Skeletal Anomalies in Wild Common Dab

SKELETAL ANOMALIES IN WILD COMMON DAB

Word count: 17 911

Christian von den Driesch Student number: 01710623

Supervisor: Prof. Dr. Koen Chiers Supervisor: Dr. Maaike Vercauteren

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Veterinary Medicine

Academic year: 2019 - 2020

Ghent University, its employees and/or students, give no warranty that the information provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any infringement of third-party rights.

Ghent University, its employees and/or students do not accept any liability or responsibility for any use which may be made of the content or information given in the thesis, nor for any reliance which may be placed on any advice or information provided in this thesis.

IMPACT OF THE SARS-COV-2 PANDEMIC

In March 2020, when the SARS-Corona-Virus-2 reached the European Union and Belgium, most of the practical work for this thesis was already done. We had plans to take five more radiographs, to have an equal amount of affected and control fish, and to let the otoliths of affected fish analyze to get their estimated age. Furthermore, we even wanted to do a computer tomography scan of one of the fish with skeletal anomalies to get a better understanding of the three-dimensional configuration of combined vertebral column deviations. Although none of these plans could be carried out, the crisis did not significantly affect the outcome of this work. The communication was switched to electronical forms without problems and without implications for the quality of mentoring and reporting.

PREFACE

About 20 months ago, when I had to choose a topic for my thesis, I was strongly motivated to write about skeletal anomalies in flatfish, although, or rather because, I did know nothing about flatfish then. I was very much drawn to broaden my horizon, by writing about something only slightly related to my studies of veterinary medicine.

“I do not know what I may appear to the world, but to myself I seem to have been only like a boy playing on the sea-shore, and diverting myself in now and then finding a smoother pebble or a prettier shell than ordinary, whilst the great ocean of truth lay all undiscovered before me.”

Sir Isaac Newton

Reading his quote, Newton’s pebbles and shells appear to be like cows and sheep for me, only a small fraction of the animal kingdom, which can be seen as the “great ocean of truth”. My studies have equipped me with a broad base of knowledge and veterinarians are taught to think comparatively, figurately taking me to the seashore, ready to – literally – take a look into the “ocean”. Isaac Newton, without any doubts one of the most important scientists of the past centuries, seems to have been quite aware of the fact, that even he himself could never fully study and understand everything, but only small pieces at a time. For my thesis this means that, although we only focused on one certain kind of anomaly, in only one species, in a very small part of the Atlantic Ocean, many small pieces can contribute to understand the greater picture and are therefore necessary and important.

Writing this thesis, as well as planning and performing the practical parts of it, were great experiences for me. I got the chance to decide what exactly I wanted to focus on, learned a lot about all elements of scientific research, and had the opportunity to work together with very experienced scientists.

Therefore I want to thank my supervisors Prof. Dr. Koen Chiers and Dr. Maaike Vercauteren. Maaike helped me a lot with designing the study, performing it, and putting it all on paper. We could discuss every part of the work during regular meetings, either in person or online and I was able to ask for help with all difficulties or uncertainties I found myself confronted with.

Also, I want to thank Dr. Annemie Van Caelenberg (Department of Veterinary medical imaging and small animal orthopedics) for helping us with taking the radiographical images and thoroughly analyzing them afterwards. It was very pleasant for me to work in such a professional and enthusiastic environment.

In November 2019 we were able to arrange a meeting with my supervisors, Dr. Van Caelenberg, and Dr. Ana de Azevedo (University of Santiago de Compostela) to discuss the results we had found by then. Dr. de Azevedo did research on skeletal anomalies in Senegalese sole and her articles had a strong influence on how our own study was designed. Therefore, I am very grateful that she was willing to share her expertise with us.

I was not the only student this year who wrote his thesis about the common dab. Since the beginning of our work I have been in regular contact with Roxanne Billiet and Sebastian Martelli and we could often help each other with finding suitable literature or discussing problems we all faced. For that reason I want to also thank both of them.

Last but not least, I want to express my gratitude towards my girlfriend Jana. She motivated me when I was not, reassured me when I was stressing out, and proof-read my whole thesis.

CONTENTS

SUMMARY | SAMENVATTING ...... 7 1 LITERATURE REVIEW ...... 8

1.1 TELEOSTEI ...... 8 1.1.1 Introduction ...... 8 1.1.2 General morphological and physiological differences with mammals ...... 8 1.1.3 The fish skeleton and its differences with mammalian bone ...... 9 1.2 SKELETAL ANOMALIES IN FISH ...... 14 1.2.1 Historical overview ...... 14 1.2.2 Wild versus aquaculture ...... 14 1.2.3 Importance of studying skeletal anomalies in fish ...... 15 1.2.4 Diagnostic techniques ...... 15 1.2.5 Terminology ...... 15 1.2.6 Types of skeletal anomalies...... 16 1.2.7 Tissues and cells ...... 18 1.2.8 Causes ...... 18 1.3 TELEOSTEI: FLATFISHES...... 20 1.3.1 Systematics ...... 20 1.3.2 History of flatfish research ...... 20 1.3.3 Phyletic history ...... 21 1.3.4 Distribution...... 22 1.3.5 Morphology ...... 22 1.3.6 Physiology ...... 23 1.3.7 Development ...... 23 1.3.8 Exploitation ...... 25 1.3.9 Threats ...... 25 1.3.10 Flatfishes in the North Sea ...... 27 1.3.11 Common dab (Dutch name: Schar) ...... 28 2 AIMS OF THIS STUDY ...... 30 3 RESEARCH...... 31

3.1 MATERIALS AND METHODS ...... 31 3.1.1 Sampling areas ...... 31 3.1.2 Examination and processing of specimens ...... 31 3.1.3 Data processing and graphical exploration ...... 32 3.1.4 Statistics ...... 32 3.1.5 Radiographic imaging ...... 33 3.1.6 Meristic counts and measurements ...... 33 3.2 RESULTS ...... 34 3.2.1 Data analysis ...... 34 3.2.2 Radiographic analysis...... 36 3.3 DISCUSSION ...... 38 3.3.1 Prevalence ...... 38 3.3.2 Radiography ...... 38 3.3.3 Possible risk factors ...... 39 3.3.4 General condition of specimens ...... 39 3.3.5 Catch data ...... 40 3.3.6 Measurements and ratios ...... 40 3.3.7 Number of vertebrae ...... 40 3.3.8 VCDs ...... 41 3.3.9 Severity ...... 41 3.3.10 Causal factors ...... 42 3.3.11 Limitations ...... 42 3.3.12 Perspective / future research ...... 42 4 CONCLUSION ...... 43 5 REFERENCES ...... 44 APPENDIX 1: OTHER FLATFISHES OF THE NORTH SEA ...... 53 APPENDIX 2: LIST OF FACTORS TESTED IN THE REGRESSION ANALYSIS ...... 57 APPENDIX 3: PREFERENCES FOR RADIOGRAPHS PER SPECIMEN ...... 58

LIST OF ABBREVIATIONS in alphabetical order

• aK – abdominal kyphosis • AMO – Atlantic Multidecadal Oscillation • BNS – Belgian part of the North Sea • C – control fish • cK – caudal kyphosis • CPUE – catch per unit effort • CTD – conductivity-temperature-depth • DV – dorsoventral • EMODnet – European Marine Observation and Data Network • FAO – Food and Agriculture Organization of the United Nations • ICES – International Council for the Exploitation of the Sea • ILVO – Flanders Research Institute for Agriculture, Fisheries and Food • iK – intermediary kyphosis • IUCN – International Union for Conservation of Nature and Natural Resources • K – kyphosis • L – lordosis • LRM – linear regression model • MRI – magnetic resonance imaging • NAO – North Atlantic Oscillation • pv – precaudal vertebrae • RL – RtLeL – right-left-lateral • S – scoliosis • SA – fish with skeletal anomalies • SD – standard deviation • sH – standard height • sL – standard length • T3 – triiodothyronine • T4 – thyroxine • TAC – total allowable catch • TL – total length • VBA – vertebral body anomaly • VCD – vertebral column deviation • vL – length of the vertebral column • VLIZ – Flanders Marine Institute

SUMMARY

Under the supervision of the Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), regular surveys have been undertaken since 1985 in the Belgian part of the North Sea to monitor fish diseases. In the course of this, skeletal anomalies in wild common dab (Limanda limanda, L. 1758) were observed occasionally. The presence of skeletal anomalies is a well-known problem in many wild and cultured fish species worldwide. It has huge economic consequences for the aquaculture-, and, to a lesser extent, the fishery industry. Research on morphology, development and causes of skeletal anomalies in fish has been done increasingly during the last decades, although the real causes often remain a mystery. During surveys, every two months from 2016-2019, 4,785 dabs were caught within eight specific areas in the Belgian part of the North Sea. Furthermore, information on fish-related (e.g. length, weight, health) and environmental factors (including anthropogenic factors) was collected. The correlation between these factors and the presence of skeletal anomalies was tested and 20 affected fish were radiographically examined, together with 15 controls. There was no significant correlation between the tested factors and the presence of skeletal anomalies. This makes it plausible, that the development of skeletal anomalies most likely occurs during pre-metamorphic life stages. The total prevalence was 0.726 % and the most important anomaly was severe kyphosis affecting the last nine vertebrae seen in several fish and often combined with other vertebral column deviations. A light kyphosis-like deviation was seen in nearly all analyzed fish (x-ray), including the control group. This thesis has successfully shown, that severe skeletal anomalies are present in wild common dab.

Keywords: common dab (Limanda limanda), skeletal anomalies, North Sea, radiography

SAMENVATTING

Onder de leiding van het Vlaams Instituut voor Landbouw-, Visserij- en Voedingsonderzoek (ILVO) werden sinds 1985 regelmatig vissen in het Belgisch deel van de Noordzee voor het monitoren van visziekten gevangen. Daarbij werden er occasioneel skeletafwijkingen bij de schar (Limanda limanda, L. 1758) vastgesteld. De aanwezigheid van skeletafwijkingen is een goed gekend probleem bij vele wilde en gekweekte vissoorten wereldwijd. Het heeft enorme economische gevolgen voor de aquacultuur en, in mindere mate, de visserijsector. Onderzoek omtrent de morfologie, het ontstaan en de oorzaken van skeletafwijkingen bij vissen is sterk toegenomen gedurende de laatste decennia, alhoewel de ware oorzaken vaak een mysterie blijven. Tijdens regelmatige vangsessies om de twee maanden tussen 2016 en 2019 werden in totaal 4,785 scharen gevist binnen acht specifieke gebieden in het Belgisch deel van de Noordzee. Bovendien werd er informatie over vis- (vb. lengte, gewicht, gezondheid) en milieugebonden factoren verzameld. De correlatie tussen deze factoren en de aanwezigheid van skeletafwijkingen werd getest en 20 afwijkende vissen werden radiografisch onderzocht, samen met 15 controlevissen. Er was geen significante correlatie aanwezig tussen de geteste factoren en de aanwezigheid van skeletafwijkingen. Dit maakt het waarschijnlijk, dat de ontwikkeling van skeletafwijkingen tijdens levensfasen vóór de metamorfose zou gebeuren. De totale prevalentie bedroeg 0,726 % en de meest belangrijke afwijking was ernstige kyfose ter hoogte van de laatste negen wervellichamen, dat bij verschillende vissen te zien was, vaak in combinatie met andere deviaties van de wervelkolom. Een lichte kyfose-achtige deviatie werd bij bijna alle geanalyseerde vissen gezien (RX), inclusief de controlegroep. Deze thesis heeft succesvol aangetoond, dat erge skeletafwijkingen aanwezig zijn bij wilde scharen.

Sleutelwoorden: schar (Limanda limanda), skeletafwijkingen, Noordzee, radiografie 7

1 LITERATURE REVIEW 1.1 Teleostei 1.1.1 Introduction More than fifty percent of all known vertebrate species are fishes and every year many new species are being discovered together with new information concerning morphology, physiology and evolution (Nelson et al., 2016). Teleost fishes form one subdivision within the class of bony fishes (Osteichthyes) representing 96 % of all living fishes (Webb et al., 1981; Helfman et al., 2009). The number of species and their variety exceeds that of any other group of vertebrates with more than 29,500 known species (63 orders, 469 families, 4,610 genera) (Fig. 1). They inhabit a broad variety of aquatic habitats where they usually are the dominating ecological group. The diversity in environmental factors evolutionary led to the existence of a broad variety of habitat-adapted fishes (Webb et al., 1981; Wootton, 1990; Nelson et al., 2016; Betancur et al., 2017).

Many marine teleost fishes, like Atlantic salmon (Salmo salar), yellowfin tuna (Thunnus albacares) or the flatfishes halibut (Hippoglossus hippoglossus) and plaice (Pleuronectes platessa), play an important role for the human diet in many regions of the world. In 2018 4.5 million tons of fish (product weight) were landed in the European Union and 1,367,820 tons were produced in European aquaculture in 2017, worth € 7.3 billion and € 4.4 billion, respectively (Eurostat (European Comission), 2018).

Figure 1. Simplified cladistics of the subdivision Teleostei within the kingdom Animalia and its link with the infraclass Tetrapoda. Based on the phylogenetic relationships described in Nelson et al., 2016.

The latest common ancestor of fish and mammals (together with amphibians, reptiles, and birds) lived approximately 360 million years ago, in the late Devonian. Ever since, the groups have evolutionary developed independently, resulting in major morphological and physiological differences, as they adapted to aquatic or terrestrial life, respectively (Wittbrodt et al., 1998; Finn et al., 2014). 1.1.2 General morphological and physiological differences with mammals The most important characteristic all mammals share is the presence of mammary glands to milk feed their offspring and this also gave this group of animals its name. Apart from three species, they are all viviparous. In contrast, Teleostei can either be viviparous, ovoviviparous or oviparous, but the latter form is most present (Kunz, 2004a; Densmore, 2019). Unlike mammals, the organogenesis exceeds the embryological stage in most fish larvae (Haga et al., 2011). The approximately 4,600 mammal species also have a neocortex as part of their cerebrum and a single-bone lower jaw (Kemp, 2005). Compared to that, fish brains are not only smaller in relation to their body size, they also lack the presence of a neocortex and their telencephalon is much more rudimental (Densmore, 2019). These are only a few

general differences between higher teleost fish and mammals. Given the topic of this thesis, the following paragraphs will focus on fish osteology and its differences with mammals. 1.1.3 The fish skeleton and its differences with mammalian bone

Figure 2. Teleost osteology illustrated by the example of the little tunny (Euthynnus alletteratus). From Romeo and Mansueti (1962). A, cranium, vertebral and appendicular skeleton; B, neurocranium; C, pectoral girdle; D, vertebra 23.

Like in all other vertebrates, the Teleostean skeleton (Fig. 2A) is an organ taking part in many physiological processes as well as providing physical protection to the cranial and abdominal organs and serving as an axis for locomotion. By that, it enables fish to actively catch prey and to feed. It also plays a role in the mineral metabolism (hormone-production), in the perception and production of sounds and serves as a storage place for lipids (Witten and Hall, 2015; Rolvien et al., 2016).

