<p>Koi and goldfish have been released into rivers in South Africa since the 1800&rsquo / s for food and sport fish and have since spread extensively. These fish are present in most of the river systems in South Africa and pose an additional threat the indigenous cyprinids in the Western Cape. Monogenean parasites of the genus Gyrodactylus are of particular concern, as their unique biology renders them a possible threat. Gyrodactylus kherulensis and G. kobayashii were identified from koi and goldfish respectively imported from Asia, Europe and locally bred fish. Morphometrics and the use of statistical classifiers, which includes univariate (ANOVA and Kruskal-Wallis), bivariate (Pearson&rsquo / s correlation) and multivariate (Principal Component Analysis) placed the two species within their respective groups. There was some intraspecific variation among the different populations collected from the various locations, especially in the hamulus and ventral bar features, but the marginal hooklets, however, remained static for both helminth species.</p>

<p>Koi and goldfish have been released into rivers in South Africa since the 1800&rsquo / s for food and sport fish and have since spread extensively. These fish are present in most of the river systems in South Africa and pose an additional threat the indigenous cyprinids in the Western Cape. Monogenean parasites of the genus Gyrodactylus are of particular concern, as their unique biology renders them a possible threat. Gyrodactylus kherulensis and G. kobayashii were identified from koi and goldfish respectively imported from Asia, Europe and locally bred fish. Morphometrics and the use of statistical classifiers, which includes univariate (ANOVA and Kruskal-Wallis), bivariate (Pearson&rsquo / s correlation) and multivariate (Principal Component Analysis) placed the two species within their respective groups. There was some intraspecific variation among the different populations collected from the various locations, especially in the hamulus and ventral bar features, but the marginal hooklets, however, remained static for both helminth species.</p>

Magister Scientiae (Biodiversity and Conservation Biology) / The expansion of the South African aquaculture industry coupled with the lack of effective parasite management strategies may potentially have negative effects on both the freshwater biodiversity and economics of the aquaculture sector. Koi and goldfish are notorious for the propagation of parasites worldwide, some of which have already infected indigenous fish in South Africa. Koi and goldfish have been released into rivers in South Africa since the 1800’s for food and sport fish and have since spread extensively. These fish are present in most of the river systems in South Africa and pose an additional threat the indigenous cyprinids in the Western Cape. Monogenean parasites of the genus Gyrodactylus are of particular concern, as their unique biology renders them a possible threat. Gyrodactylus kherulensis and G. kobayashii were identified from koi and goldfish respectively imported from Asia, Europe and locally bred fish. Morphometrics and the use of statistical classifiers, which includes univariate (ANOVA and Kruskal-Wallis), bivariate (Pearson’s correlation) and multivariate (Principal Component Analysis) placed the two species within their respective groups. There was some intraspecific variation among the different populations collected from the various locations, especially in the hamulus and ventral bar features, but the marginal hooklets, however, remained static for both helminth species. This illustrates again the importance of the minor variations in the marginal hook features in gyrodactylid taxonomy. Infection trials conducted by co-habitation of infected koi and goldfish with two indigenous redfin minnow species, Pseudobarbus burchelli and P. phlegethon showed that both G. kherulensis and G. kobayashii could successfully transfer and establish themselves on P. phlegethon, where the infection increased rapidly initially, but remained relatively constant thereafter. P. burchelli appeared to be inherently resistant as the parasite population growth rate initially remained steady, until the infection died off. The wild-caught indigenous fish were however not infected with any exotic Gyrodactylus species, but a new species, G. burchelli n. sp. described from the body surfaces of P. burchelli. / South Africa

