Foram coletadas amostras de água de piscinas térmicas e não térmicas na cidade de Porto Alegre, RS, Brasil entre os meses de maio de 2006 e março de 2007, com o objetivo de determinar a presença do gênero Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e trofozoítos e na amplificação por PCR com oligonucleotídeos gênero-específico. O potencial patogênico foi avaliado usando testes de osmotolerância e termotolerância. Das 65 amostras analisadas, 13 (20%) foram positivas para amebas de vida livre e identificados morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 9 possuíam características compatíveis com o grupo morfológico II e 4 com o grupo III. Todos os isolados identificados morfologicamente quando submetidos à Reação de PCR, confirmaram pertencer ao gênero Acanthamoeba e 38% (5/13) dos isolados foram considerados potencialmente patogênicos a partir dos testes de osmotolerância e termotolerância. Neste estudo, o método molecular de RAPD ("Random Amplified Polymorphic-DNA") foi utilizado para investigar a relação genética entre os 13 isolados de piscinas e dois isolados de referência da ATCC. De 10 oligonucleotídeos decaméricos testados, quatro foram selecionados por gerarem produtos de amplificação passíveis de análise. A similaridade entre os isolados foi calculada utilizando-se o coeficiente de Jaccard e o dendrograma construído pelo método da média das distâncias entre grupos ("Average Linkage"). Quatro grupos distintos (G1-G4) de isolados foram formados de acordo com a similaridade genética entre eles. Sugeriu-se que os isolados do G1 por agruparem-se aos isolados de referência de A. castellanii (ATCC 30010 e 50492) possam pertencer a esta espécie. Os dados fenotípicos, tais como, morfologia e testes de tolerância foram relacionados aos dados genotípicos de RAPD e permitiram a caracterização dos isolados. Os resultados deste primeiro estudo de isolamento e caracterização de Acanthamoeba de água de piscinas na cidade de Porto Alegre-RS, Brasil confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana. / Water samples were collected from both heated and unheated swimming pools in the city of Porto Alegre, RS, Brazil between May 2006 and March 2007, to determine the presence of Acanthamoeba in the water of swimming pools as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Escherichia coli. The identification of the isolates was based on the trophozoites and cysts morphology and on the amplification through PCR with genus-specific oligonucleotides. The potential pathogenic was assessed by osmotolerance and temperature tolerance assays. From the 65 samples analyzed, 13 (20%) were positive for free-living amoebae, and the isolates morphologically identified as belonging to the genus Acanthamoeba. Out of these, 9 presented characteristics compatible with morphological group II, and 4 with group III. All the morphologically identified isolates, when submitted to PCR, were confirmed as belonging to the genus Acanthamoeba, and 38% (5/13) of the isolates were considered potentially pathogenic according to osmotolerance and temperature tolerance assays. In this study, the molecular RAPD method (Random Amplified Polymorphic-DNA) was used to investigate the genetic relationship among 13 isolates from swimming pools and two strains from the ATCC reference. From the ten decameric oligonucleotides tested, four were selected for generating products of amplification possible to be analyzed. The similarity between isolates was calculated using the Jaccard coefficient and the dendrogram constructed by using the method of the average distances between groups ("Average Linkage"). Four distinct groups (G1-G4) of isolates were separated according to genetic similarity between them. It was suggested that the isolates from G1 once group up with the reference isolates of A. castellanii may belong to this species. The phenotypic data such as morphology and tolerance tests were related to RAPD genotypic data and led to the characterization of isolates. The results of this study about isolation and characterization of Acanthamoeba in swimming pools water at Porto Alegre, RS, Brazil confirm the presence of potentially pathogenic isolates which can present risks to human health.

