Towards selective breeding of Atlantic salmon for sea louse resistance: approaches to identify trait markers

Several species of sea lice, in particular Lepeophtheirus salmonis (Kr?yer), affect the welfare and condition of farmed salmon, with an estimated annual cost to the Scottish industry of 15-30 Pounds million. In Atlantic salmon, some stocks show resistance to L salmonis. Such natural resistance could be utilized for stock improvement using molecular genetic technologies. The development of molecular markers linked to resistance genes, allowing the identification of resistant fish, could increase the efficacy of selective breeding programmes. Various approaches to achieve this goal are described. One way to identify genes conferring resistance is to develop screens for salmon genes that are activated upon louse infection. One such screen--differential display--requires no previous knowledge of gene sequences, involves no preconceptions about which gene families are involved and can therefore identify novel genes. Preliminary results of comparative gene expression in sea-louse-challenged and control fish illustrate the application of differential display.     

Wild salmonids and sea louse infestations on the west coast of Scotland: sources of infection and implications for the management of marine salmon farms

The sea louse (Lepeophtheirus salmonis Kr?yer) is a major health problem for both farmed and wild salmonids. This paper investigates louse epidemiology and management in the salmon-farming zone of western Scotland. Based on a review of the marine ecology of wild salmon (Salmo salar L) and sea trout (Salmo trutta L), and catch and farm production statistics, best estimates were made for numbers of wild and farm hosts present in coastal waters in March-June 2000. Applying data for ovigerous female louse infections and fecundity, the sources and risks of larval transmission to wild salmon and sea trout were modelled. Farm salmon in the second spring of production were the primary host group (98% of fish), while numbers of wild salmonids (< 1%) and escaped farm salmon (2%) were relatively insignificant. Farm salmon produced 97% of louse eggs at high levels (eight ovigerous lice per fish), and 78% at low levels (one per fish). Wild salmonids produced < 1% of eggs under both scenarios, but escaped farm salmon produced 3% and 21%, respectively. All hosts potentially cross-infect one another, but farm salmon are more likely to infect wild and farm smolts, and also other farm salmon. Monitoring of lice on sea trout in June 1998-2000 by the Association of West Coast Fisheries Trusts corroborated the model's conclusions. Localised epizootics occurred every year and coincided with the presence of ovigerous lice on local farms. In areas of mixed-year class production on farms, epizootics were evident every spring, but occurred every second spring in areas of single-year class production. In 1998-2000 at least 14-40% of sea trout were infected with potentially lethal infestations of lice. Ovigerous louse levels of < 0.005 per fish were required on farm salmon in the spring of 2000 to produce less eggs than those emitted by wild salmonids. With the industry's continued expansion, and thus increased numbers of farm salmon, a target of zero ovigerous lice will be required on farms to minimise impacts on wild salmonids. Due to the limited long-term efficacy and availability of louse medicines, management strategies are discussed which will improve control, including single-year class production over large areas, alternate S1-S1/2 smolt inputs, and 11-month production cycles.     

Sea louse control in Scotland,past and present

Methods for farming the Atlantic salmon (Salmo salar L) in Scotland were developed at the Unilever Research Laboratory facility at Lochailort on the west coast of Scotland in the late 1960s. By the spring of 1972 a fledgling commercial company had been set up and Marine Harvest, as it was known, received its first year class of smolts at Lochailort. Sea lice (Lepeophtheirus salmonis Kr?yer) from passing wild fish infected the smolts but did not cause any significant damage until the summer of 1976 when intervention was required. The Scottish industry has since grown to become a vital source of employment for fragile rural economies and produced about 130,000 tonnes of salmon in 2000. This paper gives an overview of how fish welfare has been maintained by the development of treatments and management strategies aimed at minimising the impact of sea lice.     

