British Columbia salmon aquaculture operations use the chemotherapeutant emamectin benzoate (EMB trade name SLICE®), a synthesized avermectin compound, delivered through feed to decrease sea lice (Lepeophtheirus salmonis) parasite abundance on production fish. Avermectins bind to ion channels in crustaceans and disrupts nerve impulse transmission. Detectable amounts of EMB can accumulate in the depositional area around farms during SLICE® treatment periods, thus presenting potential for exposure to populations of proximate non-target species. The distribution of spot prawn (Pandalus platyceros), an economically important crustacean, overlaps with areas of intensive salmon farm activity. The primary objective of this research was to determine if EMB exposure had a measurable biological effect on spot prawns in the field and in the laboratory. The field component was conducted in the Broughton Archipelago, BC, to determine if emamectin benzoate residues could be detected near actively treating salmon farms, and whether farm proximity affected spot prawn size distribution. Three laboratory experiments tested the mortality, molting and behavioural response of spot prawns to SLICE® feed pellet exposure and acute exposure to EMB through sediment over ten, 30 and 45-day durations. Measurable amounts of EMB was detected in the marine sediment near five farm sites during the field survey and was found to persist between treatment periods. Male and transitional stage spot prawns captured near farm sites attained a greater size and had better body condition compared to reference sites, indicating prawns may benefit from direct or indirect farm food subsidies. However, at several farm sites the size distribution of prawns changed over the sampling period, a trend not observed at reference sites, demonstrating that farm activity may alter prawn population dynamics. Laboratory results indicated that only prawns that had been starved prior to exposure would initially consume SLICE® pellets, but feeding rates declined with subsequent exposures. Depressed consumption rates was not a residual effect of EMB, but rather an aversion to the SLICE® pellet diet as prawns resumed feeding when offered a preferred diet. Sediment EMB exposures to doses 808 µg kg-1 and greater increased prawn mortality, largely due to the inability of molting individuals to successfully complete ecdysis. Exposed individuals accumulated EMB in their abdomen tissue with levels increasing with exposure dose. Prawns exposed to EMB through sediment at concentrations 1419 and 3330 µg kg-1 displayed a significant reduction in olfactory detection and orientation behaviours to food stimuli. This research highlights that spot prawns may avoid SLICE® pellets for preferential food sources, and that only short term EMB exposure 50 to 200 magnitude greater than levels present in the marine environment elicited a measurable response in spot prawn mortality rates, molting success and behaviour. However, preliminary trends in the field survey data indicate that there may be population differences occurring in spot prawns inhabiting areas near treating salmon farms that are not observed in reference populations. These results signify the inherent pitfalls in current management policy that base decisions on short-term acute toxicity laboratory exposure results that may not be indicative of the response of marine populations near active salmon farms to long-term chronic EMB exposure. / Graduate / 0792 / 0329 / 0768

<p>The German cockroach (<em>Blattella germanica</em>) is one of the most critical urban pests globally due to the health risks, such as asthma, it imposes on people. Insecticides are known to manage their large population sizes, but the rapid rate at which cockroaches develop resistance is a continuing problem. This can be expensive and time-consuming for both the consumer and the pest management professional (PMP) applying the treatment. Each cockroach population is unique because different strains have different resistance profiles, so resistance profiles must be considered. This thesis addressed this little-studied issue in a controlled laboratory setting. Cockroach strains from Indianapolis, Indiana, Danville, Illinois, and Baltimore, Maryland, were used. Fifteen insecticide active ingredients most used by consumers and PMPs were selected for testing in vial bioassays to establish resistance profiles. No choice and choice feeding assays with four currently registered bait products were performed to assess the impacts of competing food and circadian rhythms on bait resistance