rearing and weaning of burbot, investigation of a specific

Faculty of Bioscience Engineering

Academic year 2011 – 2012

Rearing and weaning of burbot, investigation of a specific head deformity and histological development of the digestive tract.

Diederik Vanheule

Promotor: dr. ir. Nancy Nevejan

Tutor: dr. Alireza Shiri Harzevili

A thesis submitted to Ghent University, Belgium, in fulfillment of the requirements of the degree of Master of Science of Aquaculture.

Copyright

The author and the promoter give permission to put this thesis to disposal for consultation and to copy

parts of it for personal use. Any other use falls under the limitations of copyright, in particular the

obligation to explicitly mention the source when citing parts out of this thesis.

Gent, 3 May 2012

Promoter Tutor Author

dr. ir. Nancy Nevejan dr. Alireza Shiri Harzevilli Diederik Vanheule

i

Acknowledgement

I would like to thank several persons:

Firsts of all, my promoter dr. ir. Nancy Nevejan for here good guidance and dedication, dr. Shiri Harzevili

for his advice during the experiments and all the employees of the Laboratory of Aquaculture and

Artemia Reference Center.

Also I want to thank the employees of the KAHO Aqua-ERF institute, Wouter Meeus and Jurgen Adriaen

and of the INBO, Daniel De Charleroy and Johan Auwerx for their cooperation with the project.

I would like to thank Prof. dr. Annemie Decostere, Prof. dr. Koen Chiers, Prof. dr. Dominique Adriaens, dr.

Anamaria Rekecki and Annelies Declerq for their contribution in the investigation of diseased larvae.

Last, but not least my parents and girlfriend for their support during this Master study.

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Table of Contents

Acknowledgements ........................................................................................................................ i Table of Contents .......................................................................................................................... ii List of Figures ................................................................................................................................. iv List of Tables .................................................................................................................................. iv List of Graphs ................................................................................................................................. iv Abstract ......................................................................................................................................... v

Introduction ................................................................................................................................... 1

Objectives ...................................................................................................................................... 3

1. Literature .................................................................................................................................. 4

1.1. General aspects of the burbot ............................................................................................. 4

1.1.1. Classification and distribution ....................................................................................... 4

1.1.2. Morphology and growth under natural conditions ...................................................... 5

1.1.3. Biology and life cycle ..................................................................................................... 6

1.1.4. Reproduction in hatcheries ........................................................................................... 8

1.1.5. incubation of eggs .......................................................................................................... 9

1.2. Rearing and weaning of burbot larvae ................................................................................ 9

1.2.1. Food and feeding regimes, feeding trials....................................................................... 9

1.2.1.1. Properties of formulated feed ................................................................................ 10

1.2.1.2. Problems of siphoning and controlling good hygienic conditions .......................... 11

1.2.2. Parameters influencing larval rearing .......................................................................... 11

1.2.2.1. Temperature ........................................................................................................... 11

1.2.2.2. Light ......................................................................................................................... 12

1.2.2.3. Stocking density and feeding intensity ................................................................... 12

1.2.2.4. Interactions between different environmental factors .......................................... 12

1.2.3. Survival after the weaning ........................................................................................... 13

1.2.4. Rearing and weaning of cod larvae .............................................................................. 14

1.3. Cranial deformity/ hydrocephalus ..................................................................................... 14

1.3.1. Normal anatomy of the brain ...................................................................................... 14

1.3.2. Heavy metals leading to deformities ........................................................................... 15

1.3.2.1. Copper ..................................................................................................................... 15

1.3.2.2. Lead ......................................................................................................................... 18

1.3.2.3. Other heavy metals and combinations of heavy metals ......................................... 19

1.3.2.4. Chlorine and other chemicals ................................................................................. 19

1.3.3. Swim bladder problems leading to deformities........................................................... 19

1.3.4. Hydrocephalus and other brain anomalies in teleosts ................................................ 20

1.4. Histological development of the digestive tract ................................................................ 25

2. Materials and methods ........................................................................................................... 26

2.1. Rearing and weaning ......................................................................................................... 26

iii

2.1.1. Water quality parameters ............................................................................................ 26

2.1.2. Live feed phase ............................................................................................................ 27

2.1.3. Weaning experiment ................................................................................................... 28

2.1.4. Determination of total length and dry weight and statistics ....................................... 30


2.2.1. Heavy metal analysis of the water and larvae ............................................................. 31

2.2.2. Microbial and histological investigation of the bumps ................................................ 31


3. Results ..................................................................................................................................... 32


3.1.1. Water quality parameters ............................................................................................ 32

3.1.2. Live feed phase ............................................................................................................ 32

3.1.2.1. Survival .................................................................................................................... 32

3.1.2.2. Total length ............................................................................................................. 33

3.1.2.3. Dry weight ............................................................................................................... 33

3.1.3. Weaning experiment ................................................................................................... 34

3.1.3.1. Survival .................................................................................................................... 34

3.1.3.2. Total length ............................................................................................................. 34

3.1.3.3. Dry weight ............................................................................................................... 35


3.2.1. General aspects of the abnormality ............................................................................. 35

3.2.2. Analysis of the water.................................................................................................... 35

3.2.3. Macroscopic view of the cranial deformity ................................................................. 35

3.2.4. Microbiological and histological investigation ............................................................. 36


4. Discussion ................................................................................................................................ 43


4.1.1. Live feed phase ............................................................................................................ 43

4.1.1.1. Survival .................................................................................................................... 43

4.1.1.2. Growth .................................................................................................................... 43

4.1.2. The weaning experiment ............................................................................................. 44

4.1.2.1. Survival .................................................................................................................... 44

4.1.2.2. Growth .................................................................................................................... 45

4.1.2.3. Parameters influencing the weaning ...................................................................... 45

4.1.3. Economic feasibility of burbot farming ........................................................................ 46



5 References ................................................................................................................................ 50

6 Appendices

iv

List of Figures

Figure 1: The circumpolar distribution of the Lota lota ................................................................ 4

Figure 2: Adult burbot with the clearly visible barbell .................................................................. 6

Figure 3: Three different incubator types ..................................................................................... 9

Figure 4: Catfish embryo with irregular head shape after exposure to lead .............................. 18

Figure 5: Induced hydrocephalus in zebrafish embryo ............................................................... 21

Figure 6: Histology of hydrocephalus of channel catfish fry ....................................................... 23

Figure 7: Histology of normal and EMS salmon fry ..................................................................... 24

Figure 8: Deformed burbot larvae ............................................................................................... 25

Figure 9: Züger bottles for the weaning of the larvae ................................................................. 29

Figure 10: The automatic feeder ................................................................................................. 30

Figure 11: Macroscopic view of a clinically normal burbot larvae .............................................. 36

Figure 12: Macroscopic view of different intensities of the hydrocephalus ............................... 36

Figure 13: Histological section of the deformed head of a burbot larva .................................... 37

Figure 14: Histological detail of dilated ventricle of the hydrocephalus .................................... 38

Figure 15: Histological section of the deformed head of a burbot larva .................................... 39

Figure 16: Histological section of the head of a clinically normal larvae .................................... 40

Figure 17: Histological coupes of the digestive tract of a burbot larva ...................................... 41

Figure 18: Histological coupes of the digestive tract of a burbot larva ...................................... 42

List of Tables

Table 1: The different weaning combinations used by Trabelsi et al. (2011) ............................. 13

Table 2: The treatments during the live feed phase of burbot larvae ........................................ 27

Table 3: The sampling scheme during the live feed phase ......................................................... 28

Table 4: The survival rates after three weeks of the live feed phase.......................................... 32

Table 5: The total length of the larvae at different moments during the live feed phase .......... 33

Table 6: The dry weight of the larvae at different moments during the live feed phase ........... 33

Table 7: Cannibalism and survival rate of the larvae after the weaning trial ............................. 34

Table 8: The total length of the larvae after the weaning trial ................................................... 34

Table 9: The evolution of the dry weight of the larvae during the weaning ............................... 35

List of Graphs

Graph 1: The evolution of the total length during the live feed phase ....................................... 33

Graph 2: The evolution of the dry weight during the live feed phase ........................................ 34

v

ABSTRACT

Achieving a good rearing and weaning protocol of burbot larvae offers perspectives for restocking and

farming activities. This study was meant to improve the larval weaning protocols, but a three weeks after

the start of the live feed phase, the larvae developed cranial deformities. A high number of larvae was

affected and most of them died. These deformities were identified as hydrocephalus. Further histological

and bacteriological investigation could not identify the causative agents. In the culture water, a slightly

elevated copper concentration was found (4.6µg/L). Copper is known to cause deformities, but it is never

reported as the cause of hydrocephalus. A low number of larvae survived the live feed phase, but the

highest survival (5.5%) after three weeks was observed in the group that was continuously fed with

enriched (S-Presso, INVE Technologies) Artemia nauplii Instar II. During the subsequent weaning, the

larvae fed the artificial feed Aglonorse (Tromso Fiskeindustri) lost weight (-47%) and had a lower survival

than the larvae in the control group.

During the first 20 days post hatching (dph), the development of the digestive tract was investigated.

Mucous cells in the esophagus could be detected 8dph. Lipid vacuoles in the enterocytes were seen in

the anterior part of the intestine since food particles were present.

Keywords: Lota lota, burbot, larval rearing, weaning, hydrocephalus, histology, digestive tract.

1

INTRODUCTION

The burbot (Lota lota) is the only member of the cod family Gadidae which passes its complete life cycle

in freshwater. It has a wide geographical distribution in Europe and North America, but the natural

populations are declining. In many European countries the species is endangered. Several reasons for the

decline of the populations are mentioned: pollution, global warming and loss of habitat because of

altered management of the water bodies (Harzevilli et al., 2003, Jensen et al., 2008a). In Belgium, the

burbot has disappeared since the 1970’s and no natural recolonization is possible. This is due to the lack

of populations in the nearby water bodies and to the presence of migration barriers. It was decided to

create a reintroduction program. Based on genetic similarity, the populations living in the northern part

of France seems to be the best for restocking in the Flemish water (Dillen et al., 2005). Reintroduction of

yolk sac larvae did not lead to recapturing during the summer, indicating that this attempt has failed. If

juveniles were reintroduced during the fall, recapturing during the winter time was possible. The

juveniles were in a good condition and had grown fast. Only low numbers of juveniles could be captured,

this indicates that several reintroductions over several years will be needed to reinstall a population

(Dillen et al., 2008). The North American burbot population declined very fast in the Kootenai River and

Kootenay Lake. In the 1970s and 1980 the population collapsed. Until then this fish was important for

the traditional sustainable commercial fisheries and sport fishing (Jensen & Cain, 2009). This was mostly

due to alteration and loss of habitat. In contrast to declining populations in most habitats, Finland

maintains a commercial fishery on the burbot.

The disappearance of the burbot in many European waters created the necessity to improve the

technology of reproduction and larval rearing, both for farming and restocking purposes (Zarski et al.,

2009, Harzevilli et al., 2003). The inland coldwater aquaculture in Europe is mainly dominated by

rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), boor trout (Salvelinus fontinalis)

(Trabelsi et al., 2011) and carp (Cyprinus carpio) (Watson, 2008). For many fish farmers, diversification is

needed. Burbot seems to be a good candidate for many reasons: (1) it is a cold stenothermal species, (2)

it has a fast growth rate and a relative high market value, (3) the burbot has a good image to the

consumers, mostly because it belongs to the cod family, (4) it has white, boneless and tasty meat with

little fat (less than 1g per 100g edible portion) (Trabelsi et al., 2011) and (5) the liver of burbot is rich in

fish oil. In some countries, like in Canada (Thompson, 2008) and Scandinavia

(http://www.wildsight.ca/files/ling_cod_gone.pdf), the liver is considered a delicacy.

Aquavlan is an Interreg III project between Flanders and The Netherlands to stimulate aquacultural

activities in this region. Different institutes work on several aspects of fish, mollusc and salty vegetables

culture. One of the objective is to investigate the potential of burbot farming with respect to cultivation

techniques, marketing and economic feasibility. Our study contributes to the achievement of this

objective.

The big bottleneck for farming this species is the larval rearing. Little information is available on the

nutritional needs of the larvae. Feeding trials with commercial feeds were not very successful, although

commercial feeds would decrease the labor cost, space and disease transmission (Jensen et al., 2008a).

2

Jensen & Cain (unpublished data) reported high losses during the rearing of the larvae in hatcheries. It

was often seen that the chronic mortality persists into the juvenile stage. This is not uncommon for a

highly fertile species (Polinski et al., 2010b). On the other hand, survival of yolk-sac larvae in ponds till

autumn fry does not exceed 6% to 8% (Wolnicki et al., 2001).

Very little is known about specific diseases of burbot. Wolnicki et al. (2002) reported the parasitic

protozoan Chilodonella sp. during a weaning test. Some adult burbots collected in Norwegian lakes are

found to be infected with Mycobacterium salmoniphilum. Infected fish were characterized with external

lesions including skin ulceration, petechiae, cataract and exophthalmia. Granulomas could be found

mainly in mesenteries, spleen, swim bladder and heart (Zerihun et al., 2011). Dillen et al. (2005) made an

overview of the known parasites and fungi infecting the burbot (see appendix). Polinski et al. (2010a)

investigated the potential susceptibility and carrier status of the burbot towards five important fish

pathogens. Two viruses were challenged via immersion and three bacteria were injected in the young

fish. Burbot seems to be susceptible for the infectious hematopoetic necrosis virus (IHNV) and for

Aeromonas salmonicida. After injection of Renibacterium salmoninarum the burbot appeared to be an

asymptomatic carrier. After challenging to Flavobacterium psychrophilum and infectious pancreatic

necrosis virus (IPNV), the burbot seems to be refractory.

During a burbot larvae weaning experiment in the Laboratory for Aquaculture & Artemia Reference

Centre (Ghent University) last year, bumps on the head were macroscopically visible in most of the

larvae after some weeks of cultivation, but no further steps were taken to investigate this problem which

led to massive mortality. In the current study, the same problem manifested itself. Three weeks after the

introduction of the larvae, head deformities were seen in some larvae in all treatments. The affected fish

had difficulties to control their equilibrium but were still eating. Most of the affected larvae died after

several days, although some of the affected larvae recovered without any visible signs of abnormality. All

the deformations were similar and are characterized by a hemispherical semitransparent protrusion of

the cranium.

3

OBJECTIVES

(1) Nowadays restocking is often based on culturing young burbot with natural feed before restocking

them in rivers and lakes. Rearing young burbot with artificial feed would be a cost effective way to

facilitate the culture of burbot at higher densities and under more controlled conditions. The burbot is a

high quality fish with a good meat taste and quality. It is a possible candidate for aquaculture, but more

research is needed to obtain a good weaning and rearing protocol, before one can think of commercial

production. This study is meant to rear and wean the burbot larvae under different conditions to obtain

a good protocol to facilitate burbot culture for restocking and aquaculture purposes.

(2) Until now, the cranial deformity is only seen in burbot larvae who were reared in the Laboratory for

Aquaculture of the Ghent University for two consecutive years. A lot of the affected larvae died and this

interfered with the rearing. This problem is investigated in order to try to identify the causative agent(s).

If the etiology is known, measures can be made in order to prevent repetition and spreading to other

nurseries.

(3) Investigating the histological development of the digestive tract is a very important step in the

understanding of the functional relationship between the feeding, the digestion and the uptake of

nutrients. Histological changes during the ontogeny of the digestive system can help to understand the

feed requirement of the larvae. This investigation is the first description of the histological development

of the burbot larvae. This is a first step in the understanding of the onset of the feeding and the weaning

of burbot larvae.

4

1. LITERATURE REVIEW

1.1. GENERAL ASPECTS OF THE BURBOT

1.1.1. Classification and distribution

The burbot (Lota lota) is the only freshwater member of the otherwise marine family Gadidae (cods)

(Scott & Crossman, 1973). Burbots are distributed circumpolar (above 40°N) (Figure 1), there are two

subspecies: Lota lota lota with a Eurasian distribution and the North American Lota lota maculosa (Van

houdt et al., 2003).

Figure 1: The circumpolar distribution of the Lota lota (McPhail & Paragamian, 2000).

5

There is evidence that the North American burbot population has two different phenotypes. One with a

long and low caudal peduncle, while the second phenotype has a short and high caudal peduncle.

Although these 2 phenotypes can be found in different geographical areas, these different phenotypes

may be due to evolutionary adaptation to different water bodies. This can be concluded by the fact that

burbot with lower body condition can be found in rivers and reservoirs when compared to lakes (Fisher

et al., 1996).

The European burbot population can be divided into 3 subgroups: a Northern, a Western and Central

European group. This classification is based on the differences of the mitochondrial d-loop marker (Maes

et al., 2000). By using this classification, no differences are made between populations living in lakes and

populations from rivers, because the latter differentiation has probably occurred after the last ice age

(Dillen et al., 2005).

1.1.2. Morphology and growth under natural conditions

The burbot has an elongated, laterally compressed body with a flat head. There is one barbel at the tip of

the chin (Figure 2). Each nostril has a small single tube-like projection. The fish has two dorsal fins, the

cranial one is short and low, while the caudal is much longer. The anal fin is low and almost as long as the

second dorsal fin. The caudal fin is rounded. The mouth is wide and the lower and upper jaws contain

small teeth. Young burbot are often black, while adult fishes have olive to black vermiculations on their

dorsal side with a crème to white belly. The body is covered with very small cycloid scales. The scales can

not be used for age detection. They have a low swimming endurance because of their long cylindrical

shape. Large fish can not maintain themselves in a current velocity of more than 25cm/s for more than

10 minutes (McPhail & Paragamian, 2000).

Figure 2: Adult burbot with the clearly visible barbel (Coeck et al., 2008).

6

The American burbot can grow up to 8kg and reach a length of 1m. In Siberia, Muus & Dahlstrom (1971)

reported burbots of 25 to 30kg and ages of 15 to 20 years. In Slovakia the burbot will reach 25cm (100-

110g) in the first 13 months. This is in contrast to Alaska where in 12 months, the burbot will only grow

up to 10-13cm (Dillen et al., 2005).

The just hatched larvae have a slow growth until they start with the exogenous feeding. Thereafter,

during the first summer the growth was rapid before it is decreasing again towards the autumn. During

the following summer, the age-1 fish grew fast again. The growth of the age-0 burbot depends heavily on

temperature, as it increases with temperature of 4 to 18°C. The growth of older age burbots was less

temperature-dependent and their growth varied between the years (Kjellman & Eloranta, 2002).

1.1.3. Biology and life cycle

The burbot is adapted to lotic and lentic water bodies (Breeser et al., 1988). The adult burbot is a

nocturnal species which lies hidden under stones and roots or in holes during the day. At night they leave

their shelter to forage (Harzevilli et al., 2004). Muddy bottoms are avoided. The burbot is a benthic fish

that can reach extreme depths (until 700m). In the Alps, burbot can be found up to 2000m above sea

level (Beelen, 2009).

Burbot prefer water with a low alkalinity, a slightly low pH and low concentrations of potassium, calcium

and magnesium. The concentration of nitrogen, phosphorus and sodium may be elevated. Even small

oxygen depletions (due to relative high chemical and biological oxygen demand) can be withstood (Dillen

et al., 2000). The minimum requirement for oxygen is 4 to 6mg/l (Beelen, 2009).

Burbot can survive temperatures over 18°C. Although the burbot will lose weight when the temperature

is higher than 14°C. Spawning occurs in winter time at a temperature of 1 to 4°C (Harzevilli et al, 2004),

often under the ice cover. The semi-buoyant eggs are released in the depth of 0.5 to 1.5m (Zarski et al.,

2010). The eggs of the burbot are translucent and small (0.5-1.7 mm). Females reach their maturity at

the age of four years old. Males mature one year earlier. However the age of sexual maturations

depends on gender and geographical conditions. Generally, the northern populations reach their

maturity (4 to 7 years) later than southern populations (3 to 4 years) (McPhail & Paragamian, 2000).

The fecundity depends on the size of the female (Zarski et al., 2010). Beelen (2009) reports a productivity

of 600,000 eggs/kg body weight, while other authors report a much lower egg productivity of 100,000 to

220,000/kg (Baily, 1972) and 375,610 eggs/kg (Kouril et al., 1985). The temperature of the water must be

near or below 7°C to hatch. Hatching occurs after 140 to 150 degree days (°d) within the range of 0.5-6°C

(Aschenbrenner, Fischereilicher Lehr-und Beispielbetrieb Lindbergmühle, Germany, personal

communication). The embryos have a total length at hatching of 3.0 to 4.0 mm (Beelen, 2009). It takes

more than one week before the mouth starts to open and the digestive track to be active (Jensen et al.,

2008b). The overall survival up to the benthic juvenile stage is about 1%. This low survival is mainly due

to predation by other fish as well as by juvenile burbot and several other causes (unfertilized eggs,

diseases...) (Dillen et al., 2005).

7

Burbot spawn in lakes and rivers. In lakes, they spawn mostly in shallow reefs and shoals. There are some

suggestions that deep water spawning occurs in big lakes. In rivers, they spawn in areas with a low

velocity and behind deposition bars. The spawning substrate is sand, gravel, cobbles and also fine silt in

rivers (McPhail & Paragamian, 2000).

Not all adult burbot will reproduce each year. Several studies have been performed about this

concluding that 10-15%, 38%, 93% and even up to 100% of the adults don’t reproduce. This occasional

sterility is a natural phenomenon. A reason for this is that during these non-reproducing years the fish

are able to enhance their body fat mass. Another hypothesis explain that this phenomenon is due to

sterility caused by disease or chemicals (Dillen et al., 2005).

Under normal conditions the burbot will complete its life cycle in freshwater. In some areas it lives in

brackish lagoons and estuaries. The burbot enters the sea only rarely (McPhail & Paragamian, 2000).

Spawning is frequently proceeded by a long migration.

Larval burbot hatch between March and May in lakes in the northern hemisphere. The hatching occurs in

the profundal zone. After a few days, the larvae emerge to the water surface to fill their swim bladder.

After this the larvae will feed on plankton in the pelagic zone. The larvae spend two to three months in

this pelagic zone. Then they will settle to the profundal zone (Probst & Eckmann, 2009). In a German lake

the main food source for larvae were copepod nauplii, other zooplankton species like Bosmina,

Leptodora and Bythotrephes can exceptionally be eaten by the larvae. Burbot can begin to eat Daphnia

when they are about 20mm (Wang & Eckmann, 1998).

During this feeding in the pelagic zone, the larvae perform a daily vertical migration pattern. During the

day the larvae can be found in the deeper part of the water, while at night they are closer to the surface.

There are several explanations for this behaviour: (1) larvae follow their zooplankton prey (Miler &

Fischer, 2004), (2) larvae become negative phototactic to counteract the predation risk related to

increasing body size and pigmentation (Probst & Eckmann, 2009), (3) this migration can be assumed as a

kind of transition between the pelagic and benthic life style (Miler & Fischer, 2004). As the summer

progressed, the mean depth of the fish increased. The minimum depth of the larvae was 4.5m. As the

young-of-the-year (YOY) grew from May to August, they experience decreasing dial maximum light

intensities. At the end of August YOY can reach 70m (Probst & Eckmann, 2009).

For adult burbot the optimal temperature for growth is estimated on 14.4°C. This is the same

temperature as for maximum food uptake (Hofmann & Fischer, 2003). Higher temperatures have a

positive effect on growth, but negative effects on the digestion because of the increased cost of

digestion (may even exceed the energy obtained from the food) and the post-prandial increase in oxygen

consumption. Adults were still observed to feed at a temperature of 23.4°C. So it is possible that adult

burbots are able to feed in warmer waters during the summer (Pääkkönen & Marjomäki, 2000). Juveniles

have the highest growth and food consumption at slightly higher temperature as for adults (Hofmann &

Fischer, 2003).

In lakes, during the month August, the benthic juveniles appear in the littoral zone. It is during that time

that the juveniles settle in the stony areas (Hofmann & Fischer, 2001). Fischer & Eckmann (1997) showed

8

that the burbot use this littoral habitats only during a short part of their life cycle. The most fish who are

bigger that 14cm (burbot of 1 year old) migrate during the summer to the more deeper profundal zone,

where they mature.

Burbots are most active during the winter. Although it’s a poor swimmer, it is an efficient predator

especially in the winter. High temperatures during the summer are linked to a reduction in food uptake

and to starvation. During the summer the diet shifts from fish to invertebrate dominated. This coincides

with a decrease in gut content and a dissipation of the energy stores. Starvation causes a lower energy

turnover, in order to withstand the metabolism-activating summer temperatures (Binner et al., 2008).

This is in contrast to diet studies in a subarctic lake in Finland. It was concluded that the burbot prefer to

eat more molluscs, insect larvae and benthic crustaceans when the lake was covered with ice. During the

open water season (July-October) more fish and detritus was ingested. The bigger the burbot, the more

it likes to eat fish (Tolonen et al., 1999). Adults are capable to hold large amounts of food in their

stomach (>30% of their body mass) (Hofmann & Fischer, 2003). The quantity of gastric acids secreted by

the burbot is strong temperature dependent. At a temperature of 1°C the quantity is much larger that at

10°C (Zarski et al., 2010). Subadult burbot grow fast until the onset of the sexual maturation. After this

the growth rate declines. (McPhail & Paragamian, 2000).

1.1.4. Reproduction in hatcheries

Reproductive processes in fish are controlled by environmental conditions (e.g. photoperiod and the

temperature of the water). For many teleosts the photoperiod is the most important factor in the

reproductive cycle, while the temperature plays a dominant role in the maturation of the gametes,

ovulation and spawning. Artificial reproduction in many freshwater fish need a hormonal injection to

induce the final maturation of the gametes. In hatcheries spontaneous spawning of female and male

burbots can be done, after which fertilized eggs can be siphoned out of the tank. Artificial spawning can

be done by a gently pressure on the abdomen of matured females. The males can also be stripped and

fertilisation can take place after mixing the sperm with the eggs after which water is added (Zarski et al.,

2010).

The temperature of the water of the burbot should be held continuously at a quite high level to prevent

spontaneous breeding in the tanks. Adult burbot behave calm at a low temperature (2°C), but an

important disadvantage of holding fish at low temperatures is the long period of maturation. This is very

exhausting for the fish and every day small portions of eggs were collected and therefore several

incubation devices are needed. If each proportion of eggs is hatched at different moments,

differentiation in growth (and food competition and cannibalism) can be seen. Holding fish at low

temperature for a long period may cause difficulties in many fish farms. Synchronisation can easily be

done by hormone treatment or thermal stimulation. This can improve the work load during the spawning

season.

