Definition and morphology
While there is no unambiguous definition of “protozoa”, a general agreement is to consider protozoa as heterotrophic protists. Protists are defined by Adl and others (2005) as “eukaryotes with a unicellular level of organization, without cell differentiation into tissues”. FLP comprise protists which do not have an obligate parasitic life cycle, although some species such as A. castellanii, Balamuthia mandrillaris, and Naegleria fowleri occasionally cause infections in humans (Marciano-Cabral and Cabral 2003; Khan 2006; Visvesvara and others 2007).
FLP are single-celled microorganisms. Two life stages are usually distinguished: a trophozoite (vegetative cell) and a cyst (also: resting or dormant cyst). However, not all species produce cysts. The trophozoite is the life stage in which the cell feeds and multiplies. Sizes of trophozoites range from a few μm to a few mm. The variety in cell shape is virtually inexhaustible and forms the basis of morphological identification. The body surface consists of a cell membrane but can be covered by scales or be surrounded by a test or lorica (Sleigh 1989). FLP are mostly solitary but some species form colonies. Most FLP are motile (for example, swimming, crawling, or gliding), others float passively in the water column. Several species are attached to surfaces (for example, stones, submerged plants, detritus, and aggregates) by means of a stalk or trailing flagellum. Bacterivorous FLP feed on bacteria. Other modes of nutrition are feeding on protozoa, fungi, algae, or small invertebrates (for example, rotifers), growth on detritus, or absorption of organic material or molecules. Omnivorous FLP use more than 1 nutritional strategy. Some FLP are mixotrophic and combine heterotrophic feeding with photosynthesis. Many FLP have at least 1 contractile vacuole, which is an osmoregulator that pumps, at regular frequency, excess water and dissolved waste products out of the cell. FLP multiply mostly by binary cell division although several species have sexual cycles. Cyst formation (encystment) leads to a fundamental change in trophozoite morphology and physiology, and it can be induced by overcrowding, nutrient depletion, accumulation of certain metabolites, or unfavorable environmental conditions including desiccation, changes in pH, osmolarity, oxygen level, or temperature (Corliss and Esser 1974; Corliss 2001; Gutiérrez and others 2001; Hausmann and others 2003). Cysts are nonmotile and generally smaller than trophozoites and usually have a thick, often double- or multilayered wall (Corliss 2001). The cyst wall is composed of proteins, glycoproteins, and carbohydrates such as cellulose or chitin (Corliss 2001). The cyst shape may vary (for example, spherical, ovoid, pyriform) and several cysts have an outer surface ornamentation (Corliss 2001), which can aid for dispersal. Cysts can survive harsh conditions and, for example, cysts of Acanthamoeba spp. resist gamma and UV irradiation (Aksozek and others 2002), heat (Storey and others 2004) and disinfectants (Kilvington and Price 1990; Storey and others 2004; Coulon and others 2010), and they can remain viable for many years (Mazur and others 1995; Sriram and others 2008). Excystment is the transformation from cyst to trophozoite and is triggered by favorable conditions or chemicals (Hausmann and others 2003).
Based on morphology and locomotion, FLP are divided into the amebae, the flagellates, and the ciliates (Figure 1). This division is used very often for convenience, but it is strongly discouraged by protistologists because it does not take into account the phylogenetic relationships between these organisms (Adl and others 2005). However, for the sake of simplicity and to present a general overview, the FLP-morphogroups amebae, flagellates, and ciliates will be used throughout this review. In the rest of the text, only organisms nonparasitic to humans (“free-living”) are covered. Indeed, each morphogroup contains a few human parasites such as the flagellate parasites Giardia duodenalis (causing giardiasis) and Trypanosoma brucei (causative agent of sleeping sickness), the ameba Entamoeba histolytica (amebic dysentery), and the ciliate Balantidium coli (balantidiosis). Obviously, other obligate parasitic protozoa, such as Plasmodium spp. (causative agents of malaria) and foodborne pathogens such as Cryptosporidium parvum, Cyclospora cayetanensis, Sarcocystis hominis, and Toxoplasma gondii, all organisms classified in the past in the Sporozoa, will also not be discussed in this review.
Figure 1. Examples of morphotypes of free-living protozoa. (A) Acanthamoeba (an ameba), (B) Chilomonas (a flagellate), (C) Tetrahymena (a ciliate). Organisms not drawn to scale. Pictures taken from http://pinkava.asu.edu/starcentral/microscope/. Drawings made by Stuart Hedley and David Patterson, licensed to MBL (micro*scope). Reprinted with permission.
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Amebae are characterized by the possession of pseudopodia (Greek: false feet), which are transient cytoplasmic extensions of the cell. Pseudopodia are used for locomotion and feeding. However, small- and medium-sized amebae often move as a whole without forming pseudopodia (Smirnov and Brown 2004). Amebae are roughly divided into naked amebae, testate amebae, and actinopods. Naked amebae are surrounded by a plasmamembrane. The typical ameboid movement on surfaces results in temporal changes of the cell shape. Some species have a temporally flagellated life stage (for example, N. fowleri). Testate amebae have a plasmamembrane covered with a test (shell). The test is composed of organic material, inorganic salts, or agglutinated particles. Actinopods are star-like amebae with stiffened pseudopodia radiating out of a spherical cell. Some representatives of naked amebae are Acanthamoeba, Amoeba, Hartmannella, and Vannella. Examples of testate amebae are Arcella and Difflugina. The actinopods include the Heliozoa (for example, Actinophrys) and the marine Radiolaria which have a mineral skeleton.
Flagellates possess at least 1 flagellum. If there is more than 1 flagellum, they may be unequal in length and can be differently oriented (Patterson 1998). A flagellum is used for locomotion, feeding, or attachment to a surface, and it emerges near or at the anterior of the cell (Sigee 2005). The structure of a eukaryotic flagellum differs fundamentally from a prokaryotic flagellum. A cross-section of the eukaryotic flagellum shows the typical arrangement of 9 outer doublet microtubules that encircle 2 singlet microtubules (9 × 2 + 2 pattern). Flagellates swim or glide, and the flagellum can beat with a sine wave or be held stiffly, sometimes only moving with the tip (Patterson 1998). Other flagellates are sessile (for example, choanoflagellates) or are temporarily attached to the surface (for example, Bodo saltans). Many species are very small, and cells with a size between 2 and 15 μm are usually described as nanoflagellates, and those of less than 2 μm as picoflagellates (Boenigk and Arndt 2002). Some flagellate groups contain photosynthetic members (for example, Euglena). Therefore, in older literature sources, the division into zooflagellates and phytoflagellates was made. Bodo, Cercomonas, Chilomonas, Goniomonas, Heteromita, Notosolenus, Petalomonas, and Spumella are all examples of flagellate genera commonly found in the environment.
Ciliates have cilia that are structurally identical to flagella but are shorter, occur in higher numbers over the cell body, and differ in beat pattern (Sigee 2005). Cilia are used for locomotion, feeding, attachment, or sensing (Lynn 2008). Cilia are often arranged in distinct rows and they beat in a coordinated fashion. The cell size of ciliates is mostly much larger compared with amebae and flagellates (most ciliates are between 20 and 200 μm in length) (Sigee 2005). Ciliates are motile (crawling, swimming) or are attached to a substrate. Ciliates have 2 different nuclei: a small, diploid micronucleus necessary for reproduction and a large, polyploidy, transcriptionally active macronucleus, which controls the organism's phenotype (Lynn 2008). Ciliates feed by filter-feeding or predation. Filter feeders concentrate prey through the action of cilia, which are sometimes grouped to form special membrane-like structures. Many ciliates have an oral cavity with a cell “mouth” (cytostome) where food propelled through the action of cilia is concentrated into a food vacuole. Undigested material is excreted at the cytoproct which usually is located at the posterior side of the cell. Predatory ciliates catch their food (for example, other ciliates) through specialized organelles such as extrusomes. A special category is the Suctoria, a group of sessile ciliates that lack cilia in their adult stage and capture prey with cytoplasmatic tentacles. The ciliates are a morphologically very heterogeneous group. The textbook example of the ciliates is the slipper animal Paramecium, but others such as Colpoda, Colpidium, Cyclidium, Glaucoma, Tetrahymena, and sessile forms such as Epistylis and Vorticella are easily isolated from nature.
