THE BENEFITS OF HINDSIGHT: ANOTHER LOOK AT SEA LICE BIOLOGY

Alan Pike

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

Having recently completed a review of current sea lice literature I will discuss areas of particular interest and incomplete knowledge. I would like to raise in particular the development rates of sea lice and also the data on copepodid ecology and behaviour.

STRESS AND THE HOST-PARASITE INTERACTION BETWEEN ECTOPARASITIC LICE AND SALMONIDS: IS STRESS NECESSARILY MALADAPTIVE?

Declan T. Nolan, Neil M. Ruane and Sjoerd E. Wendelaar Bonga

Department of Animal Physiology, University of Nijmegen, 6500 GL Nijmegen, The Netherlands.

The stress response of an animal enables it to deal with a variety of stressful stimuli. Limited research has been carried out on host-parasite interactions and the stress response of the host. For some sea lice, this relationship has co-evolved over time, so infestation might induce a specific response directed at protecting the host from the effects of the parasite. In farmed salmonids, the sea louse Lepeophtheirus salmonis has become problematic, which may indicate some disturbance of the host-parasite relationship, enabling the parasite to overwhelm the host population. The infective stage does not impact heavily on the host, but the most damaging life stages for the host fish are the pre-adult and adult stages. We have carried out a series of studies looking at the effects of the ectoparasitic lice on the host fish. We have experimentally exposed groups of fish to infective or adult life stages. Direct effects of the parasite on the host are well known and are caused by attachment and feeding. However, indirect effects are less well known and are induced by parasite infection evoking a stress response in the host. In this presentation, the integrated stress response of teleost fish will be introduced. The direct and indirect effects of L. salmonis infection on the host will be presented, including data showing for the first time effects induced by the infective stage.

FEEDING REGIME AND LICE LOADS

J.P.G.Toovey and A.R.Lyndon,

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

Sea-lice loads were studied for half a year at a working Scottish fish farm site in the Lochaber area. Set up at this site were two types of feeding system, a spray feeding system which propels feed pellets onto the surface of a fish cage using compressed air and also the Aquasmartä system. Lice loads were found to be generally lower in the Aquasmartä fed cages. This supports the theory of the Aquasmartä system that if fish can be kept lower in the water column with more regular feeding, then the fish can avoid the upper layers of water, where larger numbers of infective lice are usually found and thus avoid heavy infection.

EFFECTS OF INFECTIVE LIFE STAGES OF CRUSTACEAN ECTOPARASITES ON THE SKIN OF THE HOST FISH AT SITES DISTANT TO THE INFECTION: IMPLICATIONS FOR SECONDARY OPPORTUNISTIC INFECTION

Silvia A.M. van den Braak, Angela van der Salm, Yvonne van der Heijden, Declan T. Nolan & Sjoerd E. Wendelaar Bonga

Department of Animal Physiology, University of Nijmegen, 6500 GL Nijmegen, The Netherlands.

As sea lice feed on the mucus, epithelial cells and blood of the host fish, the localised response has been well described. In salmonids, the sea louse Lepeophtheirus salmonis is a serious parasite and tissue damage by feeding sub-adult and adult life stages induces mortality through osmoregulatory disturbance. By contrast, attachment by the infective stage induces localised effects and is understood to have little other impact on the host. By experimentally exposing groups of fish to infective life stages of L. salmonis, we have demonstrated effects of this life stage on the skin at sites distant from the attachment site of the parasites. Experimental infection induced stress-related changes in the epidermis (ultrastructural disruption, alterations in the mucous cell populations). To examine whether this is related to the induction of an integrated stress response, we have used administration of low levels of the stress hormone cortisol alone, or in combination with exposure to the infective stage of the louse Argulus foliaceus. We studied overall effects in the skin epidermis and demonstrated that similar effects induced by infection of the parasites are likely to be mediated by cortisol. Thus, infectives of these lice are capable of inducing epithelial effects which could compromise the health of the host by widespread disruption of epithelial integrity that can be considered as maladaptive effects of increased circulating cortisol levels.

REMOVING THE EFFECTS OF SAMPLING ON THE CORTISOL RESPONSE IN TROUT

Russell Poole¹, Declan Nolan² and Oliver Tully³

¹ Salmon Research Agency, Furnace, Newport, Co. Mayo, Ireland.

² Department of Animal Physiology, University of Nijmegen, The Netherlands.

³ Department of Zoology, Trinity College Dublin, Dublin 2, Ireland.