9

1.1.5. Incubation of eggs

Jensen et al. (2008a) found that the best survival could be obtained when Imhoff cones were used to

incubate the eggs. This type of incubator performed better than mini egg-hatching jars and much better

than McDonalds-type jar (Figure 3). Controlling the flow in the incubator is a crucial aspect. When the

flow in these jars is too low, sedimentation and contamination can be seen, but if the flow is too high

eggs could escape these jars. Similar results with these jars were seen with semi-buoyant eggs of grass

carp (Ctenopharyngodon idella) (Jensen et al., 2008a).

Figure 3: Three different incubator types used by Jensen et al. (2008a).

To control fungi of the family Saprolegniacea during egg incubation 2 chemicals can be used. Formalin at

a concentration of 1,667mg/l and hydrogen peroxide at a concentration of 500mg/l. Both treatments

take 15 minutes per day. If using one of these products, the egg survival can increase up to 200%.

Concentrations of 1,000mg/l formalin and 250mg/l hydrogen peroxide also increase the egg survival, but

are insufficient to control complete fungal development on the eggs (Polinski et al., 2010b).

1.2. REARING AND WEANING OF BURBOT LARVAE

1.2.1. Food and feeding regimes, feeding trials

Under natural conditions, larvae of 3 to 10mm long fed mainly on copepods and cladocerans. Those of

11 to 20mm long prefer zooplankton and dipterans. Larvae of 21 to 30mm eat mainly zooplankton (60%)

and amphipods (30%). Those of 31 to 40mm eat mainly amphipods (85%). Larger fingerlings prefer

amphipods and insects (Ryder & Pesendorfer., 1992). If burbot larvae are fed a mixture of the globular

rotifer Asplachna sp., spined rotifers and copepod nauplii, they will select the Asplachna, probably

10

because the copepod nauplii move too fast and the protuberances of the spined rotifers may hinder

their uptake (Nunn et al., 2007).

The mouth opening of the larvae is relative big in comparison with their small body (Wolnicki et al.,

2002), which makes it possible for them to feed on small Artemia nauplii as first feed under hatchery

conditions. Jensen et al. (2008a) started feeding the larvae when the complete alimentary tract (from

mouth to anus) was apparent and after yolk sac absorption. An important issue to start the feeding is

using larvae either on the tenth days post hatching (dph) or after the yolk sac resorption (Zarski et al.,

2009).

Harzevilli et al. (2003) tried different larval feeding regimes with natural feed: Chlorella sp. (green algae),

Brachionus calyciflorus (10 rotifers/ml) and Artemia nauplii (4 nauplii/ml). Highest survival (69,20%) after

35 days of the feeding experiment was recorded in the group which received 3 days green water and

rotifers, followed by only rotifers (day 4 to day 10) and Artemia (day 10 to day 35). Lowest survival

(24,90%) was observed in the group receiving Artemia from the beginning. Similar results were also

obtained by other authors (Kujawa et al., 1999, Jensen et al., 2008a). These results indicate strongly that

the quality of the starter food is crucial for the later development of the burbot larvae and that small

preys are essential for first feeding. In the gut of the larvae, a green mass was observed in the first 3

days, demonstrating their preference for phytoplankton in the first days (Vachta, 1990). This is similar to

cod (Gadus morhua), where filter feeding of phytoplankton is seen by drinking and using visceral arches

as a trap (van den Meeren, 1991).

Adding microalgae to the larval tanks has positive effects: these algae can supply essential nutrients,

stimulate the digestive system or influence the microflora of the larvae (Howell et al, 1998). But one of

the major disadvantages of using the microalgae is the labour-intensive and expensive cultivation

(Harzevilli et al., 2003).

Weaning trials done by Jensen & Cain (2009) indicate that weaning can be done after 30 days of feeding

live diets (about 45 dph). Feeding Artemia beyond that 30-day period resulted in a greater weaning

success. First feeding of larvae with artificial feed or decapsulated Artemia cysts resulted in high

mortality and deformations (Zarski et al. 2009).

Similar results were obtained with two feeding trials with the North-American burbot L. Lota maculosa.

Highest survival and growth was obtained with larvae fed only live preys during the first weeks. Weaning

must be proceeded by a live feed phases. This indicates that the larvae have a high level of live-prey

dependence and early use of artificial diets will have a very low level of success (Jensen et al., 2008a).

1.2.1.1. Properties of the formulated feed

Wocher (2010b) investigated three different commercial feeds: Gemma Micro (Skretting), Aglonorse and

AlgoNorse Extra (Tromso Fiskeindustri) after an Artemia phase of 25 days. The larvae fed Gemma Micro

had the lowest chance to survive, but these larvae performed the best growth (final dry weight and total

11

length). Highest survival and lowest growth was obtained with Aglonorse. No ideal artificial feed without

compromising survival and growth could be obtained.

If juvenile cod (about 2.5 month old) were fed a mixture of the same feed with 5 different dry weight

contents (35, 42, 54, 65 and 95%), the highest ingestion rate could be found with the diet containing the

highest water content, even when the gut contents are expressed in term of dry weight. There are two

main hypotheses to explain this behavior: (1) moist feed has a higher palatability and (2) the moist feed

is more available for the fish because of a lower sinking rate. Also a moist feed has a softer texture which

resemble more to the natural live prey. If this experiment was repeated with smaller larvae, the dietary

moisture content did not influence the ingestion rate, but the growth was better in the group fed the

diet with the highest moisture content (Ottera et al., 2003). These findings are in contrast to Trabelsi et

al. (2011): the conclusion was made that moist feed is not adequate for burbot larvae. Although cod is a

closely related species, there are some fundamental differences in the rearing process.

One of the main problems associated with artificial is the high leaching rate. Measurements of leaching

done in commercial and experimental feed showed leaching of proteins from 18% to 42% within two

minutes. This probably corresponds to the water soluble fraction of the protein in the feed. The free

amino acids leached at a higher rate, because of their lower molecular weight. Another problem is the

form of the nutrients. It is difficult for digestive enzymes to reach the intact non-water soluble proteins if

there is no pepsin activity and acidification (like in stomachless larvae). A larger fraction of the proteins

in live feed is water soluble and has a lower molecular weight. This increases the efficiency of the

digestion process. This problem can be solved if hydrolysed proteins are supplemented to the artificial

feed. But hydrolysation of the protein increases the water solubility and thus enhance the leaching

(Hamre et al. 2009).

1.2.1.2. Problems of siphoning and controlling good hygienic conditions

When Jensen et al. (2008a) used commercial feeds, high waste accumulations and subsequent growth of

fungus, chronic larval mortality and gill disease were observed. It took 6 to 8 hours per day to clean a 17

150l tanks via siphoning. This was in contrast to the work (1 to 2h) needed to clean the same tanks when

solely Artemia was given. A high feeding rate combined with their bottom dwelling behavior and high

rearing densities caused an accumulation of waste feed, feces and fungus on the bottom of the tanks.

The mortalities seen during the weaning period were typically emaciated larvae with flared opercula.

Also a lot of juveniles with lordosis and scoliosis were seen.

1.2.2. Parameters influencing larval rearing

1.2.2.1. Temperature

Temperature is a very important factor in the development of young fish. An incubation temperature of

less than 7°C (with an optimal temperature of between 2-6°C) is needed to have a good development of

12

the embryo (Harzevilli et al., 2004). In contrast to that narrow range of low temperatures, larvae can be

kept at a wider range of temperatures, even up to 20°C. This suggest that embryos and larvae have

different physiological tolerances. Burbot larvae start to eat above 8°C. .

Some tests dealing with different temperatures during the larval rearing have been performed. It is

difficult to compare these results because the experiments were performed under different conditions

(feed, light scheme...). In general, the lower the temperature within the range 12-20°C, the higher the

survival of the larvae during the first feeding (Harzevilli et al., 2004; Wolnicki et al., 2001; Wolnicki et al.,

2002). There is an interaction of light: Harzevilli et al. (2004) obtained a higher survival under continuous

light conditions, this effect was more important at higher temperatures. While at lower temperatures

the survival of the larvae is higher, at higher temperatures the growth (total length and wet weight) is

higher. The highest larval length was found at 20°C under complete darkness (Harzevilli et al., 2004).

1.2.2.2. Light

Continuous light conditions are very important during the first weeks of the exogenous feeding. This has

a positive influence on the survival and growth (Harzevilli et al., 2004; Wocher et al., 2010a). This can be

explained by the fact that the larvae are visual hunters and under light conditions they have a greater

opportunity to see and catch their prey. Young larvae are positive phototactic (up to 4cm), but at a later

stage of development they become negative phototactic (Girsa, 1972). This correspond with Trabelsi et

al. (2011): a shorter photophase increased the survival with older larvae. This may be due to the fact that

Harzevilli et al. (2004) and Wocher et al. (2010a) performed tests with much younger larvae with possibly

less developed eyes, thus perceiving the light different than for older individuals (Trabelsi et al., 2011).

1.2.2.3. Stocking density and feeding intensity

Higher growth can be achieved when the stocking density is lower. A lower stocking density can be the

result of increased previous mortality (Harzevilli et al., 2004). Higher stocking densities have also a

beneficial effect on the feed intake. Uptake of a prey or feed particle by one larva can induce the same

effect with others.

The higher the feeding intensity, the higher the growth and survival (Wocher et al., 2010a). The more

feed available in the water, the greater the chance that a larvae is able to catch it and therefore the

larvae need to spend less energy. But one must pay attention that an excess of feed will lead to spoilage

which is economically not favorable and which lead to deteriorating water quality.

1.2.2.4. Interactions between different environmental factors

It is still not clear if co-feeding has a positive effects on survival and growth of burbot larvae. The effect

of a co-feeding period is inextricably dependent on several factors such as length of the co-feeding

13

period, type of artificial feed. Meeus et al. (2011) reported equal survival and specific growth rate after a

comparative experiment with co-feeding and non-co-feeding groups. These finding are in contrast to

Trabelsi et al. (2011) who reported better performances after a longer co-feeding phase.

Trabelsi et al. (2011) performed a fractional factorial design with 12 different factors: seven different

feeding factors (co-feeding, feeding period, food texture, feeding frequency, food origin, feeding rate

and weaning duration), four different environmental factors (photoperiod, light intensity, salinity and

wall color) and different stocking densities. 16 different combinations were tested (Table 1). The

experiment started with two month old larvae (182mg). Some correlations were found. The salinity (5%)

and food texture acted synergistically on the survival rate. The longer the weaning period (gradually

decrease of Artemia and increase of artificial feed) the better the survival. Two different artificial feeds

were tested Lansy (INVE) and Aglonorse. Although they have a similar biochemical compositions, the

rates of cannibalism were 5% respectively 21%. Lansy must have other features that increase the

acceptability. These features include: residence time in the water, palatability, ingestion rate and

nutrient leaching. One of the reasons why Lansy contributes to a lower rate of cannibalism, is by

decreasing the heterogeneity in size and mouth gape.

Table 1: The different combinations of factors studies by Trabelsi et al. (2011).

1.2.3. Survival after the weaning

Jensen et al. (2008a) obtained a survival rate of 9% after a weaning period of 56 days when started with

larvae of 41dph. Trabelsi et al. (2011) obtained better results with some of the tested combinations, this

is probably because the weaning period started with older larvae (two months old). Zarski et al. (2009)

tried to wean the larvae much earlier, resulting in a lower survival. During a period of four years, Wocher

(2010b) performed several experiments for weaning the larvae. The maximum survival of the weaning

period obtained in one year is 31.0%.

At the end of the weaning period, when the larvae were about 5g, the average survival (from hatched

larvae onwards) was 16%. This is a global result obtained by several experiments over two years. This

survival is similar by those obtained in weaning cod and pikeperch (Sander lucioperca) (Wocher et al.,

2010a).

14

1.2.4. Rearing and weaning of cod larvae

Cod is a close related fish species that has important economic perspectives. More research has been

performed on this species than on burbot. The freshly hatched cod larvae are about 4 to 5 mm long. In

intensive culture, the most used method of rearing this larvae is by feeding them rotifers until 20 to 30

dph. After this period, they are fed Artemia nauplii and/or formulated feed. Under semi-intensive and

extensive conditions, the larvae mainly prey on copepod nauplii and will switch to older copepod stages

as they grow. The growth rates under intensive conditions are lower than those under extensive

conditions. Finn & van der Meeren (unpublished) found an average growth rate of 10% for the first 50

day period of exogenous feeding. While for the same period a growth rate of 13% can be obtained under

extensive conditions (Hamre et al., 2006). Puvanendran et al. (2006) investigated if cod larvae could be

weaned faster when higher temperatures were used. Because the live feed production is costly and

labour intensive. Their results suggested that a possible optimal weaning temperature for larval cod is

between 11.5 and 14.5°C.

1.3. CRANIAL DEFORMITY/ HYDROCEPHALUS

Malformations have important economically implications, because of lower larval performances

(swimming, growing, survival…) and a negative effect on the commercialization of the final product,

because the shape of the fish will differ from wild-catch fish (Boglione et al., 2001). The most common

described body malformations are: vertebral, craniofacial, cardiovascular and yolk sac deformations

together with edema and hemorrhages (Jezierka et al., 2000).

