# Morphology, morphometry and infraciliature of four new species of Clevelandellida de Puytorac and Grain, 1976 (Ciliophora: Armophorea) from the digestive tube of hydromorphic earthworms in the Cameroonian coastal zone

Paul Alain Nana, Nectaire Lié, Nyamsi Tchatcho, Fils Mamert Onana, Zéphyrin Fokam, Claire Stéphane Metsopkeng, Pierre Ngassam, Thomas Efole Ewoukem, Estelle Masseret, Moïse Nola, et al.

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Paul Alain Nana, Nectaire Lié, Nyamsi Tchatcho, Fils Mamert Onana, Zéphyrin Fokam, et al. Morphology, morphometry and infraciliature of four new species of Clevelandellida de Puytorac and Grain, 1976 (Ciliophora: Armophorea) from the digestive tube of hydromorphic earthworms in the Cameroonian coastal zone. Protistology, 2022, 3 (4), pp.187-198. 10.21685/1680-0826-2022-16-3-4. hal-03865301

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ORIGINAL ARTICLE

# Morphology, morphometry and infraciliature of four new species of Clevelandellida de Puytorac and Grain, 1976 (Ciliophora: Armophorea) from the digestive tube of hydromorphic earthworms in the Cameroonian coastal zone

Paul Alain Nana<fnref n="1" />*, Nectaire Lié Nyamsi Tchatcho<fnref n="2" />,
Fils Mamert Onana<fnref n="2" />, Zéphyrin Fokam<fnref n="3" />, Claire
Stéphane Metsopkeng<fnref n="4" />, Pierre Ngassam<fnref n="4" />, Thomas
Efole Ewoukem<fnref n="2" />, Estelle Masseret<fnref n="5" />, Moïse Nola<fnref n="4" /> and
Télesphore Sime-Ngando<fnref n="6" />

Submitted May 12, 2022 | Accepted June 28, 2022 |

Summary

Based on morphological, morphometric and infraciliature criteria, as evidenced
by the ammoniacal pyridinated silver carbonate technique and 4',6-diamidino-
2-phenylindole (DAPI) staining, we contribute to the knowledge of four new
ciliated protozoa species from the digestive tract of earthworms (Oligochaete:
Glossoscolecidae), collected on the banks of the Nkam River (Cameroon). These new
species belong to the Class Armophorea, the genera *Nyctotherus* and *Nyctotheroides*.
In the genus *Nyctotherus*, three species are described: *N. nkamensis* n. sp. (ovoid,
85–138 µm × 52–115 µm), *N. emini* n. sp. (ovoid, 66–185 µm × 50–95 µm), and
*N. renimorphus* n. sp. (kidney form, 125–220 µm × 75–180 µm). They have in
common the presence of an apical secant system on their right side. In the genus
*Nyctotheroides*, the species *N. ndogbeleensis* n. sp. (elongated form, 75–120 µm ×
25–40 µm) is characterized by the existence of three secant systems on both sides:
an apical secant system on the right side and two secant systems (the apical and the
caudal) on the left side. Contrary to known congeners, the species described in this
study have a very pronounced polymorphism.

**Key words:** Ciliophora, earthworms, Nkam, *Nyctotherus*, *Nyctotheroides*

<footnote n="1">¹ Department of Oceanography, Institute of Fisheries and Aquatic Sciences, University of Douala, P.O. Box 7236, Douala, Cameroon</footnote>

<footnote n="2">² Department of Aquatic Ecosystems Management, Institute of Fisheries and Aquatic Sciences, University of Douala, P.O. Box 7236, Douala, Cameroon</footnote>

<footnote n="3">³ Department of Biology, Higher Teacher Training College, University of Bamenda, P.O. Box 39, Bamenda, Cameroon</footnote>

<footnote n="4">⁴ Laboratory of Hydrobiology and Environment, Faculty of Science, University of Yaoundé 1, P.O. Box 812, Yaoundé, Cameroon</footnote>

<footnote n="5">⁵ MARBEC, University of Montpellier, IRD, Ifremer, CNRS, Montpellier, France</footnote>

