No description available.

Pumkin seedlings (Cucurbita maxima) were inoculated with the fungus Sclerotinia sclerotiorum (Lib.) de Bary. Center regions of non-infected and infected cotyledons of various lengths were cut into 1 x 2 mm strips and processed for ultrastructural investigations. Complementary portions of opposing non-infected cotyledons were also processed as control tissue, and contained fewer storage products in cells than cotyledons of comparable size from non-infected plants. Young non-infected cells contained distinct lipid bodies, protein bodies and electron dense inlusions which were translocated out of older cells by abundant plasmodesmata. Initally, infected cells became plasmolyzed, followed by breakdown of the cytoplasm. Host cells were generally affected only 4-6 cells from the infection front. Many infected cells had abundant rough endoplasmic reticulum and all organells were eventually completely disrupted. Action of enzymes from fungal cells did not appear to be limited to the area immediatedly adjacent to hyphal cells.

No description available.

No description available.

Lernaeocera branchialis (Linnaeus, 1767) is a parasitic copepod possessing a complex dual-host lifecycle. The “definitive” gadoid hosts, including Gadus morhua (Atlantic cod), Melanogrammus aeglefinus (haddock) and Merlangius merlangus (whiting), are infected by the fertilised female, which penetrates the host’s ventral aorta or bulbus arteriosus whilst undertaking extensive metamorphosis and a haematophagous lifestyle. The pathogenic effects of this activity upon the host have been well documented and mortality may occur, especially when multiple parasites are present. These negative impacts on cod, particularly juveniles, by L. branchialis have the potential to adversely affect cod aquaculture in the future, and already vulnerable wild cod stocks. This PhD project therefore, investigated the immune response of wild haddock and cultured-cod post-infection by L. branchialis, and the possible mechanisms by which the parasite modulates/evades the host’s immune response. The systemic immune response of both wild haddock and cultured-cod post-infection by L. branchialis depended on the maturation stage of the parasite, and in the former host species, upon the infection intensity. Wild haddock harbouring fully metamorphosed females showed an increase in circulating thrombocytes and a decrease in serum protein levels however; if multiple mature L. branchialis were present the haddock possessed reduced circulating monocytes, and increased circulating thrombocytes and serum anti-trypsin activity. Infection by L. branchialis was also associated with a suppressive effect on haddock serum spontaneous haemolytic activity. These responses were thought to be due to the host trying to counteract the increased damage caused by the massive increase in size and the feeding of the mature parasite, which is more pronounced when multiple parasites are present, resulting in the increase in some parameters and the ‘consumption’ of others. However, the effect of parasite-derived secretions and other pathogens due to observations on wild fish could not be discounted. The laboratory-infection of cultured-cod from two different sources was also performed in order to study the immune response over time. The two groups of cod showed differences in their immune response to L. branchialis. The first group showed suppressed respiratory burst activity of phagocytes, as the parasite reached the early penella sub-stage, whilst no suppression in phagocyte respiratory burst activity was found in the second group. The parasite was found to migrate along the afferent branchial artery of the cod where a thrombus formed and was present throughout its migration into the ventral aorta. At 14 d post-infection, leukocytes expressing Interleukin 8 mRNA were observed within the free-flowing blood at the periphery of the organising thrombus within the lumen of the ventral aorta. This was speculated to aid the recruitment and activation of leukocytes to the site, and the maturation and neovascularisation of granulation tissue. The infection of the second group subsided with the death of the parasite, and none of the parasites metamorphosed past the early penella sub-stage. The live parasites infecting the first group of cod did not possess IgM or complement component C3 binding on their cuticle, however, both IgM and C3 binding occurred on the dead parasites in the second