The digestive system of ballan
wrasse (Labrus bergylta), a marine agastric carnivorous teleost
December 2020
Master's thesis
2020Steinar Flaten NTNU Norwegian University of Science and Technology Faculty of Natural Sciences Department of Biology
(Labrus bergylta), a marine agastric carnivorous teleost
Steinar Flaten
Ocean Resources
Submission date: December 2020 Supervisor: Elin Kjørsvik
Co-supervisor: Tora Bardal and Per-Arvid Wold
This thesis completes my Master’s degree in the field of Ocean Resources at the Norwegian University of Science and Technology. The work of this thesis was conducted at NTNU Sealab and completed in the fall of 2020. The study is part of the LeppeProd project, an effort to successfully cultivate ballan wrasse as a cleaner fish in Norwegian aquaculture.
I would like to thank my supervisor Elin Kjørsvik for her guidance and patience and for contributing with valuable feedback on countless drafts. I would also like to thank my lab supervisor Tora Bardal, her inspiration and assistance helped to ensure the quality of the microscope images presented in this study. I would like to thank Per-Arvid Wold as well, for providing a second input during his contribution as assisting supervisor.
Lastly, I would like to thank my father Trond Peder Flaten for additional assistance during the last period of writing in the difficult year of 2020.
Even lastlier, I would like to give a shout-out to all the nice people I have got to know during my work at NTNU Sealab.
Trondheim, December 30, 2020
Steinar Flaten
The knowledge of the digestive system of teleosts is limited, especially when considering the huge variation in morphology. There are particularly few studies of the digestive system of agastric (stomachless) teleosts such as the ballan wrasse (Labrus bergylta), one of several species currently used as cleaner fish in Norwegian fish farms. They are known for their ability to graze on parasitic sea lice (Caligidae) which target salmonids specifically.
This study was conducted with the goal of creating a comprehensive descriptive and
quantitative overview of the ballan wrasse digestive system. A new method was developed to estimate total intestinal surface area (including microvilli) from serial microscope sections of the digestive tract and accessory tissues. These were removed as one unit and fixated with the goal of preserving the organs' natural position in the buccal cavity. The tissue volume of digestive organs was estimated as well (intestine, liver, pancreas and adipose tissue).
Additionally, this study included an analysis of mucus-secreting cells along the digestive tract, specifically a histochemical analysis of mucin content (oesophagus and intestine) and estimates of goblet cell density (intestine).
The digestive tract of ballan wrasse was characterized by the adaptation of the pharyngeal teeth and foregut/oesophagus towards mechanical digestion (pharygnaty) rather than a handling/swallowing function. The rounded molariform pharyngeal teeth are used for crushing the outer shells of exoskeleton-bearing invertebrates (durophagous feeding behaviour).
The mucous oesophagus was lined with a simple cuboidal epithelium, folded into thin blood- filled branches to form a feather-like pattern. This epithelial type (simple, rather than
stratified) has not been previously described in the teleost oesophagus. Contrary to what is common for marine teleosts, the ballan wrasse oesophagus did not contain an “oesogaster”, a columnar, microvillous epithelium, believed to function in osmoregulation. It is possible that the feather-like mucosa and thin epithelium serve a similar osmoregulatory function in the absence of the oesogaster.
Although the intestine was short and lacked pyloric caeca, the intestinal surface area of ballan wrasse was, in fact, larger than any of the species that were compared in this study, including gastric and agastric teleosts of various diets/trophic levels and environments. The large intestinal surface area of ballan wrasse can be attributed mainly to the extensive mucosal folding and large microvilli surface area as well as the inclusion of a distended intestinal bulb.
DI Distal Intestinal segment Third of three intestinal segments (3rd limb) G Goblet cell count Number of goblet cells
GD Goblet cell Density Number of goblet cells per unit of intestinal surface area, not including microvilli
GL Gut Length Length of intestine (oesophagus to anus)
Gtot Total goblet cell count Total number of goblet cells contained in the intestine IC Intestinal Circumference Length around the outer perimeter of the intestinal tract LM Light Microscopy Used for the histological and stereological analysis of the
digestive system MC Mucosal Circumference
(mean) Length around the inner perimeter of the intestinal tract MI Middle Intestinal segment Second of three intestinal segments (2nd limb)
MIC Microvilli surface area
(mean) Surface area of microvilli per unit of intestinal surface area
MV Microvilli Intestinal microvilli / number of microvilli PI Proximal Intestinal segment First of three intestinal segments (1st limb) RGL Relative Gut Length Gut length divided by fish length
RGLSL Relative Gut Length
(Standard Length) Gut length divided by fish length (standard length) RGLTL Relative Gut Length (Total
Length) Gut length divided by fish length (total length) Scon Convoluted intestinal Surface
area Surface area of the intestinal mucosa, not including microvilli
SL Standard Length Length of fish between the tip of the snout and the posterior end of the last vertebra (not including the caudal fin)
Ssimp Simple intestinal surface area Outer surface area of the intestinal tract (serosa) Stot Total intestinal Surface area Surface area of the intestinal mucosa, including
microvilli TEM Transmission Electron
Microscopy Used for measurements that require higher magnifications (intestinal microvilli)
TL Total Length Length of fish between the tip of the snout and the posterior end of the caudal fin
Acknowledgements ……… i
Abstract ………. ii
List of abbreviations ……… iii
1. Introduction ……… 1
1.1. Background ……… 1
1.2. A shift from wild catches to farmed ballan wrasse ……… 3
1.3. Challenges of ballan wrasse farming ………. 4
1.4. Aim of study ……….. 6
2. Materials and methods ……….. 7
2.1. The fish samples ……… 7
2.1.1. Sampling for histological and stereological analysis ……… 8
2.1.2. Measuring gut length ………. 8
2.2. Sectioning and histology ………. 11
2.2.1. Embedding and sectioning for light microscopy (LM) ……… 11
2.2.2. Evaluating the histological staining methods ………... 12
2.3. Stereological analysis ……….. 17
2.3.1. Serial sectioning ………... 17
2.3.2. Tissue volumes ………. 18
2.3.3. Two-step analysis of intestinal surface area ………. 19
2.3.4. Tract size ……….. 22
2.4. Further analysis of the digestive tract ……….. 23
2.4.1. Goblet cell analysis ……….. 23
2.4.2. Intestinal mucosal folding ……… 24
2.4.3. Other measurements in the alimentary canal ……… 24
2.5. Estimating sections with damaged tissue ……… 26
2.6. Statistical analysis ………... 27
3. Results ………. 29
3.1. Structure of the digestive system ………. 29
3.2. Organ morphology ………... 41
3.2.1. Oesophagus ……….. 41
3.2.2. Intestine ……….... 42
3.2.3. Mucosal folding ………... 46
3.2.4. Mucous cells ……… 48
3.2.5. Exocrine pancreas ……… 51
3.2.6. Liver ………. 53
3.2.7. Biliary tract ……….. 56
3.3.2. Microvilli surface area ………. 60
3.3.3. Intestinal surface area ……….. 61
3.3.4. Goblet cell density and quantity ……….. 64
3.3.5. Lack of sections from the intestinal bulb in one fish ………... 65
3.3.6. Pathology ………. 66
3.3.7. Overview of the ballan wrasse digestive system (fig. 3.30) ……… 69
4. Discussion ………... 71
4.1. Findings ………... 71
4.1.1. The intestinal mucosa of ballan wrasse ……… 71
4.1.2. The oesophagus of the ballan wrasse ……….. 78
4.2. Digestive functionality ……… 81
4.3. Insight into stomach loss and durophagy in teleosts ………... 84
4.4. Methods ………... 85
4.5. Conclusions ………. 86
References ……….. 87
Appendices ………. 93
A.1. Feed production and feed recipe ……… 93
A.2. Tissue embedding procedure ………. 94
A.3. Staining methods ……… 95
A.4. Effects of four histological stains on three digestive organs ……….. 96
A.5. Choosing the inter-sectional distance "T": test of accuracy ………... 98
A.6. Random number generator ……….. 98
A.7. Grid size calculations ……….. 99
A.8. TEM ……….. 100
A.9. Tissue damage ……….. 101
A.10. Morphology ……… 103
A.11. Intestinal diameter ……….. 109
A.12. Gut length ……… 110
A.13. Tissue volumes ……… 111
A.14. Intestinal surface area ………. 111
A.15. Intestinal surface area comparisons ……… 112
A.16. Oesophagus mucosal folding pattern ……….. 116
1. Introduction
1.1 Background
The ballan wrasse (Labrus bergylta) is one of several species currently used as cleaner fish in Norwegian fish farms. They are known for their ability to graze on parasitic sea lice
(Caligidae) which target salmonids specifically (Skiftesvik et al. 2016).