Temporal,environmental and management factors influencing the epidemiological patterns of sea lice (Lepeophtheirus salmonis) infestations on farmed Atlantic salmon (Salmo salar) in Scotland

In association with industry, an extensive database has been established on measurements of sea louse counts on farmed Atlantic salmon for the years 1996 to 2000 from 33 Scottish fish farms. These data include extensive counts on the sea louse species, Lepeophtheirus salmonis, at various stages of the life cycle and in particular the chalimus and mobile stages. There has been considerable speculation as to what factors might affect the abundance of sea lice, much of which is based on limited evidence. Our analyses show that there is tremendous variation in sea louse infestation patterns from year to year, whereas stock type, geographical region and coastal exposure do not appear to affect mean levels of abundance. In contrast, treatments lead to pronounced cycles of sea louse infestation with peaks and troughs at 3-week intervals, and these interventions are important if the sea louse levels on fish are to be controlled. There was no evidence of water temperature affecting the mean annual abundance of sea louse infestation.     

A review of potential pathogens of sea lice and the application of cleaner fish in biological control

There are many examples of successful biological control of pest populations in aquatic environments. This approach to sea louse control has environmental benefits and is cost-effective. The range of possible pathogens of lice is reviewed and epibionts recorded from sea lice, including the monogenean Udonella caligorum and ciliates, are examined. Baculoviruses when ingested by insects form occlusion bodies resulting in severe damage to the digestive system and subsequent death, and this may be a promising approach. Cleaner wrasse (Labridae) have been stocked commercially with farmed salmon since 1989, and recent work on improving the method is reviewed. Wrasse are sourced from a wild fishery and stocked at ratios of 1 to 25-150 salmon. Over 5 million wrasse are stocked annually in Norway and c 30% of smolts in Scotland were stocked with wrasse until 1998, when an outbreak of infections salmon anaemia (ISA) deterred many farmers from transferring wild fish to cages. A case study is given showing that salmon in cages stocked with wrasse had a burden of one to eight lice through the first year compared with up to 40 lice per fish on unprotected and untreated fish. Electivity indices were used to compare the relative composition of lice developmental stages on salmon in stocked and unstocked cages, and adult male and female lice were found to comprise only 6% of the population in cages with wrasse, compared with 49% adults on fish in control cages. Measures to improve the efficacy of wrasse as a way of cleaning salmon in the second production year include the use of refuges to assist over-wintering survival, and stocking ballan wrasse. Health hygiene includes sourcing wrasse in the farm locality, testing for pathogens, vaccination of wrasse and ultimately rearing wrasse for stocking. The role of wrasse in an IPM strategy is described.     

Analysis and management of resistance to chemotherapeutants in salmon lice,Lepeophtheirus salmonis (Copepoda: Caligidae)

In Northern Europe and Canada, the salmon louse, Lepeophtheirus salmonis (Kr?yer), seriously affects the marine phase of salmon production. Although the problem is long-standing, the development of sustainable methods of pest management has been unable to keep pace with the intensification of production, leading to large-scale reliance on very few chemotherapeutants. This runs the risk of selecting for genetically determined resistance in target organisms. There are many examples of similar evolutionary adaptations in arthropod pests of arable crops, livestock and human health. Several hundred pest species are now documented as being resistant to one or more chemical classes of insecticides and acaricides. Many of these compounds are identical or closely related to ones currently employed against salmon lice. It is, therefore, opportune to consider what lessons have been learnt from contending with resistance in terrestrial organisms, the implications for sustainable use of chemotherapeutants in aquaculture, and the potential for developing effective resistance management strategies. An EU-funded project named SEARCH (QLK2-CT-2000-00809) has been initiated to explore in more detail the diagnosis, incidence, dynamics and management of resistance to chemotherapeutants in L salmonis.     

