Tesis sobre el tema "Sea lice; Salmon farms"

Thesis (Ph.D.)--Aberdeen University, 2009.
Title from web page (viewed on June 26, 2009). With: Spatial and temporal distribution of Lepeophtheirus salmonis (Krøyer) larvae in a sea loch containing Atlantic salmon, Salmo salra L., farms on the north-west coast of Scotland / M. J. Penston ... et al. Journal of fish diseases. 2008: 31, 361-371. With: Reduced Lepeophtheirus salmonis larval abundance in a sea loch on the west coast of Scotland between 2002 and 2006 / Michael J. Penston, Colin P. Millar, Ian Davies. Dis. Aquat. Org. 2008: 81, 109-117. Includes bibliographical references.

Sea lice Lepeophtheirus salmonis are the major pathogen affecting the salmon farming industry. Current methods of control, especially in the UK, are largely ineffective as they do not affect the chalimus stages of L. salmonis and are toxic to fish. Greater control of L. salmonis through improved management, more effective compounds, disease control and stock selection were examined. The epidemiology of L. salmonis on Atlantic salmon Salmo salar production sites was examined at a number of farms within Loch Sunart. Intensity of infection was found to be influenced by both management practices and seasonal variations. By instigating a series of co-ordinated, synchronous, strategic treatments throughout the loch system during the winter, initial chalimus levels during the spring were significantly reduced by 90% (p<0.001). Lice numbers for the rest of the production cycle were significantly lower (p<0.01). In addition there was a reduction in the number of treatments needed, an increase in the interval between treatments, reduced fish mortalities and improvements in fish harvest quality. There was no significant reduction in mean chalimus numbers observed at the control site where treatments were not co-ordinated. The strategic treatments were repeated in the Loch Linnhe system during 1997 resulting in an 87% (p<0.01) reduction in initial chalimus numbers. The efficacy of the synthetic pyrethroid, cypermethrin (Excis RTM) was assessed against L. salmonis chalimus. Exposure of the chalimus to 5mug I-1permethrin for 1 h resulted in a significant reduction (83%, p<0.001) in the mean number of chalimus 12 d post-treatment. The developmental stages observed in the untreated group were predominantly pre-adult II males. The majority of the remaining lice observed in the treated group were chalimus III indicating a profound effect of cypermethrin on the rate of development of L. salmonis. At 1 pg I-1permethrin for 1 h there was no significant reduction in the mean number of lice compared to controls (p=0.161) although there was a significant delay in the rate of development. 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. 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. Variability in susceptibility to settlement and survival, as well as damage caused by L. salmonis was also observed between families of Atlantic salmon.

Yeast cell wall (YCW) extracts have long been regarded as beneficial supplements in aquaculture feeds, especially with respect to immunomodulation. Previous studies have shown that supplementing salmon diets with YCW extracts increases resistance to sea lice. During the series of three trials, different sea lice challenge models and YCW extracts were studied. In Trial 1, fish fed a diet containing 0.4% YCW #a exhibited a significant reduction in sea lice burden of 23% compared to the control diet. The skin and skin mucus samples from Trial 1 were hence used for high-throughput screening of altered transcripts and proteins, while searching for candidate biomarkers for YCW extracts. Samples from Trials 2 and 3 were used for testing the candidate transcripts in independent biological samples, hence confirming or otherwise their suitability as biomarkers. Due to the lack of skin-specific libraries in the salmonid EST database, Roche-454 sequencing was used to describe the skin transcriptome of Atlantic salmon and form the basis of successive experiments. The resulting transcriptome assembly included 4,108 transcripts that were previously unknown for salmon, which include both skin-specific and low-expression transcripts. Analysis of the mucus proteome by 2D gels of Trials 1 and 2 samples showed that 27 and 49 proteins, respectively, exhibited a change in expression due to dietary treatment (p<0.05). Of these, calreticulin-like protein showed greatest potential as a biomarker since its expression was changed in both Trials 1 and 2 at the proteomic level and Trials 2 and 3 at the transcriptomic level. RNAseq was carried out on Trial 1 skin samples and 375 transcripts were significantly altered in expression across dietary treatments (p<0.05). Validation of this data by qPCR was successful and yielded similar fold changes to the RNAseq output. However, when the expression of a selection of genes was studied in Trial 2 samples, no correlation was found and hence, no candidate biomarker molecules were discovered. While further work is required to deduce the underlying mechanisms of immunomodulation due to dietary YCW extracts, this project has yielded two skin-specific transcriptomic databases which may be used to enhance the genomic data for salmonids.

The objective of the work described in the current thesis was to provide a better understanding of some of the key factors associated with sea louse, Lepeophtheirus salmonis, infection of farmed Atlantic salmon. In Chapter 2, initial work focused on establishing the vertical and horizontal distribution of sea lice copepodids and spatial patterns of on-farm infections. The louse distribution was investigated along the main current gradient across adjacent salmon production pens at three commercial sites. A depth profile for the distribution of larval lice was also established for the top 12 m of the water column at three different locations in close proximity to commercial salmon farms. Within all multi-pen sites there were clear patterns of distribution and infection along the main water current gradient with the abundance of lice in end pens at each site appearing to be different from the central pens. The vertical distribution pattern of free swimming L. salmonis larvae (nauplii and copepodids) showed that the surface 6 m harboured 85.5 ± 1.6 % of the lice present in the water body analysed (0 – 12 m depth), irrespective of sampling date and location. In Chapter 3, further environmental effects / influences on attachment success of the copepodids were analysed using controlled infection challenges. A flume with adjustable flow rates, and controlled light conditions was designed for this study. Flume current velocity was observed to be a significant factor in infection success, with higher infection levels observed at lower current velocities, while higher current velocities were demonstrated to reduce settlement success. At fixed velocity, higher copepodid exposure levels gave rise to higher infection levels, this having a linear relationship suggestive of a lack of competitive effects for space on the fish. Light was also shown to play an important role in host settlement. A positive correlation between increasing light intensity and higher louse attachment success was found for all tested light spectra / wavelengths (white - Halogen, blue 455 nm, green 530 nm and red 640 nm). Observation of an infecting cohort of copepodids showed maximal infection at four days post-moulting with a tail-off of infection by six days post-moulting. However, even under the optimal conditions represented by a flume challenge, including linear water flow, the constraint of copepodids to pass close to the salmon host and the very high exposure levels of copepodids per fish, louse attachment success was still relatively low. Chapter 4 examined implementation of a possible management approach based upon some of the environmental influences observed. This chapter described a study in which environmental manipulation of salmon swimming depth was employed on-site in an attempt to reduce farm infection of Atlantic salmon. The effects of submerged artificial lighting in combination with submerged feeding were tested with respect to salmon swimming depth and sea lice infection, following the hypothesis that L. salmonis infection in a commercial salmon population could be reduced through exposure to deep lighting and feeding. The results of the study suggest that swimming depth manipulation can indeed be used at a commercial scale to reduce salmon lice burdens on Atlantic salmon by physically minimising spatial interactions between the two animals. In the final research chapter (Chapter 5), this thesis examines the question of whether ploidy of the host impacts on sea louse infection levels and whether susceptibility of individual fish is consistent between replicate infections. Results showed that triploid salmon are not subject to higher sea louse infection levels under experimental challenge and farm infection conditions compared to diploid hosts. In addition, triploid fish subject to initial infection, did not become more or less resistant to infection compared to diploids when comparing repeated sea louse infections. In summary, this thesis describes work conducted to analyse key infection pathways and factors influencing infection of Atlantic salmon by sea lice and suggestions made as to how findings may be exploited to reduce louse burdens in Atlantic salmon farming. The practical solutions presented to exploit the results found in this work are currently under consideration by the Scottish salmon industry.