In 2018, Norwegian farmers exported 1.1 million tonnes of Atlantic salmon (Salmo salar) and 46 400 tonnes of rainbow trout (Oncorhynchus mykiss), accounting for roughly 72% (6.4 billion EUR / 63.8 billion NOK) of the total value of seafood exported from the country (Norwegian Sea Food Council 2019). Along with the expansion of the industry, pressure from some diseases has increased, particularly that of sea lice infections (Torrissen et al. 2013).
Further expansion of the industry is now highly dependent on the development of more effective methods of combating sea lice (Skiftesvik et al. 2016). The species most commonly infecting Norwegian fish farms is known as the salmon louse (Lepeophtheirus salmonis).
They graze on the skin, mucosa and blood of their hosts, potentially causing increased stress, reduced swimming performance, changes in blood sugar levels, reduced haematocrits and erosion of the skin in severe cases (Torrisen et al. 2013).
On average, an infestation over a typical Norwegian central region spring-release cycle generates damages equivalent to ca. 9% of farm revenues (Abolofia et al. 2017). In 2017, the total costs related to sea lice infections in Norway was estimated to increase to over 532 million EUR / 5.3 billion NOK (Barstad 2017).
Additionally, sea lice from escaped farmed salmon can infect wild salmon stocks. This may have devastating effects on Norwegian wild stocks which have declined significantly over the past 30 years. Escaped farmed salmon, salmon lice and infections from salmon farming are generally thought to be the greatest anthropogenic threats to Norwegian wild salmon (Forseth et al. 2018).
There are several methods of combating sea lice infestations: Chemical agents can be added through bath treatments or as in-feed medicines. Non-medicinal delousing methods include
Skirts, bubble walls, snorkels and electrical fences can be used as physical barriers between the salmon and the lice (Breck 2013). At night-time, submerged lights can be used to herd the fish away from the zone/depth where the lice gather (Wright et al. 2015). There has been recent development in the use of land-based aquaculture systems such as RAS (Recirculation Aquaculture System). Here, the salmon can be kept for extended periods before they are transferred to marine farms, delaying the initial exposure to sea lice (Warrer-Hansen 2015).
There are several problems with some of the common delousing methods; Over time, the use of chemical treatments has led to increasing resistance in the parasites (Murray 2016). Other methods are problematic in regard to animal welfare: Any treatment where the fish has to be physically handled, will cause unwanted stress (Breck 2013; Olesen et al. 2016).
Lice control by cleaner fish
Norwegian fish farmers are developing alternative control methods for sea lice that are environmentally sustainable and cause minimal stress to the fish. The use of cleaner fish such as ballan wrasse has proved to be a well-working solution, used in about 60% of Norwegian fish farms (Skiftesvik et al. 2016). The use of wrasse as a delousing agent was initiated by the Norwegian Institute of Marine Research in the late 1980s along with attempts of
cultivation/farming of ballan wrasse (Costello 2006; Espeland et al. 2010).
Different species of cleaner fish each have different attributes that make them suited for certain tasks leading to the use of combinations to best suit the needs of the farm. The ballan wrasse stands out as the most effective at grazing on sea lice. It is also able to clean larger salmon than any of the other species (Grefsrud et al. 2018). Several smaller species of wrasse are used, such as goldsinny wrasse (Ctenolabrus rupestris), corkwing wrasse (Symphodus melops) cuckoo wrasse (Labrus mixtus) and rock cook (Centrolabrus exoletus). They are well suited to clean younger salmon in a matching size range (Blanco Gonzalez & de Boer 2017).
Besides wrasse, the lumpfish (Cyclopterus lumpus) has been used successfully in Norwegian fish farms and production of cultivated lumpfish is already well established. They are easier to rear/handle and are more tolerant to lower temperatures where ballan wrasse become sluggish (Skiftesvik et al. 2016).
1.2 A shift from wild catches to farmed ballan wrasse
The majority of wrasse used in Norwegian aquaculture has been supplied by wild catches, deploying about 20 million wild-caught wrasses in 2016. However, there is a need among farmers for a shift away from wild catches, towards cultured ballan wrasse: Fish farms are highly dependent on a stable and predictable source of cleaner fish and there are several issues associated with a supply of wild caught cleaner fish (Skiftesvik et al. 2016):
Supply: Wild ballan wrasse is a resource in need of regulation, and the recent rise in demand has led to increased capture and deployment. Overexploitation and depletion of wild stocks has become an issue, with signs of declining stocks being reported in certain areas (Ottesen et al. 2012; Skiftesvik et al. 2016; Abotnes 2017; Piccinetti et al. 2017).
Welfare: Wrasse are relatively fragile and susceptible to stress. The logistics associated with the use of wild caught cleaner fish (transport, handling, storage) can be extensive and may cause unwanted stress or spread disease among the fish. This can have severe negative effects on fish health/welfare, especially if proper protocols are not followed (the removal of dead fish from tanks in particular) (Olesen et al. 2016).
