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

PLAGIOPYLA NARASIMHAMURTII SP. NOV.

( FIGS 6–11; TABLES 4, 5)

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Diagnosis: Body about 81.8 ± 6.7 × 41.3 ± 4.8 µm after silver staining; cells yellowish to beige colour in vivo; 51–69 rows of somatic monokinetids; striated band present (44.5 ± 1.4 µm long on average); single macronucleus, one compact-type micronucleus; single contractile vacuole with 2 or 3 pores; cytoproct forming a suture (long 24.0 ± 2.3 µm on average) with 3 dense cytoproct-related ciliary rows on its left; two types of extrusive organelles: straight, multi-layered extrusomes (3.7 µm in length on average), and curved homogeneous extrusomes (1.6 µm in length on average); habitat freshwater in the hypoxic/anoxic part of the water column, nearby the sediment.

Type locality: Kolleru Lake in the proximity of Malki Mohammed Puram , a small village in Bhimadole Mandal , in West Godavari District of Andhra Pradesh, Southern India (N 16°43’42.0” E 81°24’30.0”). 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 ‘2017-2 ’.

Etymology: We dedicated this new species to Professor Chatti C. Narasimhamurti (1931–93), from Andhra University ( India), for his contribution to the development of protistological study in India.

G e n e s e q u e n c e: T h e 1 8 S r R N A s e q u e n c e o f P. narasimhamurtii has been deposited in NCBI GenBank database with the following accession number: KY563721 View Materials .

Description: Cells dorso-ventrally flattened, with an elongated oval shape ( Figs 7, 8, 9, 10). Live specimens about (Х ± SD) 91.9 ± 9.8 × 70.4 ± 11.5 µm. After silver staining about 81.8 ± 6.7 × 41.3 ± 4.8µm ( Table 5). Length/ width ratio approximately 1.3 in vivo and 1.9 in stained specimens. Negative phototaxis behaviour, swimming in anticlockwise direction with respect to longitudinal body axis when viewed from the posterior end.

Silver-stained specimens showing about 51–69 rows of somatic monokinetids (27–36 dorsal rows, 24–33 ventral rows) ( Fig. 9A, B). All somatic kineties reach posterior end of body, except for 3 or 4 rows on dorsal side, terminating near cytoproct ( Figs 7A, 9A, D). Somatic ciliature not in continuity with oral one, leaving a small gap around upper and lower oral lips ( Figs 7B, 9B). Buccal region bearing longer and denser cilia than somatic ciliature ( Figs 8B, D, 10D). Oral region folding inward, forming a deep buccal-pharyngeal cavity, curving to left with respect to longitudinal body axis, with oral cilia reaching the end of pharyngeal tube ( Figs 7B, 9C, 11C).

Striated band arising from right margin of body and running along dorsal side ( Figs 7A, 8C, 9A, 10A, E). Striated band measuring about 44.5 ± 1.4 µm in length after silver staining and terminating in correspondence to cytoproct, separated from it by four parallel kineties ( Fig. 9A). Cell length/striated band ratio 1.8. During cell division, early stage of duplication of striated band detected ( Fig. 10C). Cytoproct about 24.0 ± 2.3 µm, forming a longitudinal suture beneath equatorial region of the cell ( Figs 7A, 9A, D). On left side of cytoproct, 3 dense short rows of narrowly spaced cilia ( Figs 7A, 9A, D).

Contractile vacuole in posterior body portion, opening on dorsal surface with 2–3 pores ( Figs 7A, 8B, C, 9A, 10G).

After SEM analysis, presence of a putative mucous layer secreted by the cell, all over the body ( Fig. 10D), probably with defensive function,

MIN: minimum value; MAX: maximum value; X: arithmetic mean; SD: standard deviation; CV: coefficient of variation (%); N: number; n: number of specimens analysed; SI: silver impregnation; SEM: scanning electron microscopy; FE: Feulgen staining.

as already reported by Fenchel and colleagues in P. frontata and Sonderia vorax ( Fenchel et al., 1977) .

Macronucleus ovoid (15.1 × 14.3 µm on average after Feulgen staining), one micronucleus (diameter of 1.5 µm on average after Feulgen staining) of compact-type ( Fokin 1997, 2010 /2011) ( Figs 7A, 8G).

Numerous, roundish cytoplasmic inclusions of unknown function present all over the body, especially abundant at the margins of the cortex underneath the cell membrane ( Fig. 8E, F). Two types of extrusive organelles ( Fig. 8E) uniformly distributed along the cortex: straight, multi-layered extrusomes (E1, 3.7 µm in length on average), and curved homogeneous extrusomes (E2, 1.6 µm in length on average), ( Fig. 11A, B).

TEM analysis provided information on two different morphotypes of endosymbiotic organisms associated with hydrogenosomes: the first corresponds to a short rod with electron-dense, homogeneous cytoplasm and a central long loop of DNA (average size: 1.7 × 0.4 μm) ( Fig. 11D, E); the second is rod-shaped, with one blunt end and a clearer cytoplasm (average size: 2.3 × 0.4 μm) ( Fig. 11F). Bacterial locomotory structures, such as flagella, not retrieved; cytoplasmatic roundish structures, possibly vesicles containing mineral crystals useful for redox reactions ( Pflugfelder et al., 2005), sometimes observed ( Fig. 11F). Endosymbionts associated with hydrogenosomes in the classical ‘sandwich-like’ fashion, sometimes even completely surrounded by these organelles ( Fig. 11F, G). Roundish, light structures additionally detected interpreted as a putative early stage during hydrogenosome development ( Fig. 11G). FISH analysis on this species showed positivity to universal Archaea probe ARCH915 ( Fig. 11H), suggesting the presence of methanogens. No signals detected with EUB338 probe (data not shown).

Molecular characterization and phylogeny: From molecular analysis of the 18S rRNA encoding gene of P. narasimhamurtii we obtained a sequence of 1722 bp. Identity values as compared with a database published Plagiopylidae sequences are reported in Table 4 and range between 97.9% ( L. mystax ) and 95.9% ( P. nasuta ). The identity value with P. ramani sequence was 97.4%.

In the phylogenetic analysis P. narasimhamurtii was positioned within the Plagiopyla clade, clustering together with P. ramani ( Fig. 6).