestine ( Sundh et al., unpublished result). It could be due to higher mRNA expression of claudin-15 and claudin-25b, which are both specific intestinal isoform (Tipsmark et al., 2012). These proteins line the pore that is formed between the adjacent cells, which constitute the passage way for molecules using the paracellular pathway (Sundell & Sundh., 2012). For this reason the expression of claudin isoforms has been suggested to be the main determiners of TJ ion and size selectivity (Sundell & Sundh., 2012). Further, increased intestinal mRNA expression of tricellulin and occludin has been demonstrated in the intestine of SW-salmon, indicating enforcement of tight junctions (Tipsmark et al., 2012). So far, claudin-15, claudin-25b, tricellulin and occludin have not been observed in protein level in the intestine of Atlantic salmon. In summary, the paracellular Na+ permeability in the intestine of Atlantic salmon in SW involves tight junctions and it’s believed to be down-regulated in mature Atlantic salmon and has been observed to be up-regulated in immature Atlantic salmon.

5.6. Further studies

Further works will be focused on studying the distribution of NKA in the membrane of enterocytes lining the intestine from pyloric caeca trough the anus. We will perform IHC using sections of formalin-fixed waxed embedded tissue of both proximal and distal intestine from mature vs immature Atlantic salmon in SW. The immature Atlantic salmon will be used as a control. In the current study Western blot was performed to quantify the expression of NKA, and as an extension to this work we would want to relate the result according to their distribution in the intestine. This will help us to exactly distinguish the difference in NKA localization and their abundance in proximal and distal intestine between mature vs immature Atlantic salmon in SW. Number of individuals and replicates used in the experiment should increase to ensure the results are reproducible in other individuals of Atlantic salmon. It will also reduce the variance and improve the significant of the results. Whether the expression of NKA is increased or not in mature vs immature Atlantic salmon remains to be investigated by Western blot.

6. Conclusion

The abundance of NKA in proximal and distal intestine of mature Atlantic salmon couldn’t be quantified through detection with antibodies. Although, previous studies have observed increased short circuit current suggesting increase activity of NKA, a continued experimentation of NKA by Western Blot is needed to get a result. In short, to ensure successful experimentation with Western Blot, better preparation and performance of the method is required.

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7. Acknowledgements

I would like to thank Henrik Sundh for letting me work with this project and his feedback on writing this thesis. I would to thank Linda Frank for her instructions and assistant in the laboratory. I would like to thank the University of Gothenburg and the Department of Biological and Environmental Sciences.

8. References

Gonzalez RJ. 2012. The physiology of hyper-salinity tolerance in teleost fish: a review. J Comp Physiol B 182:321-329.

Grosell M. 2011. Intestinal anion exchange in marine teleosts is involved in osmoregulation and contributes to the oceanic inorganic carbon cycle. Acta Physiol, 202, 421-434.

Madsen SS, Olesen J-H, Bedal K, Engelund M-B, Velasco-Santamaria Y-M, Tipsmark CK. 2011. Functional characterization of water transport and cellular localization of three aquaporin paralogs in the salmonid intestine. Aquatic Physiology, Volume 2 Article 56.

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Tipsmark CK, Sorensen KJ, Hulgard K, Madsen SS. 2010. Claudin-15 and -25b expression in the intestinal tract of Atlantic salmon in response to seawater acclimation, smoltification and hormone treatment. Comparative Biochemistry and Physiology, Part A 155 361-370.

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Atlantic salmon (Salmo salar - Göteborgs universitet · Atlantic salmon is an anadramous salmonid, which means its life cycle involves both freshwater (FW) and seawater (SW). They

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