M.Sc. / A general introduction provides the foremost morphological characteristics of the genus. A breeding colony of Argulus japonicus was kept under laboratory conditions in order to study sperm transfer. Pairs in copula were studied with histology and scanning electron microscopy. Sections of copulating pairs revealed sperm on the accessory copulatory structures of the male; and scanning electron microscopy showed that sperm transfer occurs in three phases which can be differentiated to ten different stages. Sperm transfer occurs via a spermatophore in A. japonicus. This is the first observation of a spermatophore in Argulus. For the second part of this study, seven specimens of an unknown freshwater ectoparasitic crustacean were collected from red tilapia fish, kept for consumption at the “Langat Fishing, Seafood and Beer Garden” Restaurant just off the Langat River in Selangor, Malaysia. Initial investigation showed that the specimens were of the genus Argulus. Light and scanning electron microscopical studies were subsequently used to identify the species. A comparison with all Argulus species formerly described from Asia and the surrounding islands was conducted. The species was identified as Argulus coregoni, due to the presence of the roughly triangular shaped anterior respiratory areas and the kidney bean shaped posterior respiratory areas. Additionally, the abdomen with sharply pointed terminal ends as well as the presence of characteristic accessory protrusions on the second ii swimming leg of the male specimens confirmed this identification. This species has not previously been described from Malaysia. The final chapter of this dissertation contains an overall summative discussion of the different parts of this study and highlights future possible research avenues.

Ph.D. / The Branchiura is a group of ectoparasitic crustaceans which infect mostly fishes. The subclass contains four genera including Argulus, Chonopeltis, Dolops and Dipteropeltis. Despite their reputation as pests our understanding of their biology is meager. In the first part of the study the anatomy and physiology is described. The species studied include Argulus japonicus, Dolops ranarum and Chonopeltis australis. Live specimens of A. japonicus, D. ranarum and Chonopeltis australis were collected, fixed in Todd‟s fixative and processed for transmission electron microscopy. The results gleaned from A. japonicus and D. ranarum indicated that the ultrastructure and physiology of the cells were similar to the digestive epithelia of free-living copepods. The anterior midgut consists of Resorptive cells (R cells) and the enteral diverticula consist of two cell types including R and Fibril cells (F). The R cells contain lipid droplets and F cells contain rough endoplasmic reticulum indicating that the absorption of nutrients and digestion occurs in the anterior midgut and enteral diverticula. The lipid droplets in both species are large in size and number. Blister cells/B cells and R‟ (apostrophe) cells occur in the posterior midgut and are involved in the processing of digestive waste. The results from C. australis were different from A. japonicus and D. ranarum and reveal the possible presence of F/R cells with multiple functions of both absorption and enzyme secretion. The cells contain few lipid droplets and instead contain many hexagonal-shaped crystalline structures that are interpreted to be proteinaceous. The differences in diet are thought to be influenced by morphological differences in the feeding appendages. The second part of the study involved the supraoesophageal ganglion or brain of C. australis. This genus is unique among the Crustacea since the antennules are absent. This trait poses implications regarding the brain morphology since particular parts of the brain impart nerves that innervate certain appendages in arthropods. Specifically the deutocerebrum or midbrain innervates the antennules. A study was conducted to discover whether the antennular nerves were absent. Specimens of C. australis were fixed in AFA, sectioned and stained with Heidenhain‟s AZAN. Results showed that the antennular nerves were absent and the deutocerebrum was smaller in comparison to the other brain segments indicating degeneration. Moreover, C. australis possesses a reduced number of sensory sensilla on its external surface compared to related genera. The loss of antennules does not affect its sensory capabilities in host searching. However, the reduced numbers of sensilla and its feeble swimming capabilities are more likely to reduce its efficiency in host searching.