levels. Selected population growth characteristics were compared in virgin females and nymphs in each cockroach strain to determine if certain population traits were associated with insecticide resistance. The results indicate that emamectin benzoate is the most effective active ingredient in causing the highest mortality in all strains in vial bioassays. No choice assays confirmed vial assay results the best, with Optigard (emamectin benzoate) being the most effective bait in all strains. The time a female carries its egg case and takes for a nymph to become an adult was significantly different across all strains, suggesting possible fitness costs for higher-level multi-resistance. The results acquired from these studies can help develop rapid tests to use in the field based on the no choice feeding assay while also adding more information supporting current resistance and cross-resistance evolution theories.</p>

Many studies have investigated the impacts of marine cage fish farming on seabed sediments. Most of these studies have focused on organic loading or toxic chemicals used for the treatment of disease, normally for a single or a small number of sites over short time periods. Only very rarely has there been the opportunity to use large data sets consisting of a large number of fish farm sites over a long time scale. In Scotland, localised nutrient impacts have been well documented for marine cage salmon farms, but mixed effects of nutrient and chemicals such as SLICE (the active ingredient of which is emamectin benzoate) have not been investigated in the long term. The aim of this project was to investigate the ecological impacts on sediments from farming activities using very large spatial and temporal data to investigate the long term effects of nutrient and chemical waste. This was achieved using a metadata set collected from 403 sampling stations at 31 fish farms on the west coast of Scotland over a 9 year period. Data consisted of sediment macrofauna, carbon and nitrogen levels, redox potential, particle size for sediment characterisation and sediment concentrations of SLICE. The data was analysed for trends using statistical and multivariate analysis to look for changes in sediment community and related conditions, and the relationships between these parameters were investigated. At sampling stations that were less than 50 metres from the sea cages, 72% of the macrofauna communities were correlated with regard to their species composition and abundance. A significant relationship between the concentration of SLICE and sediment characteristics was represented as: SLICE= 0.000644*(median size particle size) + 0.0311*(C %) – 0.00213*(redox potential) + 1.453. Annelids were the most sensitive to the presence of emamectin benzoate, with the sipunculid Phascolion strombi, the echinoderm Ophiura affinis, and the custaceans Iphinoe, Diastylis and Iphimedia also showing sensitivity. During the data period, there was a clear change in species composition associated with improved seabed conditions. This correlated with biomass changes at the relevant sites, where there was a consequent decrease in nutrient input and SLICE usage. The statistical comparison of the AMBI and ITI indices indicated a 68.9% correlation, but they differed in their ability to indicate levels of organic disturbance. AMBI was shown to correlate more closely with conditions and thus a more reliable index when working with large databases. Univariate and multivariate analysis indicated that a combination of abundance (N), Shannon Wiener (H’) and AMBI, as biological indices for describing the status of the ecological level associated with the carbon percentage and redox potential of sediments gave the most reliable representation of environmental change over a series of sampling stations. In conclusion, the overall results suggest that, in the long-term, sampling stations which contained significant levels of SLICE had a higher impact status than those affected only by nutrient inputs. The accuracy of multiple regression models were increased by adding biotic and abiotic parameters, though fish biomass at the sites were not considered be as important factor for the prediction of impacts. However, this model could be sensitive to natural environmental conditions and variations. In light of these results and conclusions, recommendations can be made both for updating the existed environmental regulation of marine fish farms and in the development of meaningful models to relate sediment conditions to accurate estimations of overall environmental impacts.