<footnote n="6">⁶ Laboratoire Microorganismes: Génome et Environnement, UMR CNRS 6023, Clermont Auvergne Université, BP 80026, 63178 Aubiure Cedex, France</footnote>
**Introduction**

Protists are the dominant eukaryotes in the bio-
sphere where they play key functional roles. Pro-
tozoan ciliates constitute a branching of the protists
widely distributed in the most diverse biotopes, both
in the fresh and salt waters as well as in the bodies
of many animals. Free-living ciliates are either
pelagic and planktonic (Sanchez Rodriguez et al.,
2011; Santoferrara and McManus, 2017) or littoral
(Wang et al., 2009; Doherty et al., 2010). They are
either attached to algae and aquatic metazoans
(Šimek et al., 2016; Sparvoli et al., 2018) or float
on the bottom of dead leaves or in the middle of
algal agglomerations (Šimek et al., 2016). Some are
catharobes and live in clear water, clean and well
oxygenated; others are more or less saprobionts,
common in putrid waters that are very changable in
organic compounds. In addition, there is a rich and
very specialized fauna of ciliates in the sand (Jiang
et al., 2013). This fauna is called interstitial because
it lives in gaps between grains of sand. Recent
results have shown that there is a huge biomass
of ciliates in soil and sediments in edaphic form
(Dong et al., 2016; Li et al., 2018). Ectoparasites
forms have been found in marine molluscs (Xu et
al., 2000; Xu and Song, 2008). In Cameroon, the
commensal forms of ciliated protists were found in
the rectal bulb of the Batrachians and in the digestive
tract of the Oligochaete Annelids (Fokam et al.,
2013; Fokam et al., 2014; Nana et al., 2018). This
category of protists, identified in the digestive tract
of oligochaetes, is very variable and can be classified
in three groups. The first group is the Clevelandellida
that have a buccal apparatus housed in the anterior
pole of the cell (Ngassam, 1983; Fokam et al.,
2014). The second group, Hysterocinetidae, is
characterized by a suction-cup type of fixation
device located at the anterior pole of the cell and a
buccal apparatus that has migrated to the posterior
pole (Nana et al., 2012). The third group, Astomes,
are ciliates without buccal apparatus (Nana et al.,
2010; Fokam et al., 2012; Obert and Vďačný, 2021).
The group, which interests us in this study, is the
Clevelandellida that colonize and cohabit the rectal
bulb of the Batrachians, but also are found in the
digestive tract of oligochaetes Glossoscolecidae and
Megascolecidae. Knowledge about the diversity of
this group of ciliated protozoa remains rudimentary.
The existing data are those of Ngassam (1983),
Affa'a et al. (2004), and Fokam et al. (2014). Based
on morphological, morphometric and infraciliary
analyses, our study aims at contributing to the

knowledge of biodiversity of this poorly known and
largely underestimated protozoan group.

**Material and methods**

STUDY REGION, COLLECTION AND IDENTIFICATION OF
EARTHWORMS

Earthworms were collected on the banks of the Nkam River in Ndogbélé (4°45′11″N; 9°96′64″E) at Yabassi, the administrative zone of Littoral Region in Cameroon (Fig. 1). This locality extends on a surface area of approximately 3080 km² between 4°27′ and 4°40′ of latitude North and between 9°57′ and 10°10′ of longitude East. This area is one of the least populated districts in the Littoral region with 4.77 inhabitants per km². The 85% of the surface area are occupied by a dense primary forest composed of large trees. Yabassi is watered by an equatorial climate of the Guinean type and the Cameroonian coastal sub-type. The climate is of two seasons, including a long rainy season, from March to November, with rainfall generally ranging between 4000 mm and 5000 mm per year, and a short dry season, from December to February. Air temperatures vary between 23 °C to 33.5 °C (Suchel, 1972).

Worms were identified as belonging to the family Glossoscolecidae, the genus *Alma*, according to the keys presented by Sims and Gerard (1999).

