Thesis (M.S.) in Marine Biology--University of Maine, 2002. / Includes vita. Includes bibliographical references (leaves 48-51).

No description available.

This study has been carried out to investigate the biological and environmental parameters influencing the settlement and post-settlement survival of the infective stages of Lepeophtheirus salmonis Kroyer 1837. The abiotic factors investigated were temperature and salinity. Temperature was found to have a significant effect on the settlement success of the copepodids with an inverse relationship between temperature and settlement. Survival of the louse at 10 days post infection showed a decrease at the reduced temperature. Temperature was also shown to have a direct relationship on lice development; higher seawater temperatures resulted in faster development. Regression analysis of temperature and settlement shows a significant correlation. A constant reduced salinity, 24%, resulted in a reduced ability of the copepodid to infect its host compared with 34%. Post-settlement survival in 24%, at approximately 13°C resulted in 5.8% survival of lice to day 10 post-infection compared to 79% in 34% salinity. When this experiment was repeated but with elevated seawater temperatures of up to 18°C, survival at the reduced salinity was found to be 75.3%, higher than the ambient control group. The developmental rate at day 10 post-infection of L.salmonis larva at 24% was shown to be slower than development at 34%o. Distribution of the L.salmonis copepodid on its host showed the highest settlement on the gills and on the fins, particularly the pectoral and dorsal fins. Examination of L.salmonis survival at day 10 post-infection indicated the highest losses on the gills and the pelvic, caudal and dorsal fins. Settlement on the pectoral fins showed the highest settlement and the greatest survival. The infective copepodid has a reduced ability to infect its host after 7 days following the moult from nauplius 2, compared to copepodids aged 1 and 3 days following the nauplius 2 moult. For copepodids of all ages, once settlement had been achieved, survival at 10 days post-infection was approximately 50% in all groups. Copepodids of all ages did not show any difference in the development rate at 10 days post infection. Highest settlement was found to be on the gills and pectoral and dorsal fins. The effects of varying dose rates of copepodids, has shown that a finite percentage of lice settle and survive the first five days post-infection. Settlement distribution was found to be highest on the body, gills and pectoral and dorsal fins. In serial infections of fish there was a reduced settlement count with second infections, possibly through intraspecific competition. Experiments using different host stocking densities showed that with an increased number of hosts the intensity of the infection of individual fish was reduced. Smaller fish appear more susceptible to settlement of L.salmonis than larger fish, and this is associated with the relatively greater fin area of those fish compared to larger fish. L.salmonis exhibits a preference for the fins as an area of settlement in all sizes of fish. Comparison of copepodid settlement on salmon and sea trout showed that in single populations of fish salmon had the highest intensities of infection whilst in mixed populations of fish sea trout had a higher intensity. Settlement distribution of L.salmonis on salmon showed greatest settlement on the body, pectoral and dorsal fins, whilst on sea trout settlement was highest on the body, pectoral, pelvic, caudal and dorsal fins. The comparative development of L.salmonis between the two species of host fish showed an increased rate of development on salmon. The calculated energy levels for L.salmonis larval stages show a decrease in available energy within each developmental stage. After 6 days from the nauplius 2 moult the copepodid starts to show a sharp decline in energy levels which coincides with the reduced ability of the copepodid to infect the host. Post-settlement energy levels remain constant even though the copepodid is actively feeding, as seen by SEM examination at 2 days postinfection. The principal lipid class found within L.salmonis larval stages as energy reserve is triacylgylcerol (37.6% of the total lipid). A preliminary epidemiological model for sea lice population dynamics is proposed. This is a differential equation compartmental model that has been designed to examine the flow of L.salmonis developmental stages on the host. The model was able to predict the timing of the maximum number of pre-adult 1 lice stages to within one day. The difference between the observed data and the model output is probably due to the considerable variability in the parameters used in the model construction.

