JPR Advance Access published December 5, 2006

JPR Advance Access published December 5, 2006 The copepod parasite (Lepeophtheirus salmonis (Krøyer), Caligus elongatus Nordmann) interactions between wild and farmed Atlantic salmon (Salmo salar L.) and wild sea trout (Salmo trutta L.) a mini-review CHRISTOPHER D. TODD GATTY MARINE LABORATORY, UNIVERSITY OF ST ANDREWS, St ANDREWS, FIFE SCOTLAND, KY16 8LB Suggested running head: Salmonid-parasitic copepod interactions The Author 2006. Published by Oxford University Press. All rights reserved For Permissions please e-mail: journals.permissions@oxfordjournals.org

Abstract Ectoparasitic copepods are major pathogens of farm and wild salmonids throughout the North Atlantic. Since the early 1990s there has been controversy regarding the extent to which infective larvae of Lepeophtheirus salmonis originate from aquaculture sites and impact wild salmon (Salmo salar) and sea trout (Salmo trutta). Because of the impracticality of tracking individual planktonic larvae from hatching to final host colonization, reliance has been placed on non-genetic and genetic experimental approaches. Microsatellite analyses show that L. salmonis comprises a single panmictic population throughout the Atlantic; gene flow between parasites on wild and farmed hosts is sufficiently high to prevent population genetic differentiation by random drift. However, because of this lack of significant differentiation, no estimates of the levels of gene flow between farm and wild are possible. The possible evolution of resistance to chemotherapeutants by caligids is of especial concern to the aquaculture industry. Decreased efficacy has been reported for numerous compounds and identification of a point mutation of a sodium channel gene might be indicative of knockdown resistance to pyrethroids. An additional concern is that the more host generalist C. elongatus might become an especially severe pathogen to both salmonid and gadid host populations as the emerging cod (Gadus morhua) aquaculture industry develops. 2

INTRODUCTION Copepods comprise an extremely species-rich and diverse crustacean subclass with at least 11,500 named species (Boxshall and Halsey, 2004). Their ecological importance is perhaps attributable largely to the key roles of planktonic cyclopoids and calanoids as primary consumers in pelagic freshwater and marine systems respectively; but parasitic copepods do exert important ecological and economic impacts on a wide array of invertebrate and fish species (Rohde, 1993). For example, caligids of the Order Siphonostomatoida are especially important ectoparasites of wild and farmed salmonids alike (Pike and Wadsworth, 1999; Johnson et al., 2004; Boxaspen, 2006). Farming of Atlantic salmon (Salmo salar L.) commenced in the 1970s and worldwide production has increased steadily to >1.1 million tonnes (ICES, 2004). Lepeophtheirus salmonis (Krøyer) is a specialist ectoparasite of salmonids in their marine phase and infests all species of wild salmonid in both the North Pacific and North Atlantic (Pike and Wadsworth, 1999, Nagasawa, 2001), and quickly became a major pest at aquaculture sites. Furthermore, farmed and wild Atlantic salmon in the North Atlantic also are impacted by the host generalist Caligus elongatus Nordmann (Wootten et al., 1982; Schram, 1998), which has been recorded from >80 species of elasmobranch and teleost hosts (Kabata, 1979). These two species together are the most economically important metazoan pathogens to S. salar aquaculture in the North Atlantic, and estimates of the worldwide costs to the industry of caligids range up to 80 million p.a. (Johnson et al., 2004). For the Scottish industry alone, the total cost is at least 38 million p.a. (Rae, 2002). In NW Europe there has been a long-standing controversy regarding the extent to which cultured salmon are a significant source of sea louse infestations of both wild sea trout (Salmo trutta L.) and S. salar populations (McVicar et al., 1993; Butler, 2002; Todd et al., 2004). Various experimental and analytical approaches have been applied in attempts to derive an objective means of ascribing the infestation source(s) of sea lice impacting wild fish, and these are briefly reviewed here. Irrespective of that debate, a clear understanding of the reproductive ecology, population dynamics, demography, population genetic structure and sources of infestation of sea lice impacting farmed salmonids has obvious commercial and management significance regarding their interventory control. L. salmonis can cause stress, pathological damage and death of the host fish (Nolan et al., 1999; Wells et al., 2007), and the small post-smolt stage is perhaps 3