Salmon aquaculture can be a potential solution to bridge the gap between declining capture fisheries and increasing seafood demand. However, the environmental impacts it creates have generated criticism. The overall objectives of this dissertation are to examine the economic consequences of environmental issues associated with salmon aquaculture, and to explore policy implications and recommendations for reducing environmental impacts. These objectives are addressed in five main analyses. The growth of salmon aquaculture is analyzed based on farmed salmon production in the four leading producing countries and the sector as a whole. Analyses indicate that salmon aquaculture is unlikely to continue to grow at its current pace. A joint production function approach is used to estimate pollution abatement costs for the salmon aquaculture industry. Results reveal that pollution abatement costs vary among observations and models. On average, pollution abatement cost is estimated at 3.5% in terms of total farmed salmon production, and 6.5% in terms of total revenue of farmed salmon. The ecological and economic impacts of sea lice from salmon farms on wild salmon population and fisheries are also studied. Analyses suggest that these effects are minor when the sea lice induced mortality rate is below 20%, while they can be severe if the mortality is greater than 30%. Sea lice have greater ecological and economic impacts on pink salmon than on chum salmon. These effects are greater under a fixed exploitation rate than under a target escapement policy. The economic performance of open netcage and sea-bag production systems for salmon aquaculture is compared. Netcage systems appear to be more economically profitable than sea-bag systems when environmental costs are either not or only partially included. Sea-bag systems can be financially profitable only when the salmon they produce can achieve a price premium. Finally, policy implications are explored and recommendations are made for sustaining salmon aquaculture in a holistic manner based on the results from previous chapters. Technologies, economic-based instruments and more stringent environmental policies can be employed to reduce environmental impacts. However, there is no single solution to solve these environmental impacts, and a combination of policy options is needed.

Background Atlantic salmon (Salmo Salar) is a key aquaculture species in several countries. Since its critical role in economic sector and scientific research, this species has been relatively extensively investigated, in comparison with other farmed and wild aquatic species. However, the genetic components associated with growth and fillet-related traits are lack consistency, and the issue of sea louse disease in both wild and famed salmon is still unsolved. Objectives Overall aim of this project was to understand the genetic basis of growth-related traits and host resistance to sea lice using three large commercial farmed salmon populations. Specifically, the method of quantitative trait loci (QTL) mapping, genome-wide association study (GWAS), and genomic prediction (GS) were utilized to dissect the genetic architectures associated with traits of interest in our experimental populations. Prior to this, linkage mapping was performed to construct a high-density linkage map for Atlantic salmon. Results Linkage map A linkage map was firstly constructed underlying a SNP array containing 132 K validated SNPs. 96,396 SNPs were successfully assigned to 29 chromosomes that correspond to the linkage group number of European Atlantic salmon. 6.5 % of unassigned contigs, which was equal to 1 % of recent whole genome reference assembly (GCA_000233375.4) anchored to exist chromosomes by referring to linkage mapping result. Genetic components associated with growth traits Heritabilities of growth-related traits were about 0.5 to 0.6 in adult and juvenile farmed salmon. The QTL mapping and GWAS suggested the growth-related traits are likely a polygenic genetic architecture with no major QTL segregating. The prediction accuracy estimated by genomic prediction showed that approximately 5,000 SNP markers could achieve the highest accuracy in body weight and length in juvenile salmon within population. Genetic components associated with lice resistance The heritability of lice resistance was 0.22 to 0.33 using pedigree and genetic relationship matrices respectively. GWAS indicated that the host resistance to sea lice was likely polygenic with no individual SNP surpassed the genome-wide significance threshold. Genomic prediction showed that about 5 to 10 K SNPs was able to achieve the asymptote of accuracy in closely related animals, while the greatest advantage of genomic prediction was observed in non-sibling test within population. Conclusions As the growth-related traits and lice resistance are both likely polygenic and population-specific, the genomic prediction is an efficient approach to capture the genetic variances of the traits in selection candidates in experimental population, especially for traits with low heritability such as flesh colour and lice resistance. Family-based selection method is the better choice than mass selection to accumulate the genetic effects in corresponding SNP platform. Given the high cost of genotyping and field data collection, the genotyping-by-sequencing and genotype imputation are likely the way to make significant improvements in relevant research.