infection trial. This may highlight the importance of these opsonins and the cytotoxic effect of phagocytes in the elimination of L. branchialis by some cod. However, the first infection was terminated as the parasite reached the early penella sub-stage due to a loss of stock cod prior to the study, so the long-term success of the infection can not be concluded. Therefore, the immune response to infection needs to be determined over the entire metamorphosis of L. branchialis to determine whether the infection was successful or not, and preferably in populations with varying susceptibility to L. branchialis. This will not be possible without further studies into the resistance of different stocks of cultured-cod. Many arthropod parasites, such as ticks and salmon lice, have been previously documented to produce pharmacologically active secretions, aiding host invasion and parasite feeding, preventing the host immune response from working effectively against the parasite, all aimed at improving survival of the parasite. Therefore, the effects of the secretory/excretory products (SEPs) produced during the initial infective stage and by the mature, fully metamorphosed female on the immune response of cultured-cod in vitro, and the location of exocrine glands associated with the oral region of the parasite were investigated. The SEPs from the infective stage of the parasite were found not to affect the intracellular hydrogen peroxide (H2O2) production of phagocytes. The practical difficulties in collecting large quantities of the SEPs from the infective stage meant that their effects could not be tested on the other host immune parameters studied. The SEPs from fully metamorphosed female L. branchialis, however, had a number of suppressive effects on the host immune response in vitro including: 1) suppression of the intracellular production of cytotoxic H2O2 during the respiratory burst of phagocytic leukocytes post-PMA stimulation, 2) suppression of the production of macrophage activating factor by leukocytes with a priming effect on naïve phagocyte function, and 3) suppression of the chemo-attraction ‘power’ of zymosan activated cod serum, i.e. anaphylatoxin activity, on head kidney-derived leukocytes. These effects were dose-dependent, and highlight the capacity of L. branchialis to suppress its host’s innate immune response at the local feeding area. Further work is required to establish the mechanisms by which the parasite-derived SEPs suppress these host immune parameters, and to identify which molecules produced by the parasite are responsible. The correlation between these in vitro results, and systemic immune parameters measured from laboratory-infected Atlantic cod and wild infected haddock are discussed. Host immuno-modulation by other arthropod parasites is mediated by pharmacologically active secretions produced by exocrine glands. Therefore, the exocrine glands of the infective and fully metamorphosed female L. branchialis were also investigated in order to identify those that might be responsible for the secretion of host-modifying products. Adult female exocrine glands were mapped using diaminobenzidine (DAB), most commonly known to stain peroxidases and catalases. These compounds are known to be involved in the neutralisation of harmful free radicals which are released during the respiratory burst and tissue damage. Such products may therefore be important protective secretory components at the site of feeding / infection. Exocrine glands were located in the infective stage associated with the oral region, one pair termed the anterior gland complex (AGC), and the other pair extending either side of the oral cone termed the circum-oral glands (CG). These were further investigated using light microscopy and transmission electron microcopy. The AGC and CGs possessed multi-component secretions and they possessed secretory vesicles, abundant and highly active rough endoplasmic reticulum and Golgi apparatus suggesting that protein is an important component of the secretory products. These glands were also observed in the fully metamorphosed females where they had increased in size within the cephalothorax post-metamorphosis. It is hoped that the identification of these glandular structures, which are thought to secrete within the local vicinity of the oral cone, will aid future studies regarding the identification and secretion kinetics of parasite-derived molecules during the infection and feeding process.

