Morphological and Morphometric Characterization of Eodinium posterovesiculatum (Ciliophora, Ophryoscolecidae) in Brazilian Cattle

Abstract The morphological variability of Eodinium posterovesiculatum (Ciliophora, Trichostomatia) has been interpreted in different ways: four distinct species or four morphotypes of the same species. The present study aims to perform morphological and morphometric comparative analysis of the four morphotypes found in cattle from the southeastern region of Brazil. Ruminal content samples were obtained from four Holstein x Gir cattle and fixed at 18.5% formalin for morphological analysis. Morphometry was performed based on individuals stained with Lugol's solution [1]. The infraciliary bands were stained using silver carbonate impregnation technique [2]. Morphological and morphometric characterizations, supported by literature, suggest that the four morphotypes of E. posterovesiculatum are actually a single polymorphic species due to small morphometric differences and mostly identical morphological characters, with the format of the caudal processes being the only morphological characteristic that sets them apart.

Using in situ flow cytometry images of ciliates and dinoflagellates for aquatic system monitoring / Usando imagem de citometria de fluxo in situ de ciliados e dinoflagelados para o monitoramento de sistemas aquáticos

Abstract Short-period variability in plankton communities is poorly documented, especially for variations occurring in specific groups in the assemblage because traditional analysis is laborious and time-consuming. Moreover, it does not allow the high sampling frequency required for decision making. To overcome this limitation, we tested the submersible CytoSub flow cytometer. This device was anchored at a distance of approximately 10 metres from the low tide line at a depth of 1.5 metres for 12 hours to monitor the plankton at a site in the biological reserve of Barra da Tijuca beach, Rio de Janeiro. Data analysis was performed with two-dimensional scatter plots, individual pulse shapes and micro images acquisition. High-frequency monitoring results of two interesting groups are shown. The abundance and carbon biomass of ciliates were relatively stable, whereas those from dinoflagellates were highly variable along the day. The linear regression of biovolume measures between classical microscopy and in situ flow cytometry demonstrate high degree of adjustment. Despite the success of the trial and the promising results obtained, the large volume of images generated by the method also creates a need to develop pattern recognition models for automatic classification of in situ cytometric images.

Resumo A variabilidade de curto período em comunidades do plâncton é pouco documentada, especialmente as variações que ocorrem em grupos específicos das assembleias por causa das análises tradicionais serem muito trabalhosas e demoradas. Além disso, não permitem que a alta frequência amostral necessária para a tomada de decisão. Para superar esta limitação, nós testamos o CytoSub, um citômetro de fluxo submersível. Este aparelho foi ancorado a aproximadamente 10 metros de distância da linha de maré baixa a uma profundidade de 1,5 metros por 12 horas para monitorar o plâncton em um sítio da reserva biológica da praia da Barra da Tijuca, Rio de Janeiro. A análise dos dados foi realizada a partir de gráficos de dispersão bidimensionais, pelas assinaturas ópticas individuais escaneadas (pulse shape profile) e aquisição de micro imagens. Resultados do monitoramento de alta frequência de dois grupos interessantes são apresentados. A abundância e a biomassa de carbono de um grupo de ciliados foram relativamente estáveis, ao passo que o grupo de dinoflagelado, foi altamente variável ao longo do dia. O modelo de regressão linear das medidas de biovolume entre a clássica microscopia e a citometria de fluxo in situ apresentou alto grau de ajustamento. Apesar do sucesso deste ensaio e dos resultados promissores obtidos, o grande volume de imagens geradas por este método também gerou a necessidade de se desenvolver modelos de reconhecimento de padrões para a classificação automática de imagens de citometria in situ.

Quantitative analysis of nucleolar chromatin distribution in the complex convoluted nucleoli of Didinium nasutum (Ciliophora)

We have earlier shown that the typical Didinium nasutum nucleolus is a complex convoluted branched domain, comprising a dense fibrillar component located at the periphery of the nucleolus and a granular component located in the central part. Here our main interest was to study quantitatively the spatial distribution of nucleolar chromatin structures in these convoluted nucleoli. There are no "classical" fibrillar centers in D.nasutum nucleoli. The spatial distribution of nucleolar chromatin bodies, which play the role of nucleolar organizers in the macronucleus of D.nasutum, was studied using 3D reconstructions based on serial ultrathin sections. The relative number of nucleolar chromatin bodies was determined in macronuclei of recently fed, starved D.nasutum cells and in resting cysts. This parameter is shown to correlate with the activity of the nucleolus. However, the relative number of nucleolar chromatin bodies in different regions of the same convoluted nucleolus is approximately the same. This finding suggests equal activity in different parts of the nucleolar domain and indicates the existence of some molecular mechanism enabling it to synchronize this activity in D. nasutum nucleoli. Our data show that D. nasutum nucleoli display bipartite structure. All nucleolar chromatin bodies are shown to be located outside of nucleoli, at the periphery of the fibrillar component.

Improvement of silver impregnation technique (Protargol) to obtain morphological features of protists ciliates, flagellates and opalinates

The research on ciliates, flagelates and opalinates have been widespread by the utilization of techniques employing silver impregnation (Protargol), modified by several authors. However, these are time consuming and its results are variable. The present work is a variant of the technique described by Tuffrau (1964, 1967) showing some adaptations made in our laboratory. The organisms can be preserved by different fixatives (alcoholic Bouin, Stieve's fluid, 2.5 percent glutaraldehyde and others) and then rinsed in destilled water followed by a fast clarification by 3 percent sodium hypochloride. If the organism is very sensitive to hypochloride, 4 percent sodium lauryl sulfate may be used and then washed 3 times in distilled water. The protista can be adhered to the glass slides with Mayer's glycerinated-albumin (1 glycerin vol. to 1 or 2 albumin vol.), diluted in water at a proportion of 1:10 Cv/v., or with 1 percent polylysine followed by fast washes with distilled water. After the slide preparation, they were covered with a layer of 0,8 percent Silver proteinate. Right after that, the slide has to be placed in a glass tray lined with moist tissue and covered to prevent the proteinate to dry. The tray was placed in a incubator at 40º-50ºC for 30 minutes. The slides are rinsed for 1 minute. with warm (35ºC) distilled water. The development of the material should be done with 0.4 percent hydroquinone with a maximum incubation time of 1 minute. It should be developed gradually, controlling the silver impregnation intensity by observation under optical microscope. Next, rinse in distilled water for 1 minute, and then, fix in 2,5 percent Sodium thiosulfate. Rinse the slide for two minutes before dehydrating it in an alcoholic serial 50-100º. Finally rinse the slides in xylene. Mount the slides with Entellan MerckTM or Canada balsam