The ectoparasitic monogenean fluke, Gyrodactylus salaris, is a parasite known to be highly pathogenic to Atlantic salmon (Salmo salar). Although present in the environment of several neighbouring European countries, the UK is thought to be G. salaris-free, but, if national contingency plans to control this parasite are to be effective, it is vital that we understand the factors underlying its transmission from host to host. This study demonstrates that the majority of parasites transferring to new hosts are mature parasites that have reproduced at least once. Since, exploration and host transfer strategies pose a risk to survival; the parasite will endeavour to pass on its genes before attempting to transfer from one host to another. This study has also shown that when pregnant parasites are forced to leave their hosts, their offspring are aborted prematurely to ensure the survival of the mature parasite. Gyrodactylids do not possess a free-swimming stage in their life cycle, which allows for their migration between hosts. In spite of this, they are able to rapidly colonise naïve hosts, even in non-shoaling populations of fish. This study investigates the transmission strategies employed by detached parasites in the colonisation of new hosts. Observations of gyrodactylids collected from 3-spine sticklebacks, Gasterosteus acuelatus, suggest that their activity increases as a stickleback approaches, alerting the host to its presence. The parasite is then ingested directly by the prospective host. A time series of experimental exposures and specimens prepared for Scanning Electron Microscopy (SEM) suggest that once ingested, the parasites attach to the lining of the buccal cavity and then migrate out to their preferred colonisation site on the outer surface of the fish. It is proposed that this may be an alternative route for host infection. Similarly, direct ingestion by the scavenging on infected hosts by 3-spine sticklebacks suggests another route of infection of new hosts. Although these routes of transmission may be of lesser significance, infections in the buccal cavity may be an important indicator for detection of infection and those personnel involved in screening fish for gyrodactylids should be aware that this is an area in which infections can occur. This study also demonstrated that the use of the anaesthetic 2-phenoxyethanol does not affect the number of gyrodactylids which leave the host to colonise a new host. Additionally, observations of the transmission process suggest that turbulence produced by the movement of the fish’s fins may facilitate the transfer of detached parasites from the substrate. While this hypothesis appears to be supported by video evidence and photographic stills gathered throughout the duration of this study, further work should be conducted using particle tracking techniques to determine the efficacy of using a vortex effect as a means of colonising new hosts. Field sampling processes may have an effect on this type of research, giving rise to problems with the accurate diagnosis, management and control of gyrodactylids in a variety of fish. Gyrodactylus infected specimens of 3-spine stickleback (Gasterosteus aculeatus L.), minnows (Phoxinus phoxinus L.) and stone loach (Barbatula barbatula L.) from one Scottish river were cohabited. The study found that small numbers of Gyrodactylus do transfer to atypical hosts. This study highlights that personnel involved in fish disease surveillance programmes should be aware of the consequences of transporting multiple species in the same transport vessel as gyrodactylids may infect species previously thought to be resistant. Equally, diagnosticians should be aware of the fact that atypical species may act as temporary hosts and that their gyrodactylid fauna should not be assumed. Non-feeding life-cycle stages, such as the dispersal stages of parasites, are dependant for survival upon finite energy reserves gathered during feeding phases. Thus, those individuals with more limited reserves will die sooner and consequently have less time available to find a new host once detached. At this stage, the principal energy reserves in gyrodactylids are stored as large lipids droplets. Confocal laser scanning microscopy (CLSM) has been used to investigate the distribution of lipid droplets in Gyrodactylus, which have migrated off their fish host, testing the hypothesis that these droplets function as a proxy for the nutritional state. This study, demonstrated that the lipid droplets were particularly associated with the gut and that there is a significant variability in the volume of stored lipid carried out by each individual. Transmission Electron Microscopy (TEM) showed that gyrodactylids carry lipid droplets at all stages of their life cycle, including at release from the birth pore. It is likely that transferring worms require stored energy reserves to survive in the event of failure to establish contact with a new host. These reserves could allow the parasite to survive without a host for several days. As gyrodactylids appear to respond to a range of stimuli including vibration and chemicals released from the host, the presence or absence of such cues may have consequences on the rates of Gyrodactylus transmission. If these chemical stimuli can be identified and then mimicked or blocked, then this may offer potential opportunities for the control of gyrodactylid behaviour and for disrupting their transmission to new hosts. Baseline gyrodactylid behaviour, in the absence of a host, was determined under white light and infrared. This was achieved using a specially constructed arena and purpose written image analysis software to analyse parasite movement under different lighting conditions. The study found that gyrodactylids were more active in the dark than in light conditions, typically displaying longer, more sinuous tracks under red light than under white light. To begin investigating the effect of chemical presence on gyrodactylid behaviour, the activity of octopaminergic agonists and antagonist which bind to muscle receptors and alter muscle activity, were assessed. The impact of octopamine hydrochloride, clonidine hydrochloride, amitraz and, a toxic reference, chlordimeform, over a range of concentrations (0.2 to 3.2µM/L) were assessed on gyrodactylid behaviour. All of the four chemicals affected Gyrodactylus and produced muscle tetanus, causing muscle spasms when extension was attempted. Prolonged exposure resulted in death. Only the highest concentration of chlordimeform, the toxic reference, affected 100% of Gyrodactylus after 24 hours. After 48 hours, all of the Gyrodactylus treated with chlordimeform were either affected, moribund or dead. Amitraz was more toxic than chlordimeform with 80% of Gyrodactylus being dead after 24 hours at the highest concentration. After 48 hours 100% of Gyrodactylus exposed to 3.2 µm/L amitraz were dead, and up to 80% were dead in those exposed to lower concentrations; with no parasites being left unaffected. Although these particular compounds are toxic to fish, the effect of these agonistic chemicals on Gyrodactylus behaviour and survival is interesting and suggests that a closely related compound that is safe for use against fish may offer a potential treatment for the control of G. salaris infections in rivers. An ultrastructure study was undertaken to contribute to the current understanding of gyrodactylid ultrastructure. The findings of this research require broad understanding of gyrodactylid behaviour for their interpretation. Photographic evidence was gathered using transmission and electron microscopy. From these results, it is clear that Gyrodactylus gasterostei on a three-spine stickleback host will respond to a range of stimuli (i.e. vibration or chemical cues released from the host) in their assessment of host suitability.

This thesis presents an investigation into Gyrodactylus species recognition, making use of machine learning classification and feature selection techniques, and explores image feature extraction to demonstrate proof of concept for an envisaged rapid, consistent and secure initial identification of pathogens by field workers and non-expert users. The design of the proposed cognitively inspired framework is able to provide confident discrimination recognition from its non-pathogenic congeners, which is sought in order to assist diagnostics during periods of a suspected outbreak. Accurate identification of pathogens is a key to their control in an aquaculture context and the monogenean worm genus Gyrodactylus provides an ideal test-bed for the selected techniques. In the proposed algorithm, the concept of classification using a single model is extended to include more than one model. In classifying multiple species of Gyrodactylus, experiments using 557 specimens of nine different species, two classifiers and three feature sets were performed. To combine these models, an ensemble based majority voting approach has been adopted. Experimental results with a database of Gyrodactylus species show the superior performance of the ensemble system. Comparison with single classification approaches indicates that the proposed framework produces a marked improvement in classification performance. The second contribution of this thesis is the exploration of image processing techniques. Active Shape Model (ASM) and Complex Network methods are applied to images of the attachment hooks of several species of Gyrodactylus to classify each species according to their true species type. ASM is used to provide landmark points to segment the contour of the image, while the Complex Network model is used to extract the information from the contour of an image. The current system aims to confidently classify species, which is notifiable pathogen of Atlantic salmon, to their true class with high degree of accuracy. Finally, some concluding remarks are made along with proposal for future work.

