AQUATIC MICROBIAL ECOLOGY Vol. 44: 85–96, 2006 Published August 16 Aquat Microb Ecol Acidotropic probes and flow cytometry: a powerful combination for detecting phagotrophy in mixotrophic and heterotrophic protists Wanderson F. Carvalho*, Edna Granéli Marine Science Department, University of Kalmar, 391 82 Kalmar, Sweden ABSTRACT: Studies with phagotrophic organisms are hampered by a series of methodological con- straints. To overcome problems related to the detection and enumeration of mixotrophic and hetero- trophic cells containing food vacuoles, we combined flow cytometry and an acidotropic blue probe as an alternative method. Flow cytometry allows the analysis of thousands of cells per minute with high sensitivity to the autofluorescence of different groups of cells and to probe fluorescence. The method was first tested in a grazing experiment where the heterotrophic dinoflagellate Oxyrrhis marina fed on Rhodomonas salina. The maximum ingestion rate of O. marina was 1.7 prey ind.–1 h–1, and the fre- quency of cells with R. salina in the food vacuoles increased from 0 to 2.4 ± 0.5 × 103 cells ml–1 within 6 h. The blue probe stained 100% of O. marina cells that had R. salina in the food vacuoles. The acidotropic blue probe was also effective in staining food vacuoles in the mixotrophic dinoflagellate Dinophysis norvegica. We observed that 75% of the D. norvegica population in the aphotic zone pos- sessed food vacuoles. Overall, in cells without food vacuoles, blue fluorescence was as low as in cells that were kept probe free. Blue fluorescence in O. marina cells with food vacuoles was 6-fold higher than in those without food vacuoles (20 ± 4 and 3 ± 0 relative blue fluorescence cell–1, respectively), while in D. norvegica cells were 4.5-fold brighter than the ones without food vacuoles (291 ± 155 and 64 ± 23 relative blue fluorescence cell–1, respectively). The use of acidotropic probes can prevent fix- ation artifacts such as regurgitation of food vacuoles and changes in the cellular characteristics. The combination of flow cytometry and an acidotropic probe proved to be an efficient tool in detecting phagotrophy in mixotrophic and heterotrophic marine phytoplankton species. KEY WORDS: Mixotrophy · Phagotrophy · Acidotropic probes · Flow cytometry Resale or republication not permitted without written consent of the publisher INTRODUCTION cent microspheres (Smalley et al. 1999), fluorescently labeled bacteria and algae have been used to investi- Studies in which phagotrophy is addressed are still gate feeding behaviors and to measure ingestion rates seriously hampered by tedious and often time-consum- of planktonic protists (Li et al. 1996). ing methods (Riemann et al. 1995). Over the last Although the use of fluorescently labeled prey has decades, many methodologies have been developed to resulted in an increased understanding of phagotro- detect phagotrophy in heterotrophic and mixotrophic phy, advances in this field have been slow (Granéli & phytoplankton species. Visually detected and manu- Carlsson 1998). Usually a large number of cells must ally counted tracer-based techniques are the most be counted manually with the help of a microscope in common approaches for estimating phagotrophy in order to achieve statistically significant ingestion rates, algae (Havskum & Riemann 1996, Granéli & Carlsson a monotonous and lengthy procedure. In addition, epi- 1998, Legrand et al. 1998, 2001, Pålsson 2001). Surro- fluorescence microscopy has a low resolution with gate prey, including dye and starch particles, fluores- regard to detection of ingested fluorescent prey, espe- *Email: [email protected] © Inter-Research 2006 · www.int-res.com 86 Aquat Microb Ecol 44: 85–96, 2006 cially in pigmented mixotrophs that exhibit high auto- the analysis is to use automatic particle counting fluorescence (Riemann et al. 1995, Havskum & Rie- (Marie et al. 2004). One of the most successful auto- mann 1996, Hansen 1998, Hansen & Calado 1999, mated cell counting and analysis methods is flow Setala et al. 2005). Techniques that radioactively label cytometry, which is increasingly being used to charac- the prey (Nygaard & Tobiesen 1993, Granéli et al. terize the microbial planktonic community structure 1997) have the advantage of being analytically sensi- in aquatic systems (del Giorgio et al. 1996, Cucci & tive. However, these techniques have limitations, such Sieracki 2001). In the last decade, flow cytometry has as short-term incubations and the large amount of han- been successfully applied to enumerate bacteria (del dling required (Smalley et al. 1999). Moreover, these Giorgio et al. 1996, Marie et al. 1997, Troussellier et approaches may have several sources of contamina- al. 1999), marine viruses (Marie et al. 1999), small