The digestive system of Atlantic salmon (Salmo salar ...

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The digestive system of Atlantic salmon (Salmo salar L.) - Ontogeny and response to soybean meal rich diets –

Christian Sahlmann

Thesis for the degree of Philosophiae Doctor (PhD)

Norwegian School of Veterinary Science Department of Basic Sciences and Aquatic Medicine Oslo 2013

© Christian Sahlmann, 2013 Series of dissertations submitted to the Norwegian School of Veterinary Science No. 147 ISSN 1890-0364 ISBN 978-82-7725-261-2 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission.

Cover: Akademika Publishing Printed in Norway: AIT Oslo AS. Produced in co-operation with Akademika Publishing. The thesis is produced by Akademika publishing merely in connection with the thesis defence. Kindly direct all inquiries regarding the thesis to the copyright holder or the unit which grants the doctorate.

Table of contents Acknowledgements ........................................................................................ I List of abbreviations and terms .................................................................. III List of articles ................................................................................................ V Summary...................................................................................................... VII Sammendrag................................................................................................. IX 1. Background ................................................................................................ 1 1.1 1.2 1.3

Life cycle of Atlantic salmon and ontogeny of the gastrointestinal tract ................ 3 Structure and function of the mature gastrointestinal tract of Salmo salar .............. 6 Soybean meal-induced enteritis as a model .......................................................... 11

2. Aims and strategies .................................................................................. 15 3. Methodology ............................................................................................. 17 3.1 3.2 3.3 3.4 3.5 3.6 3.7

Overview ................................................................................................................ 17 Histology ................................................................................................................ 18 Immunohistochemistry ........................................................................................... 18 Enzyme activity assay ............................................................................................ 21 RNA extraction and cDNA synthesis ..................................................................... 22 Quantitative real-time polymerase chain reaction (qPCR) ..................................... 23 Microarray .............................................................................................................. 25

4. Summary of seperate papers .................................................................. 31 5. Main results and discussion .................................................................... 35 5.1 5.2

Ontogeny of the digestive system of Atlantic salmon ............................................ 35 Response to soybean meal-based diets ................................................................... 40

6. Conclusions and perspectives ................................................................. 49 6.1 6.2

Conclusions ............................................................................................................ 49 Perspectives ............................................................................................................ 50

7. References ................................................................................................ 51 8. Publications.............................................................................................. 67

Acknowledgements I would like to thank sincerely my supervisors Prof. Åshild Krogdahl and Assoc. Prof. Anne Marie Bakke for giving me the chance to do the PhD at the Norwegian School of Veterinary Science and for their help, constructive and insightful criticism and for their patience throughout this period. Prof. Ben Koop from the University of Victoria for giving me the opportunity to work in his lab. The work conducted at UVic is a major part of this PhD thesis and I am grateful for Prof. Koop’s generous support and valuable input during my time at the Centre for Biomedical Research. my fellow PhD colleagues Elvis Chikwati and Fredrik Venold. You went through the highs and lows of this PhD with me. And a big thanks to the new PhD student Karina Gajardo for her help during the last stretch! the “second floor” scientists Trond Kortner for the guidance and motivation in the lab, your great support during the final strech of the thesis, great discussions and coffee times; Jinni Gu for the great help in the lab, good talks (not just scientific), many laughters and some chinese lessons; Michael Penn for good discussion, helpful advice and for being a great colleague. Jim Thorsen (former second floor scientist) for the enthusiasm and guidance during the beginning of my PhD. especially the lab technicians! Ellen Hage, Gunn Østby and Elin Valen. What would I have done without you? Thank you for building me up when I was down and of course for all the hard work you put in! sincerely Ben Sutherland from the Koop lab for the huge support during times of crisis and for the great amount of time and effort you were spending on helping me while you had your hands full with you own work. the Koop lab: Kris, Erik, Amber, Jong and Stuart. Thank you for making me feel welcome and helping me finding my way around in the lab and in Victoria. all the colleagues from the Aquaculture Protein Centre for the great meetings, good discussions and fun activities. the staff at Nofima in Sunndalsøra for their professional handling of the feeding trials and for making the sampling trips very enjoyable. Ich möchte besonders meiner Familie für die großartige Unterstützung danken. Danke Andrea! Wir haben das gemeinsam geschafft! I

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List of abbreviations and terms ANF BBM

Antinutritional factor Brush border membrane

SNV ST

Supranuclear vacuole Stomach

DAMP DEG

Damage-associated molecular pattern Differentially expressed gene

TLR

Toll-like receptor

DI

Distal intestine

DPH ECM ES FM GALT GIT

Days post hatch Extracellular matrix Esophagus Fishmeal Gut associated lymphoid tissue Gastrointestinal tract

IHC MI

Immunohistochemistry Mid intestine

mRNA

Messenger RNA Pathogen-asscociated molecular pattern

PAMP PAR

Proteinase-activated receptor

PI PRR

Proximal intestine Pathogen recognition receptor

qPCR

Quantitative real-time polymerase chain reaction Soybean meal (Standard solvent-extracted) Soybean meal induced enteritis Soybean trypsin inhibitor

SBM SBMIE SBTI

Definition of developmental stages Juvenile:

The period from hatch until adaptation to seawater (smoltification).

Alevin:

The period from hatch until first-feeding. Also called yolk sac fry or free

swimmimg embryo. The yolk sac is visible externally. The period from first feeding until the appearance of dark stripes on the lateral line (parr marks). In this stage, the yolk sac is absorbed and no longer visible outside the body. Parr: The period from the appearance of parr marks until adaptation to seawater (smoltification) Smolt: The period of transition to seawater. Parr marks are no longer visible. Post-smolt: The period after transition to seawater. Fry:

III

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List of articles

Paper I Ontogeny of the digestive system in Atlantic salmon (Salmo salar L.) and effects of soybean meal from first-feeding. Sahlmann C, Gu J, Kortner TM, Lein I, Krogdahl Å, Bakke AM. (manuscript).

Paper II Early response of gene expression in the distal intestine of Atlantic salmon (Salmo salar L.) during the development of soybean meal induced enteritis. Sahlmann C, Sutherland BJG, Kortner TM, Koop BF, Krogdahl Å, Bakke AM. 2013. Fish and Shellfish Immunology 34: 599-609.

Paper III Alterations in digestive enzyme activities during the development of diet-induced enteritis in Atlantic salmon, Salmo salar L. Chikwati EM, Sahlmann C, Holm H, Penn MH, Krogdahl Å, Bakke AM. 2013. Aquaculture, 402-403: 28-37

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Summary Commercial farming of Atlantic salmon (Salmo salar L.) started in the 1970s and is a rapidly growing industry today. Yet knowledge gaps regarding basic biological aspects of salmon still exist. This includes specific requirements for many nutrients, basic understanding of functional ontogeny and physiology at various stages of development, as well as long term implications of substituting marine feed ingredients with alternative protein and lipid sources. The inclusion of proteins from plants and other alternative feed ingredients in commercial fish feed, for example, is an important factor for the sustainable development of aquaculture as it reduces the dependence on fishmeal from wild stock fisheries. However, plant products (e.g. soybean meal, SBM) can negatively affect growth, feed utilisation and intestinal health of several fish species, including post-smolt Atlantic salmon. The SBM-induced enteritis (SBMIE) in the distal intestine of seawater-adapted (post-smolt) S. salar caused by >5-10% full fat or defatted (extracted) SBM in diets is relatively well characterised on a morphological level, and represents a promising model for the study of diet-induced intestinal inflammation. Morphologically, signs of the inflammation are apparent after only a few days of SBM feeding. Yet the early stages of development of SBMIE have not been exhaustively described from SBM introduction into the diet. How pre-smolt salmon and fry are responding to SBM has not been investigated. This thesis aimed to address current knowledge gaps regarding the ontogeny of the gastrointestinal tract (GIT) of salmon and the effects of SBM inclusion in diets for firstfeeding salmon fry on the structural, physiological as well as molecular level. An additional goal was to get a better understanding of the initial stages of the development of the soybean meal-induced enteritis (SBMIE) model by investigating immediate responses of molecular and biochemical parameters in the GIT of post-smolt Atlantic salmon during the onset of the inflammation. The results presented in paper I showed that one week after hatch, pancreas and liver were present and appeared to be functional in alevins. At the time of first feeding pyloric caeca started to form and a stomach with gastric glands was present. Molecular analyses revealed an up-regulation of genes with functions related to appetite regulation, digestion, nutrient transport, and immune response approximately one week before exogenous feeding. The study presented in paper I also revealed that growth, histomorphological development, gene expression and digestive enzyme activities in the GIT were not altered in juveniles fed a diet with 17% soybean meal (SBM) inclusion. The results presented in paper II revealed that the transcriptome of the distal intestine was affected immediately after the introduction of SBM in the diet and the early response appeared to be initiated by genes related to immune function. Subsequently, the expression of genes with important epithelial functions was altered during early stages of SBMIE (paper II) and protein expression of carbonic anhydrase 12, indicating VII

Summary degree of differentiation and maturation, progressively decreased (paper III), indicative of epithelial barrier dysfunction. Activities of the distal intestinal brush border membrane (BBM) enzymes maltase and leucine aminopeptidase were significantly reduced on day 1-2 of SBM exposure, showed a brief recovery on day 3, but then continued to decrease progressively with increasing SBM exposure time (paper III). The early response in enzyme activities, however, could also be related to the change of diet and/or variation in feed intake. Contrary to other BBM enzymes, transcripts of the potential pro-inflammatory mediator PAR-2 (proteinaseactivated receptor 2) were up-regulated on day 2 (pilot study). In conclusion, in our work presented in paper I we describe the development of the GIT of Atlantic salmon and its accessory digestive organs from hatch to the juvenile stage for the first time by using histological, biochemical and molecular methods. We could show that the digestive system is potentially functional about one week before complete yolk sac absorption. Furthermore, we found that juvenile salmon may be able to tolerate SBM during the first three months. Our study (paper II, paper III) is the first to describe the immediate response of the immune system in the distal intestine after switching to a diet containing SBM during onset of SBMIE, which subsequently appeared to result in disruption of epithelial barrier function and dysfunction of digestive processes in the GIT. With a better understanding of the development of the juvenile digestive system, salmon smolt robustness may be improved by adjusting feeding and rearing of alevins and fry. A better understanding of SBMIE in post-smolts as a model for diet-induced inflammation may provide insight into basic immune functions of salmon. On the other hand, the continuing research on SBMIE may eventually identify a way to ameliorate the condition and thereby continue towards the goal of introducing more sustainable alternative protein sources in fish feeds.

