Hartmannella, Alexeieff, 1912

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 : 68-72

Hartmannella / Copromyxa

The 18S rDNA sequences of the two strains most closely related to Hartmannella / Copromyxa were highly divergent (p -distance 9.4%, 113 differing positions). In the ML phylogeny ( Fig. 4A ) they were most closely related to and had a basal position in the lineage including Copromyxa protea , Copromyxa / Hartmannella cantabrigiensis and an unidentified strain (‘Tubulinida sp.’) (p -distances 9.7–15.2%, 117–197 differing positions).

The locomotive amoebae (mainly seen in starved cultures, see Figs 4B 1 , 3 , 7 ) of strain A2 JEPDK were monotactic with anteriorly a short-lived but well pronounced hyaline cap ( Figs 4B 7 , 10) of about 3 µm long and 6 µm wide that became rapidly filled with granuloplasm prior to the formation of a new lateral lob-like pseudopodium ( Figs 4B 2 , 5 , 6 , 11). These alternating lateral sprouting lobes gave the locomotion a sort of eruptive-like appearance and with any change of direction transformed monopodial movement swiftly in a bipodial ( Fig. 4B 4 ) or multipodial (several irregularly located pseudopodia, Fig. 4B 6 ) state. Pseudopodia were up to 13 µm long and 5 µm wide at the base.At times an eruptive lobe could be seen rippling down on one side of the amoeba ( Figs 4B 8 , 9). No differentiated uroidal structures were observed. Length of limax-shaped amoebae was 20–59 µm (mean 33 µm), width 7–17 µm (11) and length/width ratio 2–4.9 (3.1). The locomotion rate was estimated at 0.35–0.88 µm s –1. Length during non-oriented movement was 10–43 µm (24), width 6–25 µm (13) and length/width ratio 1.0–4.3 (1.9). One vesicular oval nucleus of 5–10 µm long and 3–8 µm wide was present with one central to slightly eccentrically placed globular nucleolus of about 2–3 µm in diameter ( Figs 4B 1 , 5 ). Usually one small contractile vacuole posteriorly, rarely up to 3 vacuoles in very large individuals. The cytoplasm always contained 8–22 opaque crystals (e.g. arrow in Fig. 4B 1 ) with a length and width of respectively 1.1–7.2 µm (average 2.4) and 0.7–3.4 µm (1.7), and several smaller, refractive spheres. The crystals could be single or paired and of irregular, truncated bi-pyramidal, cylindrical, centrally inclined cube-like or plate-like (sometimes divided in 4 equal squares) shape. Spherical floating forms showed a high resemblance with those of Saccamoeba limax ( Page 1988, p. 65) . They were occasionally observed after re-inoculation and had a diameter of 15–20 µm and radially displayed 5–10 small hyaline lobes of about 3 µm in diameter ( Fig. 4B 15). Cysts were spherical with a diameter of 9–20 µm, a cell wall of about 1 µm thickness and sometimes displayed an inner wall ( Fig. 4B 16). These double-walled cysts are similar to so-called sphaerocysts observed in Copromyxa protea which may be part of a (para) sexual cycle ( Brown et al. 2011). Empty cysts contained a round pore of about 5 µm in diameter (not shown).