Atlantic salmon, SaI1M salar. L., have been shown to be more susceptible to infections by the caligid copepod Lepeophtheirus salmonis (Kreyer, 1837) than other salmonid species. Atlantic salmon exhibit a reduced cellular immune response to the attached parasite which has led to the hypothesis of the presence of sea louse associated compounds that depressed the fish's normally efficient inflammatory mechanisms. The aim of the current study was to test this hypothesis. A biochemically defmed in vitro culture system was developed that would allow collection of the secreted/excreted products of the copepodid larvae of L. salmonis, and avoid their contamination by metabolites of the host from their collection, in vivo. Available tissue culture methods proved inadequate in supporting copepodid culture because of the louse requirement for a seawater maintenance medium that was osmotically unsuitable for cultured cells. Tissue engineering technologies developed in the construction of human living skin equivalents were successful in the development of an Atlantic Salmon Skin Equivalent (ASSE). ASSE is a novel organotypic tissue culture substrate that was constructed from Atlantic salmon fibroblasts (AS-6) and primary cultures of Atlantic salmon epithelial cells. Cells were supported in a matrix of collagen fibres, acid extracted from the tails of rats, and combined using a layering technique to create a substitute salmon skin. ASSE has a fibroblastic dermal equivalent overlaid by an epidermis-like layer and a layer of collagen, and was maintainable in a seawater media. During its development, the cells within ASSE showed signs of differentiation that included stratification, increased fibronectin production by cultured fibroblasts, and the formation of a basement membrane-like layer at the junction of the dermis and epidermis. In this environment, ASSE allowed the survival of copepodid larvae for an average of 12 days, a period approximately 5 days longer than that of their free-swimming counterparts. Furthermore, cultured copepodids were observed feeding, exhibiting the normal range of settlement behaviours, and also showed increases in their length. However, metamorphosis to the chalimus I stage did not occur and was not stimulated by the supplementation of ASSE with salmon mucus, salmon peptone or DL methionine. Nevertheless, copepodid maintenance on ASSE allowed the collection of the compounds they released into the culture media. These were assayed for their effects on the immunological functioning of salmon macrophages. These assays demonstrated that the chemotaxic ability of macrophages was significantly reduced following treatment with louse culture supernatants . (LCS), as was their phagocytic ability. In both assays, the activity of each function was approximately 50% lower than that recorded in untreated cells. Intracellular respiratory burst and the phagocytic index of active phagocytes, however, was not affected. The biological activity of LCS was inhibited following heating, dilution, and treatment with proteinase K, indicating that the active immunosuppressive compounds were either themselves proteins, or required protein to be effective. The active substance was designated Louse Immunomodulatory Factor (LIF). The enzyme profiles of culture supernatants were investigated using API ZYM test strips. The profiles of LCS supernatants were significantly different to those of control supernatants from the early stages of louse incubation with ASSE. LCS supernatants showed elevated levels of leucine aminopeptidase, C4 and C8 esterases, alkaline phosphatase, P-glucuronidase, and Nacetyl- j-glucosaminidase. The involvement of these enzymes in the digestive processes of arthropods is well documented. However, the presence of the chitin hydrolysing enzyme Nacetyl- Beglucosaminidase, may also be suggestive of the preparation of copepodids for moulting. This, and the involvement of these enzymes in digestion in copepodids, and as possible immunomodulatory compounds is discussed. Gel filtration chromatography identified 14 proteins in the LCS that were not present in the control supernatants. These proteins were in the molecular weight range <1 kOa to 2665 kOa. No biological activity was attributed to these proteins when isolated by chromatography and assayed for their effects on macrophage chemotaxis. This lack of activity may be associated with their dilution during the chromatography process in which the supernatants were diluted approximately 5000 times. Experimental evidence showed that the activity of LIF was absent when supernatants were diluted to 1:1000, and so methods of concentration may be required in order to establish the immunological activity of these proteins. This study has developed an Atlantic salmon skin substitute that could have a great number of applications in the study of salmonid metabolism, cellular communication, immunology, and drug and chemical testing. Here, it supported the extended maintenance of sea lice larvae in vitro and allowed the collection of the products of their culture. The findings have shown that the copepodid stage of L. salmonis produces substances, LIF, that depress the chemotaxic and phagocytic activity of salmon macrophages, in vitro. These active substances may also be responsible for the depression of the inflammatory responses of sea lice infected Atlantic salmon, in vivo.

No description available.