the most vulnerable (Finstad et al., 2000). Notwithstanding their impacts on the welfare of cultured salmon, concerns about the possible detrimental impacts of caligids on populations of wild salmonids first arose from observations in Ireland of stock collapses of S. trutta in the late 1980s/early 1990s, and apparently associated epizootics of L. salmonis, particularly on juvenile hosts (Whelan, 1993; Tully et al., 1993). Heavily infested fish often showed premature migratory return to freshwater, were in poor condition and bore heavy infestations of the sessile, larval chalimus stages of the parasite. The evidence is now persuasive that farm-origin caligids do indeed impact wild salmonids, and almost certainly have at least contributed to recent wild salmonid stock declines proximate to salmon farms in areas of Norway, the British Isles, and also W Canada (Bjørn and Finstad, 2002, Butler, 2002, Todd et al., 2004, Krkošek et al., 2005). Perhaps the most indicative empirical field data are from Loch Torridon, W Scotland (McKibben and Hay, 2004; Penston et al., 2004). Time-series plankton surveys revealed considerable spatial and temporal heterogeneity of the distribution of caligid nauplius and copepodid stages. Larvae could be captured consistently only close to the outfalls of local rivers, and in such locations wild juvenile S. trutta are especially vulnerable to infestation. S. salar are cultured on a two-year production cycle and L. salmonis infestations are especially problematic on farms during the second year (Revie et al., 2005). Over a five-year period, McKibben and Hay (McKibben and Hay, 2004) recorded planktonic larvae in their samples only when there were gravid females present on local farms, and always during the second year of the production cycle. Here, I focus on experimental studies of L. salmonis and C. elongatus in the North Atlantic, because it is there that the aquaculture industry has been established the longest and the impacts of these sea lice on wild salmonids are the most long-standing and acute. The evidence of farm-wild parasite interactions is generally indirect and primarily circumstantial or correlative, due to the intractability of physically tracking individuals of the planktonic, larval sea lice stages from initial hatching to ultimate settlement on a host fish. I provide a brief overview of the range of analytical, non-genetic and genetic methods that have been applied in attempts to ascertain either a farm or wild provenance for individual parasites established on particular host fish. I argue that the most appropriate analytical approach to this generic issue involves the deployment of molecular genetic techniques and my focus, therefore, also is on recent advances in the application of genetic techniques to other general and more specific taxonomic, systematic and ecological problems concerning sea lice. 4

Life cycles and reproductive biology of Lepeophtheiurus salmonis and Caligus elongatus In view of its economic and ecological importance, the life cycle, population dynamics and epidemiology of L. salmonis have been intensively investigated (Pike and Wadsworth, 1999; Revie et al., 2002a, 2003, 2005). By contrast, and largely because it is a host generalist, the population dynamics of C. elongatus on wild fish species are less well understood, although progress has been made in modeling its epidemiology on farmed salmonids (Revie et al., 2002b; McKenzie et al., 2004). Fertilization of L. salmonis and C. elongatus is internal and the extruded eggstrings are retained by the female until the hatching, planktonic nauplius I stage is attained. The nauplius II moults to the infective planktonic copepodid stage and this attaches to the host fish and moults to the first of four sessile chalimus stages. Planktonic development of all these non-feeding larval stages is temperature-dependent and may require 2-9 d for L. salmonis (Johnson and Albright, 1991) and 38 degree-days for C. elongatus (Piasecki and MacKinnon, 1995). The chalimus IV of L. salmonis gives rise to the first of two mobile pre-adult stages, prior to the definitive moult to the adult male or female. By contrast, there is no pre-adult stage in the life cycle of C. elongatus, which moults directly from chalimus IV to the adult (Piasecki, 1996). Several studies had indicated that female L. salmonis are monogamous (Hull, 1998; Ritchie et al., 1996a,b; Pike and Wadsworth, 1999). Adult males actively seek out and clasp onto a pre-adult I or II female and the male remains attached in a characteristic mate-guarding position (Hull, 1998). Once the female has undergone the definitive adult moult the male transfers and cements a pair of spermatophores on her genital complex. Tubules extend from the spermatophores, cross over and enter the opposite genital pores to allow transfer of the spermatozoa and their internal storage in the receptaculum seminis. The combination of active (pre-copulatory) and remote (post-copulatory) male mate-guarding by the cementing of durable, persistent spermatophores, the tubules of which occlude the genital pores on the female s genital complex, all pointed to effective monogamy of L. salmonis (Pike and Wadsworth, 1999). As discussed below, the analysis of microsatellite DNA variation is a powerful means of assessing population structure, genetic differentiation and gene flow, but microsatellite analysis does also remain the molecular tool of choice in parentage studies (Fleischer, 1996; Strassman et al., 1996). 5

