Cochliopodium, Van Wichelen & D’Hondt & Claeys & Vyverman & Berney & Bass & Vanormelingen, 2016

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 : 74-76

Cochliopodium

The lens-shaped strain A1 HEB was placed in the monophyletic genus Cochliopodium ( Fig. 7A ), but the 18S rDNA sequence differed from other representatives of the genus by no less than 19.7–23.5% (143–301 bp).

Locomotive amoebae of strain A1 HEB were round, oval or triangular (with the base directed anteriorly), had a length of 15–40 µm (mean 26 µm), a width of 15–54 µm (30) and a L/W ratio 0.7–1.2 (0.9) ( Fig. 7B ). The central mass of granuloplasm was always entirely surrounded by a hyaline sheet that covered 12–40% (23) of the body length and was often much narrower posteriorly (e.g. Figs 7B 3 , 5 ). Locomotive amoebae contained 1–14 pointed subpseudopodia of maximally 10 µm long, that were never furcated and mostly situated at the lateral and anterior hyaloplasmic margin ( Figs 7B 1–4 ). Uroidal structures were rarely visible posteriorly, either consisting of one to a few trailing adhesive filaments of maximally 10 µm long ( Figs 7B 4 , 5 ) or rarely plicate with a length of maximal 13 µm ( Figs 7B 8 , 9). The slightly ovoid vesicular nucleus was 3–5 µm in diameter and contained one globular nucleolus of 1–2 µm. Only one, centrally located contractile vacuole was visible in each trophozoite. The granuloplasm contained many, 1–2 µm long, opaque granules and up to 20 irregular, sometimes plate-like shaped crystals of 1–6 µm long. With bright field illumination easily visible micro-scales were about 0.9 µm long and 0.6 µm wide. These entirely covered the tectum, forming a regular punctuation of the hyaloplasm up to the cell’s margin ( Fig.7B 8 ). The trophozoites could also adopt a globular to ovoid bell-shaped form that had a length of 8–30 µm (13) and a width of 8–22 µm (11). These were predominantly seen attached when feeding on Microcystis colonies ( Fig. 7B 11). The clear peripheral hyaloplasmic sheet around the cell was then completely retracted with the exception of a small zone posteriorly where the hyaloplasm was clearly folded towards the substratum (for instance a Microcystis cell). When a potential food item was detected, this fringe of folded hyaloplasm did produce some small hyaline subpseudopodia that started surrounding the object of interest ( Figs 7B 6 , 7 ). Under a coverslip, this bell-shaped form could swiftly transform into a locomotive form with the re-formation of the peripheral hyaloplasmic sheet. The posterior fringe of folded hyaloplasm was then still visible for a while when it slowly retracted ( Figs 7B 8 , 9). Floating amoebae were globular to ovoid with a diameter of 8–20 µm (13), containing 2–8 long thin pseudopodia of maximal 36 µm long ( Fig. 7B 12, 13). No cyst stage was observed. Although this strain initially grew well with Microcystis offered as food it suddenly died after several weeks. Due to this early loss, no TEM analysis could be carried out to retrieve detailed information on the scale structure.

Around 20 mainly morphologically defined Cochliopodium species currently exist ( Geisen et al. 2014), 8 isolated from freshwater, 2 from fresh to brackish water, 3 from brackish, 4 from marine and 3 from terrestrial habitats. Of the 3 freshwater strains without molecular data, C. minutum was assigned a nomen dubium ( Kudryavtsev 2006), C. vestitum is more than twice as large (L: 39–70 µm, mean 59; W: 48–74, mean 65) and has with LM clearly visible spines on the scales ( Kudryavtsev 2005) and C. granulatum ( Penard 1902) , apart from also being much larger (generally> 50 µm), has many large subpseudopodia during locomotion and seems to preferably graze on diatoms. C. minutoidum , isolated from terrestrial habitats ( Kudryavtsev 2006), is only half as large (L: 8–20 µm (mean 14), W: 9–24 (17)). Given the molecular difference, the absence of the above-mentioned morphological features ( Fig. 7B ) and its specific ecology, our Microcystis -associated strain is undoubtedly a new species of Cochliopodium . However, since we lacked detailed information on the scale structure, we opted not to give this taxon an official name.

Figures

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. 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. 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. 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. 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.