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Restricted – Commercial (When completed)

LINK AQUACULTURE - PROJECT COMPLETION FORM

Project Ref. No. Project Start Date Project Completion Date

FIN22 1st May 1999 31st July 2003




Name of LINK Project: Rearing Protocols for Atlantic Halibut Larvae During Transition from Endogenous to Exogenous Nutrition.


Name of LINK Programme: AQUACULTURE

Please enter details in the boxes below:


Project Leader’s Name: Dr James Buchanan
Organisation: British Marine Finfish Association
Address: Overton, 2a Manse Road, Roslin, Midlothian EH25 9LF

Tel: 0131 4402116 Fax:      Email: Jim.Buchanan@onyxnet.co.uk







Project Costs (£)/Effort




Government Department

Research Council

Industry

Total

Approved Spend



293569

295879

589448

Actual Spend



304149

325349

629498

Approved Staff Input*




7.05

8.38

15.43

Actual Staff Input*




5.88

3.47

9.35

* Staff years

Participants

List all project participants by the following categories:

Industry

Research Base

Other


Large Enterprises

Small and Medium Sized Enterprises*

Higher Education Institutes


Other Research Base Partner

Other





     


British Marine Finfish Association

University of Glasgow

Seafish Aquaculture

     

     

     

     


Scottish Association for Marine Science

     

     

     

     


NERC (until 31/03/01)

     

     

     

     


     

     































* Less than 500 employees and with an annual turnover of less than £30 million





Section 2 – Objectives

THEME 1 Characterisation of larval attributes in commercial versus experimental rearing systems

Studies of structural, behavioural and microbiological attributes will be carried using larvae reared at the SFIA hatchery, to provide baseline information on relevant aspects of larval biology and to determine the timing and nature of problematic events in commercial systems.



Objective 1a Investigate sensory organ development and characterise the structural damage and developmental abnormalities of halibut larvae in commercial versus experimental rearing systems.

Objective 1b Describe the vertical distribution of yolk sac larvae in rearing tanks.

Objective 1c Describe the foraging behaviour of larvae in commercial rearing tanks, at critical phases of the rearing process.

Objective 1d Characterise the microbial communities in the tank water, in live feeds and in the larval gut.
THEME 2 Tolerance of larvae to physical stressors.

Theme 2 of the project will investigate whether tolerance to mechanical handling, or temperature change, vary according to developmental stage. The aim is to determine the most appropriate developmental stage for tank transfer and temperature acclimation, and to devise less damaging handling and acclimation protocols.



Objective 2a. Compare effects of mechanical handling at different developmental stages.

Objective 2b. Determine responses of larvae to temperature increases at different developmental stages and at different rates.

Objective 2c. Evaluate recommended handling and acclimation protocols in commercial-scale rearing systems.
THEME 3 Interactions between environmental conditions in rearing tanks and behaviour of larvae.

Theme 3 of the project will investigate whether the behavioural attributes of halibut larvae are responsive to differences in physical, chemical and biological variables. The aim is to identify appropriate environmental conditions that enable yolk sac larvae to be retained within the water column in commercial rearing tanks and that promote foraging behaviour during the feed initiation phase.



Objective 3a Determine the behavioural responses of different-aged larvae to vertical salinity and temperature gradients.

Objective 3b. Determine optimal environmental conditions for successful foraging, through manipulation of microalgal and lighting conditions. The responses of larvae to different lighting conditions, and to physical and chemical aspects of “green water” will be investigated.

THEME 4. Microbial control in larval rearing systems



Theme 4 will build on the findings obtained within the current BHA-funded research project on “Microbiology of Larval Halibut Rearing”. To date, a microbiological profile has been built up of the SFIA halibut hatchery and work is ongoing to characterise bacteria from other UK hatcheries (Theme 1 activity). During the final experimental phase of the BHA project (Year 1 of the LINK project), bacteria isolated from commercial hatcheries will be tested for their effects on halibut larvae, using a model rearing system. The emphasis of research work within the LINK project will then be to test methods of microbial control within the hatchery environment, including the control of total bacterial numbers and the establishment of benign (“probiotic”) bacterial communities.

Objective 4a Investigate the interactions between bacteria isolated from halibut larvae.

Objective 4b Determine the effects of individual bacterial isolates on larvae.

Objective 4c Quantify the effects of microbial water quality on yolk sac larvae in commercial rearing systems.

Objective 4d Evaluate the effects of microalgae (“green water”) and live diets on gut flora during the feed initiation phase.

Objective 4e Determine whether selected bacterial strains can be established in commercial rearing systems and evaluate their effects on rearing performance.


Milestones


Target Date

Milestones Met?