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Sammendrag Norsk lakseoppdrett som ble etablert på 1970-tallet, har utviklet seg til en rask voksende industri. Til tross for lakseoppdrettets lange historie er kunnskap om grunnleggende biologiske aspekter hos laks fortsatt begrenset. Spesielt mangler mye kunnskap om næringsstoffbehov og om laksens utvikling, strukturelt og funksjonelt, fra embryostadiet til fysiologisk moden fisk, og om langsiktige konsekvenser av å erstatte marine fôringredienser med alternative proteinog lipidkilder. Økt bruk av plantebaserte og andre alternative proteinkilder i fôr til oppdrettsfisk blir ansett som en viktig faktor for en bærekraftig utvikling av havbruksindustrien, siden det vil redusere industriens avhengighet av fiskemel. Imidlertid kan økt bruk av planteprodukter (f.eks. soyamel) gi redusert vekst og dårligere tarmhelse hos en rekke fiskearter, inkludert Atlantisk laks. Betennelsesreaksjonen (enteritten) som oppstår i baktarmen hos postsmolt laks som gis fôr som er tilsatt mer enn 5-10 % fullfett eller ekstrahert soyamel er relativt godt beskrevet på morfologisk nivå, og representerer en god modell for å studere fôrindusert tarmbetennelse. Morfologisk kan en betennelsesreaksjon observeres etter kun få dager med fôring på soyamel, men de aller tidligste stadiene i utviklingen av betennelsesreaksjonen er til nå ikke beskrevet i detalj. Hvordan presmolt laks og yngel reagerer på soyamel er ikke kjent. Denne avhandlingen tok sikte på å styrke kunnskapen om den tidlige utviklingen av magetarmkanalen i laks og effektene av soyamel i fôr til lakseyngel fra startfôring både strukturelt, fysiologisk og molekylært. I tillegg var det et mål å øke forståelsen av mekanismene bak de tidlige stadiene i utviklingen av soyamelindusert enteritt ved å undersøke umiddelbare reaksjoner i molekylære og biokjemiske parametere i mage-tarmkanalen til postsmolt Atlantisk laks. Resultatene presentert i paper I indikerer at hos Atlantisk laks er både bukspyttkjertel og lever funksjonelle én uke etter klekking. Magesekk med funduskjertler og blindsekker så ut til å være ferdig utviklet ved startfôring. Uttrykk av gener med funksjoner relatert til appetittregulering, fordøyelse, næringsstofftransport og immunrespons var oppregulert omtrent én uke før startfôring. Arbeidet som er presentert i paper I viste også at vekst, histomorfologisk utvikling, genekspresjon og aktivitet av fordøyelsesenzymer ikke ble påvirket i yngel som ble fôret et fôr med 17% soyamel. Resultatene presentert i paper II viste at genekspresjon i baktarmen til postsmolt laks ble påvirket umiddelbart etter at soyamelfôringen startet, og den tidlige responsen syntes å være initiert av gener relatert til immunfunksjon. Videre ble uttrykket av gener med viktige epitelfunksjoner endret i tidlige stadier av enteritten (paper II) og proteinuttrykket av karbonsyreanhydrase 12, som indikerer grad av differensiering og modning, avtok gradvis (paper III). Disse resultatene gir indikasjoner om nedsatt epitelbarriere i baktarmen. Aktiviteten av børstesømenzymene maltase og leucine aminopeptidase var også betydelig redusert etter kun 1-2 dagers fôring med soyamelholdig fôr, og aktiviteten IX

Sammendrag fortsatte å avta gradvis med økende eksponeringstid for soyamel (paper III). Det kan være en sammenheng mellom den raske responsen i enzymaktivitet og økningen i genuttrykk for den proinflammatoriske reseptoren PAR-2 (proteinase-aktivert reseptor 2) som så ut til å sammenfalle på dag 2 (pilot study). Resultatene som er presentert i paper I gir den første publiserte beskrivelse av utviklingen av laksens mage-tarmkanal, lever og bukspyttkjertel fra klekking til 5 grams størrelse ved hjelp av histologiske, biokjemiske og molekylære metoder. Hovedkonklusjonene er at laks ser ut til å være i stand til å fordøye fôr fra omtrent en uke før plommesekken er fullstendig absorbert. Arbeidene viser også at lakseyngelen kan være mer robust mot variasjoner i fôrets sammensetning enn mer utviklet fisk. Studiene som er presentert i paper II og III, er de første som viser hvordan immunforsvaret i laksens baktarm påvirkes umiddelbart etter bytte av fôr fra et fiskemel-basert til et med 20 % soyamel, med endringer på både transkripsjons-, translasjons- og enzymatisk nivå. Dette arbeidet har styrket kunnskapen om den tidlige utviklingen av laksens fordøyelsessystem, noe som kan gi mulighet for å justere fôring og stell av yngel og dermed gi grunnlag for en mer robust laksesmolt. Det gir også en bedre forståelse av soyamelindusert enteritt i postsmolt laks som en modell for fôrindusert betennelse og kan gi ny og grunnleggende innsikt i tarmens immunforsvar. Videre forskning på soyamelindusert enteritt vil kunne gi kunnskap om hvordan en slik fôrindusert betennelse kan forhindres, noe som vil være et viktig skritt på veien mot økt benyttelse av bærekraftige proteinkilder i fiskefôr.

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1. Background Atlantic salmon (Salmo salar L.) have been farmed on a commercial scale in Norway since the 1970’s and has rapidly grown into a large industry in several countries. However, salmon aquaculture faces several challenges that must be met for further development. Salmon production needs to progress in an environmentally sustainable manner, while ensuring both animal welfare and high quality food standards for human consumption. One aspect of sustainable development is the optimisation of Atlantic salmon rearing and the minimisation of losses of salmon smolts during and shortly after seawater transfer. Research is underway to increase the robustness of salmon smolts (e.g. Research Council of Norway funded havbruk project no. 225219: “Improving Atlantic salmon smolt robustness to reduce losses in sea by development of screening tests, exercise regimes and markers”), however, only limited information on the early ontogeny of salmon is available today. Another aspect which is important for sustainable development concerns the choice of feed ingredients. Until recent years, fishmeal was the major source of protein in salmon feeds. Despite the rapid growth of aquaculture over the last 20 years, fishery catches of key species for fishmeal production, such as anchovy (Engraulis ringens), manhaden (Brevoortia spp.; Ethmidium spp.) and capelin (Mallotus villosus) as well as various species of shellfish, have remained relatively stable (FAO, 2012b). World catch quotas need to be carefully managed in order to avoid overfishing. Because many of these fish populations are at their exploitation limit or beyond, and prices of fishmeal have increased due to increased demand, research efforts began to focus on the use of alternative protein sources, mostly plants, in the diets of S. salar and other farmed carnivorous fish species. As a result, compound feeds used in aquaculture today contain increasing amounts of plant protein sources (FAO, 2011; Table 1.1). Research on other protein sources, such as bacterial and krill meal, is also on-going (Storebakken et al., 2004; Hansen et al., 2011; Romarheim et al., 2011). Many fishmeal replacements from plant sources may not be suitable as a primary ingredient due to imbalanced amino acid, fatty acid and mineral profiles, as well as the presence of antinutritional factors (ANFs). Thus plant-based diets need to be supplemented to meet the nutritional needs of the fish. Furthermore, the use of some plant ingredients, especially legumes such as soybean meals and pea meals, in feeds for carnivorous fish have resulted in adverse effects, for example decreased growth, decreased nutrient digestibility and compromised health (Gomes et al., 1995; Regost et al., 1999; Aslaksen et al., 2007; Knudsen et al., 2008; Krogdahl et al., 2010; Yun et al., 2011). These negative effects have been related to ANFs of which plant-based feeds contain various types (Francis et al., 2001; Krogdahl et al., 2010). Antinutritional factor is a broad term for substances that are synthesized by plants and may serve various functions in the plant such as protection from foraging animals, insect pests or microbial pathogens. These substances may therefore also affect the health of animals and 1

Background Table 1.1: List of common ingredients in compound feed in finfish aquaculture and their respective inclusion level ranges (adapted from Tacon 2011) Feed ingredients

Inclusion level in compound aquafeed [%]

Plant protein meal Soybean meal Wheat gluten meal Corn gluten meal Rapeseed/canola meal Cottonseed meal Groundnut/peanut meal Mustard oil cake Lupin kernel meal Sunflower seed meal Canola protein concentrate Broad bean meal Field pea meal

3–60 2–13 2–40 2–40 1–25 ≈ 30 ≈ 10 5–30 5–9 10–15 5–8 3–10

Plant oil Rapeseed/canola oil Soybean oil

5–15 1–10

humans when ingested by interfering with various physiological processes in the intestine (Francis et al., 2001). Treatment of plant ingredients, also during feed production, may remove or inactivate antinutrients and increase nutrient digestibility. For example, heat treatment may reduce proteinase inhibitor activity and thereby increase protein digestibility (Romarheim et al., 2005) while lactic acid fermentation can be used to remove indigestible carbohydrates (Refstie et al., 2005). Enzyme treatment can also be used to eliminate ANFs and thereby increase nutrient digestibility and availability (Storebakken et al., 1998). The introductory chapters in this thesis give an overview of the present knowledge with relevance to the experimental work conducted. The life cycle of Salmo salar and the ontogeny of the gastrointestinal tract (GIT) of marine species and freshwater species is presented in chapter 1.1. A summary of present knowledge regarding structural and functional aspects of the mature GIT of S. salar is given in chapter 1.2. Current knowledge regarding the effects of plant proteins with a focus on soybean meal-induced inflammation are also presented in chapter 1.3.

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Background Table 1.2: List of antinutritional factors of plants and possible treatment for inactivation/removal (from Krogdahl et al. 2010) Antinutrient Proteinase inhibitors

Sources

Type of treatment

Legumes

Heat, methionine

Amylase inhibitors Lipase inhibitor Lectins

Peas Beans All plants seeds

supplementation Heat Heat Heat, supplementation with

Phytic acid Fibre Tannins

All plants All plants Rape seed,

specific carbohydrates

beans Saponins Sterols

Legumes Legumes

Oestrogens Gossypol

Beans Cotton seed

Oligosaccharides Quinolozidine alkaloids Goitrogens

Legumes Lupins Rape seed

Mineral supplementation Dehulling Dehulling, restriction of heat treatment Alcohol extraction Alcohol/non-polar extraction, cholesterol supplementation Alcohol/non-polar extraction Non-polar extraction, iron supplementation Alcohol/aqueous extraction Aqueous extraction Iodine supplementation

The work of this thesis aimed to address the current knowledge gaps regarding the ontogeny of the GIT of juvenile salmon (chapter 5.1; paper I) and the effects of soybean meal on the ontogeny of the GIT of juvenile salmon (chapter 5.2; paper I). In addition, immediate responses of molecular and biochemical parameters in the GIT of post-smolt Atlantic salmon during the development of soybean meal-induced enteritis were characterised (chapter 5.2; paper II, III, pilot study)

1.1 Life cycle of Atlantic salmon and ontogeny of the gastrointestinal tract The life cycle of Atlantic salmon can be divided into six stages: Egg, alevin, fry, parr, smolt and post-smolt salmon (Fig. 1.1) (Allan and Ritter, 1977). In contrast to many marine fish species, salmonids do not undergo a metamorphosis from a larval to juvenile stage. Salmon alevins hatch with a large external yolk sac that supplies nutrients during the first weeks of their life. In nature, alevins hide in the gravel and emerge to feed when the external yolk sac is no longer visible. It seems important for the survival of juveniles to have a certain time window concerning the emergence from the gravel and the beginning of exogenous feeding. This enables salmon to adapt to changing conditions in their natural habitat such as the presence of predators or the abundance of competitors. (Brännäs, 1995; Jones et al., 2003). 3

Background

Figure 1.1: Life cycle of Salmo salar L. (source: www.atlanticsalmontrust.org) After the transition to exogenous feeding, salmon are referred to as fry. Fry develop into parr, recognised by dark stripes or “parr marks” along the lateral line. Parr prepare to adapt to seawater (smoltify), which results in drastic changes in behaviour, morphology, physiology and biochemistry (review by Folmar and Dickhoff, 1980; Wedemeyer et al., 1980) and are subsequently termed smolts. Smolts travel downstream and eventually enter the ocean. Postsmolts continue their life cycle in seawater before returning to their home streams for breeding in the following years. Some aspects of early life stages of Atlantic salmon have been intensively studied due to the commercial importance of this species. A wealth of data can be found describing general aspects of the physiology and morphology of eggs and early stages of development (Cowey et al., 1985; Gorodilov, 1996), the effect of environmental parameters (Daye and Garside, 1977; Peterson et al., 1977; Gunnes, 1979; Daye and Garside, 1980; Wedemeyer et al., 1980), effects of toxins (Rombough and Garside, 1982; Peterson et al., 1983; Peterson et al., 1989; Knoph, 1992) and stress (Fenderson and Carpenter, 1971; Schreck, 1982). The ontogeny of the digestive system of Atlantic salmon, however, has received far less attention since fry readily ingest compound feed from first feeding, minimizing the effort needed to successfully rear salmon juveniles. Compared to salmon juveniles, most marine fish larvae rely on live feed, such as Artemia and rotifers (Planas and Cunha, 1999), and are more difficult to rear 4

Background during early stages of development. Therefore, numerous studies on the functional ontogeny of the digestive system have been conducted in order to understand and characterize digestive capacity at first-feeding stages of various marine larvae (reviewed by Cahu and Zambonino Infante, 2001; Zambonino Infante and Cahu, 2001; Hoehne-Reitan and Kjørsvik, 2004; Rønnestad et al., 2007; Zambonino et al., 2008). Information from studies of the ontogeny of the digestive system of marine and some freshwater fish species elucidate key points that are currently not well characterised in Atlantic salmon. Organ development in fish seems to be largely dependent on the developmental stage and the availability of endogenous nutrient supplies (Hoehne-Reitan and Kjørsvik, 2004). At hatch, the GIT of most fish species is a straight tube (Zambonino et al., 2008). In many small marine fish larvae, the morphology of the GIT appears to be less differentiated at hatch compared to newly hatched, anadromous and freshwater fish (Lazo et al., 2011). Stomach and pyloric caeca are in many fish species the last structures to develop (Zambonino et al., 2008; Lazo et al., 2011). These regions are of major importance for optimal digestion (stomach) and nutrient absorption (pyloric caeca), but the timing of the functional ontogeny varies greatly among fish species (Zambonino et al., 2008). Some species, such as chum salmon Oncorhynchus keta W. (Takahashi et al., 1978) and wolffish Anarhichas lupus L. (Falk-Petersen and Hansen, 2001), have a functional stomach at first feeding, while in others, such as Atlantic halibut Hippoglossus hippoglossus L., a functional stomach develops as late as 50 days post firstfeeding (Luizi et al., 1999). Identifying the timing of apparent functional ontogeny of the stomach and other digestive organs may therefore indicate its role during the ontogeny of different species and certainly be of importance in developing diets to match the functional ontogeny and physiological capacity of the fish. The accessory organs, liver and pancreas, are differentiated early after hatch in several fish species (Hoehne-Reitan and Kjørsvik, 2004). Pancreatic enzyme activity has been observed in marine fish larvae before external feeding was initiated, indicating that digestion in an alkaline environment is of great importance at the onset of exogenous feeding (Zambonino Infante and Cahu, 2001). In terms of digestive function, earlier theories stated that the digestive system of marine larvae may be less developed at first feeding in marine fish larvae than in salmonid species (Dabrowski, 1984). However, the advances in molecular biology have facilitated more detailed studies and partially revised this hypothesis. The GIT of marine fish larvae is probably more developed and functionally capable than previously thought (Zambonino et al., 2008; Lazo et al., 2011).

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Background In summary, some key points for the assessment of the state of the digestive system at firstfeeding is the sequential ontogeny of a functional stomach, PI with pyloric caeca as well as accessory digestive organs. Since the current knowledge regarding this in farmed Atlantic salmon was limited, these key points were addressed with the use of histomorphological, biochemical and molecular techniques as part of this thesis (paper I).

