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.

The importance of protozoan bacterivory in a subtropical environment (Lobo-Broa Reservoir, SP, Brazil) / A importância da bacterivoria por protozoários em um ambiente subtropical (Reservatório do Lobo-Broa, SP, Brasil)

This study evaluated the importance of heterotrophic nanoflagellates (HNF) and ciliates bacterivory in a mesotrophic subtropical environment (Lobo-Broa Reservoir, Brazil) by the quantification of their ingestion rates. The in situ experiments using fluorescently labelled bacteria (FLB) were carried out bimonthly over one year (three surveys in the dry season and three in the rainy one) at the sub-surface of two sampling points that have different trophic degrees. The ingestion rates for both ciliates and HNF were higher in the meso-eutrophic region (point 2) due to the higher water temperatures, which accelerate the metabolism of protozoans and the higher bacteria densities. Concerning total protozoan bacterivory, the HNF had the greatest grazing impact on bacterial community, especially the HNF <5µm. The data showed that HNF grazing, in addition to regulating the bacteria abundance, also induced changes to the bacterial community structure, such as increasing size and numbers of bacterial filaments. The ciliates were also important to the system bacterivory, especially in point 2, where there were high densities and ingestion rates. The protozoan bottom-up control was more important in the dry season and the top-down control was more important in the rainy season, so, these two forces are equally important to the bacterial abundance regulation in this reservoir in an annual basis.

Este estudo avaliou a importância da bacterivoria por nanoflagelados heterotróficos (NFH) e ciliados em um ambiente subtropical mesotrófico (Reservatório do Lobo-Broa, Brasil) pela quantificação de suas taxas de ingestão. Os experimentos in situ utilizando bactérias marcadas por fluorescência (FLB) foram realizados bimestralmente durante um ano (três estudos no período seco e três no período chuvoso) na superfície de dois pontos de coleta que apresentam graus de trofia diferentes. As taxas de ingestão, tanto para os ciliados quanto para os NHF, foram maiores no ponto 2 (P2) devido provavelmente as maiores temperaturas da água, que aceleram o metabolismo dos protozoários, e as maiores densidades de bactérias. Em relação à bacterivoria total por protozoários, as populações de NFH causaram maior impacto de predação sobre a comunidade bacteriana do reservatório, principalmente os NFH menores que 5µm. Os dados indicaram que a predação por NFH, além de regular a abundância de bactérias, provavelmente também induziu modificações na estrutura da comunidade bacteriana, como o aumento do tamanho e da quantidade de filamentos bacterianos. Os ciliados também contribuíram significativamente para a bacterivoria no sistema, principalmente no P2, onde apresentaram altas densidades e taxas de ingestão. O controle bottom-up teve maior importância no período seco e o controle top-down maior importância no período chuvoso, assim, estas duas forças são igualmente importantes na regulação da abundância bacteriana neste reservatório em uma base anual.

Proliferaciones algales 2000-2001 en Bahía de Banderas, México según el sensor SeaWiFS

Algal blooms at Banderas Bay, México (2000-2001), from SeaWiFS-sensor-data. Algal blooms for the period of 2000 and 2001 at Banderas Bay, México, were analyzed from SeaWiFS-ocean-color-sensor derived information. These events were related with the maximum chlorophyll week anomalies (ASi; a proxy variable constructed for the analysis of chlorophyll temporal variation in the bay). The winter-spring blooms were multispecific, while the summer-fall blooms were monospecific. Two proposals are made: 1) Winter-spring blooms are strongly related with mesoescale processes, due to their apparent temporal synchrony with the high chlorophyll levels in the coastal region from Sinaloa to Jalisco states; 2) Cochlodinium polykrikoides (Margalef 1961) blooms during the summer-fall periods could be induced by local conditions and the influence of previous events on the ecosystem. Rev. Biol. Trop. 56 (4): 1653-1664. Epub 2008 December 12.