Number

Title




In full

On time


01/04

commercial efficacy of microbial control methods

01/03/00

Yes

Yes

04/01

characterisation of bacteria from commercial hatcheries

01/04/00

Yes

Yes

01/01

structural characteristics of larvae in commercial systems

     

     

     

02/01

Vertical distribution of larvae in commercial rearing systems

     

     

     

03/01

Foraging behaviour of larvae in commercial first feeding tanks

     

     

     

01/02

Effects of handling larvae and recommendations

     

     

     

02/04

Relative contributions of exogenous inputs to larval gut flora, and on efficacy of probiotic approach

     

     

     

02/02

Effect of temperature acclimation and commercial recomendations

     

     

     

03/02

Commercial scale trials










01/03

Use of salinity/temperature gradients in moderating larval behaviour










02/03

Foraging behaviour in relation to microalgae













Final Report













Executive Summary of Research and Results (Include targets and objectives indicating progress made towards achieving them, or reasons for those not achieved. Also include highlights, outputs, deliverables and any unexpected benefits)
Executive Summary
This collaborative project, coordinated by the British Marine Finfish Association, brought together research teams from Seafish Aquaculture, Dunstaffnage Marine Laboratory and the University of Glasgow to study Atlantic halibut larvae (Hippoglossus hippoglossus), a critical period in their early life history transition between endogenous (yolk-sac) and exogenous feeding. The final aim of the study was to improve larval rearing protocols for this species.
The study has focused on reducing damage (both due injury and pathogens) to larvae in the yolk-sac stage and during the stress of transfer to feeding tanks in order to maximise the number of larvae able to feed. The first feeding stage has also been studied with particular emphasis on the use of “green water” when microalgae are added to encourage feeding. The mechanism by which green water stimulates feeding was not known before this project.
Our research has demonstated that damage to larvae can be reduced by having a more stable salinity regime in the yolk-sac silos together with a low salinity surface layer, which will reduce interactions with surfaces and a result in a more even distribution of larvae within the tank. Damage to larval sensory systems, in particular neuromasts (mechanoreceptors) will be reduced increasing the number of larvae able to feed. Mortalities due to microbial pathogens depends not on numbers but on the species composition of the flora which can be made more stable by using recirculation systems. An optimum transfer protocol both temperature increase regime and developmental stage has been shown to reduce mortalities and deformities at the first feeding stage.
Three species of microalgae (Nannochloris, Isochrysis and Pavlova) were used in our studies of the first feeding stage. Larvae fed and survived better when Nannochloris was used and that higher algal densities than had been used in the past produced better survival and growth. This does not appear to result from a strong chemosensory stimulus effect nor is it a nutritional effect but is merely due to the physical presence of the algae; inert particles can successfully substitute for Nannochloris. Further research is needed establish the precise mechanism.

Some of the results of our research were used as the basis for successful commercial rearing trials conducted at Seafish, Ardtoe in 2002.







Executive Summary of Research and Results/continued:
Objective 1a: Investigate sensory organ development and characterise the structural damage and developmental abnormalities of halibut larvae in commercial versus experimental rearing systems.

This part of the project is a collaboration with a UHI Millennium Institute Postgraduate Student, David Nairn. The primary focus of this work is on the development of mechanoreception. Larval fish have superficial neuromasts which, later in development, are enclosed in the lateral line canals. These organs detect water displacement and are responsible for detecting near field vibrations and water velocity but are vulnerable to damage. The gelatinous cupula which is deformed by water displacement is prone to breakage and may not reform properly or may result in malformation of the neuromast itself.. Neuromast development has been described and damage to both the cupulae and absence or malformation neuromasts has been observed. The large neuromasts that first appear on hatching are broken in 50% of larvae. The neuromast arrays surrounding the olfactory epithelia are frequently damaged or some absent in larvae that fail to feed.


Objective 1b: Describe the vertical distribution of yolk sac larvae in rearing tanks. And Objective 3a Determine the behavioural responses of different-aged larvae to vertical salinity and temperature gradients.

The distribution of yolk-sac larvae in rearing silos was observed using infra red illumination and video recording. These experiments have confirmed the preliminary results which showed that the distribution of larvae within the silo depends upon salinity and not temperature variation. The response of larvae to salinity variation changes during development. Larvae can be found at all depths in the tank throughout the yolk sac phase but mean depth decreases during this period. Early in the yolk-sac phase larvae tend to move lower in the tank during bouts of low salinity rising again when salinity increases. Later larvae are less likely to sink when salinity is low but tend to move towards the surface temporarily when salinity increases again. Neuromasts are likely to be damaged when larvae interact with surfaces (walls or surface tension layer) or when there is considerable shear stress due to turbulence. Experiments with a ¼ scale glass model silo have shown that when water is flowing into the bottom of the silo at reduced salinity convection currents cause mixing of the water redistributing the larvae. Even when water salinity is stable flow at the base of the conical part of the silo is turbulent rather than laminar. Water flow in the silos is, therefore, likely to be the cause of neuromast damage to the larvae.