Unlike mammals, fish skeletons consist of two separate entities: the endoskeleton and the exoskeleton. 1.1.3.1 Endoskeleton: development and anatomy

1.1.3.1.1 Cranium The most cranial part of the axial fish skeleton is the cranium, consisting of neuro- and branchocranium, that protects the brain and most sense organs. It contains a Figure 3. Left lateral view on the neurocranium of a 17.9 mm long gilthead seabream (Sparus aurata). much higher amount of skull bones compared to Modified based on Faustina and Power (2001). Small mammals. While the human skull holds 28 bones, for letters indicate dermal bones, capital letters cartilage example, some actinopterygian species have skulls built bone. green, ethmoid region; orange, orbital region; blue, otic region; red, basicranial region; Bas, up out of more than 150 bones, although there is an basisphenoid; Bo, basioccipital; E, ethmoid; Eo, evolutionary trend towards fusion and thus reduction of exoccipital; Ep, epiotic; f, frontal; Ic, intercalar; LE, that number (Helfman et al., 2009; Witten and lateral ethmoid; n, nasal; p, parietal; pa, parasphenoid; Pro, prootic; Pto, pterotic; Pts, Huysseune, 2009). The neurocranium (Figs. 2B, 3) can be pterosphenoid; Sp, sphenotic; So, supraoccipital; v, subdivided, following its embryonical origin, into the vomer.

chondrocranium and the dermatocranium. The first one is derived from cartilaginous capsules formed around the embryological sense organs, before being ossified (endochondral ossification), whereas the latter consists of dermal bone. This latter type of bone develops through intramembranous ossification where cartilage structures develop only secondarily (Helfman et al., 2009; Boglione et al., 2013a). Otoliths in the inner ear Figure 4. Left lateral view on the branchocranium can be subdivided into larger sagitta and smaller lapillus with sclerotic and infraorbital bones (neurocranium) and asteriscus. They contain calcium carbonate crystals of a 17.9 mm long gilthead seabream. Modified and gelatinous matter and can be used to estimate the based on Faustino and Power (2001). Small letters indicate dermal bones, capital letters cartilage bone. age of fish (Knorr, 1975; Lythgoe and Lythgoe, 1976). The green, mandibular arch; red, palatine arch and brancho- or visceral cranium, on the other hand, is suspensorium; yellow, neurocranial part; blue, derived from splanchnic mesoderm and consists of opercular apparatus; a, angular; d, dentary; ec, bones that originally formed to support the gill arches. It ectopterygoid; en, entopterygoid; H, hyomandibula; i, interopercle; io2+6, infraorbitals; la, lachrymal; Mp, can be divided into five parts, four of which are shown metapterygoid; mx, maxilla; o, opercle; P, palatine; on Figure 4. The fifth part is the branchial complex, that pmx, premaxilla; po, preopercle; Q, quadrate; Sc, is located ventrally and contains four pairs of gill arches sclerotic; so, subopercle; Sy, symplectic. with rakers and pharyngeal tooth plates (Helfman et al., 2009).(Romeo and Mansueti, 1962; Faustino and Power, 2001)

1.1.3.1.2 Vertebral column The notochord not only induces the development of the neural chord during gastrulation (Kunz, 2004b), it is also the main support axis for embryonic and early post-hatching stages of fish. It serves as a stiff structure where muscles can attach to, to allow the fish’s first movements. During larval development, the notochord stiffens by secreting collagens (type II) and some of its cells differentiate into notochordoblasts that create a sheath of collagen type II around the notochord (Boglione et al., 2013a). Contrary to mammals, the direct mineralization of that sheath, without the presence of cartilaginous precursors (intramembranous ossification), leads to the development of vertebral bodies (Witten and Huysseune, 2009). Independently from the vertebral bodies, neural and haemal arches develop based on cartilaginous blocks called arcualia (endochondral ossification). Each vertebra is formed by the fusion of four cartilaginous pieces: basidorsal, interdorsal, basiventral, interventral arcualia (Helfman et al., 2009; To et al., 2015; Witten and Hall, 2015). During further allometric growth, the vertebral bodies grow by peripheral and terminal bone apposition, primarily without the necessity of bone resorption. Remodeling, including resorption of cartilage and involving the presence of osteoclasts, is, in fact, necessary for the growth of neural and haemal arches (Witten and Huysseune, 2009; To et al., 2015).

The whole vertebral column can be divided into three parts: an abdominal region dorsal to the coelomic cavity, a caudal region and a caudal complex that runs into the tail. The vertebrae of the abdominal region are called precaudal vertebrae (pv) and have small ventral processes called parapophyses, to which ribs are attached (not on pv1 and pv2). Caudal vertebrae, on the other hand, have no ribs, but elongated haemal spines (Fig. 2D). Both types of vertebrae consist of a vertebral body (or vertebral centrum), haemal and neural arches (protecting the dorsal aorta/caudal artery and the spinal cord, respectively) and neural spines (Helfman et al., 2009). Surrounding the vertebral column and divided into myomeres, there are epaxial, hypaxial and lateral red muscles that are further separated by vertical and horizontal myosepta (Densmore, 2019).

1.1.3.1.3 Tail The most caudal vertebral bodies of higher Teleostei species fuse to become the urostyle, and their neural and haemal arches regress or become bone plates called epurals, parhypural and hypurals that support the fin rays (lepidotrichia) of the caudal fin. There are usually five hypurals, but fusion leads to a great diversity among fishes. More cranially, it is possible that pre-ural vertebrae fuse as well, but that is very species-dependent. These developments are responsible for the externally symmetrical, though Figure 5. Alcian Blue and Alizarin Red cleared and stained skeletons (a1, internally asymmetrical appearance of b1) and schematic representations of caudal fin organization (a2, b2) in zebrafish (Danio rerio; 28 mm; a) and spotted gar (Lepisosteus oculatus, the caudal fin in teleosts like goldfish 493 mm; b) as examples for homo- (a) and heterocercal (b) tail types. (Carassius auratus), for instance, called Modified based on Desvignes et al. (2018). a1/b1: arrowhead, hypural homocercal tail (Fig. 5a) (Witten and Hall, 1; arrow: hypural diastema; af, anal fin; df, dorsal fin; cfr, caudal fin rays; dorsal fin; ecfr, epichordal caudal fin rays; ep, epurals; fub, basal fulcra; 2015; Desvignes et al., 2018). The most hd, hypural diastema; hs, haemal spines; oc, opisthural cartilage; na, significant difference between a neural arches; ns, neural spine; phy, parhypural; pcta, anterior plate of homocercal tail and a heterocercal tail, as connective tissue; pctp, posterior plate of connective tissue; un, seen in sturgeons (Acipenseridae) for uroneural; una, ural neural arch; ust, urostyle. a2/b2: light grey, notochord; dark grey, haemal elements; black, first hypural; green, example, is that the notochord does plates of connective tissue; black arrow, hypural diastema; blue, caudal hardly pass the most caudal skeletal vasculature; red, caudal lepidotrichia. element in the first case whereas it runs dorsally within the tail in the latter case (Fig. 5b) (Desvignes et al., 2018).

1.1.3.1.4 Girdles and fins Next to the vertebral column, and unlike the situation in mammals, the skull is also connected to the pectoral girdle. The epioccipital and intercalar bones (Fig. 3) articulate with the posttemporal bone. Dorsal to this bone, there is/are supratemporal bone(s) that partly carry the lateral line, whereas the actual connection with the pectoral girdle is formed by the supracleithrum ventrally. The girdle consists of the dorsoventrally located cleithrum, the scapula (connecting the cleithrum with the radials) and the long coracoid caudally (Fig. 2C). The pectoral fin rays are connected to the girdle by actinosts and small distal radials. In higher teleosts, the pelvic girdles only contain one bone on each side, the basipterygia, on which the fin rays of the pelvic fins are attached (Helfman et al., 2009).

The unpaired, median fins of teleosts, anal and dorsal fins, are supported by elastin-containing ceratotrichia during larval life. These are then replaced by scale-derived pterygiophores, that support the fin rays pairwise. In some basal teleosts, there are even three pterygiophores for every fin ray, while in many higher advanced fishes, there is a one-on-one configuration in which the pterygiophores are called interneural (dorsal fin) or interhaemal bones (anal fin). In some species, the dorsal fin can be separated into two fins or one large fin plus additional smaller dorsal finlets caudal to the first. The same configuration of one big fin and one or few smaller finlets can be seen for the anal fin. Different orders of teleost fishes, like Salmoniformes (trouts and salmons) and Siluriformes (catfishes) for example, also have an adipose fin, caudal to the dorsal fin (Helfman et al., 2009).

1.1.3.2 Exoskeleton

In addition to an endoskeleton, that can also be found in mammals, fish have an exoskeleton in the form of scales, teeth, and fin rays. Its elements are derived from the ectoderm and its underlying mesenchyme and are formed, like the dermal bones of the cranium, by intramembranous ossification (Sire and Huysseune, 2003). 1.1.3.3 Fish bone classification

Compared to mammals where skeletal tissues can be either bone or cartilage and furthermore be split into categories, in fish there are several types of bone, cartilage and intermediates, but also intermediate forms between bone and connective tissue, for example (Boglione et al., 2013a). Chondroid bone is one example for these intermediate forms: it consists of chondrocyte-like cells, but its surrounding matrix is very similar to bone matrix (Witten and Huysseune, 2009). 1.1.3.4 Macroscopic characteristics

Another important general difference between the osteology of bony fish and mammals is the content of the bones. In mammals, bone marrow is the main source of blood cells as it contains hematopoietic tissue whereas in teleost it is filled with adipose and some vascular and connective tissue (Boglione et al., 2013a). The hematopoietic tissue of fish is situated in the head kidney, an organ that is only seen in teleost fish. It is comparable to the mammalian adrenal gland as it also contains Figure 6. 3-D structure of the operculum of blue interrenal cells that produce cortisol, as well as tilapia (Oreochromis aureus) obtained through the catecholamine-producing chromaffin cells, but is not serial surface view technique of focused ion beam organized into a cortex and a medulla. Moreover, it is also scanning electron microscopic images by combining stacks of slices in silico. White arrows, a preferential organ for thyroid follicles making it a thyroid- lamellar layers. From Atkins et al. (2015). Scale hormones-producing organ and thereby having an bar: 1 µm. influence on, among others, the immune system (Geven and Klaren, 2017). 1.1.3.5 Microscopic characteristics

Osteocytes are not present in the bones of higher Teleostei and their bone tissue is built up out of complex lamellar structures. It can thus be described as acellular / anosteocytic lamellated bone. The lamellae are separated by orthogonally orientated, dense arrays of collagen bundles. In comparison with their surrounding matrix, these seem to be hypomineralized (Fig. 6). This organization of fish bone makes it more compliant and Figure 7. Bone tissue and osteoclasts of teleosts: lower jaw of a juvenile Nile tilapia (Oreochromis much tougher than mammalian bone (Horton and niloticus); glycol methacrylate embedding, TRAP Summers, 2009; Atkins et al., 2015). Unlike mammals, most staining and Hematoxylin; asterisks, acellular Teleost’s osteoclasts are mononucleated, small and do not bone; red, mononucleated osteoclasts connected by long cell processes. Modified based on Witten leave behind lacunae after bone resorption (Fig. 7) (Witten and Huysseune (2009). Scale bar: 15 µm. and Hall, 2015).

1.1.3.6 Growth

Bony fish continue growing during their entire life and so does bone remodeling by resorption and de novo formation, but also by transdifferentiation (see below). Furthermore, cartilage can be formed by subdivision of existing cartilage through dedifferentiation of chondrocytes into fibroblasts during the development of the pectoral, dorsal and anal fins (Witten and Huysseune, 2009; Boglione et al., 2013a). Although the growth rate generally decreases with increasing age, growth can be completely intermitted in adult fish under suboptimal circumstances (Witten and Huysseune, 2009). 1.1.3.7 Mineral metabolism

While the mammalian bone metabolism is primarily calcium based and the skeleton is an active part of the calcium homeostasis, this is not the case in teleost fish. Calcium from their skeleton is only mobilized when there is an extreme deficiency and, in that case, primarily from the postcranial exoskeleton (scales). For their calcium homeostasis they obtain calcium present in the surrounding water by intestinal resorption in marine species or, in freshwater fish, by active transport through their gills. Also, their mineral metabolism is driven by phosphorous rather than calcium (Witten and Huysseune, 2009; Witten and Hall, 2015). 1.1.3.8 Bone dynamics

Like in other vertebrates, bones in teleost fish can adapt to changing mechanical loads. This happens already in early life stages and thereby, the mechanical loads have an influence on skeletal development. For example, the time of ossification of the tail skeleton seems to be dependent on activity, just like the formation of other cartilaginous and bony structures. Furthermore, other factors such as vitamin and fatty acid intake or even the development of the gut flora, can influence the rate of skeletal development (Witten and Hall, 2015). Unlike mammals, fish larvae have to feed themselves once their yolk sacs are completely utilized and skeletogenesis is still ongoing (Haga et al., 2011).

Transdifferentiation from one cell type into another is not only a reaction to changing mechanical loads, but also part of the normal development of skeletal elements in many teleost species. Given a compressive mechanical environment, osteogenic cells of the vertebral growth zone have been described to pathologically transdifferentiate into chondrogenic cells in several species. The same can happen with chordoblasts and intervertebral ligament cells, combined with or without cell division. Other types of transdifferentiation have also been described for example from connective tissues and muscles into bone (Boglione et al., 2013a). The fish skeleton is so dynamic that it can even adapt to severe anomalies, like the fusion of vertebral bodies resulting in one normal shaped and structured vertebral body. It can also adapt to changed mechanical forces due to lordosis (L), for example, by changing the bone architecture and increase bone formation (Witten and Hall, 2015).

With hardly any osteocytes in their bones, advanced teleost species must rely on other signaling mechanisms to detect changing mechanical loads, compared to mammals where these cells sense stress with their cell processes (Witten and Huysseune, 2009). One hypothesis states that this task is

fulfilled by osteoblasts and bone lining cells on the surface of the bones in advanced bony fish (Witten and Hall, 2015). Another consequence of anosteocytic bone is that fish have no cells within the bone matrix that can react to, for instance, micro- / fractures, resulting in the recruitment of periosteal osteocytes for bone modelling and remodeling. Fracture repair is principally possible, in contrast to sharks for example (Boglione et al., 2013a). However, the results can be much poorer in fish, compared to mammals, especially under calcium deficient Figure 8. Radiographic image of a segment of the caudal vertebral circumstances (Horton and Summers, 2009). column (vertebral bodies 13-20) of a red porgy (Pagrus pagrus; In general, elements of the dermal skeleton 41.7 cm sL). From Witten and Hall (2015). White arrowheads show beginning hyperostosis at haemal arches and haemal spines. tend to have a higher regenerative capacity than the endoskeleton (Witten and Huysseune, 2009; Boglione et al., 2013a).

