THE EFFECTS OF AZAMETHIPHOS ON SURVIVAL AND SPAWNING SUCCESS IN FEMALE AMERICAN LOBSTERS (HOMARUS AMERICANUS )

Les Burridge, Kats Haya & Susan L. Waddy

Fisheries and Oceans Canada, Biological Station, St. Andrews, New Brunswick, Canada E0G 2X0.

In southern New Brunswick, Canada the period of peak sea lice infestation and treatment is the summer and autumn. This time is coincident with spawning of the American lobster (Homarus americanus). Salmonid aquaculture sites and lobster nursery areas share the same water, resulting in a situation in which lobsters may be exposed to effluent from sea lice treatments. The only pesticide registered for use against sea lice in Canada is the organophosphate azamethiphos, an acetylcholinesterase inhibitor.

Preovigerous female American lobsters (N=72) were divided into three groups (two treatment and a control) and ovarian maturation and spawning was induced using elevated water temperature and short day length. Lobsters were exposed four times for one hour to either 10 or 5 m g/L of azamethiphos. These concentrations represent 10 and 5 % of the recommended treatment concentration. Treatments were separated by two weeks. Survival and success of spawning were monitored. After the fourth exposure to 10 m g/L azamethiphos 43% (10) of the lobsters had died, only one was dead after the third treatment. In contrast, only 8% (2) of those exposed to 5 m g/L died (only after the final treatment) and there were no deaths amongst the controls. In a separate experiment the activity of acetylcholinesterase (AChE)in the muscle of exposed lobsters was measured. These data suggest a possible cumulative inhibition of this enzyme. Alternatively, increased sensitivity of the lobsters during the fourth treatment compared to earlier treatments may be related to seasonal differences in physiology or to the endocrine state of preovigerous and spawning females.

Spawning success, as assessed by the presence of extruded eggs, was also affected by exposure to the highest concentration of azamethiphos. Eight (57%) of the surviving lobsters exposed to 10 m g/L failed to spawn, while 2 (9%) of the surviving lobsters exposed to 5 m g/L of azamethiphos and only 1 (5%) of the control lobsters failed to spawn.

EFFECTS OF HYDROGEN PEROXIDE ON SUBSEQUENT FECUNDITY OF SEA LICE CHALIMUS STAGES

J.P.G.Toovey & A.R.Lyndon

Heriot-Watt University, Riccarton, Edinburgh, U.K.

Hydrogen peroxide treatments are becoming more popular within the fish farming industry due to their reduced environmental impact compared to organo-phosphates. Previous studies have reported that peroxide treatments are highly effective against adults but have not noted any impact on chalimus stages of the lice. This study observed the impact of peroxide treatments on a fish farm. It was found that peroxide as previously recorded, had no noticeable effect on the chalimus stages. However, when these chalimus stages became adults it was found that their reproductive capacity, in terms of egg viability and copepodid production, was significantly reduced compared to untreated lice later in the sampling period.

THE EFFECT OF IMMUNE MODULATION OF ATLANTIC SALMON SALMO SALAR L., UPON THE SETTLEMENT AND SURVIVAL OF LEPEOPHTHEIRUS SALMONIS (KRØYER, 1837) (COPEPODA: CALIGIDAE)

Simon Wadsworth¹, Harry Birkbeck² & Chris Secombes³

¹ Marine Harvest McConnell, Lochailort, Inverness-shire, Scotland, PH38 4LZ.

² Glasgow University, Division of Infection and Immunity, Glasgow, Scotland, G12 8QQ .

³ Aberdeen University, Zoology Dept. Tillydrone Avenue, Aberdeen, AB9 2TN.

A number of immunosuppressants were administered to Atlantic salmon and their effect examined upon the settlement and survival of Lepeophtheirus salmonis. Corticosteroid was found to increase the mean numbers of L. salmonis observed post-challenge by 59% (p<0.05) compared to controls. Administration of immunosuppressive factors isolated from Aeromonas salmonicida induced a 70% (p<0.01) increase in mean numbers of L. salmonis observed. There was no effect on the rate of development, nor tissue distribution of L. salmonis observed on the test fish compared to controls. The control of L. salmonis on production sites is examined in relation to disease status of the fish as well as the potential for effective immune modulation.