In an additional experiment (not in the original plan of work) we have used a density gradient column to test the density of yolk-sac larvae during development and have revealed that density increases during this period. The observation that larvae move nearer the surface later in the yolk sac stage can therefore only be explained by an increase activity or other changes in swimming behaviour. Video recordings will be reanalysed to address this issue.

It is clear that for hatcheries with seawater supply prone to salinity variation the salinity of water supplied to yolk sac silos needs to be controlled. To this end a salinity controlling system has been designed by the project team and applied in our most recent experiments.


Objective 1c Describe the foraging behaviour of larvae in commercial rearing tanks, at critical phases of the rearing process.

A manual frame by frame analysis of in situ video recordings was use during the early part of the project to describe foraging behaviour. The development of suitable automated analysis software has limited the application of this type of analysis to video recordings from “green water” experiments. New software has recently been purchased by colleagues in Italy who we are working with in an EU funded project and will be evaluated for use in this project.


Objective 1d Characterise the microbial communities in the tank water, in live feeds and in the larval gut.

The major objectives were completed in the first year of the project. In larval halibut there was a defined progression in the development of the bacterial gut flora through the egg yolk sac phase and first feeding. In the yolk sac phase the gut was colonised by low numbers of bacteria of random types but on first feeding large numbers of bacteria colonised the gut, originating from the live feed. Colonisation was similar in different hatcheries and was highly selective, maturing such that vibrios and pseudoalteromonads not found initially came to dominate the flora of mature feeding larvae. The work has resulted in 2 publications – see below.





Executive Summary of Research and Results/continued:


Objective 2a: Determine effects of mechanical handling on larvae at different developmental stages





Experiments in 1999 demonstrated the suitability of small-scale static water rearing systems (3 l bowls) for raising yolk-sac larvae (with a mean survival rate at first-feeding of 14%, compared with 2.8% in a conventional yolk-sac silo). A handling trial in 2000 included standardised handling treatment (gently transferring larvae from one bowl to another with a beaker ) at 150, 174, 192, 222, 246 and 270 degree days (four bowls per treatment, 200 larvae per bowl). Data on percentage survival, length, dry weight and degree of deformity due to handling was obtained. There were no significant differences in end survival at 298 dd (mean end survival = 10 %; F(5,18) = 0.34, p = 0.881). However, larvae handled before 200 dd were significantly larger than larvae handled post 200 dd (F(1,15) = 6.81, p < 0.05). Samples were also preserved in formalin after each handling treatment and at the end of the experiment for electron microscopical analysis of sensory organ damage (Objective 1a).

Following the monitoring meeting in 2001, this experiment was successfully repeated in 2002, using a more robust handling method in which the contents of one bowl were simply poured into another, providing a greater stress. Survival results were similar to the original experiment (no significant difference; F5,25 = 0.70, p = 0.632). However, unlike the above findings, there was also no significant effect on larval mass at first-feeding (F5,24 = 0.08, p = 0.995).

Objective 2b: Determine the response of different larval developmental stages to a range of temperature acclimation regimes

Phase I: this objective is similar in design to 2a, with temperature increase (from 6 to 10oC at a fixed rate) instead of standardised handling, at 150, 190, 230 and 270 (control) dd. As with the above objective, the suitability of the design has already been tested, and water baths (to raise the temperature at predetermined developmental stages) were installed in a dedicated facility. Similar data to Objective 2a were collected. There was no overall effect on end survival, although survival was lowest at 150 dd and highest at 190 dd. Although neither result was significantly different from the control, they were significantly different from each other (F(1,8) = 5.25, p = 0.05). Temperature increase at different stages had no effect on larval size at the end point (290 dd).

Phase II of this objective involved raising the temperature at the least vulnerable developmental stage but at varying rates. On the basis of the above data, the temperature was raised at 190 dd at three different rates (1, 2 and 4 oC.d-1), and compared with a control of a temperature increase of 1oC.d-1 at 270 dd This experiment was carried out using yolk-sac larvae from Ardtoe’s June/July 2001 spawning stock, and performance parameters assessed. The ‘high’ stress group (4oC.d-1) showed significantly higher mortality. The ‘low’ and ‘medium’ stress groups did not significantly differ from the control. However, the control group lost more mass per degree-day and subsequently weighed less than the experimental groups, with the ‘medium’ stress group (2 oC.d-1) performing best in terms of mass at first-feeding.