COLLECTION AND STAINING OF CILIATES

Fragments of the digestive tract pertaining to
each of the three main portions (fore-, mid-, and
hindgut) were incised under a binocular dissecting
microscope Wild M5 (Heerbrugg, Germany) in
a Petri dish containing a physiological Ringer's
solution. The ciliates were removed from the gut
using micropipettes, counted, washed out, and
then observed *in vivo* before fixation for cytological
studies. Their shape and mobility were registered.
Silver staining was performed using the ammoniacal
pyridinated silver carbonate technique described by
Fernandez-Galiano (1994). The nuclear apparatus
was fluorescently stained using 4',6-Diamidino-
2-Phenyl Indole (DAPI) (Johnson et al., 1982).
Double staining was performed using DAPI and
FITC-conjugated anti-tubulin antibody as des-
cribed previously (Diogon et al., 2001). Stained cells
were observed under a Leica DMR epifluorescence
microscope coupled to a Q-FISH (quantitative
<figure><img src="image_2.png" /><figcaption>Fig. 1. Collection site for earthworms.</figcaption></figure>

fluorescence in situ hybridization) image acquisition system. All measurements were made with a calibrated ocular micrometer. The following parameters were calculated: arithmetic mean; standard deviation; minimum and maximum values. Morphometric data were generated after the examination of groups of 30 stained cells of each species. Abundance and frequency of infestation was obtained from the 30 worms analyzed from the study site. The cells were manually drawn under a Wild M20 microscope equipped with a camera Lucida. A digital camera was used for light micrographs. Ciliate classification follows Lynn (2008).

# Results

## TAXONOMIC DESCRIPTION

- Phylum Ciliophora Doflein, 1901

- Sub-phylum Intramacronucleata Lynn, 2008

- Class Armophorea Lynn, 2008

- Order Clevelandellidae de Puytorac and Grain, 1976

- Family Nyctotheridae Amaro, 1972

- Genus *Nyctotherus* Leidy, 1849

In this genus, while grouping ciliates characterized by the presence of an apical secant system on their right side, we found three new species.

## *Nyctotherus nkamensis* n. sp.

**General morphology and morphometry** (Table 1). *Nyctotherus nkamensis* n. sp. cohabits with the Hysterocinetidae ciliates in the midgut of earthworms, *Alma nilotica* and *A. emini*. On average, there are 10 cells of ciliates per infested worm. The cells are ovoid, with the posterior pole less rounded than the anterior pole (Fig. 2). Cell measurements are 85-138 µm in length and 52-115 µm in width. The macronucleus has an irregular shape measuring 20-25 µm in length and 10-14 µm in width, located in the first half of the cell and arranged obliquely towards the dorsal side. This macronucleus is surmounted by a spherical micronucleus of about 3 µm in diameter. The buccal apparatus consists of a peristome almost as long as the infundibulum. The peristome starts behind the apex and continues along a curvilinear trajectory. The infundibulum, very characteristic, is heart-shaped. The adoral fringe has an average of 22 adoral fibers at the peristome and 33 – at the infundibulum.