1.2 Structure and function of the mature gastrointestinal tract of Salmo salar 1.2.1 Structure The mature GIT of S. salar shares the general morphology of teleost fishes. Starting from the mouth, it can be subdivided into esophagus (ES), stomach (ST), proximal intestine (PI) with adjacent pyloric caeca, mid (MI) and distal intestine (DI) (Fig. 1.2). The ES of teleost fish has been described as a straight, thick walled tube with longitudinal folds, which allow an increase in diameter when large amounts of food are ingested (Wilson and Castro, 2010). The mucosal epithelium is stratified and includes numerous mucus secreting cells (Langille and Youson, 1985). No clear border or sphincter at the transition of ES to ST is visible in most fishes (Wilson and Castro, 2010). The ST of teleost fish is enveloped in smooth muscle and can be divided into cardiac, fundic and pyloric region (Zambonino et al., 2008; Lazo et al., 2011). Some authors do not discriminate between cardiac and fundus but describe the whole anterior part as cardiac region (Harder, 1975). In this thesis we use the term cardiac for the anterior part, fundic for the region where gastric glands are present, and pyloric for the region adjacent to the pyloric sphincter. Gastric glands of fish contain secretory oxynticopeptic cells that secrete both hydrochloric acid, as well as pepsinogen (Barrington, 1957). The pyloric sphincter marks the transition to the intestine. The PI of S. salar has fingerlike, blind appendages called pyloric caeca, which greatly increase the surface area of the proximal intestine (Fig. 1.2). Pyloric caeca and PI are surrounded by mesenteric adipose

Figure 1.2: The gastrointestinal tract of S. salar. Esophagus (Es), stomach (ST), proximal intestine (PI) with pyloric caeca, mid (MI) and distal intestine (DI). (Photo: Åshild Krogdahl)

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Background

Lu BBM

SNV

N

Figure 1.3: Structure of the distal intestinal epithelium. A: Histological section of the distal intestinal epithelium (photo: Michael Penn). B: Schematic drawing of a single enterocyte. Lu: lumen; BBM: brush border membrane; SNV: supranuclear vacuoules; N: nucleus; LP: Lamina propria tissue with interspersed, diffusely organised endocrine and exocrine pancreatic tissue. No clear morphological separation between the PI and MI is visible in S. salar other than the lack of pyloric caeca and the arrangement of mucosal folds (Løkka et al., 2013). The transition from MI to DI (Fig. 1.2) is marked by an increase in diameter, darker color of the mucosa, and the presence of complex folds. The majority of cells of the intestinal epithelium in all these regions are enterocytes. Enterocytes are columnar cells with an elongated nucleus (Fig 1.3). These cells have an increased luminal surface area due to the finger-like extensions of the membrane called microvilli, which make up the brush border membrane (BBM; Fig 1.3). Enterocytes of S. salar differ somewhat in appearance between intestinal regions. Enterocytes of the DI are characterized by large supranuclear vacuoles (SNVs) in the apical region of the cell, while these large vacuoles are absent or much smaller in enterocytes of the PI and MI. Other cells of the intestinal epithelium include mucus-secreting goblet cells, endocrine cells and intraepithelial lymphocytes. The basal membrane of the enterocytes forms a border to the lamina propria (Fig. 1.3). The lamina propria and submucosa are made up of connective tissue that contains blood vessels, nerves and various resident immune cells.

1.2.2 Digestive function The function of the esophagus is mainly to pass food from the mouth to the stomach, where digestion is initialised in an acidic environment by the digestive enzyme pepsin. When proteins, carbohydrates and lipids are passed on into the PI, the release of pancreatic enzymes such as trypsin, chymotrypsin, elastase, α-amylase and lipases, as well as bile from the liver via the gall bladder, is triggered (Holmgren and Olsson, 2009). Increased mucus and bicarbonate secretion 7

Background from the pancreas and bile buffers the acidic chyme from a pH of 4.5 in the stomach to 8 in the PI of Atlantic salmon (Nordrum et al., 2000b), which presents an optimal environment for pancreatic enzymes. Brush border membrane enzymes are responsible for final digestion of peptides and digestible carbohydrates. The majority of nutrients are absorbed in the PI with the adjacent pyloric caeca and to a lesser extent in the subsequent regions including the distal intestine (Krogdahl et al., 1999; Bakke-McKellep et al., 2000a). Nutrients can be absorbed through the epithelium by apparent paracellular transport and different modes of transcellular transport. Transcellular nutrient absorption includes endocytosis, passive diffusion as well as transporter-mediated uptake, including active, ion gradient- or energy-dependent transporters. The DI has been described to be the principle site of intact protein uptake through endocytosis (Sire and Vernier, 1992), which is indicated by the formation of the large SNVs (NoaillacDepeyre and Gas, 1973; Stroband and Kroon, 1981). Another important function of the GIT is the regulation of food intake, digestion, absorption and osmoregulation, which is accomplished by endocrine cells of the GIT and associated organs through chemical signalling (Takei and Loretz, 2011). Similar to mammals, fish possess several gastroenteropancreatic signalling peptides of which three were investigated in this thesis: Ghrelin, choleocystokinin and peptide yy. The gastric signalling peptide ghrelin is present in two isoforms in S. salar and functions as a stimulator for food intake (Murashita et al., 2009; Moen et al., 2010). Choleocystokinin, on the other hand, inhibits food intake and stimulates pancreatic enzyme secretion and gall bladder contraction and is released by intestinal endocrine cells (Liddle, 2000; Takei and Loretz, 2011). Peptide yy is a signalling peptide secreted by the pancreas and has been described to inhibit pancreatic secretion (Deng et al., 2001).

1.2.3 Barrier function The GIT of fish is not only responsible for nutrient digestion and absorption but is also a major site for host defence. Mucosal surfaces such as the skin, gills and the gastrointestinal epithelium are constantly exposed to a variety of pathogens and toxins from the external environment, and thus act as a first line of defence by creating physical, chemical and immunological barriers between the external and the internal environments (Cain and Swan, 2011). Pathogens and toxins that may enter the body through the mouth have to pass through the acidic stomach, which destroys many bacteria and denatures many toxins before they can enter the intestine. The physical barrier of the intestine is formed by enterocytes, which form the tight, one-cell layer thick intestinal epithelium (Fig. 1.3). This barrier protects the body from pathogens and toxins by tightly regulating potential inter- and intracellular entry. Furthermore, the epithelium is covered by a film of mucus secreted by mucus-producing cells within the epithelium, which presents an additional physical barrier by restricting the motility and attachment of pathogens and toxins. The mucus itself also contains antimicrobial peptides, lysozyme and immunoglobulins and hence acts as a connection between the physical, 8

Background chemical and immunological barriers. The immune cells and signalling substances within the epithelial and mucosal tissues act as a more complex barrier (see below). This barrier has multiple functions, including: 1) monitoring any entry of pathogens and toxins across the epithelial surfaces, 2) locally protecting the rest of the internal environment should pathogens or toxins enter, and 3) preventing the immune system from mounting an undesired response to a multitude of non-pathogenic microbes and antigens.

1.2.4 Immune function The mucosal immune system of the GIT plays an important role in the protection of the host against pathogens as it has to successfully recognise self from non-self, as well as pathogenic from non-pathogenic (commensal) microbes and food antigens. Within the intestinal epithelium of fish, the gut-associated lymphoid tissue (GALT) seems to be diffusely distributed in the mucosa (Abelli et al., 1997; Rombout et al., 2011). Distinct aggregations of immune cells as found in mammals (Peyer’s patches) have not been described (Rombout et al., 2011). Nor has a distinct lymphatic system with lymph vessels and lymph nodes been identified in fish. Otherwise, the immune system of fish, as in higher animals, can be divided into the innate and adaptive systems and most of the players – immune cells and their various effector and signalling substances – involved in the immune system of higher animals appear to also be present in fish. Innate immunity The innate immune response is a fast, unspecific and relatively temperature independent response that can be mounted against a range of pathogens and antigens (Ellis, 2001). Because the response is unspecific, it does not require previous exposure to a pathogen. The innate immune system of fish is comprised of cellular and humoral components. Cell-mediated innate immunity involves phagocytes (neutrophil granulocytes, monocytes/macrophages, dendritic cells), mast cells, basophil granulocytes, eosinophil granulocytes and non-specific cytotoxic cells (Cain and Swan, 2011). Neutrophils enter infected tissue and destroy invading pathogens by engulfing them (phagocytosis) and killing them with the aid of antimicrobial substances stored in their cytoplasmic granula. Monocytes enter infected tissue and differentiate into large phagocytic cells (macrophages), which then extend long pseudopodia that attach to pathogens and also destroy them by phagocytosis. Dendritic cells are also phagocytes but their most important role appears to be as antigen-presenting cells that probe the environment for antigens, which they present to other cells of the immune system. Dendritic cells can also probe the luminal side of the epithelium by sending membrane extensions between epithelial cells. However, dendritic cells do not seem to be abundant in fish mucosae (Rombout et al., 2011). Upon activation, mast cells as well as eosinophil and basophil granulocytes release an array of cytotoxic proteins into their environment that can attack the cellular membrane 9

Background of pathogens. Mast cells of salmon do not appear to contain histamine (Reite and Evensen, 2006), as they do in higher animals, but may contain other cytotoxic substances such as serine proteinases and proteoglycans (Stone et al., 2010) as well as other chemical effectors resembling mammalian mast cell histamine (Reite and Evensen, 2006). Non-specific cytotoxic cells seem to be similar to mammalian natural killer (NK) cells, however distinct NK cell lines have also been discovered in catfish (Yoder, 2004). Non-specific cytotoxic cells can eliminate infected cells rather than attacking the pathogen directly. Humoral components of the innate immune system include the complement system, cytokines, interferon, transferrin, C-reactive protein, lectins and lysozymes (Secombes, 1997). Adaptive immunity The adaptive immune system is a slower but highly specific immune response, which can take days to develop but as a result can cause the individual to develop immunity towards later exposures of the same pathogen. The adaptive immune system of fish is also comprised of cellular and humoral components (Uribe et al, 2001). Cell-mediated immunity involves B and T cells (lymphocytes) while immunoglobulins (antibodies) and cytokines, are part of the humoral immunity. Several classes of T cells can be distinguished. Cytotoxic T cells (TC), recognised by the expression of the transmembrane glycoprotein Cluster of Differentiation 8 (CD8) on their surface, interact with antigens presented by major histocompatibility complex I (MHC I) from antigen presenting cells and eliminate an infected cell. T helper cells (TH cells), recognised by the expression of CD4 on their surface, interact with MHC II and can subsequently activate TC cells as well as B cells. Regulatory T cells (Treg) can suppress the immune response to prevent damage of healthy tissue and are very important in maintaining tolerance to self-antigens as well as preventing excessive immune responses to non-pathogenic microbes and antigens. B cells differentiate into memory B cells or plasma B cells. Memory B cells are long lived cells that can rapidly be activated upon a second exposure to an antigen, while plasma B cells are short lived cells that secrete antibodies against a specific antigen. Inflammatory response In concert, cells of the innate and adaptive immune system can trigger inflammation at a site of injury or pathogen invasion in a tissue. Upon contact with a pathogen, basophil granulocytes present in the tissue release proteoglycans and proteolytic enzymes, which aid in increasing tissue permeability (Reite and Evensen, 2006). Increased permeability allows more immune cells such as neutrophil and eosinophil granulocytes and non-specific cytotoxic cells to reach the site of injury. Increased permeability also results in increased bloodflow at the site of injury, often observed as redness (rubor) and swelling (tumor) of inflamed tissue. Furthermore, damaged cells release chemokines the can guide immune cells to the site of injury by chemotaxis. Phagocytes infiltrating the tissue can be activated by interaction with pathogenassociated molecular patterns (PAMPs) present on the cell membrane of pathogens (Bianchi, 10