Microsatellite analysis of the paternity of embryos within the eggstrings of identifiable females demonstrated that polyandry is, in fact, common in L. salmonis infesting wild S. salar (Todd et al., 2005). The likelihood is that most females on wild hosts will be polyandrous during their ovigerous lifetime of several months, despite male attempts to prevent their multiple mating. Nonetheless, because of regular, interventory, chemotherapeutant treatment of sea lice infestations of cultured salmon, there must be a strong likelihood of females on farmed salmon hosts not surviving sufficiently long to be multiply mated and to display polyandry. Accordingly, seven gravid adult female L. salmonis bearing the typical pair of cemented spermatophores were randomly sampled from harvested salmon at a W Scotland farm in March 2005. Each adult and 6-20 embryos from one of the pair of eggstrings were genotyped for one of the microsatellite loci (LsalSTA5) used in the previous paternity study (Todd et al., 2005). The offspring of two of these seven farm females (Numbers 3 and 7, Fig. 1) showed dual paternity. In order to confidently determine the proportion within a population of farm females that show multiple paternity, screening of larger numbers of females for multiple loci would be necessary. But qualitatively, the important outcome is that L. salmonis infesting both farm and wild fish do show polyandry, despite the considerable investment of males in pre- and postcopulatory mate-guarding; these findings have implications for the continued development of sea lice management and control strategies in the aquaculture industry (Todd et al., 2005). Non-genetic techniques to discriminate populations of sea lice Phenotypic variation. Nordhagen et al. (Nordhagen et al., 2000) noted that adult L. salmonis sampled from wild salmon were significantly larger than those sampled from cultured salmon, but that larvae from these two sources developed in the laboratory to similar sizes. Moreover, larvae developed at 9 C gave rise to larger adults than did those developed at 12 C. Previously, Sharp et al. (Sharp et al., 1994) had reported apparent geographic variations in size of L. salmonis and C. elongatus infesting S. trutta from the east and west coasts of Scotland, with larger parasites found on the east coast. Their analysis did not, however, take account of the size of the host fish. The same criticism extends to an earlier investigation of size variation in L. salmonis infesting a cage population of S. salar in Ireland (Tully, 1989). Tully et al. (Tully et al., 1989) placed 6

smolts at sea in May in a single, unreplicated cage and sampled monthly up to January the following year. Lengths of male and female L. salmonis both were minimal in summer/autumn (July-September), when temperatures were highest, and parasite size increased progressively from September to January as temperatures fell. It is likely that temperature does exert a general influence on adult sizes, but host size and species do also have predictable effects on adult size of L. salmonis infesting wild S. salar and S. trutta (C.D. Todd and A.M. Walker, CEFAS, Lowestoft, unpubl.): mean sizes of adult L. salmonis on S. trutta always are smaller than those from S. salar, and larger host fish of either species bear larger adult L. salmonis. Moreover, L. salmonis adults sampled from S. salar are larger than those sampled from S. trutta of comparable size. As affirmed by Nordhagen et al. (Nordhagen et al., 2000), it is very clear that size of adult L. salmonis is highly plastic and size variation is of no utility in distinguishing a farm or wild origin of the initial infective copepodid (Sharp et al., 1994). ICPMS, pigment analysis, stable isotope analysis. Shinn et al. (Shinn et al., 2000a) applied conventional nebulisation ICPMS (Inductively Coupled Plasma Mass Spectrometry) to analyze adult female L. salmonis from four salmon farms and three wild commercial net fisheries in Scotland. Data for as few as 16 elements allowed sea lice from the seven sites to be distinguished by discriminant analysis, but the general applicability of this technique in attributing a farm or wild provenance of individual parasites remains unclear. The elemental signature of the parasite will, for example, depend upon both host-related and extrinsic seasonal and environmental physicochemical factors. An alternative chemical approach was to analyze tissue pigment complements. The pink coloration of the flesh of wild salmon is largely attributable to the natural carotenoid pigment astaxanthin. In order to achieve a similar coloration of cultured salmon flesh, the feed pellets are treated with synthetic pigments, such as canthaxanthin, as well as astaxanthin. Pigment complements of sea lice potentially provide an analytical tool for distinguishing individuals from farmed and wild salmon (Noack et al., 1997). Although they could not unequivocally identify canthaxanthin by HPLC, Noack et al. (Noack et al., 1997) reported that canthaxanthin-like peaks were distinguished from astaxanthin and that the ratios of the two components were shown to differ between adult sea lice taken from wild and farm fish. But such a tool is of no value in determining the provenance or origin of the infective larvae impacting a given wild (or farm) fish. Even assuming that the tissues of nauplius larvae released by farm lice initially carry a 7

