Copemetopus mystakophoros, Fokin & Serra & Gammuto & Allievi & Petroni & Modeo, 2023

Fokin, Sergei I., Serra, Valentina, Gammuto, Leandro, Allievi, Alessandro, Petroni, Giulio & Modeo, Letizia, 2023, Multidisciplinary characterization of the new species Copemetopus mystakophoros and its symbionts with a proposal for the new class Copemetopea (Alveolata: Ciliophora), Zoological Journal of the Linnean Society 198, pp. 1171-1200 : 1175-1187

COPEMETOPUS MYSTAKOPHOROS SP. NOV.

( TABLES 1 AND 2; FIGS 1–12)

ZooBank registration (family): urn:lsid:zoobank.org:act:8CCADA5A-654E-4DD3-BC78-03E733C1300E

Zoobank registration (species): urn:lsid:zoobank.org:act:D0B08C02-C63C-497A-A778-D3915329E63B

Diagnosis: Size (all quantitative data are expressed as an average) in vivo, 275 μm × 120 μm; size after silver nitrate staining, 250 μm × 103 μm; cell generally resembling a club or a bowling pin in shape (i.e. presenting an expanded front end and an elongated rear end); oral aperture-to-body length ratio of 1:2.2. Macronucleus: usually in two equally elongated nodules, 41 μm × 24 μm in size and connected by a thin karyoplasmic isthmus. Micronuclei: 11, roundish, of the compact type, 3.8 μm in diameter. Contractile vacuole: single, in the posterior end of the cell, without collecting canals. Cytoplasmic features: invariably several hundreds of elongated symbiontophorous vacuoles (length, 6–8 μm) containing two types of bacteria [i.e. Alphaproteobacteria plus, probably (according to morphology), spirochaetes]. Cortical granules: present, appearing as strips lying between the ciliary rows and consisting of three to five irregular rows of roundish granules each. Somatic kineties: 92; moustache (i.e. a row of long, whip-like ciliary units): 15 units, inserted on an outer border of the buccal cavity over the adoral zone of membranelles (AZM), without having any connection with it, and consisting of cilia with a length of up to half the cell length; dorsal brush (i.e. dense rows of longer somatic cilia beneath the anterior cell pole, on the right side): five or six rows; distinctive preoral suture. AZM: 45 membranelles; paroral membrane (PM): three ciliary rows, although sometimes not obvious. Swimming behaviour: ciliate, mainly rotating to the right and, less frequently, to the left direction. Habitat: brackish or marine water (salinity, 8–46‰), oxic–anoxic border in the bottom sediments.

Type locality: The sampling site of the type population, IPS3-1 , of Copemetopus mystakophoros was the permanent small brackish-water pond located along the Ligurian sea coastline close to the mouth of the Serchio River ( Parco Naturale di Migliarino San Rossore Massaciuccoli , Migliarino, Pisa district, Tuscany, Italy (43°47ʹ39″N, 10°16ʹ4″E), sample no. 1 ( IPS3-1 ); sampling date 16 October 2005; collector S. I. Fokin) .

Type material: One slide of the type population (IPS3-1) of Copemetopus mystakophoros with silver nitrate-impregnated holotype specimen (registration no. CAMUS _2022-1), indicated by a circle of ink on the coverslip, plus a paratype slide with permanent Feulgen-stained specimens (registration no. CAMUS _2022-2) have been deposited in the collection of the ‘Museo di Storia Naturale e del Territorio dell’Università di Pisa’ (Calci, Pisa, Italy).

Etymology: The name is derived from Ancient Greek, μουστάκι (moustáki), meaning ‘moustache’, and from -phóros and -phoros, from the stem of φέρω (phérō), meaning ‘to carry, bearing’, because the species is characterized by long frontal ciliary whip-like units forming a sort of moustache inserted on an outer border of the buccal cavity over the adoral zone of the membranelles.

Voucher material: The total genomic DNA of Copemetopus mystakophoros obtained from cells of the type population ( IPS3-1 ) and the WGA products obtained from population OALG11 are available at the Department of Biology of the University of Pisa (Pisa, Italy), Unit of Zoology–Anthropology .

Gene sequences: The 18S rDNA sequences of Copemetopus mystakophoros were deposited in NCBI GenBank database under the following accession numbers: OM955238 (from IPS3-1 population; 1773 bp long), OM955239 (from OALG11_1; 1738 bp long) and OM955240 (from OALG11_3; 1738 bp long).