Pink salmon (Oncorynchus gorbuscha) enter seawater (SW) following gravel emergence at a body mass of 0.2 g. Two hydromineral challenges associated with this remarkable early ocean entry were investigated: (1) initial exposure to a hyper-osmotic environment and (2) sea louse (Lepeophtheirus salmonis) parasitism. To survive SW, pink salmon were hypothesized to develop hypo-osmoregulatory abilities as larval alevins prior to natural SW entry as post-larval fry. To test this, alevins and fry were transferred from freshwater (FW) in darkness to SW under a simulated natural photoperiod (SNP). Ionoregulatory status was assessed at 0, 1 and 5 days post-transfer. Alevins showed no evidence of hypo-osmoregulation, marked by a loss of water balance, a 35% increase in body [Cl-], and no change in gill Na⁺/K⁺-ATPase (NKA) activity. Conversely, fry maintained water balance and increased gill NKA activity by 50%. Fry gill NKA activity also increased by 50% following exposure to SNP in FW, providing the first evidence of photoperiod-triggered smoltification for pink salmon. A 15% increase in fry body [Na⁺] was observed as well, perhaps representing a novel mechanism for maintaining water balance during ocean entry. Physical damage to the host epidermis is a primary proximal effect of louse infection. Such damage may exacerbate existing hydromineral flux in SW. To test this, ionoregulatory status was measured in pink salmon of varying size with and without attached-stage lice. In laboratory-infected fish (~1 wk SW; 0.2-0.4 g), body [Na⁺] increased by 12% when infected with 1 chalimus IV louse, and by 23% with 2-3 chalimus III lice. Mortality was 6%. In wild-infected fish (~4-12 wks SW; 0.5-1.5 g), body [Na⁺] did not differ from controls. Combining data sets revealed a “no effect” fish size threshold of 0.5 g for 1 chalimus IV louse. This threshold is partly due to increasing hypo-osmoregulatory ability. Pink salmon thus appear to possess a novel hypo-osmoregulatory strategy where ion balance is sacrificed to maintain water balance prior to maximum ion excretion capacity. Out-migrating fish are particularly vulnerable to sea louse parasitism at this time, and as such, BC fish farms have relocated to minimize interactions during this critical period.

Pink salmon (Oncorhynchus gorbuscha Walbaum) stocks in the Broughton Archipelago BC have seen a general decline in recent years. This is thought to be due to parasitism by sea lice, Lepeophtheirus salmonis (Krøyer, 1837), on pink salmon during early marine life stage. To investigate this, I measured swimming performance, an integrated measure of fish health, and post-swim body ion concentrations, a secondary stress response, of control and sea lice infected juvenile pink salmon (mass < 3.0 g). Using five different protocols (ranging in duration from 8 – 112 min), four constant acceleration tests (rates between 0.005 - 0.053 cm s₋²) and a repeated critical swim speed test, it was found that the final swimming speed of juvenile pink salmon (mass <5.0 g) at baseline was independent of the swim protocol (P > 0.05). Given this finding, estimates of swim performance in juvenile pink salmon can be accurately measured with an acceleration test lasting < 10 minutes. Using a repeated, constant acceleration (0.05 cm s₋²) protocol, the effects of sea lice on swimming performance and post-swim body ion concentrations were measured in artificially infected river-caught (RC-fish, mean body mass 0.3 + 0.05 g) and ocean-caught infected (OC-fish, mean body mass 1.1 + 0.1 g) juvenile pink salmon. Infection levels ranged in intensity (1 - 4 sea lice per fish) and development stage (chalimus 1 - adult). Swimming performance of RC-fish was not affected by lice intensity (P>0.05) but was affected by lice stage with swimming performance decreasing at chalimus 3 stage (-20.4%) and even further at more advanced sea lice stages (chalimus 4, -26.5%; motile, -37.9%). Sea lice parasitism had no significant effect on the swimming performance of larger OC-fish when compared to control. The absence of an additive effect on swimming performance of 1 to 3+ sea lice per fish suggests drag forces induced by the ectoparasite was not a major factor. In contrast, post-swim body Na⁺ and Cl- concentrations were typically elevated in infected compared to control RC-fish (P < 0.05), but not OC-fish (P > 0.05).

The study investigated biological and ecological parameters controlling and influencing the production and distribution of the free-swimming larval stages of Lepeophtheirus salmonis (Kroyer, 1838), and to a lesser extent Caligus elongatus Nordmann 1832, in the natural environment The reproductive output of L. salmonis was influenced by seasonal effects. The number of eggs produced per brood showed an inverse relationship with increasing temperature. The number of eggs per brood was also influenced by adult female body size (cephalothorax length), which in itself exhibited an inverse relationship with increasing temperature. Photoperiod had no significant effect upon the number of eggs produced or on adult female size. Mean egg size of L. salmonis varied significantly over the year; larger eggs were produced during the summer months and smaller eggs over the winter. However, factors controlling the size of the eggs were not elucidated. The proportion of viable eggs per L. salmonis ovisac remained constant throughout the year. Large variations in egg number per egg string were found in both L. salmonis and C elongatus populations sampled at one point in time. These were attributed in part to phenotypic variation in adult female size and also the number of broods individual females had produced. Egg viability was not correlated with brood size, but mean egg size was related to the number of eggs per brood. Experimental studies indicated that hatching and development of L. salmonis was highly variable. The percentage of eggs hatched and the time period over which hatching occurred varied markedly, even when held under constant and optimal environmental conditions. Temperature did not affect hatching success or viability of the nauplius I stage, although at higher temperatures the period over which hatching occurred was reduced. Low and medium salinities caused a significant decrease in both hatching success and nauplius viability. Photoperiod had no effect on initiation of hatching. Hatching occurred in a manner similar to that observed in free-living copepods. The nauplii were enclosed by two egg membranes, the outer one bursting within the ovisac, the inner one after the ovisac membrane has split. Swelling of the egg and its subsequent hatching was attributed to osmotic effects, with water being taken up from the external environment. Development was also highly dependent upon both temperature and salinity. At 5'C, nauplius 11 stages failed to enter the moult to the copepodid stage. At 7.5'C, although moulting was initiated, in a large proportion of cases it was not successfully completed. At I O'C, development to the copepodid stage was successful. Nauplii only developed successfully to the copepodid stage at salinities of 25%o or greater. Copepodids raised under optimal conditions then exposed to a range of salinities had a greater salinity tolerance than nauplii. Biochemical analysis of the eggs of L. salmonis revealed that lipids constituted a large proportion of their dry weight. Naupliar stages contained a discrete area containing lipid which decreased in size over time, suggesting that the free-swimming larval stages utilised this as an energy reserve. Rate of depletion was faster in nauplii held at higher temperatures. Longevity, activity and infectivity of the infective stage decreased with age. However, both spontaneous and stimulus dependent activity ceased many hours before death and both activity and longevity were affected by temperature. Infectivity of I day old L. salmonis copepodids was higher than 7 day old larvae, and was considered to be related to the size of the energy reserves. The settlement and distribution pattern of copepodids did not change with age of copepodid, the majority being recorded from the fins. All three L. salmonis free-swimming larval stages demonstrated a "hop and sink" swimming pattern. The velocity and duration of both passive sinking and active swimming was recorded for both nauplii and copepodids. Although greater periods of time were spent passively sinking, the speeds obtained during both upward spontaneous and stimulated swimming meant that a net upward movement of larvae in the water column occurred. At higher temperatures spontaneous swimming activity increased, whilst low salinities caused a cessation of such ability. L. salmonis larvae were positively phototactic and negatively geotactic. As well as their positive responses to light intensity, the nauplius 11 and copepodid stages reacted positively to blue-green spectral wavelengths. Moulting times were relatively short, although the larvae were not able to swim during such periods. No relationship was found between the level of lipid reserves and the overall buoyancy of the larvae. Naupliar stages of both L. salmonis and C. elongalus were obtained from the water column as a result of a plankton sampling programme at a commercial Atlantic salmon farm. No copepodid stages of either species were found. There was no difference in the vertical distribution of the two L. salmonis naupliar stages. Live larvae tended to aggregate between 0 and 5m in depth, with no diurnal vertical migration. Dead nauplii, and those with low lipid reserves, were found deeper in the water column. Naupliar stages, and in particular the first larval stage, were concentrated in number within cages indicating that the cages have a retentive characteristic. A novel control method in the form of a commercially available light lure was tested. Though increasing the numbers of free-living copepods captured, it had no effect on the numbers of L. salmonis naupliar or copepodid stages obtained in plankton samples. The present study has therefore provided valuable data concerning the biology and ecology of the free-swimming larval stages of sea lice, in what was a comparatively poorly understood area.

The use of carotenoid pigments as an archive of feeding behaviour and thus as environmental markers was tested using eggs and juveniles of sea trout and brown trout (Salmo trutta L.) invertebrates from the River Don and ectoparasitic lice (Lepeophtheirus salmonis Kroyer) of Atlantic salmon (Salmo salar L.). Carotenoids were analysed by normal phase high performance liquid chromatography (HPLC) and the resulting peaks examined in multivariate analyses. Analysis of trout eggs of known parentage suggested that the pigment profile of each egg reflects the migratory history of the maternal fish and it is also representative of the entire egg batch. With the same method, eggs of unknown or disputed parentage could be identified as those of sea trout, brown trout or salmon. Diagnostic properties permitting identification were high amounts of astaxanthin in sea trout eggs; while the presence of lutein, zeaxanthin and a greater number of carotenoid peaks were indicative of brown trout eggs. In hatchery experiments it was established that the diagnostic maternal pigment fingerprint is identifiable for some 1300°d post fertilisation. In this time the majority of carotenoids are metabolised to astaxanthin esters and exogenous feeding will have begun. Muscle tissue carotenoids of juvenile trout in the River Don clearly separated 0+ trout from 1+ fish. The former arrange in a single discrete cluster on the basis of pigmentation, suggesting a common diet throughout the river catchment. Older parr show a specialist diet typical to each region but different from fry, indicating a shift in diet acquisition throughout early life stages. Pigment analysis of stream-living invertebrates and one terrestrial invertebrate revealed that all, except the terrestrial ear-wig, Rhabdiopteryx sp., Gammarus sp. and Leuctra sp., provide a homogeneous pigment profile. In contrast, Gammarus sp. was found to be the supplier of the greatest relative amount of astaxanthin, lutein and zeaxanthin. The diagnostic potential of carotenoids in sea lice was explored in samples taken from wild and farm Atlantic salmon fed on an artificial diet. Astaxanthin and canthaxanthin, which are pigments diagnostic of a natural and synthetic diet respectively, were detected. The ratio of canthaxanthin-like pigment to astaxanthin (C:A ratio) was 45:1 in farm lice and 8:1 in wild lice. Carotenoid content therefore could potentially be used as a tracer of origin of sea lice in epidemiological investigations.

Caligid copepods, also called sea lice, are ectoparasites of marine fish, with Lepeophtheirus salmonis (Krøyer, 1837) emerging as a problem for mariculture of Atlantic salmon (Salmo salar Linnaeus, 1758) in the northern hemisphere. Annual costs of sea lice to global salmon farming was estimated to be in excess of €300 million in 2006, with the majority of this accounted for through expenses accrued from chemical treatments. Only a limited range of anti-sea louse drugs are available and licensed for the treatment of fish, and the continued use of only a few compounds creates a situation potentially favouring the development of drug resistance. Emamectin benzoate (EMB) is currently used as a salmon delousing agent, being employed as a 0.2 % in-feed pre-mix (SLICE®). Atlantic salmon farmers have reported increased incidence of reduced L. salmonis sensitivity to SLICE®, which has highlighted the requirement for further research into the molecular mechanisms controlling salmon louse resistance to EMB. Genomic and transcriptomic research concerning L. salmonis drug resistance mechanisms has not often been reported, with previous transcriptomic studies using candidate gene approaches and genetic studies focussing on population genetics. Drug resistance in ecdysozoan invertebrates is associated with a variety of molecular mechanisms including target site mutations and changes in the expression of components in drug detoxification pathways. The research reported in this thesis was aimed at the exploration of mechanisms employed by L. salmonis to reduce the toxicity of EMB exposure, following a transcriptomic approach that utilised custom oligonucleotide (oligo) microarrays and a genetic approach that utilised Restriction-site associated DNA sequencing (RAD-seq) to identify Single Nucleotide Polymorphism (SNP) markers. An EMB-resistant (PT) and drug-susceptible (S) L. salmonis laboratory-maintained strain were to be used as a model for this research, as these two strains differ in EMB susceptibility (~ 7-fold) and show stable susceptibility profiles through multiple generations, suggesting that this drug resistance phenotype may be a heritable trait. Sequence resources available for salmon lice are limited as an annotated L. salmonis genome is currently under construction. Therefore, a significant amount of this study involved creating new resources to facilitate the analysis of EMB susceptibility. Suppression subtractive hybridisation (SSH) was used to enrich for transcripts that were differentially expressed between strains PT and S, which provided sufficient target sequence for the development of 15K oligo microarrays when combined with sequences assembled from existing L. salmonis ESTs. Additionally, transcripts were generated through sequencing a pooled sample representing key developmental stages of the L. salmonis life cycle, which were later used in the construction of a 44K oligo microarray. The toxicity of EMB and other avermectins (AVMs) against ecdysozoan invertebrates is reported to be based mainly on their interaction with ligand-gated ion channels (LGIC), specifically glutamate-gated chloride channels (GluCl). However, -aminobutyric acid (GABA)-gated chloride channels (GABA-Cls) are also believed to be targeted by AVMs and neuronal acetylcholine receptors (nAChRs) can be allosterically modulated by the AVM compound ivermectin. Transcriptional responses in PT and S salmon lice were investigated using custom 15K L. salmonis oligo microarrays. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains. GABA-Cl and nAChR subunits showed significantly lower transcript levels in PT compared to S lice, which was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR using RT-qPCR, suggesting their involvement in AVM toxicity in caligids. Although, salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. RAD-seq analysis of both genders from L. salmonis strains S and PT identified 15 RAD-markers that show complete association with salmon louse strain, although these preliminary results will need further analysis to confirm marker association with reduced EMB susceptibility. Additionally, RAD marker Lsa101901 showed complete association with sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay, this SNP association pattern was further confirmed for three unrelated salmon louse strains. Marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. In conclusion, the identification of decreased transcript abundances for LGIC subunits in EMB-resistant salmon lice, and polymorphic SNP markers showing complete association with L. salmonis strains S or PT, provides suitable candidates for further investigation into their association with reduced EMB susceptibility. Further analysis will also be required to confirm whether EMB-induced mechanisms are not associated with reduced EMB susceptibility in L. salmonis. Additionally, the identification of sex-linked SNP Lsa101901 suggests that sex determination in the salmon louse is genetic and follows a female heterozygous system, with marker Lsa101901 providing a tool to determine the genetic sex of salmon lice. Improved knowledge of L. salmonis biology and the mechanisms potentially involved in EMB resistance, obtained during this study, may provide molecular markers that contribute to successful monitoring and management of this commercially important parasite of Atlantic salmon.