In general, developing embryos can be considered as particularly sensitive to all kinds of environmental

pollution and natural stressors. The larvae show a wide range of gross and minor morphological

malformations (Osman et al., 2007). More specifically, these malformations during embryonic

development can be due to (1) heavy metals, (2) low oxygen levels, (3) altered pH, (4) irradiance by

gamma-rays (Jezierka et al., 2000) and (5) to high water flow (Jensen et al., 2008b).

Fish can react on stress by adapting to the new situation. It can change its behavior or physiology or

both. This adaptation is a response to compensate the changed environment, which favors the survival.

The time to adapt the new environment is depending on species and environmental stressor. But in

general it takes about three weeks for an organism to acclimate to a changed environmental situation

(Block, 1977).

1.3.1. Normal anatomy of the brain

As with other vertebrates, the brain of teleosts can be divided into five different main regions:

telencephalon, diencephalon, mesencephalon, metencephalon and myelencephalon. Differences in

relative proportion of the regions exists when compared to mammals. The telencephalon mainly consists

out of the olfactory lobes and cerebrum. The lateral ventricles are small. The diencephalon has a saccus

15

vasculosis and saccus dorsalis, highly vascularized structures producing cerebrospinal fluid (CSF). Optic

lobes and the cerebellum form the main part of the mesencephalon respectively the metencephalon.

Unique secretory proteins (ependymins) are produced by the meninges of all teleosts. These form the

main protein component of the cerebrospinal fluid and play a role in neuronal regeneration (Speare &

Frasca, 2006).

1.3.2. Heavy metals leading to deformities

1.3.2.1. Copper

Copper is a naturally occurring essential metal for all organisms. But is becomes toxic when the natural

concentration (<0.05µmol/l = 3.2µg/l) is exceeded. Higher concentration of copper interfere with the

branchial ion transport, alter plasma ion concentrations, hematologic parameters and enzyme activities.

Also immune suppression, vertebral malformations and neurological problems can occur due to too high

levels (Stouthart et al., 1996).

The 90-percentil values (the concentration that is exceeded in 10% of the samples) for copper in the

Dutch part of the Rhine, Meuse and Scheldt for the year 2004 were respectively 7.7, 9.8 and 6.9 µg/l

(Compendium, 2005). In uncontaminated water bodies, the concentration of dissolved copper is ranging

from 0.5 to 1.0 µg/l. The Environmental Quality Standards (EQS) for the European Union gives the

maximum limit of the copper concentration to protect fresh waters from acute contamination. Copper

toxicity is water hardness related. For soft freshwater the EQS is 1µg/l (Handy, 2003). Copper

concentrations of 10µg/l to 150µg/l or more under soft water conditions can cause acute toxic effects.

Chronic toxicity concentrations for freshwater fish vary between 2µg/l and 14µg/l (Brix et al., 2001).

Copper is a noble metal. The main toxic form of copper is the cupric ion. This ion can complex with

organic and inorganic compounds. This reduces the cupric ion concentration and its toxicity. Copper is a

naturally occurring element and is widely distributed. The emissions from human activities are

substantial. The main human emission source is the disposal of coal ash and the spreading of industrial

and municipal wastes on land (Alberta Environmental Protection, 1996). Copper is an essential trace

metal. It is a structural component in metalloenzymes and in respiratory pigments (hemoglobin). There

are several methods to determine copper in samples: (1) atomic absorption spectrometry (AAS), (2)

inductively coupled plasma atomic emission spectrometry (ICP-AES), (3) inductively coupled plasma mass

spectrometry and (4) colorimetric methods.

All the possible physiological reactions due to copper intoxication can be related to: (1) up-regulation of

metabolic processes or enzymes, (2) altered cellularity in organs and (3) altered hematopoietic

responses. After copper exposure, a loss of circulating lymphocytes is seen. This can not be due to

hemodilution because an increase in circulating neutrophils occurs. A controlled redistribution of white

blood cells in the immune system is probably due to cortisol release (Handy, 2003).

Having a different sensitivity towards metals, like copper, is inherent to the species. Three main reason

can explain this: (1) copper is an essential metal and the aquatic organisms have developed strategies to

16

regulate the internal copper level. These homeostatic regulatory strategies can be a storage or active

regulation of the excretion of the excess of copper. Most species are using a combination of storage and

excretion. (2) Some organisms can reduce the membrane permeability if the copper level has increased.

(3) Allometric considerations such as the quantity of permeable membrane relative to the body volume

(Brix et al., 2001).

The central compartment for the regulation of the whole body copper concentration is the liver. It

controls the circulation and excretion of copper. Chronic sub-lethal exposure leads to a stimulation of

the hepatic excretion (Handy, 2003). Regional differences can be found in the copper accumulation in

the body in fish (Kalay et al., 1999). Differences in accumulations are even found in the brain (De Boeck

et al., 1995).

Brix et al. (2001) assessed the sensitivity of aquatic organisms to copper using species sensitivity

distributions (SSDs). With this method, possible models to chemical exposure and effects are fitted. This

is done to estimate the number of aquatic organisms potentially at risk and expand on existing

probabilistic risk assessments methods. But for fish, they were unable to identify trends in the relative

sensitivity according to feeding habit or phylogenetic relationship. As an exception, a general trend was

seen that temperate cold water fish species are more sensitive than temperate warm water species,

which in turn are more sensitive than tropical species.

Cellular and physiological changes due to elevated levels of copper are not a random cascade of actions,

but are a temporally ordered and synchronized series of events. These events are integrated in the

bodies in order to enhance survival of metal toxicity. Copper is also involved in other neurohormonal

controlled processes: copper can chelate with melatonine and thus influence the circadian rhythm

(Handy, 2003).

Copper is a disruptor of the osmoregulation in fishes. The acute toxicity of copper is characterized by a

reducing influx of sodium due to the inhibiting activity of copper on the Na+/K+-adenosine triphosphatase

pump on the gill chloride cells. Cu2+ also replace the calcium ion at the tight paracellular junction

between the gill cells. This results in an increased efflux of sodium. There is thus a net loss of sodium,

which can lead to mortality due to cardiovascular collapse. A 30% drop in plasma sodium can be

associated with mortality. The whole-body sodium concentration is a common used parameter to detect

osmoregulatory malfunctions, like those due to heavy metals and low pH. Toxicity towards copper

depends on 2 important parameters: concentration and duration of the exposure. Fish can acclimate to

low concentration, chronic copper exposure. But fish can also recover from acute toxicity: copper

concentrations of 0.47µM (29.6µg) can induce a whole body sodium loss in fathead minnow (Pimephales

promela) of about 70% within 12h of exposure. Within a period of 48h the fish is able to recover its

whole body sodium concentration (Zahner et al., 2006).

Incubation of heavy metals like copper and lead at toxic levels induce a wide range of deformities.

Common carp (Cyprinus carpio L.) eggs incubated with copper and lead concentrations of 0.2mg/l and

2.0mg/l respectively are able to induce malformations in larvae. More than 80% of the malformations

were vertrebral anomalies. Only a small part of the larvae showed craniofacial, cardiovascular and yolk

sac malformations or edema. Larvae originating from different parents showed differences in

17

malformations. Probably those differences can be related to parental factors or by the conditions of

artificial spawning. The malformations may be caused during the embryonic development or can be due

to the laborious hatching. Malformations were also seen in the control group, but at a much lower

prevalence (Jezierka et al., 2000).

Copper is also known to cause brain disorders in fish. Handy & Kay (unpublished data) reported brain

damage with edema, vacuolization of the tectum close to the hypothalamus and vacuolization and

appearance of a few necrotic cell bodies throughout the brain. These lesions were seen in rainbow trout

that has been exposed to 1000mg copper per kg of feed during six weeks. Chronic copper exposure can

affect epithelia with an increase of goblet and mucous cells, vacuolization in the sensory epithelium. This

was seen after exposure of several months at 22µg/l (Saucier et al., 1991). Beside these more specific

symptoms and lesions, several other abnormalities can be seen: reduced feeding and swimming

performances, increased respiratory stress, lethargy, cramps, ataxia.

For most species, embryos seems to be less sensitive than larvae and early juveniles (McKim et al., 1978).

It is reported that a copper concentration of 4.6µg/l can have an effect on fry. Rainbow trout fry exposed

to that concentration had reduced growth (in weight and length) and an elevated body copper

concentration after 20 days (Marr et al., 1996). A virtual calculated decrease in growth with 1% can be

seen in rainbow trout fry after a 56 day exposure experiment with a concentration of 1.1µg/l (Hansen et

al., 2002). While only a decrease in length for the sockeye and pink salmon alevins during the yolk sac

phase was seen at 6µg/l. After yolk absorption, no effect could be seen (Servizi & Martens, 1978). No

deformities were reported with these exposure experiments.

Whole body copper concentrations are positively correlated with copper exposure concentration:

rainbow trout fry contained 5.82µg copper per gram dry weight after twenty days of copper exposure to

4.6µg/l. Twenty days later, the concentration increased to 8.90µg/g. Fry from the control group (<0.9µg

Cu/l) had a body copper concentration of 3.45µg/g and 3.68µg/g after twenty respectively forty days of

exposure. Fish body weight is negatively correlated with the copper exposure concentration. The

reduced growth caused by sub-lethal copper exposure can be due to the metabolic need to detoxify the

metal and the decline in feed uptake (Marr et al., 1996).

There is a very fast uptake from the water by fish. In rainbow trout, copper is rapid transferred to blood

plasma. The liver and gills are the organs with the most accumulation in rainbow trout and carp. Also in

the muscles of rainbow trout, higher concentrations can be found (Alberta Environmental Protection,

1996).

The clearance of copper is a fast process, the halftime was 1.6 to 4.8 hour in pumpkinseed sunfish. The

bigger the fish, the faster the clearance (Anderson & Spear, 1980). Copper loss from the liver takes

longer than from gills, as was seen in stone loach and the sunfish (Alberta Environmental Protection,

1996). The elimination of copper out of carp was very fast if the carp was brought in an EDTA solution

(Muramoto, 1983). A possible pre-exposure to copper to rainbow trout reduced the toxicity. But within

seven days, this tolerance was lost (Dixon & Sprague, 1981).

18

There is a complicated effect of the pH on the toxicity of copper. If the fish species is sensitive to pH,

there will be a higher toxicity at a low pH. But if the species is not sensitive to pH, the toxicity will be

higher at higher pH values. This is due to the diminished competition of protons and Cu2+ ions at the

receptors sites compared to competition at a lower pH value (Alberta Environmental Protection, 1996).

When the pH is neutral or basic, most of the copper will be connected with carbonate and hydroxide. But

when the pH decreases, the amount on free copper ions will increase. At pH 7.6 32% of the total copper

is present as Cu2+, while at pH 6.3 this amount increases to 92%. Lowering the pH clearly increases the

toxicity of copper (Stouthart et al., 1996).

In general, the harder the water, the lower the toxicity of the copper. Increasing the alkalinity reduced

the toxicity to channel catfish (Straus & Tucker, 1993), but didn’t affect the copper toxicity to fathead

minnows (Nelson et al., 1986). Increasing copper toxicity occurs at higher temperatures for several fish

species (e.g. goldfish) and at lower dissolved oxygen levels. A decrease in toxicity can be seen at higher

H2S and suspended solids levels (Alberta Environmental Protection, 1996).

1.3.2.2. Lead

An irregular head shape could be detected in African catfish (Clarias gariepinus) larvae of which the eggs

were exposed to increased lead concentrations in the water (500mg/l). The malformed heads had

swelling-like protrusions on the lateral and anterior side. This malformation was lethal and the larvae

died soon after hatching. Three other major categories of obvious abnormalities were seen: pericardial

edema, yolk sac edema and notochordal effects. The latter one was characterized by different forms of

spinal cord curvatures: lordosis, kyphosis and scoliosis (Osman et al. 2007). The maximum acceptable

toxicant concentration for trout fry under soft water conditions is 14.6µg lead/l (Davies et al., 1976).

Figure 4: Newly hatched catfish embryo with irregular head shape after exposure to 500µg/l lead nitrate.

YS: yolk sac, S: swelling. Scale bar = 1mm (Osman et al., 2007).

19

1.3.2.3. Other heavy metals and combinations of heavy metals

Combinations of heavy metals can have additive or less additive effects depending on fish species and

age (Alberta Environmental Protection, 1996). Malformation of the vertebral curvature, body and yolk

sac are non specific for any metal, these malformations were seen in fish exposed to other heavy metals

(Lugowska, 2007). Enhanced levels of heavy metals can also affect gills: from mild degenerative lesions

to necrosis and loss of filament with hemorrhages can be seen (Pandy et al., 2008). Since other heavy

metals are probably not the cause of the cranial deformity, no further attention is paid on this topic.

1.3.2.4. Chlorine and other chemicals

Chlorinated tap water was used in the experiment. The threshold levels for chlorine induced killing are

0.04 and 0.05 mg/l for the fathead minnow (Zillich, 1972). For the protection of most aquatic organisms,

the chlorine level should not exceed 2µg/l in water bodies continuously treated with chlorine (Brungs,

1973). Intolerant cold water fish like trout is very sensitive to chlorine: after seven days incubation, half

of the population died at 10µg/l to 80µg/l (Roseboom & Richey, 1977). The effect of chlorine to the fish

is dependent of temperature, time of exposure and dose (Block, 1977). Residual chlorine can be

detoxified by adding sodium thiosulfate. It is reported that pesticides (like carbaryl and endosulfan),

aflatoxins and brevetoxin are known to induce lesions is brain (Speare & Frasca, 2006), but there are no

indications that these chemicals could be present in the experimental unit.