disproportionately high level of canthaxanthin, this farm pigment marker will be lost as larval development and growth of the sessile and mobile stages proceeds on a host fish. The same argument extends to the use of stable isotopes in discriminating populations of sea lice. Consumers (predators, parasites) generally show enrichment of the heavier (less labile) isotopes over those in their diet (McCutchan et al., 2003). Enrichment of consumer 13 C arises from differential respiratory and excretory loss of assimilated 12 C in respiratory CO 2, although lipids tend to be depleted in 13 C relative to carbohydrates and proteins. Similarly, excreted nitrogen typically is depleted in 15 N relative to assimilation. However, isotopic trophic shift for N can vary amongst species according to whether the primary nitrogenous excretory product is ammonia, urea or uric acid, and depending upon nutritional status (Vanderklift and Ponsard, 2003). Trophic step enrichment is, moreover, sensitive to rates of consumer feeding, post-absorption excretion, defaecation and the extent of isotopic discrimination. For example, Olive et al. (Olive et al., 2003) have shown that isotopic ratios can change within a matter of days of starvation, or of switching the consumer to a new food source. Butterworth et al. (Butterworth et al., 2004) applied C and N stable isotope analysis to L. salmonis sampled from farmed S. salar from the Pacific and Atlantic coasts of Canada, and from wild Pacific coho salmon (Oncorhynchus kisutch). Their objective was to validate the stable isotope technique as a means of discriminating between adult parasites from wild and farmed fish. Notwithstanding the foregoing limitations of the stable isotope technique, it is clear that this methodology will never be applicable to studies of the provenance, or infection origin, of sea lice infesting (wild or cultured) host fish. As for pigment analyses, even if the stable isotope signatures between adult sea lice (and their larvae) on farmed and wild hosts differ significantly, these differences will be lost as the larva develops and grows to the feeding chalimus and adult stages on the host fish. Genetic studies of caligid sea lice Given our inability to directly track individual larvae from hatching to host encounter and colonization, genetic approaches would appear to offer the most effective indirect means of ascribing a wild or farm source of sea lice infecting given fish. Low levels of gene flow (here, larval cross-infection) between two populations can result in those populations becoming differentiated as a result of random genetic drift. Conversely, 8

selection at farm sites, perhaps as a result of exposure of sea lice to chemotherapeutants, might also lead to population genetic divergence. If there is sufficient genetic differentiation of populations, the rates of gene flow can be estimated (Wright, 1969) and individuals can be objectively allocated to populations by any one of several assignment methods (Hansen et al., 2001). High levels of gene flow will, however, maintain genetic homogeneity of populations and this will preclude both assignment of individuals to a source and the quantification of gene flow amongst farm and wild populations. Mitochondrial sequence analysis. Because it provides a large character base, and owing to its high rate of evolution compared to nuclear DNA (Brown et al., 1979), analysis of mtdna often has been successfully used in generating tractable hypotheses and robust predictions of the phylogeny of species. The molecular approach offers indispensable tools for taxonomists and ecologists alike, especially in distinguishing closely-related, cryptic or sibling species. The mtdna molecule is double-stranded, circular and typically encodes 37 genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes); whilst the number and identity of the genes comprising mtdna is highly conserved, their order is not, at least in invertebrates (Machida et al., 2002). The non-coding control region (Displacement loop) is often used in taxonomic and phylogenetic studies, but analytical outcomes can be markedly influenced by the kinds of markers (e.g. RFLPs) investigated, and when there are high levels of DNA polymorphism (Grant et al., 1998). At present there are 33 complete crustacean mtdna sequences available (GenBank), of which 18 are for malacostracans and only two, Tigriopus japonicus (Machida et al., 2002) and L. salmonis (Tjensvoll et al., 2005), are for copepods. The mtdna sequence of T. japonicus is 14,628 bp long, but none of the other reported gene orders are comparable to T. japonicus. Also, unlike most metazoa, T. japonicus is exceptional in having all the genes on the one strand. Roehrdantz et al. (Roehrdantz et al., 2002), for example, investigated arthropod phylogeny by analysing gross gene rearrangements of mtdna. Their data included two malacostracans and a single isopod and pointed to the crustaceans as being a sister lineage to insects; whether the inclusion of the unique gene arrangements for L. salmonis, or for T. japonicus, will further reinforce this pattern is unclear. Other unusual features of the T. japonicus mtdna genome include the size reductions in the RNA genes, resulting in this being one of the smallest mtdna genomes in the arthropod lineage. 9