Efficient control of sea lice is a major challenge for the sustainable production of farmed Atlantic salmon (Salmo salar (Linnaeus, 1758)). These marine ectoparasites feed on mucus, skin and blood of their hosts, thereby reducing the salmon’s growth rate and overall health. In the northern hemisphere, the most prevalent species is Lepeophtheirus salmonis (Krøyer, 1837). In 2006, global costs of sea lice infections are estimated to have exceeded €300 million, with the majority spent on a limited number of chemical delousing agents. Emamectin benzoate (EMB; SLICE®), an avermectin, has been widely used since its introduction in 2000, due to its convenient administration as an in-feed medication and its high efficacy against all parasitic stages of L. salmonis. However, over-reliance on a single or limited range of medicines favours the emergence of drug resistance and, as a result, the efficacy of this compound in treating L. salmonis has decreased in recent years, as reported from e.g. Chile, Norway, Scotland and Canada. Declining efficacy underlines the need for an improved understanding of the molecular mechanisms underlying EMB drug resistance in L. salmonis. Elucidation of these mechanisms would allow for improved monitoring tools, earlier detection of developing resistance, extended usability of current delousing agents and development of new parasiticides. The work described in this thesis sets out to examine the molecular mechanisms underlying EMB resistance in L. salmonis. In earlier studies, research in nematodes and arthropods has linked drug efflux transporters belonging to the family of ATP-binding cassette (ABC) transporters to ivermectin (IVM) resistance, a parasiticide with high chemical similarity to EMB. ABC transporters such as permeability glycoprotein (P-gp), transport a wide range of substrates, including drugs, and have been suggested to provide a potential molecular mechanism through which EMB resistance might be mediated in sea lice. As an example of such mechanisms, increased expression of P-gp is one of the causative factors for drug resistance in human cancer cells and avermectin resistance in nematode parasites such as Caenorhabditis elegans or Haemonchus contortus. Initial research involved screening for novel salmon lice P-gps that might contribute to EMB resistance. A novel P-gp, SL-PGY1, was discovered using a combined bioinformatic and molecular biological approach. The expression was compared in two well-characterised L. salmonis strains differing in their susceptibility to EMB (S = susceptible, R = resistant). Prior to EMB exposure, mRNA levels did not differ from each other, while, after 24 h exposure, a 2.9-fold increase in SL-PGY1 mRNA expression was observed in the R strain. SL-PGY1 appears not to be a major factor contributing to reduced EMB susceptibility, although it could play a role, as expression levels increased upon exposure to EMB. A further four additional drug transporters (ABC C subfamily) were also discovered showing high homology to multidrug-resistance proteins (MRP). The relative expression levels of each MRP was compared in the strains S and R, before and after exposure to EMB. No significant changes were found in their expression patterns. If ABC drug transporters mediate the efflux of EMB and thereby reduce the intracellular concentrations of the drug in exposed animals, the inhibition of those ABC drug transporters was expected to lead to higher intracellular levels of EMB. This could result in an enhanced toxic effect when EMB is co-administered with an inhibitor. Two known inhibitors of human P-gps and MRPs, cyclosporin A (CSA) and verapamil (VER), were co-administered with EMB. CSA increased the toxic effect of EMB in both tested strains, implying that the targets of CSA are expressed at comparable levels and that they may be part of the mechanism conferring EMB resistance. VER increased the toxic effect of EMB in the R strain, but had no significant effects on the S strain. This implies that the expression of factors inhibited by VER differs between the two L. salmonis strains. It is hypothesised that a number of ABC transporters with distinct, yet overlapping patterns of inhibitor specificity are affected by those inhibitors. The search for drug-resistance conferring genes was complemented with a systematic, genome-wide survey of ABC transporters in L. salmonis to find additional members of this important gene family. Next-generation high-throughput RNA sequencing (RNA-seq) was employed to assemble a reference transcriptome from pooled total RNA of salmon lice at different development stages. The transcriptome was assembled against the L. salmonis genome and annotated. Thirty-nine putative ABC transporters were found. Of further interest were transcripts of the subfamily B, C and G, as they contain drug-transporting ABC proteins. For the ABC B subfamily, one full (SL-PGY1) and three half transporter transcripts were found. Only full transporters are known to transport drugs and SL-PGY1 is apparently not a major factor contributing to EMB resistance. Fourteen ABCC sequences were found – 11 MRPs and 3 homologues to sulfonylurea receptors. Of interest are MRPs, as they contribute to drug detoxification in humans and invertebrates. Four MRPs had been identified previously and their expression ratios did not differ between S and R strain parasites. Seven sequences belonging to ABCG subfamily were found. However, none of the L. salmonis ABCG transcripts identified showed sufficient homology to known drug transporters in other species. With the currently limited understanding of the mechanisms conferring EMB resistance, monitoring the susceptibility of L. salmonis subpopulations is essential. Dose-response bioassays are currently widely used. Tests with pre-adult II or adult parasites requires relatively large numbers of parasites (~150) to conduct this type of bioassay, which may not always be available. Addressing this issue, we tested the feasibility of a single-dose bioassay (requiring fewer test animals than dose-response bioassays) to discriminate between L. salmonis strains with differing EMB susceptibility. This alternative approach uses time-course toxicity analysis, where the toxic effect of EMB is monitored over time. After clearly defining the effect criteria, we found that it is possible to discriminate between those L. salmonis strains. However, while requiring fewer test animals, time course toxicity analysis is more labour-intensive, but the alternative design can be suitable under certain circumstances. The work reported here has provided new knowledge concerning the mechanisms of EMB resistance in sea lice. Several novel putative drug transporters have been identified, an important first step toward unravelling the complex interactions of genes involved in EMB resistance in this commercially important parasite.