O benzoato de emamectina, um antiparasitário da classe das avermectinas, é amplamente utilizado em diversos países para o tratamento de infecções causadas por helmintos intestinais e ectoparasitas, os quais estão relacionados a grandes prejuízos na produção aquícola. Apesar de sua eficácia ser conhecida na aquicultura, seu uso não está regulamentado no Brasil. Diante da demanda nacional por uma maior variedade de medicamentos veterinários regulamentados para uso em espécies aquícolas, o presente projeto visou estimar um período de carência para o antiparasitário benzoato de emamectina, a partir de um estudo experimental de depleção residual desse fármaco em tilápia (filé com pele em proporção natural), a espécie de maior importância comercial para a produção aquícola no Brasil. Para tanto, fez-se necessárias as seguintes etapas: (i) incorporar o benzoato de emamectina na ração através da técnica de revestimento polimérico em equipamento de leito fluidizado; (ii) desenvolver e validar métodos analíticos para a determinação do benzoato de emamectina na ração e em filé de tilápia empregando cromatografia líquida de alta eficiência acoplada à espectrometria de massas (LC- MS/MS); e (iii) realizar um ensaio com tilápias para avaliar a depleção residual de emamectina B1a no filé. O revestimento da ração com 1,5% de etilcelulose permitiu incorporar o benzoato de emaectina na ração sem alterar a estrutura física da mesma, o que torna o processo vantajoso, visto que a ração mantém suas características nutricionais e de flutuabilidade. Além disso, o processo foi homogêneo (CV de 2,1%) e não apresentou taxa de lixiviação quantificável do fármaco para a água, contribuindo para uma maior segurança em relação à dose de tratamento a ser administrada e em relação ao risco potencial ao meio ambiente. A emamectina B1a foi extraída da ração por processo sólido-líquido e do filé de tilápia pelo método QuEChERS modificado. Os métodos foram avaliados mediante os seguintes parâmetros: seletividade, curva analítica, linearidade, precisão, exatidão, limite de detecção (LD) e de quantificação (LQ). Todos os parâmetros avaliados se encontraram conformes às recomendações dos guias de validação tomados como referências, qualificando os métodos como adequados para os objetivos propostos no presente trabalho. Para avaliar a depleção de emamectina B1a no filé de tilápia, os peixes receberam o fármaco via ração em dose de 50 ?g/kgPV/dia durante sete dias consecutivos. As curvas de depleção residual de emamectina B1a se ajustaram ao modelo exponencial de primeira ordem. Tomando como referência o limite máximo de resíduo de 100 ?g/kg recomendado pelo Codex Alimentarius, não houve a necessidade de se propor um período de carência mínimo para o abate dos peixes, considerando as condições experimentais empregadas. / Emamectin benzoate, an antiparasitic of the class of avermectins, it is widely used in several countries for the treatment of infections caused by intestinal helminths and ectoparasites, which are related to great losses in aquaculture production. Although its known effectiveness for aquaculture, its use is not regulated in Brazil. The national demand for a greater variety of regulated veterinary drugs for use in aquaculture species,the current project that aimed to evaluate a withdrawal period for the antiparasitic emamectin benzoate, based on an experimental study of residual depletion of this drug in tilapia ( fillet with skin in natural proportion) the species of major commercial importance for aquaculture production in Brazil. To do so, it was necessary to: (i) incorporate emamectin benzoate into the feed through the polymer coating technique in fluidized bed equipment; (ii) to develop and validate the analytical methods for the determination of emamectin benzoate in the feed and in tilapia fillet using high-performance liquid chromatography coupled to mass spectrometry (LC-MS / MS) and (iii) perform a tilapia test to evaluate the depletion of emamectin B1a in fillet. The feed coating with 1.5% ethylcellulose allowed to incorporate the emaectin benzoate into the feed without altering the physical structure, which makes the process advantageous, since the feed retains its nutritional and buoyancy characteristics. In addition, the process was homogeneous (CV 2,1%) and did not present quantifiable leaching rate of the drug into the water, contributing to a greater safety in relation to the dose of treatment to be administered and in relation to potential risk to the environment. Emamectin B1a was extracted from the feed by solid-liquid process and from tilapia fillet by the modified QuEChERS method. The methods were evaluated using the following parameters: selectivity, analytical curve, linearity, precision, accuracy, limit of detection (LD) and quantification (LQ). All the evaluated parameters were in accordance with the recommendations of the validation guides taken as references, qualifying the methods as adequate for the objectives proposed in the present work. To evaluate the depletion of emamectin B1a tilapia fillet, the fish received the drug via feed at a dose of 50 ?g / kgPV / day for seven consecutive days. The residual depletion curves of emamectin B1a were fitted to the first order exponential model. Taking into account the maximum residue limit of 100 ?g / kg recommended by the Codex Alimentarius, there was no need to propose a minimum withdrawal period for the slaughtering of fish, considering the experimental conditions used.

