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DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to resreports@defra.gsi.gov.uk
Project title
Studies on Aquaculture and Fish Diseases (Sandwich Students)
DEFRA project code
FC1135
Contractor organisation
and location
Dr Matthew Longshaw
CEFAS Weymouth Laboratory,
Barrack Road
The Nothe
Weymouth,
DORSET
DT4 8UB
Total DEFRA project costs
Project start date
£ 304,000
01/09/99
Project end date
Executive summary (maximum 2 sides A4)
CSG 15 (Rev. 6/02)
1
30/09/04
Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
Scientific report (maximum 20 sides A4)
This project had one main objective, which was to utilise students to carry out studies that
complimented and fed into the main R&D approaches undertaken at CEFAS Weymouth. This
involved employing students from the University of Aston for a period of one year, running from
September to September every year. Students selected were those that had completed their second
year at University, most often in human biology disciplines. Students were selected by open
competition an annual basis, with CEFAS staff designing a suitable project for the students. Students
were interviewed, mainly in the Easter break, and selected on the basis of their aptitude and interest,
as well on the recommendation of their university supervisor. Overall, the calibre of the students was
high, with an average of 10 students per year applying for five posts at CEFAS Weymouth. Most of
the students wished specifically to be employed by CEFAS, even though the subject area was
outwith there immediate sphere of experience. The main reason for them wishing to work at CEFAS
appears to have been that they had been informed by previous sandwich course students that the
laboratory was a good place to work and that job prospects after graduation were significantly
improved as a result of working at a government establishment. In addition, the majority of the
students that had spent a year at the laboratory returned to University armed with practical
experience that proved useful in their final year’s lectures. The vast majority of the students that
graduated the following year after their placement year did so with an upper second or first class
honours degree. Very few, if any, graduated with a lower second, and no student graduated with a
third. Thus, whilst the provision of students to CEFAS Weymouth was extremely beneficial to
CEFAS, as will be described in more detail below, it is clear that the students also benefited form this
arrangement.
Overall, the project was deemed to be highly successful with a number of new project ideas being
generated during the lifetime of the project. In addition, through the generation of new data and new
insights, several publications were forthcoming from the project. The project was flexible enough to
allow the development and exploration of new ideas, which were considered important to CEFAS or
Defra, which may not have been possible under existing projects with defined milestones,
deliverables and direction. With an average cost of around £12,000 per student, per year and low
overheads, with five students per year, it is clear that this represents a very cost effective method to
carry out research. Another advantage to having the students has been that we have the ability to
assess any potential new recruits for up to a year that the students, rather than necessarily in a short
interview. However, this is not so say that we would exclusively employ only those that had spent a
year at the laboratory, nor does it negate the need for an interview process. The final advantage of
the projects has been that it has allowed dedicated time to an area of research by a student for a
year, which under normal circumstances would not have been possible with a member of staff. This
has included the investigation of avenues of research that have proved less than fruitful.
The students spent their year’s placement within selected teams. These included Pathology and
Parasitology, Virology and Molecular Genetics, Microbiology and Physiology. Whilst the majority of
students were placed solely within a specific team, many spent time working in other teams in order
to allow cross-discipline projects to take place and to provide the students with training in a wide
variety of techniques and methodologies. This also had the advantage of promoting cross-pollination
between established staff at CEFAS Weymouth. There now follows a breakdown of the various
areas of work carried out by the students by team.
CSG 15 (Rev. 6/02)
2
Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
Pathology and Parasitology:
Overall, studies in the Pathology and Parasitology team were aimed at developing new methods and
introducing established, but previously unutilised techniques in histology and electron microscopy into
the team. Ultimately it was anticipated that these methodologies would become established as
routine in the team to be used in the identification of pathogens and diseases of fish and shellfish and
in many cases, this has occurred. In the main, the way this was achieved was to provide the students
with a project subject, such as material collected as part of a survey, then to apply new methods to
investigate certain aspects of the pathogen. This has a two-fold effect – the students were able to
develop methodologies on these tissues and CEFAS was able to use the results of the survey in the
production of papers and in support of other Defra funded projects.
