Delphinapterus leucas (Pallas, 1776)

Jin, Didi, Qu, Zhishuai, Wei, Bojue, Montagnes, David J. S., Fan, Xinpeng & Chen, Xiangrui, 2021, Two parasitic ciliates (Protozoa: Ciliophora: Phyllopharyngea) isolated from respiratory-mucus of an unhealthy beluga whale: characterization, phylogeny and an assessment of morphological adaptations, Zoological Journal of the Linnean Society 191, pp. 941-960 : 949-952

Host: Delphinapterus leucas (beluga whale).

Voucher material: Four slides with protargol-stained specimens (indicated with a black circle of ink on each coverslip) have been deposited in the collection of Ningbo University (registration numbers: LJ-II- 20170502-01, 02, 03 and 04).

General morphology and ciliary pattern of Ningbo population: The cell size is 55–80 × 40–50 μm in vivo and 50–80 × 32–47 μm after protargol staining. The body is laterally flattened in shape with a length– width ratio of about 3: 2 in lateral view ( Figs 6A, 7A). From lateral view, the cell is discoid or ovate in outline; the anterior end is slightly pointed and the posterior margin is broadly rounded ( Fig. 6A, C). The dorsal margin of the ciliated right region is sculptured by a C-shaped band (or groove) ( Fig. 7A, M). This band extends from the cell apex to the posterior end. Some long bands (23 out of 35 individuals observed) curve around the posterior end and anteriorly on to the left of the podite. This structure is visible as a deep groove in vivo and as an argentophilic band (AB) in protargolimpregnated specimens ( Fig. 6D, E). The left surface is not regular in shape ( Figs 6B, 7B). The oral cavity is broad and located at the anterior quarter of the cell. The cytostome is located posteriorly in the oral cavity ( Figs 6F, 7G, I, M). The cytoplasm is colourless,

contained materials or connected with the outside, double-arrowhead marks the rich cytoplasmic vesicles in the cytoplasm beneath the folds and near the sacs. Abbreviations: Co, circumoral kineties; CT, cytopharyngeal tube; FV, food vacuole; MIT, mitochondria; NS, non-ciliated stripe; PCMT, postciliary microtubules; PS, parasomal sacs; Scale bars = 10 μm (A), 0.5 μm (B–D, H, I) or 2 μm (E, G), 1 μm (F).

Abbreviations: CV, coefficient of variation in %; Max, maximum; Mean, arithmetic mean; Min, minimum; n, number of specimens examined; SD, standard deviation; SE, standard error.

containing multiple food vacuoles consisting of unidentified amorphous material. The single contractile vacuole is up to 6 μm across and positioned at the left of mid-body in ventral view; contraction occurs with an interval of 20–30 s; the contractile vacuole pore is located between the second and third right kineties in mid-body ( Fig. 6A, E).

The macronucleus is ovoid and heteromerous, located in mid-body ( Figs 6F, 7M). The micronucleus is ellipsoid and adjacent to the macronucleus ( Fig. 6D). The podite is broadly cone-shaped, situated in the posterior quarter of cell, about 4–6 μm in length and 3–8 μm across at the base ( Figs 6A, E, 7A). Individuals are often attached to the substrate by the podite and rotate through viscous medium with the cilia beating in a regular pattern.

Cilia are about 8 μm long in vivo. The ciliature is shown in Figs 6E, F, 7F–M. Kinetosomes in somatic kineties are densely arranged. Somatic kineties are divided into three parts: right, left and kinetofragments. The right field comprises 41–58 kineties; the leftmost has 15–25 relatively short kineties, extending from the level of the oral field to the level of the podite; remaining kineties originate from the apex of the cell and extend posteriorly to the cell end and bend to the left ( Fig. 6E). Three to four left kineties are located on the left of the oral cavity, originate from near the apex of the cell and terminate posteriorly at the level of the posterior margin of the cytostome, about two-fifths of the cell length ( Figs 6E, 7G, H). Four short kinetofragments are located on the anterior-left of the podite and are often curved to the right. ( Figs 6F, 7J, K, L). Equatorial fragment are not detected.

Oral ciliature is composed of a pre-oral kinety (seldom two), two parallel circumoral kineties and nine to 13 infundibular kineties ( Fig 6F). Circumoral kineties are located on the anterior of the cytostome ( Figs 6F, 7I). The pre-oral kinety is located on the anterior-left of the circumoral kineties, consisting of one or two closely set kinetosomes ( Figs 6F, 7H). Infundibular kineties are positioned in an arc of circumoral kineties ( Fig. 7H). The cytopharynx extends below mid-body and curves posteriorly; no nematodesmal rod is found ( Figs 6F, 7G, M).

MOLECULAR DATA AND PHYLOGENETIC POSITION

( FIGS 8–10)

The GenBank accession numbers, lengths and G+C contents of sequences (SSU and LSU rDNA, and ITS1-5.8S-ITS2) of Kyaroikeus paracetarius and Planilamina ovata from this study are provided in Table 2. The topologies of the BI and ML trees are almost identical, thus, only the ML tree is presented here, with support values from both of the algorithms indicated on branches.

SSU rDNA ( Fig. 8): Planilamina ovata is sister to Trochilia petrani Dragesco, 1966 with low support values (ML/BI, 65%/0.80) and their clade then clustered with Kyaroikeus paracetarius with good support. The clade of Trochilia / Kyaroikeus / Planilamina in turn clusters with Microxysma Deroux, 1976 (ML/BI, 80%/1.00) and then with the core of Dysteriidae species represented by Dysteria Huxley, 1857 , Mirodysteria Kahl, 1933 and Spirodysteria Gong et al., 2007 , with moderate support values (ML/BI, 78%/0.98).

ITS1-5.8S-ITS2 ( Fig. 9): Planilamina ovata clusters with Kyaroikeus paracetarius (ML/BI, 67%/0.82), forming a clade with Dysteria derouxi Gong & Song, 2004 (ML/BI, 88%/0.97).

LSU rDNA ( Fig. 9): Kyaroikeus paracetarius clusters with Planilamina ovata with good support, and then this branch forms a clade with Dysteria derouxi with good support.

Concatenated genes ( Fig. 10): The phylogenetic tree based on the concatenated dataset is different from the SSU rDNA tree; i.e. Kyaroikeus paracetarius , Planilamina ovata and Trochilia petrani group into a clade with 100% bootstrap support in the ML tree, but are not resolved by BI. The families Chilodonellidae and Lynchellidae are located in different positions: Lynchellidae is closer to Chlamydodontidae in the concatenated gene tree, while in the SSU rDNA tree Chilodonellidae is closer to Chlamydodontidae , and Lynchellidae is in a peripheral position.