O. Roger Anderson

The application of molecular phylogenetics to research on protists has substantially transformed our understanding of their evolution and systematics. More recently, advances in molecular technology, including high throughput sequencing, has opened new avenues for genomic analyses that elucidate major aspects of protistan biology across all levels of biological organization from cellular to ecosystems. This is a review of recent advances (particularly in the last two decades) of transcriptomic research on heterotrophic and autotrophic protists within three major topics: (i) Physiology and metabolism, (ii) Development and life cycles, and (iii) Environmental and ecological studies. Emphasis is placed on selection of representative research that highlights findings across diverse taxonomic groups within each of the three topics. Examples are drawn from parasitic as well as free-living taxa to provide a broad overview of some of the research strategies, and major findings, that have emerged from application of transcriptomics and related techniques in advancing our understanding of protistan biology.

Marshes bordering rivers and estuaries are productive ecosystems that interact dynamically with the adjacent water mass. This is a multi-year study (2019–2022) of seasonal changes in the density of naked amoebae in monthly samples from the surface water of the Hudson estuary near Piermont, N. Y. with relationships to key environmental variables (surface water temperature, salinity, Secchi depth representing turbidity, and enterococcus bacterial counts). During the colder months (November to March), when decayed leaves and litter from the deciduous marsh grass produced organic matter in the sediment surface, the mean abundance of active amoebae ± standard error of the mean (SEM) was higher (3.07 ± 0.99 × 10⁴/ L, N = 7). In warmer months (May to September) the abundance of amoebae was lower (1.35 ± 0.29 × 10⁴ / L, N = 10). A multivariate linear regression analysis was performed relating amoeba abundance to four major water mass variables, resulting in the following statistically significant equation (p = 0.03): A_D = 0.121 × T + 0.301 × L – 0.047 × S + 0.359 ×  C,  where: AD = active amoebae density (× 10 /L), T = temperature ( C), L = tide level (m), S = Secchi disc depth (cm) and C = bacterial enterococcus concentration (number/ml). In general, given the increasing evidence of the potential importance of amoeboid protists in aquatic ecosystems, further research is warranted on their role in food webs and the carbon biogeochemical cycle within heterotrophic estuarine and coastal waters.

Cochliopodium megatetrastylus n. sp. is described based on light microscopy, fine structure and molecular genetic evidence. Amoebae are broadly oval to somewhat triangular during locomotion with average length of 37 μm and breadth of 50 μm, and surrounded by a hyaloplasm margin, somewhat narrow when at rest but more expanded during locomotion (~ 5–10 μm at the anterior). Sparsely occurring subpseudopodia, barely emergent from the hyaloplasm, are blunt and finger-like, occasionally becoming adhesive laterally or at the posterior. Cysts develop after 2–3 weeks in culture and are round with a distinct margin, decreasing in size from 20 to 5 μm during maturation. The granuloplasm contains refractile crystals. A vesicular nucleus (~ 6 μm), containing a nucleolus (2–3 μm), is variable in shape from somewhat lenticular in section to irregularly rounded with undulating or lobed margins. Surface scales (~ 0.3 μm in height) have an apical deeply concave funnel-like collar (~ 0.15 μm deep), without a spine, composed of radial fine rays and concentric filaments forming a finemesh, supported on four non-cross-linked styles (~ 0.2 μm apart) attached to a round to broadly angular base plate (0.6–1 μm) with a fine gridtexture. Cysts are rounded and enclosed by an organic wall bearing remnants of the scales on its outer surface. Both concatenated analysis of SSU-rDNA and COI genes and comparative morphologies support the designation of Cochliopodium megatetrastylus n. sp. as a new species.