Hyperostosis (Fig. 8) is a non-pathological process exclusively seen in teleost fish, where excess bone is formed on skull and spine bones of old individuals (Witten and Hall, 2015; Soto et al., 2019). Unlike most bone tissue of the endo- and especially exoskeleton, this bone does contain osteocytes and multinucleated osteoclasts. Witten and Huysseune (2009) also considered, that the presence of osteocytes has a direct influence on the presence of large, multinucleated osteoclasts instead of mononucleated osteoclasts, as seen in acellular bone. 1.2 Skeletal anomalies in fish 1.2.1 Historical overview Unlike hyperostosis, many pathological skeletal anomalies can be seen regularly in fish. Deformations of the jaw, the opercular apparatus and the vertebral column appear in many fish species worldwide. Studies on skeletal anomalies in farmed fish date back to the 1970s when the first aquaculture species have been studied (Boglione et al., 2013a). Since then, skeletal anomalies have been described in many farmed species, like common carp (Cyprinus carpio; Wunder, 1981), rainbow trout (Oncorhynchus mykiss; Aulstad and Kittelsen, 1971; Gislason et al., 2010), Atlantic salmon (McKay and Gjerde, 1986; Witten et al., 2005), and gilthead seabream (Sparus aurata; Andrades et al., 1996; Afonso et al., 2000), as well as in the flatfishes Atlantic halibut (Lewis et al., 2004; Lewis and Lall, 2006), Japanese flounder (Paralichthys olivaceus; Haga et al., 2011), turbot (Scophthalmus maximus; Tong et al., 2012) and Senegalese sole (Solea senegalensis; Gavaia et al., 2002; de Azevedo et al., 2017a, 2017b) for example. In wild fish, fewer studies were carried out focusing on skeletal anomalies (e.g. haddock (Melanogrammus aeglifinus; Jawad et al., 2018) and Chinook salmon (Oncorhynchus tshawytscha; Munday et al., 2016, 2018)). 1.2.2 Wild versus aquaculture Nevertheless, several studies during the last years have studied common cultured species in the wild to compare the prevalence of skeletal anomalies. In general, this prevalence is lower in wild fish and the anomalies are often less severe (Castro et al., 2008; Gavaia et al., 2009; Boglione et al., 2013a). In aquaculture up to 100 % of fish can be affected by skeletal anomalies, with interspecies and ontogenic

differences (Gavaia et al., 2002, 2009; Georgakopoulou et al., 2010; Gislason et al., 2010; de Azevedo et al., 2017b), whereas in the wild, the highest reported prevalence is 33 % in haddock and 19 % in Senegalese sole larvae, but often lower (2 - 6 %) (Gavaia et al., 2009; Hansen et al., 2010; Jawad et al., 2018). 1.2.3 Importance of studying skeletal anomalies in fish Especially for the growing aquaculture-industry, the presence of skeletal anomalies can be a huge problem as it decreases the economic value of the fish with an annual loss of more than €50,000,000 for the European market (Afonso et al., 2000; Boglione et al., 2013b; de Azevedo et al., 2019). Deformed fish can only be used to produce filets or fish meal but never be sold in one piece. This also implies that manual work is necessary, not only to sort out deformed fish after the hatchery stage, but also because modern machinery can only process normal shaped fish (Boglione et al., 2013b). The affected fish have a decreased food efficiency, delayed growth and they are more susceptible to diseases, infections, and stress. Furthermore skeletal anomalies affect the morphology of the animal and possibly its welfare, although the presence of pain or discomfort has not yet been proven (Andrades et al., 1996; Gavaia et al., 2009; Hansen et al., 2010; de Azevedo et al., 2017b, 2019). 1.2.4 Diagnostic techniques Live fish are routinely checked for skeletal anomalies by visual examination and palpation, but radiographs are becoming more popular at hatchery farms to sort out deformed fish as soon as possible, because the sensitivity of classical methods has been proven to be low (Witten et al., 2009; Boglione et al., 2013b; Losada et al., 2014; de Azevedo et al., 2017a).

To visualize skeletal anomalies in larvae and small Figure 9. Lordosis (left arrow) and kyphosis (right juveniles, a whole mount double-staining technique arrow) in a 30 days old Senegalese sole (Solea senegalensis) larvae. From Pimentel et al. (2014). (Fig. 9) with Alcian blue for cartilage and Alizarin red for bone, combined with potassium hydroxide and hydrogen peroxide treatment for a higher transparency of the surrounding tissues, has been widely used in studies (Gavaia et al., 2002; Losada et al., 2014; de Azevedo et al., 2017a). Contrary to the classical methods mentioned above, fish have to be euthanized in advance to stain them. Other researchers additionally have used more advanced methods on euthanized fish, like synchrotron microcomputer tomography and computer tomography scans, histology and histopathology, or histochemistry and immunohistochemistry (Witten et al., 2009; Boglione et al., 2013b). 1.2.5 Terminology Although there have been many studies on skeletal anomalies in fish, they often lack a common terminology which makes comparison between species and different studies very difficult as numerous terms describe the same anatomical anomaly, or the same term is used for different anomalies (Boglione et al., 2013b). Given the diversity and the possible combinations of anomalies within one specimen, several authors have found more than 35 different skeletal anomalies (Afonso et al., 2000; de Azevedo et al., 2017a).

1.2.6 Types of skeletal anomalies 1.2.6.1 Jaw deformities

Jaw deformities have been described in many species, for example in Atlantic salmon where an ankylosis of the mandibular articulation fixates the lower jaw in an open mouth position (“screamer disease”) (Afonso et al., 2000; Lumsden, 2019). 1.2.6.2 Operculum abnormalities

A congenital or acquired loss of bone and/or epithelial tissue can result in operculum deformities leading to a reduced respiratory efficiency (Lumsden, 2019). In gilthead seabream an inside-folding process of the operculum and sub-operculum has been described by Georgakopoulou et al. (2010). 1.2.6.3 Vertebral anomalies

The most occurring and most studied skeletal anomalies in fish are vertebral anomalies. To standardize the terminology of these, Witten et al. (2009) proposed a classification system based on radiographic images of Atlantic salmons, that can be adapted for many species (Table 1).

Table 1. Type definitions of a classification system for vertebral anomalies proposed by Witten et al. (2009).

type description type description

1 decreased intervertebral space 11 pronounced biconcave

2 homogenous compression 12 hyper-radiodense

3 1 + 2 13 hyper-radiodense with flat end plates

4 compression without X-structure 14 lordosis

5 one-sided compression 15 kyphosis

6 compression and fusion 16 scoliosis

7 complete fusion 17 vertically shifted

8 fusion center 18 irregular internal structures

9 elongation 19 internal dorsal or ventral shift

10 widely spaced and undersized vertebrae 20 severe multiple malformations

The prevalence of vertebral anomalies differs between species, environment (wild or farmed) and developmental stages. The lowest prevalence was found in adult, wild Atlantic cod (Gadus morhua) (2 - 6 %) and the highest in hatchery reared Senegalese sole juveniles (75 %) (de Azevedo et al., 2017a; Jawad et al., 2018). Contrary to this diversity in prevalence, many studies found the caudal region and the caudal complex to be a predilection site for vertebral anomalies (Chatain, 1994; Lewis et al., 2004; Hansen et al., 2010; Haga et al., 2011; Losada et al., 2014; de Azevedo et al., 2017b; Jawad et al., 2018). Vertebral anomalies can be divided into two main categories: vertebral body anomalies (VBAs; types 1-13; 17-20) and vertebral column deviations (VCDs; types 14-16) (de Azevedo et al., 2017a).

1.2.6.3.1 Vertebral body anomalies The most common VBAs are compressions and fusions of vertebrae. In Atlantic salmon alterations within the vertebral growth zone lead to the compression of vertebrae throughout the whole vertebral column (Fig. 10). Thereby the fish are shorter and have an increased standard height (sH), a morphological appearance known as “short tail” (Witten et al., 2005). Compression can lead to complete fusion of vertebrae (Fig. 11) (Witten and Hall, 2015). These vertebral fusions have Figure 10. Compressed vertebrae in a ‘short also been described in many species and do not have to be tail’ Atlantic Salmon (Salmo salar). From Witten et al. (2005). pathological, like the fusion of the first one or two vertebrae with the basioccipital bone that is considered to be non- pathological in Osteichthyes (de Azevedo et al., 2017b). Furthermore the fusion of two vertebral bodies can result in one normal sized and shaped vertebral body that can only be recognized by the presence of two neural and haemal spines (Witten et al., 2006).

1.2.6.3.2 Vertebral column deviations In cultured fish, especially L, but also kyphosis (K) and scoliosis Figure 11. Fusion of two vertebral bodies in (S) (VCDs) are the most frequently seen skeletal anomalies Atlantic Salmon (Salmo salar). Modified (Chatain, 1994; Kranenbarg et al., 2005; Haga et al., 2011). based on Witten et al. (2006). L describes a ventral deviation of the vertebral column, K a dorsal deviation (Fig. 9) and S a single or multiple lateral deviation (Fig. 12), all resulting in an abnormal, non-linear shape of the vertebral column (Kranenbarg et al., 2005).

Figure 12. Dorsoventral radiographic image of a juvenile Senegalese sole with scoliosis. Modified based on de Azevedo et al. (2017a). Like for vertebral anomalies in general, L and S are most present in the caudal region (Andrades et al., 1996; Tong et al., 2012; de Azevedo et al., 2017a, 2017b). Combinations of multiple VCDs and / or VBAs are common and can involve large parts of the vertebral column (Afonso et al., 2000; de Azevedo et al., 2017a). For example, “saddleback syndrome” describes a combination of L and K in Figure 13. Lateral radiographic image of a gilthead seabream affected seabass (Dicentrarchus labrax) by the LKS-syndrome. From Afonso et al. (2000). (Kranenbarg et al., 2005). Afonso et al. (2000) found a specific combination of repeating L, K and S (Fig. 13) that could be linked to a certain family in reared gilthead seabream, but, to a lesser extent, could also be seen in non-related fish (0.2 % versus 6.5 %). In Senegalese sole the first three to four vertebrae form an arch to the basioccipital bone that is not pathological (de Azevedo et al., 2017a). VBAs and

VCDs can also imply secondary anomalies like fused or bent spines, fused ribs and hypercalcification (Andrades et al., 1996). 1.2.7 Tissues and cells On a cellular level the development of skeletal anomalies always requires bone resorption and remodeling, either as the primary cause or in response to a changed mechanical load. In VBAs there is metaplasia of bone forming cells in vertebral endplates and the intervertebral space into chondrocytes before the produced cartilage is mineralized (Witten and Huysseune, 2009; Boglione et al., 2013a; de Azevedo et al., 2019). Associated with VCDs, Munday et al. (2016) have found a higher degree of fibrosis within the connective and muscle tissues connected to the vertebral column in affected Chinook salmons compared to non-affected. This perivertebral fibrosis occurs unilateral in most cases and its extend is significantly correlated with the severity of the VCD. 1.2.8 Causes Although a lot of research on skeletal anomalies has been done, the causative factors often still are unknown. Taking into consideration that one specific anomaly can have different etiologies in different or even within the same species and that combinations of genetical and environmental factors coexist, many possible causative factors have been brought forward (Castro et al., 2008; Lumsden, 2019). 1.2.8.1 Genetics

Some species have been shown to be more susceptible to the development of skeletal anomalies than others, both in natural and aquaculture environments (de Azevedo et al., 2019). As mentioned earlier, the prevalence is higher in cultured fish which could be a result of inbreeding as fish from a certain family often show a higher prevalence than unrelated individuals (Afonso et al., 2000; de Azevedo et al., 2019). Furthermore, higher growth rates have been linked to the development of such anomalies as well as triploidy (Witten et al., 2005; Fraser et al., 2013; Munday et al., 2018). Although earlier researchers have found a low heritability for L in gilthead seabream, Negrín-Báez et al. (2016) could identify four quantitative trait loci associated to the LKS-complex mentioned above (Fig. 13). 1.2.8.2 Anthropogenic factors

Anthropogenic factors like vaccination, trauma to eggs and larvae by manipulation in hatcheries or electric shocks have also been reported to cause skeletal anomalies (McKay and Gjerde, 1986; Castro et al., 2008). Additionally, humans have an important influence on other environmental factors in many cases. In nature, the changing temperature and acidification of the ocean have been proven to increase the prevalence of skeletal anomalies (Pimentel et al., 2014), whereas on farms, the influence of temperature also plays a significant role (Georgakopoulou et al., 2010). Environmental contamination is responsible for the high prevalence of skeletal anomalies in polluted areas (Gavaia et al., 2009; Jawad et al., 2018). Pollutants can form compounds with the sediments on the seabed and accumulate in polychaetae worms leading to a high exposure to flatfish and other benthic life (Kerambrun et al., 2012; Jawad et al., 2018). 1.2.8.3 Swim bladder non-inflation

Associations of skeletal anomalies with a non-inflating swim bladder have been found in several species like European seabass, gilthead seabream (Chatain, 1994) and red seabream (Pagrus major) (Kitajima et al., 1981). Without a functional swim bladder, hatched larvae have a negative buoyancy and must use the pectoral fins to remain and move inside the pelagic zone. This can lead to an overuse of the pectoral fin muscles and result in vertebral anomalies as the mechanical load is increased 18

(Boglione et al., 2013b). On the other hand, Andrades et al. (1996) have reported that L is already present in gilthead seabream larvae before the time inflation normally would occur. Furthermore, although the L stays present, surviving adults were observed to have a functional swim bladder in this study. A similar overuse of perivertebral muscles can also be caused by high water currents, with the same results, although temperature also seems to play an important role (Georgakopoulou et al., 2010; de Azevedo et al., 2017b). 1.2.8.4 Nutritional factors

Early stages of cultured Atlantic cod fed with a natural diet have been found to develop skeletal anomalies less often than fish on the commercial diet usually used (Gavaia et al., 2009). This shows the importance of nutritional factors for the ontogeny of fish and many deficiencies and excesses have been associated with the development of skeletal anomalies. Vitamin A is essential for embryological development (Haga et al., 2011) and deficiency can reduce collagen synthesis and bone formation resulting in bone loss and the development of S (Lumsden, 2019). An excess of vitamin A, on the other hand, can also lead to S and other anomalies due to precocious mineralization and has further been connected to the loss of the caudal fin and supernumerary caudal fin rays in Japanese flounder as well as loss of the pelvic fin in European seabass (Gavaia et al., 2002; Haga et al., 2011; Lumsden, 2019). Vitamin C is required for the hydroxylation and maturation of collagen, normal osteoblast function and bone mineralization. A deficiency can upset the collagen metabolism and cause VCDs with broken vertebrae (“broken back disease”), whereas an excess can lead to operculum deformities (Gavaia et al., 2002; Lumsden, 2019). An excess of vitamin D and a deficiency of tryptophan can both cause a mineral imbalance, possibly leading to bone resorption (Chatain, 1994). Phosphorous is sparse in water, and a low dietary intake or anorexia can lead to a deficiency, resulting in resorption and softening of bones and thereby deformities of the skull, the vertebral column, and vertebral spines (Witten and Huysseune, 2009; Lumsden, 2019). 1.2.8.5 Infection and inflammation

Bacterial infections with Flexibacter psychrophilus can cause destruction of axial muscles and thereby lead to L and K (Cipriano and Holt, 2005), whereas parasitic infections with Myxobolus cerebralis have also been reported as a possible cause of bone resorption and thus skeletal anomalies due to parasitic replication within the cartilage (Baldwin et al., 2000; Witten and Huysseune, 2009). The perivertebral fibrosis in Chinook salmon, found by Munday et al. (2016), is associated with inflammation of the original tissues, although it is not clear whether this inflammation is primary, or develops secondarily in response to changed mechanical loads. 1.2.8.6 Age

Some vertebral fusions and comparable anomalies can occur later in life, but most skeletal anomalies begin to develop during early larval stages when osteo- and chondrogenesis take place (Boglione et al., 2013b). In many species, like Atlantic halibut, Senegalese sole and gilthead seabream a high percentage of the hatched larvae have skeletal anomalies (Afonso et al., 2000; Lewis and Lall, 2006; de Azevedo et al., 2017b). This percentage decreases in wild post-metamorphic fish but stays high in farmed fish and anomalies even increase in severity (Gavaia et al., 2002; de Azevedo et al., 2017a, 2017b; Munday et al., 2018). The variety of skeletal anomalies is also reported to increase with increasing length of the fish (Lewis et al., 2004; Lewis and Lall, 2006).

1.3 Teleostei: flatfishes Although they share the general osteology of teleost fish (Fig. 14), described in the first section, only few researchers have studied skeletal anomalies in flatfish species (order Pleuronectiformes) – and even less studies were done on wild flatfish.

Pleuronectiformes are the most asymmetric group of vertebrates on earth (Schreiber, 2013; Betancur- R and Ortí, 2014). During their post-embryological development, these fish develop from a pelagic, laterally symmetrical, and fusiform larva to a demersal, laterally compressed juvenile. Thereby they change their posture by 90 degrees and lay on what used to be a lateral side.