Mistletoes are a group of hemiparasitic plants that grow on a wide variety of host trees and differ in their degree of host specificity, ranging from specialists to generalists. Mistletoes can also be locally host specific where host preference varies geographically, i.e. at a given location a mistletoe species may infect only part of its overall host set. The mistletoe Agelanthus natalitius parasitises at least 11 tree genera distributed throughout South Africa. However, there is geographic variation in infection patterns over the parasite’s range, suggesting that A. natalitius may be locally host specific. We quantified the degree of host specificity and tested the mechanisms that direct host specialisation in two distinct mistletoe populations at Highover and Mtontwane (about 110 km apart) in KwaZulu-Natal, South Africa. We investigated the distribution, abundance and community composition of woody species that host the mistletoe. We also assessed the effect of light on germination and early survival of the mistletoes in a greenhouse experiment. We conducted field reciprocal transplant experiments at both sites to investigate the compatibility of these mistletoes with their hosts Acacia karroo and A. caffra during early development. We then analysed the nutrient and water contents of the mistletoe-host pairs to investigate the role of nutrient and water status in directing host specificity in mistletoes. We further studied avian dispersal in the field and in captivity to investigate optimal dispersal distance and germination success, and evaluated their role in determining mistletoe host specificity. At both study sites, five host species were recorded as being parasitised by the mistletoe A. natalitius. A. karroo and A. caffra appear to be the two most common host species in the region; both grow abundantly at the study sites and were recorded with high infection by A. natalitius. However, A. karroo is the most abundant host species and the mistletoe showed a high degree of host specificity on A. karroo. Infection by mistletoes was positively correlated with tree size, and was highly aggregated, both individually and locally. Field observations and greenhouse shade experiments showed that light can influence mistletoe distribution. Germination of mistletoe seeds was independent of host species and site. However, hypocotyls (the structures that develop into haustoria) grew longer when placed on their source host species within their locality. Additionally, they showed preference for the most abundant host species, A. karroo. Water and nutrient status of the host species A. karroo and A. caffra had no significant effect. Thus, host nutrient and water content may not account for host specificity in this mistletoe species. Mistletoes accumulated more nutrients and maintained more negative than their host trees. We also investigated the mistletoes’ use of passive nutrient uptake (from host xylem) and active nutrient uptake (from host phloem) by using the N:Ca ratio as an index of nutrient access. Mistletoes growing on A. caffra had a ratio > 1, i.e. the mistletoe actively accessed nutrients from the phloem of host trees. However, mistletoes on A. karroo had a N:Ca ratio < 1, which implies that they passively accessed nutrients from the xylem. The difference in mechanism of nutrient acquisition on different host species may reflect the level of compatibility between mistletoe and host. Several bird species were frequently observed to feed on mistletoes, many of which were used in our captivity studies. Although birds did not consume mistletoe fruits in captivity as they do in the field, they were effective in removing the pulp cover of mistletoe fruits and exposing seeds in germinable condition. In captivity, the Red-winged Starling ingested whole fruits and regurgitated seeds, deliberately wiping their bills on twigs to remove the sticky seeds. As a result, germination success of mistletoes processed by Red-winged Starlings was higher than any other bird species tested in captivity. Overall, there appears to be host specificity in morphologically identical mistletoes. Understanding the mechanisms that result in host race evolution are potentially important to the process of speciation in hemiparasitic mistletoes. We need to take into account genotypic matching in conserving these different forms of mistletoes and their host Acacia genotypes. Further research into the mechanisms of host specificity and patterns of genotypic matching is warranted. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Argulus foliaceus (Linnaeus, 1758) is a member of the branchiuran family Argulidae and has a worldwide distribution, causing major economic impacts for freshwater aquacultured fish species worldwide. In the UK, it has economic impacts for both aquaculture and sports fishing industries. Previous studies observed haemorrhagic and inflammatory responses after Argulus infection, which has been taken to support the idea that the parasite secretes chemicals during the feeding process to assist with the ingestion of blood and epithelial tissue. The present study suggests that the blood-feeding ectoparasite of fish, A. foliaceus, may use similar mechanisms for evading host immune responses to those used by sea lice and other haematophagous arthropods. No previous studies have directly investigated the nature of the bioactive compounds / proteins, assumed to be released from these ectoparasites, and which are considered to contribute to feeding processes and host-parasite interactions during infection. Thus, the work described in this thesis was undertaken with the objective of identifying, describing and characterising the secretory components that have previously been suggested to be secreted from glandular cells associated with the feeding appendages of Argulus foliaceus. The current study applied transcriptomic and proteomic techniques in conjunction with in situ methods to investigate known immunomodulatory genes that may serve a function in parasite-host interactions. Overall, the findings of this project have generated considerable additional knowledge concerning the biology of Argulus spp. and have provided a list of proteins that may be used by the parasite to facilitate feeding processes by secreting these active molecules into the host and hence modulating their immune defence mechanisms. This information can be used as a baseline for developing freshwater lice control strategies to help prevent the spread of Argulosis in aquaculture by applying vaccination as means of control using the candidate antigens described in this study to specifically target Argulus spp. Knowledge generated by the work described in this thesis can also contribute to the development of drugs for controlling Argulus or functional components of feed that may serve to protect fish against this parasite. Furthermore, data from this thesis enhances the knowledge of the distribution of toxin/venom or venom-like substances in crustaceans and arthropods in general.

This work allowed the first characterization of the three members of the chloride channel CLC family in T.brucei. The TbCLCs are expressed in the two proliferative stages of the parasite and two of their members appear non-essential. The three TbCLCs act as chloride transporters in X.laevis oocytes and some of their biophysical properties were determined. Furthermore, TbCLC-b appeared to be involved in lysis by the human innate immunity protein apoL-1<p>A novel function of T.brucei adenylate cyclases was discovered in their ability to suppress expression of the innate immunity protein TNF-α. The suppression of the innate response occurs before the first peak of parasitemia and reduces the host ability to control the parasite.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Call number: LD2668 .T4 1979 C66 / Master of Science

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.