One of the areas that has benefited from the use of students has been in the development of
methods for the preparation of samples for scanning electron microscopy (SEM). One of the big
issues surrounding disease agents is host specificity. Failure to correctly identify a fish host, can lead
to erroneous results when determining such host specificity. Identification of hosts is usually carried
out at a gross level, or in some cases, using light microscopy. More in-depth studies of e.g.
pharyngeal bones, fin ray counts or indeed molecular studies can provide a definitive answer but can
be time consuming and/or costly. Methods requiring the use of light microscopy, whilst less timeconsuming, have the disadvantage of loss of resolution at higher magnifications and of difficulties in
interpretation of certain structures. One such example is in the identification of marine goby species,
whereby discrimination of certain species in the genus Pomatoschistus can be based on the
arrangement of pores in the head region. This is also true for juvenile fish species in freshwater,
such as cyprinids, which have a similar body shape and overall morphology to each other, especially
in the first few months of life. In order to confirm the speciation of juvenile cyprinids examined under
FC1137 and FC1138, studies were carried out to determine whether it would be advantageous to
utilise SEM to visualise the pore structures on the head region of these fish as a means of
discriminating them. In order to confirm that this was possible, gobies were collected from two
estuarine sites in the northeast England and in the northwest England.
Two sympatric
Pomatoschistus species are known to occur in estuaries in the northeast England, namely P. lozanoi
and P. minutus, whereas only P. minutus is thought to occur in the northwest. The reason gobies
were selected was because one of the most useful methods for discriminating the two species is via
the pore structure. This is normally carried out at the light microscope level and thus in order to
demonstrate that the use of pore structures at the SEM level would be possible, these species were
examined. Results showed that we were clearly able to discriminate the two species of goby via this
route. Subsequently, the methods wee applied to juvenile cyprinids collected in Yorkshire and
Humberside in support of FC1137 and FC1138. Samples of fish, namely chub, dace, minnow and
roach, were prepared for SEM using standard protocols and examined using SEM. No specific
features of the pore structure or arrangement were identified to allow discrimination of different
cyprinid fry from each other. This was in part due to the lack of published data on pore structure and
arrangement these species. In addition, it was found that if fish were suitably preserved in NBF and
more than 3 months of age, discrimination at the light microscope level was possible. The
techniques may have applicability to earlier life stages, although this was not pursued during this
study.
Samples prepared for light microscopy and wax histology are normally fixed in NBF and embedded in
wax and are thus not generally amenable to examination under an SEM, which is used to examine
surface structures. Methodologies developed in the medical field have allowed the examination of
samples by SEM which have previously been prepared for wax histology. These methods were tried
on samples collected at CEFAS Weymouth. Whilst at a simple level they were suitable for cursory
examinations, the quality was poor. Thus, one of the students was tasked with improving the
CSG 15 (1/00)
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Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
methods. By increasing the length of time taken to complete the process of de-waxing and preparing
for the SEM, as well as by incorporating an osmium tetroxide step in the process, better quality
samples were obtained which allowed for more accurate identification of surface structures to be
made. This methodology has been applied to fish and shellfish material previously prepared for light
microscopy studies.
Whilst the SEM has been traditionally used to examine the surface structures of organisms and
objects, it can also be used to improve the resolution of objects visible at the light microscope level.
One such example is the marginal hooks of ectoparasitic monogeneans, in particular Gyrodactylus
spp. The main problem however, is that these hooks are buried within parasite tissues and therefore
are impossible to visualise using normal SEM techniques. Methods developed elsewhere have
provided the means to extract the marginal hooks (a major taxonomic feature) from the surrounding
parasite tissue and thus allow examination of these hooks in a relatively clean way. In so doing, the
clear features of the marginal hooks can be easily discriminated. Three conflicting methodologies
have been described in the scientific literature and thus one student was tasked with utilising all three
methodologies and to determine which method was most suitable. The method proposed by Dr
Andrew Shinn, University of Stirling, was found to be the most efficient in order to isolate hooks from
its surrounding tissues. This method has now been adopted in the lab. One further refinement was
in the development of a grid system that allowed the exact position of each hook to be noted at the
light microscope level, and thus readily located at the SEM level. This new approach has ensured a
reduction in the amount of time taken to evaluate samples at the SEM level as less time is spent
searching for the isolated hooks at high magnifications.