Pacific salmon (Oncorhynchus spp.) are key elements of ecological systems, and play an important role in the cultural foundation of human societies. All species of wild salmon face multiple, simultaneous threats, with habitat degradation likely playing a key role in survival. Open net-pen salmon farms can degrade important nursery marine habitat for wild juvenile salmon by disrupting natural salmonid host-parasite dynamics. The first two chapters in this thesis examine louse parasitism of wild juvenile chum (Oncorhynchus keta), pink (O. gorbuscha), and sockeye salmon (O. nerka) in relation to their marine migration past salmon farms. I compare sites of low and high exposure to salmon farms, and include two areas without farms on British Columbia’s central and north coasts to assess baseline infection levels. Louse prevalence and abundance were lowest and most similar to natural baseline levels at low exposure sites, and highest at high exposure sites in all farm regions. A significantly greater proportion of the lice infecting juvenile chum and pink salmon were Lepeophtheirus salmonis at high exposure sites. Caligus clemensi was the principal louse species infecting all juveniles in areas without salmon farms, and at low exposure sites within salmon farm regions; C. clemensi was also the dominant louse to infect juvenile sockeye that migrated past farms. Mixed-effects modelling results showed that exposure to salmon farms was the most consistent factor to explain the variation in louse infection levels, and support my hypothesis that salmon farms are a major source of sea lice on juvenile wild salmon in regions with salmon farms. I discovered that juvenile sockeye at one particular location within the Georgia Strait hosted unusually high lice levels; this location was situated at a distance from salmon farms, but near a farm salmon processing facility. Upon further investigation, I found live sea lice, Lepeophtheirus salmonis, mucus, and fish tissue in effluent discharged from the processing facility. Sea lice transmitted from this source may pose a threat to wild salmon populations, and the release of potentially untreated offal, including blood water, is of considerable concern. These results form the third chapter in my thesis. Given the challenges facing juvenile salmon in general, and sockeye from the Fraser River in particular (i.e., 2009 was the lowest return on record), and because poor habitat conditions within Georgia Strait are considered the major cause of the recent decline in Fraser River sockeye, this raises the question as to whether food limitations are a factor. The final chapter in my thesis examines the prey assemblage, diet composition, and foraging selectivity of juvenile sockeye, and investigates whether food limitations can be detected during early migration through Georgia Strait. Juvenile sockeye demonstrated high prey diversity, with preference for particular prey. Prey were more concentrated in the north, which may help explain migratory behavior of juveniles through the study region, and temporal similarities in sockeye foraging success may reflect short-term food resource stability. Moreover, I could not find evidence of food limitations that might suggest juvenile sockeye were strongly food deprived during the years of this study. Finally, my thesis explores how best to conserve salmon populations given the multitude of stressors. Because stressors often interact to produce compound effects and unpredictable results, ranking the overall threats in order of severity may not be useful. Instead, the most successful ranking system may be in terms of reducing harm where possible. For juvenile salmon during their early marine migration, risks posed by salmon farms can be more easily mitigated than the far-reaching effects on ocean productivity of climate change and ocean acidification, or predator removal. I recommend we begin here.
Graduate

The issue of sea lice (Lepeoptheirus salmonis and Caligus clemensii) transfer from salmon farms to wild salmon is a controversial topic in British Columbia (BC). A series of sea lice epizootics (four in five years) on juvenile chum (Oncorhynchus keta) and pink (0. gorbuscha) salmon in the Broughton Archipelago (BA), an area with the highest density of salmon farms on the west coast, have caused significant concern among conservationists, local First Nations, and the general public over the possible impacts of salmon farms on wild salmon. Key to the debate has been a lack of data on ambient sea lice infection rates on juvenile salmon in the absence of the influence of salmon farms. This work represents one of the first attempts to empirically examine ambient sea lice infection rates on juvenile salmonids. Objectives were to test hypotheses including: geographic variability is a significant factor in sea lice population dynamics, ambient sea lice infection rates on juvenile salmon are low, juvenile salmon susceptibility to sea lice infection, and the influence of salmon farms on ambient sea lice infection rates. Over a three year period, samples of juvenile chum and pink salmon (n=13.874) were collected using a beach seine net in the central coast of British Columbia (Klemtu/Bella iv Bella), a vast area with limited salmon farming activity. and with geography that allowed for simultaneous assessment in the same region of the natural interaction between sea lice and juvenile salmon and the influence of salmon farms on the interaction. Sampling was also conducted in other areas without salmon farms (Southern Gulf Islands: n=3847) and with salmon farms (Broughton Archipelago; n=3911). The results of the field experiments were also used in conjunction with laboratory experiments to examine the susceptibility of juvenile chum vs. pink salmon to infection by sea lice. The ambient lice infection rates for juvenile chum and pink salmon were up to 32% prevalence, less than one louse per fish and less than two lice per gram (prevalence: 2.0 (1.0) - 32.0 (19.0), mean lice per fish: 0.02 (0.01) - 0.67 (0.22), mean lice per gram: 0.56 (0.08) - 1.93 (0.13)). This result was found to be consistent across geographic areas with no salmon farming activity suggesting that geographic variability was not a significant factor in the natural interaction between sea lice and juvenile salmon. Salmon farms were found to strongly influence the relationship between sea lice and juvenile chum and pink salmon. Sea lice infection rates of juvenile salmon collected near salmon farms were significantly higher than non-salmon farming regions ranging from 3 - 150 times higher in the BA and from 2 - 14 times higher in the Klemtu region. Infection levels near salmon farms were variable in intensity from year to year. The extent to which the sea lice-salmon relationship was affected by salmon farms was dependent on farmed species, farm location, within year variability in fish size, and the scale of salmon farming activities within the region. The results from the laboratory and field studies demonstrate that juvenile chum salmon were more susceptible to infection by sea lice than juvenile pink salmon. However, the exact mechanism for the observed differences was not identified. Possible reasons for the observed differences could be related to genetically determined susceptibility, fish mucous differences, lethal lice infection tolerances. or other factors not examined. The results of this study suggest that the elevated sea lice infection rates observed in the BA and other areas present a significant risk to the health of wild salmon and that salmon farms are the most likely cause based on the biology and ecology of sea lice. In order to better understand the potential for salmon farms to affect wild salmon populations, it is suggested that investigations into farm level sea lice contributions be conducted in the BA and other areas where salmon farms operate. In addition, investigation into the lethal lice infection rates for juvenile salmon at early marine life size should also be conducted.