Amebas de vida livre (AVL) do gênero Acanthamoeba estão amplamente distribuídas no ambiente e podem tornar-se amebas patogênicas ao homem. O objetivo deste trabalho foi isolar em de água de torneira amebas de vida livre do gênero Acanthamoeba, identificá-las e classificá-las. Um total de 132 amostras de água de torneira foi coletado de escolas estaduais e municipais entre os meses de março a novembro de 2009. As amostras passaram pelo processo de filtração e as membranas foram semeadas em ágar não-nutriente 1,5% coberto por uma suspensão de E. coli inativadas pelo calor. Todas as amostras positivas para AVL foram submetidas à clonagem celular e identificadas como pertencentes ao gênero Acanthamoeba, através da morfologia dos cistos e trofozoitos e pela PCR utilizando oligonucleotídeos gênero-específicos que amplificam a região ASA.S1 do gene 18S rDNA. Ensaios fisiológicos de termo e osmotolerância foram utilizados para avaliar a patogenicidade dos isolados. Vinte sete isolados foram positivos para AVL e 10 foram identificados como pertencentes ao gênero Acanthamoeba tanto pelas características morfológicas quanto pela análise molecular. Destes, nove isolados apresentaram características do grupo II e um do grupo III, segundo Pussard e Pons (1977). A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição no grupo genotípicos T2 (40%), T2/T6 (40%), T6 (10%) e T4 (10%). Nos ensaios de termotolerância e osmotolerância 50% dos isolados obtiveram um baixo potencial patogênico. Os resultados indicaram a presença do gênero Acanthamoeba em água tratada no estado do RS, revelando sua importância epidemiológica e a necessidade de mais estudos para determinar sua distribuição no ambiente e seu potencial patogênico. / Free-living amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can become human pathogenic amoebae. The aim of this study was to isolate from tap water in free-living amoebae of Acanthamoeba, identify them and then classify them. A total of 132 samples of tap water was collected from state and municipal schools between march and november 2009. The samples passed through the filtration process and the membranes were seeded in non-nutrient 1.5% covered by a suspension of E. coli heatinactivated. All samples of AVL were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites by PCR using primers and genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Tests of physiological thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. Twenty seven isolates of AVL and 10 were identified as belonging to the genus Acanthamoeba through the morphological and molecular analysis. Nine of the isolates showed characteristics of group II and one isolate showed characteristics of group III, according Pussard and Pons (1977). The sequencing analysis by comparing the sequences submitted to GenBank, showed that genotype distribution in group T2 (40%), T2/T6 (40%), T6 (10%) and T4 (10%). In tests of thermotolerance and osmotolerance 50% of isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in the RS, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.

Cryptococcus gattii é um dos principais agentes de infecção oportunista em todo o mundo. Esse fungo está presente no meio ambiente e, por isso, pode infectar diversos hospedeiros, inclusive seres humanos, nematóides e células ameboides. Acanthamoeba spp. são protozoários de vida livre que fagocitam diversos organismos, especialmente bactérias e fungos. Apesar de macrófagos e amebas serem evolutivamente distantes, eles compartilham diversas etapas comuns no processo de fagocitose e eliminação do patógeno. Além disso, existem teorias de que amebas e macrófagos possuem um ancestral comum. Para averiguar se essas duas células fagocíticas apresentam estratégias antifúngicas similares, nós analisamos o mecanismo de imunidade nutricional. Essa estratégia imunológica reduz a disponibilidade de nutrientes essenciais para o patógeno, inclusive metais de transição como o zinco. Neste trabalho, nós analisamos se há modulação da homeostase de zinco em Acanthamoeba castellanii durante sua interação com C. gattii. Testes de fagocitose e taxa de replicação intracelular (IPR) realizados através da interação de amebas com a linhagem selvagem (WT) e mutante do gene ZIP1 de C. gattii. O mutante utilizado (zip1Δ) é caracterizado pela sua incapacidade de crescer sem a presença de zinco. Nós observamos que a linhagem mutante foi mais fagocitada por células de A. castellanii comparado com WT. Também, o teste de IPR mostrou que a atividade antifúngica das células hospedeiras apresentou-se mais efetiva contra as células mutantes. Entretanto, a sobrevivência de zip1Δ foi maior quando zinco extracelular (10 M) foi adicionado ao meio de interação. Esses resultados sugerem que as células criptocócicas internalizadas podem estar sofrendo uma privação da disponibilidade de zinco no interior do fagossomo. Para analisar alterações nos transportadores de zinco das células hospedeiras durante sua interação com C. gattii, análises de PCR quantitativo em tempo real (RT-qPCR) foram realizadas para os transportadores de zinco das famílias ZIP e ZnT. Uma intensa modulação de alguns genes foi observado