Disease: Wild caught cleaner fish run the risk of introducing pathogens to the farm and formalin treatment with four weeks of quarantine is recommended for wild caught ballan wrasse brood stock (Helland et al. 2014). Much is unknown about the susceptibility of the ballan wrasse to pathogens (viral agents in particular) and consequently, the demand for farmed, vaccinated fish has been increasing (Olesen et al. 2016).
Stability: Wild catches are unpredictable regarding quantity, size and species. When cleaner fish are introduced to a salmon pen, they should, ideally, be of an even size, fitting the size of the salmon. Therefore, a wide range in size is needed to match the salmon as it grows from young smolt to harvest-ready fish. Additionally, each species of cleaner fish thrives within a certain temperature range, where they are the most effective at lice grazing. Seasonal changes and varying climates along the Norwegian coastline create a demand for available cleaner fish, suited to the right climate, at the right time (Skiftesvik et al. 2016).
As of August 2017 there were four active producers of juvenile ballan wrasse in Norway but
1.3 Challenges of ballan wrasse farming
Ballan wrasse are altricial, meaning that the larvae require an initial live feeding period until they are able to handle formulated feed. Traditional altricial larval feeds with enriched rotifers and Artemia have shown variable results in ballan wrasse, while the use of nauplii from copepods such as Arctica tonsa has shown more promise (Gagnat et al. 2016; Øie et al. 2017).
Especially weaning onto on-growing diets have been challenging with traditional fishmeal- based diets resulting in high mortality rates in ballan wrasse. The wrasse have shown a lack of appetite and unwillingness to consume this type of feed, leading to death from starvation.
Healthy growth has been largely dependent on shrimp meal in the diet during the first days before introducing any fish meal or krill. However, these ingredients are expensive and there are still issues of deformities and poor performance in the early life stages (Kousoulaki et al.
2014).
A thorough understanding of the digestive system of the ballan wrasse is key to the development of an optimal cultivation process of this and similar species.
Digestive system of the ballan wrasse
The ballan wrasse is a stomachless (agastric) fish, meaning that the oesophagus releases directly into the intestine. The gut is curled into a single loop (Z-shape) and lacks pyloric caeca. The intestinal lumen is slightly alkaline, ranging from pH = 7.7 in the proximal to pH = 8.2 in the distal end (Krogdahl et al. 2014). Relative gut length (RGL; gut length divided by total length (TL)) was estimated at 0.62 in wild caught ballan wrasse (Dipper et al. 1977).
Earlier estimates of RGL relative to standard length (SL) in teleosts have ranged from 0.5–2.4 in carnivores, 1.0–4.2 in omnivores and 3.7–4.2 in herbivores (Al-Hussaini 1947). This places the ballan wrasse at the very bottom range for carnivores (RGLTL > RGLSL).
The ballan wrasse fits into a group of agastric teleosts that have short, tubular intestines.
Species in this group show a wide variety of diets/trophic levels and mainly belong to the families Labridae (incorporating Odacidae and Scaridae), Blenniidae, Hemiramphidae and Atherinopsidae (Horn et al. 2006). Unlike most agastric fish, which are herbivores or detrivores (Kryvi & Poppe 2016), the ballan wrasse is a carnivore, feeding mainly on echinoderms, crustaceans and molluscs (Quignard & Pras 1986).
Lobel (1981) suggested that species of the Labridae family that have a diet rich in calcium carbonate (i.e. shellfish) would experience a neutralization of acid contents of the stomach leading to the loss of gastric peptic digestion. Having no stomach, they are unable to secrete hydrochloric acid or pepsinogen which is needed to break down the complex, globular proteins of fish and other vertebrates (Grabner & Hofer 1988; Lie et al 2018). Rather, the ballan wrasse has specialized in feeding on invertebrates, while the harder-to-digest proteins have been phased out of the diet along with the stomach (Farrell et al. 2011; Lie et al. 2018).
The driving mechanisms behind this are not fully understood, but it does remove the most energy-consuming part of the digestive system: The gastro-intestinal tract and associated organs may account for up to 40% of an animal's metabolic rate, most of which is used for secretion and neutralization of gastric juice (Cant et al. 1996).
The digestive tract of agastric carnivore fish has not been studied to the same degree as those of traditionally more important species like salmon, carp (Cyprinus carpio) and cod (Gadus morhua) (Horn et al. 2006). Few estimates of intestinal surface area have been made in adult teleosts. No estimates have been made for any agastric species, carnivore species or marine species which also include surface enlargement by microvilli (total intestinal surface area), so the present study will provide new knowledge in this respect. A comprehensive analysis of the digestive tract and accessory glands (liver and pancreas) of ballan wrasse would aid in the understanding of the digestive process and help further improve cultivation/production as well as the general understanding of teleost digestive systems.
1.4 Aim of study
The main goal of this study was to perform a qualitative and quantitative analysis of the ballan wrasse digestive system. The analysis consisted of the following steps:
1. A histological description of the digestive tract and the accociated glands and tissues (liver, pancreas, adipose tissue)
2. Estimates of the intestinal surface area (including microvilli) and the tissue volume of the digestive organs.
3. A description of the mucous cells of the alimentary canal with estimates of quantity/density.
The data are compared to other teleost species, both agastric and gastric, in order to explore and understand the functionality of the ballan wrasse’s digestive system.
2. Materials and methods
The study took place at the Norwegian University of Science and Technology (NTNU), Centre of Fisheries and Aquaculture (Sealab). The sampling and most of the laboratory work was performed during 2011 and 2012.
2.1 The fish samples
One and two year old L. bergylta (fig. 2.1), reared at the NTNU Centre of Fisheries and Aquaculture (NTNU Sealab) were used in this study. As larvae, they were start fed cultivated copepods (Acartia tonsa) nauplii and enriched Artemia sp. nauplii, before being weaned to a formulated diet (Appendix A.1). They were transferred to 500 litre fiberglass tanks (1 m2, grey) at 60 days after hatching, and kept at 12-14 °C with a 16:8 hour day/night cycle. The tanks were enriched with black plastic structures, mimicking macroalgae, which were replaced every two weeks. They were fed a commercial cleaner fish diet (Labrus 1.5 mm pellets, Skretting) from automatic continuous feeders (Appendix A.1).
Figure 2.1 One year old L. bergylta juvenile (fish #0)
2.1.1 Sampling for histological and stereological analysis
Eleven one-year old L. bergylta juveniles (table 2.1), mean size 20.7 g wet weight and 9.7 cm standard length were randomly sampled from a rearing tank by netting and killed by an overdose of the sedative tricaine methane sulfonate (Finquel MS222, Argent Labs) at 100 mg/L seawater. The fish wet weight was measured with a Mettler PM 4800 DeltaRange Balance. Total length (TL) and standard length (SL) was measured with laminated millimetre graph paper (SL = the distance from the tip of the snout to the posterior end of the midlateral portion of the hypural plate).
