Brazilian Journal of Biology http://dx.doi.org/10.1590/1519-6984.05016 ISSN 1519-6984 (Print) Original Article ISSN 1678-4375 (Online) Using in situ flow cytometry images of ciliates and dinoflagellates for aquatic system monitoring G. C. Pereiraa*, A. R. Figueiredoa and N. F. F. Ebeckena aPrograma de Engenharia Civil, Núcleo de Transferência de Tecnologia – NTT, Instituto Alberto Luiz Coimbra de Pós-graduação e Pesquisa de Engenharia – COPPE, Centro de Tecnologia, Universidade Federal do Rio de Janeiro – UFRJ, Cidade Universitária, Av. Athos da Silveira Ramos, 149, Ilha do Fundão, CEP 21941-909, Rio de Janeiro, RJ, Brazil *e-mail: [email protected] Received: April 1, 2016 – Accepted: October 4, 2016 – Distributed: May 31, 2018 (With 4 figures) 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. Keywords: aquatic systems, microbial ecology, in situ flow cytometry, method. Usando imagem de citometria de fluxo in situ de ciliados e dinoflagelados para o monitoramento de sistemas aquáticos 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. Palavras-chave: sistemas aquáticos, ecologia microbiana, citometria de fluxo in situ, método. 1. Introduction The distribution of marine plankton is highly variable and the habitat factors determining community assemblages in space and time (Bricaud et al., 2012; Batchelder et al., contribute to this variability. Informations about such 2012). The occurrence of geographical variation in nature variability are obtained from different sampling approaches. 240 240/247 Braz. J. Biol. 2018, vol. 78, no. 2, pp. 240-247 Image in flow for aquatic monitoring These approaches involve a range of classical techniques and biomass variation of both groups given the great (Utermöhl, 1958; Hobbie et al., 1977 or Brito at al., 2013) importance in trophic dynamics of pelagic systems, as and devices, including vessel-based equipment, anchored key organisms connecting the microbial loop with the instruments used for in vivo chlorophyll measurements and classical food chain. satellite observations from which the colour of the sea can be determined (Del Giorgio et al., 1996; Dickey, 2001). 2. Material and Methods Phytoplankton populations, for example, display metabolic diversity and a high degree of physiological plasticity (Platt, 2.1. Study site 1981), but their buffering capacity is low (Smayda, 1998). The study was conducted at a fixed site Thus, these populations respond quickly to external forcing (23°00`51.62``S, 43°23`45.10``W) in the biological reserve factors. This responsiveness allows them to serve as good of Barra da Tijuca beach, western of Rio de Janeiro city. It is indicators of environmental conditions. However, our located within the boundaries of the Marapendi ecological understanding of phytoplankton population dynamics and park. This park offers a very attractive natural landscape the factors regulating their populations at meso and micro (Figure 1) displaying white sandbars deposited during the scales remains limited due to our inability to measure the past 5100 years (Roncarati and Menezes, 2005) covered by species composition, size distribution and growth rates of restinga fixing vegetation. The beach, used recreationally phytoplankton (Sosik et al., 2003). The composition and by the population, is on a spit is crossed by Sernambetiba size structure of plankton, for instance, helps to determine Avenue, which provides access to the area. The coastal the carbon and nutrient flows through the ecosystem and seawater is always green. Due to the coastal drain and the can be an important indicator of the response of coastal effects of submarine outfalls, the water shows a degree of environments to anthropogenic disturbances, such as nutrient eutrophication. The seawater temperature, measured by or pollutant inputs. Harmful algal blooms or outbreaks of a reversing thermometer, ranged from 19.3 °C to 20.5 °C toxic organisms (Anderson et al., 2002) commonly coexist during the day, whereas the salinity remained at 35.7 ppm. with species that have similar ecological requirements and The salinity was measured with a Milwaukee MA887 digital environmental tolerances, later called functional groups refractometer (Milwaukee, WI, USA). (Nair et al., 2008). 2.2. Data acquisition However, with the exception of the information furnished by several laboratory experiments (Tillmann, 2004; Rosetta For cytometric data acquisitions, the standard and McManus, 2003; Alonso et al., 2000), little is known CytoSense flow cytometry instrument (CytoBuoy bv, about the functional characteristics of protozooplankton Worden, The Netherlands) was be made submersible (ciliates and microflagellates) and of metazoans or the (CytoSub) by adding a specially designed stainless steel interactions occurring in the overall assemblage, e.g., the housing. The device was connected to a unit converter for interactions among zooplankton, phytoplankton, bacteria power and data transfer from a simple kiosk on the shore and viruses. The first prerequisite for a more realistic in the late summer (March). A long, high-quality cable understanding of the interactions among the factors that is used to attach the device to the converter (Figure 2a). regulate species composition is the availability of detailed The instrument can be connected to the Internet for remote and continuous long-term observations. This type of operation and can detect and record large suspended observation has become possible because new instruments particles (> 1-1000 mm diameter) in relatively large were developed and made available for use in the continuous volumes of water (more than 4 cm3 per sample). The water monitoring of specific organisms or groups Pereira( et al., is pumped into a flow chamber to avoid external turbulence. 2016; Bergkvist et al., 2012, Campbel et al., 2010). However, The flow is controlled by a peristaltic pump. The speed these instruments produced substantial amounts of data. of the pump is adjustable and was set at 7.8 mm3 s-1 for In this context, models based on cytometric signatures have this experiment. The sheath fluid “runs” at a rate of been successfully developed and applied (Embleton et al., 80 cm3 min-1. Each suspended particle crosses a 5 µm 2003; Kenneth et al., 2008; Pereira and Ebecken, 2011). blue laser beam (Coherent Saphyre, 488 nm, 15 mW). The in situ scanning flow cytometry (SFC) technique, The laterally scattered light signals (side scatter, SWS) in conjunction with an add-on image-in-flow module, and the signals from fluorescence within the chamber are represents one of the most promising technologies for dispersed by a concave holographic grating and collected addressing the gap of knowledge in this area. This approach with a hybrid photomultiplier device (HPMT). This optical can be used to study the microscopic constituents of design