**Infraciliature.** There are 95-110 kineties unevenly distributed on the two lateral faces of the ciliate. On the right side, 45-50 tightly packed kineties cover the cell. The apical secant system is well marked, the preoral one is very well developed and is characterized by the presence of short ciliary striae (Fig. 2, C). On the left side, there are 35-40 regularly spaced kineties (Fig. 2, D).
<table><caption>**Table 1.** Main characteristics of the new and some known species of the genus *Nyctotherus*.</caption><thead><tr><th></th><th>Nyctotherus nkamensis<br>n. sp.</th><th>Nyctotherus emini<br>n. sp.</th><th>Nyctotherus renimorphus<br>n. sp.</th><th>Nyctotherus ngassami<br>Fokam et al., 2014</th><th>Nyctotherus hoyoi Tuzet, Manier et<br>Jolivet, 1957</th><th>Nyctotherus ovalis Leidy,<br>1850</th></tr></thead><tbody><tr><td>Cell form</td><td>Ovoid</td><td>Ovoid</td><td>Kidney</td><td>Kidney</td><td>Globular, Ovoid</td><td>Ovoid</td></tr><tr><td>Cell length (µm)</td><td>85-138</td><td>66-185</td><td>125-220</td><td>153-269</td><td>85-280</td><td>57-185</td></tr><tr><td>Cell width (µm)</td><td>52-115</td><td>50-95</td><td>75-180</td><td>102-148</td><td>48-230</td><td>48-100</td></tr><tr><td>Kineties left side</td><td>35-40</td><td>30-35</td><td>39-55</td><td>32-43</td><td>43-45</td><td>54-61</td></tr><tr><td>Kineties right side</td><td>45-50</td><td>32-38</td><td>45-60</td><td>32-43</td><td>43-45</td><td>55-62</td></tr><tr><td>Macronucleus length (µm)</td><td>20-25</td><td>23-27</td><td>32-36</td><td>16-31</td><td>37</td><td>40</td></tr><tr><td>Macronucleus width (µm)</td><td>10-14</td><td>12-16</td><td>12-17</td><td>36-53</td><td>20</td><td>15</td></tr><tr><td>Micronucleus diameter<br>(µm)</td><td>3-4</td><td>3-4</td><td>3-5</td><td>3-5</td><td>4-5</td><td>2-3</td></tr><tr><td>Peristome (fibers)</td><td>22</td><td>36</td><td>55</td><td>29</td><td>30</td><td>25</td></tr><tr><td>Infundibulum (fibers)</td><td>33</td><td>23</td><td>20</td><td>48</td><td>50</td><td>34</td></tr><tr><td>Frequency (%)</td><td>14</td><td>12</td><td>10</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Average abundance</td><td>10±3</td><td>7±1</td><td>15±5</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Type host</td><td>Alma nilotica and A. emini<br>(Earthworms)</td><td>Alma emini (Earthworms)</td><td>Alma nilotica and A. emini<br>(Earthworms)</td><td>Eupolytoreutus sp.<br>(Earthworms)</td><td>Spirostreptus virgalor, Spirostreptus<br>multisulcalus, Ophiisreplus digitulatus<br>occiduus, Scaphiostreptus<br>Aculiconus (Myriapods diplopods)</td><td>Blatta orientalis (Insect)</td></tr><tr><td>Type locality</td><td>Ndogbélé (Littoral<br>Cameroon)</td><td>Ndogbélé (Littoral<br>Cameroon)</td><td>Ndogbélé (Littoral<br>Cameroon)</td><td>Bambili and Bambui<br>(North-West Cameroon)</td><td>Zaire (Democratic Republic of Congo)<br>and Maboké (Central African Republic)</td><td>Moroccan strain in<br>breeding in Rennes</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** *Nyctotherus nkamensis* n. sp. A – General morphology after double staining by DAPI (blue) and immunofluorescence microscopy (IF) using FITC-conjugated anti-tubulin antibody (green); B – general morphology (drawings after silver staining); C – ciliature of the ventral face; D – ciliature of the dorsal face. *Abbreviations:* Cy – cytopyge, In – infundibulum, Ma – macronucleus, Mi – micronucleus, Pe – peristome, SCL – short ciliary lines, SS – secant system. Scale bars: 20 µm.</figcaption></figure>

**Diagnosis.** Commensal of the digestive tract of *Alma nilotica* and *A. emini*; ovoid cell with the posterior pole less rounded than the anterior pole: 85-138 ×52-115 µm; 95-110 kineties unevenly distributed on both sides; peristome: 22 fibers; infundibulum: 33 fibers; frequency: 14%; average abundance: 10 cells per infested worm; locality of collection of the host: Ndogbélé at Yabassi.

**Type host.** Hindgut in earthworms *Alma nilotica* and *A. emini*.

**Type locality.** Ndogbélé (4°45'11"N; 9°96'64"E) at Yabassi, Littoral Region, Cameroon.

**Etymology.** The species is named “nkamensis” in reference to the “Nkam” river, place of collection of the hosts.

**Type material.** Slides of the holotype (MNHN-IR-2017-0002) are deposited to the protist collection of the National Museum of Natural History, Paris, France.

