Vannella

Van Wichelen, Jeroen, D’Hondt, Sofie, Claeys, Myriam, Vyverman, Wim, Berney, Cédric, Bass, David & Vanormelingen, Pieter, 2016, A Hotspot of Amoebae Diversity: 8 New Naked Amoebae Associated with the Planktonic Bloom-forming Cyanobacterium Microcystis, Acta Protozoologica 55 (2), pp. 61-87 : 65

Vannella

The vannellid strains belonged to two lineages in different parts of the Vannella ML phylogeny ( Fig. 1A ). The first lineage had low bootstrap and moderate PP support and contained four Microcystis -associated strains interspersed between 3 Vannella simplex and 1 uncultured clone sequence (p -distances 0.3–3.5%, number of differing positions 4–63). Its sister taxon was Vannella persistens (2.0–3.6%, 24–68). The second lineage (upper part of the phylogeny) had moderate bootstrap and maximal PP support and consisted of six Microcystis -associated Vannella strains (p -distances 0.8–3.0%, 13–28 differing positions). Its closest relatives were unidentified Vannella strains or uncultured clones from natural samples (2.4–9.1%, 32–98).

The locomotive cells of the vannellid strains A17 WVB, A1 SMB, A6 HEB and A7 HEB, all closely related to V. simplex ( Fig. 1C ), shared a mainly flabellate to semicircular, rarely spatulate shape, a length/ width ratio of 0.4–1.6 (mean 0.8) and an average coverage with hyaloplasm about half of the cell length anteriorly, but differed in average size. Locomotive forms of strains A17 WVB, A1 SMB and A6 HEB /A7 HEB were respectively 38–79 (mean 49), 20–56 (35) and 15–60 (31) µm long and respectively 45–83 (65), 20–61 (43) and 20–85 (41) µm wide. On several occasions, trophozoites of strain A17 WVB were observed containing a flagellum-like pseudopodium, either very long and straight or contracted and helical in appearance ( Fig. 1C 17–19). The trophozoites of strain A17 WVB were always covered with fecal pellets. For the other 3 strains this feature disappeared after several months of cultivation, however fecal pellets of about 9 µm in diameter could always be observed freely dispersed in the culture medium.

Locomotive amoebae of strains A4P4 ZHB, A2 SMB, A2 FBB, A1 DVDPB, A2 DVDPB, A3 DVDPB, all belonging to the second vannellid lineage, were fan-shaped however with considerable variations. They could adopt a semicircular ( Figs 1B 1–2 ), flabellate ( Figs 1B 4–5 ) or spatulate shape containing a pronounced tail up to 21 µm long ( Fig. 1B 3 , 8 ). Feeding amoeba attached on Microcystis colonies ( Fig. 1B 9) had a greatest linear dimension of 15–50 µm (mean 31). The hyaloplasm formed a broad margin around the cell and covered 13–71% (40%) of the cell anteriorly. When floating cells settled and became locomotive, the frontal hyaline area could display a wavy front edge ( Figs 1B 2 , 4 , 6 ) and sometimes contained longitudinal ridges ( Fig. 1B 7 ) as a result of transverse wave formation. The vesicular nucleus was 2–7 µm in diameter and contained a centrally located nucleolus of 1–5 µm in diameter (arrowheads in Figs 1B 1–4 ). Usually one small contractile vacuole was present posteriorly (black arrows in Figs 1B 1 , 7 , 8 ), with up to 3 vacuoles in some individuals. Floating amoebae were either radial shaped, 10–35 µm in diameter, symmetric, displaying radially 5–11 pointed and basally thickened (up to 6 µm in width) pseudopodia up to 50 µm in length, or sometimes irregularly shaped with only one to a few thick and round-tipped pseudopodia ( Figs 1B 10–11) or in a few cases even without pseudopodia. Fecal pellets, varying in size between 5 and 13 µm, were present in the culture medium but did not tend to adhere posteriorly. Elliptical to ovoid cysts were observed a few times in old cultures. These had a thin wall (0.5 µm) and a diameter of 11–32 µm. Each had either a round opening of about 6 µm in diameter (white arrow in Fig. 1B 12) or a bone-shaped narrow slit of about 11 µm in length (white arrow in Fig. 1B 13).

Thirty Vannella species are currently recognized, 24 from marine and brackish, 4 from freshwater and 2 from soil environments ( Smirnov et al. 2007). Our phylogenetic analysis placed 4 of our vannellid strains in a clade containing several strains of Vannella simplex , a species with a wide geographically distribution, isolated from both freshwater and brackish habitats and genetically rather variable ( Smirnov et al. 2002, Nassonova et al. 2010). Since our strains also share the relatively large cell size and the coverage by fecal pellets, both typical for V. simplex isolates ( Smirnov et al. 2002), we believe they indeed belong to this species. Our remaining vannellid strains were much smaller in size, lacked the coverage by fecal pellets and formed a separate clade with uncultured clones or unidentified Vannella ’s as their closest relatives. They were clearly separated from two other freshwater species, V. miroides and V. lata . No sequence information is available for V. cirifera , the only other freshwater Vannella species known, however this species was announced a nomen dubium by Smirnov et al. (2002, 2007). For this reason and because of its specific ecology we declared the strains of our second lineage as belonging to a new species, Vannella planctonica .