Occurrence of the microsporidian parasite Nucleospora salmonis in four species of salmonids from the Massif Central of France

Nucleospora salmonis (Hedrick, Groff et Baxa, 1991), an intranuclear microsporidian parasite of marine and freshwater fish, causes diseases mainly in salmonid species. Losses have been reported in stocks of salmonid fish reared in the region of Auvergne (France). The cause of chronic mortalities in the local host species raised in aquaculture and destined for supplementation of the river system Loire-Allier was examined. The presence of N. salmonis was confirmed by PCR and histology in Salmo salar L. previously and in newly investigated salmonid species, Salmo salar, Salmo trutta fario L., Thymallus thymallus (L.) and Salvelinus alpinus (L.), present in European streams. The infection by N. salmonis was consistent in all cases with characteristic symptoms of the disease in deceased or moribund fish. The small subunit ribosomal DNA from N. salmonis was partially sequenced and compared to previously characterised N. salmonis isolates. As a result, a genotype, or clonal entity, was attributed to N. salmonis among Atlantic salmon found along the Northern Atlantic coastal lines and other salmonid species co-inhabiting or co-cultivated in the Auvergne region.     

Development of vaccines against sea lice

A review of efforts to develop a vaccine against sea lice is presented together with analysis of the rationale behind the approaches and potential future directions. Vaccines against the caligid copepod, Lepeophtheirus salmonis, have the potential to be a cost-effective means of controlling the infection and avoid many of the disadvantages of medicine treatments. However, research towards such vaccines is in its infancy and approaches so far used have met with little or no success. Most strategies for sea louse vaccines have adopted methods used for vaccines against other ectoparasites. A vaccine against the cattle tick (Boophilus microplus) is in field use while other vaccines such as the sheep blowfly (Lucilia cuprina) vaccine are at an earlier stage of development. These haematophageous parasites ingest host antibody as part of a large blood meal which can target antigenic sites in the gut. However, the assumption that arachnid and insect physiology are directly comparable with that of sea lice is not proven, and this may partly explain the slow progress this approach has had with sea lice. Success in developing a louse vaccine will depend upon a better understanding of louse digestive biology, particularly an evaluation of whether the cattle tick model is applicable to the development of a louse vaccine. If the louse gut is to be targeted immunologically, critical antigens will need to be identified and evaluated, bearing in mind that an economic vaccine must include recombinant proteins or be a DNA vaccine. Alternatives to the louse gut as a target are also worth consideration. Antibodies could target critical host-parasite interactions that are amenable to disruption, although no such targets have been identified.     

Differential host utilisation by different life history stages of the fish ectoparasite Argulus foliaceus (Crustacea: Branchiura)

In this study we examine differences in the occurrence of life history stages of the destructive fish ectoparasite Argulus foliaceus (L., 1758) on eight fish species (stickleback, rudd, roach, gudgeon, bream, tench, crucian carp and common carp) sampled from a mixed-species recreational fishing lake on nine occasions during late spring and summer. Total numbers ofA. foliaceus, as well as the number of larval, juvenile and adult parasite stages, from each fish were recorded along with the fish species. Lice generally exhibited an aggregated distribution approximating a negative binomial distribution. Significant differences in the prevalence, intensity and intensity frequency distribution were observed between life history stages and between host species. In general, all life history stages of A. foliaceus exhibited an over-dispersed distribution. However, larval lice did show some degree of aggregation particularly within the stickleback samples. Infection data for parasite larval stages suggested that sticklebacks are more likely to be infected than other host species. For adult lice, however, carp appeared to be the main host. We propose that A. foliaceus infection characteristics are predominantly determined by the level of host exposure to the parasite and its life history stages (larval, juvenile and adult) rather than by an innate difference in host susceptibility related to individual host factors such as immune responses. We conclude that host exposure is determined by the parasite-host behavioural interplay related to species-specific ecology and behavioural traits such as microhabitat preference and normal swimming speed.     

Using observed load distributions with a simple model to analyse the epidemiology of sea lice (Lepeophtheirus salmonis) on sea trout (Salmo trutta)

Sea lice are ectoparasites of salmonids that have been associated with the recent decline in sea trout numbers in north-west Europe. Observed patterns of louse load distribution between sea trout in the seas surrounding the UK, Ireland and Norway and a simple model have been used to analyse the epidemiology of lice. Loads are aggregated and deviate strongly from the Poisson distribution, although less than is observed with many other parasites. The louse numbers on fish from offshore sites are slightly less variable than for fish from coastal sites with comparable mean loads. Analysis of louse development stages and sexes shows that selection between hosts by sea lice plays a limited role. If host selection is absent, then associated poor condition would be caused by, not the cause of, high louse burdens; however the absence of such selection is not proved. Scenarios with infection that is patchy in space and time best generate the aggregated load patterns observed; these patches accord with observed swarms of copepodids. Prevalence patterns may indicate the movement of trout between environments. Control of copepodids in infection 'hot spots', either directly or through control of louse egg production in their catchment, may reduce louse loads on wild sea trout and, in particular, extreme and damaging loads.     