Figure 14. RtLeL radiographic image of a common dab (16.35 cm sL; 40kV / 6.3 mAs) to illustrate the osteology of flatfishes. A, abdominal region; aF, anal fin; aFr, anal fin ray; C, caudal region; CC, caudal complex; cF, caudal fin; cFr, caudal fin ray; coe, coelomic cavity; cv6, sixth caudal vertebra; dF, dorsal fin; dFr, dorsal fin ray; dp, distal pterygiophore; ep, epural; ha, haemal arch; hp1+2/hp3+4, fused hypurals; hph, haemapophysis; hs, haemal spine; lE, left eye; N, neurocranium; na, neural arch; ns, neural spine; p, parapophysis; PF, pectoral fin; pF, pelvic fin; Pg, pectoral girdle; pg, pelvic girdle; ph, parhypural; pp, proximal pterygiophore; pu, pre-ural vertebra; pv6, sixth pre-caudal vertebra; r, ribs; rE, right eye; S, sagitta; sH, standard height; sL, standard length; TL, total length; u, urostyle; scale bar: 10 mm. 1.3.1 Systematics The three defining features of this monophyletic order are (1) eye migration during ontogeny, resulting in cranial asymmetry, (2) a dorsal fin that extends far rostral over the head and (3) the presence of protrusible eyes (Schreiber, 2013; Betancur-R and Ortí, 2014). The order contains more than 800 extant species divided into 14 families (Fig. 15), making it the third most diverse order of marine teleosts (Munroe, 2015a; Nelson et al., 2016). Although flatfishes are widespread around our globe and appear worldwide, the species living in temperate climate zones of the Northern and Southern hemisphere have been studied far better than their relatives in the tropics and deep sea. There is reason to believe that many species, especially small ones, that are of no importance for fisheries, have not yet been identified (Munroe, 2015a). 1.3.2 History of flatfish research Flatfishes’ unique metamorphosis, as well as the ability to change their color-pattern according to their surroundings are unique characteristics that have fascinated scientists for many decades (Kyle, 1923; Geffen et al., 2007; Schreiber, 2013; Gibson, 2015; Wirtz and Davenport, 2017).

Apart from scientists, flatfish are known to a broader public as being an important part of the human diet in many areas of the world. In the European Union, for instance, flatfish are economically important as landings account for 8.7 % of the total fishery value, although their total weight is only 3.4 %1. The history of flatfish consumption reaches back many hundred years as prehistoric rock carvings and archeological findings confirm (Gibson, 2015). Cunningham (1895, 1893), among others, already studied the life history and pigmentation of flatfishes in the late 19th century (Ahlstrom et al., 1984; Geffen et al., 2007). At that same time first approaches to control flatfish fisheries have been made to avoid overfishing that ultimately led to the formation of organizations such as the International Council for the Exploitation of the Sea (ICES) (Gibson, 2015). Historical and modern research, rearing fish for restocking purposes as well as aquaculture studies during the last three decades have thereby mainly focused on the most exploited species, such as common sole (Solea solea), Atlantic halibut and Japanese flounder (Hoshino and Amaoka, 1998; Lewis et al., 2004; Geffen et al., 2007; Gibson, 2015).

Figure 15. Simplified cladistics of several flatfish species mentioned in this thesis and their interspecies relationships. Continuation of Fig. 1. Based on the phylogenetic relationships described in Nelson et al., 2016. 1.3.3 Phyletic history On the other hand, only few flatfish fossils have yet been discovered (Byrne et al., 2018; Cheung and Oyinlola, 2018). This missing link between fusiform fishes and flatfish fossils has led to evolutionary debates in the past. †Heteronectes chaneti and †Amphistium paradoxum are two fossil spiny-rayed fish species (Eocene epoch, 53 million years ago) that are described as the most primitive flatfishes ever found. They share many characteristics with modern pleuronectiforms, for example an asymmetrical skull, yet their eye migration was incomplete. They are assumed of having lived benthic lives. This supports the hypothesis that eye migration evolutionary followed changing from a pelagic to a benthic lifestyle (Friedman, 2008; Schreiber, 2013; Munroe, 2015a). The most primitive extant flatfishes (genus Psettodes) still show some ancient characteristics, for example the migrating eye only moves to the dorsal crest of the body and eye-sidedness (sinistral and dextral individuals) is randomly developed among individuals of the three species in this genus (Suzuki and Tanaka, 2015; Nelson et al., 2016).

1 EUMOFA, 2020. Fishery – Landings: Time series at EU and country levels. URL http://www.eumofa.eu/fl-ts-at- eu-and-ms-levels (accessed 20.4.20). 21

1.3.4 Distribution In general flatfishes are carnivorous, demersal fishes that inhabit a broad variety of habitats throughout the world and from pole to pole. These habitats are usually shallow marine waters, but some species can be found near deep thermal vents or even in freshwater rivers. Ten species are known to fulfill their entire lifecycle in freshwater. Only in the deepest parts of the oceans, flatfishes have not yet been observed. The lowest diversity is found in the Antarctic seas whereas the highest diversity is found in subtropical and tropical waters (74.9 %), especially in the Indo-West Pacific (Ahlstrom et al., 1984; Helfman et al., 2009; Schreiber, 2013; Gibson, 2015; Munroe, 2015b). 1.3.5 Morphology Although flatfishes appear to be a morphologically unique order within the animal kingdom, lateral compression (body depth < body height) is a common hydrodynamical characteristic of many teleost fishes - unlike rays, for example, that are dorso-ventrally compressed (Heessen et al., 2015a). Contrary to juveniles and adults, the larval stages of flatfishes are similar to other members of the subdivision Teleostei in shape, size and anatomical variety (Geffen et al., 2007). 1.3.5.1 Camouflage

As mentioned above, most flatfishes can camouflage themselves by mimicking color and pattern of the sediment they rest on. Visual stimuli are thereby translated into rapid nervous and slower hormonal signals that stimulate chromatophores in the skin on the ocular side (Gibson, 2015). This process can take up to one day, which the animals spend buried in the sediment. Ultimately both burying and Figure 16. Scanning electron microscopic camouflaging are important characteristics of flatfish to avoid images of different types of scales. From predation (Spinner et al., 2016). Compared to other fish, Spinner et al. (2016). A: blind side sole; B: flatfish have a relatively small coelomic cavity that is limited ocular side sole; C: blind side E. flounder; D: ocular side E. flounder; E: blind side dab; F: to the cranial half of the body (Bayliss, 1935; Knorr, 1975). ocular side dab; G: blind side plaice; H: ocular side plaice; scale bars: 1 mm. 1.3.5.2 Blind versus ocular side

Many unique morphological characteristics in adult flatfishes can be linked to their asymmetry resulting in differences between their two (originally) lateral sides. The ocular side has a more convex shape than the blind side and is pigmented (Heessen et al., 2015a) with larger olfactory organs and telencephalon (Schreiber, 2006). Contrary to that, the blind side is usually non-pigmented and often lacks a lateral line (Ahlstrom et al., 1984). In larvae chromatoblasts of neural crest origin settle at the base of fins and one fraction of these cells differentiates into larval melanophores for larval pigmentation. Another fraction migrates to the skin of the ocular side through myosepta and differentiates into melanophores and xanthophores responsible for the adult pigmentation pattern in juveniles and adults (Suzuki and Tanaka, 2015; Vroman, 2019). 1.3.5.3 Scales and fins

Squamation does not only differ among flatfish species, from embedded cycloid scales to overlapping ctenoid scales, but also between the two sides of one individual, with more scales and less ctenial

spines on the blind side (Fig. 16; Spinner et al., 2016). Pelvic and pectoral fins are often shorter on the blind side and the latter can be differently placed compared to the ocular side. The dorsal fin extends cranially at least up to the level of the eye in all families apart from Psettodidae and the anal fin reaches the abdominal region cranially but can also extend even further (Ahlstrom et al., 1984). 1.3.6 Physiology Given their morphological differences, it may be clear that locomotion differs in flatfishes compared to other fish. The basic method of movement close to the sediment is undulating the caudal part of their bodies together with the dorsal and anal fin by contractions of axial muscles. Faster movements from the bottom to the water column are carried out by strokes of the tail combined with undulations of the body and using the pectoral fins as a rudder. These movements are usually followed by slower swim and glide behavior back to the bottom (Gibson et al., 2015). Wirtz and Davenport (2017) have described a third type of movement seen in several flatfish species: fin-crawling. Wavy movements of the dorsal and anal fin in which the axial muscles are not involved. This allows the fish to move fore- and backwards and even rotate within their own body length. 1.3.7 Development 1.3.7.1 Spawning

Avoiding competition plays a key role in flatfish development as the habitats of many species overlap. By spawning at separate times of the year and in slightly different habitats interspecies competition is reduced. Larvae of European flounder (Platichthys flesus), for example, can be found in estuaries with low salinity whereas common dab (Limanda limanda) larvae prefer a higher salinity (Barbut et al., 2019). Furthermore, intraspecies competition between different life-stages is reduced, among other factors, by different locations of spawning and nursery grounds (Duffy-Anderson et al., 2015). Nevertheless, hybrids are common between some flatfish species (Garrett, 2005). 1.3.7.2 Pelagic stages

The mostly pelagic eggs of flatfishes are round, contain homogenous yolk and oil globules in many cases (usually not in Pleuronectinae) and vary in size between 0.5 and 4.5 mm (Ahlstrom et al., 1984; Geffen et al., 2007). Compared to species with smaller eggs, larvae in species with larger eggs are more developed at the time of hatching, with pigmented eyes, a functional mouth and pectoral fins (Ahlstrom et al., 1984). Like the eggs, hatched larvae are also planktonic and are transported by currents until the beginning of their metamorphosis (Barbut et al., 2019). Unlike members of the subfamily Pleuronectinae, halibut larvae float upside down beneath the water surface during the first hours of their lives, because their yolk contains an oil globule (Al-Maghazachi and Gibson, 1984). The utilization of yolk and growth rate during the first stages of development are both positively correlated with temperature (Al-Maghazachi and Gibson, 1984; Geffen et al., 2007). The pelagic stages show a very low and variable survival rate (~0.1 %) as they passively must be transported to suitable nursery grounds with shallow water and soft sediment (Barbut et al., 2019). 1.3.7.3 Metamorphosis

The larval stage ends with the process of metamorphosis, in which larvae undergo several morphological changes and become juveniles (Fig. 17) (Geffen et al., 2007). It usually begins around the time of first feeding and flexion of the caudal notochord can be seen (Lewis and Lall, 2006). At this point the size of larvae is 10 – 25 mm. During metamorphosis, larval spines are lost, and their function is taken over by elongated rays. Gut protrusions are brought into the body cavity, the swim bladder is

lost, and the fish develop juvenile pigmentation patterns. Ossification of the vertebral column and other structures is completed, and scales are formed (Ahlstrom et al., 1984).

Furthermore one larval eye migrates to the contralateral side of the body, creating dextral (both eyes on the right side; usually in Poecilopsettidae, Pleuronectidae, Samaridae, Achiridae, Soleidae) or sinistral (both eyes on the left side; usually in Psettodidae, Citharidae, Bothidae, Achiropsettidae, Scophthalmidae, Paralichthyidae, Cynoglossidae) individuals (Ahlstrom et al., 1984; Byrne et al., 2018; Castellini et al., 2018). The development of right- or left-sidedness can differ between populations of the same species and even within one population. A higher incidence of “reversals” has been reported in aquaculture fish compared to wild specimens (Gibson, 2015; Heessen et al., 2015a; Schreiber, 2013). The eye migration also affects the laterality of the optic nerve, the olfactory organs and other internal organs that follow the same direction (Ahlstrom et al., 1984; Suzuki and Tanaka, 2015). Figure 17. Metamorphosis of flatfish larvae (plaice (Pleuronectes Eye migration begins with asymmetric platessa)). From Geffen et al., 2007. a: beginning metamorphosis; b, intermediary metamorphosis and beginning neurocranial remodeling in which ethmoidal settlement; c, end of metamorphosis and settlement. Scale bars: and frontal bones grow asymmetrically and 1 mm. twist towards the future position of the migrating eye. The cartilaginous supraorbital bars on both sides are differentially resorbed and dermal fibroblasts proliferate into a dense cell layer under the migrating eye. Deposition of osteoblasts within this layer leads to the formation of a cranial flange of frontal bone (postlateral ethmoid) that fills up the area where the eye used to be (Hoshino, 2006; Schreiber, 2013). The non-migrating eye rotates 90 degrees and, like the migrating eye, is positioned extra-orbital. This enables juveniles and adults to burrow into the sediment and still see their surroundings, especially what is in front of them but also behind and above (Schreiber, 2013; Gibson et al., 2015).

Metamorphosis is thyroid hormone driven and, like in mammals, T4 must first be metabolized to active T3 in the liver. The development of thyroid follicles as well as the biosynthesis of T4 depend on the dietary intake of vitamin A. Concentrations of thyroid hormones are highest around the peak of metamorphosis (Schreiber, 2013; Suzuki and Tanaka, 2015; Fernández et al., 2017). Furthermore, the nodal pathway, in which genes are activated that influence the laterality of organs in all vertebrates, has been found to also regulate eye-sidedness and pigmentation in flatfishes (Suzuki and Tanaka, 2015).

Metamorphosis is an energy-demanding process during which both feeding and growth are reduced in many species. Therefore, it is necessary for the larvae to have enough energy stored in the liver before the onset of metamorphosis which could be a limiting factor to start it rather than size (Geffen et al., 2007).

Al-Maghazachi and Gibson (1984) have defined five stages of development of flatfish larvae to juveniles, although these are not necessarily followed by one another:

1.) Larvae symmetrical, yolk sac present 2.) Larvae symmetrical, development of spines and swim bladder (not in all species) 3.) Appearance of fin rays, straight notochord 4.) Asymmetry and eye migration, notochord caudally bent dorsally 5.) Completion of eye migration, spines and swim bladder resorbed ➔ Completion of metamorphosis: 1) upper eye away from dorsal margin, 2) head spines completely resorbed 3) swim bladder completely resorbed (if present) 4) dorsal fin extends to above upper eye

A more detailed staging (29 stages) and description from fertilization until the completion of metamorphosis has been presented by Martinez and Bolker (2003) in summer flounder (Paralichthys dentatus). It has to be noted, that the duration and order of these events as well as the size before and after metamorphosis varies among species (Geffen et al., 2007). 1.3.7.4 Settlement

The adoption of a demersal lifestyle is called settlement and usually occurs in shallow, coastal waters (Barbut et al., 2019). In several flatfishes, juveniles continue their pelagic life after metamorphosis, and continue feeding on pelagic prey until settlement is completed. Juvenile Pacific Dover soles (Microstomus pacificus), for example, continue their pelagic lifestyle for many months before settling. Thus, metamorphosis and settlement are two independent processes (Geffen et al., 2007). 1.3.8 Exploitation Many flatfish species are commercially important to fisheries worldwide. Although the amount of landed flatfish has been declining during the last 50 years, around two million tons of flatfish have been caught annually in the last decades (Cheung and Oyinlola, 2018; Barbut et al., 2019). On the other hand, the use of flatfishes in aquaculture has increased. Japanese flounder, turbot, halibut, common sole, Senegalese sole and European flounder have become important fish in farms all over the world (Haga et al., 2011; Geffen et al., 2015; Cheung and Oyinlola, 2018). Aquaculture has provided scientists with the opportunity to study cultured species very closely whereas studies in the wild provide essential information for aquaculture (Geffen et al., 2015). 1.3.9 Threats 1.3.9.1 Climate change

Cheung and Oyinlola (2018) have shown that climate change can have negative impacts on both fisheries and aquaculture, mainly due to rising temperature of the sea water (1.1 - 6.4° C by 2100) (Pimentel et al., 2015). The dispersal of eggs, hatching and survival rates and thus recruitment success as well as whole benthic communities will change together with changing temperatures and wind altering the currents in the North Sea (Hinz et al., 2005; Pimentel et al., 2014; Lacroix et al., 2018).

Another threat for marine life is the acidification of the seas. As the concentration of carbonate dioxide in the atmosphere increases, more of it dissolves in surface waters decreasing its pH (Δ 0.3-0.4 by 2100). This results in a decreasing availability of carbonates necessary, for example, for the exoskeleton of many invertebrates. Although there are interspecies differences, fish appear to be relatively resilient to acidification (Pimentel et al., 2015; Hurst et al., 2016).