Background 2007). Individual PAMPs have commonality within a large group of microbes, which allows phagocytes to recognise a broad range of pathogens (Bianchi, 2007). The recognition of these molecular motifs is accomplished by pathogen recognition receptors (PRRs) present on the surface of phagocytes, such as toll-like receptors (TLRs). In addition to PAMPs, mammalian studies recently showed that TLRs may also recognise endogenous distress signals (damageassociated molecular patterns, DAMPs) released by host cells as a response of the breakdown of cellular structures, such as the extracellular matrix (Matzinger, 2002; Bianchi, 2007). This type of DAMP recognition and pro-inflammatory mediation has also been indicated in fish (Castillo-Briceño et al., 2009). The binding of TLRs to a pathogen induces the release of messenger cytokines such as the interleukins IL-1β and IL-18. Cytokines are important signalling substances for and between all types of immune cells and important in modulating the type of immune response. Phagocytes and dendritic cells also present antigens to lymphocytes and thus can activate an adaptive immune response. Naïve TH cells interact with the antigenpresenting cells and, if the antigen is recognised, are activated. The resulting effector TH cells can subsequently initiate a variety of immune responses such as the stimulation of B cells to proliferate, secrete antibodies and form memory B cells, mast cell degranulation, and eosinophil activation. Antibodies secreted by plasma B cells can directly attach to antigens on the surface of pathogens. Pathogens are thereby marked for elimination, which is mediated by cytotoxic T cells as well as various professional phagocytes. Degranulation of mast cells, basophils and eosinophils results in the release of pro-inflammatory mediators, such as cytokines as well as cytotoxic factors that can directly help eliminate pathogens. Mammalian studies showed that during an inflammatory response, components of the innate and adaptive immune system can induce intestinal epithelial dysfunction, which may play a critical role in the development and exacerbation of intestinal inflammatory conditions (Groschwitz and Hogan, 2009). Inflammation-induced dysregulation can be mediated by cytokines, T cells, mast cells or eosinophils (Groschwitz and Hogan, 2009). The intestinal immune system of salmon is not well understood and further investigation could provide valuable information regarding inflammatory processes in Atlantic salmon.

1.3 Soybean meal-induced enteritis as a model As S. salar is carnivorous in nature, the inclusion of plant materials in their diet exposes the digestive system to substances that are not typically part of their natural diet (Francis et al., 2001; Knudsen et al., 2007; Krogdahl et al., 2010). Fullfat and solvent (hexane) extracted soybean meal (SBM) was initially expected to be a good alternative source of protein because of its high protein content, better composition of amino acids than most other plant proteins and its lower price compared to fishmeal (Fig 1.4) (Olli et al., 1994b; FAO, 2010, 2012a). However, at inclusion levels higher than 5-10% Atlantic salmon develop inflammation (enteritis) in the distal part of the intestine (van den Ingh et al., 1991; Olli et al., 1994b; Baeverfjord and 11

Background



  

 





 



























   !"    

Figure 1.4: Global fish meal prices from 2005 until 2012 (adapted from FAO Food Outlook - November 2012) Krogdahl, 1996; Krogdahl et al., 2003). Therefore, standard, solvent (hexane)-extracted SBM is not used in commercial aquafeeds for Atlantic salmon today. Soy protein concentrate, on the other hand, is produced by alcohol extraction and does not trigger enteritis (Olli and Krogdahl, 1995). Soybean meal-induced enteritis (SBMIE) is, however, a useful model to study dietrelated effects on intestinal health and more specifically, basic digestive physiological and immunological functions in the intestine of Atlantic salmon. The wide array of ANFs also makes SBM a suitable model to study the effects of ANFs on digestive processes. This inflammation is not only triggered by soybean meal as similar effects have been demonstrated with some other legumes, such as pea protein concentrate (Penn et al., 2011). The etiology of this inflammation is not yet understood, however, ANFs present in SBM may play an important role. Antinutritional factors found in SBM include proteinase inhibitors, lectins, phytic acid, saponins, phytoestrogens, antivitamins, phytosterols and antigens/allergens (Francis et al., 2001) as well as possibly more, not yet identified substances. Recently, saponins were found to be a possible trigger of inflammation (Knudsen et al., 2008; Chikwati et al., 2012; Kortner et al., 2012b). Saponins are heat-stable glycosides, which are known ANFs present in SBM and other legumes (Francis et al., 2001; Krogdahl et al., 2010). Multiple studies to characterise SBMIE in S. salar have been carried out since the first observations of the condition. Morphologically (Fig. 1.5), SBMIE is characterized by decreased height and complexity of the distal intestinal mucosal folds, decreased size and/or amounts of SNVs, widened lamina propria and submucosa with increased leukocyte infiltration, as well as increased amounts of intraepithelial leukocytes and diffuse immunoglobulin M (IgM) (van den Ingh et al., 1991; Baeverfjord and Krogdahl, 1996; Bakke-McKellep et al., 2000b; Bakke-McKellep et al., 2007a). Morphological changes are visible after 2-5 days of 12

Background

Figure 1.5: A: Distal intestinal mucosal folds of FM fed fish B: Distal intestinal mucosa of fish with severe soybean meal-induced enteritis. feeding SBM (van den Ingh et al., 1991; Baeverfjord and Krogdahl, 1996). After 7 days of feeding SBM, all of the individuals may show signs of inflammation and after 21 days the typical characteristics are exacerbated. The inflammation in the DI is usually accompanied by impaired epithelial barrier function, increased cellular permeability (Nordrum et al., 2000a; Knudsen et al., 2008) and decreased macromolecular uptake (Urán et al., 2008). Furthermore, loss of digestive function is indicated by alterations of digestive enzyme activities in the distal intestine. The activity of BBM enzymes are strongly reduced while pancreatic enzyme activities are increased (Bakke-McKellep et al., 2000b; Lilleeng et al., 2007; Krogdahl et al., 2010). Most of these functional studies describe the responses during chronic stages of SBMIE. The immediate response of molecular and biochemical parameters in the intestine of post-smolt Atlantic salmon during the development of SBMIE has not yet been characterised in detail. In this thesis we addressed this knowledge gap by characterising alterations in the transcriptome and digestive enzyme activities in the GIT of post-smolt Atlantic salmon during the development of soybean meal-induced enteritis (paper II and paper III, pilot study). Furthermore, it was previously unknown if SBM would trigger the same condition in salmon fry from first-feeding. Therefore this was investigated in our study presented in paper I.

1.3.1 Proteinase-activated receptor 2 as potential pro-inflammatory mediator Proteinase inhibitors are ANFs that are present in many plants and several studies have concentrated especially on the effects of soybean trypsin inhibitor (SBTI) in mammals as well as in fish (Liener, 1970; Krogdahl et al., 1994; Olli et al., 1994a). The most abundant inhibitor present in soybeans is the Kunitz-type inhibitor, which can bind to trypsin and 13

Background chymotrypsin (Kunitz, 1947). The presence of moderate amounts of SBTI may stimulate trypsin secretion from the pancreas in order to compensate for the enzymes blocked by the inhibitor (Olli et al., 1994a). Feeding trials with SBM-based diets in Atlantic salmon showed normal or reduced trypsin levels in the proximal intestine but increased trypsin-like activity in the tissue and contents of the distal intestine (Krogdahl et al., 2003; Lilleeng et al., 2007. In mammals, serine proteinases (e.g. trypsin and tryptase) have been identified to be activators of membrane-bound G protein-coupled receptors, e.g. proteinase-activated receptors (Schmidlin and Bunnett, 2001). In mammals, proteinase-activated receptor 2 is expressed in endothelial cells, colonic myocytes, enterocytes, enteric neurons, terminals of mesenteric afferent nerves and immune cells (Nystedt et al., 1995; Böhm et al., 1996). Thorsen et al. (2008) cloned and characterized two full length PAR-2 transcripts in Atlantic salmon and further demonstrated that distal intestinal PAR-2 mRNAs levels changed significantly in Atlantic salmon fed a 20% SBM diet. In mammals, PAR-2 has been shown to mediate pro-inflammatory effects in colonic tissue, such as granulocyte infiltration, tissue damage, changes in the cellular permeability and subsequent bacterial translocation (Schmidlin and Bunnett, 2001; Cenac et al., 2004). A recent study indicated that purified trypsin from salmon, sardine and king crab may activate the PAR-2 signalling pathway in humans (Larsen et al., 2011). In order to further investigate the potential role of PAR-2 during SBMIE in salmon, we conducted a pilot study to identify changes in PAR-2 protein and mRNA expression during early stages of SBMIE.