One of the main duties of the Pathology and Parasitology team is to be able to readily and accurately
identify pathogens in tissue sections prepared by standard histological techniques. In many cases,
this is relatively straightforward. Identification of a pathogen can be based on a recognised pathology
associated with a particular organism or on the visualisation of the pathogen in tissue section. This
visualisation however, relies on the fact that the pathogen or the surrounding tissue is subjected to
differential staining and thus allows the pathogen to be discriminated form the host tissue. This forms
the basis of all current dye-based staining methods. The most commonly used dye-based method is
Haematoxylin and Eosin (H&E). All currently available dye-based stains are for the most part robust,
but can require considerable expertise and an element of trial and error in order to ensure consistent
results with the stains. A student was thus tasked to utilise and refine a number of recognised, but
under-utilised methods including the Russell-Movat Pentachrome stain. There is a requirement for
the methods of each “new” stain to be manipulated according to local conditions and the material
being examined. Thus the student carried out these trials over a period of several months. In
addition, in order to provide new stains that might provide discrimination of pathogens from the
surrounding tissues, the student attempted to use a number of different dyes, including over-thecounter fabric dyes. The most successful was Mexican Red which was selective for the hard parts of
monogeneans parasites. It was useful in demonstrating the presence of the hooks in tissue sections
and in staining up these structures on whole mount specimens thus allowing more detailed
examinations to be made. Overall, these novel dye based stains provide a robust and cheap method
to visualise pathogens in tissue.
The use of biotinylated lectins has been used previously to demonstrate the presence of selected
pathogens in tissue sections, including the agent for salmonid proliferative kidney disease (PKD),
Tetracapsuloides bryosalmonae. The method relies on the lectins binding to selected sugars on the
parasite and in some cases, this has been found to be specific to certain pathogens. In the case of
T. bryosalmonae, the lectin GS-1 appears to be specific to the parasite, but only at selected life
stages. A panel of 30 lectins were tried on a range of pathogens, including T. bryosalmonae, and
whilst the data generated provided new information on the types of sugars present in some of these
CSG 15 (1/00)
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Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
pathogens, no particular lectin was found to be more effective at discriminating pathogens from
surrounding tissues when compared with the less labour intensive, and cheaper methods of
established dye-based stains.
The use of antibodies raised to selected pathologies or pathogens has been use successfully for a
number of years in disease studies in a wide range of animal groups and in particular in mammalian
studies. Their use in understanding of disease pathogenesis in fish is, however, restricted to a small
number of studies. The utility of antibodies, is that they are useful to identify particular pathogens on
the basis of an immune response, or they can provide data on the types of responses that the animal
produces to a particular environmental or pathogen insult. A number of commercially available kits,
originally developed for mammalian tissues, were selected to see if they could be used on fish
material. It was expected that the majority of these kits would prove ineffectual as they were
designed to be used in mammalian tissues. To demonstrate their usefulness, the kits purchased
were those predominately developed for the discrimination of selected tumour types in mammals.
There were two reasons for this. Firstly, CEFAS has a wide selection of different liver tumour types
collected over a number of years predominately from marine fish disease cruises, and a small
number from freshwater disease outbreaks. Secondly, it was expected that there would be a degree
of conservation in the antibody responses to in both mammals and fish and thus the chances of
success were considered to be greater. The kits were more successful than expected and were used
to show that the degree of conservation between mammalian tumour types and fish tumours was
very close. The results obtained are to be used to submit a project proposal to the medical research
council and Defra to further refine the methods and to provide insights into mammalian cancers. As
well as examining tissues at the light microscope level, the antibodies were labelled with gold and
used on samples prepared for transmission electron microscopy (TEM). This allowed for the
localisation of the antibodies within cells and cell organelles, confirming the highly specific nature of
the antibodies. The development in the use of antibodies has provided data on host responses at the
cellular level and allowed the greater understanding of the pathogenesis of disease in aquatic
animals.