It has been shown that the combination of acidification and increased temperature can reduce metabolic rates in flatfish larvae (Pimentel et al., 2014). This also leads to an increased heat shock response and activity of antioxidant enzymes (catalase and glutathione S-transferase), but not to an extent that would be necessary to counter future environmental conditions of oxidative stress. Furthermore, digestive enzymes (pancreatic and brush border intestinal) are decreased under such circumstances in post-metamorphic larvae and have been correlated with decreased growth and survival and increased incidence of skeletal anomalies (+32 % including S, L and K) (Pimentel et al., 2015). 1.3.9.2 Fishery

Bottom trawling is the traditional and most widely used technique to catch demersal fish. By pulling tickler chains along the seabed, fish are forced to leave the benthic and enter the pelagic zone, where they are caught with the trawl. This way of fishing threatens flatfishes in the southern North Sea by disturbing the seabed and its benthic communities, for example by decreasing abundance and biomass of infauna and causing shifts to smaller faster-growing opportunistic life forms. This has been shown to change the diet of plaice and the distribution of common sole. With a reduced diversity of prey it can also lead to an increased competition with small non-commercial flatfishes (e.g. Buglossidium luteum and Arnoglossus laterna) that normally feed on those smaller taxa. These small flatfishes are usually not caught by fisheries and profit from the increasing population of smaller prey as well as from global warming as they prefer warmer temperatures. Thereby the abundancy of these fishes has increased since the 1970s, increasing their dietary competition with juveniles of targeted species like sole and plaice (van Hal et al., 2010; Schückel et al., 2012; Eggleton et al., 2018; van der Reijden et al., 2018).

Fishing of undersized fish often leads to discarding them, but studies have shown that, especially very small fish, often do not survive being caught and released and die within the following days and weeks or are immediately caught by sea birds when put back into the sea. The European Union reacted to that by banning the discarding for pelagic fisheries (landing obligation). Since 2019 all quota regulated species, like plaice and sole, must be landed and may not be discarded, although fishers are still allowed to discard fish with a high chance of survival (Uhlmann et al., 2016; van der Reijden et al., 2017).

Pulse trawling is one alternative to traditional beam trawling to get demersal fish in the nets using an electrical stimulus instead of tickler chains. The electric shock immobilizes fish that can then be collected in the trawl without having to disturb the seabed. Other advantages are reduced fuel consumption, less discards and a higher survival rate of undersized fish, although the presence of spinal injuries has been reported (Boute et al., 2018). 1.3.9.3 Pollution

Coastal waters, that serve as nursery grounds for many flatfish juveniles and as a habitat for adults in some species, are also the most polluted areas of the seas, due to the inflow of contaminated water from estuaries (van Beek et al., 1989; Cameron et al., 1992).

1.3.10 Flatfishes in the North Sea 1.3.10.1 General geographical information

The North Sea is part of the northeast Atlantic Ocean and covers an area of 570,000km² (Walsh et al., 2015). It extends up to 4°W and 62°N in the North, is connected to the Baltic Sea through Skagerrak and Kattegat and to the English Channel by the Dover Strait which also forms its southern limit and connection to the main part of the Atlantic Ocean (Heessen et al., 2015b; ICES, 2016). It is a shallow sea with an increasing depth from the estuaries towards the deeper (200m) regions in the North. The salinity in coastal regions is strongly influenced by the estuaries of large European rivers (Rhine, Thames, Scheldt, Tees, Tyne, Weser, Elbe, the Wash, Humber) Figure 18. The North Sea: ICES statistical areas (IIIa, IIIb, IIIc, IVa, IVb, IVc, VIId, with an average salinity of 29 psu VIIe) and ecoregions (CNS, central North Sea; EEC, eastern English Channel; SNS, southern North Sea). From Barbut et al., 2019. compared to northwestern regions where the salinity is higher (35 psu) and dependent on Atlantic currents (Heessen et al., 2015b). The mean temperature of the southern part of the North Sea is 11.7° C but varies significantly between winter (7.3° C) and summer (17.2° C). Currents are tide and wind dependent in the southern North Sea and water usually circulates northwards – although there is a high variability linked to the North Atlantic Oscillation (NAO) (Barbut et al., 2019). It is part of the Food and Agriculture Organization of the United Nations’ (FAO) major fishing area 27 and subdivided into northern (IVa), central (IVb) and southern (IVc) North Sea by the ICES as shown on Figure 18 (FAO, 2017). 1.3.10.2 Fish

In the North Sea, flatfishes represent 29 % of the total biomass of demersal fishes (Piet et al., 1998). Pleuronectoidei is the largest suborder of the order Pleuronectiformes (Fig. 15) (Chapleau, 1993) and contains all six most exploited species in the North Sea (turbot, brill (Scophthalmus rhombus), common sole, European flounder, European plaice and common dab) (Braber and de Groot, 1973; Barbut et al., 2019). Dab and plaice are the most abundant flatfishes in the North Sea followed by sole and flounder. Turbot and brill are rare, but, like flounder, are valuable bycatch in the targeted fishery for sole and plaice. Dab is of low commercial value and therefore often discarded (Barbut et al., 2019). As mentioned above, they are all found in the same part of the ocean, but trophic, spatial and temporal segregation, especially in juveniles, decreases interspecies competition (Piet et al., 1998; Schückel et al., 2012). A more detailed description of five of the these six species mentioned above, is provided in Appendix 1, whereas the following paragraph will concentrate on the common dab, the most abundant flatfish species in the North Sea and subject to our studies (see 2 and 3) (Bolle et al., 1994; Hinz et al., 2005; Goldsmith et al., 2015).

1.3.11 Common dab (Dutch name: Schar) 1.3.11.1 Morphology

Figure 19. General external morphology of the common dab. Modified based on Hillewaert (2020)2.

Dab is a member of the family Pleuronectidae (right-eyed flounders), that is morphologically characterized by the absence of spines in the fins, eyes on the right side of the body, with the left optic nerve always dorsal to the right one, and the lower jaw protruding beyond the upper (Sakamoto, 1984; Chapleau, 1993; Goldsmith et al., 2015). Within that family, the subfamily Pleuronectinae is defined by the presence of a neural arch on the first abdominal vertebral body (Chapleau, 1993).

Dabs can reach a length of 45cm, have a small mouth and exposed ctenoid scales, with more ctenoid spines on the ocular side than on the blind side (Fig. 16 E, F; Goldsmith et al., 2015; Spinner et al., 2016). Like in other Pleuronectinae the lateral line runs in a curve over the pectoral fin and is visible on both sides. The ocular side is sandy brown with darker freckles and often orange or green to black spots (Fig. 192) (Goldsmith et al., 2015). Like all flatfishes, dabs have a highly asymmetric skull built up out of many bones in neuro- and branchocranium. Their vertebral column usually consists of 40 vertebrae, 10 precaudal and 30 caudal, including the caudal complex (Jordan and Goss, 1889; Sakamoto, 1984). 1.3.11.2 Distribution

Dabs inhabit marine habitats from Iceland to the Biscay Bay as well as the western Baltic Sea and White Sea but not the Mediterranean Sea. They are most abundant in inner shelf waters, especially those of the southern North Sea, with a relatively constant density over a depth range of 0 – 80 m (Hinz et al., 2005; Goldsmith et al., 2015). After sprat and sandeels, dab is the fish with the third highest biomass in the North Sea (Goldsmith et al., 2015). 1.3.11.3 Reproduction

Female dabs grow faster and get larger than males but mature later (3-5 years versus 2-3 years). Their two ovaries mature at different rates to ensure spawning over a prolonged period (Rijnsdorp et al., 1992; Goldsmith et al., 2015; Barbut et al., 2019). Intersex fish regularly occur and are externally

2 Hillewaert, H., 2020. Kliesche, Scharbe. fischlexikon.eu. URL https://www.fischlexikon.eu/images/fischlexikon/galerie/kliesche-01.jpg (accessed 2.5.20). 28

identified as males in most cases. Spawning occurs offshore and from winter (December/January) to summer (August; till June in the North Sea (Knijn et al., 1993)), with a peak in March and April (Rijnsdorp et al., 1992; Goldsmith et al., 2015; Barbut et al., 2019). Like in other flatfishes, females have a higher food consumption than males and unlike males, feeding does not decrease during winter months (Lozán, 1992; Hinz et al., 2005). 3,300 eggs per gram bodyweight are spawned in batches by females every year, resulting in a fecundity of 80,000 to 246,000 eggs (Knijn et al., 1993; Goldsmith et al., 2015). Dab eggs are the most abundant fish eggs in the southern North Sea, especially in winter (Cameron et al., 1992).

After the larvae have hatched they can be transported several hundred kilometers to their nursery grounds, leading to a high level of exchange between different dab populations (Barbut et al., 2019). At the nursery grounds larvae begin their post-metamorphic demersal life at a length of 13 to 20 millimeters and then rapidly grow from approximately three centimeters in June to seven centimeters in December (Knijn et al., 1993; Bolle et al., 1994). During their first year of life juveniles can be found in shallow coastal and offshore waters around Dogger Bank and the eastern central North Sea and far less in the Wadden Sea and the Scheldt estuary for example (Bolle et al., 1994; Goldsmith et al., 2015). As a consequence of overlapping habitats, hybrids between dab and flounder or plaice can occur (Goldsmith et al., 2015). 1.3.11.4 Diet

Larval dabs feed on copepods and other crustacean plankton, whereas juveniles and adults prefer mollusks, polychaetas (Owenia fusiformis), decapods (Liocarcinus spp.), brittle-stars as well as small sea urchins and fish (Braber and de Groot, 1973; Schückel et al., 2012; Goldsmith et al., 2015). Compared to other flatfishes, dabs have a relatively small esophagus and stomach and their intestines have four pyloric caeca (De Groot, 1971). Like Scophthalmidae and other Pleuronectidae, dabs are visual feeders and use fast acceleration from the seabed to catch prey (Schückel et al., 2012). 1.3.11.5 Exploitation

Common dab is often caught as bycatch in trawl fisheries but less valuable than other flatfishes and therefore discarded in great amounts (Goldsmith et al., 2015; Miller and Verkempynck, 2016; van der Reijden et al., 2017). Although there is no limited total catch, the European Union publishes an annual total allowable catch (TAC) for dab together with flounder that has never been exceeded (Miller and Verkempynck, 2016). In the International Union for Conservation of Nature and Natural Resources’ (IUCN) red list of threatened species, common dab is ranked with “Least Concern” and populations in the North Sea remain stable or even increase in size. Dabs already spawn most eggs before the main fishing season starts. This, and the fact that trawling likely increases prey availability (together with the eutrophication of the sea) and decreases the density of predatory fish species, makes, that dab could actually profit from fishery interventions in the North Sea (Hinz et al., 2005; Miller and Verkempynck, 2016). In 2016, 399 tons of dab have been landed by Belgian fisheries and the average price at Belgian harbors was 0.81€/kg (Department of Agriculture and Fisheries, 2017). 1.3.11.6 Common pathologies

Disease monitoring for North Sea fishes has been done for nearly 40 years (Lang et al., 2017). Dab is a suitable bioindicator species for monitoring surveys, because these fish live a benthic life, closely connected to the sediment where pollutants accumulate. Furthermore, they have a relatively static lifestyle, with the exception of the spawning period, so that possible causative factors present at a certain location can be linked to the presence of a disease. Dabs are highly abundant with a low

commercial value and thereby economically more interesting for research than valuable flatfishes, like turbot or sole. Some diseases, like skin ulcerations for example, can develop rapidly and can be easily recognized (ICES, 1996; Kerambrun et al., 2012; Lang et al., 2017; Vercauteren et al., 2020).

Diseases related to the integument can be frequently seen in common dab. Pigmental disorders, like, for example, ambicolouration, where there is pigment on the blind side, and hypomelanosis, describing a deficiency of pigment cells on the ocular side, can be highly prevalent in some areas (Lang et al., 2017; Castellini et al., 2018; Noens, 2018). Skin ulcerations, fin rot, lymphocystis and epidermal hyperplasia/papilloma are other skin diseases affecting the wild common dab. Furthermore, several types of non- and neoplastic liver lesions have been described (Lang et al., 2017; Vercauteren et al., 2018, 2020). 2 AIMS OF THIS STUDY

The research on skin ulcerations in wild common dab in the Belgian part of the North Sea (BNS) has led to a closer examination of specimens of this species. Among other abnormalities, externally visible skeletal anomalies were present in several specimens (Fig. 20). Although skeletal anomalies have been described in a lot of fish species today, to my knowledge, there have not been any radiographical studies on deformations of the vertebral column in wild common dab.

Figure 20. Dorsal view of a common dab caught during a disease monitoring survey in 2015. Skeletal anomalies are clearly visible in the caudal half of the body, altering the trajectory of the lateral line and the posture of the tail.

The main objective of this study was to determine the prevalence of skeletal anomalies in wild common dab from the BNS. Furthermore, we aimed to find and describe these anomalies in detail by taking and analyzing radiographic images of affected and control fish. As the causes for skeletal anomalies are still unsolved in many cases, several possible causal factors were taken into consideration to study whether they affected the presence of skeletal anomalies in common dab. On a broader scope, this thesis aims to highlight the presence of severe vertebral deformities in wild fish and thereby should serve as a note for future research on skeletal anomalies in both, wild and reared fish.

3 RESEARCH 3.1 Materials and Methods 3.1.1 Sampling areas Fish were caught every two months, between 2016 and 2019, on well described sampling areas around eight scattered locations within the BNS (Fig. 21). The BNS is part of the southern North Sea and covers a total area of 3,454km² with a coastline of 67km (Department of Agriculture and Fisheries, 2018). It is a part of the FAO’s major fishing area 27 and ICES statistical area IVc (FAO, 2017).

The research vessel (‘Simon Figure 21. The locations around which the eight sampling areas within the BNS are Stevin’), with which the situated; L1, 51°29'24.00"N / 2°47'13.20"E - depth: 22m; L2, 51°31'12.00"N / 2°51'18.00"E - depth: 22m; L3, 51°30'36.00"N / 2°52'40.80"E - depth: 23m; L4, sampling was carried out, is 51°28'12.00"N / 3° 4'40.80"E - depth: 14m; L5, 51°17'60.00"N / 2°35'34.80"E - depth: equipped with a 3m-beam 19m; L6, 51°16'12.00"N / 2°36'57.60"E - depth: 13m; L7, 51°14'24.00"N / 2°43'37.20"E trawl (mesh-size: 22mm) - depth: 8m; L8, 51°10'48.00"N / 2°42'0.00"E - depth: 4m. and a conductivity-temperature-depth (CTD) probe (Seabird 19plusV2, Sea Bird Electronics, USA) with which seawater temperature, salinity, oxygen-concentration and depth were measured at the sampling areas. The pH of the water collected at the seabed on all locations was also measured (Hanna Instruments, Belgium). Moving with a speed of three to four knots the beam trawl was towed during approximately 20 minutes per haul. 3.1.2 Examination and processing of specimens Immediately after sampling, the total length (TL; cm) of all common dabs was measured from snout to the end of the tail, and they were individually weighted (W; g). Based on these values, the Fulton body condition score was determined (100 x (W/L3) (Fulton, 1904). The results were recorded together with sex, pH, and the results from the CTD probe (sea water temperature, depth, turbidity, oxygen saturation and salinity). The specimens were divided into groups according to their externally visible state of health, considering the presence or absence of skin ulcerations and skeletal anomalies. The fish were then humanely euthanized on board with an overdose of benzocaine (Ethyl 4- aminobenzoate, 200 mg L-1; Merck, Germany) or tricaine methane sulfonate (MS-222; 500 mg L-1; Sigma Aldrich N.V., Belgium).