após 3 e 24 horas pós-infecção. Além disso, análises de citometria de fluxo mostraram que os níveis de zinco livre das amebas estavam reduzidos devido a presença do fungo. Esses resultados sugerem que amebas podem modular a disponibilidade de zinco, afim de prejudicar o patógeno. / Cryptococcus gattii is one of the most important agents of opportunistic infections worldwide. They are found in the environment, where it can interact with different host types, including humans, nematodes and amoebic cells. Acanthamoeba spp. are free-living protozoa that basically feed on bacteria and yeast through phagocytosis. Macrophages and amoebae, although evolutionarily distant, share conserved mechanisms related to steps of phagocytosis and microbial killing. In addition, it has been hypothesized that amoeba and macrophage have a common ancestor. To investigate if there are similar antifungal strategies between both cellular types, we analyzed the nutritional immunity mechanism. It is a process defined as a reduction of essential nutrients availability to the pathogen, such as zinc. In this context, we investigate if amoeba cells are able to modulate zinc homeostasis during the interaction with C. gattii. Phagocytosis and intracellular replications (IPR) analysis performed through the interaction between amoebae and wild-type (WT) and mutant for the ZIP1 gene (zip1Δ) strains of C. gattii. The mutant is unable to grow in absence of zinc. We found that zip1Δ strain is more readily engulfed by A. castellanii cells compared to WT. In addition, IPR analysis showed that the antifungal activity of such host cells was more effective against the mutant cells. However, the mutant strain survival was increased when additional extracellular zinc (10 M) was added to the interaction medium. This data suggests that engulfed cryptococcal cells might have been experiencing a deprivation of zinc inside the phagosome. To further evaluate alterations of zinc transporters in host cells due to cryptococcal infection, RT-qPCR analysis was performed for the ZIP and ZnT zinc transporter families. An intense modulation of some genes was found after 3 and 24 hours’ post-infection. Furthermore, flow cytometry analysis showed that free zinc levels from amoebae are reduced by the cryptococcal presence. These results indicate that amoebae are able to modulate zinc availability to harm the pathogen.

Amebas de vida livre (AVL) são distribuídas mundialmente no solo e na água. Um número pequeno delas é considerado importante para a saúde dos seres humanos: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris e Sappinia diploidea. Algumas das infecções são oportunistas, ocorrendo em indivíduos imunocomprometidos, enquanto outras são não oportunistas. Amostras de água foram coletadas de banheira de hidromassagens e piscinas térmicas na cidade de Porto Alegre, RS, Brasil, com o objetivo de determinar a presença de Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e na amplificação por PCR com oligonucleotídeos gênero-específico. De 72 amostras analisadas, 20 (27,77%) foram positivas para amebas de vida livre, e identificadas morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 11 possuíam características compatíveis com o grupo morfológico II e 9 com o grupo III. Entre os isolados, 11(55%) foram considerados potencialmente patogênicos a partir de testes de osmotolerância e termotolerância. Somente 9 isolados quando submetidos à Reação da PCR, confirmaram pertencer ao gênero Acanthamoeba. A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição nos grupos genotípicos T3 (11,1%), T5 (11,1%), T4 (33,3%) e T15 (44,4%).Os resultados obtidos com este confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana nos ambientes de banheiras de hidromassagem e piscinas térmicas. / Free-living amoebae (FLA) are widely distributed in soil and water. A few number of them are implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts, while others are non opportunistic. Water samples were collecyed from both hot tubs and thermal swimming pools in the city of Porto Alegre, RS, Brazil, to determine the presence of Acanthamoeba in the water as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Eschererichia coli. The identification of the isolates was based on the cysts morphology and PCR amplification using genus-specific oligonucleotides. From 72 samples analyzed, 20 (27,77%) were positive for free-living amoebae, and the isolates were morphologically identified as belonging to the genus Acanthamoeba. Out of these, 11 presented morphological characteristics compatible with group II, and 9 with group III. Among the isolates, 11 (55%) were considered potentially pathogenic according to osmotolerance and temperature assays. The isolates when submitted to PCR reaction only 9 were confirmed as belonging to the genus Acanthamoeba. The sequences analysis when compare to the sequences in the GenBank, showed that genotype distribution in group T3 (11,1%), T5 (11,1%), T4 (33,3%) and T15 (44,4%). The results of this study confirmed the presence of potentially pathogenic isolates of free living amoebae in hot swimming pool and spas which can present risks to human health.

Amebas de vida livre (AVL) do gênero Acanthamoeba estão amplamente distribuídas no ambiente e podem tornar-se amebas patogênicas ao homem. O objetivo deste trabalho foi isolar em de água de torneira amebas de vida livre do gênero Acanthamoeba, identificá-las e classificá-las. Um total de 132 amostras de água de torneira foi coletado de escolas estaduais e municipais entre os meses de março a novembro de 2009. As amostras passaram pelo processo de filtração e as membranas foram semeadas em ágar não-nutriente 1,5% coberto por uma suspensão de E. coli inativadas pelo calor. Todas as amostras positivas para AVL foram submetidas à clonagem celular e identificadas como pertencentes ao gênero Acanthamoeba, através da morfologia dos cistos e trofozoitos e pela PCR utilizando oligonucleotídeos gênero-específicos que amplificam a região ASA.S1 do gene 18S rDNA. Ensaios fisiológicos de termo e osmotolerância foram utilizados para avaliar a patogenicidade dos isolados. Vinte sete isolados foram positivos para AVL e 10 foram identificados como pertencentes ao gênero Acanthamoeba tanto pelas características morfológicas quanto pela análise molecular. Destes, nove isolados apresentaram características do grupo II e um do grupo III, segundo Pussard e Pons (1977). A análise do sequenciamento através da comparação das sequências dispostas no GenBank, demonstrou a distribuição no grupo genotípicos T2 (40%), T2/T6 (40%), T6 (10%) e T4 (10%). Nos ensaios de termotolerância e osmotolerância 50% dos isolados obtiveram um baixo potencial patogênico. Os resultados indicaram a presença do gênero Acanthamoeba em água tratada no estado do RS, revelando sua importância epidemiológica e a necessidade de mais estudos para determinar sua distribuição no ambiente e seu potencial patogênico. / Free-living amoebae (FLA) of Acanthamoeba genus are widely distributed in the environment and can become human pathogenic amoebae. The aim of this study was to isolate from tap water in free-living amoebae of Acanthamoeba, identify them and then classify them. A total of 132 samples of tap water was collected from state and municipal schools between march and november 2009. The samples passed through the filtration process and the membranes were seeded in non-nutrient 1.5% covered by a suspension of E. coli heatinactivated. All samples of AVL were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites by PCR using primers and genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Tests of physiological thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. Twenty seven isolates of AVL and 10 were identified as belonging to the genus Acanthamoeba through the morphological and molecular analysis. Nine of the isolates showed characteristics of group II and one isolate showed characteristics of group III, according Pussard and Pons (1977). The sequencing analysis by comparing the sequences submitted to GenBank, showed that genotype distribution in group T2 (40%), T2/T6 (40%), T6 (10%) and T4 (10%). In tests of thermotolerance and osmotolerance 50% of isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in the RS, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.

