Plagiopyla ramani, Nitla & Serra & Fokin & Modeo & Verni & Sandeep & Kalavati & Petroni, 2019

PLAGIOPYLA RAMANI SP. NOV.

( FIGS 1–6; TABLES 3, 4)

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Diagnosis: Body about (arithmetic mean ± SD, Х ± SD) 74.1 ± 6.1 × 38.3 ± 6.9 μm after silver staining; cells yellowish to beige colour in vivo; 41–51 rows of somatic monokinetids; striated band long 42.5 ± 4.4 µm on average; single macronucleus, 1–5 vesicular-type micronuclei; single contractile vacuole with 2–5 pores; cytoproct forming a suture (long 21.8 ± 2.4 µm on average) with two dense cytoproct-related ciliary rows on its left; curved extrusomes (long 1.9 μm on average); habitat freshwater in the hypoxic/anoxic part of the water column, nearby the sediment.

Type locality: Kolleru Lake , in the proximity of the back of Sri Peddintlamma Thalli temple, Kolletikota, Andhra Pradesh, Southern India (N 16°36’48.0” E 81°18’32.0”). This species inhabits freshwater sites with oxygen-depleted water, in presence of decomposed sediments (sapropel) and is mainly a consumer of sapropel-dependent bacteria and diatoms GoogleMaps .

Type material: The slide with the silver-stained holotype specimen (indicated with a black circle of ink on the coverslip) and some paratype specimens has been deposited in the collection of the ‘ Museo di Storia Naturale dell’Università di Pisa’ (Calci, Pisa, Italy) with registration number ‘2017-1 ’.

Etymology: We dedicate this new species to Professor Emeritus Akkur V. Raman from Andhra University ( India), for his contribution to the development of protistological study in India.

Gene sequence: The 18S rRNA sequence of P. ramani has been deposited in NCBI GenBank database with the following accession number: KY563720 View Materials .

Description: Cells small, non-contractile and dorso-ventrally convex, with ovoid-reniform shape ( Figs 1–4). Cells in vivo about (Х ± SD) 84.0 ± 2.5 × 58.5 ± 6.4 μm, after silver staining 74.1 ± 6.1 × 38.3 ± 6.9 μm (body size reduction about 10–15%) ( Table 3). Length/width ratio approximately 1.4 for live cells and 1.9 for stained specimens.Live cells yellowish or beige in colour.Negative phototaxis behaviour, swimming in anticlockwise direction with respect to the longitudinal body axis when viewed from the posterior end. Anterior and posterior end rounded ( Figs 1–4). Somatic cilia equally distributed around body surface ( Figs 2A–C, 3A, B), arranged in about 41–51 rows of somatic monokinetids (22–25 dorsal rows and 19–26 ventral rows) ( Fig. 1A, B). Not all somatic cilia reach posterior end of cell dorsal side: 3 rows terminating near opening of cytoproct, remaining ciliary rows terminating at posterior end, meeting each other and forming a suture ( Figs 1A, 3A).

On antero-ventral side characteristic oral apparatus opens with slit-like aperture, inclined toward inner part of body as a transverse groove ( Figs 1B, 2B, D, 3B, 4C–E).

Oral region folding inward, forming a deep buccal-pharyngeal cavity, curving towards left with respect to longitudinal body axis ( Figs 1A, 3C). Buccal region formed by two lip-like structures (upper lip and lower lip), composed of oral polykinetids ( Figs 1B, 4D). Oral cilia not in continuity with somatic ones, which terminate before, leaving a small gap in between ( Figs 1B, 4D, E). Oral lips bearing longer and densely arranged cilia than somatic kinetids ( Fig. 4D, E).

Striated band running along right margin of dorsal side and terminating close to cytoproct region ( Figs 1A, 2C, 3A, 4A, F). Striated band length 42.5 ± 4.4 µm on average after silver staining, composed of thick, transverse ridges ( Fig. 4G). Transverse ridges perpendicular to vertical axis of the body. Cell length/striated band ratio 1.7.

Cytoproct located on dorsal side of body, in sub-equatorial position, forming a longitudinal suture, about 21.8 µm long ( Figs 1A, 3A, D, 4H). On the left side of cytoproct, two short rows of narrowly spaced cilia ( Figs 1A, 3A, D, 4H, J). Cytoproct terminating slightly before contractile vacuole opening region.

Contractile vacuole in posterior body portion ( Fig. 2B, E). It opened dorsally with 2–5 distinct pores, aligned in series following cell longitudinal axis ( Figs 1A, 2C, 3D, 4H, I)

A single ovoid macronucleus (on average 15.1 × 12.4 µm after Feulgen staining) with uniformly arranged chromatin, situated in the equatorial region of the cell beneath the buccal region ( Figs 1A, 2B), and 1–5 small micronuclei (diameter of 1.8 µm on average after Feulgen staining) of vesicular-type ( Fokin, 1997, 2010 /2011), close to the macronucleus ( Fig. 2H).

In cortical region, presence of uniformly distributed, slightly curved extrusomes ( Figs 2F, G, 5A), measuring around 1.9 μm in length, and roundish, electron-dense cortical granules (diameter around 0.3 μm) ( Figs 2E, 5B).

In TEM preparations ectosymbionts detected, lying on the cell surface in ciliate cortex invaginations ( Fig. 5B), appearing as rods of about 0.80 × 0.20 μm. Ectosymbionts not observed in SEM preparations ( Fig. 4). Two different forms of endosymbiotic organisms detected inside the cytoplasm. First form: short rods with tapered ends, surrounded by a lighter layer of cytoplasm, a sort of ‘halo’ ( Fig. 5C–E), about 1.20 × 0.31 μm in size. Their cytoplasm, more electron-dense in the periphery, contains a long loop of DNA ( Fig. 5C, E). Second type of endosymbionts: rod-shaped, about 1.28 × 0.32 μm in size; surrounded by a halo, encircled by a double membrane ( Fig. 5F). All endosymbionts flanked by hydrogenosome-like structures ( Fig. 5C, D), scattered in the cytoplasm throughout the host cell. No bacterial flagella retrieved.

Hydrogenosomes showing an electron-dense matrix and an irregular body shape when associated to endosymbionts ( Fig. 5C). Some roundish and lighter structures could be interpreted as an early stage of hydrogenosome formation ( Fig. 5G), showing a clear matrix with several membranous foldings without cristae ( Fig. 5H).

Fluorescence in situ hybridization (FISH) analysis confirmed the presence of Archaea inside P. ramani : host cells were positive to Archaeal universal probe ARCH915 and, later, to the species-specific probe, Methanoc512 ( Fig. 5I). The positivity to the probe Methanoc512 ascribed at least one type of these symbionts to the methanogenic Archaea genus Methanocorpusculum ( Methanomicrobiales , Euryarchaeota). No signals detected with EUB338 probe (data not shown).

Molecular characterization and phylogeny: From molecular analysis on 18S rRNA encoding gene of P. ramani , a sequence of 1715 bp was obtained. Identity values to database published Plagiopylidae sequences are reported in Table 4 and range between 97.4% ( Plagiopyla narasimhamurtii , see later) and 96.1% ( P. nasuta ).

The phylogenetic position of P. ramani was within the clade of the Plagiopylidae family, more precisely in one of the two subclades including also Lechriopyla mystax , P. narasimhamurtii (see later), and some sequences of unidentified Plagiopyla species ( Fig. 6). A more detailed description of the phylogeny will be presented in the last paragraph of the ‘Results’ section.