The efficacy of a range of anti-parasitic chemotherapeutic agents against the salmon louse Lepeophtheirus salmonis following topical application was studied in vitro and in vivo. In general, adult and preadult lice were susceptible to a wide range of compounds with dose rates, following 1 hour exposures, ranging several orders of magnitude (10.0 - 0.001 mg/L). Overall the pyrethroid compounds which were tested were found to have the widest therapeutic ratios, indicating the potential of this group of chemotherapeutants for sea lice control. Resistance to the organophosphorus (OP) compounds dichlorvos and azamethiphos was detected in isolated populations of lice. Field trials with azamethiphos indicated that the compound was highly efficacious against sensitive lice (@ 0.1 mg/L; however, where resistance was present, efficacy (@ 0.2 mg/L) was highly variable. When used at the above dose rates, azamethiphos was found to be well tolerated by fish as indicated by a lack of significant brain acetylcholinesterase inhibition. Results on cross resistance (between pyrethroids and OPs) were inconclusive which was believed to be, primarily, due to the overall high toxicity of the group; but also to the variable responses from exposed lice. In a series of preliminary trials, one of the pyrethroid compounds, PHRDL-D, was found to effectively remove lice when administered orally to infected salmon, indicating the potential of pyrethroids as oral chemotherapeutants. A comparison of the relative toxicity of azamethiphos (OP), resmethrin (pyrethroid), ivermectin (avermectin) and the structurally similar compound SKB7 (milbemycin), indicated that chalimus stages were only susceptible to ivermectin and SKB7 following topical and intra-peritoneal injection to lice infected fish. In contrast, azamethiphos and resmethrin were found to be non toxic to chalimus larvae at dose rates which were highly toxic to both adult lice and treated fish. Preliminary studies on the uptake of r4C]azamethiphos in adult lice indicated that uptake was both concentration and time dependant, reaching a plateau at the onset of toxicity. Uptake appeared to be primarily associated with frontal plates, 1st antennae and anus. The fmdings indicated that several compounds/compound classes are highly active against lice and, given the limited number of compounds available for sea lice control and the development of resistance to OPs, might be considered as alternatives. In light of these findings, the potential of chemotherapy for the future control of sea lice is discussed.

A study has been carried out to investigate some aspects of the feeding and digestion of the parasitic copepod Lepeophtheirus salmonis (Kreyer, 1837), a serious pathogen of wild and farmed marine salmonids. The alimentary canal consists of a cuticularised foregut and hindgut and a midgut, the latter comprising most of the length of the alimentary canal. It consisted of an anterior diverticulum, and anterior midgut, mid midgut and posterior midgut. All the midgut is lined in the luminal side with a monolayer of digestive epithelium. The latter is supported by a porous basal membrane. Mesenteries suspend the gut in the haemocoel and attach to the basal membrane by means of extensions of their cytoplasm that interlock with the pores of the basal membrane and are intimately associated with the cellular basal membrane of the enterocytes. Four types of cells could be recognised ultrastructurally and were named according a widely accepted nomenclature for the types of digestive epithelial cells found in decapod crustaceans. R-cells were the most abundant type and were believed to be involved in the absorption of nutrients, storage of lipids and probably excretion of waste material. Once the R-cells have finished their useful life, it is proposed that they finally die after passing through a stage, called A-cell stage, comparable to apoptosis or programmed cell death, after which the A-cell is finally sloughed off the epithelium and discarded in the faeces. F-cells constituted the second cell type, in charge probably of the secretion of digestive enzymes. Once this stage is finished, F-cells are believed to transform into the third type of cells, the B-cells, which engage in intracellular digestion of nutrients. At the end of their useful life they are sloughed off the epithelium. The last cell type, the E-cells, are believed to be embryonic cells that differentiate either into R- or F-cells. A revised mode of feeding is proposed, in which the louse scoops up strips of host skin epithelium of relatively constant size and shape, by means of the structures associated with the mouth cone. Analysis of the gut contents and the faecal pellets revealed that the main food item was host skin epithelium. The feeding activity usually reaches the dermal layer, suggested by the common occurrence of host melanin in the gut and in the faecal pellets. Two types of faecal pellets were found. The first type was a cylindrical one which was made of the ingested host's epithelial cells with little signs of having being digested. Cylindrical pellets could be clear or dark depending on the amount of host melanin present in them. The second type of pellet was a tape- or ribbon-like gelatinous pellet produced mainly when the lice were not eating (as during starvation) or sometimes associated to the end of the cylindrical pellets. Pellets were always surrounded by a delicate peritrophic envelope composed of several peritrophic membranes. Blood was believed to be another important food item. Blood feeding was commonly associated with the production of lipids in the R-cells and it is proposed (but not confirmed) that this alternative food item could be associated with some physiological process that requires lipogenesis, like vitellogenesis. A method for manually feeding the lice was devised. Salmon blood and milk were fed in this way. No sign of digestion of these food items was observed suggesting that possibly an endogenous factor different from the presence of food in the gut is necessary to trigger the digestive process. An analysis of type of gut contents, peritrophic membranes and cell type occurrences during a digestive period was carried out. The results suggested that some digestive processes take place preferentially in one or another gut region. Mid and posterior midgut are probably associated with the production of faecal pellets, production of digestive enzymes, intracellular digestion and excretion of waste materials judging by the higher occurrence of peritrophic membranes, B- and A-cells in these regions. Absorption of nutrients probably takes place throughout the midgut. The morphological changes in the topography shown by the midgut epithelium suggest that it is a very active and plastic tissue. The results of this study were discussed and compared with similar studies of other crustaceans, including other parasitic and free living copepod species. Suggestions were made of the implications of the present findings in the search for a control method for this parasite.