cili- tion: transfer of radioactivity from one organism to ates (Lindström et al. 2002), and most recently to ver- another without effective ingestion, labeled bacteria ify phagotrophy in heterotrophic cells based on aci- can be attached to the theca of mixotrophs or hetero- dotropic probe fluorescence (Rose et al. 2004). In trophs instead of being ingested, and the cell mem- contrast to the time-consuming microscopy-based branes can also become contaminated by the isotopes methodologies (Lepesteur et al. 1993, Granéli & Carls- (Legrand et al. 1998). son 1998, Lindström et al. 2002), flow cytometry Thus, results obtained by the use of artificial or allows single-cell analysis of thousands of cells within manipulated prey coupled with microscopy can be minutes (Avery et al. 1995, Collier 2000, Marie et al. affected by a number of errors. (1) Organisms that 2004), providing statistically significant data (Marie et can actively select their prey might reject artificial or al. 1997, 1999, Collier 2000). The precision of flow surrogate prey and phagotrophy may go undetected cytometric counts are on average 10 times higher than or be underestimated (Bockstahler & Coats 1993a,b, epifluorescence counts, and sample processing time Havskum & Riemann 1996, Legrand et al. 1998, can be reduced by 10- to 20-fold (del Giorgio et al. Smalley et al. 1999, Stoecker 1999, Hammer et al. 1996). 2001). In addition, artificial prey does not allow Although flow cytometry is a versatile tool, it faces growth and adaptation of predators to prey (Hammer the same optical problem as microscopic analysis when et al. 2001) and some protists can egest the particles 2 analyzing heterotrophs that have fed on colorless prey or 3 min after ingestion (Weisse 2002), leading to or mixotrophic cells that have fed on autotrophic prey. unrealistic ingestion rates. (2) Human counting errors Due to interference from the strong autofluorescence (Marie et al. 2004) enhanced by the difficulties of of chlorophyll and accessory pigments of the mixo- identifying phagotrophy in algae species (Porter trophs, it is virtually impossible to detect the ingested 1988, Legrand et al. 1996, Bouvier et al. 1998, Granéli prey, as both have the same fluorescence. The problem & Carlsson 1998, Ishii et al. 2002, Rose et al. 2004), is even worse for heterotrophic prey, which lack and questionable detection of phagotrophy (Stoecker pigments. 1999) also lead to under- or overestimations of the To overcome the difficulties described above, we occurrence of phagotrophy among phytoplanktonic developed a method that combines the flexibility and species. speed of flow cytometry techniques with an acido- The food vacuole method, mostly used to estimate tropic probe (LysoSensor™ Blue DND-167, Molecular grazing by heterotrophic dinoflagellates and ciliates, Probes) that stains acidic organelles such as food vac- has also been extended to estimate phagotrophy in uoles. The few published studies using LysoSensor™ mixotrophs (Bernard & Rassoulzadegan 1990, Dolan & probes can be found in medical journals (Lin et al. Coats 1991, Bockstahler & Coats 1993a). This method 2001). is based on observations of food vacuole content cou- Our main objective was to develop a fast and reliable pled to experimentally determined digestion rates (Li method for detecting phagotrophy in phytoplanktonic et al. 2001). Although the content of food vacuoles in species. The use of flow cytometry in combination with larger protists may be identifiable, this technique is pH-specific probes might solve some of the method- inadequate when studying small flagellates (Riemann ological constraints cited above. Furthermore, our et al. 1995). Another problem is determining whether methodology allows real-time observation of the the particle inside a cell is a prey, parasite or gamete changes in the behavior of heterotrophs and (Granéli & Carlsson 1998, Legrand et al. 1998). Conse- mixotrophs under different environmental conditions quently, the presence of particles inside the cells does such as nutrient sufficiency and deficiency, and differ- not necessarily imply phagotrophy (Porter 1988, ent densities of co-occurring species of prey. This Granéli & Carlsson 1998, Legrand et al. 1998). approach might help to more accurately assess the role An effective way of accelerating cell counting, mini- of mixotrophic and heterotrophic predators in aquatic mizing human error and improving the sensitivity of food webs. Carvalho & Granéli: Detection of phagotrophy in mixo- and heterotrophic protists 87 MATERIALS AND METHODS 16 ± 1°C and a light intensity of 100 μE m2 s–1, with a 16:8 h light:dark cycle, till they reached high densities Flow cytometer (FCM) setup. All samples were ana- (4.5 × 103 and 8.5 × 105 cells ml–1 for O.