Figures

Fig. 1.A – 18S rDNA maximum likelihood phylogeny of Vannella, including Microcystis–associated strains (in bold). ML bootstrap values respectively posterior probabilities are shown at the nodes. GenBank accession numbers are given together with the species names. B – LM pictures of V. planctonica (strainsA2FBB: 2, 4, 5, 9–13 andA4P4ZHB:1, 3, 6–8) showing locomotive trophozoites (1–8), a grazing amoeba with a Microcystis cell inside a food vacuole (9), floating forms (10–11) and a cyst stage (12–13). C – LM pictures of V. simplex (strain A17WVB) showing the floating form (14), locomotive amoebae (15–18) with the presence of food vacuoles containing partly digested Microcystis cells (17), posteriorly adhered fecal pellets (15–16), a long flagellum-like pseudopodium encircling a Microcystis cell (17–18) and grazing amoebae on a colony of Microcystis aeruginosa with expanded (arrow) or contracted (arrowhead) flagellum-like pseudopodia visible in some of the trophozoites (19). The presence of a contracticle vacuole, a nucleus or a cyst opening is indicated by a black arrow, a black arrowhead or a white arrow respectively. Scale bars: 20 µm.

Fig. 4. A – 18S rDNA maximum likelihood phylogeny of the family Hartmannellidae, including Microcystis-associated strains of Copromyxa (in bold). ML bootstrap values respectively posterior probabilities are shown at the nodes. GenBank accession numbers are given together with the species names. B – LM pictures of C. microcystidis showing locomotive trophozoites (1–7, 10) containing a pronounced hyaline cap (white arrowheads), a vesicular nucleus with globular nucleolus (white arrows), big crystals (black arrows) and Microcystis cells inside food vacuoles (black arrowheads), non-oriented moving trophozoites (8–9, 11), a grazing amoeba capturing a Microcystis cell (12–14), the floating form (15) and a double-walled cyst stage (16). C – LM pictures of C. vandevyveri showing locomotive trophozoites (1, 6–9), trophozoites during non-oriented movement (2–5, 10–12) and floating forms (13–15). Black arrows indicate the presence of small, refractive crystals in the cytoplasm, the white arrow shows the vesicular nucleus with a globular lacuna-containing nucleolus, white arrowheads indicate a pronounced hyaline cap. Ingested Microcystis cells are visible in (9) and (11). Scale bars: 20 µm.

Fig. 5. LM pictures of Schoutedamoeba minuta showing trophozoites in non-oriented movement (a–f) and the limax-shaped locomotive form (g–i). The presence of a pronounced hyaline cap, small adhesive uroidal filaments and tiny granules in the cytoplasm are indicated with white arrowheads, black arrows and a white arrow respectively. Scale bars: 20 µm.

Fig. 3. TEM pictures of the scales on the cell surface of Korotnevella jeppesenii (a, c) and K. pelagolacustris (b, d) trophozoites viewed from top (a, b) and side (c, d). Scale bars: 500 nm.

Fig. 8. A – 18S rDNA maximum likelihood phylogeny of Angulamoeba. Sequences new for this study are indicated in bold. ML bootstrap values respectively posterior probabilities are shown at the nodes. GenBank accession numbers are given together with the species names. B – LM pictures of Angulamoeba microcystivorans (strains A1WVB (1, 2, 4) and A4WVB (3)) showing the characteristic rusty-colored plasmodium-like mucilage matrix with embedded trophozoites devouring a culture of Microcystis aeruginosa colonies (1–2), multi-vacuolated trophozoites that are seemingly connected by their filopodia (3) and amoeba-flagellates in different stages of transition (4). Scale bars: 200 µm (1), 100 µm (2), 20 µm (3, 4).

Fig. 2. A – 18S rDNA maximum likelihood phylogeny of Korotnevella, including Microcystis-associated strains (in bold). ML bootstrap values respectively posterior probabilities are shown at the nodes. GenBank accession numbers are given together with the species names. B – LM pictures of K. jeppesenii (strain A1JEPDK) showing trophozoites in non-oriented movement (1–5), during locomotion (6, 8) and during grazing on Microcystis aeruginosa cells (7) with the arrow indicating a lacuna-containing nucleolus. C – LM pictures of K. pelagolacustris (strains A8WVB (7, 12), A16WVB (6, 8, 11), A21WVB (1, 13), A54WVB (3–5) and A1LMS (2, 9, 10)) showing trophozoites in different stages of non-oriented movement (1–4, 6), the locomotive form (8–12), grazing amoebae with Microcystis cells inside food vacuoles (5, 10), the cyst stage (7) and the floating form (13).Arrows indicate the nucleus with clearly visible nucleolus (in 8, 10, 12). Scale bars: 20 µm.

Fig. 6. A – 18S rDNA maximum likelihood phylogeny of Vexillifera, including the Microcystis-associated strain (in bold). ML bootstrap values respectively posterior probabilities are shown at the nodes. GenBank accession numbers are given together with the species names. B – LM pictures of locomotive (1–7, 12–13) and floating (8–11) amoebae. Microcystis cells inside food vacuoles are visible in (1–2) and (13). The arrow in (5) points to a temporal, uroid-like structure sometimes visible during locomotion. Scale bars: 20 µm.

Fig. 7. A – 18S rDNA maximum likelihood phylogeny of Cochliopodium, including the Microcystis-associated strain (in bold). ML bootstrap values respectively posterior probabilities are shown at the nodes. GenBank accession numbers are given together with the species names. B – LM pictures of locomotive amoebae clearly showing the surrounding hyaline sheet (black arrows) punctuated with microscales (best visible in 8), a few small subpseudopodia (black arrowheads) and trailing adhesive uroidal filaments (white arrows) (1–5), a tropho- zoite during and just after grazing on Microcystis cells showing a prominent fringe of folded hyaloplasm (white arrowheads) (6–9), the bell-shaped form on colonies of Microcystis aeruginosa (10–11) and floating amoebae (12–13). Scale bars: 20 µm.