Description: Type population IPS3-1: Size of the ciliate variable, 200–450 μm × 90–150 μm in vivo; after fixation and silver staining, 188–328 μm × 78–135 μm (249.7 μm × 102.6 μm on average; Figs 1–4; Table 1). Length-to-width ratio close to 2.4:1 ( Figs 1–6; Table 1). Cell size after SEM procedure: 100–205 μm × 65–135 μm (163 μm × 93.7 μm on average), with a cell shrinkage of 35% and 8.7% compared with in vivo length and width, respectively ( Fig. 7). Cell shape elongated (oblong), sometimes ovoid, usually with an expanded cell anterior and a tapered posterior end; cell shape resembling a club ( Figs 1–7A). Both cell ends rounded in vivo ( Fig. 1). Shape changing from ovoid to club- (most commonly) or bowling pin-shaped observed in some cells in the population, possibly as a consequence of the cell growing up process. Unfortunately, cell division and post-division processes were not observed. Generally, cells are spherical in cross-section, although most of the larger cells have a slight dorsoventrally flattened appearance.

Macronucleus (Ma) consisting of two oval nodules of equal size (41 μm × 24 μm in size each) connected by a thin karyoplasmic isthmus ( Figs 1D, 2, 5, 8A, C); 11 roundish micronuclei (Mi; 3.8 μm in diameter) of the compact type distributed close or, sometimes, far from the Ma ( Figs 2, 5; Tables 1 and 2; see below for ultrastructural details on nuclear apparatus).

A single large contractile vacuole (CV) with a single pore is located in the posterior end of the ciliate. Apparently, the CV pulsates with long breaks, hence its activity is difficult to detect ( Figs 1E, 2). Cytoproct not visible.

In living conditions, many ciliates showed in the cytoplasm several relatively large food vacuoles (phagosomes) with a green content ( Fig. 1A, B) and unknown cytoplasmic inclusions ( Fig. 1E); neither bacteria nor diatoms were observed inside the phagosomes. In the cytoplasm, several hundreds of slightly elongated symbiontophorous vacuoles (SVs) were invariably observed ( Fig. 8; see below for ultrastructural details).

Species Salinity Size in vivo Somatic AZM, Somatic Moustache, Kinetosomal Mi,

(‰) (µm) kineties, number of ciliature, number of rows in DB, number number membranelles number of whip-like number cilia per unit ciliary units

CV, pos- Cortical Mucocysts ition granules, number in the strips

Copemetopus 33? 300– 90–100 32? 9? 8–10 Middle 2–3 subsalsus 400 × 100–

Villeneuve- 125

Brachon, 1940

Copemetopus 130 200–400 × 70–102 (88) a 43–47 (44) 1 b 9–10 6–7 9–15 (11) Posterior ? subsalsus sensu 65–130

Al-Rasheid

(2001) *

Copemetopus verae 17 120–208 × 60–88 (72) 31–44 (37) 2–4 c 10 d 6–11 2–17 (7) – – Capello-Nunes 43–85 (8)

et al., 2022

Copemetopus nd nd nd 35 e nd 19 e nd 3 e nd nd chesapeakensis

Small & Lynn,

1985

Copemetopus 15 200–450 × 85–103 (92) 42–48 (45) 2–6 (4) 8–21 (15) 5–6 7–18 (11) Posterior 3–5 mystakophoros 90–150

Present study

(type population)

+

?

–

nd

–

* Published as Copemetopus subsalsus ( Al-Rasheid, 2001; see main text for details);

aarithmetic means;

breported in the text but, according to figs 18–23, p. 192 ( Al-Rasheid, 2001), in each ciliary unit there are two or even three cilia;

cnumber reported in the text only;

dnumber reported only according to Fig. 4C;

edata according to illustration.

Abbreviations: AZM, adoral zone of membranelles; CV, contractile vacuole; DB, dorsal brush; Ma, macronucleus; Mi, micronucleus; nd, no data; Ref., reference;?, uncertain description; +, character present; –, character not present.

The kinetome structure was visible in silver-stained ciliates and both in some living cells and in cells treated for FISH (see Fokin, 2016) thanks to the pattern of cortical granules (CGs). In FISH samples, the kinetome was mapped by strips highlighted by both Eubacteria and Alphaprotobacteria probes ( Figs 3C, 6C) and consisted of CGs distributed in irregular rows (see below for ultrastructural details). According to FISH results, each strip consisted of between one and five CGs across, depending on the body region of the ciliate ( Fig. 6C).

