Korotnevella, Goodkov, 1988

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 : 67-68

Korotnevella

All the dactylopodial strains were placed in the genus Korotnevella , which appeared monophyletic with good bootstrap and posterior probability support ( Fig. 2A ). The Microcystis -associated Korotnevella strains fell into two lineages, containing two and five strains respectively, without representatives of known species. P -distances were 4.9–5.9% (75–99 differing positions) between both lineages and 0.0–1.7% (0–23) within a lineage.

Locomotive amoebae belonging to the first lineage, strains A4 DVDPB and A1 JEPDK, were triangular, elongated or irregularly shaped ( Fig. 2B ). No differentiated uroidal structures were observed. Feeding amoebae (on Microcystis colonies) were spherical to cylindrical without dactylopodia ( Fig. 2B 7 ) but sometimes with a very long and thin sub-pseudopodium when maintained in starved conditions. Length of the locomotive form was 16–41 µm (mean 28), width 6–20 µm (11) and length/width ratio 1.2–6.0 (2.6). One vesicular globular nucleus about 2–3 µm in diameter was present containing one slightly eccentrically located globular nucleolus of about 1–2 µm, occasionally showing a highly refractive central pore-like area (lacuna) under phase-contrast illumination (arrow in Fig. 2B 7 ). Floating forms were spherical with a diameter of 10–16 µm (14) and displaying 4–10 long, hyaline, pointed pseudopodia of maximally 25 µm long. Large spherical planktonic aggregates of up to several hundred individuals were observed occasionally in older cultures. Cysts were never observed. The scales, covering the entire cell surface, consisted of an elliptical disc with upwards curved edges and a centrally located cone-like spine giving it a ‘witches hat’ appearance ( Fig. 3a, c ). The length of the scales observed at top view ( Fig. 3a ) varied from 193–327 nm (277), the width from 124– 203 nm (172) and the L/W ratio from 1.2–2.1 (1.6). The spine length observed in cross-section ( Fig. 3c ) was 47–97 nm (76), width at the base 56–124 nm (80) and L/W ratio 0.5–1.5 (1.0).

The locomotive amoebae from the second lineage, strains A8 WVB, A16 WVB, A21 WVB, A54 WVB and A1 LMS, were all triangular, elongated or irregularly shaped due to the formation of short to deeply inclined pseudopodia on one or both ends or all around the cell body ( Figs 2C 3 , 4 , 8 –12). No differentiated uroidal structures were observed. Amoebae during non-oriented movement were elongated ( Figs 2C 2 , 6 ) or irregularly shaped ( Fig. 2C 1 ), containing several small, sometimes bifurcated, round-tipped pseudopodia. Feeding amoebae (on Microcystis colonies) were spherical to cylindrical without clear pseudopodia ( Fig. 2C 5 ). Starved amoebae had a similar cell shape but sometimes contained a very long and thin sub-pseudopodium. Cell length during locomotion was 20–65 µm (mean 37), width 6–32 µm (14) and the length/ width ratio 1.2–6.0 (2.8). One vesicular globular nucleus about 6 µm in diameter was present containing one slightly eccentrically located globular nucleolus of about 3 µm (arrows in Figs 2C 8 , 10, 12), occasionally showing a highly refractive central pore-like area (lacuna) under phase-contrast illumination (faintly visible in Fig. 2C 8 ). Floating forms ( Fig. 2C 13) were of radial type, spherical, with a diameter of 10–25 µm (15) and displaying 1–20 (9) long, hyaline, pointed pseudopodia with a length of 20–100 µm (52). The entire cell surface was covered with scales, only visible with electron microscopy ( Figs 3b, d ). In cross-section ( Fig. 3d ), these were generally oriented in a roof-tile pattern (imbricated). The scales consisted of an elliptical disk with upwards curved edges and a centrally located cone-like spine giving it a ‘witches hat’ appearance. The length of the scales observed at top view was 260–391 nm (333), the width 153–206 nm (180) and the length/width ratio 1.5–2.6 (1.9). The spine length measured on scales observed in cross-section was 98–163 nm (121), the width measured at the base of the spine 61–131 nm (91) and the length/width ratio 0.8–2.2 (1.4). Starved amoebae tended to float and aggregate into large, spherical colonies of several tens to hundreds of individuals. A few times, a globular cyst-like body with a diameter of 15– 20 µm was observed in the cultures ( Fig. 2C 7 ) .

Korotnevella is a genus of scale-bearing amoebae all characterized by the absence of parasomes ( Smirnov et al. 2011). Currently 8 Korotnevella species are recognized – 4 from freshwater, 3 from marine habitats and 1 from terrestrial origin – that are mainly distinguished on the base of their scale morphology ( O’Kelly et al. 2001, Udalov 2015). The Korotnevella species without 18S rDNA sequence data are K. nivo , a marine species with crown-like scales ( Smirnov 1996 /97), 3 freshwater species, being K. bulla with basket-like scales ( Page 1981), K. diskophora with disc-like scales (Smirnov 1999) and K. limbata having both scale types ( Udalov 2015) and the terrestrial K. heteracantha also bearing both scale types ( Udalov 2015). TEM analysis of the Microcystis -associated Korotnevella strains from both lineages showed the presence of disc-like scales ( Fig. 3 ) very similar to the scales of K. diskophora . Apart from some small differences in scale dimensions and spine length, our strains mainly differed from K. diskophora , by having a spherical floating form with radially many pointed pseudopodia (irregular with few pseudopodia in K. diskophora ) and by their preference for Microcystis and thus planktonic occurrence (bacterivore and benthic lifestyle in K. diskophora ). Apart from the phylogenetic divergence, both Microcystis -associated Korotnevella lineages also differ in their morphology since the size of the trophozoites and of the scales and spines is smaller in strains of the first lineage (A4 DVDPB and A1 JEPDK). Based on the above-mentioned differences between both Microcystis -associated Korotnevella lineages and with K. diskophora , we described both lineages as new species, K. pelagolacustris and K. jeppesenii .

Figures

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. 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.

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. 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. 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. 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. 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. 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.