The mitochondrial cytochrome oxidase I (mtcoi) gene evolves particularly rapidly and sequence comparisons of this gene have often been used in distinguishing closely related taxa. For example, Bucklin et al. (Bucklin et al., 2003) found that analysis of a 639 bp region of mtcoi allowed the reliable distinction of 34 calanoid copepod species embracing ten genera and two families. With specific reference to the ectoparasitic caligid C. elongatus, Øines and Heuch (Øines and Heuch, 2005) exploited the variability of mtcoi in separating two distinct haplotypes. Whether or not these are separate species remains to be confirmed, but morphologically they are virtually indistinguishable and the two are intermixed on various host fish species. Certainly, from both ecological and economic standpoints, because C. elongatus is abundant on both wild (Todd et al., 2007) and farmed Atlantic salmon (McKenzie et al., 2004), it will be essential to ascertain if these different haplotypes/species are associated with particular host fish species (Øines et al., 2006) Shinn et al. (Shinn et al., 2000b) investigated partial sequences of the 18S rrna gene and the ribosomal internal transcribed spacer (ITS-1) region obtained from mtdna for individual L. salmonis. Their objective was to assess the utility of mtdna sequence data in perhaps distinguishing parasites from wild and farmed Atlantic salmon. They sampled L. salmonis from eight localities (three salmon farms and five wild commercial net fisheries) in Scotland. Eight individual L. salmonis (two each from two farms and two wild sites) were sequenced for a 900 nucleotide fragment of the 18S rrna gene. That gene, however, is highly conserved and showed no sequence variation. Thirteen individual L. salmonis, again from two farms and two wild sites, were sequenced for a 454 nucleotide fragment of ITS-1. Overall there was 92% similarity of sequences, with variation concentrated in one hypervariable region. They reported greater sequence similarity for ITS-1 within rather than between farm and wild lice, and proposed this technique to provide a means of assigning lice to either farm or wild populations. Furthermore, on the basis of this result they (Shinn et al., 2000b) suggested that the causal process may be attributed to restricted gene flow within lice populations at S. salar cage sites. But this is counter-intuitive, given the obligatory 2-9 d duration of the free-swimming planktonic larval stages, during which period the nauplius I and II stages are incapable of colonizing host fish. Planktonic export (and hence gene flow from farm to farm and between farm and wild host populations) of nauplius I larvae from cage sites is inevitable: there is no possibility of developing nauplii being retained within cages for the required time period. Re-importation of fully-developed copepodid larvae, and hence 10