Somatic cilia were ~10 μm long in vivo; caudal cilia were not detected ( Figs 1, 2, 7). Somatic cilia units were ciliferous on both ventral and dorsal sides of the cell, mainly consisting of three or four cilia (3.6 on average in line), but with unit composition ranging between two (dikinetids), five and six (polykinetids) in the set (see below for ultrastructural details; Figs 7, 9–11; Table 2). No special ciliary fields with an identical organization to most of such units were observed ( Figs 4, 7). Somatic ciliary rows: ~85–103 (93.8 on average; Table 1).

A single row of long, whip-like ciliary units, forming a sort of moustache, is present at the anterior region of the oral cavity near to AZM on its outer perimeter. The moustache (number of whip-like ciliary units, 10–21; 15 on average) is located along the membranelles of AZM, but independent from them ( Figs 1C–F, 2, 3D, E, 4C, 7, 9A, B); at their own base (SEM width, ~2.8 µm), moustache units consisted of a combination of two or three tightly located layers of eight to ten rows of long cilia ( Figs 7, 9A, B). Moustache extends up to half of the body of the ciliate (i.e. 100–150 μm), with units becoming thinner from their insertion to the tip ( Figs 2, 7, 9A, B) and clearly not participating in AZM beating activity, but performing one or more different functions yet to be elucidated. Some living cells in calm conditions (i.e. without movement) repeatedly showed a fan-like or nimbus-like moustache position with respect to the cell body ( Figs 1E, 2), whereas during movement they showed a ‘folded’ moustache (i.e. located in parallel along the body; Fig. 1C).

The preoral suture is conspicuous, presenting an empty space and running from the upper right side of the buccal aperture almost to the right anterior part of the cell dorsal side ( Fig. 7). On the dorsal side, part of five or six kinetosome rows beneath the anterior pole on the right side consisting of closely located sets of kinetosomes, the so-called dorsal bristle. Cilia of the region are longer (~15–20 μm in vivo) than the rest of the dorsal somatic kineties cilia, therefore forming a kind of tuft, clearly visible in living cells and in some fixed cells ( Figs 1A, 3B, 4A, 7).

Length of the oral aperture reaching almost half of the body length (45.5% on average, based on silver nitrate-stained specimens); oral aperture ovalelongated, usually oblique, with a slightly expanded frontal part located along the middle axis of the ventral side of the ciliate and a narrowed rear part lying closer to its right edge with an angle of ~20° ( Figs 1, 3, 4, 7). Oral aperture always positioned close to the frontal edge of the cell, but with an inclination with respect to the cell longitudinal axis sometimes higher (i.e. ≤50°). Paroral membrane consisting of three closely located rows (not always very distinct from each other) of short cilia (length, ~6 μm in vivo; Figs 2, 3D, E, 4B, C, 7) running on the right side of the oral cavity. Distinct whirling S-shaped AZM, with 42–48 membranelles (44.6 on average), occupying the left side of the oral groove ( Figs 1E, 2, 3C, D, 4C, 7).

Ultrastructural features: Rod-shaped bacterial ectosymbionts can sometimes be detected on the cell surface in living and fixed ciliates, both after staining with DAPI dye ( Fig. 6A, B) and after the TEM procedure ( Fig. 10A); in TEM sections, ectosymbionts are tangentially and perpendicularly oriented with respect to the cell surface. The SEM investigation did not show any ectosymbiotic bacteria on the cell surface, possibly for technical reasons connected to the SEM procedure performed ( Figs 7, 9).

In the cortex: (1) under the plasma membrane, flat alveoli and many non-homogeneous, roundish to slightly elongated to even irregularly shaped CGs (length, ~0.5–0.8 μm) sprout in a single, irregular layer (more commonly) but are sometimes distributed in several layers ( Figs 6C, 10A, B, E–G); and (2) regarding the somatic infraciliature, both dikinetids ( Figs 10A–C, 11) and polykinetids ( Fig. 10D, E) were observed and their ultrastructure investigated in both cross-section ( Fig. 10C, D, F) and longitudinal section ( Fig. 10A, E). Electron-dense material surrounds the somatic dikinetids and forms a desmose linking the two kinetosomes to each other; from the desmose, a roundish spur of electron-dense material arises anterior to the left ( Fig. 10C). As for the fibrillar associates, the anterior kinetosome shows a tangential ribbon consisting of six or seven transverse microtubules plus, in front of triplet 4, an isolated couple of additional microtubules with an apparently perpendicular orientation with respect to the tangential ribbon; the posterior kinetosome shows a striated kinetodesmal fibre ( Fig. 10C, F, G) oriented towards the right or slightly posteriad and contacting the anterior kinetosome; an electron-dense structure arises posteriad to the right, with between six and 15 long postciliary microtubules curving posteriad and forming flat ribbons; no stacking of the ribbons was observed (i.e. postciliodesma were not detected; Fig. 10F, G). In between kinetosomes, there are no additional microtubules ( Fig. 10C). In polykinetids, such as triplets of kinetosomes, the anteriormost and posteriormost kinetosomes do not diverge from corresponding kinetosomes in dikinetids; the middle-located kinetosome presents transverse microtubules and is linked by a desmose to the posterior one ( Fig. 10D). Along the kinetosomes, longitudinal microtubules, underlined by a microfibrillar network system, run in parallel ( Fig. 10A, E, F).