This is a review of over 400 published research papers on free-living, non-testate amoebae during the approximate last half century (1965-2017) particularly focusing on three topics: Biogeography, Ecology, and Physiology. These topics were identified because of the substantial attention given to them during the course of the last half century, and due to their potential importance in issues of local and global expanse, such as: aquatic and terrestrial stability of habitats, ecosystem processes, biogeochemistry and climate change, and the role of eukaryotic microbes generally in ecosystem services. Moreover, there are close epistemological and thematic ties among the three topics, making a synthesis of the published research more systematic and productive. The number of reviewed publications for each of the three individual topics is presented to illustrate the trends in publication frequencies during the historical period of analysis.  Overall, the number of total publications reviewed varied somewhat between 1965 and early 2000 (generally less than 10 per year), but increased to well over 10 per year after 2000. The number of Biogeography and Ecology studies identified in the online citations increased substantially after the mid 1990s, while studies focusing on Physiology were relatively more abundant in the first decade (1965-1974) and less were identified in the 1985 to 2004 period. Citations to the literature are listed in tables for each of the three topics for convenience in retrieving references to specific aspects, and representative examples of the cited research in the tables are reviewed in the text under subheads dedicated to each of the three topics. Biogeographic studies largely focused on the geographic distribution and localized patterns of occurrence of amoebae, with more recent studies incorporating more attention to likely correlates with environmental and biotic factors in the distribution and community composition of amoebae. Ecological studies reviewed in the later decades tended to focus more on community dynamics, the effects of environmental variables on communities (including climate-related topics), a trend toward more physiological ecology studies, combined field-based and experimental studies, and incorporation of newer methodologies such as molecular genetics. In general, physiology studies in the first decades of the review tended to focus on topics of cell physiology such as basic biochemistry, enzyme assays, mechanisms of cell division and development, encystment, and motility. Later studies examined broader topics such as osmoregulation, nutrition, fine structure evidence of cellular changes during the life cycle (including encystment and excystment), and issues related to asexual and sexual reproduction, with increasing substantial evidence of evolutionary patterns and phylogenetics based on molecular evidence.  A final section on Conclusions and Recommendations summarizes the findings and presents some potentially productive approaches to future research studies on Amoebozoa within the three designated topics of analysis.

The ecology of tundra terrestrial environments has gained increasing attention due to potential major changes resulting from global warming and climate change. However, the composition of terrestrial microbial communities and their role in the biogeochemical carbon cycle are less well studied. This is the first report of the C-biomass of bacteria, fungi, and representative protozoa (heterotrophic nanoflagellates, naked amoebae, and testate amoebae) in Alaskan tundra soil samples, and the effects of glucose solution enrichment in laboratory studies simulating release of soluble organic compounds as may occur during permafrost melt and increased plant root exudates due to global warming. The data for three moss-rich surface samples, two in spring and one in summer (2011), are reported for C-supplemented (8,000 μg glucose-C) and non-supplemented treatments in laboratory culture. Seven days after supplementation, fungal C-biomass in the glucose-treated and untreated samples were similar in the range of 5 to 11 mg g–1 soil dry weight, the highest values in the summer samples. The bacterial C-biomass was the next highest in the range of 20 to 120 μg g–1 soil dry weight, followed by heterotrophic nanoflagellates (2 to 14 μg g–1 soil dry weight). The naked amoebae (0.13 to 0.94 μg C g–1 soil dry weight) and testate amoebae (2 to 20 ng C g–1 soil dry weight) contributed the least C-biomass. All of the bacterial and protozoan treatments showed increased biomass with glucose supplementation. Based on size, and C-biomass estimates, the phagotrophic protozoa appear to be organized in a classical bacterial-based trophic hierarchy (i.e. bacteria – nanoflagellates – naked amoebae – testate amoebae, in ascending order). Correlations of the C-biomass of bacteria to each of the protozoa, provided further evidence of a trophic pyramid; bacteria vs. nanoflagellates (r = –0.986), indicating top-down control by predatory flagellates, bacteria vs. naked amoebae (r = –0.361) and bacteria vs. testate amoebae (r = –0.131), each of decreasing magnitude as would be predicted for higher level consumers. Estimates of bacterial predation indicated strong predatory pressures on bacteria by the protozoa, greater with C-supplementation compared to the non-supplemented treatments.