A more recent refinement of visualisation techniques has been the development of DNA-based
methods known as in-situ hybridisation (ISH). In essence, this is a PCR reaction carried out on
tissue sections. Whilst the method is relatively well established, as with the dye based stains, it
requires an element of refinement and optimisation for each new probe developed which can take a
number of months and trials to complete. The main use of the ISH technique by the students was in
the development of the method for the visualisation of the PKD agent in tissue sections. Initial trials
with ISH on tissue containing the PKD agent demonstrated that the probes were effective when large
numbers of the parasite were present, but were much less effective when few parasites were
present. This, obviously was unacceptable, as it was anticipated that the method was to be used to
visualise the parasite in tissues as it entered the fish host. Thus, the student was tasked with
optimising the method to ensure that it was able to detect single cells in tissue sections. This was
successfully done and as a result, the methods were then applied to tracking the route of infection of
host under Defra contract FC1138 with a subsequent publication arising form this work.
For a number of years, data has been collected on the parasites and pathogens of juvenile cyprinids
in Yorkshire and Humberside. As reported in FC 1137 and FC1138, these data have provided
information on the pathogens present in these fish and demonstrated, using multiple regression
models, that disease may have an impact on fish population success. As a stand alone method, the
multiple regression models are considered robust enough that it is clear that disease is an important
factor in recruitment success. However, to provide further support, and to examine some ecological
aspects of disease transmission, a student was employed to carry out further analyses of the data.
These studies were complementary to those carried out under FC1137 and FC1138 and confirmed
CSG 15 (1/00)
5
Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
the results obtained by the multiple regression approach that disease was an important factor. This
included the application of new and novel statistical methods such as principal component analysis,
non-metric multidimensional scaling and methods developed under marine monitoring programmes
to demonstrate, that even in areas where there were limited data sets, disease may be an importantly
factor. When using regression modelling techniques, there is a need to have continuous data sets to
demonstrate a correlation. By using other non-parametric methods, we were able to show that
certain populations clustered together on the basis of disease status. Thus, we were able to infer
that some of these populations were subjected to similar pressures and may therefore be impacted
by disease. In addition, the student applied a number of methodologies normally used in free-living
population ecology studies to the data to show that the parasite populations were subjected to similar
selection pressures as their free-living counterparts. This included the demonstration that some
parasite populations were highly nested, as well as over-dispersed in any given population.
Furthermore, the student was able to show using these methods in combination with regression
models, that these aspects of parasite ecology may in themselves be responsible for the mortality of
juvenile fish.
Virology and Molecular Genetics (VMG)
The main purpose of students seconded to the VMG team was to develop and refine methods and to
characterise unknown viruses of concern to Defra and the industry. In essence, this meant that the
student provided support and added value for Defra contract FC1136. Overall, the students
characterised new and emerging viruses by a number of methods, including serological methods and
molecular biology and refined PCR primers for use in virus identification. By utilising students to
carry out the initial characterisations and refinement of PCR primers, this allowed staff to concentrate
on other duties and more in-depth studies appropriate to the Defra contracts.
One of the first steps in virus identification is the isolation of putative viruses on susceptible cell lines.
The production of a cytopathic effect (CPE) in cell lines seeded with fish viscera tends to indicate that
viruses are present in the tissue sample. However, for each cell line there are clearly defined
environmental parameters that need to be elucidated in order to ensure that the virus grows under
optimal conditions. For each cell line, these parameters will vary but include pH and temperature.
One student was employed to consider the effect of altering pH and temperature on the viability of
selected cell lines. It was found that pH needed to be kept within relatively tight tolerances in order
for CPE to be noted in the cell lines. In addition, as expected, the temperature at which the cell lines
were grown was critical to the success of the cell line. For each virus that is tested for by CEFAS,
there is a particular cell line that is used. In order to confirm that the correct cell line was being
utilised for the correct virus, trials were initiated to attempt to grow different viruses on different cell
lines. It was found that in the majority of cases, the cell lines currently used for selected viruses were
the best. The data provide confirmation that the current cell lines are the most appropriate and
provided information of those cell lines that were most suitable for isolating new and emerging
viruses. Once any new and emerging viruses have been isolated, there is a need to carry out
primary tests on these isolates to elucidate some of the basic biological characteristics and to confirm
that the CPE seen in tissue culture is caused by a virus. For any viruses that were reported, the
student carried out a number of these tests, including neutralisation and serological tests. Data
obtained was used under FC1136 to further characterise the viruses isolated. One such example
where the use of students proved to be extremely useful was in the development and use of
serological methods for virus identity. Students were used to carry out initial characterisation studies
on aquabirnaviruses from ornamental fish imported from Singapore. These studies demonstrated
that these viruses represented a new serogroup of aquabirnaviruses, which was subsequently
confirmed by molecular methods.