In the cytoplasm:

1. There are a huge number of medium-sized to large lipid droplets ( Figs 10E, F, 12A, C).

2. Invariably, there are several hundreds of slightly elongated SVs (6–8 μm in length; Fig. 8) containing two types of bacteria: type I, elongated rod-shaped bacteria, 3.0–3.5 μm × 1.0–1.2 μm in size; and type II, twisted bacteria, 2.0–3.0 μm × 0.15–0.20 μm in size ( Fig. 12A, B). According to FISH analysis, type I, targeted by the ALF1 b probe, is a representative of Pseudomonadota ( Fig. 8D); according to their general morphology and shape, type II are probably spirochaetes ( Fig. 12A, B). Both bacterial types do not manifest distinctive nucleoids. Within each SV, there are usually around ten representatives of each bacterial type present simultaneously; SVs are often in contact with the rough endoplasmic reticulum cisternae of the ciliate ( Fig. 12A, B). On a few sections, a third type of rod-shaped cytoplasmic bacteria within individual vacuoles can be observed near the ciliate cortex layer ( Fig. 10H) .

3. The nodules of Ma show dense chromatin bodies and several nucleoli, whereas Mi contain a less packed chromatin ( Fig. 12C, D).

4. There are two types of energy-related organelles: in the oral cavity region, closer to the cell surface, there are classical mitochondria, with a size of 1.5 μm × 2.0 μm and tubular cristae ( Fig. 10H); and more deeply in the ciliate cytoplasm, among the SVs, there are many hydrogenosome-like organelles (1.2 μm × 0.6 μm), with a homogeneous, more electron-dense core (matrix) surrounded by several peripheral tubular–vesicular (swollen) cristae-like structures ( Figs 10J, 12A, B).

Notes on biology and ecology: The ciliate was observed near the bottom of the water bodies, in the water– sediment interface or even in the upper loose layer of bottom sediments. All samples containing Copemetopus mystakophoros smelled strongly of hydrogen sulfide. The oxygen level in water of this sediment layer varied between 0.6 and 1.1%. During the collection of living cells from the native sample into the depression slide in the laboratory, oxygenation was not tolerated by Copemetopus mystakophoros cells, which disappeared in <1 h. In contrast, the change of salinity conditions turned out not to be critical for survival of the ciliate when salinity remained at least in a brackish range or increased to 60‰. During most of the previous ciliate study period in our laboratory (i.e. 2005– 2008), the salinity of the water in the sampling place remained within the brackish water range (8–15‰). Unfortunately, in 2008, after a severe storm and flood in the Serchio River mouth coastline, the ecology of the water reservoir, where representatives of Copemetopus mystakophoros had been collected previously, was completely destroyed. The salinity decreased to almost zero, and the entire set of characteristic ciliate species in the bottom layer disappeared. However, during the recent study period (2020–2021), the water in the collecting place (i.e. the Laguna di Levante in Orbetello) always showed a higher salinity (34, 36, 39 and even 46‰) with a significant presence of hydrogen sulfide, indicating that these sampling conditions are suitable to sustain the survival of Copemetopus mystakophoros .

The swimming rotation of the ciliate was in a clockwise direction with respect to the longitudinal body axis, viewed from the posterior end. In a few cases, an anticlockwise direction was also observed.

It is noteworthy that during the entire study we have not been able to detect any dividing cell of the ciliate. However, this also applies to the other ciliate representatives of this ecological station studied in parallel, such as Metopus , Parablepharisma , Plagiopyla and Sonderia .

Marine or brackish water samples, according to our observations, are more stable than freshwater ones, and the ecological succession of organisms in the former (should it occur) usually proceeds more slowly than in the latter samples. Apparently, Copemetopus mystakophoros can survive adverse conditions by forming cysts. Repeated checks of the samples taken from Laguna di Levante, Orbetello, showed that the ciliate disappeared in the native/original samples a few days after the opening of the Falcon tubes in the laboratory (in July 2020 and November 2021), appearing again after 3–4 months in the same samples kept in closed conditions, but disappearing again a few days later.