Classification and diversity of FLP
The artificial division of FLP in amebae, flagellates, and ciliates has no phylogenetic value but is often used in teaching. The classification of microscopic eukaryotic organisms has undergone many revisions and it was already clear from the beginning of the 19th century that single-celled eukaryotes were a difficult part in eukaryotic systematics (Scamardella 1999). The eukaryotes were divided according to Whittaker into the Kingdoms Animalia, Plantae, Fungi, and Protista (Whittaker 1969). The classification of protozoa (“animal-like” protists), photosynthetic organisms other than plants and cyanobacteria (“plant-like” protists or (micro)algae), and slime molds (“fungi-like” protists) in the Kingdom Protista served for many years. Several fundamental revisions of Whittaker's classification scheme have followed (for example, Woese and others 1990; Cavalier-Smith 2004) and the idea of protists as a separate group was abandoned. Currently, eukaryotic organisms are classified into 5 supergroups based on ultrastructural, biochemical, and molecular phylogenetic data (Adl and others 2012): (i) the Amoebozoa (including naked and many testate amebae, slime molds), (ii) the Opisthokonta (including Metazoa [sponges, animals], fungi, yeasts, choanoflagellates, Mesomycetozoea, Nuclariids), (iii) the Excavata (including several flagellate genera and the ameba group Heterolobosea), (iv) Sar, a cluster of Stramenopiles (including the chrysophytes, diatoms, brown algae, some fungi-like organisms), Alveolata (including ciliates (Ciliophora), dinoflagellates, Apicomplexa), and Rhizaria (including Cercozoa, Foraminifera, Radiolaria, some testate amebae), and (v) the Archaeplastida (including Glaucophyta, plants, and red and green algae).
The global number of protist species is still hotly debated. Recent estimates range from 16600 to 300000 species (Fenchel and Finlay 2006; Foissner 2008), but it is not unlikely that even these numbers are underestimates. While until recently most protist species were considered to be cosmopolitan, there is increasing evidence that microorganisms (including protists) display biogeographic patterns (Hanson and others 2012; Bates and others 2013). For example, the ciliate Tetrahymena thermophila, a species that is a commonly used as a eukaryotic model organism in genetic and molecular biology studies and bacteria–FLP interaction studies (see later) has recently been shown to have a distribution which is restricted to the eastern United States (Zufall and others 2013). The different opinions on global protist species richness and biogeography can be largely explained by the absence of a generally accepted species concept, the occurrence of cryptic species (defined by Caron 2009 as “morphospecies of protists that contain strains possessing different physiological abilities or mating incompatibilities”), and inadequate methodology such as undersampling of habitats, difficulties to exhaustively characterize communities (underreporting) and in determining dispersal rates (Schlegel and Meisterfeld 2003; Foissner 2008; Caron 2009; Boenigk and others 2012).
Ecological importance of FLP
Bacterivorous feeding by FLP is, together with viral-mediated lysis, one of the main regulatory processes of bacterial biomass (Sherr and Sherr 2002; Pernthaler 2005). Especially heterotrophic nanoflagellates such as bicosoecids and Chrysophyceae (for example, Spumella spp.) and small ciliates are very important bacterial consumers in marine and freshwater ecosystems (Boenigk and Arndt 2002). Numbers of heterotrophic nanoflagellates vary between hundreds to several tens of thousands cells/mL, depending on the trophic system and season (Boenigk and Arndt 2002). Amebae are important grazers of biofilms and are probably, together with flagellates, the most important group of soil protozoa (Ekelund and Rønn 1994; Foissner 1999). Protozoan grazing is often prey-selective, and medium-sized bacterial cells (0.4 to 1.6 μm) are preferably consumed (Pernthaler and others 1996; Hahn and Höfle 2001). Ingestion rates vary from several hundreds to more than 1000 bacteria/protozoan cell/h (Parry 2004). However, food selection is not only determined by prey size. Physiological state of the prey (with preference for bacteria in stationary phase compared to exponentially growing bacteria) (Ayo and others 2009) and chemicals secreted by the prey (Hamels and others 2004) have also been shown to influence prey selectivity. Protozoan characteristics such as feeding forms (for example, phagocytosis and filter feeders), cell size (for example, small flagellates versus large ciliates), and behavior are important as well. Remarkably, recent feeding history of the protozoan had an influence on the response toward prey types (Ayo and others 2009).