1.3.3. Swim bladder problems leading to deformities

Deformation of the notochord due to pressure from the swim bladder is a malformation that occurs

frequently in intensive cod farming systems. The malformation is characterized by an slight upward tilt of

the head. Consequently, the dorsal body contour at the transition of the head and trunk is indented. The

whole pathological process is associated with malformation of the neurocranium, the cranial region of

the vertebral column and the cranial part of the epaxial lateral muscles. Histological research showed a

deformation of the notochord. The deformation process begins in the transition period to live feed

(approximately 7dph). In most cases this deformation is caused by an increased pressure from the swim

bladder on the notochord. This can be caused by hyperinflation of the swim bladder or pathologically

expanded abdominal organs (Grotmol et al., 2005). Hyperinflation of the swim bladder can be due to (1)

hypersaturation of N2. Degassing columns can prevent this (Planas & Cunha, 1999). (2) A sudden

decrease of the ambient pressure, this can be due to upwelling aeration. If the larvae are caught in the

current, there will be a rapid upwards vertical migration causing a drop in pressure (Grotmol et al.). (3)

Infection of the swim bladder (Rekecki et al., 2011).

20

1.3.4. Hydrocephalus and other brain anomalies in teleosts

Hydrocephalus is the brain anomaly characterized by abnormal flow of the cerebrospinal fluid (CSF)

causing pathological magnification of the ventricles. There are two types of hydrocephalus:

communicating and non-communicating. The former one is caused by an increase of the production or

decrease of the absorption of the CSF resulting in an overall increase of the CSF quantity. The latter one

is caused by an obstruction (e.g. tumor, infection…). Hydrocephalus can be the result of genetic

deviations, congenital malformations, infections, tumors and hemorrhages (Crews et al., 2004). There

are different pathogeneses of a hydrocephalus: (1) a stenosis or obstruction in the ventricular system, (2)

a failure in the formation of the dorsal cranium, (3) a developmental failure of the formation of

fontanels, (4) an increased production or decreased absorption of the cerebrospinal fluid (Muench et al.,

1997).

Pathological events like hemorrhages, infections and brain trauma can induce the formation of

hydrocephalus via oxidative stress. The endogenous antioxidant defense and repair mechanisms are

exceeded by the production of reactive oxygen species and free radicals. Because of that, irreversible

damage to lipids, DNA and proteins occur (Socci et al., 1999).

The development of a chronic hydrocephalus in humans is associated with higher levels of ferritin and

inflammatory cells after a subarachnoid hemorrhage. Metabolism of blood in the subarachnoid space

results in the formation of bilirubine and iron. The latter is then detoxified to ferritin. The ferritin levels

are not correlated with the amount of blood, but with the intensity of the inflammatory reaction (Suzuki

et al., 2006). After intraventricular hemorrhages similar events are seen, also iron was hypothesized as

playing a role in the formation of hydrocephalus and brain damage (Chen et al., 2010).

Deformities can be caused by parasites and several stressors like nutritional deficiencies, extreme oxygen

or temperature fluctuations and chemicals. But none of them are being associated with hydrocephalus in

teleosts (Muench et al., 1997). Infectious diseases affecting the brains can be regional or be a part of a

systemic infection. Pathogens can use a retrograde way via cranial nerves to enter the brain. Direct

inoculation of the agents into the brain and the hematological route of infection are other possibilities of

entering the brain.

1.3.4.1 Reported cases of hydrocephalus in teleosts

A congenital hydrocephalus has been reported in Atlantic salmon alevins. The fish came from four

hatcheries. The fish were lethargic and mortality was significant. Gross physical research concluded

swollen cranium with exophthalmos. This deformity was characterized by an increased space between

the meninges and the optical lobes of the brain (hydrocephalus externus). The diseased fish in the four

hatcheries were originated from one particular stock. Virology was done and no pathogenic virus was

found. Some of the alevins had hemorrhages in the subarachnoid space or in the optic lobes. Obvious

necrotic cells were found in the meninges and on the outer surface of the optic lobe. The diagnosis of a

21

congenital hydrocephalus was stated in this case, because of a lack of an infectious etiology along with

the young age of the fish. (Rodger & Murphy, 1991).

Kramer-Zucker et al. (2005) could induce hydrocephalus in zebrafish embryos after knocking down genes

responsible for the expression of the moving particles of the microtubules in cilia or flagella (Figure 5).

The loss of the capability to have flow of fluid leads to accumulation of fluid. Also other malformations

were seen: pronepfric cysts, pericardial edema and ventrally curved bodies. Most embryos died on day

5-6.

Figure 5: Top: induced hydrocephalus is zebrafish embryo (**) by knocking down fluid flow regulating

genes. *: kidney cyst. Bottom: normal zebrafish embryo (Kramer-Zucker et al., 2005).

Cranial deformities in four week old channel catfish (Ictalurus punctatus) larvae were seen in a

commercial hatchery. The larvae had also serious swimming difficulties, stopped eating and died finally.

About 0.5 to 3% of the population in all tanks were affected. Five of the affected fry were examined. All

ventricles of the brain were marked dilated (hydrocephalus internus). The tectum opticum was thin and

22

was displaced to dorsal, due to the pressure of the very dilated ventricle of the mesencephalon. In two

of the five fry, the distended fourth ventricle was filled with a pale eosinophilic fluid containing moderate

numbers of red blood cells (Figure 6). There were no other abnormalities observed in the region outside

the brain. One week later, the incidence of hydrocephalus was dropped below 0.05% in the hatchery.

Ten of the hydrocephalic fry were homogenized and inoculated on several cell lines. After two weeks no

cytopathic alterations, indicative for the presence of a virus, were seen. A herbicides assay on other fry

was negative. There was a lack of evidence for an toxic, infectious, nutritional and environmental

etiology. A possible cause is a congenital (possible genetic) defect, because of the young age of the fry

and the low number of affected fry.

Figure 6: A transverse section of the head of channel catfish fry. The arrows indicate the dilated fourth

ventricle with eosinophilic fluid admixed with red blood cells. M: medulla oblongata, E: esophagus, L:

liver, bar = 400µm (Muench et al., 1997).

In pike fry, hydrocephalus has been reported after infection with Pike Fry Rhabdovirus (PFR). There is an

excess fluid accumulation in the third ventricle (Speare & Frasca, 2006). This virus cause an acute

hemorrhagic infection with high mortality. This virus has properties in common with the spring anemia

virus of carp (Chen et al., 2009). In infected pike fry also other symptoms were seen: petechial

23

hemorrhages in the brain, eye, spleen and kidney. In the kidney and liver, necrotic lesions were found.

These lesions were not seen in the catfish fry with hydrocephalus (Muench et al., 1997).

Also another rhabdovirus is reported to cause hydrocephalus as the main symptom in pike fry (<20mm):

Viral Hemorrhagic Septicaemia Virus (VHSV) (Enzmann et al., 1993). This virus is known to cause serious

problems in the European rainbow trout farming. Some natural infections of nonsalmonid species, like

pike are reported (Meier et al., 1994). The virus causes a generalized state of disease within young pike.

This viral disease is, like PFR, associated with high mortality. Most of the pike fry died within 24h after

the onset of the symptoms (between day 3 and 5 post infection) (JØrgensen et al., 1993). The optimum

temperature for an outbreak was 13 to 15°C. Experimental transmissions of the virus from pike to

rainbow trout and vice versa, and from brown trout to rainbow trout were demonstrated (Enzmann et

al., 1993).

In Salmonids living in the Great Lake and other inland lakes (Canada), the early mortality syndrome (EMS)

is a non-infectious disease in fry characterized by a neuropathy involving hyperexcitability and a loss of

equilibrium. Prior to death, hydrocephalus, anorexia and reduced yolk-sac utilization can occur. There is

a strong association between the occurrence of EMS and low thiamine (vitamin B1) levels in the eggs of

the lake trout (Salvelinus namaycush) (Fritzsimons et al., 2001). These low levels may be due to the

consumption of forage containing thiamine degrading factor (thiaminase) (Elliot, 2005). High levels of

thiaminase can be found in some prey fish (Brown et al., 2005). Other studies conclude that this

syndrome is probably due to a multifactorial etiology. Involvement of contaminants like PCBs is possible,

because its thiamine degrading effect in rats (Fitzsimons et al., 2001). There are two other similar

syndromes associated with low thiamine levels: the ‘Cayuga Syndrome’ in larval landlocked Atlantic

salmon (Salmo salar) in several lakes in the neighborhood of New York and M74 of Atlantic salmon from

the Baltic Sea (Fischer et al., 1995). Both syndromes also cause 100% mortality and can induce a

hydrocephalus.

Histological investigation of EMS fry concluded that most lesions occur in the brain. Basophilic foci are

present in the molecular layer of the cerebellum (Figure 7). In some cases, cell degeneration and necrosis

with nuclear degeneration and karyorhexis can be seen in cerebellum and mesencephalon. Hemorrhages

can be found in some regions of the brain. An apparent separation of the granular and molecular layer of

the cerebellum can be visible (Elliot, 2005).

24

Figure 7: Top: Normal histology of the cerebellum of a coho salmon (Oncorhynchus kisutch) fry. Bottom:

Apparent separation of the granular and molecular layer can be seen. A basophilic focus is shown in the

box. HE stain (Elliot, 2005).

25

In coho salmon fry, a focal swelling on the head due to a cranial defect is described. The etiology is

unknown, but exposure to teratogenic products like malachite green is the most likely. Malachite green

is known to target cranial tissue. A similar pathology is seen in salmon fry: the acquired external

hydrocephalus was caused by edema of the middle layer of the endomeninx resulting in an outpouching

of the cranial vault (Speare & Frasca, 2006). Hydrocephalus with fin necrosis was seen in rainbow trout

fry after exposure to a cleaned-up extract of fly ash (Helder et al., 1982).

There is only one reported case of deformed burbot larvae: Jensen et al. (2008b) found that an increased

water flow can affect the embryonic development of burbot. This resulted in an increased amount of

abnormal larvae.

Figure 8: Live deformed burbot larvae collected by Jensen et al. (2008b).

1.4. HISTOLOGICAL DEVELOPMENT OF THE DIGESTIVE TRACT

The weaning to formulated feeds is a very crucial stage in the larviculture. Inadequate knowledge about

the functional development of the digestive system and nutritional requirements decline the success of

larviculture. Trying to understand the development of the digestive system may contribute to reduce the

26

bottlenecks of the larval rearing (Hamlin et al., 2000). The digestive structure of fish larvae is correlated

with the feeding habit (Elbal et al., 2004). Obvious histological changes can give information about the

way the larvae digest their food.

At hatching the digestive tract is a straight undifferentiated tube for many fish species like sea bream,

sole and haddock (Ribeiro et al., 1999; Elbal et al. 2004; Hamlin et al;, 2000). During the larval

development, the length of the digestive tract increases, this causes formation of loops.

The onset of the exogenous feeding coincide with the presence of mucous cells in the esophagus for

some fish species, while in other fish species these mucous cells appear later in the ontogeny. The

number of mucous cells increase with larval development (Ribeiro et al., 1999). These mucous cells

contain neutral and acid mucines. Neutral mucines are responsible for the digestion of easy digestible

products like short chain fatty acids and disacharids. The acid mucines play a role in protection against

invader viruses or bacteria. The mucines help swallowing the food particles. Their abundance indicates a

pre-gastric digestion. Via this mechanism, live preys can be digested, but not artificial feeds, because live

preys contain enzymes contributing to the digestion. Efficient digestion of artificial feeds needs effective

mechanical and enzymatic stomach activities (Tindemans et al., 2010).

The presence of big vacuoles in the enterocytes just after the onset of the exogenous feeding is

indicative for temporarily fat storage when the absorbance of fats exceeds the exporting capacity

(Tindemans et al., 2010). As a result of pinocytotic absorption and intracellular digestion, clear

acidophilic inclusions can be detected in the gut of several teleosts. This has been suggested as an

alternative pathways to digest proteins as in teleost larvae the enzymatic digestion is not well developed

(Sarasquete et al., 1995). The disappearance of these inclusions indicate gastric activity or starvation

(Tindemans et al., 2010).

2. MATERIALS AND METHODS

2.1. REARING AND WEANING

The weaning and rearing experiments were conducted at the Laboratory for Aquaculture and Artemia

Reference Center of the Ghent University.

2.1.1. Water quality parameters

The larvae were held in tap water. The water was aerated before entering the fish tanks to remove

residues of chlorine (concentration of free chlorine <60µg/l). The concentrations of ammonia and nitrite

were determined with the commercial NH4+ and NO2

- test kit (JBL). The oxygen concentration was

measured with the SCHOTT Fieldlab Oxy meter. Temperature was monitored with an analogue

thermometer. These parameters were controlled every day. The hardness of the water and the pH were

27

controlled every month. The former was measured with the commercial JBL water hardness test kit and

the latter with the Sartorius Basic pH meter PB-20. The tanks were cleaned twice daily by siphoning and

the biofilm was removed with a sponge twice weekly.