MOLECULAR AND PHYLOGENETIC ANALYSIS

The three obtained 18S rDNA sequences of Copemetopus mystakophoros (IPS3-1, OALG111 and OALG11-3) were substantially identical except for one nucleotide in position 639 of the type sequence (IPS3-1). In this position, each electropherogram of Copemetopus mystakophoros from Orbetello presented a double peak, always involving A and G, which we interpreted as a polymorphism of the species.

The BLAST search on NCBI of the IPS3-1 Copemetopus mystakophoros sequence gave the following best hits: Copemetopus verae MZ 441076 and MZ441075 , showing 98.39 and 98.37% identity, respectively (23 mismatches, three or four gaps); three ‘uncultured eukaryotes/ciliates’, KT346288, KJ760065 and KT346292, showing 88.66, 88.62 and 88.52% identity, respectively; and Parablepharisma bacteriophora ( MN319554 ) and Meseres corlissi Petz & Foissner, 1992 ( EU399528 ) showing 88.53 and 88.35% identity, respectively.

From the identity matrix (Supporting Information, Table S1), the average identity of Copemetopus species with selected representatives of the phylum Ciliophora was 83.78%.

In our phylogenetic tree ( Fig. 13), sequences of Copemetopus mystakophoros clustered together with those of Copemetopus verae with high statistical support (BI/ML: 0.97/99), showing that they represent two different Copemetopus species. Moreover, in all our phylogenetic analyses the Copemetopus mystakophoros sequences clustered together inside the Intramacronucleata clade, branching basally to the Armophorea –Litostomatea–Odontostomatea– Cariacotrichea –Muranotrichea– Spirotrichea group ( Fig. 13; Supporting Information, Figs S1–S 3).

As an additional sister group to the Copemetopus clade, besides the other aforementioned classes, there was the Protocruziea clade. All the other Intramacronucleata [(Plagiopylea, Prostomatea, Oligohymenophorea, Colpodea) and (Phyllopharingea, Nassophorea)] formed a sister group to them all ( Fig. 13).

Statistical values were often not sufficiently robust in our analysis, especially for dataset 1 ( Fig. 13), probably owing to the presence of different evolutionary rates among ciliate clades (e.g. Odontostomatea, Licnophora ) that determine long branches in the phylogenetic tree. For this reason, we performed extra analyses using reduced databases (databases 2–4), which provided more robust statistical support, especially for the nodes involving Copemetopus sequences: 36, 49 and 86% of bootstrap, respectively (Supporting Information, Figs S1–S 3).

SCREENING OF THE SYMBIONTS

Preliminary FISH results showed positive signals in response to the ALF1b probe ( Fig. 8D) and to ethidium bromide ( Fig. 8C) in the symbiontophorous vacuoles of IPS3-1 cells. Staining with DAPI also highlighted the presence of prokaryotes associated with the ciliates' cell surface ( Fig. 6A, B).

From the screening performed on the IPS3-1 population using molecular methods, we retrieved two 16S rDNA consensus sequences, operational taxonomic unit (OTU) #1 and #2 ( Table 3). From NGS methods on the OALG11 population, we retrieved another four sequences, OTUs #3–#6 ( Table 3). All the 16S rDNA sequences obtained in the present work were deposited in NCBI GenBank database, under the accession numbers given in Table 3.

We found six different 16S rDNA sequences, belonging to different bacterial classes and even bacterial phyla and domains: a bacterium belonging to a still undescribed clade related to Planctomycetota (incertae sedis, Bacteria: Planctomycetota), OTU #1, and a spirochaete bacterium (Bacteria: Spirochaetota), OTU #2, in the IPS3-1 population; and in the OALG11 population we found members of the following: (1) Asgard group ( Archaea : Crenarchaeota), OTU #3; (2) Desulfobacterales (Bacteria: Thermodesulfobacteriota), OTU #4; (3) ‘ Candidatus Cloacimonetes’ [Fibrobacteres–Chlorobi– Bacteroidetes (FCB) group; Bacteria], OTU #5; and (4) Campylobacterales (Bacteria: Campylobacterota), OTU #6. The results of the BLAST analyses on the retrieved sequences are shown in detail in Table 3.

Testing in silico to compare the retrieved 16S rDNA from the IPS3-1 population with the oligonucleotide sequence of ALF1 probe showed only a partial matching in both the OTUs (seven nucleotides for OTU #1; seven to nine nucleotides for OTU #2, out of 17).