Fish with skeletal anomalies were frozen and stored at -20°C within labelled plastic bags until further examination. Fish with skin ulcerations and control fish were primarily used in a different study (Vercauteren et al., 2018) and therefore necropsied, where abdominal organs, skin ulcerations and otoliths were removed. These specimens were also frozen and stored under the same circumstances

after that study was finished and included in the present study to serve as controls for the radiographic approach. 3.1.3 Data processing and graphical exploration Based on the measurement results, obtained during the sampling sessions, a dataset was created and further filled with data on the unsmoothed Atlantic Multidecadal Oscillation (AMO)-index (downloaded from ESRL-PSD3), representing an index of the North Atlantic sea surface temperatures for every month. The online database of the European Marine Observation and Data Network (EMODnet) was consulted for information regarding the substrate of the seabed4, mean shipping density and mean fishing vessel density ((hours/km2) x month-1) at the time of sampling and one month prior to that, respectively, for the latter two factors5. Data on pollution, such as the concentration of heavy metals and polychlorobiphenyls, in the sediment (granular fraction < 63 µm) was provided by The Flanders Research Institute for Agriculture, Fisheries and Food (ILVO). The degree of pollution for the sampled locations was assessed based on five pollution cluster zones in the BNS presented by Lagring et al. (2018). Information about anthropogenic activities, such as dredging, offshore energy and maritime transport for the studied areas was gathered from the Flanders Marine Institute’s (VLIZ) ‘Coastal Portal’6. Distance from the shore (perpendicular) was calculated based on the coordinates of the sampling areas.

Using the program R Studio 1.2.1335 (RStudio Inc., 2019), entries for fish with abnormalities other than skeletal anomalies were ruled out. The total prevalence of skeletal anomalies was calculated as the number of fish with skeletal anomalies divided by the sum of affected and healthy fish. In R Studio the ‘dplyr’ package was used to compare the affected with the non-affected fish regarding fish-related and environmental factors. For TL and condition, the minimum, maximum, mean, standard deviation (SD), median and variance values were determined for the healthy and affected groups. The total prevalence, and the prevalence per year, for each location was calculated by dividing the number of affected specimens by the sum of affected and healthy fish. The sex ratio of affected fish was calculated by dividing the total numbers of male and female individuals with skeletal anomalies by the total number of male/female fish. The total prevalence for each of the four seasons was calculated by dividing the number of affected fish by the total number of fish per season. Diagrams for prevalence per season and sex frequencies of healthy and affected fish per season were made using the ‘ggplot’ package. 3.1.4 Statistics Using RStudio, Wilcoxon rank sum tests were performed for TL, weight, and condition. For sex, a Chi- squared test was used, whereas for season and location, with some samples being small (n < 5 ), a Fisher’s exact test was performed. A linear regression model (LRM) using foreword selection was used to study the association between the presence of skeletal anomalies and 19 possible causative factor candidates (three fish-related factors, four related to anthropogenic usage and 10 other spatial and temporal environmental factors; see Appendix 2). These factors were chosen after the Pearson correlation coefficient was determined for each combination of two factors. Collinearity of any of these combinations led to the exclusion of the factor considered to be biologically less relevant (length –

3 ESLR-PSD, 2019. Climate Timeseries: AMO (Atlantic Multidecadal Oscillation) Index. URL https://www.esrl.noaa.gov/psd/data/timeseries/AMO/ (accessed 07.09.19). 4 EMODnet, 2019a. Seabed habitats. URL https://www.emodnet-seabedhabitats.eu (accessed 21.09.19). 5 EMODnet, 2019b. Human Activities: Vessel Density 2017. URL https://www.emodnet-humanactivities.eu/view- data.php (accessed 21.09.19). 6 VLIZ, 2019. The Coastal Portal. URL http://kustportaal.be/en (accessed 19.09.19). 32

weight / length – age / depth – distance to shore / season – sea water temperature / season – fishing intensity). To assess the population density, catch per unit effort (CPUE) was used. The null hypothesis was defined as the absence of a causative correlation between these factors and the presence of skeletal anomalies. p-values < 0.05 were considered statistically significant whereas p-values between 0.05 and 0.10 were considered a trend. 3.1.5 Radiographic imaging Digital radiographs of 35 (20 affected, 15 controls) frozen fish were taken in dorsoventral (DV) and right-left-lateral (RtLeL; simplified to RL in the following) projections, using a stationary x-ray machine (EDR6CANON, type CXDI-50G, flat panel detector, scintillator and amorphous silicon Sensor LANMIT 4, Santa Clara, California, USA). The fish were kept inside plastic bags and supported by a radiographic foam positioner for the DV projection. X-ray preferences, TL, and the presence of skeletal or other anomalies were noted. Tube voltage (kV) and tube current and exposure time product (mAs) were adjusted as little as possible, but in accordance to the size of the fish within a range of 40.0-52.0 kV and 0.8-14.0 mAs (exact preferences per fish are provided in Appendix 3). The images were stored in Digital Imaging and Communications in Medicine (DICOM) and JPEG formats. 3.1.6 Meristic counts and measurements The radiographs were viewed and analyzed with RadiAnt DICOM Viewer 5.5.0 (2019) software. The standard length (sL; on DV and RL projections) and sH (on RL projection) were measured. To obtain the Figure 22. RtLeL radiographical image of a common dab (17.3 cm sL; 40 kV / 8 mAs) illustrating approximate length how the vL and angles of vertebral column deviations were measured using RadiAnt Dicom Viewer of the vertebral 5.5.0 ; scale bar: 10 mm. column (vL) it was divided into more or less linear parts of which the length was measured separately (Fig. 22) and then summed up, both on DV and RL projections. The mean values for sL and vL ((RL+DV)/2) were calculated afterwards. The vL:sL ratio was calculated, as well as the sL:TL ratio that provides information on the relative length of the tail. The sL:sH ratio was calculated and used as a value for the cranio-caudal compression of fish’ bodies. The number of vertebrae per region (abdominal, caudal, caudal complex) was counted to see whether affected fish have an altered amount of vertebrae compared to healthy fish. The number of vertebrae included in abdominal (aK) and caudal (cK) kyphosis (if present) and number of sites with K (type 15), L (type 14) and S (type 16) were also counted for each deformed fish as a measure of the local and total complexity of anomalies. The angles of aK, intermediary kyphosis (iK), cK and S were measured in all fish (Fig. 22) and translated into a severity score, according to the ranges suggested by Munday et al. (2016): no deviation = 0, <20° = 1, 20°-40° = 2, >40° = 3. For S, the sum of all angles was used for the score of each affected fish. All measurements and meristic counts were brought together into a second dataset and the mean and SD were calculated for both groups using RStudio software. The significance of the differences between each of these values was tested with a Wilcoxon rank sum test.

3.2 Results 3.2.1 Data analysis Of 4,785 fish caught, dataset entries with abnormalities other than skeletal anomalies were rejected to avoid cases of comorbidity, decreasing the number of observations to 4,545. Out of these, 4,512 fish were healthy and 33 showed skeletal anomalies, resulting in a total prevalence of 0.726 %.

The TL of the fish was 16.2 ± 3.4 cm and 15.9 ± 4.5 cm in the affected and healthy groups, respectively, not differing significantly (p=0.9548). 91.02 % of the healthy specimens and all the affected had been weighted, resulting in a mean weight of 48.9 ± 38.3 g and 53.0 ± 37.6 g for the two samples, respectively. Neither weight (p = 0.4157) nor body condition (1.07 ± 0.647 versus 1.09 ± 0.207; p=0.0543) were significantly different between the groups, although, for condition there was a trend.

Contrary to the affected specimens, where the sex could be determined in all cases, in 4.82 % of the healthy individuals this was not possible. In general, more female fish (n = 2,492) were caught than male ones (n = 1,834), with no significant difference in frequency between the two groups (p=0.8624; see Table 2). The frequency of both sexes per season of the year is given on Figure 23 to visualize possible differences between spawning and non-spawning periods.

In all of the eight areas, fish with skeletal anomalies were caught. The exact frequencies per location are shown in Table 2. For each combination of year and location with a prevalence of skeletal anomalies higher than zero, the absolute and relative frequencies are provided in Table 3.

Most fish, both with (n = 19) and without (n = 2,070), skeletal anomalies were caught in spring, although the highest prevalence (1.23 %) was seen in summer (Fig. 24). There were no significant differences in frequencies between affected and non-affected fish for all locations (p=0.3493), but a trend was visible for the seasons (p = 0.09607).

The results of the first step of the LRM showed that none of the tested factors could be significantly associated with the presence of skeletal anomalies. Thereby no factor could be further used to build up the model (p > 0.05; exact p-values are provided in Appendix 2).

Table 2. Absolute and relative (%) frequencies of affected fish of both sexes and among the eight sampling areas. *the percentage for sex describes the relative frequency of each sex within the group (healthy / affected).

healthy affected healthy affected

female 2472 20 57.6* 60.6*

sex male 1821 13 42.4* 39.4*

L1 486 5 98.98 1.02

L2 129 1 99.23 0.77

L3 666 1 99.85 0.15

L4 542 4 99.27 0.73

L5 344 4 98.85 1.15 location L6 617 4 99.36 0.64 L7 1073 11 98.99 1.01

L8 655 3 99.54 0.46

Figure 23. Sex frequencies of healthy (H) and affected (S) fish in every season.

Table 3. Prevalence of skeletal anomalies per location per year.

location year n prevalence (per year)

2018 3 2.01 L1 2019 2 0.93

L2 2018 1 1.75

L3 2018 1 0.19

2016 2 0.8 L4 2019 2 2.04

L5 2018 4 2.9

2016 1 1.28

L6 2017 1 0.27

2018 2 1.85

2017 4 0.83

L7 2018 5 1.35

2019 2 1.17

2016 2 2.67 L8 Figure 24. The prevalence of skeletal anomalies in the caught fish during all four 2018 1 0.5 season.

3.2.2 Radiographic analysis The control fish, categorized as such after visual external examination, generally presented with a well aligned vertebral column on the radiographic images, although the abdominal region of 93.3 % of these specimens showed a light dorsal curvature (aK). Contrary to that, multiple curvatures were visible within the trajectory of affected fish, both on DV and RL projection. 3.2.2.1 Measurements

The TL of affected fish (n = 20) was 16.0 ± 2.76 cm and thereby significantly different from that of the control group (n = 15; TL = 20.0 ± 2.48; p = 0.00033) (Table 4). The same was true for sL with 13.9 ± 2.40 cm for the deformed fish and 17.5 ± 2.27 cm for the control group (p = 0.00020). The fish with VCDs also had a significantly lower sH (6.35 ± 1.14 cm versus 7.37 ± 0.94 cm; p = 0.00537) and vL (11.0 ± 2.09 cm versus 13.5 ± 1.80; p = 0.00103). 3.2.2.2 Ratios

The mean vL:sL ratios were 79.5 ± 4.03 % and 77.4 ± 1.50 % and the values differed significantly (p = 0.00353). On the other hand, their sL:TL ratio did not differ significantly from that of the controls (86.7 ± 2.92 % versus 87.3 ± 1.75 %; p = 0.1994). The sL:sH ratio was significantly lower in the affected group, compared to the control group, with mean values of 2.19 ± 0.154 and 2.38 ± 0.0967, respectively (p = 0.0002984).

Table 4. Results of the performed measurements on 20 fish with skeletal anomalies (SA) and 15 controls (C) using radiographs. Aak/AiK/AcK/AS stands for the measured angles of abdominal, intermediary and caudal kyphosis, as well as the sum of angles of scoliosis. The values presented for “all SA” or “all C” are based on the complete samples; “aff. SA” / ”aff. C” only takes the specimens into account that show the respective anomaly.

measurement group mean ± SD median min max SA 16 ± 2.76 16.0 10.0 21.8 TL (cm) C 20 ± 2.48 20.0 15.0 24.0 SA 13.9 ± 2.40 14.0 8.3 18.9 sL (cm) C 17.5 ± 2.27 17.6 12.8 21.2 SA 6.35 ± 1.14 6.4 4.0 8.7 sH (cm) C 7.37 ± 0.936 7.3 5.2 9.0 SA 11.0 ± 2.09 11.0 6.8 15.1 vL (cm) C 13.5 ± 1.80 13.4 9.9 16.7

SA 79.5 ± 4.03 80.0 66.9 88.5 vL:sL ratio (%) C 77.4 ± 1.5 77.3 72.9 79.1 SA 86.7 ± 2.92 86.8 79.9 93.7 sL:TL ratio (%) C 87.3 ± 1.75 87.4 84.0 90.8

all SA 17.3 ± 10.30 16.0 7.1 54.1 AaK (°) all C 5.55 ± 2.33 5.7 0.0 9.1 aff. C 5.94 ± 1.82 6.1 3.1 9.1 all SA 5.35 ± 8.93 0.0 0.0 33.7 AiK (°) aff. SA 15.3 ± 8.67 12.2 8.6 33.7 AcK (°) all SA 35.8 ± 9.44 34.4 20.8 58.6 all SA 33.5 ± 30.8 28.2 0.0 105.0 AS (°) aff. SA 41.9 ± 28.8 32.7 13.9 105.0 36

3.2.2.3 Meristic counts

All affected and 14 control fish showed aK. The mean severity score of the control group was 0.833 ± 0.309, while that of the fish with additional VCDs was 1.25 ± 0.550 (Table 5), and the values were significantly higher (p = 0.005278). iK, affecting the cranial part of the caudal region of the vertebral column, was seen in seven of the affected (35.0 %) and none of the control fish with a mean severity score of 1.14 ± 0.378 for the fish affected by this type of K. cK was the most severe, affecting all deformed specimens (severity score = 2.35 ± 0.489), followed by S found in 16 fish (80.0 %; severity score = 2.19 ± 0.834). The mean numbers of included vertebrae were 9.7 ± 0.923 and 8.8 ± 0.894 for aK and cK, respectively.

Table 5. Meristic counts for 20 fish with skeletal anomalies (SA) and 15 controls (C) based on radiographs. NK, number of kyphosis sites; NvaK, number of vertebrae included in abdominal kyphosis; NvcK, number of vertebrae included in caudal kyphosis; NL, number of lordosis sites; NS, number of scoliosis sites; SaK, severity of abdominal kyphosis; Sik, severity of intermediary kyphosis; ScK, severity of caudal kyphosis; SS, severity of scoliosis; The values presented for “all SA” or “all C” are based on the complete samples; “aff. SA” / ”aff. C” only takes those specimens into account that show the respective anomaly.

meristic count group mean ± SD median min max

NK all SA 2.40 ± 0.50 2 2 3 NvaK all SA 9.70 ± 0.92 10 8 11 NvcK all SA 8.80 ± 0.89 9 8 11 NL all SA 0.35 ± 0.59 0 0 2 NS all SA 2.45 ± 1.76 2 0 6

all SA 1.25 ± 0.55 1 1 3 SaK all C 0.83 ± 0.31 1 0 1 aff. C 0.89 ± 0.21 1 0.5 1 all SA 0.40 ± 0.60 0 0 2 SiK aff. SA 1.14 ± 0.38 1 1 2 ScK all SA 2.35 ± 0.49 2 2 3 all SA 1.75 ± 1.16 2 0 3 SS aff. SA 2.19 ± 0.83 2 1 3

A detailed visual analysis of VCDs in all control and deformed specimens is provided in the Supplementary File.