Lichens are widely recognized as important examples of a fungal-algal or fungal-cyanophyte symbiosis; and in some cases they are a major food source for some animal grazers such as caribou (Rangifer tarandus), especially in the Arctic during winter. However, relatively little is known about the ecology of their co-associated bacterial and protistan communities. This is one of the first reports of an analysis of microbial communities associated with rock-dwelling foliose lichens (Flavoparmeliasp.), including a more detailed analysis of the microbial communities associated with segments of the shield-like, radially arranged lobes. Samples were taken from lichens on granite boulders beneath an oak and maple tree stand on the Lamont-Doherty Earth Observatory Campus, Palisades, N.Y. The bacteria and protist members of the lichen associated microbial communities are comparable to recently reported associations for foliose lichens growing on tree bark at the same locale, including the presence of large myxomycete plasmodial amebas, heterotrophic nanoflagellates, and naked and testate amebas. To obtain evidence of possible differences in the microecology of different portions of each radial lobe, three segments of the radial lobe in the shield-like lichen were sampled: 1) inner, more mature, central segment; 2) middle section linking the central and peripheral segments; and 3) outer, peripheral, usually broader, less closely attached segment. The mean densities (number/g) and biomasses (µg/g) of bacteria and heterotrophic nanoflagellates were highest in the older central segment and lowest in the peripheral segment of the radial lobes, especially when expressed on moist weight basis. Large myxomycete plasmodial amebas were typically located in the outermost segment of the radial lobe. The proportion of vannellid amebas (Vannella spp. and Cochliopodium spp.) were significantly more abundant in the samples of the inner lobes compared to non-vannellid amebas that were more prevalent in the outer lobes. The outer segment of the thallus lobe was typically more spongiose and absorbed more water per unit weight (based on a wet/dry-weight ratio) than the innermost segment. In general, patterns of densities and taxonomic composition of bacteria and eukaryotic microbes intergraded from the inner most segment to the outer part of each lobe – indicating a possible microecological gradient, coincident with the age-related and morphological radial gradations of the lobe. Overall, the evidence shows that the radial variation in the morphology and age-related variables of the three lobe segments may affect the microenvironment of the lobe segments and hence influence the organization of the microbial communities within each segment.

Naked amoebae are among the most abundant soil protists, especially in highly productive soils. Their capacity to produce cysts during unfavorable growth periods, especially drying, enhances their survivability as resting stages and promotes dispersal by wind and air currents. However, the dynamics of their cycles of encystment and active growth are poorly documented. Using a recently developed culture observation method, including a dried preparation stage to detect encysted amoebae, data are presented on the ratios of active and encysted stages of naked amoebae based on field samples from diverse terrestrial sites differing in plant cover and moisture content during spring and summer months 2008 at a location in northeastern U.S.A. Percentage of encysted amoebae varied between 32% and 100% depending on locale and moisture content. Carbon content of the cysts (estimated from recently excysted individuals) relative to trophic stages varied between 22% and 100% at these same locales. Laboratory experimental studies of winter soil samples, that were cultured at 25oC to promote amoeba community growth, indicated that a dynamic relationship exists between active and encysted stages during proliferation with varying ratios depending on the moisture content and qualities of the soil at the collection site, thus suggesting a revised model as presented here of the encystment-excystment cycle for populations during a growth succession.

Our best evidence is that life arose in the marine environment, and over many millennia of evolutionary proliferation, punctuated by occasional massive extinctions, marine protists have developed remarkably elegant and sometimes complex relationships with prokaryotic and eukaryotic symbionts. Current evidence of the range of marine protist taxa possessing symbionts, including their diversity and physiological functional relationships, is reviewed within an ecological context. Some perspectives are presented on potential opportunities for new avenues of research in unraveling the remarkable adaptive value of two or more genetically diverse marine unicellular organisms living in a close structural and physiological relationship.

Respiratory CO2 emissions from laboratory freshwater microcosms enriched with organic C (glucose and amino acids) and kept in the dark at 24°C were compared to control microcosms without C enrichment for two different freshwater pond sources. The purpose was to estimate experimentally the rate of respiratory CO2 emission from organically polluted freshwater ecosystems compared to non-enriched water. Experiment One, used pond water collected at the Lamont-Doherty Earth Observatory campus; and Experiment Two used pond water, inoculated with natural detritus, obtained from North Carolina. At peak respiration, the net efflux of CO2 (enriched minus control) to the atmosphere was ~ 90 nmol min–1 L–1 (Day 7, Experiment One) and ~ 240 nmol min–1 L–1 (Day 3, Experiment Two). The corresponding net efflux of C to the atmosphere was 25 nmol C min–1 L–1 (Day 7, Experiment One) and 65 nmol C min–1 L–1 (Day 3, Experiment Two). Peak CO2 emissions from the organic-enriched microcosms expressed as μmol m–2 s–1 (as more typically reported for natural bodies of water) were 0.20 for Experiment One, and 0.42 for Experiment Two, at a surface layer depth of approx. 20 cm, i.e. the microcosm depth. There was a relatively large correlation between respiratory CO2 emission and bacterial densities in the organic-C enriched microcosms (r = 0.76), but a smaller correlation (r = 0.32) in the non-enriched, control microcosm. Further broad scale research, robustly sampling natural bodies of organically polluted water, is needed to confirm and better establish the results of the research reported here using microcosms.