**Remarks.** *Nyctotherus nkamensis* n. sp. differs from the other ovoid-shaped species by the presence of a caudal secant system and a somewhat rectilinear disposition of the buccal apparatus. Although the general morphology of *N. nkamensis* reminds the one of *N. ngassami* Fokam, Nana, Ngassam, Bricheux, Bouchard, Vigues and Sime-Ngando, 2014 and the ovoid form of *N. hoyoi* Tuzet, Manier and Jolivet, 1957, the area occupied by the infundibulum differs in these two latter species. Beside, *N. dupouyi* Ngassam, 1983 and *N. hoyoi* present a massive macronucleus hiding the micronucleus while in *N. nkamensis*, the macronucleus is crescent in shape with a micronucleus situated anteriorly in it concavity.

*Nyctotherus emini* n. sp.

**General morphology and morphometry** (Table 1). *Nyctotherus emini* n. sp. lives together with *Ptychostomum elongatum* Njiné and Ngassam, 1993 and *Proptychostomum commune* Ngassam and Grain, 1997 (Hysterocinetidae) in the midgut of earthworms *Alma emini*. The cell is ovoid, with the anterior pole more rounded than the posterior pole (Fig. 3). It measures on average 110 µm (66-185 µm) in length and 75 µm (50-95 µm) in width. The long macronucleus, located close to the anterior pole of the cell, is transversal. It measures on average 14 µm in length and 25 µm in width, and is surmounted by a micronucleus averaging 3 µm in diameter. The peristomal gutter starts above the apex and is 35 µm long. The infundibulum, slightly curved, is 24 µm in length; it opens at the level of the equatorial plane. The adoral fringe has on average 36 membranelles at the peristome and 23 – at the level of the infundibulum. Cytopyge is a well-marked slit.

**Infraciliature.** The somatic ciliature consists of 62-73 kineties covering the cell. There are 32-38 kineties arranged longitudinally on the right side where the secant system is well marked (Fig. 3, C). On the left side, 30-35 kineties are arranged obliquely (Fig. 3, D).

**Diagnosis.** Commensal of the digestive tract of *Alma emini*; ovoid cell with anterior pole more rounded than the posterior pole: 66-185×71-76 µm; 50-95 kineties evenly distributed on both sides; peristome: 36 fibers; infundibulum: 23 fibers; frequency: 12%; average abundance: 7 cells per infested worm; locality of collection of the host: Ndogbélé at Yabassi.
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** *Nyctotherus emini* n. sp. A — General morphology after double staining by DAPI (blue) and immunofluorescence microscopy (IF) using FITC-conjugated anti-tubulin antibody (green); B — general morphology (drawings after silver staining); C. — ciliature of the ventral face; D — ciliature of the dorsal face. *Abbreviations:* Cy — cytopyge, In — infundibulum, Ma — macronucleus, Mi — micronucleus, Pe — peristome, SS — secant system. Scale bars: 20 µm.</figcaption></figure>

**Type host.** Midgut, in earthworms *Alma emini*.
**Type locality.** Ndogbélé (4°45'11''N; 9°96'64''E) at Yabassi, Littoral Region, Cameroon.

**Etymology.** The species is named “emini” in reference to the specificity of the host.

**Type material.** Slides of the holotype (MNHN-IR-2017-0004) are deposited to the protist collection of the National Museum of Natural History, Paris, France.

**Remarks.** By its general morphology, *Nyctotherus emini* n. sp. could at first sight be confused with *N. ovalis* Leidy, 1850, if it were not for the number and very tight arrangement of the kineties on its two faces. This species differs from *N. hoyoi* and *N. dupouyi* by its slightly curved infundibulum and the presence of oblique cinetia on the left side.

*Nyctotherus renimorphus* n. sp.

**General morphology and morphometry** (Table 1). This ciliate is commensal of the digestive tract of *Alma nilotica* and *A. emini* where it cohabits the midgut and hindgut with the Hysterocinetidae of the genus *Ptychostomum* Stein, 1860. This species has a frequency of 10% and an average abundance of 15 ciliates per worm. The cell is kidney-form with the anterior pole more rounded than the posterior pole (Fig. 4). It averages 185 µm (125-220 µm) in length and 98 µm (75-180 µm) in width. The macronucleus is irregularly shaped and obliquely arranged, straddling the first and second upper thirds of the cell. It measures on average 34 µm in length and 15 µm in high. The micronucleus, located above the macronucleus near the right side, measures between 3 and 5 µm in diameter. The buccal apparatus is always constituted by a peristome and an infundibulum. The peristome begins near the right face; it is 80 µm in length on average and made up of 55 adoral organelles. The infundibulum carries about 20 fibers. The cytopharynx is curvilinear. The cytopyge is presented by a well-marked V-shaped piece.