Systems biology methods can provide novel insight into the responses of an organism to a suboptimal environment, an infection or exposure to a xenobiotic. In the interaction of salmon and salmon lice, there are several areas requiring further research. These include the impacts of lice infection on wild salmon, response mechanisms of different salmon species or life stages to lice infections, effects of environmental conditions on lice stress, and mechanisms underlying the emergence of resistance to important parasiticidal chemicals. Here, I combine global gene expression analyses with phenotypic and physiological responses of salmon or salmon lice to further our understanding of these topics. In the first chapter, I introduce the work by discussing relevant background material on the current knowledge of salmon and salmon lice interactions, salmon immunity, the state of salmon and louse genomics and the emerging field of ecological genomics. I also discuss how these approaches are applied to the study of non-model organisms and sustainable aquaculture development and fisheries conservation. In the second chapter, I present the first large-scale transcriptome profiling of a Pacific salmon to a salmon lice infection, identifying transcript signatures associated with an infection in a sensitive life stage of pink salmon Oncorhynchus gorbuscha. In the third chapter, I present the results of multiple co-habitation infections of three species of Pacific and Atlantic salmon to compare physiological and transcriptomic responses at the local (skin) and systemic levels (anterior kidney). In the fourth chapter, I explore louse transcriptome functioning during temperature and salinity perturbations to characterize the molecular stress response and coping strategies of lice, as well as provide stressor context to response genes. In the fifth chapter, I evaluate sensitive Pacific and resistant and sensitive Atlantic lice responses to emamectin benzoate, an important compound for louse control which has recently been evaded by the louse through resistance development in multiple regions worldwide. In the sixth and final chapter, I conclude with a synthesis of what was learned about knowledge gaps discussed above and how to best apply this information by providing some approaches for future research to address remaining challenges.
Graduate
0369
0792
0718
bensutherland7@gmail.com

The objective of this thesis was to investigate the physiological changes that sea lice (Lepeophtheirus salmonis) cause in Atlantic salmon (Salmo solar). Sea lice are parasitic copepods that naturally infect salmonids in low numbers across the Northern Hemisphere with infrequent epizootics. The increase in the period of these epizootics in wild salmonids has mirrored the rise of the aquaculture industry in coastal areas. Therefore, it is important to answer basic physiological questions about parasite-host interactions such as sub-lethal lice levels required to affect performance, and the behavioural implications of increased lice infection. Two preliminary experiments were performed using rainbow trout (Oncorhynchus mykiss) to refine the techniques used to measure fish physiological variables. The first was designed to determine which anaesthetic, between clove oil and MS-222, better minimised fish stress during surgery and recovery. Both clove oil and MS-222 reduced Cortisol levels compared to non-anaesthetised controls within 10 minutes of blood sampling. However, fish exposed to MS-222 had elevated Cortisol levels 1 hour after sampling. Based on this result, few other differences occurring between the two anaesthetics, and the fact that MS-222 is a human retinotoxicant, clove oil was used for all subsequent experiments. The second preliminary experiment was performed to determine proper recovery and interval times for critical swimming speed tests (Ucrit), using hypoxia to stress fish in place office. Four hours of recovery following minor invasive surgery did not hinder the swimming performance of fish. Both 10 and 20 minute time intervals were found to be valid for measuring Ucrit, blood and cardiac variables. During exercise, cardiac output (Q) and heart rate (ƒH ) of normoxic fish increased (70 % and 25 % respectively) while those of hypoxic fish did not. A trend of a compensatory increase in stroke volume (Vs) with decreased ƒH of hypoxic fish also was evident. Hypoxia reduced the Ucrjt of trout and lactate accumulation is not one of the physiological mechanisms. Blood variables were measured in both studies, while cardiac variables and Ucrit of fish also were measured in the latter. The succeeding two experiments involved the artificial infection of Atlantic salmon with sea lice. In the first, the physiological impact of two sub-lethal levels of sea lice was measured on Atlantic salmon to determine when the health of fish is compromised. Fish with infection levels of 0.13 lice g-1 suffered a significant decrease (19 %) in Ucrit and impaired osmoregulatory ability. Infection levels of 0.05 lice g-1 did not affect the swimming performance of salmon compared to controls. The second infection study was designed to determine if the observed behaviour of heavily infected wild salmonids in seawater (SW) returning early to native freshwater (FW) streams had a physiological basis. The Ucrit of infected fish in SW decreased significantly (22 %) compared to controls, similar to the preceding study. However, salmon exposed to FW for 4 hours did not differ from controls with respect to Ucrit. Evidence of the osmoregulatory ability of infected fish in SW being compromised did not occur. It is likely that energy use increased in the latter group to maintain osmotic balance. Blood and cardiac variables, and Ucrit were measured in both studies. The final study had an experimental and theoretical component. Lice with visibly ingested blood were first collected from infected fish, then their gut contents weighed. Juvenile trout then had repeated blood samples taken of two known quantities for one week and then had their Ucrit measured. Gut contents and decline in performance due to blood loss were used to predict the possibility of blood loss due to sea lice infection decreasing the performance of their host. It was predicted that 5-10 % of the tissues consumed by lice is blood. At an infection level known to decreases Ucrit (0.1 lice g-1) lice would consume only 1 % of host blood, while 8 % loss is required to decrease Ucrit- Significant reductions in Ucrit purely due to blood loss would not occur until lethal infection levels (0.75+ lice g-1) are reached. This series of studies has helped to answer some basic physiological questions about the interaction between sea lice and salmonids. It is of great concern that sea lice can significantly affect salmonid physiology at infection levels lower than previously found on wild fish in areas of intensive aquaculture (0.5-2.1 lice g-1). These findings support the implementation of continuous lice removal programs at all aquaculture sites to reduce infection levels in intensive aquaculture areas to those found in pristine areas (0.02 lice g-1) that do not impair Atlantic salmon performance.