Foram coletadas amostras de água de piscinas térmicas e não térmicas na cidade de Porto Alegre, RS, Brasil entre os meses de maio de 2006 e março de 2007, com o objetivo de determinar a presença do gênero Acanthamoeba, bem como realizar a caracterização fenotípica e genotípica dos isolados. Amebas foram isoladas em cultivo monoxênico com Escherichia coli. A identificação dos isolados foi baseada na morfologia dos cistos e trofozoítos e na amplificação por PCR com oligonucleotídeos gênero-específico. O potencial patogênico foi avaliado usando testes de osmotolerância e termotolerância. Das 65 amostras analisadas, 13 (20%) foram positivas para amebas de vida livre e identificados morfologicamente como pertencentes ao gênero Acanthamoeba. Destas, 9 possuíam características compatíveis com o grupo morfológico II e 4 com o grupo III. Todos os isolados identificados morfologicamente quando submetidos à Reação de PCR, confirmaram pertencer ao gênero Acanthamoeba e 38% (5/13) dos isolados foram considerados potencialmente patogênicos a partir dos testes de osmotolerância e termotolerância. Neste estudo, o método molecular de RAPD ("Random Amplified Polymorphic-DNA") foi utilizado para investigar a relação genética entre os 13 isolados de piscinas e dois isolados de referência da ATCC. De 10 oligonucleotídeos decaméricos testados, quatro foram selecionados por gerarem produtos de amplificação passíveis de análise. A similaridade entre os isolados foi calculada utilizando-se o coeficiente de Jaccard e o dendrograma construído pelo método da média das distâncias entre grupos ("Average Linkage"). Quatro grupos distintos (G1-G4) de isolados foram formados de acordo com a similaridade genética entre eles. Sugeriu-se que os isolados do G1 por agruparem-se aos isolados de referência de A. castellanii (ATCC 30010 e 50492) possam pertencer a esta espécie. Os dados fenotípicos, tais como, morfologia e testes de tolerância foram relacionados aos dados genotípicos de RAPD e permitiram a caracterização dos isolados. Os resultados deste primeiro estudo de isolamento e caracterização de Acanthamoeba de água de piscinas na cidade de Porto Alegre-RS, Brasil confirmam a presença de isolados potencialmente patogênicos que podem representar um risco à saúde humana. / Water samples were collected from both heated and unheated swimming pools in the city of Porto Alegre, RS, Brazil between May 2006 and March 2007, to determine the presence of Acanthamoeba in the water of swimming pools as well as perform the phenotypic and genotypic characterization of the isolates. Amoebae were isolated in monoxenic culture with Escherichia coli. The identification of the isolates was based on the trophozoites and cysts morphology and on the amplification through PCR with genus-specific oligonucleotides. The potential pathogenic was assessed by osmotolerance and temperature tolerance assays. From the 65 samples analyzed, 13 (20%) were positive for free-living amoebae, and the isolates morphologically identified as belonging to the genus Acanthamoeba. Out of these, 9 presented characteristics compatible with morphological group II, and 4 with group III. All the morphologically identified isolates, when submitted to PCR, were confirmed as belonging to the genus Acanthamoeba, and 38% (5/13) of the isolates were considered potentially pathogenic according to osmotolerance and temperature tolerance assays. In this study, the molecular RAPD method (Random Amplified Polymorphic-DNA) was used to investigate the genetic relationship among 13 isolates from swimming pools and two strains from the ATCC reference. From the ten decameric oligonucleotides tested, four were selected for generating products of amplification possible to be analyzed. The similarity between isolates was calculated using the Jaccard coefficient and the dendrogram constructed by using the method of the average distances between groups ("Average Linkage"). Four distinct groups (G1-G4) of isolates were separated according to genetic similarity between them. It was suggested that the isolates from G1 once group up with the reference isolates of A. castellanii may belong to this species. The phenotypic data such as morphology and tolerance tests were related to RAPD genotypic data and led to the characterization of isolates. The results of this study about isolation and characterization of Acanthamoeba in swimming pools water at Porto Alegre, RS, Brazil confirm the presence of potentially pathogenic isolates which can present risks to human health.

Amebas de vida livre (AVL) são protozoários amplamente distribuídos na natureza e em ambientes artificiais. Alguns gêneros são oportunistas ou patogênicos, com característica anfizóica, são esses: Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., e recentemente Vermamoeba spp. AVL podem servirem de reservatórios de várias bactérias patogênicas, uma vez que se alimentam dessas no ambiente por fagocitose, algumas espécies de bactérias após internalizadas escapam da via de degradação sobrevivem e multiplicam-se no interior das amebas. Face ao exposto, o presente trabalho objetivou isolar e identificar AVL e as bactérias associadas presentes em águas de torres de resfriamento, cuja temperatura da água varia entre 25 a 30°C (ideal para o desenvolvimento de diversos microrganismos). Amebas foram isoladas em cultivo monoxênico com Escherichia coli inativadas pelo calor. Das amostras positivas, foram realizadas Reações em Cadeia de Polimerase (PCR), utilizando oligonucleotídeos específicos para cada gênero. Os produtos das PCR foram sequenciados e as sequências geradas foram comparadas com sequências parciais de cada gene alvo depositadas no GenBank. Das 36 amostras, 33 (91,66%) foram positivas para presença de AVL, destas 15 (45,4%) foram positivas para Acanthamoeba spp., 12 (36,3%) para Naegleria spp., 6 (18,2%) para Vermamoeba spp. Dos endossimbiontes pesquisados, somente Pseudomonas spp. foram identificadas em 13 (39,3%) isolados de AVL. Os resultados obtidos no presente estudo confirmam a presença de amebas de vida livre potencialmente patogênicas e carreadoras de bactérias patogênicas oportunistas que podem representar um risco à saúde humana. / Free-living amoebae (FLA) are protozoa widely distributed in natural and artificial environments. Some genus are opportunistic or pathogenic, with anfizoic feature : Acanthamoeba spp., Naegleria spp., Balamuthia spp., Sappinia spp., and recently Vermamoeba spp. AVL may serve as reservoirs for various pathogenic bacteria, since they feed by phagocytosis in the environment, some species of bacteria after internalized escape to the pathway of degradation surviving and multiplying within the amoebae. The aim of this study was to isolate and identify FLA and their associated bacteria present in water from cooling tower, whose temperature water varies between 25 to 30 °C (ideal for the development of different organisms). Amoebae were isolated in monoxenic cultivation with Escherichia coli inactivated by heat. Of the positive samples Polymerase Chain Reactions (PCR) were performed using specific primers for each genus. The PCR products were sequenced and the sequences were generated compared with partial sequences of each gene target deposited in GenBank. From the 36 samples, 33 (91.66%) were positive for the presence of FLA. Of these, 15 (45.4%) were positive for Acanthamoeba spp., 12 (36.3%) for Naegleria spp. and 6 (18.2 %) for Vermamoeba spp. Of the amoeba isolated, 13 (39.3%) were associated to bacteria wich belong to Pseudomonas genus (100%). Another bacteria investigated were absent. The results of this study confirm the presence of potentially pathogenic free-living amoebae as vehicles of opportunistic/pathogenic bacteria that can pose a risk to the human health.