14

2. Aims and strategies The main aim of this PhD thesis was to provide new knowledge that could contribute to securing fish health in a situation of rapidly increasing use of alternative plant protein sources in commercial salmon feeds. The gained knowledge of this research could facilitate the development of a more sustainable aquaculture production. Extracted soybean meal was used as a model plant protein source since it induces inflammation in the distal intestine of salmonids and is therefore considered a worst case scenario. The following specific strategies were employed to address the main aim: •

To characterise the ontogeny of the GIT and accessory digestive organs (pancreas and liver) from hatch to 5 g size by analysis of histomorphological, biochemical and molecular parameters (paper I).



To characterise histomorphological, biochemical and molecular parameters during the ontogeny of the GIT in juvenile Atlantic salmon fed a soybean meal-containing diet from first-feeding (paper I).



To characterise immediate changes in digestive enzyme activities in the GIT as well as the distal intestinal transcriptome in post-smolt salmon during the onset of soybean meal-induced inflammation (papers II and III).



To examine immediate changes in the protein and mRNA expression of PAR-2 as a potential pro-inflammatory mediator in post-smolt salmon during the onset of a soybean meal-induced inflammation (pilot study).

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16

3. Methodology 3.1 Overview In our strategies to address the main aim of this thesis we used methods that allowed us to investigate effects on multiple levels of biological organisation. Feeding experiments were the basis for this research and thus it is of utmost importance to minimise causes of variation during the course of the experiment as well as during sampling. The feeding trials for this work were conducted at Nofima at Sunndalsøra, Norway. Several factors may influence the sample quality and variation between individuals, such as differences between feed batches, fish handling and sampling procedures. Nofima routinely runs feeding trials, which is beneficial for standardisation of procedures and results in highly reproducible outcomes. Furthermore, monitoring procedures for feed intake, mortality, water temperature and oxygen saturation allow good quality control during trial runs. The experiment described in paper I was conducted to follow the ontogeny of the GIT and accessory digestive organs – liver and pancreas – in juvenile S. salar from hatch to 5 g size. Effects of a SBM-containing diet (16.7% inclusion level) compared to a fishmeal-based (FM) control diet on fry development from first feeding at 46 dph (days post hatch) was also investigated. Fish were sampled at numerous time points from 3 to 144 dph. The experiment described in paper II and paper III was designed to analyse alterations in gene and protein expression and enzyme activities during the development of SBMIE from day 0, when the fish had been fed a FM-based diet, to day 21 of SBM exposure (20% inclusion level) in postsmolt S. salar in seawater. Selected methods used for the analysis of the fish and their tissues sampled from the feeding trials are described in detail below. On the morphological level, histological analysis was used to describe morphological alterations during juvenile development from hatch and for the evaluation of the development of SBMIE. On the protein level, immunohistochemistry (IHC) and biochemical assays were used. Immunohistochemistry was used to describe protein expression of a cell differentiation marker (carbonic anhydrase 12) and a potential pro-inflammatory mediator (PAR-2). Biochemical assays were used to characterise enzyme activity patterns. On the transcriptional level, quantitative real-time polymerase chain reaction (qPCR) and microarray were used to identify changes in gene expression. As there are many regulatory steps between the expression of a gene to the synthesis and allocation of a functional protein, analysing the up or down-regulation of gene expression can only be used as an indicator for increased or decreased protein abundance and enzyme activity in a tissue. Combining analyses on different levels of biological organisation can aid in identifying possible underlying mechanisms responsible for the observed functional and morphological changes. 17

Methodology

3.2 Histology During standard histological preparations, formalin-fixed tissue was cut into 5 μm thin sections and placed on a microscopic slide. The sections were then stained with a hematoxylin and eosin (H&E) stain. The basic dye hematoxylin binds to acidic substances, such as chromatin and stains it with a blue/purple colour. The acidic dye eosin stains basic substances in the cytoplasm, connective tissue and collagen with a red/pink colour. The colouration allows identification of cellular structures within a tissue sample. Other stains may also be used to more specifically identify tissue components and chemical substances (e.g. ions, proteins, lipids, polysaccharides). In the study presented in paper I we used histological evaluation to describe the development of S. salar juveniles from hatch until 144 dph. This study focussed on two aspects: Firstly, the description of the development of the GIT and the associated organs, i.e. mouth, esophagus, stomach, yolk sac, pyloric intestine, pyloric caeca, pancreas, liver, mid intestine, distal intestine and rectum, of fish fed a fishmeal-based diet were investigated. The results should reflect the normal development of the GIT. Secondly, the effects of a SBM-based diet on the morphological development of the GIT were evaluated. In the work presented in paper II and paper III, we used histomorphological analysis to follow the development of SBMIE in post-smolt S. salar from the first day of feeding a SBM-containing diet. The evaluation was based on earlier published characteristics of changes that occur during the development of SBMIE (Baeverfjord and Krogdahl, 1996). Histopathological analysis confirmed that signs of inflammation were evident in the distal intestine after 5 days of feeding SBM and that all fish developed inflammation during the 21 day trial.

3.3 Immunohistochemistry Fluorescent and chromogenic labelling of proteins on tissue sections can be accomplished using antibodies. The antibody specifically binds to an epitope, which is part of an antigen that is recognised by the antibody. In this way, IHC can provide information on the distribution of the protein throughout the tissue and its localisation on or within individual cells. Various labelling procedures can be used for visualization of the antibody-antigen binding, such as direct, indirect, and indirect with signal amplification. For direct labelling, the antibody is complexed (labelled) with a fluorescent or chromogenic label, which then supplies a signal visible upon microscopy (Fig 3.1B). If no such labelled primary antibody is available, indirect labelling is possible using a similarly labelled secondary antibody that specifically binds to the primary antibody (Fig 3.1C). For fluorescent labelling, the primary or secondary antibody is labelled with a fluorophore. The detection of this reporter can be observed using fluorescence microscopy. The fluorescence microscope emits light of the specific wavelength which excites the fluorophore. The fluorophore then emits light with a different wavelength, 18

Methodology

Fig. 3.1: Types of antibody labelling. A: Legend; B: primary antibody with fluorescent label; C: primary antibody labelled with seconday antibody labelled with fluorescent probe; D: primary antibody with protein A gold complex which is detected by the fluorescence microscope. On the other hand, some primary and secondary antibodies are labelled with an enzyme or an enzyme substrate, which then requires an enzymatically catalyzed color reaction for the signal to become visible (labelling with signal amplification). Commonly used enzymes for chromogenic protein detection include alkaline phosphatase (AP) and horseradish peroxidase (HRP). The colour reaction then marks the area of the antigen-antibody complex formation and can be detected by light microscopy. A quantitative approach using antibody labelling is the use of immunogold labelling. For immunogold labelling, ultrathin sections of tissue (~500 nm) are exposed to the primary antibody that is either directly labelled with a protein A gold complex (Fig 3.1D) or indirectly with a secondary antibody labelled with protein A gold complex. Colloidal gold can then be visualised as electron dense particles using a transmission electron microscope (TEM). As individual gold particles can be counted and particle density for an area can be calculated, this technique allows a better quantification (Griffiths and Hoppeler, 1986). The advantage of indirect labelling using secondary antibodies is that different applications can be used to visualise or detect the primary antibody as a greater variety of secondary antibodies with different labels are available. Secondary antibodies may also increase the signal intensity but at the same time result in increased unspecific binding. As such, the choice of antibodies is strongly dependent on the methods used and the protein of interest. For studies on mammals and certain model organisms, many primary and secondary antibodies are commercially available. For S. salar, however, the selection of available salmon-specific antibodies is much smaller. However, an increasing number of commercial, monoclonal antibodies are developed to complex with epitopes of the protein antigen that are evolutionarily highly conserved. This allows for an increasing number of antibodies that can be used across species. In any case, the specificity of an antibody for protein antigens in an animal must be validated (see below). 19