A combination of light microscopy (LM) and transmission electron microscopy (TEM) was used for the histological and stereological analysis. Because the same individual fish could not be used for both LM and TEM, they where divided into two groups:
Group 1: Six fish (table 2.1, fish #0-5) were used for LM histology and/or stereology: Using a pipette, fixative (4% formaldehyde solution in phosphate buffer, Apotekproduksjon AS vnr.
329847) was carefully injected directly into the mouth of the fish until drops of fixative were seen at the anus. This was done in order to preserve the mucosal layer inside the alimentary canal. The fish were then dissected and the digestive system was removed intact by cutting off the alimentary canal as close as possible to the posterior end of the pharyngeal teeth and the anus and gently removing it along with surrounding tissue (fig. 2.2). The samples (digestive tract + adjacent tissues) were kept in fixative for at least 24 hours.
Group 2: Five fish (table 2.1, fish #6-10) were used for TEM histology and stereology: After dissection, tissue samples were removed with a scalpel from the proximal, middle and distal intestine (PI, MI, DI) from fish #6-10 and fixed in 2.5% paraformaldehyde, 2.5%
glutaraldehyde in 0.08 M cacodylate buffer (0.5% sucrose, pH = 7.4) for at least 24 hours.
2.1.2 Measuring gut length
Five frozen one-year old L. bergylta were thawed overnight at 5 °C and measured for wet weight (table 2.1, fish #1-5). They were dissected and the gut was removed with a scalpel by cutting as close to the pharyngeal teeth and the anus as possible. The gut was carefully placed on a laminated millimetre graph paper and measured for length, to obtain gut length
measurements which could be factored into the calculations of the stereological analysis.
Table 2.1 Weight, total length (TL) and standard length (SL) of one-year-old ballan wrasse used for LM/TEM histology/stereology and frozen one-year-old ballan wrasse used for gut length analysis (GL). These fish were used for histological and stereological analyses, including analyses of microvilli and goblet cell density/content. Analyses were conducted in the intestine (X) as well as the
oesophagus, in some cases (X*)
Fish
# W
(g) TL
(cm) SL
(cm)
Gut length
(cm) Histology
Goblet cell mucin
content Stereology
Goblet cell
density Microvilli
LM
0 24.2 12.0 10.0 -‐ X X -‐ -‐ -‐
1 22.2 12.0 10.0 -‐ X* X* X X -‐
2 41.4 14.1 12.5 -‐ X X X X -‐
3 14.2 10.3 8.8 -‐ X* X* X X -‐
4 21.8 11.8 10.2 -‐ X X X X -‐
5 21.7 11.7 10.0 -‐ X X X X -‐
TEM
6 27.1 -‐ 10.6 -‐ -‐ -‐ -‐ -‐ X
7 20.0 -‐ 9.8 -‐ -‐ -‐ -‐ -‐ X
8 14.5 -‐ 9.1 -‐ -‐ -‐ -‐ -‐ X
9 12.2 -‐ 8.5 -‐ -‐ -‐ -‐ -‐ X
10 8.8 -‐ 7.7 -‐ -‐ -‐ -‐ -‐ X
GL
11 30.7 -‐ -‐ 7.0 -‐ -‐ -‐ -‐ -‐
12 25.9 -‐ -‐ 6.5 -‐ -‐ -‐ -‐ -‐
13 20.5 -‐ -‐ 6.0 -‐ -‐ -‐ -‐ -‐
14 18.3 -‐ -‐ 6.0 -‐ -‐ -‐ -‐ -‐
15 16.2 -‐ -‐ 5.0 -‐ -‐ -‐ -‐ -‐
*includes oesophagus
Figure 2.2 A: Digestive system of one year old L. bergylta. The intestine is surrounded by liver and
To our knowledge, this is the first study attempting to estimate intestinal surface area of fish from serial sections. The method used in previous studies of intestinal surface area in teleosts (Al-Hussaini 1949; Frierson & Foltz 1992) involved removal of the intestine at dissection and manually measuring the length of the (unfolded) gut. Then, tissue samples were taken from the intestine to create microscope sections containing intestinal mucosa. The distance around the inner perimeter of the tract was estimated for each section (mucosal circumference). These estimates could then be combined with the measurements of gut length to estimate the
intestinal surface area. This method provides only a limited overview of the mucosa along the intestinal length and assumes a relatively stable/unchanged surface between sections. The accuracy suffers considerably if there are unexpected changes in folding patters, not represented in the samples. The distance is known along the anterioposterior axis, so
estimates can be made for simple shapes, but unless the intestine is a straight tube, estimating the length of the non-straight parts (turns) is problematic, especially when there is variation in the curvature. Additionally, fixation may cause shrinking of the more fragile tissues like the intestine.
To solve this problem, an additional batch of fish was dissected in order to specifically analyse gut length (table 2.1, fish #11-15). Gut length (GL) in relation to fish wet weight (W) was analysed by linear regression (r2 = 0.94) (Appendix A.12), giving the following formula to calculate the gut lengths of the fish used for stereology:
𝑮𝑳= 𝟎.𝟏𝟎𝟔𝑾 + 𝟑.𝟕𝟔
2.2 Sectioning and histology
2.2.1 Embedding and sectioning for light microscopy (LM)
After fixation, the tissues were cut by scalpel into 3-4 pieces of a suitable size to be fitted into plastic cassettes (5 and 10 mm deep). The tissue was cut around the turns of the intestinal loop, minimizing damage to the intestine (fig. 2.3). The samples were dehydrated and embedded in paraffin according to the standard automated protocol at NTNU Sealab (Leica TP1020 Automatic Tissue Processor, procedure: Appendix A.2) and sectioned (thickness = 4 µm) with a microtome (Leica Jung Autocut 2055). Serial sectioning for stereological analysis was applied (see chapter 2.3.1). Microscope slides were mounted and prepared for analysis by light microscopy (Axioplan 2 plus, Zeiss). For higher quality images, a selection of slides were scanned with a NanoZoomer (Model SQ C13140-21, Hamamatsu Photonics K.K.
Japan).
Serial sections of six fish were used for a general histological description of the digestive system, for estimating the density of mucus secreting goblet cells in the intestine (chapter 2.4.2), and for measurements of mucosal folding. One of these fish was sectioned in order to both determine the parameters of serial sectioning for stereological analysis of the other five fish (chapter 2.3.1), and to evaluate optimal staining methods for the histological and
stereological analyses (chapter 2.2.2). The other five fish were sectioned accordingly and used to analyse the internal surface area of the intestine (chapter 2.3.3) and to estimate tissue volumes of the intestine, liver, pancreas and adipose tissue (chapter 2.3.2).