Se analizaron las proliferaciones algales ocurridas en Bahía de Banderas, México, durante los años 2000 y 2001 a partir de información derivada del sensor de color del océano SeaWiFS. Estos eventos estuvieron relacionados con los máximos niveles de Anomalías Semanales de Clorofila’ (As i; variable construida para analizar la variación temporal de la clorofila dentro de la bahía). Las proliferaciones ocurridas en los periodos invierno-primavera fueron multiespecíficos, mientras que en verano-otoño fueron dominadas por una sola especie. Se plantean dos propuestas: 1) las proliferaciones algales de invierno-primavera están fuertemente relacionadas a procesos de mesoescala dada su aparente sincronía temporal con altos niveles de clorofila en las costas de Sinaloa a Jalisco; 2) mientras que las proliferaciones de Cochlodinium polykrikoides durante los periodos verano-otoño son favorecidas por el efecto de otras proliferaciones algales previas sobre el ecosistema y por condiciones predominantemente locales en la bahía.

Growth, delayed fluorescence and pigment composition of four Prorocentrum minimum strains growing at two salinities

Prorocentrum minimum is a potentially harmful and widely distributed marine dinoflagellate. Several P. minimum strains have already been studied, showing phylogenetical relations of strains isolated from the same geographical regions. Similarity among the strains was further examined on the basis of their physiology. Pigment composition and concentration, as well as delayed fluorescence (DF) decay kinetics and intensity, were measured in four P. minimum strains isolated from the Baltic and Adriatic Seas. The strains were grown at two salinities characteristic of the Baltic (8 PSU) and North Adriatic Seas (32 PSU). Strain differences in DF decay kinetics and growth did not always follow their genetic relations. While two strains showed similarities to the previously described strains from the Baltic and Adriatic Seas in DF parameters, the other two strains seemed to be specific. The differences among strains isolated from the same sea could stem from adaptations to conditions in the specific habitats. Cluster analysis based on the ratio of individual carotenoid pigments concentrations to the chlorophyll a concentration or to total carotenoids were not conclusive in showing relations among the strains. Among the measured pigments, only peridinin concentration depended on salinity in all st.

Epiphytic dinoflagellates associated with ciguatera in the northwestern coast of Cuba

The spatial and temporal abundance of epiphytic dinoflagellates associated with ciguatera was studied over two annual cycles (March 1999 to March 2000 and March 2001 to March 2002) in the northwestern coast of Cuba. From 14 species of macroalgae (Phaeophyta, Chlorophyta, and Rhodophyta), 1340 samples were obtained identifying seven species as potentially noxious; five of them are new reports for Cuba's phytobenthos: Prorocentrum belizeanum Faust, P. concavum Fukuyo, P. mexicanum Tafall, Coolia monotis Meunier, and Ostreopsis lenticularis Fukuyo. ANOVA/MANOVA analysis showed significant spatial differences: lower cell abundance near the shoreline adjacent to a river inlet and higher cell abundance in the deepest area. Prorocentrum lima (Ehrenberg) Dodge 1975 was found mainly on Phaeophyta followed by Chlorophyta and Rhodophyta. Gambierdiscus toxicus was found mainly on Phaeophyta followed by Rhodophyta and Chlorophyta. All the species reported in the study area were mainly on Padina spp. (Phaeophyta). Acanthophora spicifera (Rhodophyta) did not host dinoflagellate species. Environmental conditions in summer (higher temperature, more nutrients, greater water transparency, and low wind intensity) are suitable for macroalgae development, which serves as a substrate for potentially harmful dinoflagellates, and possibly the main vector for spreading ciguatera along the coast of Cuba.

Se estudió la abundancia espacial y temporal de dinoflagelados epífitos asociados a la ciguatera durante dos ciclos anuales (marzo 1999 a marzo 2000, y marzo 2001 a marzo 2002) en la costa del noroeste de Cuba. Recolectamos 14 especies de macroalgas (Phaeophyta, Chlorophyta y Rhodophyta) y obtuvimos 1340 muestras. Identificamos siete especies de dinoflagelados potencialmente nocivas, cinco de ellas nuevos registros para el fitobentos cubano: Prorocentrum belizeanum Faust, P. concavum Fukuyo, P. mexicanum Tafall, Coolia monotis Meunier, y Ostreopsis lenticulares Fukuyo. El análisis de ANOVA/MANOVA mostró diferencias espaciales significativas: la abundancia celular más baja se encontró cerca de la desembocadura del río y la más alta en el área más profunda. Prorocentrum lima (Ehrenberg) Dodge, se encontró principalmente sobre las Phaeophyta seguido de las Chlorophyta y Rhodophyta. Gambierdiscus toxicus se encontró principalmente sobre las Phaeophyta seguido de las Rhodophyta y Chlorophyta. Todas las especies halladas en el área del estudio estaban sobre Padina spp. (Phaeophyta). No se encontró ninguna especie de dinoflagelado sobre Acanthophora spicifera (Rhodophyta). Las condiciones ambientales en verano (temperatura, nutrientes, transparencia de agua y la baja velocidad del viento) favorecen el desarrollo de las macroalgas, siendo un substrato adecuado para el desarrollo de dinoflagelados potencialmente nocivos, y posiblemente el vector principal para la extensión de la ciguatera en la costa noroeste cubana.

Decoloración en proliferaciones de microalgas como parámetro bioindicador en la Bahía de Mazatlán, México / Discoloration of microalgal blooms as a bioindicating parameter of Bahia de Mazatlan, Mexico

Hace 25 años la UNAM desarrolló un programa de observación y muestreo de mareas rojas en la Bahía de Mazatlán (1979-2003), con el propósito de conocer su evolución a través del tiempo. Se apoyó en observaciones diarias de las decoloraciones (manchas) y los resultados indican cinco tendencias: I) Años sin días de manchas, los cuales están asociados al fenómeno de El Niño; II) Años de días con manchas súbitas; III) Años con un número similar de días de manchas; IV) Años con una disminución progresiva de los días de manchas; V) Años con un aumento gradual de manchas. El promedio anual es de 25.3 días de decoloraciones. En el año 2000 hubo 96 días de decoloraciones y representa el más alto en los 25 años de registros. En marzo es donde existen mas frecuentemente las manchas de mareas rojas (174 días) incluyendo las tóxicas. Estas tendencias nos indican un comportamiento continuo de la máxima concentración de las microalgas y por lo tanto desempeñan un papel muy importante para conocer el deterioro ambiental que sufren las zonas costeras por aumento de microalgas nocivas y tóxicas

In order to verify whether harmful algal blooms increased since 1979, a long-term project for the study of harmful algal blooms in Mazatlán Bay began 25 years ago (1979-2003). Observations were done daily and results indicate five trend types: I) years without discoloration related to El Niño events; II) years with days of sudden occurrence of discoloration, related to the release of accumulated energy after El Niño events; III) years with similar number of discoloration events (days); IV) years with a progressive decrease of the occurrence (days) of discoloration events; V) years with gradual increase of discoloration events. The annual mean average of discoloration is of 25.3 days. Ninety-six discoloration events were recorded during year 2000, and represent the highest number of events in the last 25 years. Discoloration events are more common in March (174 days), harmful and toxic blooms included. This indicates continuity in the occurrence of the highest content of microalgae, and therefore, it is concluded that they play an important role for a better understanding of the environmental deterioration undergone by the coastal zone due to an increase of harmful and toxic microalgae blooms

Recent progress in understanding the temporal behavior of unicellular organisms

This survey summarizes the findings concerning endogenous oscillations of three unicellular organisms: the dinophyte Gonyaulax polyedra, the ciliate Tetrahymena thermophila and the euglenophyte Euglena gracilis. All of them behave rhythmically and show the common features of zeitgeber action, differential sensitivity and temperature compensation; however, they exhibit some species-specific peculiarities that make each of them suitable for addressing particular chronobiological questions. Although ultradian rhythms have been described for Tetrahymena thermophila and Euglena gracilis, they appear under different conditions: in the first case, a modulation of the period in relation to the concentration of nutrients is observed, whereas Euglena oscillates in an ultradian and circadian fashion simultaneously. Transitions between periodic and aperiodic states can be induced in Euglena gracilis and Gonyaulax polyedra: Euglena gracilis can enter an aperiodic state after repeated exposure to short light pulses (up to I 0 sec) given at intervals of 40 min or less, whereas in Gonyaulax polyedra the circadian oscillator is arrested at temperatures below 12 degrees Celsius. In the arrhythmic state, the oscillator might be driven into singularity within the phase space of a limit cycle attractor; re-initiation from the holding point occurs by transition to a relatively precisely defined new phase. Photoperiodism as another important chronobiological phenomenon can be studied in Gonyaulax polyedra: cells enter the dormant stage of an asexual cyst under short days and a temperature below 16 degrees Celsius. This response can be mimicked by 5-methoxylated indoleamines such as melatonin and 5-methoxytryptamine, which are synthesized by this organism. Melatonin concentration exhibits an endogenous circadian rhythm characterized by a rapid increase shortly after the onset of darkness. Encystment, as induced by indoleamines, is associated with stimulations of bioluminescence. The coupling of the two processes involves, as a common element, the release of protons from an acidic vacuole.