2.1.2. Live feed phase

The larvae were held in four white, conical 90l tanks. Before use, the tanks were washed with biological

soap and disinfected with CID 2000™. After the disinfection, the tanks were rinsed very well to remove

all chemical products. The tanks functioned as upwellers. At the outlet, a sieve (mesh size 150µm) with a

big surface was installed to keep the larvae in the tanks. The larvae were held in tap water. The water

was first put into a 150l tank where it was aerated. The water was then pumped through a cooling device

(RESUN CL650) before it was distributed to the four culture tanks. The hydraulic retention time of the

culture tanks was 1.5h. During this live feed phase, the larvae were held under continuous light

conditions.

In a first attempt, larvae with some yolk remaining (endogenous feeding) were obtained from the

Research Institute for Nature and Forest (INBO) in Linkebeek, Belgium. After a few days, the mortality

was very high (>98%) so it was decided to start all over again.

A total of 50,000 larvae (14dph) were obtained again from INBO. They had started exogenous feeding on

Artemia nauplii 5 days before. The larvae travelled for 1h to the laboratory in 30l bags inflated with pure

oxygen. Upon arrival, the larvae were acclimatized in a rectangular tank (50l) by gradually adding some

of the aerated tap water to the bags. After 3h the bags were removed. The larvae were held in that tank

overnight. The next day, the larvae were divided over the four tanks. Only the fittest larvae were

restocked. Each of the four tanks received about 8,000 larvae. The larvae stayed in these tanks for six

weeks. In week 5, an active carbon filter was installed to lower the amount of toxicants entering the

tank. After six weeks, the remaining larvae of tank 4 were restocked into seven small Züger bottles (8l) at

a density of 29 larvae per bottle (Figure 9). The larvae of tank 1, 2 and 3 were not used, because of the

low survival rate and the high incidence of the head deformity in the surviving larvae.

Treatment

Tank Name Day 0 – day 14 Day 15 – day 42

1 NED Non - enriched Artemia, discontinuous feeding

Non - enriched Artemia, discontinuous feeding

2 NEC 1 Non - enriched Artemia, continuous feeding

Non - enriched Artemia, continuous feeding

3 NEC 2 Non - enriched Artemia, continuous feeding

Non - enriched Artemia, continuous feeding

4 EC Non - enriched Artemia, continuous feeding

Enriched Artemia, continuous feeding

Table 2: The treatments during the live feed phase of Lota lota larvae.

28

The first week, the larvae were fed at a feeding ratio of 50 Artemia nauplii/larvae (4.4 nauplii/ml). After

one week the concentration gradually increased to reach 8 nauplii/ml after three weeks. This feeding

intensity was used for the rest of the live feed phase for all the tanks. During this live feed phase, there

were three treatment groups. The larvae of tank 1 were fed discontinuously with non-enriched nauplii

twice a day. The other three tanks were fed continuously with a peristaltic pump. The larvae of the tanks

2 and 3 were fed non-enriched nauplii throughout, while the larvae of tank 4 switched to enriched

nauplii two weeks after the start of the experiment. The enrichment product S-presso (INVE

Aquaculture, Dendermonde, België) was used. S-presso has a high level of DHA and a DHA/EPA ratio of 9.

Each day, cysts (SepArt OF 500µm, INVE Aquaculture, Dendermonde, Belgium) were hatched in white,

conical tanks filled with natural seawater (10l) at a density of (2.5g/l). The temperature was kept at 28°C

and there was continuous aeration and light. After 24h, the Artemia nauplii Instar I were harvested.

Since the cyst shells were coated with iron particles, the nauplii could be very well separated from the

cysts when the culture passed through a magnetic tube. The nauplii were sieved onto a 150µm sieve,

rinsed thoroughly with fresh seawater and put into a 5l beaker with seawater for cold storage in the

fridge (4°C) with continuous aeration.

Date (dph) Days of the experiment Sampling per treatment Treatment

23 February (15) 0 3 x 20 larvae Stocking

2 March (22) 7 20 larvae Continuous – discontinuous feeding

9 March (29) 14 20 larvae Continuous – discontinuous feeding

16 March (36) 21 20 larvae / counting NED / NEC 1 and 2 / EC

23 March (43) 28 20 larvae NED / NEC 1 and 2 / EC

6 April (57) 42 10 larvae / counting EC

Table 3: The sampling scheme during the live feed phase of Lota lota larvae.

During the first four weeks the larvae of all treatments were sampled each week. The larvae were first

put into a solution with clove oil for sedation and then transferred to a Bouin solution for 24h. Then they

were transferred into a 70% ethanol solution. First sampling was done during the stocking of the tanks

(three times 20 larvae). For the four other samplings, each time 20 larvae were collected out of each

treatment group. This weekly sampling for length and weight determination was stopped after four

weeks for the NED, NEC 1 and NEC 2 groups.

The daily mortality could not be recorded due to practical reasons. The larvae were counted during the

stocking of the tanks and after three weeks. After six weeks, only the larvae of the EC group were

counted.

2.1.3. Weaning experiment

After being fed for six weeks with live feed in the lab, the weaning started. Only the larvae from tank 4

were used. The larvae were inspected and four larvae with an abnormal cranium were eliminated. The

29

203 larvae were divided into seven 8l Züger bottles. Each bottle has 29 larvae. The first four bottles

(bottles A, B, C and D) were fed Algonorse. The particle size was 200-300µm. The larvae were fed at a

daily feeding intensity of 51mg/larvae. The feed was added to the bottles each hour, 24 times per day.

An automatic feeding system was made to provide the feed (Figure 10). The control group (bottles E, F

and G) was fed non-enriched Artemia nauplii for the first 7 days, then 1 day a mixture of frozen

bloodworms (Ocean Nutrition, Essen, Belgium) and Artemia nauplii and finally only bloodworms twice a

day. Feeding was done “ad libitum”. The nauplii were added continuously via a peristaltic pump.

In contrast to the live feed phase, the tap water first passed through an active carbon filter before it

entered the 150l tank. The water was than pumped into the central inlet system at the bottom the

bottles (upwellers). The hydraulic retention time was 15 minutes. At the inlet of the bottles, a stone was

put to prevent escaping larvae to go into the inlet pipe system. At the outlet, a sieve (mesh size 1mm)

was installed. The light schema was 16/8 light/dark. This experiment lasted 2.5 week. At the end of the

experiment the larvae were counted and five larvae were sampled out of every bottle and stored in 70%

ethanol.

Figure 9: 8l Züger bottles used for the weaning of Lota lota larvae.

30

Figure 10: The automatic feeding system specially made for the weaning trial. A = wheel containing 12

eppendorf tubes filled with the artificial feed, the wheel made one turn in 12h. B = continuous air flow.

C = air tube providing air just below the water surface to counteract the water tension at the surface.

2.1.4. Determination of total length and dry weight and statistics

The total length (the distance from the upper lip to the end of the tail) was measured at the Department

of Morphology (Faculty of Veterinary Science, Ghent University). This was done with the specialized

software Cell D. 12 larvae out of every sampling of 20 larvae were used for length determination. Not all

larvae could be measured because some of the fixated larvae were too curved to do an appropriate

length measurement. The larvae that were bigger than 2cm were measured with a ruler. The dry weight

determination was done at the Laboratory for Aquaculture (Faculty of Bioscience Engineering, Ghent

University). Each sample was divided into two subsamples to have replicas for the dry weight

determination. The larvae were incubated for 4h at 103°C. The data were analyzed using the statistical

functions of Excel. The analysis of variance (ANOVA) was done to determine significant differences

among the treatment groups.

31


Because of the lack of time and possibilities, only attention was paid to the histological and

bacteriological investigation of the deformity. On the other hand, the possible presence of elevated

levels of lead and copper was also identified as a possible cause since the water entered the test room

via a copper tube (diameter 1.25 inch and length 9m) and because in former times, a part of the tubes

supplying water to the laboratory were made of lead. There were no indications that feeding conditions

were involved as an inducer of the problem since a similar diet was used at Aqua-ERF research institute

(Katholieke Hogeschool Sint-Lieven, Sint-Niklaas) and INBO (Linkebeek) without any signs of

hydrocephalus. The chlorine concentration after aeration of the water was not determined, because no

such effects caused by chlorine are reported.

2.2.1. Heavy metal analysis of the water and larvae

Two weeks after the first observation (day 21) of the lumps on the head of the larvae, the concentration

of 2 metals, lead and copper, was measured by the Laboratory of Analytical Chemistry and Applied

Ecochemistry (Ghent University). The body copper concentration of larvae after 3 weeks of exposure

(time of appearance of first bumps) was determined in the same laboratory. The copper concentration of

larvae of the same age and origin, but reared in another institute (Aqua-ERF), was also determined. Two

weeks after the installation of the active carbon filter (contained 2.5kg of active coal), the copper

concentration was determined in water that came directly from the tap and water that passed through

the filter. All copper concentrations were determined with inductively coupled plasma mass

spectrometry (ICP-MS, Perkin Elmer Elan DRC). There were no indications to determine the

concentrations of other heavy metals or chemicals.

2.2.2. Microbiological and histological investigation of the bumps

This was done at the Laboratory of Veterinary Pathology (Ghent University). The larvae (38dph) were

first euthanized in a solution of MS222 (tricaine methanesulfonate) before processing. For the microbial

analysis, the bumps were cut open with the tip of a sterile needle. Some fluid out of the bumps was

collected with a sterile swab. The collected fluid was inoculated on two different agars (normal blood

agar and SHI-medium) and the plates were incubated at 25°C for five days. Other larvae were used for

the histological work. Longitudinal serial sections of larvae with a variable size of the deformity were

processed. The coupes were stained by hematoxylin and eosin (HE) or periodic acid shiff (PAS).


Eggs were obtained from the INBO in Linkebeek. The eggs were close to hatching point and were held in

a 8l conical tank with an upflow water movement. The origin of the water was the same as for the

32

rearing experiment. The larvae were fed Artemia nauplii from day 9 after hatching. Every two days five

larvae were sampled. The larvae were euthanized with clove oil and were put into Bouin fixative

solution. After 24h the fixed larvae were transferred into a 70% ethanol solution for storage. Last

sampling was done 22dph. The average temperature was 12.1°C (10.8°C – 13.4°C). The larvae were

processed at the Faculty of Veterinary Medicine, Department of Morphology, Ghent University.

Histological coupes were made of three of the five larvae of 0, 4, 8, 12, 16 and 20dph. The coupes were

stained with HE or PAS-coloration.

3. RESULTS

3.1 REARING AND WEANING

3.1.1. Water quality parameters

During the whole experiment the concentration of ammonia and nitrite was below 0.05 respectively 0.01

mg/l. The dissolved oxygen concentration was 7.02mg/l. During the first four weeks, the temperature of

the water was on average 12.1°C (10.8°C - 13.4°C). After this, the temperature gradually increased to

14.9°C (13.6°C - 16.2°C) due to improper cooling capacity. The average temperature of the water of the

Züger bottles was 16.1°C (15.6°C - 16.6°C). The pH of the water ranged from 7.58 to 7.70. The carbonate

hardness was 9°d (161.1 mgCaCO3/l).


3.1.2.1. Survival

Treatment Number of larvae Survival

NED 330 4.1

NEC 1 415 5.2

NEC 2 401 5.0

EC 436 5.5

Table 4: The survival (%) of Lota lota larvae after three weeks (initial stocking 8,000 larvae/tank).

There are two replicates in the group fed continuously non – enriched Artemia nauplii (NEC 1 and NEC 2).

There are no replicates for the other two treatments.

The survival of the larvae of the EC treatment between week 3 and 6 was 56%.

33

3.1.2.2. Total length

Time (days) NED NEC 1 and 2 EC

0 3.76 ± 0.39 3.76 ± 0.39 3.76 ± 0.39

7 4.61 ± 0.51 4.94 ± 0.62 4.94 ± 0.62

14 5.39 ± 0.70 5.76 ± 0.88 5.76 ± 0.88

21 9.06 ± 0.92 10.10 ± 1.02 10.23 ± 0.92

28 12.06a ± 1.57 13.36a ± 1.73 14.96b ± 1.09

42 - - - - 26.29 ± 2.16

Table 5: The total length (mm) of Lota lota larvae at different times during the live feed phase for the

three treatments (mean ± SD, n = 12). Different superscript letters indicate significant difference (p <

0.05).

Graph 1: The average total length (mm) of Lota lota larvae at different times during the live feed phase

for the three treatments.

3.1.2.3. Dry weight

Time (days) NED NEC 1 and 2 EC

0 0.036 ± 0.011 0.036 ± 0.011 0.036 ± 0.011

7 0.052 ± 0.018 0.067 ± 0.021 0.067 ± 0.021

14 0.195 ± 0.065 0.31 ± 0.17 0.31 ± 0.17

21 1.07 ± 0.18 1.33 ± 0.32 1.59 ± 0.39

28 2.21a ± 0.33 3.12

a ± 0.67 5.64b ± 0.88

42 - - - - 27.26 ± 5.39

Table 6: The dry weight (mg) of Lota lota larvae during the live feed phase for the three treatments

(mean ± SD, n=2). Different superscript letters indicate significant difference (p < 0.05).

34

Graph 2: The average dry weight (mg) of Lota lota larvae at different moments during the live feed phase

for the three treatments.

3.1.3. Weaning experiment

3.1.3.1. Survival

Treatment Replicates (n) Cannibalism Survival

Aglonorse 4 3.45 ± 2.83 58a ± 13.08

Control 3 1.15 ± 1.99 79b ± 3.51

Table 7: Cannibalism (%) and survival (%) of Lota lota larvae weaned for 17 days with Aglonorse or

bloodworms (control) (mean ± SD). Different superscript letters indicate significant difference (p < 0.05).