Figure 2.3 Diagram explaining how the digestive system of L. bergylta was split into
2.2.2 Evaluating the histological staining methods
A selection of slides from fish #0 was tested with four common histological stains (Appendix A.3). Hematoxylin & Eosin (H&E) was chosen because it provides a good overview of tissue structure (while also identifying structures as normal, inflamed, degeneratively changed or pathological). Masson-Goldner Trichrome (MG) was chosen for its ability to stain connective tissue. Periodic acid-Schiff (PAS) was chosen for its ability to stain mucosubstances in the goblet cells of the alimentary tract and glycogen in the liver. Alkaline Blue/PAS (AB/PAS) was chosen because of its ability to stain differently for acid versus neutral mucosubstances in the intestinal goblet cells.
Analysis of each of the four stains was done in the intestine (fig. 2.4), pancreas (fig. 2.5) and liver (fig. 2.6). The observations are described in detail in Appendix A.4.
Figu re 2.4 In tes tin al m uco sa of L. b erg ylt a. St ain ed wit h M ass on Gol dne r ( MG ), H em ato xyl in
& E osi n ( H&
E), A lci an Blu e/PA S (A BPA S) a nd Peri odi c A cid
Schi ff (PA S). A bbr evi ati ons : a m a cidi c m ucus gobl et c ell , g g oble t cells , gr g ran ula r c ells , mm mix ed m ucu s g oble t cell , n nucle i, sm su bm uco sa, v venul e
Figu re 2.5 Pa ncr eas of L. b erg ylt a. Sta ined with Mas son Gold ner (M G), H em ato xylin & E osin (H
&E ), A lcia n B lue/P AS (A BP AS ) a nd P erio dic Acid Sch iff
(PA S).
A bbre via tio ns:
e x e xoc rine pa ncre as, pd pa ncre ati c d uct , v ve in/
ven ule , z g z ym oge n g ranu les
Figu re 2.6 Li ver of L. b erg ylt a. Sta ined with Mas son Gold ner (M G), H em ato xylin & E osin (H
&E ), Al cia n B lue /P AS ( ABP AS) and P eri odi c Ac id Sch iff
(PA S).
A bbre via tio ns:
d pan cre ati c d uct /bi le duc t, s si nus oid ca pil lari es, v port al v ein
Ranking of histological stains
H&E was the only method that stained the exocrine pancreatic tissue in a satisfactory manner, differentiating it from other tissues at low magnifications. The exocrine pancreas was the most fragmented of the tissues, mostly found scattered as islets across the liver parenchyma, in the adipose tissue, as well as covering blood vessels connecting to the spleen and intestine.
H&E stained the pancreas dark blue, providing a good contrast to the lighter shades of surrounding tissues. The exception was the endocrine part of the pancreas which stained pale magenta similar to the liver but no other stain yielded any significantly better colouring of this tissue. To choose the stain best suited for histological analysis, they were ranked by effect on three different tissue types (intestine, pancreas and liver). These were then pooled into an overall final ranking:
1: H&E emerged as the favourable stain, mostly because of the staining of nuclei, which in turn revealed a lot about the cell structure (especially the epithelium). In addition, H&E provided the overall most lucid images with a clear overview of nuclei, cytoplasm and cell walls within the tissues. Most tissues were easily distinguished with the possible exception of smooth muscle and connective tissue which were sometimes hard to differentiate.
2: AB/PAS stained and differentiated the goblet cells of the intestine. However, the other tissues mostly appeared pale and greyish, providing a monotone image, especially at lower magnifications.
3: MG performed weakest of all the stains at the cellular level. Being the only stain without visible nuclei and having an overall very monotone brick red colouring within the cells, it was the only stain in which the epithelial cells of the intestine were indistinguishable. MG's
highlighting of the connective tissues however, provided good insight into epithelial structures (like epithelial height in ducts) as well as revealing collagen density.
4: PAS resulted in clustered/muddled colorization of liver tissue and glycogen granules were more clearly observable by H&E.
Conclusion: H&E was chosen as the most suitable stain for the stereological and histological analyses. Additionally, a selection of slides was stained with AB/PAS and used for further analysis of the mucous cells of the alimentary tract (see chapter 2.4). Sections already stained with MG were referred to when H&E stain was inadequate.
2.3 Stereological analysis
2.3.1 Serial sectioning
For the stereological analysis, a fixed inter-sectional distance (T) was used to create a series of parallel transverse sections through the digestive system of each fish. Each section of the series represents a slab (tissue between sections) with a length of T (Howard & Reed 1998;
Mayhew 1991; Mouton 2002). By a test of accuracy, a T = 260 sections was chosen
(Appendix A.5). Factoring in the sectional thickness of 4 µm, gave the absolute value for T:
𝑻= 𝟒 µμ𝒎×𝟐𝟔𝟎= 𝟏𝟎𝟒𝟎 µμ𝒎
The paraffin blocks of five fish (table 2.1) were sectioned until the first tissue was spotted and a random number r was created in the interval of 0-T (0-260 sections) using a random number generator (fig. 2.7, Appendix A.6). This number decided the sectioning distance to the first sectional area (A1) used to produce microscope slides (systematic random sampling) (Mouton 2002). After the first sectional area, the block was sectioned through using the set T value.
Three microscope slides were mounted per sectional area, each containing 1-3 sections. This was done in order to have a selection from which the highest quality slides could be chosen.
This method of serial sectioning made it necessary to re-embed the final part of the tissue which was inaccessible to the microtome. The new blocks were then sectioned by the same method as the original blocks until tissue could no longer be observed (fig. 2.7).
Figure 2.7 Procedure of serial sectioning. Each block was sectioned until tissue was first spotted. A random number r between 0-T determined the distance to the first sectional area A1. Inaccessible tissue was re-cast and sectioned using the same method until tissue was last spotted. Abbreviations: n number of sectional areas in series, T inter-sectional distance, r randomly generated number.
2.3.2 Tissue volumes
The slides of fish #1-5 were studied under a stereomicroscope (MZ75, Leica Microsystems, Switzerland) with a Nikon Digital Sight DS-L2 colour video camera (Nikon corp., Japan).
Images containing all tissue present in each section were captured and analysed using the counting software CAST (Olympus Denmark AS 2000). The tissue sections were analysed according to the Cavalieri principle (Mayhew 1991; Howard & Reed 1998; Mouton 2002), where a systematic array of area specific points are superimposed over photos of each section.