Several clocks may simplify the circadian system of Gonyaulax

The circadian system of Gonyaulax involves at least two pacemaking oscillators. These oscillators have been shown to be distinct at the physiological level by an examination of their rhythmic periods under different conditions, their phase response to pulses of light and darkness and their ability to compensate the rhythms for changes in temperature. It is also known that Gonyaulax can restrict synthesis of different proteins to at least three different times of day. We suggest that the two known oscillators may each control synthesis of a differently timed class of proteins.

The effect of light on the biosynthesis of beta-carotene and superoxide dismutase activity in the photosynthetic alga Gonyaulax polyedra

Daily oscillations of both beta-carotene and superoxide dismutase (SOD) activity are related to the intracellular control of reactive oxygen species (ROS). It is well established that ROS are present in all aerobic cells. We studied the marine dinoflagellate Gonyaulax polyedra which has been extensively used as a model to understand the biological clock at the molecular level. beta-Carotene, besides suppressing singlet molecular oxygen (1O2), may act as a photoreceptor pigment in many photosynthetic cells. The levels of beta-carotene during the day phase were shown to be twice as high as during the night phase. The dose-response curve for light-induced carotenoid synthesis was linear for up to 45 min of light exposure, after which night phase cells contained the same levels of beta-carotene as day phase cells. Cells exposed to light pulses at different times during the dark period displayed the highest beta-carotene induction in the middle of the night. SOD activity of cell-free extracts of G. polyedra was three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. Non-denaturing polyacrylamide gels revealed the presence of several SOD isoenzymes in G. polyedra, including CuZnSOD and MnSOD. Furthermore, G. polyedra SOD cross-reacts with a polyclonal antibody raised against SOD. In addition to being gene regulated by ROS concentration, G. polyedra SOD expression seems also to be under the control of the biological clock.

Imaging oscillations in Gonyzaulax: a chloroplast rhythm of nitrate reductase visualized by immunocytochemistry

Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.

Chronobiology of indoleamines in the dinoflagellate Gonyaulax polyedra: metabolism and effects related to circadian rhythmicity and photoperiodism

The marine bioluminescent dinoflagellate Gonyaulax polyedra is capable of producing various indoleamines. The first enzyme in their formation, tryptophan hydroxylase, exhibits a high-amplitude circadian rhythm with a maximum during photophase. Hydroxyindole-O-methyltransferase shows a biphasic pattern with a major maximum during scotophase. 5-Methoxylated indoleamines, such as melatonin and 5-methoxytryptamine, peak at the beginning and in the second half of scotophase, respectively. A drop in temperature from 20 to 15 degrees Celsius leads to dramatic increases of melatonin, up to more than 50 ng/mg protein. This effect may explain why a lower temperature sensitizes this organism to photo-periodic, indoleamine-mediated induction of asexual cysts. Melatonin can be catabolized either enzymatically or non-enzymatically. The non-enzymatic pathway involves free radicals, e.g., photooxidant cation radicals, and leads to the formation of N1-acetyl-N2-formyl-5-methoxykynuramine. Enzymatic catabolism comprises deacetylation to 5-methoxytryptamine and formation of 5-methoxytryptophol. 5-Methoxytryptamine represents a key substance acting as a stimulator of bioluminescence and a mediator of the encystment response. It opens proton channels in the membrane of an intracellular acidic vacuole system which is loaded by the action of a V-type ATPase, as shown by experiments using bafilomycin A1.