3.3 Discussion Skeletal anomalies, like deformations of the vertebral column, are an increasing problem in aquaculture facilities worldwide, but little is known on similar deformations in many wild fish species (Boglione et al., 2013a). Common dab is a highly abundant flatfish species in the BNS, that is often discarded by fisheries due to its low economical value (Hinz et al., 2005; van der Reijden et al., 2017). It has been used as a bioindicator species in numerous other studies (Lang et al., 2017) and skeletal anomalies were part of the fish disease monitoring in the past, although they are not included in the ICES fish disease index (ICES, 1996; Lang et al., 2017). Nevertheless, to my current knowledge, skeletal anomalies in common dab, including VCDs, have never been described before, based on radiographic images. The aim of the present study was to determine the prevalence of skeletal anomalies in wild common dab caught in the BNS between 2016 and 2019, and to create a detailed description of the trajectory of the vertebral column. Furthermore, we studied the correlation between the presence of these and several fish-related and environmental factors. 3.3.1 Prevalence As the most occurring vertebral deformities in fish are VCDs, it is no surprise, that these can also be seen in common dab (Chatain, 1994). The terminology of these VCDs used in the present study is in accordance with the definitions given by Kranenbarg et al. (2005). Based on visual external examination onboard of the research vessel, the total prevalence of skeletal anomalies was 0.726 %. We can presume, that this is an underestimation of the real prevalence, as the sensitivity of this visual method is rather low. Only the most severe anomalies are externally visible and the prevalence is often higher when more sensitive methods are used additionally (de Azevedo et al., 2017a). In accordance to that, 93.3 % of the fish externally declared as control fish in our study were found to have a light form of aK visible only on the radiographic images. With the focus exclusively on VCDs and no other skeletal anomalies, and taking into consideration the probable underestimation, this prevalence found in common dab is comparable to the prevalence found in other species that mostly ranges between two and six percent (Hansen et al., 2010; Jawad et al., 2018). 3.3.2 Radiography Radiographic images are not only used on hatchery farms to sort out fish with skeletal anomalies, but have also been broadly used in numerous studies on skeletal anomalies, including studies on flatfishes (de Azevedo et al., 2017a). Our study has shown, that radiographic images are a suitable diagnostic technique for the detection of VCDs in wild common dab and high quality radiographic images could be taken with a standard veterinary x-ray machine. By taking two orthogonal radiographic images for each fish, all three types of VCDs could be visualized (L, K, S). Adding the DV to the (standard) RL projection was proposed by de Azevedo et al. (2017a) to avoid underestimation of anomalies, especially because S is best visualized on DV projections. In our study it was also very evident that severe forms of S were hardly visible on RL projections whereas clearly visible on DV projections. aK was not only found in all deformed fish, but also in a great percentage of the (based on external observation) healthy ones, although the severity was significantly lower in the latter group (Fig. 25). This leads to the assumption that a certain degree of aK, that is externally invisible, could possibly be physiological rather than pathological. In Senegalese sole, a similar curvature in that region has been described, but with less vertebral bodies involved (de Azevedo et al., 2017a). There is no swim bladder in larval, juvenile or adult dabs, so a hypothetical correlation with non-inflation or resorption of it during metamorphosis, as described in seabass and seabream for example, can be ignored (Al- Maghazachi and Gibson, 1984; Chatain, 1994).

Figure 25. RtLeL radiographic images comparing the vertebral column of an affected fish (a) with a control specimen (b). a: whole body of the specimen with the largest angles for abdominal kyphosis (aK 54.1°), intermediary kyphosis (iK2 33.7°), caudal kyphosis (cK 58.6°); blue line illustrates expected normal trajectory of the vertebral column; A = abdominal region, C = caudal region, CC = caudal complex, L = lordosis; (42.0 kV / 8.0 mAs). b: detail of the vertebral column of a control specimen showing a non-pathological trajectory (16.9 cm sL; 40.0 kV / 6.3 mAs); scale bars: 10 mm. 3.3.3 Possible risk factors None of the 19 fish-related and environmental factors taken into consideration in the LRM, was significantly associated with the presence of skeletal anomalies. One should take into consideration that these factors were recorded at the time of sampling and are thereby temporally and spatially separated from the factors during the probable time of onset of the anomalies. This could lead to the conclusion, that genetic or epigenetic factors during the fish’s early development are possibly responsible, rather than factors occurring later in life. Unlike skin ulcerations, for example, that can develop within days, skeletal anomalies likely take a long time to develop and more research will be necessary to point out the influences of risk factors during different life stages. 3.3.4 General condition of specimens All specimens were completely metamorphized according to the four characteristics for that described by Al-Maghazachi and Gibson (1984). The TL values measured onboard suggest, that the studied specimens were either older juveniles or adults, as males usually mature at a length of ~11 cm and females at ~14 cm (Rijnsdorp et al., 1992). Either way, the presence of skeletal anomalies in fish of this length-range makes it more than plausible, that these fish can successfully mature, mate, and thereby produce offspring. They also did not seem to suffer from starvation or to experience difficulties in feeding behavior as their mean weight and condition were similar to that of the control fish. The intestines of some fish were observed to be filled with prey species based on the radiographic images. A normal flexibility of the vertebral column seems to be necessary to allow undulatory movements. In fish with K or L this ability could be limited, but it is possible, that processes of adaptation of bone tissue itself and surrounding connective and muscular tissues compensate for that (Boglione et al.,

2013a; Munday et al., 2016). Further studies, using magnetic resonance imaging (MRI), for example, are necessary to study possible differences in the tissues between affected and healthy fish. 3.3.5 Catch data The total amount of fish caught per location differs among the eight locations. Location 7, where most control and deformed fish have been caught, is a shallow area (depth 8m) and located relatively close to the shore (8,247m). According to Goldsmith et al. (2015), the density of dabs does not vary among depths of 0-80m. Thus, depth does not seem to be a plausible explanation for the high abundancy at location 7. A lot of other factors could have an influence on that, but were not further investigated in this study, because there was no significant difference in the amounts of healthy and unhealthy fish caught there, nor at any other location. Thus, the location where fish were caught does not seem to have an influence on the presence of skeletal anomalies.

More fish with skeletal anomalies were caught during summer than during other seasons, and although this difference was not significant, the p-value was considered a trend. Therefore, future research should look for reasons for that difference, possibly linked to migration or death of affected fish during the other seasons. 3.3.6 Measurements and ratios

Contrary to the TL of the complete groups in the dataset (nhealthy = 4,512 / naffected = 33), the TL, sL and sH did significantly differ between the two samples (nhealthy = 15 / naffected = 20) in the radiographic approach. This is possibly the result of a biased selection of control fish as these specimens were primarily used as affected and control fish in a study on skin ulcerations. The prevalence of skin ulcerations increases with TL, so the controls’ length in this study was not necessarily representative for the whole population of healthy common dabs in the BNS. Nevertheless, the differences in length and height were assumed to have had no critical impact on the study, as VCDs have been described to increase in variety of anomalies with increasing length and larger fish should therefore rather show skeletal anomalies than smaller ones (Lewis et al., 2004; Lewis and Lall, 2006).

The fact, that the vL did also differ significantly between the two groups is in accordance with the differing sL. Furthermore, the vL:sL ratio did also differ, so the vertebral column of deformed fish was relatively longer compared to the control group. The vertebral bodies had a normal shape in most cases and were not elongated, so it can be concluded, that deformed fish are relatively shorter than non-affected ones and appear to be more compressed (sL:sH). The sL:TL ratio was measured as a value for relative length of the caudal fin. The fact that it did not differ between the groups shows that the presence of L, K and/or S does not have an influence on the length of the caudal fin. 3.3.7 Number of vertebrae The total number of vertebrae was only counted in the affected group, because the mean value for healthy dabs was known from literature (n = 40) (Sakamoto, 1984). The results in fish with skeletal anomalies, as well as their variation, were in line with literature and ranged from 39 to 41 vertebrae in total: 10 abdominal vertebrae (except for one specimen with 11), 28 or 29 caudal vertebrae and two vertebrae in the caudal complex including the urostyle (data not shown) (Sakamoto, 1984; de Azevedo et al., 2017a). A variety in the number of vertebral bodies has also been described in other species like Atlantic salmon without a notable difference between healthy and affected fish (Witten et al., 2005).

Figure 26. DV radiographic images comparing the vertebral column of an affected fish (a) with a control specimen (b) a: specimen with the most severe scoliosis (cumulative angle of 104.9°); orange line illustrates the trajectory of the vertebral column (13.4 cm sL; 43.0 kV / 5.0 mAs); b: detail of the non-pathological trajectory of the vertebral column in a control specimen (16.9 cm sL; 40.0 kV / 6.3 mAs); scale bars: 10 mm

3.3.8 VCDs Contrary to aK, iK and cK were not seen in the control group. The first type affected 35 % of deformed fish and it was slightly less severe than the other types of K. The curvature in between two sites of K could have been defined as L, but in this study, this was only done, if the trajectory passed a line drawn from the first to the last vertebral body. cK, on the other hand, was the most severe type of VCDs analyzed. It usually involved the last nine (8.8 ± 0.894) vertebral bodies cranial to the caudal complex, more than two times as many vertebrae as found in other species, like Senegalese sole (de Azevedo et al., 2017a). A possible explanation for that difference is the fact, that the above mentioned article studied juvenile fish and we concentrated on adults, another possible indication that the severity and complexity of VCDs could increase with increasing age.

Only if taking into consideration that the aK may be non-pathological, the results of this study are in line with results of other studies that found that most vertebral anomalies to be present in the caudal part of the fish (Andrades et al., 1996; Tong et al., 2012; de Azevedo et al., 2017b, 2017a).

S was present in 16 deformed specimens and none of the control fish (Fig. 26). In other species S (de Azevedo et al., 2017a) and L (Chatain, 1994; Kranenbarg et al., 2005) were the most frequent VCDs, while in the present study it was K. The 16 specimens affected by S also had K in all cases. This is very similar to the LKS-complex discovered by Afonso et al. (2000) in gilthead seabream, although the pattern in the present study is less uniform among the specimens. 3.3.9 Severity In our present study we used the categorization for severity of VCDs presented by Munday et al. (2016) in their article on the correlation between L, K, and S and perivertebral fibrosis in Chinook salmon. We have shown, that this method can be used in flatfishes as well, although we slightly adjusted it to meet our subdivision of the vertebral column for the three regions in which we saw the most frequent VCDs (aK, iK, cK). As expected, the severity of fish externally classified as normal was lower than that of visibly affected ones. The results of this study for healthy and affected common dab were very much in line with the severity scores found in both healthy and affected Chinook salmon for all regions (Munday et al., 2016).

3.3.10 Causal factors Many possible causes for skeletal anomalies have been taken into consideration in recent studies, e.g. inflammation, nutritional factors (vitamin A, C and D; minerals; amino acids), toxins, and many more (Boglione et al., 2013a). As mentioned above, the factors tested in the regression analyses and the analyses of length, condition, sex, location, and season have not revealed any correlation with the presence of vertebral anomalies. Together with the relatively lower prevalence of skeletal anomalies in wild fish compared to aquaculture, they are likely congenital (Castro et al., 2008; Boglione et al., 2013a). Natural factors, like predators and weather, could lead to the death of most affected fish in the wild, while in aquaculture the survival chances are much higher (Gavaia et al., 2009). Many potential causes studied under aquaculture conditions can be ruled out for the present study, such as triploidy and incest. Healthy and affected fish share the same diet (nutritional factors) and are exposed to the same environmental and anthropogenic factors as adults. These facts are in line with our previous mentioned hypothesis, that most skeletal anomalies develop during early developmental stages. Boglione et al. (2013b) mainly found development of these anomalies in embryological and early larval stages, corroborating our hypothesis.

In contrast to that, Witten and Huysseune (2009) found, that growth can be delayed under suboptimal circumstances. It could be possible, that there is a difference in growth rate of the vertebral column compared to other body tissues, caused by certain environmental factors. This could then lead to a craniocaudal compression and the necessity for the vertebral column to expand elsewhere than its normal topographical position when it elongates. 3.3.11 Limitations In the present study, the samples of both affected and control group fish were small, compared to other studies. Therefore, the sL did differ significantly among the specimens and the power of the relative meristic results could be questioned. Although this study can serve as a template for future research on skeletal anomalies in wild flatfish species, the sample sizes should be increased.

The combination of RL and DV projections made a detailed visualization of the vertebral column possible, but in some cases, a dissection or computer tomography could have been helpful to better understand the trajectories of the deviations, especially combinations of S and K. In future studies, also the usage of MRI could be considered to gather information on how the muscles and ligaments around the deformed vertebral column are organized compared to normal fish.

As we have shown that severely affected fish are cranio-caudally compressed, and the growth rate in fish can strongly vary among individuals, the TL of the fish seems to be a suboptimal value for the selection of control fish. The otoliths of all affected and control fish should therefore be analyzed in future studies, so that the two categories can be matched by estimated age rather than length. 3.3.12 Perspective / future research There are several indications that skeletal anomalies are already present in juvenile fish and causative factors can be found earlier in the life-history. Having a look at the differences in shape and movements between pre- and post-metamorphic fish, K seems to have a smaller impact on pelagic living larvae, because their hydrodynamical shape is not altered severely by VCDs other than S, and dorsal and anal fin are not yet the most important fins to move. During metamorphosis, the muscles and connective tissues could then adapt to the changing mechanical loads with minimal negative impacts for the animal. Thus, a similar study should be set up for common dab larvae, that are also highly abundant within the ichthyoplankton of the North Sea and known to frequently be deformed, especially during

winter (Cameron et al., 1992). As an alternative to radiology, a whole mount staining technique could be used, and the trajectory of the notochord should be studied in detail. If in this future study, environmental or fish-related factors can neither be found as being causative, it should be the logical next step, to analyze the genome of affected fish and compare it with that of healthy individuals.

VBAs, like compressions, fusions and other adaptations of the vertebral bodies, have not been the topic of this study, but often occur together with VCDs, as well as anomalies of the arches, spines and caudal plates (Tong et al., 2012; de Azevedo et al., 2017b). Although the vertebral counts in the present study were in line with what is considered as normal and we thus do not expect the presence of fusions, the radiographic images of this study (Supplementary File) could be used in the future, to analyze the specimens for the presence of VBAs other than fusions and to correlate these to the results of this study. 4 CONCLUSION

This study has determined the prevalence of skeletal anomalies in wild common dab, caught in the BNS, based on a sample of 4,545 specimens caught between 2016 and 2019. The results were comparable to that of other species. Radiography was used to further describe and analyze the vertebral anomalies suspected after visual examination. A type of K affecting the region of the first ten vertebral bodies has been found in nearly all fish that were used in the radiographic approach, inclusively the control group. Further research is necessary to determine whether this deviation can be considered as non-pathological in common dab (Fig. 27, abdominal region “A”). The absence of a correlation with tested fish-related and environmental factors makes it plausible that the development of skeletal anomalies occurs during pre-metamorphic life stages, a hypothesis that should also be further studied in the future.

Figure 27. Digitally modified RtLeL radiograph of an affected common dab (18.9 cm sL; 48 kV / 12.5 mAs) highlighting different osteological structures. B, branchocranium; For other abbreviations see Fig. 14; scale bar: 10 mm.

This thesis has successfully shown, that severe skeletal anomalies are present in wild common dab. Although most studies on skeletal anomalies today concentrate on cultured species, the monitoring of wild fish regarding skeletal anomalies (among other diseases) should be obligatory. A funded understanding under natural circumstances can help to better understand similar abnormalities under farming conditions.

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APPENDIX 1: OTHER FLATFISHES OF THE NORTH SEA

Turbot (Scophthalmus maximus) Turbots are left-eyed flatfish that belong to the family Scophthalmidae. They can get up to 38 years and 100 cm and are thereby the second largest flatfish species in northwest European shelf waters. Their body has a rhomboid shape and their skin can mimic any substrate (Fig. 31). They have relatively large mouth compared to other European flatfishes and the pelvic fins are equally developed on both sides of these fish. There is no membrane between the tips of the first dorsal fin rays (Velasco et al., 2015).

They inhabit the whole inner shelf of the northeast Atlantic coast from Iceland to the Mediterranean Sea with subspecies in the Baltic (S. m. maxima) and Black Sea (S. m. maeotica). Whereas the highest abundancy Figure 28. Left lateral view of an adult turbot. From Velasco et al., 2015. of turbot is found in the Baltic Sea and Kattegat, in the North Sea they are mostly found in continental waters less than 50 m deep. Younger animals prefer more shallow waters with sandy sediments but with aging they move to deeper areas with gravel (Velasco et al., 2015). In their model on the interconnectivity of flatfish populations in the North Sea, Barbut et al. (2019) have found a limited connectivity between turbot populations from different areas.