A paleoecological study was carried out on a 4.7 m peat core from Anderson Fen on central Vancouver Island, using a multi-proxy approach. Pollen, non-pollen palynomorphs, and physicochemical analyses were used to document past vegetation, peatland developmental history, and carbon and nitrogen accumulation rates over the last 14,000 years. Lake sediment and aquatic plant remains at the base of the core indicate a shallow pond was present at the site after deglaciation. By ~11,700 calendar years before present (cal yr BP), the shallow pond became a herb-dominated wetland (marsh) through terrestrialization. Bog formation started around 10,500 cal yr BP with decreasing water levels, as indicated by high C:N, Sphagnum and fungal remains, and testate amoebae such as Archerella flavum and Heleopera. A fen developed by ~9400 cal yr BP with fluctuating water levels through the rest of Holocene. Carbon accumulation rates were highest towards the surface and during the early Holocene warm period, with an overall mean rate of 12.9 g/m2/cal yr, which is low compared to continental and northern peatlands. Pollen analysis reveals that non-arboreal communities dominated by Salix prevailed soon after deglaciation before the expansion of Pinus forests 13,200 cal yr BP. Pseudotsuga menziesii dominated forests between ~10,700 and 8400 cal yr BP under warm and dry conditions. Tsuga heterophylla rainforest was established by ~7000 cal yr BP under increasingly cool and wet conditions. Overall, Anderson fen and the surrounding area experienced major and rapid changes in environmental conditions and vegetation in response to climate change during the late glacial and early Holocene, while mid- to late Holocene changes have been more subtle and relatively gradual. / Graduate / 2020-10-25

This thesis attempts to broaden what is known about bacterial symbionts within amoebae by the use of a number of different molecular methods. Initially a number of different amoeba strains were screened for bacterial symbionts by 16S rRNA gene PCR, then the symbionts were identified by comparative sequence analysis and phylogenetic analysis. The amoeba strains containing bacterial symbionts were characterised by cell morphology, 18S rRNA gene sequencing, internal transcribed spacer sequencing and allozyme electrophoresis. Amoebae belonging to the genera Acanthamoeba, Naegleria, Ripidomyxa and Saccamoeba were identified as containing symbionts that belonged to a wide range of different bacterial genera. Relationships between bacterial symbionts and their host amoebae were analysed by the use of transmission electron microscopy and fluorescent in situ hybridisation using symbiont specific probes. Also described are attempts that were made to isolate and grow the bacterial symbionts outside of their host amoebae as well as experiments to try to transfer bacterial symbionts from one amoeba strain to another. Lastly the results from this study are discussed as a whole to put into perspective how they contribute to the body of knowledge of symbionts within protozoa.

This thesis investigates sea-level changes in order to test the hypothesis that the main contribution to early 20th century sea-level rise was Northern Hemisphere land-based ice melt. Multiproxy sea-level reconstructions were established for the Falkland Islands, a location where models suggest sea-level rise from Northern Hemisphere ice melt produces the largest signal. The Falklands reconstruction indicated sea levels in the early 20th century accelerated compared to the long-term rate, synchronous with accelerations observed globally. The magnitude of the acceleration in the Falklands reconstruction was greater than Northern Hemisphere rates, consistent with the spatial pattern from a Northern Hemisphere melt source, but likely less than in New Zealand and Australia. It is therefore not possible rule out other contributions to the observed sea-level acceleration. The Falklands reconstruction indicated a rapid sea-level jump around 8.4 ka BP, synchronous with a jump observed in the Northern Hemisphere, which has been attributed to the sudden drainage of Laurentide proglacial lake Agassiz-Ojibway associated with the 8.2 ka BP climatic downturn. A maximum estimate of 0.89 ± 0.22 m for this jump in the Falklands is considerably less than estimates from Northern Hemisphere records. This difference could indicate additional contributions from the Southern Hemisphere are being recorded in the Northern Hemisphere signal. This thesis also focused on developing testate amoebae as sea-level indicators. In the Falklands, testate amoebae transfer functions were able to reconstruct sea level with precision (±0.08 m) comparable to diatoms (±0.07 m). However, preservation issues were indicated in the fossil testate amoebae assemblages which limits their use as tools for sea-level reconstruction. In addition, contemporary distributions of salt-marsh testate amoebae were investigated over one annual cycle. Seasonal variations in the live assemblages were observed to be asynchronous between taxa. Variations in the death assemblages were also observed which were correlated with variations in the live assemblages. This observation suggests the commonly applied assumption in palaeoenvironmental studies that analysing the death population negates temporal bias is invalid. Further research is required to investigate the impact these observed variations have on reconstructive performance.