Activity levels and predator detection by amphipods infected with an acanthocephalan parasite, Pomphorhynchus laevis

The acanthocephalan parasite Pomphorhynchus laevis (Müller, 1776) uses freshwater amphipods as its intermediate host. In order to complete the life cycle, the infected amphipod must be consumed by a fish, where the acanthocephalan will mature and reproduce. Parasite transmission, and therefore fitness, could be enhanced if infected amphipods fail to detect or avoid predatory fish. We compared the activity levels of infected and non-infected amphipods, Echinogammarus stammeri (Karaman, 1931), in both the presence and absence of odours from its natural, definitive host, the fish Leuciscus cephalus (L.). Throughout the experiment, infected amphipods were more active than were non-infected individuals. The non-infected amphipods reduced their activity after the addition of fish odours, but the infected amphipods failed to show a significant decrease. The failure of infected amphipods to reduce activity levels in the presence of fish odour may reflect a parasite strategy to increase its chances of transmission by making its amphipod host more vulnerable to predation by fish.     

Identification of cDNAs induced by the organophosphate trichlorphon in the parasitic copepod Lepeophtheirus salmonis (Copepoda; Caligidae)

Lepeophtheirus salmonis is a copepod ectoparasite of wild and farmed salmonids and can cause considerable damage to infected fish. We have examined the effect of the organophosphate trichlorphon, which was one of the early treatments for sea lice as Neguvon^® on L. salmonis. Suppression subtractive hybridisation was used to prepare a cDNA library enriched for cDNAs expressed in response to the organophosphate trichlorophon and using this technique a total of 33 cDNAs were found to be differentially regulated in response to organophosphate exposure. Sequence analysis of the cDNAs revealed that many were involved in cellular stress responses and or neurotoxicity. The expression of two of these cDNAs was confirmed to be up-regulated in response to trichlorophon.     

Effect of Clerodane-Type Diterpenoids Isolated from Salvia spp. on the Feeding Behaviour of Spodoptera littoralis

Twenty-nine clerodane-type diterpenoids isolated from species of Salvia growing in Mexico, or analogues of them, were tested for antifeedant activity against Spodoptera littoralis larvae using behavioural and electrophysiological bioassays. Eight of the compounds showed potent antifeedant activity in choice and no-choice bioassays and these compounds also stimulated dose-dependent responses from neurones in the lateral styloconic sensilla of S. littoralis.     

Monooxygenase mediated pyrethroid detoxification in sea lice (Lepeophtheirus salmonis)

The role of monooxygenases in detoxification of the pyrethroids cypermethrin and deltamethrin was examined. Four strains of sea lice (Lepeophtheirus salmonis Krøyer) with normal or moderately reduced sensitivity towards the pyrethroids were tested in bioassays by exposure to the pyrethroid alone and in combination with an oxygenase inhibitor, piperonyl butoxide (PBO). The normal (baseline) sensitivity was considered as the sensitivity range for the two most sensitive strains. Pre‐treatment with PBO elevated the sensitivity (P < 0.01) compared with groups exposed to the pyrethroid only. A positive, but not statistically significant, correlation between the activity of haem peroxidases and the pyrethroid concentration immobilizing 50% of the parasites was demonstrated (ρ = 0.500 for deltamethrin and ρ = 0.310 for cypermethrin). The results indicate that cytochrome P450 monooxygenases are involved in detoxification of pyrethroids in sea lice. ¹⁴C‐Deltamethrin was absorbed in a lesser amount in a group of sea lice exposed to a mixture of the compound and PBO than in a group exposed to ¹⁴C‐deltamethrin alone. A significant difference could be demonstrated both immediately after exposure (P < 0.01) and 24 h after exposure (P < 0.05). No significant differences were found between groups pre‐treated with PBO and groups exposed to ¹⁴C‐deltamethrin only. ¹⁴C‐Deltamethrin was taken up mainly through the cuticle, especially the cuticle on the extremities of the ventral surface, and subsequently distributed throughout the body of the parasite. Copyright © 2005 Society of Chemical Industry     