Estimation of baseline circulating cortisol in wild fish populations, as an indicator of stress, is problematic because of the sensitivity of the hormonal response to all practical fish sampling methods. In this paper we demonstrate, using laboratory rainbow trout and wild sea trout, that if fish are sampled in a timed sequence then these capture effects can subsequently be removed by modelling the relationship between cortisol and the time since capture. The parameters of a fitted quadratic time response model can provide useful indications of the baseline or precapture cortisol levels, the time to maximum stress response, the rate of response to stress and the subsequent reduction in the response. This method is sufficiently sensitive to enable differences to be detected in the baseline (presampled) cortisol levels between sampling dates in wild sea trout, between sea trout infested with different numbers of sea lice and between fish that originate from different freshwater systems.

THE EFFECTS OF SALMON LICE (LEPEOPHTHEIRUS SALMONIS KRØYER) INFESTATION ON ATLANTIC SALMON (SALMO SALAR L.) POST SMOLTS: LABORATORY AND FIELD STUDIES

Bengt Finstad¹, Pål Arne Bjørn¹, Andrea Grimnes², & Nils Arne Hvidsten¹

¹ The Norwegian Institute for Nature Research, Tungasletta 2, N-7005 Trondheim, Norway.

² The Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway.

Hatchery reared one-year old Atlantic salmon post smolts (Salmo salar L.), artificially infected with salmon lice (Lepeophtheirus salmonis Krøyer) copepodids, were found to suffer from primary alterations (increased cortisol levels) at early lice stages. Secondary alterations, such as osmotic stress (increased chloride levels), first occurred after the preadult stages of the lice appeared. The increased cortisol levels measured in post smolts infested with the early larva stages of salmon lice indicate that infested fish may suffer from immunosuppression in addition to osmoregulatory stress. These results have been compared to a seven year long field study where wild post smolts of Atlantic salmon infected with salmon lice have been sampled in a fjord system in middle Norway.

SALMON LICE (LEPEOPHTHEIRUS SALMONIS KRØYER) INFECTION ON SEA TROUT (SALMO TRUTTA L.) IN AN AREA WITH AND WITHOUT SALMON FARMING; PHYSIOLOGICAL EFFECTS AND MORTALITY OF THE INFECTION

Pål Arne Bjørn, Bengt Finstad & Roar Kristoffersen

The Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway.

The Norwegian Institute for Nature Research, Tungasletta 2, N-7005 Trondheim, Norway.

Previous investigations on infection intensities of salmon lice on sea trout have sampled hosts on return to freshwater or hyposaline conditions. The present investigation sampled wild sea trout at their feeding grounds at sea as well as those returned to freshwater. The results showed that 34 % of sea trout post smolts in a farming area were infected above the level, which is expected to cause mortality. The most heavily infected fish returned to freshwater. Heavy infected sea trout had a significantly increased plasma cortisol level and tended to have increased blood glucose levels and osmotic disturbances. These results imply that salmon lice may have detrimental effects on wild sea trout in areas with salmon farming activity. In the area without salmon farming the infections were low, and negative effects of the infection were not likely to occur.

STRESS-RELATED RESPONSE IN MUCUS AND OSMOREGULATORY FUNCTION IN RELATION TO SEA LICE (LEPEOPHTHEIRUS SALMONIS) INFESTATION IN WILD SEA TROUT (SALMO TRUTTA) POST SMOLTS

G. O'Flaherty¹, R. Poole², R. Ryan¹, U. MacEvilly¹, O. Tully³ & D. T. Nolan

¹ School of Biological Sciences, Dublin Institute of Technology, Kevin street, Dublin 8.

² Salmon Research Agency of Ireland, Newport, Co. Mayo.

³ Department of Zoology, Trinity College Dublin. (Department of Animal Physiology, University of Nijmegen, Nijmegen, The Netherlands.

To determine the impact of sea lice (L. salmonis) on host sea trout (S. trutta) we analysed gill Na+K+ ATPase activity, plasma Na+ and Cl- and skin mucus protein profiles of fish which were infested with varying levels of sea lice. Lice infested fish were sampled from the Owengarve and Newport river estuaries in Co. Mayo, June 1998. Lice infested fish demonstrated a variation in gill Na+K+ ATPase activities (4 to 47(?mol Pi/mg/h) whilst total lice numbers for these fish ranged from 0 to 59. The results obtained from both estuaries indicated that there was a positive correlation (correlation coefficient of 0.61) between lice numbers and gill Na+K+ ATPase activity. Non-linear regression analysis [y = b1+ b2 x ln (lice numbers)] suggested that there was an upper maximum limit to the gill Na+K+ ATPase response to sea lice, possibly indicating that the metabolic cost of osmoregulation is increased when sea lice are present. There was no apparent correlation between increasing lice numbers and [Na+] and [Cl-], indicating that hydromineral balance was maintained through increased gill enzyme activity.