**Infraciliature.** The somatic ciliature consists of 95-110 kineties unevenly distributed on the two la-teral faces of the ciliate (Fig. 4, C-D). On the right side, these kineties are less dense and arranged longitudinally. On the left side, they are more dense and arranged obliquely.

**Type host.** Midgut and hindgut, in earthworms *Alma nilotica* and *A. emini*.

**Type locality.** Ndogbélé (4°45'11''N; 9°96'64''E) at Yabassi, Littoral Region, Cameroon.

**Etymology.** The species is named "*renimorphus*" in reference to the kidney shape of the cells.

**Type material.** Slides of the holotype (MNHN-IR-2017-0006) are deposited to the protist collection of the National Museum of Natural History, Paris, France.

**Remarks.** *Nyctotherus renimorphus* n. sp. is similar in its general somatic ciliature to *N. neocurtillae* Carini, 1938, a commensal of *Neocurtilla hexadacryla* Earl, 1972, and *Neocurtilla alpha* Earl, 1972, a commensal of the hindgut of *Gryllotalpa gryllotalpa*. However, it differs from them by the reniform morphology and its V-shaped cytopyge, bringing our ciliate closer to *Nyctotherus hoyoi* Tuzet, Manier and Jolivet, 1957. The described ciliate is morphologically close to *Pronyctotherus dragescoi* Albaret and
<figure><img src="image_5.png" /></figure>

**Fig. 4. Nyctotherus renimorphus** n. sp. A – General mor-phology after double staining by DAPI (blue) and immunofluorescence microscopy (IF) using FITC-conjugated anti-tubulin antibody (green); B – general morphology (drawings after silver staining); C. – ciliature of the ventral face; D – ciliature of the dorsal face. *Abbreviations:* Cy – cytopyge, In – infundibulum, Ma – macronucleus, Mi – micronucleus, Pe – peristome, SS – secant system. Scale bars: 20 µm.

<figure><img src="image_6.png" /></figure>

**Fig. 5. Nyctotheroides ndogbeleensis** n. sp. A – General morphology after double staining by DAPI (blue) and immunofluorescence microscopy (IF) using FITC-conjugated anti-tubulin antibody (green); B – general morphology (drawings after silver staining); C. – ciliature of the ventral face; D – ciliature of the dorsal face. *Abbreviations:* Cy – cytopyge, In – infundibulum, Ma – macronucleus, Mi – micronucleus, Pe – peristome, SS – secant system. Scale bars: 20 µm.

Njiné, 1975, with the difference that in the latter, the somatic ciliature is dense. It presents an apical secant system on the right side, which is a typical character of *Nyctotherus*.

## Genus *Nyctotheroides* Albaret, 1975

*Nyctotheroides* groups the ciliates characterized by the existence of three secant systems on their two faces (Albaret, 1975): on the right face, we note an apical secant system and on the left face – two secant systems (one apical and the other caudal). In this genus, we found a new species.

### *Nyctotheroides ndogbeleensis* n. sp.

**General morphology and morphometry** (Table 2). This ciliate is commensal in the digestive tract

of *Alma nilotica* and *A. emini* collected at Ndogbélé where it cohabits in the hindgut with the Hysterocinetidae ciliates of the genus *Ptychostomum* and *Preptychostomum* de Puytorac, 1968. *Nyctotheroides ndogbeleensis* n. sp. has a frequency of 25% and an average abundance of 23 ciliates per worm. The cell is elongate and averages 95 µm (75-120 µm) in length and 32 µm (25-40 µm) in width (Fig. 5). The constriction of the right side is more or less marked. The macronucleus is bilobed and forms two dense asymmetric masses located perpendicular to the curvature of the peristome. It measures 15-22 µm in length and 7-10 µm in width. The cytopyge, a narrow and oblique channel, opens on the ventral side of the posterior pole of the cell. The buccal apparatus comprises on average 45 adoral fibres.