The points were counted using an unbiased selection rule where the relevant tissue had to touch not only the centre of the cross but also the top and right lines (fig 2.8, right). Tissues counted were liver, exocrine pancreas, adipose tissue, intestinal tract, spleen, and gonads. The remaining tissues were grouped as “other” and included tissue like large blood vessels/ducts, kidney and unidentified tissue. An optimal area per point a(p) for the CAST point grid was determined in order to ensure an accurate count, the following method was used: A CAST grid with a relatively large a(p) was placed on a section with a good representation of all the different tissue types. The tissues were counted and the a(p) was gradually decreased in CAST by the smallest intervals the program would allow. For each count the deviation to the previous count was calculated and when this deviation was under 15% for all tissue types, the second last a(p) in the chain was chosen to be the standard grid size (73917 µm2, Appendix A.7). The tissue volumes could then be estimated using the following formula (Mayhew 1991; Mouton 2002):
𝑽𝒙 =𝑪𝑷×𝒂 𝒑 ×𝑻
Where Vx is the tissue volume contained in the slab belonging to the section that was counted (x). CP is the number of counting points falling on the relevant tissue, a(p) is area per point and T is the inter-sectional distance. Summarizing the tissue volumes of all sections in a sample yields the total volume of the tissue:
𝑽=𝑽𝟏+𝑽𝟐+𝑽𝟑…+𝑽𝒏
2.3.3 Two-step analysis of intestinal surface area
Step 1: Light microscopy: Convoluted intestinal surface area
The internal surface area of the intestine was estimated using LM images of serial sections (Mayhew 1991; Howard & Reed 1998). In CAST, a set of line segments were superimposed over the images each with an associated counting point on the leftmost end of the line (fig.
2.9). The length per line segment L(p) = 148 µm, and area per point a(p) = 129521 µm2,was decided by the same method used when counting for tissue volume (chapter 2.4.1). Two counts were made on the line segments;
1. Intersections between lines and the surface border of the intestinal epithelium (I).
2. Counting points within the bounds of the intestinal tissue (CP). The same selection rule was used for these counting points as was done counting for volume (fig. 2.8, right).
Both counts were made twice for each section (one regular and one rotated 90o), using the average of the two as the final value. The distance around the inner perimeter of the tract (mucosal circumference / MC), could then be calculated for each section by incorporating the intestinal tissue area (IA) (see chapter 2.3.2).
𝑴𝑪=𝟐× 𝑰
𝑪𝑷×𝑳 𝒑 ×𝑰𝑨
The mean mucosa circumference was then multiplied by gut length (table 2.1) to give what Frierson & Foltz (1992) called the “convoluted surface area” (Scon). This value represent the surface area of the intestinal epithelium from what is measurable by LM, and does not includine enlargement by microvilli (measurable by TEM).
𝑺𝒄𝒐𝒏 = 𝑴𝑪×𝑮𝑳
Step 2: Transmission electron microscopy: Total intestinal surface area
To estimate the total intestinal surface area (Stot), the additional surface area caused by intestinal microvilli had to be included. The average microvilli surface area per unit of area (MIC) was estimated from TEM measurements. The convoluted surface area (Scon) could then be multiplied by MIC to give Stot (Howard & Reed 1998).
Samples from the proximal, middle and distal intestine (PI, MI and DI) of five fish (table 2.1) were post-fixated, bulk stained and embedded in EPON 812 following standard procedure at NTNU Sealab (Appendix A.8.1). Ultrathin (60 nm) sections were cut using a Leica Reichert Ultracut microtome (Leica Microsystems, Germany) and contrasted for TEM with lead citrate solution (Appendix A.8.2).
Micrographs of microvilli were captured at x5000 magnification using a Jeol JEM-1011 transmission electron microscope (Jeol Ltd., Japan). Picture analysis was done in the software ImageJ 1.50c (Wayne Rasband, National Institutes of Health, USA).
Estimates of microvilli density and surface area were based on a method deployed in a study by Frierson & Foltz (1992). Microvilli were analysed from micrographs (ca 10 per fish) of the intestinal brush border (fig. 2.10 A). A sampling line with a known length of L was drawn through the microvilli at a 90o angle, counting the number of microvilli (n) across L and mea- suring their height (H) and diameter (D). The surface area of individual microvilli could then be calculated using the formula for surface area of a bottomless cylinder: 𝑨=𝑯𝝅𝑫+𝝅𝑹𝟐 The microvilli density per epithelial length was calculated (𝒏 𝑳) and the value was squared to represent microvilli density per epithelial area (𝒏/𝑳)𝟐. This was then multiplied by the average surface area per microvilli (𝑨) to give the MIC per epithelial area (fig. 2.10 B).
𝑴𝑰𝑪=𝑨×(𝒏/𝑳)𝟐
Separate MIC values were estimated for PI, MI and DI and applied to the corresponding Scon
values to give Stot.
𝑺𝒕𝒐𝒕 = (𝑴𝑰𝑪×𝑺𝒄𝒐𝒏)𝑷𝑰+(𝑴𝑰𝑪×𝑺𝒄𝒐𝒏)𝑴𝑰+(𝑴𝑰𝑪×𝑺𝒄𝒐𝒏)𝑫𝑰
Figure 2.8 The Cavalieri method: Left: A series of parallel sections were cut through the object at a fixed distance. The sections were layered with a randomly applied point grid where a known area was connected with each point. The area of a tissue could then be determined by counting the number of points hitting the tissue and multiplying with the point area. The volume was then determined by multiplying the tissue area in a section with the absolute T value. Figure by Howard and Reed (1998).
Right: Selection rule for accepting counting points: both the top and right lines of the cross as well as the centre has to be within the bounds of tissue (arrow).
Figure 2.9 Line segments in CAST superimposed over a section. The blue dots are intersections between the line and the surface border (I). The green square shows a counting point within the bounds of the intestinal tissue (CP), only the left end of the line segments was used as counting points.
Figure 2.10A: Microvilli from the proximal intestine (PI) (Fish #8, photo by Ida Anette Norheim).
Microvilli within L were counted and height/diameter was measured along with the length of L. B:
MIC represents the combined surface area of all microvilli contained in a square where L=1, which can be estimated by multiplying Ā by the number of microvilli contained in said square (n).
Abbreviations: A microvillus surface area, Ā average microvillus surface area, L sampling line
2.3.4 Tract size
The tract diameter (average of the major and minor axis) was measured (Appendix A.11) and used to estimate intestinal circumference (IC) which could then be multiplied by gut length to give the outer surface of the tract, called the simple intestinal surface area (Ssimp) (Frierson &
Foltz 1992). Ssimp, Scon and Stot were used to illustrate the degree of contribution from the different factors that make up the total intestinal surface area (tract size, mucosal folding and microvilli).
2.4 Further analysis of the digestive tract 2.4.1 Goblet cell analysis
Sections stained with H&E from five fish (table 2.1) were selected to estimate the goblet cell density of the intestinal epithelium. Three sections were selected per fish from the proximal, middle and distal intestine (PI, MI, DI). The sections were chosen to have minimal mucosal tissue damage and be located as close as possible to the middle or proximal/distal end of the intestine, while also keeping the position in the tract as consistent as possible between the fish. The goblet cells along the surface were counted under a light microscope using a KD- 8025 hand tally counter. The number of goblet cells (G) was then divided by the mucosal circumference (MC, see chapter 2.3.3). This gave an estimate of density measured in number of goblet cells per unit of epithelial length, which was squared to give the goblet cell density per unit of epithelial area (GD).
𝑮𝑫=(𝑴𝑪 𝑮 )𝟐
Three estimates of GD was made per fish representing each of the three gut segments PI, MI and DI. These were multiplied by the Scon of each gut segment and finally summarized to give the total number of goblet cells (Gtot):
𝑮𝒕𝒐𝒕 = (𝑮𝑫×𝑺𝒄𝒐𝒏)𝑷𝑰 +(𝑮𝑫×𝑺𝒄𝒐𝒏)𝑴𝑰+(𝑮𝑫×𝑺𝒄𝒐𝒏)𝑫𝑰
Mucin content
Sections from six fish were stained with AB/PAS for mucous cell content analysis (table 2.1).
Alcian blue stained the mucous vacuoles of the cells blue when positive (AB+) for hyaluronic acid sulphomucins and sialomucins. Periodic Acid-Schiff stained the mucus magenta when positive (PAS+) for neutral glycoproteins. Analysis of the oesophageal mucous cells was made where sections were available (table 2.1).
Random areas from the oesophagus and intestine were chosen and the stained, mucous parts of the cells were measured across their longest axis and once more at a 90 degree angle to calculate average diameter. The measurements were done in the viewing software NDP.view 2.4.26 (Hamamatsu Photonics 2015).
2.4.2 Intestinal mucosal folding
Four other estimates were made using measurements and analyses of intestinal mucosa in six fish (table 2.1). Three sections were used per fish, located as close as possible to the middle and the proximal/distal ends of the intestine.
1: The number of primary folds of the mucosa were counted. This did not include new secondary folds (branches), only the folds with a root/base connected to the wall of the tract (fig 2.11).
2: The lengths and heights of the folds were measured by tracing from the tunica muscularis below the base of the primary fold to the fold apex (fig. 2.11). The trace line was kept as close to the centre of the fold as possible. Each primary fold was measured once and, if secondary folding occurred, the longest branch was prioritized.
3: The width across the intestinal folds was measured. The folds started out wide at the base and quickly narrowed in, reaching the minimum width. Outwards from this point, the fold width remained relatively constant (not accurately illustrated in fig. 2.11). The measurements were done in these parts of the folds.
4: The frequency of branching was noted. Branches were (for this count) defined as all new secondary folds that split off the original, primary fold (fig. 2.11). This meant an unbranched primary fold would have a branching frequency of 0 and that one instance of branching would create two secondary folds. However, because of the great variations between fish and
difficulty in defining what constitutes a separate branch (especially with varying degrees of mucosal damage) a broader approach was chosen. The branching frequency was placed into one of three categories based on general observations: None to low (0-5 instances), medium (6-10) and high (over 10).
2.4.3 Other measurements in the alimentary canal
The following measurements were made in the alimentary canal of six fish (table 2.1):
1: The tract diameter.
2: Ephitelial height in the oesophagus and intestine.
3: Fold length and fold width in the oesophagus.
Figure 2.11: Intestinal wall. The length of the red line that traces the internal surface of the intestine is the mucosal circumference. Primary folds (P) would split into multiple secondary folds, creating branches (b), defined here as all secondary folds exept the longest (apical fold). Mucosal fold height was measured from t. muscularis below the base of each primary fold to the terminal point of the apical fold. Mucosal fold width was measured at the most narrow points of the fold (“standard width”). Intestinal diameter was measured from the outer surface at each side of the tract, as the average length across the longest and shortest axis.
2.5 Estimating sections with damaged tissue
Of all tissues analysed, the intestinal mucosa was most susceptible to damage and torn epithelium was frequently observed. This resulted in many sections deemed too damaged to count with line segments (Appendix A.9)
Estimating the intestinal surface area of these sections was achived by using the average surface areas of the two closest counted sections (fig. 2.12, left). When damaged sections were located at the terminal ends of the intestine, only one reference section could be used (fig. 2.12, right).
The accuracy of these estimates depended on several factors, including the number of sequential damaged sections between reference sections, the number of available reference sections (one at the proximal/distal ends of the intestine, otherwise two) and the variance in the width of the tract in the damaged sections.
The level of accuracy could be illustrated by dividing the number of available reference sections by the number of needed estimates. The higher the resulting score, the more accurate the estimate. For example, in fig. 2.12, X1 has two reference sections (A and B), and needs one estimate, so the accuracy level would be 2. X2 has two reference sections and needs two estimates while X3 has only one reference but also needs only one estimate making the score 1 for both. X4 has one reference point and needs two estimates resulting in the lowest
accuracy score of 0.5.
Figure 2.12 Method for estimating values from damaged sections (X) using the average of the closest good sections (A, B). When the damaged section(s) was located at the terminal end of the intestine (bottom two), only A could be used. The level of accuracy was variable: In this figure X1 is the most accurate but the accuracy drops as the number of sequential damaged sections rise (X2) and the number of reference sections drops to one (X3). The largest loss of accuracy of course happens when both of these occur (X4)
2.6 Statistical analysis
All statistical analysis was done in SigmaPlot 13.0 for Windows (2014, Systad Software Inc.) Linear correlation analysis was performed on surface areas of intestinal mucosa (Stot) and tissue volumes (V) as functions of weight and standard length. Also tested was the relationship between gut length and fish weight.
Shapiro-Wilk tests for normality were performed previous to all ANOVA tests to reveal if the data was normally distributed between the fish. The data that passed this test was analysed by one way ANOVA (assumes normality) and the data that failed the test was analysed with ANOVA on ranks (does not assume normality).
One way ANOVA tests for variance were performed on goblet density, microvilli surface density and the other microvilli data (average height, diameter and number per µm). The groups compared for variance were foregut, midgut and hindgut.
Holm-Sidak test was performed on tract diameter between gut segments. This method was implemented in cases where one way ANOVA test revealed differences between sample groups.
ANOVA on ranks: This test was performed on the average lengths of the mucosal folds as well as average number of folds. The groups compared for variance were foregut, midgut and hindgut.
The p-value/threshold for significance for all tests was P = 0.05.
3. Results
3.1 Structure of the digestive system
Gross anatomy
The ballan wrasse intestine is shaped as a simple tube (i.e no pyloric caeca) and organized into three definable segments. The proximal intestine was distended, forming an intestinal bulb, the middle intestine was curled into a single loop, and the distal intestine was simple and straight (fig. 3.1). The internal surface was observed through the intestinal wall, revealing a “web-like” pattern of zig-zagging longitudinal folds, connected to each other through frequent transverse folds (fig. 2.2). The liver grew around most of the anterior and middle intestine where the intestinal loop was located; Adipose tissue was found attached to the intestine along its entire length (fig. 2.2, fig. 3.1). The pharyngeal apparatus was located anterior to the intestinal bulb (oesophagus);it consisted of a single ventral teethed plate and a dorsal set made up of two lateral teethed plates, all rounded molars (fig. 3.1, left).
