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Arthropod parasites and micropredators, such as ticks and mosquitoes, influence terrestrial ecosystems and harm their hosts directly and indirectly by vectoring micro-organisms. Whether micropredators similarly affect marine ecosystems and hosts is not well understood. In coral reef fish communities, the most abundant micropredators are isopods, in particular, gnathiids. Our understanding of how isopods affect fishes has been restricted by a lack of information regarding basic isopod biology including; patterns of abundance, parasite identity, host specificity and host pathology. Also it is unknown if small juvenile fish are parasitised by isopods, and if this affects their survival. Sequentially, the aims of my PhD thesis were to understand the ecology of several lesser known parasites in sufficient detail to perform manipulative experiments that could test the effects of micropredation on small juvenile reef fish. All field studies took place at Lizard Island, Great Barrier Reef My first study aimed to describe temporal and spatial patterns of isopod abundance by measuring emergence rates. Gnathiid isopod juveniles emerge from the benthos into the water column to find hosts or change locations. Although diel patterns have been demonstrated, the relationship between substrate and emergence on coral reefs is not clear. I measured emergence rates of parasitic isopods (Gnathiidea and Flabellifera) in 6 “habitats” at 2 very different sites at Lizard Island. Isopods were collected from the periphery and centres of 3 sizes of reef, and from the substrate between these reefs (sand or rubble). At both sites, the most abundant gnathiid species (Gnathia falcipenis and Gnathia sp C), was exclusive to that site. Although strong diel patterns in emergence were observed, gnathiid abundance could not be predicted by habitat. However, gnathiids were larger and more often fed over reef borders than in the centres of reefs. To explain these patterns, I suggested that first stage larvae had the largest influence on total abundance and were patchily distributed in accordance with adults from which they had recently hatched. As later stage larvae also depend on fish, more successful (fed and older larvae) are found on the edges of reefs where appropriate hosts may be more abundant. Gnathiids were over-dispersed in all habitats investigated, including apparently homogeneous beds of coral rubble and sand. This indicated that gnathiid distribution may be better predicted by very fine scale differences in substrate, or that larvae are simply gregarious, and that abundance may be difficult to predict on the basis of substrate. Without reliable differences in parasite abundance among habitats at Lizard Island, subsequent studies would rely on manipulating parasite abundance via excluding wild parasites in the field (which proved very difficult) or by controlling abundance in laboratory simulations. I then investigated host specificity in the 2 most abundant gnathiid species from the previous study. Discrete species distributions between the two sites suggested that the 2 parasites may have had different diets. Host-specificity data for gnathiid isopods are scarce because the parasitic juvenile stages are difficult to identify and host-parasite contact is often brief. Engorged third stage gnathiids were photographed and permitted to moult into adults to allow identification. I compared mtDNA sequences from their blood meals to host sequences available on GenBank. The host frequency distributions used by each species were significantly different; only four host families were shared. I concluded that G. falcipenis and Gnathia sp C operate as preferential feeders. Importantly, this work showed that G. falcipenis was indeed a natural parasite of several species of damselfish (Pomacentridae) that could be collected as young recruits and used in manipulative experiments. I then used G. falcipenis as a model parasite to investigate the effects of isopods on recently recruited Dischistodus perspicillatus and small juvenile Acanthochromis polyacanthus damselfishes. Working with honours student Ms Rose Penfold, we determined that A. polyacanthus was infected by gnathiids in the wild at sizes as small as 4.2 mm SL. Laboratory infections revealed that larger A. polyacanthus were much better at eating gnathiids, a behaviour which prevented infection, and that smaller fish were more likely to die post-infection. Infection prevalence was > 3 % during the day, but we could not sample fish for nocturnal infection prevalence. A caging experiment suggested that most micropredation on damselfish took place at night. For D. perspicillatus, I found that exposure to 2 parasites each evening for 7 nights after settlement halved the growth of these fish. Lastly, numerous free-swimming cymothoid isopods were found associated with larval fish in light traps. Cymothoids have a multi-morphic life cycle composed of micropredatory stages that eventually become females, which are permanently attached to fishes. Because cymothoid taxonomy is based around female morphology, natatory-stage cymothoids can not be identified. I sequenced mtDNA from natatory and adult female life history stages and matched 4 of 11 natatory cymothoid morphotypes. Molecular data were also used to produce a phylogeny exploring the evolution of different forms of host attachment within the Cymothoidae. This phylogeny suggests that external attachment, formerly thought to be plesiomorphic, is a derived condition and has evolved several times independently. I suggest that attachment to the buccal cavity or gills is a primitive form of attachment. This research has provided information on emergence patterns and hostspecificity which is necessary for the future study of isopod vector biology. It also provides a platform for future taxonomic and phylogenetic studies on cymothoids. I demonstrate that gnathiids infect juvenile coral reef fish and suggest that they may influence survival both directly and indirectly by reducing growth and predisposing infected fish to size-selective mortality. Thus, interactions between isopod micropredators and recruiting fishes may determine the survivorship of individual fish and influence the subsequent community structure.

Arthropod parasites and micropredators, such as ticks and mosquitoes, influence terrestrial ecosystems and harm their hosts directly and indirectly by vectoring micro-organisms. Whether micropredators similarly affect marine ecosystems and hosts is not well understood. In coral reef fish communities, the most abundant micropredators are isopods, in particular, gnathiids. Our understanding of how isopods affect fishes has been restricted by a lack of information regarding basic isopod biology including; patterns of abundance, parasite identity, host specificity and host pathology. Also it is unknown if small juvenile fish are parasitised by isopods, and if this affects their survival. Sequentially, the aims of my PhD thesis were to understand the ecology of several lesser known parasites in sufficient detail to perform manipulative experiments that could test the effects of micropredation on small juvenile reef fish. All field studies took place at Lizard Island, Great Barrier Reef My first study aimed to describe temporal and spatial patterns of isopod abundance by measuring emergence rates. Gnathiid isopod juveniles emerge from the benthos into the water column to find hosts or change locations. Although diel patterns have been demonstrated, the relationship between substrate and emergence on coral reefs is not clear. I measured emergence rates of parasitic isopods (Gnathiidea and Flabellifera) in 6 “habitats” at 2 very different sites at Lizard Island. Isopods were collected from the periphery and centres of 3 sizes of reef, and from the substrate between these reefs (sand or rubble). At both sites, the most abundant gnathiid species (Gnathia falcipenis and Gnathia sp C), was exclusive to that site. Although strong diel patterns in emergence were observed, gnathiid abundance could not be predicted by habitat. However, gnathiids were larger and more often fed over reef borders than in the centres of reefs. To explain these patterns, I suggested that first stage larvae had the largest influence on total abundance and were patchily distributed in accordance with adults from which they had recently hatched. As later stage larvae also depend on fish, more successful (fed and older larvae) are found on the edges of reefs where appropriate hosts may be more abundant. Gnathiids were over-dispersed in all habitats investigated, including apparently homogeneous beds of coral rubble and sand. This indicated that gnathiid distribution may be better predicted by very fine scale differences in substrate, or that larvae are simply gregarious, and that abundance may be difficult to predict on the basis of substrate. Without reliable differences in parasite abundance among habitats at Lizard Island, subsequent studies would rely on manipulating parasite abundance via excluding wild parasites in the field (which proved very difficult) or by controlling abundance in laboratory simulations. I then investigated host specificity in the 2 most abundant gnathiid species from the previous study. Discrete species distributions between the two sites suggested that the 2 parasites may have had different diets. Host-specificity data for gnathiid isopods are scarce because the parasitic juvenile stages are difficult to identify and host-parasite contact is often brief. Engorged third stage gnathiids were photographed and permitted to moult into adults to allow identification. I compared mtDNA sequences from their blood meals to host sequences available on GenBank. The host frequency distributions used by each species were significantly different; only four host families were shared. I concluded that G. falcipenis and Gnathia sp C operate as preferential feeders. Importantly, this work showed that G. falcipenis was indeed a natural parasite of several species of damselfish (Pomacentridae) that could be collected as young recruits and used in manipulative experiments. I then used G. falcipenis as a model parasite to investigate the effects of isopods on recently recruited Dischistodus perspicillatus and small juvenile Acanthochromis polyacanthus damselfishes. Working with honours student Ms Rose Penfold, we determined that A. polyacanthus was infected by gnathiids in the wild at sizes as small as 4.2 mm SL. Laboratory infections revealed that larger A. polyacanthus were much better at eating gnathiids, a behaviour which prevented infection, and that smaller fish were more likely to die post-infection. Infection prevalence was > 3 % during the day, but we could not sample fish for nocturnal infection prevalence. A caging experiment suggested that most micropredation on damselfish took place at night. For D. perspicillatus, I found that exposure to 2 parasites each evening for 7 nights after settlement halved the growth of these fish. Lastly, numerous free-swimming cymothoid isopods were found associated with larval fish in light traps. Cymothoids have a multi-morphic life cycle composed of micropredatory stages that eventually become females, which are permanently attached to fishes. Because cymothoid taxonomy is based around female morphology, natatory-stage cymothoids can not be identified. I sequenced mtDNA from natatory and adult female life history stages and matched 4 of 11 natatory cymothoid morphotypes. Molecular data were also used to produce a phylogeny exploring the evolution of different forms of host attachment within the Cymothoidae. This phylogeny suggests that external attachment, formerly thought to be plesiomorphic, is a derived condition and has evolved several times independently. I suggest that attachment to the buccal cavity or gills is a primitive form of attachment. This research has provided information on emergence patterns and hostspecificity which is necessary for the future study of isopod vector biology. It also provides a platform for future taxonomic and phylogenetic studies on cymothoids. I demonstrate that gnathiids infect juvenile coral reef fish and suggest that they may influence survival both directly and indirectly by reducing growth and predisposing infected fish to size-selective mortality. Thus, interactions between isopod micropredators and recruiting fishes may determine the survivorship of individual fish and influence the subsequent community structure.

Three major questions were addressed in this dissertation: 1)Do immune abnormalities associated with autism primarily alter CD4+ T cell-mediated or humoral immune responses? 2) Are specific T cell clones expanded in autism? 3) Which, if any, infectious agents play a role in autism? CD4+ T cell-mediated (Th1) or humoral (Th2) immune responses can be distinguished on the basis of the cytokines expressed. CD4+ T-cells secrete interleukin type 2 (IL-2) and interferon-γ, whereas a Th2 response is associated with secretion of interleukin type 4(IL-4). mRNA extracted from peripheral blood mononuclear nuclear cells (PBMC) showed significantly increased levels of IL-2 and interferon-γ expression in 24 autistic subjects relative to 19 normal controls. IL-4 mRNA was undetectable in the same group of autistic subjects. These results indicate that a CD4+ T cell-mediated immune response is associated with autism. The expression of V-β chain mRNA was used as a marker or particular T cell clone expression. The expression of V-β 13 was significantly elevated in the study group of 11 autistic subjects, but not in 9 normal subjects. This suggests that T cell-mediated autoimmunity is a factor in the disease. Two types of human leukocyte antigens (HLA) alleles, DR4 and DR1, are associated with autism. The association between V-β 13 expressing T cell clones and autism was shown even more strongly in the subgroups expressing HLA DR4 or DR1. This result suggests a link between antigen presentation by HLA DR4 or DR1 and expansion of V-β 13 T cell clones. The potential involvement of pathogens suspected to trigger autism was investigated by examining T cell proliferation responses to peptide epitopes. As a group, the 24 autistic subjects did not show a decreased response to peptides derived from rubella virus, influenza A virus, herpes simplex virus type 1, cytomegalovirus, and Clostridium tetani. Another model of autism postulates that autism is induced by pathogens that possess epitopes identical to the hypervariable region 3 (HVR-3) of the HLA DR4 or DR1 alleles. Two antigens derived from the Escherichia coli dna J protein and the Epstein-Barr virus glycoprotein 110 peptides that contain sequences identical to the HVR-3 of the DR4 and DR1 alleles were examined for their ability to induce T cell proliferation in autistic and normal subjects. No effect of the DR4 or DR1 alleles on the response to these two antigens was detected. Therefore, both types of results do not support the model of immune tolerance in autism. However, average T cell proliferative activity was significantly lower in the same autistic subjects. This confirms many prior reports that reduced T-cell responses may shape susceptibility to autism. Further understanding of how immune abnormalities and infectious agents lead to autism should guide development of preventative and therapeutic strategies for this disease. (152 pages)

Estudos tem salientado que a expansão urbana, fragmentação do habitat, desmatamento e sobreposição das áreas de vida das populações humanas e silvestres tem constribuido para o surgimento de doenças emergentes e reemergentes nas últimas décadas. A paisagem da floresta amazônica vem sofrendo constantemente a dominância antropica e como resultado suas populações silvestres encontram-se cada vez mais expostas ao contato com populações humanas e domesticas com um alto risco de transmissão de agentes infecciosos. A cidade de Manaus, localizada na Amazônia Brasileira, tem afrontado um crescimento desordenado e vertiginoso devido ao seu desenvolvimento industrial causando uma alteração constante em sua paisagem; representando um modelo potencialmente útil para entender os mecanismos de transmissão de doenças. Os primatas são as espécies evolutivamente mais próximas dos humanos e essa proximidade filogenética tem facilitado o compartilhamento de diversos agentes infecciosos. O presente estudo propõe utilizar subpopulações de sauim-de-coleira (Saguinus bicolor) que ocupam os fragmentos urbanos de Manaus como espécie sentinela, para avaliar a presença de agentes infecciosos na interface-humano primata. A pesquisa objetiva também determinar se a perturbação antrópica dos locais de estudo estaria favorecendo a transmissão desses agentes. Entre os anos 2011 e 2014 um total de 55 sauins de coleira foram capturados em 9 fragmentos florestais urbanos e 1 area controle. Análises moleculares foram realizados para detectar Rotavirus A, Hantavirus, Coronavirus, Flavivirus, Enterovirus, Influenza A, Adenovirus, Metapneumovirus, Virus Sincitial Respiratório Humano, Parainfluenza 1, 2, 3, 4, Virus do Oeste de Nilo e Plasmodium spp. Os resultados indicaram uma prevalência para Hantavírus de (4/48), Rotavírus (9/48). Pela primeira vez é detectada a presença de hantavírus em primatas neotropicais. Nossos dados indicaram que a presença de infeção estaria associada com a existência de algum tipo de impacto antrópico nos locais pesquisados. Nenhum indivíduo resultou infectado na area controle. / In past decades, numerous studies have highlighted how urban expansion, habitat fragmentation, deforestation, and superposition of human and wildlife population areas contribute to surges in emergent and reemergent diseases. As a result of continuing anthropogenic disturbance in the Amazon, wildlife populations find themselves increasingly exposed to human populations and their domestic animals, bringing higher risks of transmission of a variety of infectious agents. Manaus, located in the Brazilian Amazon, represents a potentially useful model to understand the mechanisms of disease transmission. The city has undergone a disorganized and precipitous growth with ongoing industrial development, causing constant landscape alteration. Because non-human primates are closely evolutionary related to humans they share a diversity of infectious agents. The present study proposes to use subpopulations of Pied tamarins (Saguinus bicolor) occupying urban forest fragments in Manaus as a flagship species to evaluate the presence of infectious agents at the human-nonhuman primate interface. It will also assess whether anthropogenic perturbation at sites favors transmission of agents within this human dominated matrix. During the period of 2011-2014 a total of 55 pied tamarins in 9 urban forest fragments and 1 control area. Molecular analyses were performed for the detection of Rotavirus, Hantavirus, Coronavirus, Flavivirus, Enterovirus, Influenza A, Adenovirus, Metapneumovirus, Sincytial Human Respiratory virus, Parainfluenza 1, 2, 3, 4, West Nile Virus and Plasmodium spp. The results indicate prevalence for Hantavirus (n =4/48) and Rotavirus (n =9/48). This is the first record of Hantavirus in neotropical primates. Data indicate that the presence of infection in the study sites could be associated with anthropogenic impact. The control area resulted uninfected.