**Infraciliature.** The somatic kineties are almost equally distributed on the two lateral faces (Fig. 5,
<table><caption>**Table 2.** Main characteristics of the new and some known species of the genus *Nyctotheroides*.</caption><thead><tr><th></th><th>Nyctotheroides ndogbeleensis n. sp.</th><th>Nyctotheroides chiromantisi Albaret, 1975</th><th>Nyctotheroides njinei Albaret, 1975</th></tr></thead><tbody><tr><td>Cell form</td><td>Elongated</td><td>Kidney</td><td>Kidney</td></tr><tr><td>Cell length (µm)</td><td>75-120</td><td>148-181</td><td>95-125</td></tr><tr><td>Cell width (µm)</td><td>25-40</td><td>72-89</td><td>59-81</td></tr><tr><td>Kineties left side</td><td>10-15</td><td>45-50</td><td>32-36</td></tr><tr><td>Kineties right side</td><td>25-30</td><td>40-45</td><td>32-36</td></tr><tr><td>Macronucleus length (µm)</td><td>15-22</td><td>43</td><td>36</td></tr><tr><td>Macronucleus width (µm)</td><td>7-10</td><td>18</td><td>17</td></tr><tr><td>Micronucleus diameter (µm)</td><td>2-4</td><td>5-6</td><td>4-5</td></tr><tr><td>Peristome (fibers)</td><td>30</td><td>65</td><td>35-40</td></tr><tr><td>Infundibulum (fibers)</td><td>22</td><td>70</td><td>85-95</td></tr><tr><td>Frequency (%)</td><td>25</td><td>100</td><td>60</td></tr><tr><td>Average abundance</td><td>23 ± 6</td><td>-</td><td>-</td></tr><tr><td>Type host</td><td>A. nilotica and A. emini (Earthworms)</td><td>Chiromantis rufescens (Amphibians)</td><td>Phrynobatrachus batesi (Amphibians)</td></tr><tr><td>Type locality</td><td>Ndogbélé (Littoral Cameroon)</td><td>Maboké (Central African Republic)</td><td>Cameroon</td></tr></tbody></table>

C-D). The right side bears 25-30 kineties; 10-15 of these kineties form, together with the single dorsal kineties that terminate above the peristome, a right dorsal apical secant system. The left lateral side shows a reduced preoral secant system, formed by the confrontation of left dorsal kineties and the ends of right ventral kineties. The left apical secant system is formed by the chevron-like confrontation of 3 to 4 latero-ventral kineties at the end parallel to the peristomal groove and as many lateral kineties. This secant system is almost median (Fig. 5, D).

**Diagnosis.** Commensal of the digestive tract of *Alma nilotica* and *A. emini*; elongated cell: 75-120 µm × 25-40 µm; bilobed macronucleus forming two dense dissymmetrical masses: 15-22×7-10 µm; buccal apparatus: 45 adoral fibers; frequency: 25%; mean abundance: 23±5 ciliates per infested worm; locality of host collection: Ndogbélé in Yabassi.

**Type host.** Hindgut, earthworm (*Alma emini* and *A. nilotica*).

**Type locality.** Ndogbélé (4°45'11''N; 9°96'64''E) at Yabassi, Littoral Region, Cameroon.

**Etymology.** The species is named "ndogbeleensis" in reference to Ndogbélé, the collection place of the hosts of the ciliates.

**Type material.** Slides of the holotype (MNHN-IR-2017-0007) are deposited to the protist collection of the National Museum of Natural History, Paris, France.

**Remarks.** *Nyctotheroides ndogbeleensis* n. sp. has a general appearance quite similar to that of *N. chiromantisi* Albaret, 1975, a commensal of the rectum of *Chiromantis rufescens*, of *N. njinei* Albaret, 1975,

a commensal of the rectum of *Phrynobatrachus batesi*, and of *N. faberi* Carini, 1939, a parasite of *Hyla faber*. It differs, however, by the bilobed shape of its macronucleus located perpendicular to the anteroposterior axis of the cell. Additionally, it differs notably from *N. tejerai* Pinto, 1926 (a commensal of the rectum of *Leptopelis notatus*) by its elongated shape, but also by its peristome, which is clearly longer than the infundibulum and located behind the nuclear apparatus.