SDS-P.A.G.E analysis of skin mucus showed 51 and 61 protein bands for the Newport and Owengarve estuaries respectively. This difference may reflect environmental and/or geographical differences between the two sample locations. All the skin mucus samples showed strong protein bands in the 45kDa and 14.4kDa regions. The 14.4kDa band migrated with the same molecular mass as lysozyme. Lysozyme has previously been reported in rainbow trout (Oncorhynchus mykiss), plaice (Pleuronectes platessa), and in the kidneys of sea trout. This heavily staining band was consistently the most prominent of all the protein bands. Protein band numbers were the same for uninfested and infested fish. The intensity of staining appeared to be lower for fish with small numbers of lice. This paper presents previously unreported physiological responses of wild sea trout to lice infestation.

THE ROLE OF KAIROMONES IN HOST-FINDING BEHAVIOUR BY LEPEOPHTHEIRUS SALMONIS

Greg J. Devine¹, Anna Ingvarsdottir¹, William Mordue¹, Alan Pike¹, John Pickett², Ian Duce³ & A. Jenny Mordue¹

¹Zoology Department, Aberdeen University, Tillydrone Avenue, Aberdeen AB24 2TZ.

²Department of Life Sciences, University Park, Nottingham University, Nottingham NG7 2RD

³Biological and Ecological Chemistry Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ

Significant numbers of adult male salmon-lice transfer between hosts in sea cages, and this redistribution is probably the result of lice reattaching to hosts after being dislodged, and of male lice searching for unmated females. Part of the host-finding process may involve the use of salmon-derived kairomones by lice and, if so, these might have potential for use in management strategies.

Using simple flow chambers that allowed host–conditioned seawater to be proffered, lice were monitored for the presence or absence of behavioural responses. Using a digital tracking system, the relative strength of these responses was assessed by measuring the speed and direction of movement of the lice. In order to gauge the attractancy of these stimuli, 'Y-tube’ assays were developed which allowed lice to exhibit preference for one stimulus over another.

As a result of these studies, it was shown that male salmon lice exhibited directional and kinetic responses to salmon-conditioned water and, therefore, that kairomones of salmon origin elicited a positive rheotaxis in Lepeophtheirus salmonis. The potential for the further development and utility of a semiochemical-based control strategy for this pathogen is discussed.

CIRCALUNAR VARIATION IN LARVAL DENSITIES OF SEA LICE LEPEOPHTHEIRUS SALMONIS IN A FISH FARM ON THE WEST COAST OF IRELAND

Ger O'Donohoe, Mark Costelloe & John Costelloe

Aqua-Fact International Services Ltd., 12 Kilkerrin Park, Liosbaun, Galway, Ireland.

The sea louse Lepeophtheirus salmonis is a caligid copepod, which is ectoparasitic on salmonid fish. Its importance to marine salmonid culture stems from the extensive damage it may inflict on hosts through feeding and contact abrasion. L. salmonis has been the subject of extensive investigations by many authors. However, little is known about the spawning behaviour of L. salmonis. The number of eggs produced within an egg string has been shown to vary considerably, confirming that there are wide variations in reproductive output. The objectives of the present study were to examine the production and distribution of the larval stages of L. salmonis within a cage containing Salmo salar in order to identify specific spawning cues, frequency and intensity. The density of larvae in a cage unit was examined over the months June to August in a fish farm on the west coast of Ireland. In general, tows were taken on the flooding tide within one hour prior to high water. Highest densities of larvae were recorded on dates coinciding with neap tides. The data were statistically tested with the result that there were significantly higher numbers of larvae recovered during neap tides than those recovered during spring tides (P<0.05). The results of the present study increase our knowledge of the complex behaviour of the sea louse L. salmonis. The information provides a basis from which management strategies may be developed in order to reduce larval lice intensities on a farm and in turn possibly control the parasitic intensity on the farm in general.

SIZE VARIATION IN LEPEOPHTHEIRUS SALMONIS (KRØYER) ECTOPARASITIC ON WILD SALMON (SALMO SALAR L.) AND SEA TROUT (SALMO TRUTTA L.) IN SCOTTISH WATERS

Alan M. Walker¹, Christopher D. Todd¹, Jane E. Hoyle¹, Jeff A. Graves² & Mike G. Ritchie²

¹ Gatty Marine Laboratory, Environmental & Evolutionary Biology, University of St Andrews, St Andrews, Fife, Scotland KY16 8LB.

² Bute Medical Building, Environmental & Evolutionary Biology, University of St Andrews, St Andrews, Fife, Scotland KY16 9TS.

Samples of Lepeophtheirus salmonis (Krøyer) were obtained from wild salmon (Salmo salar L.) and sea trout (Salmo trutta L.) from coastal waters throughout Scotland between 1995 and 1998. Samples were collected in the summer and autumn months (May-September). Parasite size was measured as the cephalosome width, but excluding the marginal membrane. The effects on parasite size variation of host species, size, location, date of capture and intensity of infestation were analysed for both adult male and adult female L. salmonis. Predictable host-related patterns in size variation are detectable for these two host species, but shape (cephalosome width/length) variation was also noted. Because cephalosome length has often been used in other studies, comparability of parasite size variation is subject to this qualification. Size variation may be attributable to differential rates of development of parasites infesting the two host species, or it may reflect contrasting quality of feeding. Variation in somatic size of adult females presumably has especially important allometric influences on fecundity, the size and quality of eggs, and hence individual fitness.

REPRODUCTIVE INVESTMENT BY LEPEOPHTHEIRUS SALMONIS - CHEMICAL ANALYSIS OF NEWLY OVIPOSITED EGGS

Gordon Ritchie¹, Anne Jenny Mordue², Alan Pike² & Gordon Rae³

¹ Nutreco Aquaculture Research Centre, Sjøhagen 3, 4016 Stavanger, NORWAY.

² Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, UK.

³ Scottish Salmon Growers Association Ltd., Drummond House, Scott street, Perth PH1 5EJ, UK.

Significant seasonal variation in the number and size of eggs produced by adult female L. salmonis exists. Fewer but larger eggs are produced during summer months, suggesting these receive a greater allocation of resources and nutrients per egg. Using standard techniques, the chemical composition of newly extruded eggs was evaluated in terms of protein, lipid and carbohydrate content to determine if concentrations change in relation to egg size and season. Significant seasonal variation in the concentration of protein, lipid and carbohydrate per egg string, protein per egg and percentage dry weight of egg string as protein were observed. Eggs produced in summer had more nutritive material than those produced in winter. Adult female L. salmonis show an adaptive reproductive strategy, producing larger eggs with more yolk protein per egg in summer and smaller eggs with less protein in winter.

INVESTIGATION OF MICROSATELLITE GENETIC ELEMENTS AS TOOLS FOR THE STUDY OF LEPEOPHTHEIRUS SALMONIS ECOLOGY

Damien Nolan¹, Karen Glennon², Yvette Kelly², Sam Martin², Roy Palmer², Terry Smith² & Richard Powell¹

¹ Department of Microbiology

² National Diagnostics Centre, BioResearch Ireland, National University of Ireland, Galway.

This project seeks to determine if polymerase chain reaction (PCR)-based microsatellite assays for L. salmonis have the ability to detect genetic variation between different populations of sea-lice. Initially, L. salmonis DNA was prepared, cloned as short-DNA inserts into E. coli cells, and screened with a variety of microsatellite-encoding DNA probes. Of 3,700 clones, 1.6% contained microsatellite elements and 50 clones were chosen for subsequent DNA sequencing. 20% of these microsatellites contained sufficient flanking DNA sequence to allow the design of PCR primer-sets. 10 microsatellite PCR primer-sets were tested, of which 4 supported microsatellite amplification among individual L. salmonis sampled from different geographical locations. Currently, these 4 microsatellite PCR primer-sets are being tested for their ability to detect genetic variation in, and ultimately distinguish between, L. salmonis individuals sampled in Ireland, Scotland and Norway.

EXPRESSED SEQUENCE TAGS OF THE SALMON LOUSE LEPEOPHTHEIRUS SALMONIS AND CHARACTERIZATION OF TRYPSIN AND TRYPSIN-RELATED ENZYMES

Stewart C. Johnson, K. Vanya Ewart, Jane A. Osborne & Neil W. Ross

Institute for Marine Biosciences, National Research Council Canada, 1411 Oxford St., Halifax, Nova Scotia, Canada, B3H 3Z1.

Analysis of partial cDNA sequences "expressed sequence tags" (ESTs) is a useful approach for gene identification. For pathogens, this may result in the identification of targets for chemotherapy and vaccine development. We have generated a cDNA library for pre-adult male and female L. salmonis. To date, we have sequenced the 5' ends of 224 clones in order to generate ESTs. Of these, 56.5% have significant matches to genes from other organisms. Based on sequence matches, these include: 30 clones for ribosomal proteins, 21 clones for enyzmes, 10 clones for mitochodrial proteins, 15 clones for structural proteins and 61 others. We have identified clones for 5 enzymes within the trypsin family including 3 trypsin isoforms and 2 chymotrypsins. Complete cDNA sequences are being obtained for these clones and will be compared to sequence data from L. salmonis-derived trypsin-like enzymes purified from the mucus of infected Atlantic salmon. The potential role of trypsin and related proteases as L. salmonis virulence factors will be discussed.