Of the 430+ extant species of Gyrodactylus, ectoparasitic monogenetic flukes of aquatic vertebrates, Gyrodactylus salaris Malmberg, 1957 is arguably the most well-known. Following the introduction of this species into Norway in the 1970s with consignments of infected Atlantic salmon smolts, Salmo salar L., this species has had a devastating impact on the Norwegian Atlantic salmon population, decimating wild stocks in over 40 rivers. Gyrodactylus salaris is the only OIE (Office International des Epizooties) listed parasitic pathogen of fish and has been reported from 19 countries across Europe, though many of these records require confirmation. The UK, Ireland and some selected watersheds in Finland are currently recognised as G. salaris-free states; however, the threat that this notifiable parasite poses to the salmon industry in the UK and Ireland is of national concern. Current British contingency plans are based on the assumption that if G. salaris were to be introduced, the parasite would follow similar dynamics to those on salmonid stocks from across Scandinavia, i.e. that Atlantic strains of Atlantic salmon would be highly susceptible to infection, with mortalities resulting; that brown trout, Salmo trutta fario L., would be resistant and would lose their infection in a relatively short period of time; and that grayling, Thymallus thymallus (L.), would also be resistant to infection, but would carry parasites, at a low level, for up to 143 days. Two of the objectives of this study were to confirm the current distribution of G. salaris across Europe, and then, to investigate the relative susceptibility of British salmonids to G. salaris, to determine whether they would follow a similar pattern of infection to their Scandinavian counterparts or whether, given their isolation since the last glaciation and potential genetic differences, they would exhibit different responses. It has been almost six years since the distribution of G. salaris across Europe was last evaluated. Some of the European states identified as being G. salaris-positive, however, are ascribed this status based on misidentifications, on partial data resulting from either morphological or molecular tests, or according to records that have not been revisited. Additional Gyrodactylus material from selected salmonids was obtained from several countries to contribute to current understanding regarding the distribution of G. salaris across Europe. From the work conducted in the study, G. salaris is reported from Italy for the first time, alongside three other species, and appears to occur extensively throughout the central region without causing significant mortalities to their rainbow trout, Oncorhynchus mykiss (Walbaum), hosts. The analysis of archive material from G. salaris-positive farms would suggest that G. salaris has been in the country for at least 12 years. Material obtained from rainbow trout from Finland and Germany was confirmed as G. salaris supporting existing data for these countries. No specimens of G. salaris, however, were found in the additional Gyrodactylus material obtained from Portuguese and Spanish rainbow trout, only Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999, a morphologically similar species was found. Gyrodactylus salaris is now reported from 23 out of ~50 recognised states throughout Europe, only 17 of these however, have been confirmed by either morphology or by an appropriate molecular test, and only ten of these records have been confirmed by a combination of both methods. To assess the susceptibility of English and Welsh salmonids to G. salaris, a number of salmonid stocks of wild origin, were flown to the Norwegian Veterinary Institute (NVI) in Oslo, where they were experimentally challenged with G. salaris. Atlantic salmon from the Welsh River Dee, S. trutta fario from the English River Tyne and T. thymallus from the English River Nidd, raised from wild stock in government hatcheries, were flown out and subsequently challenged with G. salaris haplotype A. After acclimation, each fish was infected with ~50–70 G. salaris and marked, so that parasite numbers on individual fish could be followed. The dynamics on individual fish were followed against a control (Lierelva Atlantic salmon). The experiment found that the number of G. salaris on S. salar from the River Dee continued to rise exponentially to a mean intensity (m.i.) of ~3851 G. salaris fish-1 (day 40 post-infection). These salmon were highly susceptible, more so than the Norwegian salmon control (m.i. ~1989 G. salaris fish-1 d40 post-infection) and were unable to regulate parasite numbers. The S. trutta fario and T. thymallus populations, although initially susceptible, were able to control and reduce parasite burdens after 12 (m.i. ~146 G. salaris fish-1) and 19 (m.i. ~253 G. salaris fish-1) days, respectively when peak infections were seen. Although the latter two hosts were able to limit their G. salaris numbers, both hosts carried infections for up to 110 days (i.e. when the experiment was terminated). The ability of S. trutta fario and T. thymallus to carry an infection for long periods increases the window of exposure and the potential transfer of G. salaris to other susceptible hosts. The