Results from the above two objectives suggest that to maximise larval survival, mass and condition, larvae should be handled and subjected to temperature increase between 180 – 200 dd.



Objective 2c and 3b (Phase III): Application of results on a commercial scale



The above results were combined with the results of Objective 3 and compared with current Ardtoe best practice on a commercial scale (1000 L yolk-sac silos and 1300 L first-feeding tanks). Two egg batches were split into four silos (20 000 eggs per silo); one silo from each batch treated as the experimental group, the other as a control. The two experimental groups were moved at 190 dd to first-feeding tanks, and the temperature raised to 10°C over three days. Lights, algae and food were administered at 230 dd. The experimental groups received twice as much Nannochloris algae as the controls throughout (mean = 2 cf. 1× 106 cells.ml-1). The control groups were moved to first feeding tanks at 270 dd and algae, food temperature increase and light immediately administered.

By day 60 post first-feeding, growth in one experimental group was significantly greater than it’s control counterpart (68 % greater; p<0.05), with a slight increase in percentage survival (21 vs. 19). In the other experimental group, there was no improvement in growth, but a large increase in survival (24 vs. 15 %). Therefore, in terms of biomass (dry weight; g), the experimental groups weighed 28.4 and 45.5 g at day 60, compared with 11.9 and 26.7 g for the controls. Although each group started with 20 000 stocked eggs, the early removal and high algal levels contributed to improvements in production of 138 and 70 % per experimental group.






Executive Summary of Research and Results/continued:

Objective 3b. Determine optimal environmental conditions for successful foraging, through manipulation of microalgal and lighting conditions. The responses of larvae to different lighting conditions, and to physical and chemical aspects of “green water” will be investigated.

Our first experiment showed that adding Nannochloris to rearing tanks resulted in better feeding, survival and growth of the larvae when compared with Isochrysis or Pavlova. More recent experiments, concentrating on the use of this Nannochloris, have shown that increasing microalgal density increases survival and growth. Densities below 2x105 cells.ml-1 had no effect and survival, growth and feeding incidence continued increase up to the highest density used (1.2x106 cells.ml-1). This is a much higher cell density than has been used routinely in the past. These effects could not be attributed to alteration in the bacterial flora, nutrient loading or ammonia levels in the rearing tanks.

Further experiments designed to ascertain the sensory stimuli provided by “green water” that stimulate feeding have focused on chemical and physical stimuli. When algae were added to dialysis bags floating in the rearing tanks larvae did not perform any better than no algae controls suggesting that a chemical stimulus alone may not be sufficient to stimulate feeding. In the final experiment the effect of inert particles, algae and algal filtrate on survival and first-feeding response were compared. Two types of inert particles, Kaolin and Vinacryl beads (Vinamul, Ltd.), were used with sufficient particles added to match turbidity with the Nannochloris treated tanks, 5 NTU. At this turbidity level absorbance (optical density) was similar in both Kaolin and Nannochloris treatments but much lower when Vinacryl was used. An additional treatment used Vinacryl at double the density in order to match absorbance with the other treatments. Survival after 11 days was similar in the Vinacryl and algal treatments at over 50% but Kaolin resulted in only 10% survival, similar to controls (11%). Although 25% of larvae in the algal filtrate treated tanks survived feeding and growth were very poor and similar to the Controls. We can conclude that the feeding and survival enhancing effect of “green water” is not due to chemical feeding due primarily to a physical effect and that it may be possible to replace algae with an inert substitute. Very low bacterial counts were found in the algal group compared with others with barely detectable levels of bacteria. This is not wholly unexpected as some algae have pronounced anti-bacterial effects. It is possible that this accounts in part for the high survival of larvae in this group. The vibrios isolated were almost all sucrose -ve, except for those from the kaolin group. In addition, the Vinamul group contained a higher proportion of non-vibrios than other groups (17/52 isolates) compared with (8/132) from all other groups combined. The poor survival of the Kaolin treated larvae is intriguing especially since survivors had fed and grown well but may be explained by these differences in bacterial flora. Further analysis will be carried out by identifying dominant organisms of the bacterial populations for each group. This will take some time to achieve as limited resources are available for this task.



Executive Summary of Research and Results/continued:
Objective 4a Investigate the interactions between bacteria isolated from halibut larvae.

Screening of a large number of bacteria isolated from halibut and turbot has revealed three which are highly antagonistic towards other bacteria, including the major pathogen Vibrio anguillarum.