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.

The sea louse (Lepeophtheirus salmonis), whose larvae are planktonic and disseminated in the water column, is an economically important parasite of Atlantic salmon (Salmo Salar). The effect of temperature (5, 10, 15°C), diet (larvae alone, larvae plus phytoplankton), and bivalve size (small, medium, large) on the amount of L. salmonis larvae ingested by various species of filter-feeding bivalves (Pacific oysters, Pacific scallops, blue/Gallo’s mussel hybrids, basket cockles) was examined in a series of laboratory experiments. Four separate temperature/diet experiments were conducted (one for each species) in which large bivalves were individually placed in 2-L containers holding 750 ml of aerated, filtered seawater and fed one of three treatment diets: (1) phytoplankton: ~7.1 x 104 cells ml-1 of Isochrysis sp. (Tahitian strain, TISO); (2) sea lice larvae: ~431 larvae (mostly nauplii); and (3) phytoplankton and larvae (at the levels mentioned above). There was also a control treatment of phytoplankton and larvae, but no bivalve. After feeding for 1 h, the bivalve soft tissues were excised and preserved, the digestive system was dissected, and sea lice larvae were removed and counted to provide direct evidence of ingestion. The larvae remaining free swimming in the container were iv preserved and counted. The proportion missing from the container was used to estimate ingested larvae in statistical analyses. Two additional experiments investigating the effect of bivalve size (small, medium, large) on the ingestion of sea lice larvae were conducted with Pacific oysters and Pacific scallops. The heights for oysters (anterior-posterior axes) were 19.2, 44.2, and 84.0 mm, and scallops (dorsal hinges to ventral margins) were 40.3, 64.1, 102.7 mm. The methodology for the size experiments was as previously described for the temperature/diet experiments with the following changes: (1) the diet of larvae alone was not used; (2) the mean number of larvae in each container was ~498; (3) the mean concentration of TISO added to each container was ~7.8 x104 cells ml-1, and (4) the mean water temperature was 10.4°C. The data for the four temperature/diet experiments indicate that all four bivalve species ingested sea lice larvae, whether their diet included phytoplankton or not, and that temperature had no significant effect. The data for the two size experiments indicated that all three sizes of oysters and scallops ingested sea lice larvae and that there was a significant size effect. Large shellfish consumed a significantly greater proportion of the sea lice larvae than the small shellfish. Bivalves grown at salmon net pens as part of an IMTA (Integrated Multi-Trophic Aquaculture) system may be able to reduce the number of sea lice larvae as well as being an additional crop of market value. Future research, conducted at a commercial scale at a salmon farm, is warranted in order to determine if bivalves can serve in this role. / Graduate

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