Evidence for occurrence of an organophosphate-resistant type of acetylcholinesterase in strains of sea lice (Lepeophtheirus salmonis Krøyer)

Acetylcholinesterase (AChE) is the target of a major pesticide family, the organophosphates, which were extensively used as control agents of sea lice on farmed salmonids in the early 1990s. From the mid‐1990s the organophosphates dichlorvos and azamethiphos were seriously compromised by the development of resistance. AChE insensitive to organophosphate chemotherapeutants has been identified as a major resistance mechanism in numerous arthropod species, and in this study, target‐site resistance was confirmed in the crustacean Lepeophtheirus salmonis Krøyer isolated from several fish‐farming areas in Norway and Canada. A bimolecular rate assay demonstrated the presence of two AChE enzymes with different sensitivities towards azamethiphos, one that was rapidly inactivated and one that was very slowly inactivated. To our knowledge this is the first report of target‐site resistance towards organophosphates in a third class of arthropods, the Crustacea. Copyright © 2004 Society of Chemical Industry     

Homing of Gyrodactylus salaris and G. derjavini (Monogenea) on different hosts and response post-attachment

In natural European waters, the congeneric monogeneans Gyrodactylus derjavini Mikailov, 1975 and G. salaris Malmberg, 1957 are primarily found on brown trout Salmo trutta L. and Atlantic salmon Salmo salar L., respectively. Interestingly, rainbow trout, Oncorhynchus mykiss (Walbaum), originating from North America, is as susceptible as brown trout to G. derjavini. However, the mechanisms involved in this host specificity are poorly understood but may include behavioural, mechanical and chemical factors affecting parasite attraction, attachment, feeding, reproduction and host responses. In the present laboratory work, this question has been studied. Detached parasites (either G. derjavini or G. salaris) were offered a choice in small aquaria between fry of rainbow trout, Atlantic salmon and carp Cyprinus carpio L. Within 48 hours more than 90% of G. derjavini colonised rainbow trout and left salmon almost uninfected. Some parasites were found on carp. During the same time span, more than 60% of G. salaris attached to salmon, the rest infected rainbow trout and none were found on carp. Following attachment, the parasites need appropriate stimuli to initiate feeding and reproduction but even such a successful specific colonisation can be followed by a host response. Both humoral and cellular elements have been suggested to participate in these reactions but in the present work it was demonstrated by immunoblotting and immunocytochemistry that no antibodies in host mucus and host plasma bound to any parasite structures or epitopes.     

Acylated 1,3-Aminopropanols as Repellents against Bloodsucking Arthropods

Starting from a set of known repellent compounds, a general structural framework with high probability for repellent activity was developed by molecular modelling techniques. Synthesis, structure–biological activity relationships of acylated 1,3-aminopropanols on the yellow fever mosquito Aedes aegypti and the properties and activity of the new development candidate compound KBR 3023 on Ae. aegypti, the malaria mosquito Anopheles stephensi, the common house mosquito Culex quinquefasciatus and the stable fly Stomoxys calcitrans are described. The sensory effect of KBR 3023 and deet has been studied in behavioural and neurophysiological investigations on Ae. aegypti and the American cockroach Periplaneta americana. The compounds clearly reduce or even eliminate the approach behaviour towards attractants like host odours or sexual pheromones. Electrophysiological studies on the insects' olfactory receptor organs reveal that certain cell types, which are not involved in perception of the attractive odorants, respond to deet and/or KBR 3023. As soon as one of the compounds is presented together with an attractant, a new input is active in the brain, which adds to the input from other receptors activated by the attractant. This new overall pattern clearly differs from that elicited by the attractant, so that the insect is no longer able to detect the latter. The specificity and mode of action of KBR 3023 was investigated by experiments exploring second-messenger responses elicited in antennal preparations of male P. americana. KBR 3023 induced a rapid increase in the concentration of inositol triphosphate in a dose-dependent and tissue-specific manner; other second-messenger systems were not affected. These observations suggest that KBR 3023 may act via subsets of G-protein-coupled receptors in sensory neurones.     