potential role that brown trout may play in the transmission and spread of G. salaris in the event of an outbreak, needs to be considered carefully, as well as the interpretation of the term “resistant” which is commonly used when referring to brown trout’s susceptibility to G. salaris. The current British surveillance programmes for G. salaris are focused on the screening of Atlantic salmon and on the monitoring of the rainbow trout movements. The findings from this study demonstrate that G. salaris can persist on brown trout for long periods, and suggest that brown trout sites which overlap with Atlantic salmon or rainbow trout sites are also included within surveillance programmes and that the role that brown trout could play in disseminating infections needs to be factored into contingency/management plans. Throughout the course of the study, a number of parasite samples were sent to the Aquatic Parasitology Laboratory at Stirling for evaluation. Some of these samples represented Gyrodactylus material that were associated with fish mortalities, but the species of Gyrodactylus responsible appeared to be new to science. A further aspect of this study was, therefore, to investigate these Gyrodactylus related mortalities in aquaculture stock and to describe the species found in each case, which may represent emerging pathogens. The two new species, Gyrodactylus orecchiae Paladini, Cable, Fioravanti, Faria, Di Cave et Shinn, 2009 and Gyrodactylus longipes Paladini, Hansen, Fioravanti et Shinn, 2011 on farmed gilthead seabream, Sparus aurata L., were collected from several Mediterranean farms. The finding of G. orecchiae in Albania and Croatia was associated with 2–10% mortality of juvenile stock and represents the first species of Gyrodactylus to be formally described from S. aurata. Subsequently, G. longipes was found in Bosnia-Herzegovina and Italy, and at the Italian farm site, it occurred as a mixed infection with G. orecchiae, but these infections did not appear to result in any loss of stock. Unconfirmed farm reports from this latter site, however, suggest that a 5–10% mortality of juvenile S. aurata was also caused by an infection of Gyrodactylus, which is suspected to be G. longipes. Additional samples of Gyrodactylus from a gilthead seabream farm located in the north of France have been morphologically identified as G. longipes, extending the geographical distribution of this potentially pathogenic species to three countries and three different coasts. In addition to these samples, some specimens of Gyrodactylus from a Mexican population of rainbow trout were sent for evaluation.

Gyrodactylus salaris Malmberg, 1957, is a notifiable freshwater ecto-parasite that infects both wild and farmed populations of Atlantic salmon (Salmo salar, L.). It has caused catastrophic damage to wild salmon stocks in Norway since its accidental introduction in 1975, reducing salmon density in some rivers by 98% over a period of five years. It is estimated that G. salaris has cost the Norwegian salmon industry more than 500 million EUR. Currently the UK has G. salaris free status under EU law, however, it is believed that if G. salaris emerged in the UK the impact would be similar to that witnessed in Norway. The aim of this thesis is to develop mathematical models that describe the salmon-G. salaris system in order to gain a greater understanding of the possible long-term impact the parasite may have on wild populations of Atlantic salmon in G. salaris-free territories such as the UK. Mathematical models, including deterministic, Leslie matrix and individual based models, were used to investigate the impact of G. salaris on Atlantic salmon at the individual and population level. It is known that the Atlantic strain of Atlantic salmon, examples of which occur naturally in Norway and the UK, does not have any resistance to G. salaris infections and the parasite population is able to quickly grow to epidemic levels. In contrast, the Baltic strain of Atlantic salmon, examples of which occur naturally in Sweden and Russia, exhibits some form of resistance and the parasite is unable to persist. Thus, baseline models were extended to include immunity to infection, a trade-off on salmon reproductive rate, and finally, to consider interactions between populations of G. salaris and multiple strains of salmon exhibiting varying levels of immunity from fully susceptible to resistant. The models proposed predict that in the absence of host resistance or an immune response infections by G. salaris will result in an epidemic followed by the extinction of the salmon host population. Models also predict that if salmon are able to increase their resistance to G. salaris infections through mutations, salmon population recovery after the epidemic is indeed possible within 10-15 years post introduction with low level parasite coexistence. Finally, models also highlight areas where additional information is needed in order to improve predictions and enable the estimation of important parameter values. Model predictions will ultimately be used to assist in future contingency planning against G. salaris outbreaks in the UK and possibly as a basis for future models describing other fish/ecto-parasite systems.