The global survival since day 0 of the live feed phase until day 17 of the weaning trial is: 5.5% x 56% x

58% = 1.8%.

3.1.3.2. Total length.

Initial (d42) Algonorse (d59) Control (d59)

Replicates (n) 10 20 15

Length 14.96a ± 2.16 23.93b ± 2.19 32.08c ± 4.66

Table 8: The increase in average total length (mm) of Lota lota larvae during the weaning experiment

(mean ± SD). Different superscript letters indicate significant difference (p < 0.05).

The total length increased with 60% in the Aglonorse group, while in the control group this increased

with 114%.

35

3.1.3.3. Dry weight

Initial (d42) Algonorse (d59) Control (d59)

Weight 27.26a ± 6.17 14.48b ± 4.12 52.89c ± 12.38

Table 9: The dry weight (mg) of Lota lota larvae at the start and the end of the weaning experiment

(mean ± SD, n = 2). Different superscript letters indicate significant difference (p < 0.05).

During the weaning, the average dry weight per larvae declined with 47% in the group fed Aglonorse.

This is in contrast to the control group where an increase in dry weight was seen with 94% after 2.5

weeks. The uptake of an artificial feed particle was seldom seen, most larvae seemed to feed on flakes

and biofilm material hanging on the walls of the bottles.


3.2.1. General aspects of the abnormality

The bumps were seen for the first time three weeks after the introduction of the larvae. One week later

about 20% of the population in each tank was affected, except in the tank fed enriched Artemia nauplii

Instar II. In the latter tank, the prevalence of the deformity was estimated at 5%. During the next weeks

these prevalences were approximately constant: new larvae got affected and most of the affected larvae

died after several days, but some of the larvae recovered without any visible sign of abnormality. The

affected larvae had difficulties to control their equilibrium, but they still continued eating. Most of the

bumps were seen in the bigger larvae. All the deformations were similar and were characterized by a

hemispherical semitransparent protrusion of the forehead. In those protrusions red zones could be seen

macroscopically

3.2.2. Analysis of the water

The concentration of copper and lead was 4.5µg/l and below 0.2µg/l respectively. The body copper

concentration of the affected larvae was 710µg/kg wet weight. This is 1.7 times higher when compared

to the 414µg/kg concentration found in larvae from the KAHO Aqua-ERF research institute. The copper

concentration of the water passing through the carbon filter and directly from the tap was 3.35µg/l

respectively 4.74µg/l. Thus installing the carbon filter reduced the copper concentration with 29% two

weeks after the installation.

3.2.3. Macroscopic view of the cranial deformity

Clinical normal larvae had a smooth dorsal head contour (Figure 11). Deformities of the cranium could be

seen in different levels of anomaly (Figure 12).

36

Figure 11: Macroscopic view of a clinically normal fixated Lota lota larva (d21). Total length: 11.34mm.

Figure 12: Fixated Lota lota larvae (d21) with different intensities of the cranial deformity. Scale bar =

5mm.

3.2.4. Microbiological and histological investigation

No bacterial colonies were counted after 5 days of incubation of the cyst fluid at 25°C, on both media.

37

The histological coupes revealed that the deformities were due to hydrocephalus (Figure 13). The dorsal

side of the ventricles were covered with one layer epithelium. There were no indications of necrosis or

apoptosis in the cartilage. Fraying of the neuronal tissue next to the ventricles can be seen (Figure 14),

but this can also be seen in the clinically normal larvae. All the ventricles are involved in the process

(communicating hydrocephalus). The main parts of the ventricles were optical empty, although there

was a small amount of proteins and extended aggregates of red blood cells present (Figure 15). These

hemorrhages were also present in the normal larvae (Figure 16) and could be secondary due to pressure

or trauma. There were no indications for an inflammatory or infectious process. Dorsal of the cysts, the

cartilage was lacking over a wide zone. But this was also seen in clinical normal (without cysts) larvae. All

larvae had a good body condition, but had a high number of goblet cells in the skin.

Figure 13: Histological longitudinal section of the deformed head of a Lota lota larva (38dph). A: eye, B:

liver, C: gills. The red line indicates the virtual dorsal contour of a normal larva. The box give the range of

Figure 14. HE stain.

38

Figure 14: Detail of the dilated ventricles (A) of a Lota lota larva (38dph). B: Goblet cells in the skin, C:

fraying of neuronal tissue, D: cartilage. HE stain.

39

Figure 15: Histological longitudinal section of the deformed head of a Lota lota larva (38dph). Aggregates

of red blood cells (A) and proteins (B) can be detected in the cerebrospinal fluid. C: oral cavity, D:

esophagus. HE stain.

40

Figure 16: Histological longitudinal section of a clinically normal Lota lota larva (38dph). A: Fraying of

neural tissue, B: ventricle filled with protein aggregates, C: a partially lack of cartilage dorsal of the brain.

HE stain.


Just after hatching, the digestive tract is identified as an undifferentiated tube with cylindrical

epithelium. The pancreas and the cylindrical enterocytes of the intestine could clearly be identified at

4dph. The yolk sac was absorbed 8dph and an empty lumen remained. The first mucous cells in the

esophagus could be detected at 8dph in one of the three larvae while at 12dph all larvae had these cells.

At 12dph, two of the three larvae had lumen content. This was composed out of parts of Artemia nauplii

together with amorphous masses with clear eosinophilic and basophilic droplets. This coincided with the

first appearance of vacuoles in the enterocytes. These vacuoles were found in the anterior part of the

intestine. The larvae that didn’t have a content in the lumen of the intestine also lacked the vacuoles. No

stomach or eosinophilic supranuclear inclusions in the enterocytes were detected during the first 20

days.

41

Figure 17: Histological coupes of the digestive tract of Lota lota larvae. a) cylindrical epithelium of the

digestive tube (0dph), b) detail of the pancreas (4dph), c) overview of the digestive tract (4dph), d)

overview of the digestive tract (8dph), e) mucous cells in the esophagus (12dph), f) lumen content (LC)

containing parts of Artemia nauplii in the intestine (I) (12dph). E: esophagus, P: pancreas, YS: yolk sac, N:

notochord, SB: swim bladder. HE stain.

42

Figure 18: Histological coupes of the digestive tract of Lota lota larvae. a) detail of the vacuoles (V) in the

enterocytes (16dph), b) overview of the digestive tract, the intestine has a wide lumen (20dph), c) detail

of the intestine (I) with lumen content (LC) (20dph), d) detail of the acid and neutral mucous cells

(dph16), e) detail of the vacuoles in the enterocytes (dph16), f) detail of the PAS-positive brush border

(BB) (20dph). E: esophagus, L: liver, P: pancreas, SB: swim bladder (a,b,c: HE stain; d,e,f: PAS stain).

43

4. DISCUSSION

4.1. REARING AND WEANING


4.1.1.1. Survival

Two of the three treatments of the live feed phase had no replicates. The other treatment had only two

replicates. The lack of sufficient replicates is due to the fact that the larvae were reared first into four big

tanks. Although this is statistically insufficient, it can give an indication that the survival is quite similar

over the three treatments. The tank that was continuously fed enriched nauplii had a slightly higher

survival. According to Aschenbrenner (personal communication), the enriching of Artemia has only a

small impact on the survival of the larvae. The extra work and cost related to enriching should not

exceed the advantages of enriching. Similar things were also seen in African catfish: no differences in

survival between enriched and non-enriched Artemia nauplii were seen (Verreth et al., 1994), but

according to Wocher et al. (2010a) the enriching of live feed is a condition needed to obtain a

satisfactory survival.

The global survival during these first 21 days (until 36dph) was very low (about 5%). Wocher et al.

(2010a) obtained a ten times higher survival percentage under similar conditions, but from the start of

the exogenous feeding. The fact that the larvae have already started to eat before the experiment

started, makes the result of the present rearing even worse, because the start of the exogenous feeding

is a crucial moment that coincide with high mortalities. The counting of the larvae was done just before

the first cranial deformities were seen. The low survival can be due to several factors: (1) A possible pre-

clinic effect of the cranial deformity could influence the survival. (2) According to Trabelsi et al. (2011) a

higher mortality was seen if the larvae were held under normal freshwater (salinity of 0g/l) conditions. A

salinity of 5g/l increases the survival. (3) The presence of fungus flakes on the wall of the tanks. (4)

Possible effects of the use of chlorinated tap water, although the water was good aerated before

entering the tanks, it can not be excluded that chlorine entered the culture tanks. (5) Possible effects of

the slightly elevated copper concentration.

4.1.1.2. Growth

The larvae fed enriched Artemia had a significant higher growth two weeks after the start of the

enriching. A higher HUFA level in the diet clearly increased the growth. Similar results were obtained in

burbot (Wocher et al., 2010a) and other fish species like Japanese flounder (Paralichtys olivaceus)

(Izquierdo et al., 1992) and gilthead seabream (Sparus aurata L.) (Rodriguez et al., 1998).

Three weeks after the start of the live feed experiment (until 36dph), the average total length for the

three treatments was 9.80mm. Harzevilli et al. (2003) obtained at 35dph a total length of 8.87mm for a

44

similar experiment, but the water temperature was 10°C, 2°C lower than during this live feed phase. The

difference is temperature is probably the main reason for the difference in total length. It was concluded

that the length growth is positively correlated with increasing temperature in the range from 12°C to

20°C (Harzevilli et al., 2004, Wolnicki et al., 2002). Although 10°C is not within this range, it is very likely

that the growth will be higher at 12°C.

Four weeks after the start of the experiment (43dph), the dry weight of the larvae that were

continuously fed enriched Artemia was 5.64mg. The feeding intensity was high, because a fixed amount

of Artemia nauplii was added each day to the tanks, but due to the high mortality, the number of nauplii

per larva increased drastically during the experiment. Wocher (2010b) performed trials to compare the

effect of feeding intensity. In the group with the highest intensity (gradual increase of 80 to 320

nauplii/larva/day), the specific growth rate (sgr) was 10.02%. The calculated average dry weight for these

larvae at 43dph is 5.2mg, which is close to the 5.64mg found in the present experiment.

Although the difference between the continuous and discontinuous feeding is not significant at day 28 of

the experiment, it can be indicative that continuous feeding improved larval performances. This can be

due to: (1) a slightly higher concentration of the Artemia nauplii in the water column due to continuously

adding of nauplii at the water surface. (2) The continuously adding of new live nauplii, because of the

cold storage in seawater. Nauplii can survive only a short time under freshwater conditions.

4.1.2. The weaning experiment

4.1.2.1. Survival

Aglonorse is a specialized feed that is being used for cod and other marine fish species. Since cod and

burbot are close related species, it was estimated that this feed should meet the requirements of burbot

(Trabelsi et al. 2011). This feed was used before in some weaning trials: Wocher (2010b) obtained a

survival of 41% after a weaning of 56 days which started from the onset of the exogenous feeding.

Trabelsi et al. (2011) performed several weaning protocols with larvae of 2 months old. The survival

ranged between 0% and 76% after 36 days. Meeus et al., (2011) reported a survival of 16% after a

weaning period of 29 days which started with larvae of 2.5 month old. These trials experiments included

a co-feeding phase. There is a lot of variation between the different experiments which can be due to

different conditions during the weaning. The survival obtained after the present weaning trial was 58% in

the Aglonorse group and 79% in the control group. The global survival after the live feed and weaning

experiments was only 1.8%. This weaning test only lasted 2.5 weeks, if the weaning phase would lasted

longer, the survival percentages would be much lower due to the starving larvae that still lived at day 17

of the weaning phase.

45

4.1.2.2. Growth

In the weaning group, the length of the larvae increased with 60%, this was lower than the length

increase of the control group (114%). In contrast to that increase in total length, a serious decrease (-

47%) in dry weight was seen in the group that was fed the artificial feed. The final dry weight of the

larvae of the control group was 3.7 times higher than the weaning group. This indicates that larvae still

have the capacity to have an increase in length to the prejudice of their own weight under unfavorable

conditions. This negatively correlation of weight and length growth under starvation is not reported in

literature.

There are several possible reasons for the negative weight growth in the group fed Aglonorse: (1) A

possible influence of the previous deformity problem could influence this weaning trial, although the

weaning trial started after the disappearance of the cranial deformities. Most of the larvae that were

affected with the cranial deformity were the biggest larvae and most of them died. The larvae that left

over can be considered as slower growing larvae or larvae that have been recovered from the deformity.

Since the etiology of this deformity is still unknown, it is possible there are sub-clinically effects on the

weaning. (2) The particle size of the artificial feed that was used was 200-300µm, this is smaller than an

Artemia nauplii. While normally the particle size of the feed need to increase with increasing larval size.

(3) The low stocking density. In this first weaning trial each 8l bottle contained 29 larvae, this is a density

of 3.6 larvae/l. Other similar weaning experiment were performed with much higher stocking densities

(Trabelsi et al. 2011; Wocher et al., 2010a; Jensen et al., 2008a). Probably eating can be seen as a group

effect: eating larvae will attract other larvae.

4.1.2.3. Parameters influencing the weaning

Trabelsi et al. (2011) concluded that the longer the co-feeding period (gradual increase of artificial feed

and decrease of live preys), the better the performances. Also Wocher et al. (2010a) used co-feeding to

prepare the larvae for dry feed. This is in contrast to Aschenbrenner (personal communication), who

indicated that the best results can be obtained if no co-feeding was used. Co-feeding allegedly leads to

an increase in cannibalism. Meeus et al. (2011) compared weaning with and without co-feeding. Similar

larval performances were obtained in both groups.