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6
Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
More recent advances in molecular biology has meant that PCR has become much more routine.
However, this still requires the use of different primer sets for each virus or group of viruses. In order
to produce these primers, there is a requirement to know, at least in part, what the sequence of the
virus in question is. This requires the development and refinement of PCR primers. Students were
used to develop primer sets for a range of viruses and to then optimise the PCR reactions to obtain
maximum yield. In the case of the initial characterisation and in improvement of primer specificity
and PCR reactions, this long term development has freed up the staff to concentrate on other duties.
In some cases, refinement of PCR reactions can take up to six months.
Following the development of these new primers, two students in successive years were then able to
carry out initial sequence analysis of the Koi Herpes Virus (KHV) genome. Such information is vital
to understand the origin of the virus, as well as providing information that may prove useful in future
to combat the disease.
Microbiology team
Studies carried out in the Microbiology team were designed to either provide direct support to existing
Defra contracts or to act as a “pump-priming” mechanism to investigate new and emerging bacteria
or technologies. The main bacteria of interest in this area were Lactococcus garvieae and
Renibacterium salmoninarum (the causative agent of bacterial kidney disease) and in carrying out
initial trials on phage therapy.
As with studies carried out in the VMG team, some of the students time was occupied with carrying
out initial characterisations of “new” bacteria. Established biochemical and physical methods of
identification were used to characterise the bacteria and confirm their identity. Whilst these primary
test are important to ensure that the bacteria has been properly identified, it is essential that the
bacteria are then further characterised using secondary methods to confirm the differences noted by
the primary tests. In order to ensure that the correct methods were employed in characterising these
“new” bacteria, the student tasked with this duty was asked to carry out a number of trials to confirm
that these methods were appropriate. This included modifying the types of agar used to ensure that
the most appropriate methods were being utilised. It is important that new technologies are employed
wherever possible to ensure that the latest diagnostic methods are available to assist in the
identification of bacteria. A student was employed to develop new PCR primers to assist in this task.
These new primers were used to good effect during the outbreak of Lactococcus garvieae when the
primers developed by a student were used to confirm the presence of the bacteria in the fish. In
addition, the primers were also used under FC1151 to carry out further bacterial identifications.
A number of students were used in support of studies on Renibacterium salmoninarum, the causative
agent of bacterial kidney disease (BKD). This mainly involved carrying out a number of pre-studies
prior to the commencement of the Defra funded studies on BKD. One of the main objectives of the
Defra funded work was to assess the impact and distribution of BKD in wild fish. In order to
determine how widespread the disease was and to inform the study design, a student was employed
to carry out an initial survey on wild fish to assess which species were most likely to act as carriers of
the bacterium. The information gained determined the future design of the Defra funded work. It was
recognised that such filed studies would require the collection of numerous kidney samples from fish
in the wild with the concomitant problem of storage on the quality of those samples on return to the
laboratory. Thus, studies were conducted to determine the effect on PCR efficiency of multiple
freezing and thawings of BKD infected kidneys. It was founds that best results were obtained if the
kidney samples were frozen as soon as possible after collection and that after less than five freezing
and thawing cycles, the bacterium could no longer be amplified by the PCR test in these samples.
The information collected from both the distribution of the bacterium in wild fish and the impact of
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Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
storage studies was used top design the fish farm survey that was later carried out under the Defra
project. Finally, on studies relating to BKD, some initial trials comparing the efficiency of PCR with
traditional culture methods was conducted. As expected, the PCR method was far more sensitive
when compared with culture methods and results could be obtained within a much shorter time
period. However, it was recognised that in using the PCR method there was a possibility that false
positive results may have been obtained.