Ph.D. / The Branchiura is a group of ectoparasitic crustaceans which infect mostly fishes. The subclass contains four genera including Argulus, Chonopeltis, Dolops and Dipteropeltis. Despite their reputation as pests our understanding of their biology is meager. In the first part of the study the anatomy and physiology is described. The species studied include Argulus japonicus, Dolops ranarum and Chonopeltis australis. Live specimens of A. japonicus, D. ranarum and Chonopeltis australis were collected, fixed in Todd's fixative and processed for transmission electron microscopy. The results gleaned from A. japonicus and D. ranarum indicated that the ultrastructure and physiology of the cells were similar to the digestive epithelia of free-living copepods. The anterior midgut consists of Resorptive cells (R cells) and the enteral diverticula consist of two cell types including R and Fibril cells (F). The R cells contain lipid droplets and F cells contain rough endoplasmic reticulum indicating that the absorption of nutrients and digestion occurs in the anterior midgut and enteral diverticula. The lipid droplets in both species are large in size and number. Blister cells/B cells and R' (apostrophe) cells occur in the posterior midgut and are involved in the processing of digestive waste. The results from C. australis were different from A. japonicus and D. ranarum and reveal the possible presence of F/R cells with multiple functions of both absorption and enzyme secretion. The cells contain few lipid droplets and instead contain many hexagonal-shaped crystalline structures that are interpreted to be proteinaceous. The differences in diet are thought to be influenced by morphological differences in the feeding appendages. The second part of the study involved the supraoesophageal ganglion or brain of C. australis. This genus is unique among the Crustacea since the antennules are absent. This trait poses implications regarding the brain morphology since particular parts of the brain impart nerves that innervate certain appendages in arthropods. Specifically the deutocerebrum or midbrain innervates the antennules. A study was conducted to discover whether the antennular nerves were absent. Specimens of C. australis were fixed in AFA, sectioned and stained with Heidenhain's AZAN. Results showed that the antennular nerves were absent and the deutocerebrum was smaller in comparison to the other brain segments indicating degeneration. Moreover, C. australis possesses a reduced number of sensory sensilla on its external surface compared to related genera. The loss of antennules does not affect its sensory capabilities in host searching. However, the reduced numbers of sensilla and its feeble swimming capabilities are more likely to reduce its efficiency in host searching.

The farming of Atlantic salmon (Salmo salar) is challenged by parasitic infestations caused by Lepeophthreirus salmonis and Caligus elongatus. A convenient and effective way to control sea lice and treat farmed salmon is by in-feed treatments such as Slice®. A reliable, accurate and reproducible method for the determination of emamectin benzoate (EB), the active ingredient of Slice®, and its desmethylamino metabolite (DES) in sediment was developed and validated. It involved methanolic extraction, clean-up using solid-phase extraction with a strong cation exchanger, and derivatisation with trifluoroacetic acid anhydride and N-methylimidazole. Analytes were quantified following HPLC separation with fluorescence detection. The method was successfully applied to determine EB and DES in salmon flesh and skin, seawater, mussels (Mytilus edulis) and seaweed (Palmaria palmata). A laboratory study showed that EB was persistent under anaerobic conditions in two different sediments at 4 and 14 ºC. A further study also demonstrated that the growth of seaweed (P. palmata) was not affected by the presence of EB and that EB did not accumulate significantly in the seaweed. This result is encouraging in view of proposed polyculture systems involving seaweeds. Studies conducted on a working Scottish salmon farm investigated the fate of EB and DES in target and non-target matrices. For three months post-treatment, EB was detected, by mass in descending order, in the salmon flesh, skin, faeces, then mucus and sea lice with concentrations in each matrix declining steadily over the period. As EB had never been quantified in sea lice before, it was unclear whether they were a significant sink for EB in the environment, following their exposure to the medicine and dislodging from salmon after feeding. However, due to the low concentrations of EB detected in the sea lice, faeces are most probably the main route for emamectin entering the environment. Sediment collected directly below and around two active walkways, over five or six months following treatment, showed that the spatial dispersion of EB and DES was mainly limited to the area within 25 m of the cage edge, although concentrations depended on sampling location in relation to water currents. Maximum EB concentrations were recorded three months after treatment. Seven days after treatment, 6 % of the total EB input was present in the sediments within 25 m of the cage edge. Neither EB nor DES were detected in seawater, mussels, periwinkles, dogwhelks and seaweed samples collected from the walkway and the surrounding environment. This work, one of the few studies of the uptake of EB by indigenous fauna and flora of an active salmon farm, suggests that it is not significantly accumulated in matrices outwith the target organism and the sediment.

The use of wrasse (Pisces: Labridae) as cleaner fish to combat infections with the parasitic copepods Lepeophtheirus salmonis (Kroyer) and Caligus elongatus (Nordmann) (sea-lice) in the culture of Salmo salar L. (Atlantic salmon) is now common. Infections with these parasites has caused considerable losses in the industry since its formative years. The use of the wrasse species Ctenolabrus rupestris (L. ) (goldsinny), Centrolabrus exoletus (L. ) (rockcook), Symphodus melops (= Crenilabrus melops) (L. ) (corkwing) and Labrus mixtus L. (cuckoo) as cleaner fish was first suggested in 1988. The use of these species in the industry is now widespread in Scotland, Ireland and Norway. The fish used are normally caught from the wild before being stocked with S. salar smolts during their first year at sea. The fish are routinely collected from waters close to the farm sites to be stocked. As most of the S. salar sea production sites in Scotland are located on the west coast of the country, the wrasse to be used in these sites are normally collected from these waters. The movement of wild fish into farm pens presents a risk of disease transfer from wrasse to S. salar and vice versa. Prior to their use as cleaner fish, these four species of wrasse had received little attention as subjects of scientific study. As a result, there was very little information available in the literature regarding their diseases. The present study was undertaken to investigate the potential pathogens present in wild populations in Scottish coastal waters, and, in particular, which of these pathogens, if any, could be transmitted to the S. salar. The study also investigated the susceptibility of wrasse to the two major viral diseases of S. salar to which they would be exposed in pens. In order to fully assess the pathogenicity of the potential disease agents under farm conditions, it was first necessary to establish the normal morphology of the wrasse species. Hence, a study of the morphological features of wrasse, with particular emphasis on those features important in the health of the fish was undertaken. Wrasse were shown to differ in many aspects from salmonids but shared many morphological features with other perciforme fish. Major differences from salmonids were evident in the skin, fins, pancreas, intestine, gonads and heart. There were also aspects of their morphology which differed from other perciforme fish, notably the structure of the heart. These features were regarded to be adaptations to the specific demands of their feeding strategies and habitats. This study was the first of its kind undertaken for wrasse and showed some early contraindications for the use of wrasse in culture; most notable was the marked lipid accumulation in, and resultant degeneration of, the liver resulting from the consumption of high energy S. salar feeds. Once the normal morphological features were established, it was possible to examine the disease status of wrasse. Wild fish were sampled from three different locations on the west coast of Scotland. These sites were all geographically distinct and were all used as sources of wrasse for the S. salar farming industry. Samples of wrasse were also obtained from farm sites supplied with wrasse from these wild sites, and an additional number of other geographically distinct farm locations. As a comparison wrasse were also obtained from a wrasse captive breeding facility and another captive location unrelated to the S. salar industry, a public aquarium. The fish from all of these sampling sites were examined fully for the presence of parasites, bacteria and, in some cases, viruses. Histological examination was also carried out on all of the fish studied. A total of 24 new parasite host records, and two tentative ones, were recorded from the four wrasse species studied. These new parasite records included protozoa, digeneans, nematodes and crustacea. Parasite infections were found to vary in prevalence, abundance and intensity in respect to the geographical characteristics of sampling sites and also the length of time spent in S. salar pens. It was concluded that the separation of wrasse from their natural diet and habitat influenced the degree of parasitism. None of the parasites found to infect wrasse were observed to cause any significant pathology in their hosts other than localised tissue responses. The possibility of transfer of wrasse parasites to S. salar was also investigated experimentally in a series of infections in which parasites dissected from wrasse were introduced to S. salar smolts by means of a novel gavage method. None of the parasites used established in the S. salar, indicating that there is little risk of transfaunation of parasites between wrasse and S. salar. However, this aspect requires further work due to the low number of parasites available and the subsequent low numbers of S. salar infected. Bacterial isolates were obtained from wrasse held in S. salar pens but were not found in any of the fish collected from the wild. Most of the bacterial strains isolated would normally be considered as opportunistic pathogens of fish. It was concluded that the relatively high levels of stress, both environmental and physical, that wrasse are subjected to under farm conditions were instrumental in the number of bacterial infections seen in wrasse. Only one pathogenic bacterial infection was seen in any of the fish sampled. This was an isolate of Aeromonas salmonicida, the agent known to cause the disease furunculosis, isolated from a wrasse obtained from one of the farm samples. Other authors have reported that this bacterium has already caused substantial losses of wrasse under farm conditions. It was concluded that Aeromonas salmonicida will prove to be a major pathogen of wrasse held in S. salar pens. No viruses wereI isolated from any of the wrasse studied. The susceptibility of wrasse to the most significant pathogens of S. salar under farm conditions was also subjected to investigation. In addition to sea-lice infection, the industry lists Infectious Pancreatic Necrosis (IPN) and Pancreas Disease (PD) as of primary importance for further research. Both of these diseases cause substantial losses in the industry. The susceptibility of wrasse to both of these disease conditions was investigated by means of experimental infections. In the case of IPN wrasse were infected by bathing with two different infective doses, a low dose which would be expected to induce the disease in S. salar parr and a second dose substantially higher than the first. The C. rupestris used were found to be susceptible to IPN. The wrasse developed some of the pathological characteristics typical of the disease in S. salar, however, other pathological signs were peculiar to wrasse. The recovery rate from the disease seen in wrasse was far more rapid than that recorded from S. salar. Shedding of the virus in the faeces of infected C. rupestris was also demonstrated. This study has illustrated for the first time the susceptibility of wrasse to IPN and that they can shed the virus in their faeces. This suggests that infected wrasse could be a source of continual reinfection in an affected sea site. Experimental infections of C. rupestris with PD followed a standard protocol for the reproduction of the disease in S. salar. Infection was by means of intraperitoneal injection with putatively infective material obtained from S. salar affected with PD. Two infection doses were used, the lowest dose used had been proven to be effective in inducing the disease in S. salar parr while the second dose, ten times higher than the first, had been shown to be effective in reproducing PD in S. salar smolts. The C. rupestris infected did not develop any of the typical signs of the disease seen in S. salar. It was, therefore, concluded that wrasse were not susceptible to PD.