Species of Argulus are macro-, ecto-parasites known to infect a wide variety of fish, but in the UK mainly cause problems in rainbow (Oncorhynchus mykiss) and brown trout (Salmo trutta). Argulus foliaceus is estimated to have caused problems in over 25% of stillwater trout fisheries in the UK. While A. foliaceus does not usually cause high levels of mortality, the parasite affects fish welfare, and also makes fish harder to catch due to morbidity and reduced appetite. This can cause severe economic problems for the fishery, resulting in reduced angler attendance due to poor capture rates and the reduced aesthetic appearance of fish; in the worst-case scenario this can result in the closure of the fishery. Current methods of control include chemical treatment with chemotherapeutant emamectin benzoate (Slice), physical intervention with egg-laying boards which are removed periodically and cleaned in order to reduce the number of parasites hatching into the environment, and the complete draining and liming of the lake to remove all free-living and egg stages of the parasite. While these treatments have all been shown to reduce parasite numbers, none are known to have resulted in permament eradication of the parasite. There is evidence to suggest that A. foliaceus will eventually develop resistance to Slice - the only currently available chemical treatment against the infection - and egg-laying boards and the draining and liming of the lake are both time- and labour-intensive. Previous studies have shown that slow fish turnover is a risk factor with respect to A. foliaceus infections, and with a wide variety of stocking practices occurring in the UK one of the first aims of this project was to determine their impact on the host-parasite dynamics. Mathematical models provide a cost-effective way of examining the impact of such practices, and after a literature review (chapter one), in chapter two a three-compartment mathematical model was adapted for use in the A. foliaceus-trout system. Four generalised stocking methods were then incorporated and analysed, and a minimum threshold host density was found to be necessary to sustain the parasite. Including a function which reduced the capture rate as the parasite burden increased allowed the parasite to survive at a lower host density, as susceptible fish were removed from the water at a slower rate, and attached parasites also remained in the water for longer. This resulted in hysteresis in the model, as the invasion threshold for the parasite remained the same, but once established the parasite became harder to eradicate, requiring significant reductions in the host density. In chapter three the model was further developed in order to improve its biological real- ism. Several features were added and these included: natural host mortalities, a separate compartment for the parasite egg population, and parasite survival after the natural or parasite-induced mortality of its host. In chapter four seasonality was added by incorporating temperature-dependent egg-laying rates and an over-wintering period during which the parasite was unable to reproduce. The model was then fit to the available data, and estimates for the rate of parasite-induced host mortalities and the parasite’s rate of attachment to a host were found. In chapter five we returned to stocking methods, this time looking at the frequency and timing of stocking events and the impact of imposing a rod limit (whereby anglers are only permitted to capture four fish per visit); it was concluded that while current guidelines suggest that very frequent trickle stocking is recommended when dealing with Argulus spp. infections, monthly stocking does not appear to worsen the infection, and if the fish capture rate is high then less-frequent stocking may also be permissable - particularly if stocking occurs towards the end of the year when the parasite is no longer active. This practice may, however, be detrimental to the fishery due to low fish densities in the summer months. In chapter six treatment with Slice was included in the model, and it was demonstrated that with constant treatment, and in the absence of reservoir hosts and a withdrawal period from the drug prior to stocking treated fish into the fishery, the parasite was eradicated. Under current veterinary cascade guidelines, however, trout are required to undergo a withdrawal period of 500 degree days prior to being made available for human consumption. When this was included in the model the drug still decreased parasite abundance, but did not eradicate it - this is in agreement with results reported by communications with fishery managers currently treating fish with Slice. A reduction in the withdrawal period of 25% was shown to further decrease parasite abundance, but still did not result in parasite extinction. As constant treatment with Slice is not advisable due to the potential for resistance build-up, we then sought to find time at which to apply a single treatment of Slice, and found that this was in August when the temperature was highest and the parasite was reproducing and attaching to hosts quickly. Egg-laying boards were also incorporated into the model and similarly to findings by Fenton et al. [11] the success of this treatment was mostly dependent on the proportion of eggs being laid on the boards (as opposed to natural substrates). In contrast with the A. coregoni system, however, the boards would have to be cleaned and replaced more frequently that once per year, as several cohorts of A. foliaceus emerge during a single year.

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.