ultimate self-reinfestation of cage sites in semi-enclosed fjords is, however, clearly possible; but the hydrodynamic regime at any farm site will ensure initial dispersal of planktonic larval stages away from the source host population and thereby the potential for infestation of other farms and/or wild salmonids. The scale of realised larval dispersal in L. salmonis and C. elongatus, and thence gene flow and levels of genetic differentiation of populations, is therefore dependent both upon passive planktonic dispersal of the parasite larval stages and the migratory ranges of the various wild host species. Whereas S. trutta generally remain in coastal waters and close to their natal river, wild S. salar may migrate thousands of km across the North Atlantic (Klemetsen et al., 2003). Allozyme (protein) variation. In explicitly addressing the issue of scale of population genetic differentiation of L. salmonis, earlier studies of allozyme variation of adult sea lice sampled from farm and wild populations of Atlantic salmon had reported rather conflicting results. Todd et al. (Todd et al., 1997) screened two polymorphic allozyme loci (Fum, Got-2) for 403 adult L. salmonis sampled from three W Scotland farms and from wild S. salar and wild S. trutta in two river estuaries in E Scotland. Analysis of Wright s F-statistics confirmed no significant genetic differentiation between east and west coasts (geographic range ~770 km), or between farmed versus wild host fish. Neither was there significant genetic differentiation of male and female L. salmonis. By contrast, Isdal et al. (Isdal et al., 1997) screened four allozyme loci (Est, Pgi, Idh, Pgm) for L. salmonis sampled at six sites spanning a north-south geographic range in Norway of ~1100 km. Their data showed some evidence of differentiation into two ( northern and southern ) geographic groupings, but differentiation was attributable to allele frequencies at only the one locus. Although allozyme electrophoresis offers a reliable, well-established and relatively cheap means of obtaining genetic data for individuals, and thence measures of population homogeneity/heterogeneity, the overall levels of polymorphism of allozyme loci typically are very low in contrast to those that can be revealed by molecular DNA techniques. An early application of PCR technology to population analysis was the RAPD (randomly amplified polymorphic DNA) technique, which uses anonymous PCR primers. Todd et al. (Todd et al., 1997) screened samples of L. salmonis from host fish taken around the west, north and east coasts of Scotland with six primers. Hosts included wild and farmed S. salar, farmed rainbow trout (Oncorhynchus mykiss Walbaum) and 11

wild S. trutta. Analysis of molecular variance (AMOVA) indicated significant differentiation between farm and wild L. salmonis, and amongst the various farms sampled. Moreover, certain RAPD fragments appeared to be putative markers exclusive to L. salmonis sampled from farm fish, and two bands for one primer were apparently sex-linked and diagnostic of female L. salmonis. The identification of genetic markers that might permit unequivocal assignment of individual sea lice to, for example, a farm or wild population would be of value to the salmon farming industry and wild fishery managers alike. However, the weaknesses of the RAPD technique are considerable (Grosberg et al., 1996). The observer has no specific information on the fragments of DNA being amplified, and amplifications appear to be very dependent upon the precise PCR conditions. Moreover, RAPDs are dominant markers and the allelic basis of fragment variation cannot be inferred with confidence. A subsequent study (Dixon et al., 2004) of L. salmonis from around Scotland, using the same RAPD primers, indicated no systematic geographic patterns of differentiation or distinction between L. salmonis of farmed or wild origin. RAPD markers did not, therefore, provide a sufficiently informative or reliable analytical tool to address questions relating to the genetic structuring of copepod sea lice populations, or of farm versus wild provenance of parasites infesting given host fish. Although time-consuming and expensive to develop anew for a given species, microsatellite (VNTR) DNA loci currently offer an excellent opportunity of addressing such questions by providing unequivocal and highly polymorphic allelic data for presumed neutral loci (see Wan et al., 2004). For highly structured and differentiated populations, perhaps showing private alleles, microsatellite data offer the possibility of allowing assignment of individuals to populations (or, in the wider context, a farm or wild provenance if those population components are discrete and distinct). Microsatellite DNA variation. Microsatellites are rapidly-evolving, presumed selectively neutral loci in typically non-coding regions of DNA that include short tandem nucleotide repeats (Wan et al., 2004). The quantification of allelic variation in microsatellite repeat length for multiple loci provides a high resolution database for the analysis of population structuring and individual assignment (Strassman et al., 1996). For L. salmonis, GenBank presently includes ten unpublished and nine published microsatellite sequences (Nolan et al., 2000; Todd et al., 2004). No loci are available for other caligid copepods. In their preliminary analysis of small samples of L. salmonis 12