## Discussion

The described ciliated protists, collected for the first time in the digestive tract of oligochaetes of the genus *Alma*, are thus distinguished from known species by various characters. With respect to their morphology and the pattern of their ciliature (absence of a postoral secant system), the first three species described above belong to the genus *Nyctotherus* as originally described by Leidy (1849) and some later authors (Grassé, 1928; de Puytorac and Öktem, 1967; Albaret and Njiné, 1975; Ngassam, 1983; Fokam et al., 2014).

Ecologically, *Nyctotherus nkamensis* n. sp., *Nyctotherus renimorphus* n. sp., and *Nyctotheroides ndogbeleensis* n. sp. proliferate mostly in the hindgut of earthworms. It has been clearly demonstrated that the hindgut portion of the digestive tract of *Alma emini* and *A. nilotica* is alkaline, less rich in mineral elements and less viscous than the foregut (Nana et al., 2014). It is also noted that in the foregut,
the ciliated mesofauna is very mobile and active
unlike in the hindgut populated by Hysterocinetidae
(Nana et al., 2014). De Puytorac and Mauret (1956)
had already shown a stratification of Astome, endo-
commensal ciliates of the digestive tract of *Allolo-
bophora savignyi*, related to physicochemical vari-
ables. The cohabitation of *Nyctotherus emini* n. sp.
and some Hysterocinetidae in the midgut confirms
the theory of "species association", characterized
by a distribution in distinct compartments. Indeed,
species that enter into various associations, either
ecological or trophic, in various compartments,
inevitably show similar reactions to variations in
the conditions of their environment (Pinel-Alloul
et al., 1990).

Let us note that the variation in abundance is not
the only characteristic fact of the Nyctotheridae of
the digestive tract of earthworms. Gohre (1943) high-
lighted an analogous fact for the three species of
*Gregarina*, parasites of the larva of the insect *Tenebrio
molitor*. Adam (1951), studying the distribution of
ciliates in the large intestine of the horse, showed that
two successive faunas could be recognized there.
One, with *Blepharocorys uncinata* and *Cycloposthium
bipalmatum*, was characteristic of the cecum and
colon up to the pelvic curvature; the other, with
*Bundleia postciliata* and *Blepharocorys curvigula*,
was present from there to the rectum. In all the simi-
lar cases, the variation of abundance and the strati-
fication of the described species would obviously
depend on the varied physico-chemical and biotic
conditions, successively offered by the biological
environment to the ciliates, in relation to the needs
of each of them. In such a seriation, the external
environment would intervene additionally, promo-
ting a competition between the various groups of
protists. If there is such an antagonism in the case
of the ciliates that are the objects of this study,
its importance does not seem considerable under
normal conditions. Indeed, when one of the species
is missing, the place it would occupy in the portion
of the digestive tract generally remains free. These
observations do not exclude the possibility of such
competition at the origin of the settlement of the
ciliates in the digestive tract. If this was so, as is
probable, selection might have subsequently sorted
out the forms so closely adapted to certain portion
of the digestive tract as to render the antagonism
between them unnecessary.

Schmidt with coauthors (2010, 2011) showed that
the enzymes (cellulase, chitinase, and hydrogenase)
were nearly absent in the pharynx, esophagus, crop,
and gizzard of many worms, whereas they reach their

highest concentration in the anterior part of the
intestine. While the Hysterocinetidae that populate
the hindgut seem to be able to do without such
diastases, it is rather possible that the Nyctotheridae
have a great need for them. A similar case has been
proved in *Diplodinium* and *Entodinium* of the rumen
of ruminants: the representatives of the former
have cellulase and cellobiase that enable them to
transform cellulose into glucose, whereas species of
the latter genus lack them (Tracey, 1951).

Acknowledgments

This research was partially supported by the
CNRS (UMR 6023) and the University Clermont
Auvergne, Clermont-Ferrand, France. The authors
thank Dr. Bernard Vigues for the DAPI technique
application.

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