The use of phage therapy to control bacterial infections has been promulgated for a number of years
but it has only been recently that its potential use in controlling bacterial infections in fish has been
suggested. Prior to the awarding of a Defra project to CEFAS, initial trials were conducted to assess
the feasibility of using phage therapy to control Aeromonas salmonicida. The physicochemical and
biological factors which affected the growth and virulence of the phage were assessed. Once the
optimum conditions had been assessed, it was then possible to establish and enhance the in vivo
and in vitro virulence of the bacteriophage. As a result of the initial trials carried out by the student,
the most appropriate methods of administering the phage were established and methods to recover
and quantify the levels of phage in both the fish and the environment were developed.
Inspectorate team
Over a period of two years, students were employed with the express purpose of determining the
efficacy of disinfectants for use in disease control. Products were rested on the basis of their current
or planned use within the aquaculture industry and the main aim of the project was to provide
guidelines on the use of the selected disinfectants in disease eradiation programmes. It was not
intended that the results would be used to recommend any particular treatment and a distinction was
made in the study between daily use of disinfectants on the farm and their use in eradication.
The products selected were tested on a range of bacteria and viruses of concern. The initial trials
were conducted to determine the optimum concentration and length of exposure required to
inactivate the pathogen. The values selected included a wide range around those recommended by
the disinfectant manufacturers.
It was found that the most efficacious disinfectants were
hypochlorite, buffodine and virkon which inactivated the pathogens after a 5 minute exposure. These
values however were obtained under optimal laboratory conditions and thus do not reflect conditions
in the field. Thus trials were instigated to measure the effect of a number of environmental
parameters. It was found that altering the pH of the water in which the disinfectants were used had a
limited effect on disinfectant efficacy. However, increasing the organic material of the water
decreased the efficacy of all disinfectants tested, with the exception of gluteraldehyde where there
was limited impact. Finally, the longevity and stability of disinfectants were tested. It was noted that
exposure of the products to air reduced the efficacy, a point which is noted by the manufacturers
whose recommendations err on the side of caution.
Physiology team
The main purpose of the studies carried out in the physiology team was to examine selected factors
impacting on the development of the aquaculture industry. The two main factors examined were in
relation to gonad development and fin erosion.
It is important, particularly in single-sex culture, to determine the post-hatch age at which the gonad
develops. The length of time taken for sexual differentiation to occur varies between species and is
determined by a number of external and internal factors. This can include the presence of dominant
siblings, temperature and photoperiod. A project as initiated to examine the age at which Dover sole
gonad develops and at which it is possible to determine the sex of individual fish. This information is
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Project
title
Studies on Aquaculture and Fish Diseases (Sandwich
Students)
DEFRA
project code
FC1135
also important in studies to manipulate the sex of individual fish. Since there was an interest in
producing single sex cultures of Dover sole to reduce the possibility of inbreeding this information
was crucial. The data obtained allowed the determination of the minimum age (and thus the earliest
stage) at which the fish could be sexed. In addition, it provided data on the latest age at which the
sex of the fish could be manipulated. Subsequently, studies were instigated to refine the methods
currently used to manipulate sex ratios in Dover sole. These methods included pressure and heat
shock.
Finally, with the exception of disease, one of the major constraints in fish farming is the problem of fin
erosion. In addition to reducing the market value of the stock, the main concern is that fin erosion
can be seen as a welfare issue. Studies were undertaken to examine the cause of fin erosion in
Dover sole with the intention of applying the knowledge gained to salmonid aquaculture. A series of
experimental approaches were taken to manipulate the environment in which Dover sole were
reared. It was initially suspected that bacterial infection may be responsible for the fin erosion.
However, extensive studies using standard microbiology techniques failed to isolate consistent
bacteria in sufficient quantities to support this view. In addition, histological analysis of affected fins
demonstrated that although bacteria were present, they did not appear to be the primary cause of
erosion. Ultimately, it was determined that the behaviour of the fish, particularly in the juvenile
stages was most important. Fin nipping, either as a result of hierarchical structures being developed
or by accident during feeding periods appear to contribute to the erosion which is then compounded
by the introduction of opportunistic bacteria through the damaged epithelium.
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