THE VARIATION IN SETTLEMENT AND SURVIVAL OF LEPEOPHTHEIRUS SALMONIS (KRØYER, 1837) (COPEPODA: CALIGIDAE) BETWEEN STOCKS OF ATLANTIC SALMON SALMO SALAR L.

Simon Wadsworth¹ & Craig Selkirk²

¹ Marine Harvest McConnell, Lochailort, Inverness-shire, Scotland. PH38 4LZ.

²Marine Harvest McConnell, Farms Office, Blar Mhor, Fort William, PH33 7PT.

Selective breeding programs have been in operation in the salmon farming industry in Scotland and Norway for over 20 years. Selection has focused on increased growth rates, reduced frequency of early maturation, improved flesh quality and disease resistance. There have been good results to date in selection for resistance to disease, as fish immune responses appear to have a high heritibility. Different stocks and families of Atlantic salmon Salmo salar were examined for variable susceptibility to settlement and survival of L. salmonis. Differences in susceptibility of up to 55% (p<0.01) in mean lice numbers were observed between stocks. There was no significant difference in mean lice numbers observed immediately post-challenge (8 h) but there were significant differences observed by 8 d post-challenge. There was no difference in the activity of complement (CH50) between the stocks but complement did decline following challenge. Variability in susceptibility to settlement and survival, as well as damage caused by L. salmonis was also observed between families of Atlantic salmon.

A PHYSIOLOGICAL BASIS FOR SALMON LOUSE VACCINES

Alexandra. G.Roberts¹, Ian Bricknell¹, Rob Raynard¹, Peter. F. Billingsley² & Alasdair. J. Nisbet²

¹ FRS Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, AB11 9DB.

² Aberdeen University, Department of Zoology, Tillydrone Avenue, Aberdeen.

The continuing problems with salmon lice on fish farms throughout Scotland, Canada and Norway has led to the development of a variety of control methods, however none are ideal. This project aims to improve our knowledge of louse gut physiology and investigate how it may be applied in order to control lice infections.

As little is known about the digestive processes of lice, this project has investigated the gut of Lepeophtheirus salmonis for the presence of 20 enzymes, using the Api Zym test. Six enzymes were found to be present at high levels, they are, leucine aminopeptidase, valine aminopeptidase, n-acetylglucosaminidase, alkaline phosphatase, and acid phosphatase. Characterisation of these enzymes and the identification of the primary digestive enzyme is in progress and the results will be presented.

Previous work by a number of different groups has involved using gut proteins as potential protective antigens. This is used in the vaccines against parasitic infections in sheep (Haemonchus contortus) and cattle (Boophilus microplus). While ingested host antibody provided protection in these cases, the ability of fish antibodies to survive in the louse gut for long enough to produce a protective effect has not been examined. Therefore, the project will investigate this and identify the location of the six key enzymes in the louse gut, and will test the hypothesis that fish antibodies can be used to target specific gut enzymes as possible protective antigens.

If this approach does not prove viable, for example, if antibodies are digested too rapidly, or are rendered inactive in the louse gut environment, an alternative will be to investigate the use of chemicals to target a known gut enzyme and impair louse physiology. This has proven successful in the anti-chitin feed additive, produced by Trouw.

BEHAVIOURAL STUDIES OF NAUPLII AND COPEPODIDS OF SALMON LICE (LEPEOPHTHEIRUS SALMONIS KRØYER). POSSIBLE EFFECTS OF PHYSICAL PARAMETERS ON POSITION AND DISPERSAL OF LARVAE

Karin Boxaspen, Anne Berit Skiftesvik & Howard Browman

Institute of Marine Research, Austevoll Aquaculture Research Station, N-5392 Storebø, Norway.