The close proximity of net-pen salmon farms and wild Pacific salmon stocks in British Columbia (BC) is an incentive for precautionary management of the environmentally and economically damaging parasites known as sea lice. Bivalves cultured as part of an integrated multi-trophic aquaculture (IMTA) system may contribute natural, preventative louse control through the ingestion of planktonic sea lice larvae. A field trial was conducted to test sea lice mitigation by bivalves at a commercial Atlantic salmon (Salmo salar) farm in BC using Pacific oysters (Crassostrea gigas). Oysters were cultured in trays around one end of the farm and at a reference site approximately 150 m away from August 2013 until August 2014. Parasitic and planktonic sea lice (Lepeophtheirus salmonis and Caligus clemensi) were monitored before and during oyster deployment, beginning in December 2012. Parasite abundance peaked in January 2013 (6.5 lice/fish, >85% C. clemensi), and the following year in February 2014 (3.3 lice/fish, >80% L. salmonis). Larval density within cages peaked in January, both in 2013 (1.28 larvae/m3) and 2014 (0.96 larvae/m3). Parasite abundance was significantly correlated with both surface salinity (r2= 0.28, p=0.04) and sea lice larval density (r2= 0.65, p=0.01). Observed densities were significantly lower (t=3.41, p=0.009) than those calculated for the site based on water temperature and salinity, the number of adult female lice present, and the approximate number of fish. Sea lice mitigation by oysters was assessed by comparing monthly sea lice larval densities inside bivalve and non-bivalve fish cages, and by analyzing preserved oyster digestive tracts from January 2014 (when larval densities were highest) for presence of L. salmonis DNA using PCR. Using these methods, no significant evidence of sea lice mitigation was detected. Oyster growth was monitored by measuring whole wet weight, soft tissue wet, dry, and ash-free dry weight, and shell length, width, and height approximately every four months. Oysters were sampled equally across different sides of the farm and at the reference site (~150 m away from the farm) at three depths: 1, 3, and 6 m. All seven measurements increased significantly over time. Effects of side and depth varied by growth parameter; in general, oysters at 1 and 3 m were significantly larger than those at 6 m, and oysters cultured at the reference site were either significantly smaller or the same size as those cultured around the farm. Oysters from select sides were consistently, significantly larger than those from other sides and from the reference site. Overall, the findings suggest that sea lice larvae quickly dispersed away from the farm after hatching and were not significantly impacted by bivalve presence around the fish cages. Bivalves grew significantly larger over time and size was significantly impacted by both depth and side of the fish cage. While no evidence of larval sea lice reduction/ingestion by cultured bivalves was detected, this study provides information on all sea lice stages present throughout an Atlantic salmon production cycle, as well as the first detailed growth analysis of Pacific oysters cultured alongside farmed Atlantic salmon in BC.
Graduate
0792
byrneaa@gmail.com

Parasitic copepods including Lepeophtheirus salmonis have been the focus of strong concern for the health of wild and farmed salmonids in the Pacific and Atlantic Oceans. Salmon are highly valuable species from both socioeconomic and ecological perspectives. The host-parasite dynamics of Lepeophtheirus salmonis and the Atlantic and Pacific salmonids have changed over evolutionary time to the point that both Atlantic and Pacific salmon and Atlantic and Pacific Lepeophtheirus salmonis are genetically distinct. Recent human interference with the natural population dynamics of this parasite and its hosts may have altered the population genetic structure of Lepeophtheirus salmonis, particularly because salmon farms may provide more stable conditions for parasite population growth. High abundance of Lepeophtheirus salmonis on salmon farms causes damage to the farmed salmon and leads to increased infection intensities in nearby wild hosts. Some Atlantic Lepeophtheirus salmonis have developed resistance to the anti-parasitic drugs they are repeatedly exposed to. No drug resistance has yet been detected within the Pacific Ocean, where only one drug is available, and heavily relied on, to treat Lepeophtheirus salmonis infections. Control of Lepeophtheirus salmonis abundance on Pacific salmon farms is important to maintain the health of farmed salmon and is also important to protect wild salmonids from increased infections originating from salmon farms. The goal of this thesis was to characterize and employ a large suite of molecular markers to assess the population structure of Lepeophtheirus salmonis in the Pacific Ocean. Until this point, the primary focus of Lepeophtheirus salmonis population genetics research has been limited to the Atlantic Ocean and has relied on a small number of available molecular markers. Available expressed sequence tag DNA libraries were screened to identify putative polymorphic loci, which were then experimentally evaluated. We characterized 22 novel microsatellite loci and 87 single nucleotide polymorphisms within 25 nuclear loci for Lepeophtheirus salmonis. We used these genetic markers, as well as 5 microsatellite loci previously developed for use in Atlantic Lepeophtheirus salmonis population studies, to genotype 562 Lepeophtheirus salmonis that were collected from12 Pacific Ocean sampling locations. We compared Lepeophtheirus salmonis genotypes among: (1) seven wild host populations and five farmed host populations within the Pacific Ocean; (2) geographically separated wild host populations, ranging from the Bering Sea to the southwest end of Vancouver Island, British Columbia; and (3) temporally separated cohorts of farmed Atlantic salmon from two geographically distant farm locations on the northwest coast of Vancouver Island and the Campbell River area east of central Vancouver Island. Our analyses failed to resolve significant population structure among sampled Pacific Lepeophtheirus salmonis and, therefore, supports a hypothesis of high gene flow throughout the Northeast Pacific Ocean. It is important to understand the biology and population dynamics of Lepeophtheirus salmonis because it is a consequential parasite of wild and farmed salmonids in the Pacific Ocean. Both the molecular tools developed for this study and the population genetics information generated from this study have contributed to our overall understanding of the evolutionary history and population dynamics of Lepeophtheirus salmonis.
Graduate