Female turbots are larger and mature later than males (2-3 years / 34cm versus 1-2 years / 20cm). Spawning occurs from late March to August with a peak in May and June at coastal spawning grounds (4-30m deep) such as Aberdeen Bank, Turbot Bank, around Dogger Bank, Danish Coast, German Bight, and southern North Sea. Over a period of eight weeks, females spawn batches of eggs every two to four days resulting in a total annual fecundity of 1,078 eggs per gram body weight. Most energy in winter and spring is needed for reproduction leading to hardly any growth in that time of the year (Velasco et al., 2015). In comparison to flounder and plaice spawning occurs later and more coastal (Barbut et al., 2019). Hatched larvae (2.1-2.8 mm) are transported to sandy beaches by wind driven currents where larvae and juveniles (<30 cm) spend their first summer before moving to deeper grounds where adult fish live a sedentary life (Velasco et al., 2015).

Adult turbots are visual feeders that feed mainly on sandeels (Braber and de Groot, 1973; Geffen et al., 2007). The diet of larvae consists of planktonic crustaceans whereas juveniles (<20cm) prefer polychaeta worms and mysids (Velasco et al., 2015).

Although there are some targeted fisheries, turbot is mainly caught as valuable bycatch with 2,650 tons from the North Sea every year (Velasco et al., 2015) and 532 tons caught by Belgian fisheries in 2016. The average price per kilogram for turbot in Belgian harbors was 9.54 € in 2016 (Department of Agriculture and Fisheries, 2017). There have been stocking experiments to enhance natural populations and establish aquacultures, but these have not been successful yet (Velasco et al., 2015).

Brill (Scophthalmus rhombus) Belonging to the same family, brill is similar to turbot, but has a lighter appearance and a more oval shaped body (Fig. 32). Brill is also left-eyed and has no fin membrane between the first rays of the dorsal fin, its maximal length is 100 cm as well and in both species the blind side is white and translucent. Brill are usually brown with multiple darker and lighter spots and two dark spots on the midline. Its dorsal and anal fins do not continue to under the tail.

Brill can be found along the coasts of the northeast Atlantic including the Mediterranean and Black Sea. Compared to turbot, there is a strong overlap in distribution and depth, but brill is usually found less in the Baltic Sea and northern North Sea.

The larger female fish, that mature after three years (30 cm), spawn from February to August with a peak in May, releasing eggs during a period of 16 weeks. Larvae are four millimeters long when they hatch and develop very similar to turbot larvae with older fish moving to deeper waters (Velasco et al., 2015).

Like turbots, brill are visual feeders that mainly feed on sand gobies and sandeels as adults, but also on Figure 29. Left lateral view of an adult brill. From Velasco et al., 2015. squid and shrimps (Braber and de Groot, 1973; Geffen et al., 2007; Velasco et al., 2015). Larvae feed on copepod nauplii, larval decapods and mollusks (Velasco et al., 2015).

Every year 1,350 tons of brill from North Sea are landed (Velasco et al., 2015) and 355 tons by Belgian fisheries in 2016. One kilogram of brill was worth 6.74 € in Belgian harbors in 2016 (Department of Agriculture and Fisheries, 2017).

Common sole (Solea solea) As a member of the family Soleidae, soles are right eyed, slender-bodied, have a rounded head and the lower jaw does not protrude beyond the upper jaw (Fig. 33). Their dorsal fin begins already on the snout and the preoperculum is covered with scales. They have large pectoral fins, the ocular side is grey-brown, and the slightly smaller blind side is creamy white. Scales on both sides are overlapping and ctenoid and have a diameter Figure 30. Right lateral view on an adult sole. From https://upload.wikimedia.org/wikipedia/commons/c/cd/Solea_solea_1. of 1.5 millimeters (Rijnsdorp et al., 2015a; Spinner et al., jpg (accessed 22.04.2020). 2016). The maximum reported length is 70 cm and the maximum age is 42 years.

Habitats of soles can be found along the inner shelf waters of the northeast Atlantic from the African coast to Kattegat including the Mediterranean Sea. The highest abundancy can be found in the southern North Sea, the Irish Sea, the Bristol, and the English Channel. They prefer sandy to muddy sediments and move to deeper waters as they grow. Unlike all other flatfishes described so far, soles are active during the night and spend their days buried in the sand. In winter soles seek warmth by moving offshore in autumn and concentrating in deeper parts of the sea (Rijnsdorp et al., 2015a).

Like in other species females are larger than males and mature later. They spawn batches of eggs at night from May to June. Due to global warming there is a trend to earlier spawning, especially in shallow coastal waters. Annually a female produces 1,040 eggs per gram body weight. Hatched larvae (3 mm) show a vertical migration until the beginning of metamorphosis when they stay closer to the seabed (Rijnsdorp et al., 2015a, 2015b).

Whereas larvae mostly feed on copepod nauplii, juveniles and adults consume crustaceans, mollusks and polychaetas (Braber and de Groot, 1973; Rijnsdorp et al., 2015a). Unlike the other five flatfishes described here, soles are olfactory feeders (Geffen et al., 2007).

As a main targeted species, landings of sole reach 15,000 tons per year in the North Sea and 2,481 tons by Belgian vessels (2016) with a price of 11.37 €/kg (Rijnsdorp et al., 2015a; Department of Agriculture and Fisheries, 2017). 54

European flounder (Platichthys flesus) Flounders, like plaice and dab, belong to the family Pleuronectidae and are right-eyed although left-eyed reversals occur regularly (up to 30 % in the Baltic Sea). The up to 60 cm large fish can be identified by the presence of small, sharp spines along the bases of dorsal and anal fins and along the lateral line. Reddish spots can frequently been seen on the ocular side and the blind side is opaque white (Goldsmith et al., 2015). Scales on the ocular side are generally deep- embedded and cycloid but there are also tubercular scales with ctenial spines, whereas on the blind side scales are cycloid with the exception of few ctenoid scales along the Figure 31. Right lateral view on an adult flounder. From lateral line, around the operculum and along dorsal and anal https://de.wikipedia.org/w/index.php?title=Datei:Platichthys_fles fins (Spinner et al., 2016). us_1.jpg (accessed 22.04.2020).

The life of flounders contains life-stages in marine, brackish, and freshwater habitats. Juveniles of this euryhaline species can even move up rivers (Knijn et al., 1993; Goldsmith et al., 2015). This species can be found everywhere from the White to the Black Sea including the Mediterranean Sea and Baltic Sea where they are most abundant and where hybrids between flounder and plaice are common. Flounders are used to monitor pollution as pollution accumulates in estuaries, habitats where flounders are often found (Goldsmith et al., 2015).

Flounders are winter spawners and in the southern North Sea spawning takes place from mid-January until April (Goldsmith et al., 2015; Rijnsdorp et al., 2015b)There one a mature female (>35 cm) can spawn one million pelagic eggs with a diameter of 0.8-1.1 mm. In the Baltic Sea eggs are not only larger (1.5 mm diameter) but can also be demersal in coastal populations (salinity 5-7 psu) (Knijn et al., 1993; Goldsmith et al., 2015; Rijnsdorp et al., 2015b). After five to seven days 2.2-3.3mm big larvae hatch at nursery grounds with shallow water along the coast and around estuaries as well as in the Wadden Sea. Metamorphizing larvae in the southeastern North Sea use selective tidal transport. They rest on the seabed during ebb and wait for the flood before they move upwards again. Final settlement occurs in juveniles of 20-30 mm length. Their main predators then are shrimps (Crangon crangon) and crabs (Carcinus maenas) (Goldsmith et al., 2015; Link et al., 2015). Juveniles stay in coastal waters during the first years of their lives before they join the stock of mature fish. Although also mature adults spend most of their lives in relatively shallow waters (<50 m), they migrate offshore to spawn in autumn and early winter. After spawning they return to the coastal waters to feed there during the summer, storing enough energy for the winter (Goldsmith et al., 2015; Rijnsdorp et al., 2015b).

Like Scophthalmidae and other members of the family Pleuronectidae, flounders are visual feeders (Geffen et al., 2007) and feed on polychaetas, bivalves, crustaceans and small fishes (smelt (family Osmeridae) and gobies (family Gobiidae)) (Goldsmith et al., 2015).

Especially in the Baltic Sea, Kattegat and Dutch freshwater habitats, flounder is an economically important species, either as valuable bycatch or targeted fish. In the North Sea where flounders are mainly caught as bycatch in plaice and sole fisheries they are often discarded (Goldsmith et al., 2015). In 2016 242 tons of flounder have been landed by the Belgian fishing fleet with an average price of 0.68 €/kg at Belgian harbors (Department of Agriculture and Fisheries, 2017).

European plaice (Pleuronectes platessa) As a member of the same family, plaice is similar to flounder and dab in many aspects. Plaice have small jaws that only reach to the level of the eyes. Their lateral line, that is only visible on the ocular side, runs above the pectoral fin, and the edges of the anal and dorsal fins are smooth compared to flounders (Goldsmith et al., 2015). The body surface also feels smooth as plaice have deep embedded overlapping cycloid scales (Spinner et al., 2016). The ocular side is deep brown with orange spots and the blind side is white and translucent. Sometimes darker spots can be seen on the blind 55

side that are associated with trematode infections. They reach a maximum length of 90cm and a maximum age of 30 years (Goldsmith et al., 2015). Plaice are widely distributed from Iceland and the White Sea to the western Mediterranean Sea down to a depth of 100 m, though their highest density is found in southeastern North Sea. Fish smaller than 25 cm can only be found in coastal areas south and east of Dogger Bank (Goldsmith et al., 2015).

Like in other flatfishes, females are larger and mature later than male fish (4-5 years versus 2-3 years). Spawning occurs from January to March in the Figure 32. Right lateral view on an adult plaice. From Goldsmith et al., 2015. Southern Bight and can be delayed in years with lower water temperatures. Male fish are in spawning condition for 11 weeks while in females this is only 5 weeks. During that time males are more easily caught because they aggregate and are more active. As the salinity in the Baltic Sea is too low for plaice to spawn, they have to travel to Kattegat for that (salinity >15 psu) (Goldsmith et al., 2015). A 37 cm-female can spawn up to 130,000 eggs resulting in 265 eggs per gram body weight (Knijn et al., 1993). The eggs (2 mm diameter) are transported to the coast to hatch and metamorphizing larvae also use selective tidal transport. Larger larvae are less likely to be washed out of the nursery area (Fuiman and Higgs, 1997; Goldsmith et al., 2015). Settlement takes places after a total pelagic period of three to four months. At the nursery grounds, shallow estuarine areas, Brown shrimps are the juveniles’ main predator (Goldsmith et al., 2015). The Wadden Sea is the most important nursery ground for plaice, being responsible for more than 50 % of recruitment (Bolle et al., 1994). Like flounder, plaice are also winter spawners and thus rely on energy uptake during the summer months (Rijnsdorp et al., 2015b). Like the selective tidal transport, adults use similar behaviors to migrate between spawning and feeding grounds (Goldsmith et al., 2015).

The diet of plaice larvae consists of appendicularians, copepods, algae, post larval bivalves. Juveniles feed on regenerating parts of invertebrates (tail tips of lugworm and siphons of bivalves) and larger juveniles switch to polychaetas (Nephtys spp. and Owenia fusiformis), bivalves (Nucula nitidosa), and decapods (Liocarcinus spp. and Galathea spp.) (Schückel et al., 2012). Adults, that are visual feeders as well, mainly consume mollusks and sandeels, but a change has been observed during the 20th century from mollusks to worms (Braber and de Groot, 1973; Geffen et al., 2007; Goldsmith et al., 2015).

Plaice are caught by targeted bottom-trawling fisheries. Undersized plaice are also caught as bycatch in sole or brown shrimp fishing with narrow meshes (Goldsmith et al., 2015). Of the 100,000 tons of plaice totally landed from the North Sea every year (Goldsmith et al., 2015), 8,946 tons have been caught by Belgian vessels in 2016 with an average price of 1.64 €/kg in Belgian harbors then (Department of Agriculture and Fisheries, 2017).

Additional References Rijnsdorp, A.D., Goldsmith, D., Heessen, H.J.L., van Hal, R., 2015a. 76. Soles (Soleidae), in: Heessen, H.J.L., Daan, N., Ellis, J. (Eds.), Fish Atlas of the Celtic Sea, North Sea, and Baltic Sea. Wageningen Academic Publishers, Wageningen, NL, pp. 472–482.

Rijnsdorp, A.D., Van Damme, C.J.G., Witthames, P.R., 2015b. Ecology of reproduction, in: Gibson, R.N., Nash, R.D.M., Geffen, A.J., Van der Veer, H.W. (Eds.), Flatfishes: Biology and Exploitation. John Wiley & Sons, Ltd, Hoboken, NJ, USA, pp. 101–131.

Velasco, F., Heessen, H.J.L., Rijnsdorp, A.D., de Boois, I., 2015. 73. Turbots (Scophthalmidae), in: Heessen, H.J.L., Daan, N., Ellis, J. (Eds.), Fish Atlas of the Celtic Sea, North Sea, and Baltic Sea. Wageningen Academic Publishers, Wageningen, NL, pp. 429–446.

APPENDIX 2: LIST OF FACTORS TESTED IN THE REGRESSION ANALYSIS health factor factor type factor p-value

Skeletal anomaly environmental year 0.412

Skeletal anomaly fish-related length 0.703

Skeletal anomaly fish-related sex 0.736

Skeletal anomaly fish-related body condition 0.84

Skeletal anomaly environmental pH 0.747

Skeletal anomaly environmental temperature 0.141

Skeletal anomaly environmental turbidity 0.17

Skeletal anomaly environmental oxygen-concentration 0.903

Skeletal anomaly environmental salinity 0.762

Skeletal anomaly environmental AMO 0.162

Skeletal anomaly environmental sediment 0.647

Skeletal anomaly anthropogenic fishery intensity 0.635

Skeletal anomaly anthropogenic fishery intensity 1 month prior 0.172

Skeletal anomaly anthropogenic pollution 0.227

Skeletal anomaly anthropogenic anthropogenic activities 0.241

Skeletal anomaly environmental CPUE (population density) 0.176

Skeletal anomaly environmental distance from the shore 0.677

APPENDIX 3: PREFERENCES FOR RADIOGRAPHS PER SPECIMEN

general TL (cm) Control L: kV L: mAs DV: kV DV: mAs condition 18 complete X 40 6.3 40 6.3 21 organs removed X 50 14 50 14 19 organs removed X 40 6.3 40 6.3 24 organs removed X 40 12.5 40 12.5 20.5 organs removed X 40 9 40 4.5 20 organs removed X 40 6.3 40 6.3 18.8 organs removed X 40 8 42 8

21.5 organs removed X 40 10 40 10 20 organs removed X 40 10 43 10 18 complete X 40 8 42 8 24 organs removed X 40 14 40 14 23 organs removed X 40 11 40 11 20 organs removed X 40 8 40 8 15 organs removed X 40 0.8 40 0.8 17.5 X 40 5.6 40 7.1 19 organs removed 40 8 42 8

15 complete 40 3.6 40 4.5 15.5 complete 40 4.5 42 4.5 11.5 complete 40 3.6 40 3.6 18 complete, good 42 8 42 8 13.5 ok 40 3.6 40 3.6 13.5 complete 40 3.6 40 3.6 16.3 complete 40 4.5 42 7.1 16 complete 40 4.5 43 4.5 15.5 complete 40 4.5 43 5

16 complete 40 3.2 40 3.2 18 complete 40 3.6 40 3.6 19 complete 40 8 40 8 17.5 complete 40 8 42 8 10 ok 40 3.6 40 3.6 16 complete 40 3.6 40 3.6 13.5 complete 40 3.6 45 5.6 18.5 complete 42 8 42 8 21.8 complete 48 12.5 52 12.5

16 complete 42 8 42 8