Salmon lice are planktonic when they hatch as nauplius I and remain free-living through the molt to nauplius II and until the infective copepodid stage. In Norway, salmon farmers who undertake regular monitoring can observe pulses of salmon lice settlement onto hosts. These pulses appear to be connected to changes in the physical environment; they are commonly explained as being the result of the synchronous hatching of eggs (released from unidentified source populations) combined with tidal and/or coastal currents. Active swimming of salmon lice free-living stages, and the cues that affect/guide these movements, have not been studied extensively. Using three-dimensional silhouette video photography and a computer-based movement tracking and path analysis system, we set out to examine the free-swimming movements of salmon lice in response to changes in salinity, light and temperature. Path analysis revealed that activity, swimming velocity and the relative trajectory of movements were different under the various environmental conditions and that nauplii and copepodids responded differently.

FRONTAL FILAMENT DEVELOPMENT IN THE SALMON LOUSE LEPEOPHTHEIRUS SALMONIS

Pablo González¹, Stewart C. Johnson², Glenda M. Wright¹ & John F. Burka¹

¹ Department of Anatomy and Physiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, C1A 4P3.

² Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, NS, B3H 3Z1.

Chalimus stages of Lepeophtheirus salmonis attach to their host by a frontal filament. Knowledge of filament development could be useful in designing sea lice control strategies. We are investigating the morphological changes associated with filament production. Atlantic salmon were infested with copepodids. Lice were sampled at 0, 3, 5, and 7 days post-infestation (dpi) and analysed using high-resolution light microscopy. Three cell groups identified as A, B and C are thought to be involved in production of filament material (S1and S2). At 0 dpi, A and B were present. S1, S2 and C were present at 3dpi. Group C appeared to be intimately associated with S2. Group B, S1 and external filament were present at 5 dpi. A, B, C, S1, S2, and external filament were present at 7dpi. We are currently determining the histochemical characteristics of cell groups and filament material. (Supported by a grant from the NSERC / NRC Partnership Program.)

EGG PRODUCTION IN SALMON LICE (LEPEOPHTHEIRUS SALMONIS) IN RELATION TO TEMPERATURE AND ORIGIN

P.A. Heuch¹, J.R. Nordhagen ² & T.A. Schram²

¹ Fish Health Section, National Veterinary Institute, PO Box 8156 Dep., 0033 Oslo, Norway.

² Department of Biology, University of Oslo, PO Box 1064 Blindern, 0316 Oslo, Norway.

Egg production in salmon lice, Lepeophtheirus salmonis Krøyer 1837 was investigated at temperatures from 7.1 to 12.2ºC. A statistical analysis indicated that egg strings were longer and had more eggs at the lowest temperature. However, the percentage of infertile eggs in the strings simultaneously increased. The maximum life time from infection to death was 191 days for a female louse at 7.2ºC. During this period 11 pairs of egg strings were produced. Egg strings on salmon lice collected from farmed and wild Atlantic salmon have neither significantly different lengths, nor number of eggs per string.

AN OPTICAL METHOD FOR THE DETECTION OF SEA LICE, LEPEOPTHEIRUS SALMONIS

R. D. Tillett, C. R. Bull & J. A. Lines

Silsoe Research Institute, Silsoe, Bedford, MK45 4HS, UK.

Developments towards a novel video camera based system for estimating the sea lice burden of freely swimming salmon is reported.

The spectral reflectance of sea lice and salmon skin were measured over the wavelength range 400 to 1100nm. Canonical variate analysis was then used to identify a combination of reflectances which maximises the differences between skin and sea lice. This is shown to provide good discrimination when the lice are attached to the lighter underside of the fish and a degree of discrimination when the lice are attached to the darker areas.

The results of this analysis were used to develop a simple video based discrimination system. An image of the salmon is synthesised from the ratio of grey levels of images taken through narrow band pass filters at wavelengths of 700 and 800 nm. This enhances the visibility of the lice and suppresses variations in the skin colour. Further development of this technique could lead to an automated passive system for estimating lice burden.