Bacteria have developed several antipredator strategies including cell size reduction, modified cell morphology (for example, filamentous growth), modification of cell wall characteristics (for example, surface potential), high-speed motility, and production of exopolymers or toxins (Hahn and Höfle 2001; Matz and Kjelleberg 2005; Pernthaler 2005; Jousset 2012). Biofilm and microcolony formation are other well-known defense mechanisms to resist protozoan grazing, and quorum-sensing is an important communication tool to inform neighboring bacterial cells of upcoming threats (Matz and Kjelleberg 2005). Some bacteria resist digestion through escape from or modification of the phagosome (for example, L. pneumophila), or they survive because of the inefficiency of the protozoan digestive system (Thurman and others 2010).
The combination of selective grazing, protozoan community composition, and bacterial antipredator mechanisms influences the morphological structure and genotypic and taxonomic compositions of bacterial communities (Hahn and Höfle 2001; Jürgens and Matz 2002). Moreover, it is hypothesized that grazing not only selects for bacteria with traits which infer resistance to grazing, but also for traits responsible for virulence (Adiba and others 2010). It was suggested that virulence is maintained for their role in grazing resistance rather than for virulence as such (Adiba and others 2010).
Importantly, as a result of protozoan grazing, dissolved organic matter and inorganic nutrients such as nitrogen and phosphorus are released into the environment. These nutrients enter into the microbial biomass and as such become available for higher trophic levels. For example, as a result of grazing activities in soil, the release of nutrients from consumed bacterial biomass together with a shift to rhizobacterial communities (which are responsible for better nitrogen fixation) both have a beneficial effect on plant growth (Bonkowski 2004).
Finally, FLP serve as food for other organisms such as zooplankton, other small invertebrates, and fish larvae, and as such play an important role in aquatic and terrestrial food webs.
Occurrence in natural habitats and anthropogenic environments
FLP are ubiquitous in aquatic and terrestrial ecosystems. Important habitats include coastal waters, lakes, oceans, ponds, rivers, swamps, sediments, and soils. FLP are distributed all over the water column and can be found in waters with different nutrient and salt contents. In soils, FLP live in the water film around soil particles. Although FLP mainly inhabit water or moist environments, some species are able to survive in soils with very low moisture content such as deserts and the dry valleys on Antarctica. They become active, often for a very limited period, under favorable conditions such as a short rain period. FLP are found in extreme environments such as deep sea vents, geothermal hot springs, and acidic and hypersaline lakes. In addition to water and soil, air can contain viable FLP and cysts (Schlichting 1964; Kingston and Warhurst 1969; Rivera and others 1992; Rogerson and Detwiler 1999). The above-mentioned examples of habitat types illustrate that FLP can tolerate a broad range of physical (for example, temperature and humidity), chemical (for example, pH and oxygen content), and nutritional parameters. The tolerance range can be large or small for certain parameters, and tolerance limits of several species can be broadened by gradual adaptation to new conditions (Hausmann and others 2003).
Besides abiotic environments, FLP are present on or in various organisms. Insects can carry FLP on their body (Maguire 1963; Revill and others 1967a, b) and some FLP inhabit the hindgut of insects such as termites (Ohkuma 2008). Ciliates inside the rumen of ruminants such as cattle and sheep play an important role in the digestion process and contribute substantially to the energy and nutrient balance (Veira 1986; Williams 1986). Roots of plants are colonized with FLP (Bonkowski 2004), as well as the above-ground parts such as leaf surfaces (Ruinen 1961; Bamforth 1971) and bark of trees (Bartošová and Tirjaková 2008). Other examples are mosses (Anderson 2006; Glime 2007), pitchers of pitcher plants (Hegner 1926; Rojo-Herguedas and Olmo 1999), and tank water of bromeliads (Foissner and others 2003).
Given their ubiquity, it is not surprising that FLP have also been detected in various man-made constructions such as cars (Simmons and others 1999), cooling towers (Yamamoto and others 1992; Berk and others 2006; Behets and others 2007), dental unit waterlines (Barbeau and Buhler 2001; Singh and Coogan 2006), drinking water supplies (Thomas and Ashbolt 2011), eyewash stations (Paszko-Kolva and others 1991; Bowman and others 1996), homes (Marciano-Cabral and others 2003; Trzyna and others 2010; Stockman and others 2011), hospitals (Rohr and others 1998), and hospital water networks (Patterson and others 1997; Thomas and others 2006), moisture-damaged buildings (Yli-Pirilä and others 2004), spacecraft (Ott and others 2004), swimming pools (Rivera and others 1993), and wastewater treatment plants (Madoni 1994; Pauli and others 2001).