from Ireland, Scotland and Norway, Nolan et al. (Nolan et al., 2000) confirmed the potential for microsatellites to allow the detection of population differentiation in caligids. Todd et al. (Todd et al., 2004) screened 1007 Atlantic (n = 973) and Pacific (n = 34) L. salmonis for six microsatellite loci. Their Atlantic samples centred on three host species across multiple farm and wild sites around Scotland (n = 856 L. salmonis), but included material from wild sea trout in N Norway (n = 58) and farmed Atlantic salmon in E Canada (n = 59). The small outgroup Pacific sample was taken from farmed S. salar in British Columbia. The Norwegian and eastern Canadian samples were analytically especially important in the geographic context because the infestations carried by those fish would have been acquired locally; by contrast, the remaining wild salmon sampled from Scotland will have ranged migratorily to both the Norwegian Sea and the coasts of Greenland. There they will have intermixed and cross-infected with fish of Russian, European, Icelandic and North American origin (Jacobsen and Gaard, 1997; Todd et al., 2000). There were no analytical problems from null alleles and, for the North Atlantic samples, F-statistic analyses showed no significant differentiation amongst host species or between farm and wild L. salmonis. There was no evidence of isolation by geographic distance (regression slope, b ~0) over the sampled 6000 km North Atlantic range and this conformed to expectation. The levels of gene flow (larval cross-infection) between farm and wild salmonids, and between the two primary wild species in the North Atlantic (S. salar, S. trutta), are sufficiently high as to prevent genetic divergence of populations over a 6000 km range. As expected, however, there was highly significant differentiation between North Atlantic and North Pacific L. salmonis, presumably attributable to genetic drift arising from the historic isolation of North Pacific and North Atlantic L. salmonis. Although these analyses of microsatellite differentiation provide no direct evidence of infection of wild fish by farmed fish, they do conclusively demonstrate that farm sea lice populations are not genetically isolated and there is no evidence that L. salmonis throughout the North Atlantic comprises anything other than a single, panmictic population. Given the numerical imbalance between the numbers of wild and farmed salmonids in the North Atlantic, and the fact that cultured salmon are present in coastal waters year-round, the likelihood is that the bulk of the infective interaction concerning wild S. salar and S. trutta is from farm to wild, rather than wild to farm (Butler, 2002; Todd et al., 2004). It has to be emphasized that randomly chosen microsatellites will not 13

reveal selection for specific mutations that may, for example, confer resistance to chemotherapeutants. Molecular studies of chemical resistance. Many insect populations have developed genetic resistance to pyrethroid insecticides (Zhao et al., 2000) and to organosphosphates and carbamates (Walsh et al., 2001); similar responses by sea lice on salmon farms were therefore to be expected. Pyrethroid resistance involves genetic mutations of neuronal sodium channel proteins (Soderlund and Knipple, 2003), whereas organophosphate and carbamate resistance is conferred by mutations of acetylcholinesterase (AChE) genes (Walsh et al., 2001). The organophosphates dichlorvos and azamethiphos were widely used as louse chemotherapeutants from early in the development of salmon aquaculture and soon there were reports of decreased sensitivity of L. salmonis to dichlorvos in Scotland (Jones et al., 1992). Similarly, Fallang et al. (Fallang et al., 2004) reported decreased sensitivity of L. salmonis in Norway and Canada to azamethiphos. Biochemical assays showed differential sensitivities of two AChE enzymes one was rapidly inactivated, and the other slowly inactivated but the genetic basis of this remains unclear. Knockdown resistance (kdr) differs from resistance by metabolic detoxification in arising from a decrease in the sensitivity of the nervous system to pyrethroids (Soderlund and Knipple, 2003). The pyrethroids deltamethrin and cypermethrin have been widely used by the aquaculture industry in the control of sea lice, and anecdotal reports of their decreased efficacy have ranged from Ireland and Scotland to Norway (Sevatdal et al., 2005). Mutations of the para-type voltage-sensitive sodium channel gene appear to specifically confer pyrethroid resistance in arthropods and Fallang et al. (2005) have sequenced a 318 bp fragment of that gene in L. salmonis. Four of the six previously reported kdr primary resistance mutations in arthropods are located in the second of four specific regions of the protein (Soderlund and Knipple, 2003), and the mutation reported by Fallang et al. (Fallang et al., 2005) also was located in domain II. Although Fallang et al. (Fallang et al., 2005) have presently only correlated the occurrence of their mutation with pyrethroid resistance, its location in domain II does strongly suggest a causal relationship. 14