CELL CULTURE BIO-ASSAYS FOR POTENTIAL ANTI-SEA-LOUSE CHEMOTHERAPEUTANTS TO BE USED IN THE SALMON FARMING INDUSTRY

J.P.G.Toovey & A.R.Lyndon

Heriot-Watt University, Riccarton, Edinburgh, U.K.

Potential disease treatments require in-vivo trials to determine limits for effective minimum and maximum dosages and also to determine effects on the fish themselves. These trials are expensive, requiring special licences and large numbers of fish. However, this cost could be greatly reduced if some knowledge of effective dosages was available beforehand. Cell culture could provide a source of such data. Primary salmon epidermal cell cultures were used to determine both chronic and acute dosages of potential chemotherapeutants on the fish, as well as to determine the effects they have on the sea-lice copepodids, either in the water column (pre-infection) or attached (post-infection).

SEA LICE TREATMENT CHEMICALS - TARGETED ENVIRONMENTAL MONITORING USING BIOMARKER/BIOASSAY TECHNIQUES

I.M. Davies¹, G.K. Rodger², J. Redshaw³ & R.M. Stagg¹

¹ FRS Marine Laboratory, Victoria Road, Torry, Aberdeen, Scotland AB11 9DB.

² Rodger Environmental, 1 Oyne Road, Craigiebuckler, Aberdeen AB15 8HS.

³ Scottish Environment Protection Agency, West Region, 5 Redwood Crescent, Peel Park, East Kilbride, Scotland G74 5PP.

There are five types of chemotherapeutants which are currently used, or proposed, for use in the control of the treatment of ectoparasitic sea lice (Lepeophtheirus salmonis Krøyer and Caligus elongatus Nordmann) infestations on salmon (Salmo salar) in Scottish marine salmon farms. Each of these chemicals is released into the environment during use, either as an in-feed treatment or bath immersion treatment.

The Scottish Environment Protection Agency (SEPA) is responsible for monitoring the quality of Scottish coastal waters and regulating discharges, such as those from cage fish farms, which may affect the quality of these waters. Current monitoring programmes concentrate on analyses of water and sediment quality, and are primarily directed at the effects of organic enrichment and nutrient release.

SEPA has identified a need to develop monitoring procedures, which are targeted at the effects of sea lice treatment chemicals after release to the marine environment. Biological effects measurements (biomarkers and bioassays) offer the potential to separate the effects of the chemotherapeutants from those of organic enrichment at fish farms and may provide an early warning of effects. The development and application of these methods could enhance SEPA’s ability to identify and predict the effects of sea lice treatment chemicals on the marine environment.

The aim of this desk study was to identify and evaluate those biological effects techniques which may be suitable for the environmental monitoring of sea lice treatment chemicals used in marine salmon farms. The specificity and suitability of these biological effects techniques to the mode of toxic action, metabolism and environmental fate of the chemotherapeutants was considered.

USING CLEANER-FISH TO CONTROL SEA-LICE: A VIDEO PRESENTATION

Rune Vindenes¹, Per Gunnar Kvenseth² & Anne-Mette Kvenseth³

¹ Gaia Studio, Georgernes verft 3, 5011 Bergen, Norway.

² KPMG Management Consulting as - Centre for Aquaculture and Fisheries, Sandviksboder 5, N-5035 Bergen, Norway.

³ University of Bergen, Department of Fisheries and Marine Biology, High Technology Centre, Pb 7800, N-5020 Bergen, Norway.

This video is about using wrasse to reduce problems with sealice in seawater farming of Atlantic salmon. To achieve good results with cleaner-fish it is important to know the biology and behaviour of wrasse. The video also gives an introduction to the use of wrasse, from capture and transport to handling, use in fish farms, and diseases of wrasse. The basis is a combination of results from research and practical experience.

The video is meant for both salmon farmers, and people with a more general interest, who want to know more about wrasse and how they can be used to control sealice. It is a way of giving everyone working in this field an introduction to a most environmentally friendly way of reducing sealice problems.