Methodology For our work presented in paper III we used carbonic anhydrase 12 (CA12) as a marker for epithelial cell differentiation and maturation. This antibody was a monoclonal IgG2 antibody produced in mouse against human CA12, which had previously been used successfully in salmon (Romarheim et al., 2011). Two full-length sequences for PAR-2 (PAR-2a and PAR-2b) were previously cloned in our lab and published in an earlier article (Thorsen et al 2008). To be able to identify both isoforms, monoclonal recombinant antibodies against N-terminal peptides of salmon PAR-2a (clsqeteqsnadven) and b (cvvdpqdadrvtvskatadt) were ordered from AbD Serotec. For the production of the antibodies, AbD Serotec used the Human Combinatorial Antibody Library (HuCAL®) technique. A complete antibody consists of the Fab (fragment, antigen binding) region, which is responsible for antigen recognition, and the Fc (fragment, crystallising) region which possesses immunomodulatory properties. The antibody from AbD serotec consisted of only the Fab region and contained a combined c-myc and polyhistidine peptide tag at the C-terminus of the antibody heavy chain (Fig 3.2). Using only the Fab region should increase antibody specificity while the presence of two peptide tags increases the range of application that the antibody can be used in. In a pilot study, antibodies against the two isoforms of the transmembrane protein PAR-2 (PAR-2a/b) were labelled with a fluorescent marker and tested on sections of the distal intestine. Tissue sections were analysed under a confocal microscope. The antibody against PAR-2a showed a low background signal and stained structural components that were assumed to contain PAR-2 from the information taken from mammalian studies (Böhm et al., 1996). Immunogold labelling for quantitative analysis was tested on 500 nm distal intestinal tissue sections. As secondary antibody we used an antibody produced in rabbit against a myc tag. Immunogold labelling showed a high background signal and thus it was not further evaluated. For further validation, the antibodies were tested with western blot technique in our lab (not part of this thesis) but we were unable to verify the specificity. Antibody validation is a vital part when using newly designed antibodies. However no strict guidelines on antibody validation are established and laboratory practices on this matter differ. Recently, recommendations for good laboratory practices on antibody validation have been

Fig. 3.2: Fab Mini antibody (source: AbD Serotec)

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Methodology published (Bordeaux et al., 2010). The authors describe a rigorous testing scheme but also point out that IHC and western blot may be sufficient to validate the antibody given that both techniques show corresponding results (Bordeaux et al., 2010). If one of these techniques does not provide enough information, immunoprecipitation may be an additional method to consider (Bordeaux et al., 2010).

3.4 Enzyme activity assay The analysis of digestive enzyme activities can provide information on the functional status of the GIT, also following a change of diet (Krogdahl et al., 1994; Krogdahl et al., 2003). Enzyme activities were measured colorimetrically using a spectrophotometer. This technique takes advantage of the changes in light absorbing properties of synthetic substrates during enzymatic breakdown. The sample material is homogenised and a given amount of the homogenate is mixed with a synthetic substrate specific for the targeted enzyme. Natural or synthetic substrates are available for various enzyme and inhibitor activity assays. Synthetic substrates offer the advantage that they may provide higher specificity to the respective enzyme or inhibitor (Kakade et al., 1969). In our study, enzyme activities of important digestive enzymes were measured from either dissected intestinal tissue, to assess enzymes of the brush border membrane (BBM), or from intestinal contents, to assess enzymes secreted from the pancreas. The following substrates were used: Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride (L-BAPNA) for trypsin, N-benzoyl-L-tyrosine ethyl ester for chymotrypsin, succinyl (L-alanine)3 p-nitroanilide for elastase, p-nitrophenyl myristate for lipase, L-leucine-β-naphthylamide for leucine aminopeptidase (LAP) and maltose for maltase activity. The reaction was started by mixing the homogenate with the synthetic substrate. Absorbance readings are taken at the start and end point of the reaction. In our studies, pancreatic enzyme activities (trypsin, chymotrypsin, elastase, lipase) and LAP measured in intestinal content were expressed as the difference in optic density (ΔOD) per mg drymatter (U/mg = ΔOD mg DM-1). Another possibility to assess enzyme activity would be to produce a standard curve using purified enzyme and then calculating the amount of enzyme in the sample. Purified salmon trypsin was not available and thus we did not use the standard curve method. Purified bovine trypsin has previously been used as standard but it may not represent the actual concentration or activity of salmon trypsin due to differences in the efficiency of substrate breakdown (Krogdahl and Holm, 1983). Brush border membrane bound digestive enzymes LAP and maltase activities were expressed as total tissue activity per kg fish, also called capacity, and specific activity (i.e. per mg protein). The digestive capacity can be important for the interpretation of the results from BBM enzymes when comparing fish of different weights as they differ in their metabolic rate. Furthermore the intestine ususally shows reduced weight during SBMIE and thus calculating 21

Methodology the activity in relation to body weight can provide more accurate information on the effect of inflammation on digestive capacity.

3.5 RNA extraction and cDNA synthesis The first and most important step in the analyses of gene expression is the extraction of high quality RNA from the sample tissues. Typically, total RNA is extracted, containing the mRNA that is used for the quantification of gene expression. Several standard protocols are available for this process. Generally, RNA within a sample homogenate has to be separated from other cellular molecules. In our studies, we used an extraction method based on the guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987). During this method, the homogenate is separated into three phases: an aqueous phase containing RNA, an interphase containing DNA and an organic phase containing proteins and lipids. After phase separation, RNA in the aqueous phase can be precipitated, washed, DNase treated and solubilized in RNase-free water. For the work presented in paper I we used a column-based extraction method in which no phase separation was performed and all steps were performed on filter columns (Direct-zol™ Mini Prep, ZymoResearch). For the study presented in paper II, we used a column-based extraction method in which all steps after phase separation were performed on filter columns (RNeasy Mini Kit, Qiagen). Filter columns are easy to use, the process is fast and it can lead to high quality RNA. Working with RNA requires careful handling and good laboratory practice to avoid genomic DNA contamination and degradation by RNases. It is therefore important to check the quality of the extracted RNA. Purity was tested using a Nanodrop spectrophotometer (Nanodrop 1000, Thermo Scientific), which calculates an absorbance ratio

Fig. 3.3: Example of 1% agarose gel electrophoresis for RNA quality check on samples from the microarray study. Samples show clear 28S and 18S bands.

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Methodology between 260/280 nm and 260/230 nm wavelengths. The values of these ratios can be used to identify RNA purity and possible contamination. Integrity of the RNA samples was evaluated by running an aliquot of the RNA sample on a 1% agarose gel, and inspecting RNA bands (Fig. 3.3). The quality of RNA can also be evaluated by microfluidic capillary electrophoresis systems such as the Agilent Bioanalyser and BioRad Experion. If the extracted RNA is of high quality it is transcribed into cDNA. Synthesis of cDNA introduces another possible factor for sample variation as the efficiency with which the samples are transcribed may differ and may depend on the choice of the synthesis strategy. The synthesis of cDNA is accomplished by a reverse transcriptase. In our study, we used the SuperScript® III reverse transcriptase (Invitrogen). Reverse transcriptases can differ in their RNase H activity, thermostability and RNA detection sensitivity. High RNase H activity may lead to the degradation of RNA in a RNA/DNA hybrid and can affect the yield of the cDNA synthesis (Berger et al., 1983; Goff, 1990). Thermal stability is an important factor as secondary structures of mRNAs must be denatured for the cDNA synthesis. Sensitivity of RNA detection determines if mRNA with low abundance is transcribed. Negative controls are important to run in parallel during all methods in order to identify possible contamination. Another important factor is the choice of primers. Random hexamer primers are comprised of 6 random bases that align at different positions across RNA strands and will therefore hybridise to all RNA species in a sample. Oligo dT primers hybridise to the 3’ poly (A) tail of mRNAs. If the obtained cDNA will be used for quantification of one specific gene transcript only, gene specific primers can be used. In our study we used Oligo dT primers.

3.6 Quantitative real-time polymerase chain reaction (qPCR) Quantitative real-time polymerase chain reaction (qPCR) allows analysis of the amount of mRNA from genes of interest within a given sample. This technique is based on the in vitro amplification of nucleotide sequences. The assessment of changes in mRNA expression of genes of interest may indicate that the translation of mRNA into protein is also affected, i.e. higher mRNA abundance leads to higher protein abundance. However, due to the complexity of the regulation of translation and post-translational mechanisms, this may not always be the case (Laurent et al., 2010). The analysis of mRNA expression can therefore benefit from additional analyses of protein expression or enzyme activities. To measure mRNA abundance with qPCR, the extracted mRNA has to be reverse transcribed into cDNA for analysis. During the polymerase chain reaction (PCR), template DNA strands go through 3 steps: 1) Denaturation, 2) annealing and 3) elongation. Denaturation is accomplished by heating the double stranded DNA (dsDNA) to about 90-95°C. In the process, dsDNA is melted into single stranded DNA (ssDNA), which is accessible to primers for replication. Primers are short nucleotide sequences that align to the complementary 23