We describe the amoeboid isolate ATCC© 50593™ as a new taxon, Telaepolella tubasferens n. gen. n. sp. This multinucleated amoeba has filose pseudopods and is superficially similar to members of the vampyrellids (Rhizaria) such as Arachnula impatiens Cienkowski, 1876, which was the original identification upon deposition. However, previous multigene analyses place this taxon within the Gracilipodida Lahr & Katz 2011 in the Amoebozoa. Here, we document the morphology of this organism at multiple life history stages and describe data underlying the description as a new taxon. We demonstrate that T. tubaspherens is distinct from Arachnula and other rhizarians (Theratromyxa, Leptophrys) in a suite of morphological characters such as general body shape, relative size of pseudopods, distinction of ecto- and endoplasmic regions, and visibility of nuclei in non-stained cells (an important diagnostic character). Although Amoebozoa taxa generally have lobose pseudopods, genera in Gracilipodida such as Flamella and Filamoeba as well as several organisms previously classified as protosteloid amoebae (e.g. schizoplasmodiis, cavosteliids and Stemonitales) present filose pseudopodia. Thus, classification of amoeboid organisms merely by filose-lobose distinction must be reconsidered.

Naked amoeba densities, sizes, biodiversity and carbon content were examined in relation to two particle size fractions (< 200 μm and > 200 μm) of suspended matter in the water column of the Hudson Estuary at a near-shore location south of the Tappan Zee, Palisades, New York. The densities varied markedly among the two particle fractions, and therefore the mean densities were not significantly different between the larger and smaller particle fractions. In contrast, the mean sizes and mean carbon content were statistically greater on larger size suspended particles compared to smaller size particles. There was a broader size range of amoebae on the larger particles, including very large Cochliopodium, Vannella, Mayorella, and Hartmannella species suggesting a larger biodiversity, also indicated by a larger diversity coefficient for the > 200-μm-particle fraction compared to the < 200-μm-particle fraction, 4.51 and 4.18, respectively. In conclusion, the size of suspended particulates in the water column of near-shore, estuarine habitats may have a significant influence on the composition of naked amoebae communities and their ecological roles, especially the organization of particle-associated microbial food webs.
 

Current research is reviewed on aquatic and soil microbial ecology with attention to the fate of organic carbon in bacterial-based protist food webs, including some new data. Particular attention is given to the effects of pulsed sources of low-molecular weight organic sources of carbon on soil respiration, changes in bacterial, nanoflagellate, and naked amoeba C-biomass, and evidence for throughput of carbon in microbial food webs in Arctic and some low-latitude, temperate soil environments. The proportion of pulsed sources of glucose-C that is sequestered in microbial biomass relative to loss as CO2 is examined in laboratory experimental studies, and implications of the research for microbial community dynamics and global warming due to terrestrial sources of respiratory CO2 are discussed.

We describe Diplophrys parva n. sp., a freshwater heterotroph, using fine structural and sequence evidence. Cells are small (L = 6.5 ± 0.08, W = 5.5 ± 0.06 µm; mean ± SE) enclosed by an envelope/theca of overlapping scales, slightly oval to elongated-oval with rounded ends, (1.0 × 0.5–0.7 µm), one to several intracellular refractive granules (~ 1.0–2.0 µm), smaller hyaline peripheral vacuoles, a nucleus with central nucleolus, tubulo-cristate mitochondria, and a prominent Golgi apparatus with multiple stacked saccules (~ 10). It is smaller than published sizes of Diplophrys archeri (~ 10–20 µm), modestly less than Diplophrys marina (~ 5–9 µm), and differs in scale size and morphology from D. marina. No cysts were observed. We transfer D. marina to a new genus Amphifila as it falls within a mo-lecular phylogenetic clade extremely distant from that including D. parva. Based on morphological and molecular phylogenetic evidence, Labyrinthulea are revised to include six new families, including Diplophryidae for Diplophrys and Amphifilidae containing Amphifila. The other new families have distinctive morphology: Oblongichytriidae and Aplanochytriidae are distinct clades on the rDNA tree, but Sorodiplophryidae and Althorniidae lack sequence data. Aplanochytriidae is in Labyrinthulida; the rest are in Thraustochytrida; Laby-rinthomyxa is excluded.