Figure 3.1 Left: Upper set of pharyngeal teeth located dorsal to the oesophagus before removal. Right:
Alimentary canal cut from between the pharyngeal teeth and anus. Some liver tissue was removed by scalpel, exposing the Z-shaped intestinal loop and the intestinal bulb. Abbreviations: O Oesophagus, 1 proximal intestine/bulbus, 2 middle intestine (D2 first turn, B2 second turn), 3 distal intestine, lv liver
Histological description from sections
The sections were categorized into six areas along the anteroposterior axis (fig. 3.2):
Following the oesophagus (area O), the sections containing intestine were divided into five areas (area A-E) based on the layout of the intestinal loop (two at the anterior and posterior ends, two at the turns and one at the middle of the loop) (fig. 3.2, bottom).
The intestine was then further divided into three segments based on the proximodistal
position in the loop: 1: proximal (includes the intestinal bulb), 2: middle (includes both turns of the intestinal loop) and 3: distal (may include rectum). The different parts of intestine in each section could then be labelled by a combination of both the anteroposterior and the proximodistal position, for reference use (fig. 3.2).
Figure 3.2 Structure of the alimentary canal of L. bergylta. The sections following the oesophagus (area O) was divided into five areas (A-E) based on the arrangement of the intestinal tract as observed in the sections. The intestine could then be further divided into three segments (1-3) based on its position in the intestinal loop (proximal, middle and distal). The label key shows how the sections where categorized into six areas (O, A-E) and how the intestinal segments observed in these sections were labelled by a combination of both the anteroposterior and the proximodistal position
Area O: Oesophagus (fig. 3.3)
The oesophagus was surrounded by a thick circular layer of striated muscle, oriented at a skewed angle relative to the transverse sections and the GI tract. The mucosa was arranged into longitudinal folds with extensive branching. Primary folds were upright and tall, reaching almost all the way to the centre of the tract. The folds were roughly triangular in shape, resulting in a tree-like structure. The anterior end of the liver was observed here and contained islets of exocrine pancreatic tissue (hepatopancreas). The head kidney was observed here.
Transitional area O/A: Oeso-intestinal junction (fig. 3.4)
The oesophagus connected directly to the intestine through a sphincter valve (a.k.a. the lower oesophageal sphincter or cardiac sphincter). Compared to the oesophagus, the folds of the intestine were thicker and less upright. The t. muscularis of the intestine was thinner and the width was consistent around the tract as opposed to the thick irregular t. muscularis of the oesophagus. The hepatopancreas was larger at this point than in the proximal sections of the oesophagus, almost surrounding the entire tract. Adipose tissue containing exocrine pancreas, large bile/pancreatic ducts and the anterior end of the gallbladder was first observed here. The ducts and gallbladder were located at the ventral/lateral side of the tract.
Area A (fig. 3.5)
The intestinal bulb was most prominent (widest) in the proximal intestine (A1). The mucosal folds were high, often reaching into the centre of the tract, and frequently branched into secondary folds. The main lobe of the liver was located ventral to the intestine, stretching around to the lateral sides. Two smaller lobes were observed on the dorsal side in posterior sections. This area, along with the oeso-intestinal junction, contained the biggest share of liver tissue. Most of the gallbladder (fig. 3.5, bottom) was located within this area along with the major bile ducts and pancreatic ducts (fig. 3.5, top), which in turn connected to the intestine in the anterior end of area A (not shown). Endocrine pancreatic tissue was
concentrated around these ducts (fig. 3.5, top) and connected to the intestine in the anterior area A. Extrahepatic pancreas surrounding veins and embedded in adipose tissue was found in the coelom throughout this area. The anterior end of the spleen (not shown in fig. 3.5) was in one instance (fish #4) observed on the dorsal side of the tract in the posterior end of the
Area B (fig. 3.6)
This area contained the second turn of the intestinal loop (B2) and only included one or two sections. In the proximal segment (B1), mucosal folds were still tall and heavily branched but the tract was narrower than in anterior sections. There was less liver present here than in area A, usually only one lobe remained on the ventral side. As the portion of liver/hepatopancreas decreased, the portion of adipose tissue increased, mostly located in the space between the gut segments and liver. The gallbladder was still observed in some of the fish.
Area C (fig. 3.7)
Here all three segments of the intestine were present. The mucosal folds of the middle (C2) and especially the distal (C3) segments tended to be shorter than in the proximal segments (C1), with less branching. The distal segment tended to be narrower than the other two. The liver was smaller than in the preceding area but the amount of extrahepatic pancreatic and adipose tissue was larger. C3 is the most posterior area at which the gallbladder was observed in any of the fish. The spleen was observed here in most fish, and could be observed
connecting to the splenic vein. The splenic vein was attached to the pancreatic and adipose tissue in the coelom.
Area D (fig. 3.8)
In this area, the first turn of the intestinal loop (D2, proximal MI) as well as the middle DI segment (D3) was observed. The mucosa of the MI was heavily branched and the tract was wider than in the DI. The complexity of the mucosal folding varied between fish in the DI (fig. 3.8), from little/no branching (fish #3-5) to medium/high branching (fish #1-2). The amount of liver/hepatopancreas was either minimal or completely absent. The coelom was dominated by adipose tissue and extrahepatic pancreas. The distal end of the gonads could sometimes be observed. In one instance a sphincter-like structure was observed in the middle of the intestinal turn (fig. 3.8 top), but no such structure was observed in any other sections.
Area E (fig. 3.9)
The distal intestine (E3) was the segment that varied most in size and mucosal folding complexity between fish. This was especially apparent in the two fishes shown in fig. 3.9, where the fish in the top picture has a narrow tract with short folds and minimal branching while the fish in the bottom picture has a wider tract with a high level of branching (more than 10 branches). The main body of the gonads were located here dorsal to the intestine.
Adipose tissue and exocrine pancreas was present throughout the area.
Figure 3.3 Area O, Oesophagus: The tunica muscularis of the oesophageal wall was thick and made up of skeletal muscle fibres. The mucosa was heavily folded. Hepatopancreas was present as well as the head kidney. Stain: H&E. Fish #1. Abbreviations: O oesophageal canal/mucosa, k kidney, lv liver, p exocrine pancreas, tm tunica muscularis
Figure 3.4 Transitional area O/A: The oeso-intestinal junction connected the oesophagus with the intestine through a narrow passage circumscribed by the pyloric sphincter. Major bile/pancreatic ducts and the gallbladder were first observed here. Stain: H&E. Fish #3 Abbreviations: A1 proximal PI, at adipose tissue, d duct, gb gallbladder, h heart, k kidney, lv liver, O oesophagus, p pancreas, s pyloric sphincter