CONCLUSION An early resolution of the controversy surrounding the caligid infestation interactions between farmed and wild salmonids would have been readily achievable if it were possible to unequivocally determine, by direct observation, the source of infective copepodids of L. salmonis parasitizing wild host fish. With the rare exception of (visibly) large and very short-lived marine planktonic larvae such as the tadpole larvae of ascidians, which can be followed from release to settlement by SCUBA divers (Davis and Butler, 1989) it is impractical to physically track the planktonic stages of marine invertebrate larvae in the water column (Todd, 1998). The challenge therefore has been one of developing indirect methods to confirm the connection between larval sea lice production from aquaculture sites and infestations of free-ranging wild salmonids. Despite the range of experimental and analytical approaches applied, it is clear that population genetic techniques are the only plausible means of addressing general and broad scale questions relating to the management of wild-farm interactions and copepod infestations of wild salmonids. Molecular DNA techniques have confirmed that farm populations of L. salmonis are not genetically isolated from populations on wild hosts, and the plankton data of McKibben and Hay (McKibben and Hay, 2004) and Penston et al. (Penston et al., 2004) should urge wild and farmed fishery managers to be resolute in adopting a precautionary approach to the problem of farm-wild infestation interactions. A very real concern, however, must lie in the paucity of the range of chemotherapeutants available to the aquaculture industry, and the commercial expense and lead time for developing new active compounds. Presently, there is a widespread and very heavy reliance by the industry on the one major compound, emamectin benzoate (SLICE, Schering-Plough), with no replacement product close to market. Populations of Atlantic L. salmonis have been shown to be genetically open: there is no indication that there is any genetic distinction between populations infesting wild or farmed salmon on either seaboard of the North Atlantic. This feature alone dictates that eradication of L. salmonis as a pest species on salmon farms is impossible. But there are consequences also for the possible evolution of resistance of L. salmonis to chemical treatments. One of the tenets of pest management and control is that in the absence of pesticides resistant genotypes are selected against, or resistant individuals are less fit (Devonshire et al., 1998). Given the genetic openness of L. salmonis populations on farms, and high levels of gene flow between wild and farmed fish and amongst farms, it is important to understand how resistance might develop on farms because resistance 15

genes ought to be selected against on wild fish and outwith the farm environment. Nonetheless, in view of the possibility of resistance developing, the challenge to the aquaculture industry is to deploy the few currently available and licenced treatments at minimum dosages and perhaps to engage in rotation of treatment use. The fish husbandry and economic problems caused by the monogenean skin fluke, Benedenia seriolae, to the emerging kingfish aquaculture industry in Australia (Chambers and Ernst, 2005) are timely reminders that metazoan parasites other than copepods can present major environmental, economic and animal welfare challenges to intensive marine aquaculture industries. For the salmon aquaculture industry, the past focus of research into sea lice biology in the North Atlantic has necessarily been on the salmon louse, L. salmonis, but the importance of the host-generalist, C. elongatus, to both farmed and wild salmon and other teleost fish in the North Atlantic, should not be underestimated. For example, for wild 0-group cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) of only 13-63 mm length, Nielsen et al. (Nielsen et al., 1987) recorded intensities of C. elongatus at up to 35 and 15 chalimi respectively per fish in the NW Atlantic. On the basis of empirical physiological data, on the stress impacts of L. salmonis on 19-70 g post-smolt S. trutta (Wells et al., 2007), such intensities of infestation of wild gadids will almost certainly be lethal. In the farm environment, high intensity C. elongatus infestations, and consequentially severe head lesions, were reported for juvenile farmed halibut (Hippoglossus hippoglossus) early in the development of what was then a new aquaculture species (Bergh et al., 2001). Cod farming presently is expanding rapidly in the NE Atlantic. The wealth of experience and knowledge that has been garnered in relation to L. salmonis should be readily transferable to the possibly detrimental impacts on wild gadid and salmonid stocks that C. elongatus may well exert as cod farming develops (Øines et al., 2006). ACKNOWLEDGEMENTS This work was partly funded by the European Commission (SUMBAWS, Contract No. Q5RS-2002-00730). For assistance with the paternity analyses I am grateful to N. Gagne and M. Feilen. J. Graves provided insightful comments on the manuscript. REFERENCES Bergh, Ø., Nilsen, F. and Samuelson, O. B. (2001) Diseases, prophylaxis and treatment of the Atlantic halibut Hippoglossus hippoglossus: a review. Dis. Aqua. Org., 48, 57 74. 16

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Figure Legend Fig. 1. Lepeophtheirus salmonis. Proportional frequencies of paternal microsatellite (LsalSTA5) alleles within seven families of offspring. The numbers of embryos typed for each family are given above the bars. Single paternity Families 1, 2, 4-6; Dual paternity Families 3 and 7. 27

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