Objective 4b Determine the effects of individual bacterial isolates on larvae.

In several infection trials 20 isolates from halibut larval rearing trials, both those which performed well and those performing poorly, were tested for their activity when added to larvae reared in the absence of bacteria. None of the 20 isolates caused larval mortalities, in contrast to Vibrio anguillarum, which was highly virulent. A manuscript has been submitted – see below.


Objective 4c Quantify the effects of microbial water quality on yolk sac larvae in commercial rearing systems.

These experiments (5 replicates) showed that the bacterial flora of larvae could be manipulated by (a) rearing in a recirculation system with water passed through a biofilter, or (b) addition of antibiotics, in comparison with the standard flow-to-waste system. Higher survival occurred in recirculated water and in antibiotic-treated larvae than with flow-to-waste. The concentration of bacteria in the gut was greatest in the recirculated water showing that the types of bacteria not numbers was important in determining survival. A manuscript is in preparation.


Objective 4d Evaluate the effects of microalgae (“green water”) and live diets on gut flora during the feed initiation phase.

The initial work concentrated on determining whether potential probiotic bacteria could prevent mortalities on yolk sac larvae induced by V. anguillarum. None of the 3 isolates from objective 4a were capable of this and it is proposed that such studies should be limited to the first feeding phase when loading of the gut is possible by colonisation of Artemia with probiotic bacteria.

As an additional part of this section, linked to Objective 3b (Effect of microalgae on feeding behaviour) we have begun to investigate the changes in microbial flora in the larval gut under rearing conditions with different types and concentrations of microalgae. Molecular biology techniques are being evaluated to monitor changes in the gut bacterial flora. This involves surface sterilisation of the larvae, extraction of DNA, PCR amplification of the 16S rRNA genes of bacteria present and cloning the amplicons into a plasmid vector to form a clone library. From each sample we have selected 192 clones for interrogation with DNA probes specific for eubacteria and vibrios. Other probes are under development. Initial results have revealed the presence of some unusual bacteria, not likely to be detected by culture, as well as some routinely found by culture. This work will be continued over the next few months to provide a complete picture of the changes in bacterial flora induced by culture in the presence of different microalgae.









Future R&D resulting from this project (Include any other non-tangible benefits and state any Teaching Companies Scheme action if appropriate)

Our experiments on the distribution of yolk sac larvae in culture silos have highlighted inadequacies in the design of these tanks. Flow pattern and control of salinity could be improved. Trial and error would not be appropriate but with some input from physicists and modellers computational fluid dynamics could be applied to design an improved tank.


Our experiments on the effects of “green water” show that this is an important area for further study because of its application to all species of marine fish larvae, not just halibut. Some related work is being carried out by colleagues in Norway. It would be sad if this was not to continue in the UK.



Industrial relevance and plans for future commercial exploitation

     




Patents and Publications (Including those pending)

Birkbeck, T.H. and Verner-Jeffreys, D.W. (2002) Development of the intestinal microflora in early life stages of flatfish. In: Microbial Approaches to Aquatic Nutrition within Environmentally Sound Aquaculture Production Systems. (C.-S. Lee and P. O'Bryen, eds.) pp. 149-160. The World Aquaculture Society, Baton Rouge.

Verner-Jeffreys, D.W., Shields, R.J., Bricknell, I.R. and Birkbeck, T.H. (2003) Changes in the gut-associated microfloras during the development of Atlantic halibut (Hippoglossus hippoglossus L.) larvae in British hatcheries. Aquaculture, 219, 21-42.

Verner-Jeffreys, D.W., Shields, R. and Birkbeck, T.H. (2003) Bacterial influences on Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larval survival and start-feed response. Diseases of Aquatic Organisms, in press.

Verner-Jeffreys, D.W., Shields, R.J., Bricknell, I.R. and Birkbeck, T.H. Effect of different water treatment methods and antibiotic addition on larval survival and gut microflora development in Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae. Submitted to Aquaculture. March 2003.

Oral papers were presented at the British Marine Finfish Association Workshop in November 2000, November 2001 and November 2002.




To be completed by the Project Leader

Declaration


I declare that the information given has been approved by all the project participants and is correct to the best of the best of my knowledge and belief I understand that the information contained in this form may be held on a computer system.

Signed:Jim Buchanan Position:      Date:15/12/03








Thank you for completing this form. Please return to:
Dr Mark James

Programme Co-ordinator

LINK Aquaculture, FRM Ltd.,

Freshwater Fisheries Laboratory

Faskally

Pitlochry

Perthshire PH16 5LB

Tel: 01796 472 060 Fax: 01796 472 856 Email: majames@frmltd.com









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