Trabelsi et al. (2011) performed several tests to wean the burbot under very different conditions (age of

the larvae, length of co-feeding phase, larval density, different feeds...). It was not possible to propose an

ideal combination. This indicates that still a lot of factors and interactions between these factors

influencing the rearing and weaning process remain unknown and thus uncontrolled. It is possible that

total different protocols have similar results. The suitability of a combination also depends on the aim of

the producer.

In the control group, the transition from Artemia nauplii to bloodworms didn’t cause problems: the

larvae were immediately attracted to the worms and started to eat. The bloodworms were frozen, so

there was no movement of the worm themselves, but the upflow water current induced the worms to

46

curl slowly in the water column. This may have an attractive effect on the predator, because burbot

larvae are visual hunters. Girsa (1972) on the other hand observed that the larvae are positive

phototactic till the size of 4cm, while our larvae measured 4.7cm, implying that at this size, movement

of the prey is not important anymore.

In a small extra test (not mentioned in the thesis), good results were obtained when the larvae of the

control treatment (75 dph) switched from bloodworms to a commercial sturgeon feed (Aquabio,

Turnhout, Belgium) after 12h of starvation. The length of these 2.5 month old fish larvae increased with

19% in 17 days, from 47.3 (±3.1)mm to 56.6 (±8.0)mm. The starvation period can be an important factor

to motivate the larvae to eat other things than their usual bloodworms. The bloodworms can be seen as

a transition between the highly active Artemia nauplii and the pellets that only made small rolling

movements. This opens the possibility to grow the larvae extensively in natural ponds till reaching a size

of 4 cm before weaning them on artificial food. This avoids the feeding with expensive Artemia nauplii

and labor intensive cleaning of the tanks.

4.1.3. Economic feasibility of burbot farming

Wocher et al. (2010a) calculated the cost of rearing and weaning burbot larvae. The main cost of

production of the larvae until the size of 5g are: (1) broodstock, (2) Artemia cysts, (3) emulsions for

enrichment of Artemia, (4) artificial feed, (5) salt, chemicals and disinfectants, (6) fuel to heat the water

and (7) electricity. The labor costs were not included. They started with 200,000 larvae. At the end of the

live prey phase 101,500 larvae survived. The production cost until then was 0.0248€/larva. After the

weaning 31,500 larvae of 5g survived. The price of the weaning was 0.0529€/juvenile. The total cost

(without labor) was thus 0.1328€/juvenile. It is difficult to compare the results with other fish species,

because the labor cost is not included in this price of a juvenile. The production cost of 100,000 pike-

perch juveniles of 10g is 0.40€/piece and the labor cost is 26% of the total production cost of these

fingerlings. The cost of pike-perch juveniles is about 20% of the total production cost of a marketable fish

of 1.5kg (Kamstra, 2003). It is difficult to estimate the labor cost for burbot fingerling production, but this

is probably higher than for pike-perch, because Jensen et al. (2007) reported that burbot larviculture is a

labor intensive production.

Burbot can be bought in Finland: round burbot is sold on the market there for 10€/kg (Jurgen Adriaen,

Aqua-ERF, personal communication). This is a good price, but these local people are used to eat this fish,

while in Belgium this fish is still unknown. Research is needed to investigate the potential market for this

fish in Belgium.


When the fish are exposed to higher levels of heavy metals, a wide range of symptoms appears including

hydrocephalus. In larval stages, deformities and reduced hatchability are prominent while in adult fish,

47

heavy metals increase the oxidative stress (e.g. gill damage). In our case, the head deformity was the

main symptom aside from an increased number of goblet cells in the skin.

In the present experiment only the concentrations of lead and copper were determined. The maximum

acceptable concentration of copper supplied by a Belgian water company is 2mg/l. The water delivered

in the lab contains an average of 0.01mg copper/l (± 0.01mg/l) according to the TMVW water company.

The average copper level found in the water was 4.62µg/l. This level is lower than the average level of

copper in the water supplied by the water company. The Belgian standard for copper in drinking water is

quite high compared to the toxic levels for fish. Because toxicity to aquatic organisms can already be

seen at copper levels higher than 3.2µ/l (Stouthart et al., 1996) or even 2µg/l (Brix et al., 2001) for

chronic exposure. The maximum acceptable toxicant concentration for lead is 14.6µg/l for trout larvae.

This is high in contrast to the lead concentration in the present study (0.2µg/l). Lead can be excluded as

inducer of the hydrocephalus problem.

A clear difference was seen between the copper concentration in the deformed larvae of the present

experiment (710µg/kg wet weight) and non-affected larvae of the same age and batch of the KAHO

research facility (414µg/kg wet weight). Our copper content is 71% higher. Unfortunately, no water

samples of the KAHO have been investigated. The fact that the concentrations were determined on wet

weight basis can distort the results a bit due to possible differences in larval water content, so they have

to be interpreted with caution. The dry weight of larvae of two months old is 15% (Wouter Meeus,

KAHO, personal communication). The converted copper concentrations of the present experiment and

the KAHO institute are 4733µg/kg dry weight and 2760µg/kg dry weight respectively.

No specific toxicity tests of copper on the burbot have been reported. Some incubation tests with

chronic copper exposure to rainbow trout were performed with a copper concentration that was close

the concentration found in our water samples: Marr et al. (1996) obtained a body copper concentration

of 5.82µg/g after 20 days exposure at 4.6µg/l. Exposure of juveniles at water copper concentrations of

0.19µg/l (negative control) and 9.5µg/l resulted after 20 days in a copper accumulation (on dry weight

basis) in the fish of 4.42µg/g and 6µg/g respectively (Hansen et al. 2002). In the present experiment, the

copper concentration of 4.62µg/l resulted after 21 days in a body copper concentration of 4.7µg/g dry

weight. This indicates that the copper accumulation in burbot larvae is roughly similar to that in trout fry.

Only a reduced growth was observed in the trout. Copper did not cause deformities at these

concentration in trout. According to Prof. C. Janssen, Laboratory of Environmental Toxicology and

Aquatic Ecology, Ghent University (personal communication), this concentration is too low to cause any

deformation in fish larvae, but burbot is known to be a very sensitive species towards pollution (Polinski

et al., 2010), so this elevated concentration can not be excluded as a possible reason for the

development of the hydrocephalus.

Copper is known to disrupt osmoregulation by influencing the Na+/K+-adenosine triphosphate pump and

the cell adhesion in the gills. This leads to increased permeability (Zahner et al., 2006). These processes

all take place in the gills, but possibly there is also a similar effects going on in the brain region leading to

overproduction of the CSF. Copper exposure also results in a drop in the sodium concentration (Zahner

et al., 2006). Freshwater fish under freshwater conditions are hyperosmotic, so when the fish lose

48

sodium, there is a decrease in osmotic pressure. This counteracts the hypothesis that the hydrocephalus

is due to a direct osmotic problem. If the total sodium level in the body drops, then the osmolarity in the

cerebrospinal fluid is higher relative to the whole body and plasma concentration. This difference in

osmolarity can draw water to the cerebrospinal fluid.

Toxicity also varies a lot depending on the water quality: e.g. organic material and hard water can

decrease the toxicity of a certain copper concentration. Toxicity is related to the amount of free Cu2+.

The overall pH of the water was between 7.58 and 7.70. At the pH of 7.6 only 32% of the copper will be

present as free Cu2+. At lower pH values this percentage increases (Stouthart et al., 1996). This indicates

that only about one third of the total copper concentration of 4.6µg/l is biological active, but it is known

that copper can induce an effect even at very low concentrations (Brix et al., 2001). It can be assumed

that the burbot is highly sensitive towards copper, because this fish is generally seen as a pollution

sensitive fish (Polinski et al., 2010). Due to the lack of other possible causes, copper may be the inducer

or a part of the mechanism that causes the cranial deformity.

Only a few cases of diseased fish with hydrocephalus as the main symptom are described (Rodger &

Murphy, 1991, Muench et al., 1997, Enzmann et al., 1993, Speare & Frasca, 2006), but the real etiology

has never been determined (except for viral diseases in fry). Sometimes a genetic defect was concluded,

but this was only after exclusion of other possible causative agents. In other cases in which

hydrocephalus is one of the several symptoms, the etiology is mostly known (heavy metals, thiamine

insufficiency, viruses). In our study, larvae from the same original batch were reared at two other places,

where no such deformities were seen. A genetic defect as the main reason for this deformity can be

excluded.

It has been demonstrated that the consumption of forage (prey fish) containing thiaminase leads to

lower thiamine levels in the eggs and subsequently leading to hydrocephalus in Salmonid larvae.

Histological investigation concluded the presence of hemorrhages and degenerative zones in the brains

(Elliot, 2005). Hemorrhages were also seen in the larvae of the present experiment, but not the

degenerative or necrotic zones. Larvae of the same origin, but reared in other institutes, did not develop

a hydrocephalus, indicating that the thiamine level in the eggs was not abnormally low. An alternative

pathway to obtain low thiamine levels could be the ingestion of thiaminase producing bacteria. Although

the tanks were cleaned twice daily, it could not be excluded that flakes and biofilm material was present

in the tanks. The other two institutes in Belgium involved in the rearing used water with 5ppt salt to

counteract massive bacterial and fungal proliferation.

In the histological research of the larvae, abundant blood cells were seen in between the brain tissue.

The cells were not located in capillaries or other blood vessels. The presence of such blood cells is

indicative for a hemorrhage. Blood accumulation in the brain can result in hydrocephalus (Chen et al.,

2010), but in this case no signs of inflammation or necrosis that react on the presence of red blood cells

could be seen. This is indicative that the hemorrhages are recent. It is possible that these hemorrhages

occurred during the euthanasia and processing of the larvae.

If the affected larvae survived, the deformity disappeared. This could be due to several mechanisms: An

acclimatization of the larvae to the causative agent: a lower susceptibility to elevated copper

49

concentrations is seen after some weeks (Marr et al., 1996, Hansen et al., 2002) or a possible age

dependent susceptibility.

In conclusion, no clear explanation can be given for this deformity. There are several possible causes, but

none of them is typically associated with this disease. When looking at heavy metal intoxication or

bacterial diseases, a wide range of symptoms and lesions appears, while here only one obvious

macroscopic lesion could be seen. The fact that the problem appeared also the year before in the same

lab, but in no other institution in Belgium or abroad, indicate that the lesion develops due to a specific

pathogen or under specific conditions. If copper would be the main inducer of this type of deformity, the

copper concentration must be in a specific range. At higher concentrations other deformities and lesions

would also be present. This range would also depend on several factors that enhance or decrease copper

toxicity such as pH, water hardness… Aside from the chemicals and pathogens, there are some

correlations of the present pathology with the thiamine deficiency found in Salmonid larvae.

As mentioned before, as a secondary symptom, the increased presence of goblet cells was observed in

skin/gills. Saucier et al. (1991) saw an increase in the number of goblet cells after a long sublethal

exposure of copper (22µg/l) in young rainbow trout. This can be a possible explanation for the increase

of these cells in the affected burbot larvae, but the copper concentration was much lower (4.5µg/l).

Chlorine on the other hand is a oxidizer, which may also cause irritation to the fish‘s skin and thus

causing a overstimulation of the protective mucus layer (Iger et al., 1994). In our experimental unit, tap

water containing chlorine is used. This is in contrast to the other burbot rearing farms: Aqua-Erf and

INBO (Belgium), Lindberg (Germany), Salzburg (Austria), Idaho (U.S.A.) where natural water is used from

streams, lakes and groundwater. In these latter farms, no bumps or similar deformities were seen.

Although aeration was used to remove the chlorine, it can not be excluded that some chlorine residues

entered the rearing tanks. It is possible that these bumps were caused by free chlorine but in literature

no such effects of chlorine are described.

Larvae fed enriched Artemia nauplii clearly had a lower incidence of disease. Supplementing HUFA’s

could have improved larval resistance to stress. This is also seen in several other fish species (Sorgeloos

et al., 2001) although this is not true for example for African catfish (Verreth et al., 1994).

Supplementation of HUFA’s can decrease the stress possibly due to copper, chlorine, viruses... and fulfill

the nutritional requirement to have a higher growth (Aschenbrenner, personal communication).


The presence of vacuoles in the intestine coincided with the presence of a content in the lumen. These

vacuoles are an indication of temporally lipid storage. Those vacuoles are only present for a specific time

in other teleost (Ribeiro et al., 1999). Future research should focus on how long these vacuoles can be

detected in larvae fed live feeds. This should provide information if an artificial feed can be digested or

not during the phase in which these vacuoles should be present. The eosinophilic and basophilic droplet

in the lumen of the intestine possibly contain the mucines from the esophagus helping in the digestion.

50

This experiment only lasted until 20dph, more research is needed to investigate the digestive tract after

this period, because larvae can be weaned after 30 days (Jensen et al., 2008).

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I

APPENDICES

Table I: Overview of the different diseases and parasites of the burbot (Dillen et al., 2005).

II

Table II: Drinkwater investigation by the laboratory of the Flemish water supply company (TMVW)

(www.tmvw.be/documenten/modules/ss_3.pdf).

rearing and weaning of burbot, investigation of a specific

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