Methodology sequence on the template DNA and serve as a starting point for DNA-polymerases. During the second step of the qPCR (annealing), the temperature is lowered to the optimal hybridisation temperature of the respective primers, which in our study was 60°C for the majority. During the third step (elongation), the temperature is raised to 72°C. Taq-polymerase then extends the ssDNA starting from the primers in 3’ – 5’ direction, thus producing two dsDNA copies of the template. This 3 step cycle is repeated multiple times to amplify the template DNA. In a qPCR, this protocol is enhanced by introducing a fluorescent dye that binds to dsDNA in order to quantify the amount of amplified DNA. In our study we used SYBR® Green, which binds to all dsDNAs in a sample thus potentially labelling non-target dsDNA. Non-target dsDNA can be identified by running a melt-curve analysis at the end of the qPCR run. Different dsDNA strands differ in their base content and size, which ultimately defines the melting temperature. If the melt curve analysis reveals more than one temperature peak, it would point to a possible non-target amplification. In our study, primers were designed in silico by using the online software Primer3 (Rozen and Skaletsky, 2000). We designed the primers to meet the following criteria: amplicon size 70 – 150bp, primer size 18 -23bp (optimal 20bp) to ensure specificity, GC content ~50% to ensure stable binding, perfect match to the target sequence, no complementarity to other primers to avoid primer dimers, less than 3 repeating bases to avoid mispriming. Primers for the study presented in paper I were designed from salmonid sequences from Genbank (http://www. ncbi.nlm.nih.gov/genbank/) or obtained from earlier publications. For the study presented in paper II, primers were designed by using contig sequences provided by the annotation file from the microarray. Due to a duplication of the genome during salmonid evolution, several isoforms of a gene may be present, which needs to be considered when designing new primers. New primers were tested by running 2-fold serial dilutions (starting with 1:5) of samples with each primer at the calculated optimal temperature (typically around 60°C). The tested primers were then checked for primer dimer formation, amplification cycle and efficiency. Primer efficiency was calculated with the serial dilutions by plotting a standard curve. Efficiency of 100% equals a value of 2.0. As the amplification of cDNA is logarithmic, a drop of a few percent in primer efficiency can result in a large difference over the cause of several amplification cycles. Thus an efficiency as close to 100% as possible and not lower than 80% is desired. The qPCR was conducted in a thermal cycler that can detect the fluorescent dye, such as the LightCycler® 480 (Roche Diagnostics) used in our study. The fractional PCR cycle used for quantification is termed quantification cycle (Cq) and is calculated using the second derivative maximum in the LightCycler® 480 system. The expression of mRNA can be quantified by measuring absolute or relative abundance depending on the research question. If information on copy numbers of a targeted mRNA sequence is desired, absolute quantification should be used. Absolute mRNA abundance can be measured by cloning the target sequence and 24

Methodology producing a standard curve from a dilution series of known mRNA concentrations. For gene expression profiling and information on changes in mRNA expression under experimental conditions, relative quantification is commonly used. Relative quantification is achieved by normalising the Cq values of the genes of interest against the Cq values of an internal control. Commonly, reference genes that showed stable expression during a given experiment are used as an internal control. These reference genes should reflect cDNA abundance within a sample and thus normalise the data. Several programs are available to analyse the stability and the quality of a reference gene, such as geNORM, Normfinder and BestKeeper (Vandesompele et al., 2002; Andersen et al., 2004; Pfaffl et al., 2004). However, defining a stable expression may not always be a straight forward process and caution is advised when using these programs (Kortner et al., 2011b). In the work presented in paper I we tested 8 potential reference genes and finally normalised genes of interest against the geometric mean of RNA polymerase II (rnapolII), hypoxanthine-guanine phosphoribosyltransferase 1 (hprt1) and beta-actin (actb). For the evaluation of gene expression presented in paper II we normalised genes of interest against the geometric mean of 3 reference genes (glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase II (rnapolII) and (hprt1)) that were previously evaluated for our experiment (Kortner et al., 2011b). The normalisation was performed after Pfaffl (2001) using the following formula:                          

Alternatively in the ΔΔCt method (Ct = Cq), the Cq value of the target gene is subtracted from the Cq value of a reference gene (Livak and Schmittgen, 2001). This is done for a control measurement and the test measurement. The resulting ΔCq values are then subtracted from each other and the resulting ΔΔCq or ΔΔCt value is used for analysis. However, this method assumes a primer efficiency of 100%, which is not always the case and can be misleading as discussed earlier.

3.7 Microarray For our study presented in paper II, we used the microarray technique. Microarrays allow the analysis of changes in mRNA expression of thousands of genes. This profiling of changes in mRNA expression can reveal pathways that may be affected under the given experimental conditions. Microarrays can thus provide a base for further research of the identified pathways and help us to understand underlying mechanisms. Microarray technology is based on the Watson-Crick base pairing principles. Strands of DNA or RNA will selectively hybridise to their complementary strands. A microarray chip comprises several thousand nucleotide sequences (probes) spotted on a solid surface (glass slide) representing known mRNA 25

Methodology sequences of the respective organism. Strands of cDNA or cRNA from a sample are labelled with a fluorescent dye and the nucleic acid transcripts of interest (targets) will then hybridise to their corresponding spotted probes. Subsequent scanning allows quantification of the amount of the respective target hybridised to the respective probes. There are two different spotting techniques, DNA microarrays use DNA fragments or cDNAs as probes while oligonucleotide microarrays use synthetic oligonucleotide probes ranging between 20-100-mer in length. Several cDNA microarray platforms are available for S. salar today (Rise et al., 2004; Jordal et al., 2005; von Schalburg et al., 2005; Koop et al., 2008; Taggart et al., 2008) with probe numbers ranging from 73 (Jordal et al., 2005) to 32,000 (Koop et al., 2008). Salmon oligonucleotide arrays have been developed only recently: the Salmon Immunity and Quality (SIQ) arrays (Krasnov et al., 2011; Grammes et al., 2012; Castro et al., 2013; Krasnov et al., 2013) and the consortium for Genomic Research of all Salmon Project (cGRASP) salmonid oligonucleotide microarray (Jantzen et al., 2011) platforms are published so far. Commercially in situ printed oligonucleotide arrays offer the opportunity to design high density and high quality microarrays. Due to the complex nature of the microarray application and the variable ways of designing, conducting and analysing, guidelines have been established that define “Minimum information about a microarray experiment” (MIAME; Brazma et al., 2001). These guidelines define six parts of reporting a microarray experiment: Part I: Experimental design; Part 2: Array design, Part 3: Samples; Part 4: Hybridisations; Part 5: Measurements; Part 6: Normalization controls (Brazma et al., 2001). We followed the MIAME recommendations in our study and details are given in the publication (paper II). Selected parts of MIAME are presented in the following section as well as the validation procedure using qPCR in our study. Part 1 Experimental design: The microarray study was conducted on a SBM feeding experiment. In this experiment, S. salar were fed with a SBM-containing diet over 21 days. Day 0 in this experiment is referred to as control since no parallel tanks with fish fed a fishmeal control diet were used. As such, each day of SBM feeding (i.e. 1, 2, 3, 5, 7) was compared to day 0 as control. This study could have benefited from a parallel control group as day to day variations in gene expression may occur and it would be beneficial to have a baseline for expression throughout the entire experimental period. Part 2 Array design: In this work we used the 44k salmonid oligonucleotide microarray (GEO platform ID: GPL11299) developed under cGRASP (Jantzen et al., 2011). The 44k microarray comprises 43,689 probes of very low redundancy and large transcript representation (Jantzen et al., 2011). The 60-mer probes present on the array are based on contiguous, overlapping DNA segments (contigs) from S. salar (~80%) and O. mykiss (~20%) (von Schalburg et al., 2005; Koop et al., 2008; Jantzen et al., 2011). From these contigs, 84% are well annotated using hits in public databases (December 17, 2009) with an e-value cut-off at 1e-10. Further 26

Methodology details on the library construction, sequence analysis and contig assembly can be found in Koop et al. (2008) and on the cGRASP website (http://web.uvic.ca/grasp/microarray/). Part 3 Sample preparation: To produce amplified cRNA for the microarray procedure (Fig 3.4), total RNA was extracted from distal intestinal tissue. Total RNA was then transcribed into cDNA using a reverse transcriptase. Microarray experiments can be performed as one- or two-colour experiments. In a one-colour design, the samples are labelled with one colour and hybridised directly to the microarray. In a two-colour experiment, a reference is labelled with one colour and the samples are labelled with another colour. The reference and the sample are then mixed and hybridised to the microarray. We performed a two-colour experiment, thus cDNA was transcribed into cRNA with a T7 RNA polymerase and labelled with either

Fig. 3.4: Schematic of cRNA amplification (adapted from Agilent ‘Two-Color MicroarrayBased Gene Expression Analysis - Low Input Quick Amp Labeling’ Protocol Version 6.5, Mai 2010) 27

Methodology cyanine 3 (Cy3) or cyanine 5 (Cy 5) fluorescent dye. A common reference was generated by pooling samples from each time point used in the study. For the hybridisations, we chose a reference design as opposed to a direct design to minimise the amount of arrays needed. In a direct design a sample is hybridised to a sample from the experimental control group (e.g. diseased vs. healthy). Part 6 Normalisation controls: In our reference design we used three samples per time point to create a common reference pool (i.e. 18 samples). The reference design has the advantage that all present genes will hybridise to the respective spots and thus allow the comparison of all samples among each other. Reference pool cRNA was labelled with Cy3 while samples from each time point were labelled with Cy5. On each array the resulting labelled cRNA from the reference pool and cRNA from one of the six time points were hybridised. Each time point had ten replicates (6 time points = 60 arrays). For further processing, each microarray slide was scanned and the spot intensity was quantified by imaging software. The large amount of information resulting from a microarray experiment presents a challenge for the analysis. Data analysis of the microarray results is strongly dependent on available information on annotated genes. Approaches to deal with the amount of data differ between labs. Commercially available software such as Agilent present a user friendly interface but are limited by their predefined technical and analytical possibility. Open source software such as R offers a greater analytical power and more user control but requires more training to handle. In our study we used the commercial software GeneSpring GX11.5 from Agilent. In GeneSpring, all entities (spots) were filtered to retain only those entities in which 80% of the biological replicates had raw intensity values ≥500 in any one of the six conditions (i.e. time points). Entities were then filtered to retain the entities in which at least 80% were flagged as ‘present’ in six of six conditions. The remaining entities were tested for significant differential expression by comparing each of the time points (days 1, 2, 3, 5, 7) to day 0 using the MannWhitney test (p