Effect of pulp and paper mill effluent (BKME) on physiological parameters of roach (Rutilus rutilus) infected by the digenean Rhipidocotyle fennica

Physiological parameters were measured after experimental infection of roach (Rutilus rutilus L.) with Rhipidocotyle fennica Gibson, Valtonen et Taskinen, 1992 (Digenea) cercariae. The fish were caught from two lakes: a eutrophic bleached kraft mill effluent (BKME)-contaminated lake and an oligotrophic unpolluted lake. The intensity of infection was followed up to 10 days post infection (p.i.) and physiological parameters indicating non-specific stress responses and the condition of fish were examined simultaneously. The mean abundance, the number of parasites per fish, of R. fennica was significantly higher in the fish from the contaminated water during the first two days p.i., probably reflecting the decreased resistance of these fish to infection. The decrease of leukocrit, as well as the increase of the activity of transaminases (GOT and GPT) in infected fish of both groups are suggestive of pathological processes caused by cercariae penetrating the fish. A significantly lower leukocrit value, as well as higher alkaline phosphatase activity and plasma chloride levels were noted in fish originating from the contaminated lake compared to those from the unpolluted lake. No significant differences were noted in haematocrit, plasma protein and calcium values between the fish from the uncontaminated and contaminated lakes, or between the infected and uninfected control fish.     

Diurnal variation of walnut tree volatiles and electrophysiological responses in Cydia pomonella (Lepidoptera: Tortricidae)

BACKGROUND: In recent years, many studies have been carried out on the behavioural and electrophysiological responses of Cydia pomonella (L.) to host volatile emissions, to find alternative attractants to the sex pheromone for pest monitoring. These studies have focused on apple and pear, and very little has been done on walnut. In the present work, the diurnal and seasonal variation in walnut volatile emissions and the electrophysiological response of C. pomonella have been studied. RESULTS: Ninety compounds were detected in walnut emissions, mainly monoterpenes and sesquiterpenes. The most abundant compound was β‐pinene, which, together with (Z)‐3‐hexenyl acetate, (E)‐β‐ocimene, limonene, germacrene D, 1,8‐cineole, sabinene, (E)‐β‐farnesene, (E)‐β‐caryophyllene, β‐myrcene and β‐phellandrene, constituted between 81.9 and 90.5% of the total chromatographic area. Differences between seasonal periods were significant for 39 compounds, and between daytimes for 14 compounds. Discernible and consistent EAD responses were detected to 11 walnut‐origin compounds, and confirmed with synthetics to seven of them. Except for alloocimene, pinocarvone and caryophyllene oxide, all these compounds are also emitted by apple. CONCLUSION: Walnut volatile emissions differ widely from apple ones, but both share many compounds that are EAD‐active in C. pomonella. However, among EAD‐active compounds there are three walnut‐specific ones, which should be further tested in behavioural assays. Copyright © 2008 Society of Chemical Industry     

Permethrin treatments in canadian forests. Part 1: Impact on stream fish

The effects of permethrin on native and caged fish when aerially applied directly to forest streams at dosages between 8.8 and 70 g ha^?1 were studied between 1976 and 1981. None of the applications caused mortality of caged or native fish in streams. Trout and salmon diets were altered by the treatments due to effects on fish food organisms. The duration of the effects varied from several months to over a year with increasing dosage. Reductions in salmonid growth rates and reductions in fish densities in treated areas, presumably due to emigration, were documented following severe impacts on aquatic invertebrates in salmon nursery streams. Growth rates and population densities both recovered within four months after treatment.