Locomotive amoebae (mainly seen in starved cultures, Figs 4C 1 , 6 –9) of strain A5 DVDPB were monotactic, displaying anteriorly a short-lived but well pronounced hyaline cap ( Fig. 4C 9) about 5 µm long and 5 µm wide that was rapidly filled with granuloplasm just before a new lateral pseudopodium was formed. With any change of direction, monopodial movement was however easily transformed into a bipodial ( Figs 4C 6 , 9) and eventually a multipodial (several irregularly located pseudopodia) state due to these alternating sprouting (eruptive) lobes. Pseudopodia were up to 25 µm long and about 5 µm wide at their base, laterally harboring a few small, sprouting, short-living subpseudopodia ( Figs 4C 3 , 11) of which one eventually became the new leading pseudopodium. Limax-shaped amoebae were 20–55 µm long (mean 38) and 8–25 µm wide (13) with a length/width ratio of 1.4–6.7 (3.1). Amoebae during non-oriented movement (e.g. when feeding on Microcystis cells) were more irregularly shaped, 15–25 µm in diameter, with radially 5–12 small blunted, round-tipped pseudopodia ( Figs 4C 3 –5,10– 12). No differentiated uroidal structures were observed apart of a few adhesive uroidal filaments occasionally (slightly visible in Fig. 4C 9). One vesicular oval nucleus of 4–8 µm long and 4–7 µm wide with one centrally to slightly eccentrically located globular nucleolus of about 3 µm in diameter (arrow in Fig. 4C 5 ) was present. The nucleolus may contain a highly refractive central pore-like area (lacuna). Usually one contractile vacuole of about 6 µm in diameter present posteriorly (e.g. Figs 4C 9–12). The cytoplasm contained 10–20 small, spherical to irregular shaped, dark-colored, refractive, single or paired crystals of about 0.5–1.6 µm (1.0) in diameter (e.g. Figs 4C 5 , 7 , 11). Floating forms were occasionally observed after re-inoculation. They were spherical to slightly elongated, with a diameter of 15–20 µm, radially displaying 5–12 small hyaline lobes with a maximal length of 15 µm ( Figs 4C 13–15), generally resembling those of Nolandella hibernica ( Page 1980, Fig. 4 ). Spherical cysts about 20 µm in diameter were occasionally present in starved cultures.

Together with Saccamoeba , Glaeseria , Cashia and Copromyxella (for the last two, 18S rDNA sequences are not available), Hartmannella and Copromyxa belong to the family Hartmannellidae ( Smirnov et al. 2011) . Currently, Hartmannella contains only three species ( Smirnov et al. 2011). Two of these are marine – H. lobifera and H. vacuolata – and one is from freshwater, Hartmannella cantabrigiensis ( Smirnov 1996 /97, Anderson et al. 1997). Copromyxa contains only one species, C. protea , however there is ongoing discussion as to whether H. cantabrigiensis should be included in Copromyxa ( Brown et al. 2011, Smirnov et al. 2011). The trophozoites of our strains were characterized by their aquatic lifestyle and absence of fruiting-body formation in contrast to Copromyxella and Copromyxa protea that are terrestrial, dung-inhabiting, aggregative, fruiting amoebae (slime molds) ( Raper et al. 1978, Brown et al. 2011); their clearly visible and rather extended hyaline cap in contrast to Glaeseria , Cashia and Saccamoeba ( Page 1988) ; their absence of a differentiated uroidal structure in contrast to Saccamoeba ( Page 1988, Anderson et al. 1997, Corsaro et al. 2010); and their generally eruptive-like movement which is usually non-eruptive in all other members of the family, including Hartmannella ( Page 1988, Smirnov 1996 /97, Anderson et al. 1997). The last character is however not very reliable since Raper et al. (1978) observed potentially ‘explosive’ pseudopodia in trophozoites of all described Copromyxella species, Page (1980) mentioned uncharacteristic eruptive activity in some Nolandella species, Smirnov et al. (2011) observed occasional eruptions in a Hartmannella / Copromyxa strain and Watson et al. (2014) mentioned semi-eruptive movement in the recently established hartmannellid Ptolemeba .

Brown et al. (2011) emended the generic diagnosis of Copromyxa by also including aquatic, potentially non-fruiting limax-shaped amoebae ( H. cantabrigiensis , Hartmannella strain 4/3Da/10) similar in morphology to our strains. For this reason and because we also observed a double-walled cyst stage very similar to the sphaerocysts described for C. protea in one of our cultures, we placed our strains within the genus Copromyxa . The phylogenetic distance between our strains and the other taxa within this clade warranted the description of two new species (based on the large divergence in 18S rDNA sequence), C. microcystidis and C. vandevyveri .

Figures

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. 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. 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. 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. 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. 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. 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. 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).