Strains, culture conditions, and environmental samples
Five strains of Euglena sanguinea from algae collections were used in the study: SAG 1224-30 (SAG, Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen, Germany, as Euglena magnifica E.G.Pringsheim), Henderson (the strain isolated from toxic bloom from a pond in Texas), MI-20 and MI-51 (MI, Michigan isolate Triemer Lab, Department of Plant Biology, Michigan State University), and ACOI 1267 (ACOI, Culture Collection of Algae at the Department of Botany, University of Coimbra, Portugal). In the control group experiments, DNA samples from nine Euglena species were used: E. rubra A. D. Hardy (MI 103), E. splendens (MI 47), E. tristella S. P. Chu (NJ, New Jersey isolate Triemer Lab, Department of Plant Biology Michigan State University), E. sociabilis (ACOI 920), E. deses Ehrenberg (ASW08075, now available from CCAC, Culture Collection of Algae, University of Cologne, Germany as E. intermedia Matvienko CCAC 2443 B), E. clara Skuja (SAG 25.98), E. gracilis G. A. Klebs (SAG 1224-5/25), E. laciniata (SAG 1224-31), and E. viridis Ehrenberg (SAG 1224-17d). All strains were cultivated in a liquid soil–water medium enriched with a small piece of garden pea (medium 3c, Schlösser 1994) and kept in a growth chamber maintained at 17 °C and a 16:8-h light/dark cycle, ca. 27 μmol photons m−2 s−1 provided by cool white fluorescent tubes. Additionally, three environmental fresh water samples from Poland containing E. sanguinea cells were used. Environmental sample 1 was collected from a small pond in Rudawka village (53° 51′ 56.5″ N, 23° 30′ 52.6″ E) in July 2015; the other two samples were collected from field ponds near Urwitałt village: sample 2 (53° 49′ 09.5″ N, 21° 39′ 21.8″ E) in June 2011 and sample 3 (53° 50′ 43.1″ N, 21° 36′ 42.3″ E) in June 2012. From each pond, a 10-L sample was collected, and plankton nets with a mesh size of 10, 50, and 100 μm were used to increase density (up to 1 L) and exclude bigger plankton organisms and other macroscopic objects. Samples were transported to the laboratory and were centrifuged (100 mL of each sample); the sediment was suspended in 10 mL of water and split into separate Eppendorf tubes (1 mL) and stored at − 20 °C until needed for DNA isolation. The presence of E. sanguinea cells in samples was confirmed with a NIKON Eclipse E-600 microscope with a differential interference contrast, equipped with the NIS-Elements Br 3.1 software (Nikon). Also, the population density of each species was estimated as follows: (o) cells very occasionally observed in one drop (50 μL of the 10-mL sample after centrifugation), (+) 5–10 cells, (++) 11–20 cells, (+++) 21–30 cells, and (++++) over 30 cells.
Based on the alignment of all available euglenid nSSU rDNA sequences, the regions for primer design were chosen according to the following principles: (i) regions conserved for E. sanguinea (GenBank numbers: strain Argentina JQ281804, Henderson JQ281805, and SAG 1224-30 JQ281806), but dissimilar to any other species of euglenids, were chosen; (ii) intraspecific variations within the region were flanked by the primers; and (iii) the length of the PCR product had to be appropriate for efficient amplification. Two sets of species-specific primers were designed manually—the external primers sangF0/R0 (encompassing the region between helix 29 and 45 in the secondary structure of nSSU rDNA; sangF0: CTGYGGGCGCCACGCCCCCTTG, sangR0: ACGGACTTGCRGGGTTTCCCAGC) and the internal primers sangF1/R1 (between helix 30 and 45; sangF1: CGCCCCCTTGACCGAGAAATCCG, sangR1: GCCRGGGCCCRCAGAARACGAGG).
Three types of templates were used: (i) DNA isolated from cultures, (ii) DNA from lysis of a single cell/a defined number of cells, and (iii) DNA isolated from environmental samples (fresh water reservoirs). Total genomic DNA from cell cultures and environmental samples had been purified with DNeasy Tissue Kit (Qiagen) in accordance with the animal tissues protocol. Single cell lysis was performed according to the Lax and Simpson (2013) procedure, slightly modified. Single cells were isolated with a micropipette using a micromanipulator (MM-89, Narishiege) installed on a Nikon Ni-U microscope and collected in 0.2-mL PCR tubes. Probes with 1, 5, 30, and 100 cells of E. sanguinea were prepared. Liquid traces were removed by centrifuging in a Speed Vac concentrator, followed by the addition of 5 μL of the Phusion GC PCR buffer (no additional buffer was used in the subsequent PCR reaction). The cells were lysed using five freeze/thaw cycles (liquid nitrogen/heating block 95 °C) and used directly in PCR.
PCR amplification and sequencing
The annealing temperature for the two sets of primers was optimized independently in a gradient PCR reaction (50–72 °C). The final conditions were as follows: a 25-μL reaction mixture contained 0.5 U Phusion High-Fidelity DNA Polymerase (Thermo Scientific), 0.2 mM dNTPs, 1.5 mM MgCl
, 5 pmol of each primer, reaction buffer GC (Thermo Scientific), and Q-solution (Qiagen). The PCR protocol consisted of 2 min at 98 °C, followed by nine initial cycles comprising the following steps: 30 s at 98 °C, 30 s at 62 (sangF0/R0) or 60 °C (sangF1/R1), and 20 s at 72 °C, then by 39 cycles comprising steps of 15 s at 98 °C, 15 s at 62 or 60 °C, and 20 s at 72 °C. The final extension step was performed for 5 min at 72 °C. As a template, 10–50 ng of DNA was used in standard PCR reaction, but low concentrations of DNA were also tested in the range 1–0.001 pg. Nested PCR was used in order to make the reaction more sensitive and specific (sangF0/R0 primers in the first round, sangF1/R1 in the second round), particularly for amplification of DNA derived from single/defined number of cells. The conditions were as described above. In the second round as a template, 1 μL of the mixture from the first round was used. The PCR protocol for the first round was as described above (annealing 62 °C); the second round consisted of the initial step 2 min at 98 °C, followed by 39 cycles comprising 15 s at 98 °C, 15 s at 60 °C, and 20 s at 72 °C. The final extension step was performed for 5 min at 72 °C. The control PCR reactions were also performed with DNA stemming from various Euglena species. All PCR reactions were carried out in the presence of positive (DNA from E. sanguinea) and negative (water or buffer) controls. Chosen PCR products were sized on agarose gels, purified and sequenced directly from both strands using the BigDye Terminator Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems).