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Volume 2 Issue 3
Sep.  2020
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Redescription of five tintinnine ciliates (Alveolata: Ciliophora: Oligotrichea) from coastal waters of Qingdao, China

  • Corresponding author: Xiaozhong Hu
  • Received Date: 2019-12-23
    Accepted Date: 2020-02-13
    Published online: 2020-05-04
  • Yang Bai and Rui Wang have contributed equally to this work.
    Edited by Jiamei Li.
  • Tintinnine ciliates are widely distributed around the world and are characterized by the possession of a lorica that is highly diverse in structure and morphology. In the present study, five tintinnines, namely Tintinnopsis cf. radix, T. everta Kofoid and Campbell, 1929, T. mulctrella Kofoid and Campbell, 1929, Eutintinnus inflatus Marshall, 1969, and Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924, were collected from coastal waters of Qingdao, China. Three are newly recorded from China, i.e., T. everta, T. mulctrella, and E. inflatus. The morphology of the lorica and cell proper of all five species are described and compared with original and subsequent descriptions. The morphology in vivo of the lorica and cell proper of all five species are described and compared with original and subsequent descriptions. In addition, the ciliary patterns of F. ehrenbergii and T. cf. radix are revealed based on protargol-stained specimens.
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Redescription of five tintinnine ciliates (Alveolata: Ciliophora: Oligotrichea) from coastal waters of Qingdao, China

    Corresponding author: Xiaozhong Hu
  • 1. Institute of Evolution and Marine Biodiversity & Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao 266003, China
  • 2. Marine College, Shandong University, Weihai 264209, China
  • 3. Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan

Abstract: Tintinnine ciliates are widely distributed around the world and are characterized by the possession of a lorica that is highly diverse in structure and morphology. In the present study, five tintinnines, namely Tintinnopsis cf. radix, T. everta Kofoid and Campbell, 1929, T. mulctrella Kofoid and Campbell, 1929, Eutintinnus inflatus Marshall, 1969, and Favella ehrenbergii (Claparède and Lachmann, 1858) Jörgensen, 1924, were collected from coastal waters of Qingdao, China. Three are newly recorded from China, i.e., T. everta, T. mulctrella, and E. inflatus. The morphology of the lorica and cell proper of all five species are described and compared with original and subsequent descriptions. The morphology in vivo of the lorica and cell proper of all five species are described and compared with original and subsequent descriptions. In addition, the ciliary patterns of F. ehrenbergii and T. cf. radix are revealed based on protargol-stained specimens.

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Introduction
  • Planktonic ciliates are among the most diverse and numerically important members of microzooplankton and act as a trophic link in the microbial food web of aquatic ecosystems (Agatha 2011; Countway et al. 2005; Doherty et al. 2010; Dolan 2010; Orsi et al. 2011; Santoferrara et al. 2013; Song et al. 2009, 2018a, b, 2019a, b; Wang et al. 2019a, b; Worden et al. 2015; Xu et al. 2017). Within planktonic ciliates, tintinnines are easily recognized by possessing an agglomerated or hyaline lorica with divergent configuration (Agatha and Strüder-Kypke 2012). Up to now, at least 1000 tintinnine morphospecies have been discovered and their classification is based almost entirely on lorica features (e.g., Agatha and Strüder-Kypke 2012; Kofoid and Campbell 1929, 1939). However, in recent years, it is widely recognized that lorica features alone are insufficient for species circumscription and identification in this group of ciliates because of cryptic diversity and the polymorphic nature of the lorica (Laval-Peuto 1981; Santoferrara et al. 2015, 2017; Xu et al. 2013; Zhang et al. 2017). Recent studies have suggested that the tintinnine taxonomy is jointly determined by the characters of lorica, cell proper and ciliature (Agatha 2008, 2010a, b; Agatha and Tsai 2008; Gruber et al. 2018; Hu et al. 2019; Jiang et al. 2012; Santoferrara et al. 2016, 2017; Smith et al. 2018). Unfortunately, the ciliary and cytological data are available for only about 5% of known tintinnine morphospecies (e.g., Agatha and Strüder-Kypke 2012; Foissner and Wilbert 1979; Gruber et al. 2018; Jiang et al. 2012; Smith et al. 2018).

    The Chinese coastal zone includes a large number of different ecological environments (e.g. mangroves, aquaculture ponds, and beaches) that support a high biodiversity of tintinnines, i.e., about 150 species have been reported from Chinese coastal waters (Hu et al. 2019; Liu 2008; Xu et al. 2001; Xu and Song 2005; Zhang et al. 2012). In the present study, five tintinnine species, namely Eutintinnus inflatus, Favella ehrenbergii, Tintinnopsis everta, T. mulctrella and T. cf. radix, collected from coastal waters of Qingdao, China, were morphologically investigated using observations of live and, where possible, protargol-stained specimens. This study aims to expand knowledge and understanding of the diversity of this group of eukaryotic microorganisms (Table 1).

    Characters Species Min Max Mean SD CV N
    Lorica, total length Tr 190 310 243.1 41.8 17.2 8
    Te 65 95 81.6 11.4 14 8
    Tm 70 85 80 5.8 7.2 7
    Ei 95 125 111.3 9.5 8.6 8
    Fe 230 300 270.3 34.3 12.7 10
    Lorica, anterior opening diameter Tr 35 60 45.6 7.8 17 8
    Te 75 110 92.1 12.8 13.9 8
    Tm 25 35 30.7 4.5 14.6 7
    Ei 30 35 31.9 2.6 8.1 8
    Fe 70 105 83.6 12.5 14.9 7
    Lorica, posterior opening diameter Ei 5 15 11.3 2.3 20.6 8
    Lorica, collar length Te 35 50 44.9 6.1 13.6 8
    Tm 15 25 20.7 3.5 16.7 7
    Lorica, bowl width Te 40 55 46.8 5.1 10.9 8
    Tm 30 50 40.7 7.9 19.3 7
    Ei 30 45 36.9 5.3 14.4 8
    Lorica, ratio of total length: opening diameter Tr 4.8 6.3 5.3 0.5 9 8
    Te 0.8 0.9 0.9 0.1 11.1 8
    Tm 2.4 3.2 2.6 0.3 10.9 7
    Ei 3.2 3.8 3.5 0.3 7.5 8
    Fe 2.4 2.8 2.6 0.2 7.7 8
    Cell proper, length Tr 53 112 79.6 20.8 26.2 8
    Fe 71 124 85.2 6.2 7.3 9
    Cell proper, width Tr 38 51 43.3 4.4 10.2 8
    Fe 79 108 93.1 6.2 6.7 7
    Macronuclear nodules, number Tr 2 2 2 0 0 8
    Fe 2 2 2 0 0 8
    Anterior cell end to anterior macronucleus nodule, distance Tr 13 22 16.1 3.3 20.3 8
    Fe 9 21 14.6 4 27.3 9
    Ventral kinety, length Tr 20 49 30 11.5 38.4 8
    Fe 29 47 35.7 6.4 1.8 9
    Ventral kinety, number of kinetids Tr 37 46 41.8 3.2 7.8 8
    Fe 33 49 39.5 3.1 7.8 8
    Ventral kinety, distance to collar membranelles Tr 4 5 4.8 0.5 9.7 8
    Fe 2 2 2 0 0 8
    Dorsal kinety1, length Tr 44 84 58.5 14.6 24.9 8
    Fe 58 80 71.1 15.2 21.4 9
    Dorsal kinety1, number of kinetids Tr 41 54 46.6 4.4 9.4 8
    Fe 72 85 78.3 8.5 10.9 9
    Dorsal kinety2, length Fe 70 90 82.3 13.2 16 8
    Dorsal kinety2, number of kinetids Fe 80 92 85.5 6.3 7.4 8
    Posterior kinety, length Tr 15 23 18.6 2.6 14 8
    Posterior kinety, number of kinetids Tr 14 17 15.4 1.3 8.5 8
    Right ciliary field, number of kineties Tr 9 12 10 1.2 12 8
    Fe 40 58 49 5.4 11 7
    Lateral ciliary field, number of kineties Tr 17 24 20.4 2.1 10.5 8
    Fe 7 14 12 1.7 14.2 7
    Left ciliary field, number of kineties Tr 10 13 11.4 9.2 8.1 8
    Fe 27 40 33.5 3.1 9.3 9
    Adoral zone of membranelles, diameter Tr 38 51 43.3 4.4 10.2 8
    Fe 92 114 102.5 3.1 3 6
    Collar membranelles, number Tr 16 19 16.5 1.2 7 8
    Fe 16 16 16 0 0 8
    Collar membranelles, number of elongated ones Tr 4 4 4 0 0 8
    Fe 5 5 5 0 0 8
    Buccal membranelles, number Tr 1 1 1 0 0 8
    Fe 1 1 1 0 0 8
    Lorica data are based on living observations, cell data are based on protargol-stained specimens The lorica measurements were calculated from micrographs taken at×100–400 magnifications and are accurate to± 5 μm. Measurements are in μm
    CV coefficient of variation in %, Max maximum, Mean arithmetic mean, Min minimum, N number of specimens examined, SD standard deviation, Tm Tintinnopsis mulctrella, Tr Tintinnopsis cf. radix, Te Tintinnopsis everta, Ei Eutintinnus inflatus, Fe Favella ehrenbergii

    Table 1.  Morphometric data of five species

Results and comparison
  • Order Choreotrichida Small and Lynn, 1985

    Genus Tintinnopsis Stein, 1867

    Tintinnopsis cf. radix (Fig. 1a-l; Table 1)

    Figure 1.  a-l Drawings (a-d) and photomicrographs (e-l) of Tintinnopsis cf. radix in vivo (a, e-h) and after protargol staining (b-d, i-l). a Representative specimen, arrow indicates posterior opening. b, c Ciliary pattern of ventral (b) and dorsal (c) sides of same specimen. d Kinetal map of a morphostatic specimen. e Representative specimen. f Contracted specimen. g Anterior region of lorica and cell proper. h Posterior opening (arrow). i Ventral side, showing the ventral kinety, right ciliary field, and lateral ciliary field. j Anterior portion of dorsal kinety. k Dorsal kinety (arrowhead). l A middle divider. BM buccal membranelle, CM collar membranelles, DK dorsal kinety, LA lateral ciliary field, LF left ciliary field, OP oral primordium, PCM prolonged collar membranelles, PK posterior kinety, RF right ciliary field, VK ventral kinety. Scale bars: 120 μm (a), 30 μm (b, c), 100 μm (e, f)

Description
  • Lorica conspicuously elongated and narrow, 190-310 μm long (Fig. 1a, e, f). Opening is widest portion of lorica, 35-60 μm in diameter (Fig. 1a, e-g). Anterior 2/3 of lorica cylindrical, posterior 1/3 cone-shaped narrow gradually to posterior end, which often has an irregular hole near tip, about 20-50 μm across (Fig. 1a, e-h). Lorica wall thin, agglomerated with mineral particles about 2-10 μm in size (Fig. 1a, e, f).

    Cell proper obconical (Fig. 1a, e-g), measures 40-95 μm×40-65 μm in vivo, and 53-112μm ×38-51 μm after protargol staining, posterior portion progressively narrows into a peduncle that is about 150 μm long and attaches to bottom of lorica. Two macronuclear nodules, each about 15μm ×10 μm in size after protargol staining (Fig. 1j). Micronuclei difficult to recognize, because they were insufficiently stained with protargol. Tentaculoids, striae, accessory comb, cytopyge, contractile vacuole, and capsules not recognized. Cell proper retracts quickly into lorica using its contractile peduncle and posterior portion after being disturbed; when motionless oral membranelles bend towards centre of peristomial field. Cytoplasm colorless, with food vacuoles contains microalgae (Fig. 1e, g). Locomotion by rotating about main cell axis and irregular swimming.

    Somatic ciliary pattern complex, comprises a ventral, dorsal, and posterior kinety and right, left, and lateral ciliary fields (Fig. 1b-d, i-l). Kinetids of each ciliary row ostensibly connected by argyrophilic fibers. Ventral kinety commences about 5 μm anteriorly to left two or three kineties of right ciliary field, about 5 μm posteriorly to collar membranelles, 20-49 μm long, and composed of 37-46 monokinetids, densely spaced in anterior portion and progressively more widely spaced in posterior portion (Fig. 1b, d). Right ciliary field mostly commences about 5 μm posteriorly to collar membranelles, comprises 9-12 kineties, neighboring kineties about 3 μm apart, each composed of 4-11 widely spaced monokinetids and one anterior dikinetid; two leftmost kineties commence about 1 and 2 μm, respectively, below anterior end of remaining right kineties (Fig. 1b, d, i). Left ciliary field commences about 5 μm posteriorly to collar membranelles, comprises 10-13 kineties, neighboring kineties about 5 μm apart; kineties in left portion always obviously shorter than remaining kineties; each kinety composed of a single anterior dikinetid and zero to nine widely spaced monokinetids (Fig. 1b-d). Each basal body in left and right ciliary fields bears a cilium, anteriormost one in each kinety about 8-10 μm in length and others about 2 μm in length after protargol staining. Lateral ciliary field starts about 5 μm posteriorly to collar membranelles, with 17-24 monokinetidal kineties of similar length, kineties in right portion usually more closely spaced than those in left portion (Fig. 1b, d, i). Dorsal kinety 44-84 μm long, commences at same level as lateral ciliary field, about 5-10 μm away from left and right ciliary fields, and curves leftwards, terminating near posterior 1/6 of cell proper; comprises 41-54 dikinetids, and only posterior basal body of each dikinetid bears a cilium that is about 8-10 µm long after protargol staining (Fig. 1c, d, j, k). Posterior kinety, 15-23 μm long, commences below ventral kinety and extends to posterior end of cell proper, comprises 14-17 dikinetids; only posterior basal body of each dikinetid bears a cilium that is about 8-10 µm long (Fig. 1b, d, i).

    Adoral zone of membranelles closed, composed of 16-19 collar membranelles, of which four extend into buccal cavity, and 1 buccal membranelle (Fig. 1a-d, g), and cilia of membranelles up to 30-40 μm long, structure of polykinetids not recognized. Endoral membrane not recognized. A middle divider was observed, which showed oral primordium of opisthe positioned left of ventral kinety and posterior to lateral ciliary field (Fig. 1l).

  • Comparison of lorica features with original and subsequent descriptions

  • Tintinnopsis radix has been recorded on many occasions worldwide (e.g., Durán 1953; Imhof 1886; Jiang et al. 2012; Kofoid and Campbell 1929; Marshall 1969). It was first described in Imhof (1886) as Codonella radix, and redescribed by Brandt (1907) who transferred it into genus Tintinnopsis. The ciliature was first revealed by Jiang et al. (2012). Our specimens resemble original description in lorica shape (i.e., elongated cylindrical with a narrowed posterior portion and a small posterior opening) and the diameter of the opening (35-60 μm vs. 48 μm), but differs in its shorter length (190-310 μm vs. 480 μm). Compared with other descriptions, its size falls well within the recorded range (e.g., lorica length 182-524 μm and opening diameter 30-72 μm) (Balech 1959; Brandt 1907; Imhof 1886; Jiang et al. 2012; Kofoid and Campbell 1929; Marshall 1969; Paulmier 1997).

  • Comparison of ciliature

  • Tintinnopsis evertaKofoid and Campbell, 1929 (Fig. 2a-l; Table 1)

    Figure 2.  a-l Photomicrographs (a-l) of Tintinnopsis everta in vivo. a Lateral view of a representative individual. b-d, g, h Lateral views, to show variation in shape and size among different individuals (b, d, g) and during morphogenesis (c, h), to show the new lorica of proter (arrowhead). e Ventral view of a fully extended cell, arrowhead marks the collar annuli. f Details of a cell removed from the lorica, arrowheads show anteriormost cilia of the right and left ciliary fields, arrows indicate tentaculoids. i Ventral view of oral portion of cell, arrow shows the endoral membrane. j Subapical view of lorica. k Detail of the peristomial rim, showing the clavate tentaculoids (arrowhead). l Ventral side, showing the somatic cilia in vivo, arrowhead indicates the elongated anteriormost cilia of the right ciliary field. Scale bars: 30 μm

Description
  • Lorica campanulate, 65-95 μm long, composed of a sub-spherical bowl and a funnel-shaped collar that is tilted at an angle of 30° to the long axis (Fig. 2a-e, g, h). Opening 75-110 μm across with irregular rim, ratio of length to opening diameter 0.8-0.9 (Fig. 2a-e, g, h). Collar about 35-50 μm in length, with 4-6 annuli on surface (Fig. 2a-e, g, h). Bowl 40-55 μm in width with posterior end broadly rounded (Fig. 2a-e, g, h). Lorica wall densely agglutinated with refractile particles about 1-10 μm in diameter (Fig. 2a-e, g, h). During cell division, the new lorica is produced before the separation of proter and opisthe (Fig. 2c, h).

    Cell proper elongates obconical, about 50-60μm ×20-30 μm in size when fully extended, posterior portion narrows progressively forming a peduncle that is about 45 μm long and attaches to the bottom of lorica (Fig. 2a-e, g, h). Cilia of collar membranelles about 25 μm long; anteriormost cilia of kineties in right and left ciliary fields about 30 μm long, remaining cilia about 8 μm long (Fig. 2f, l, i). Kineties in right ciliary field commence progressively more posteriorly from left to right, rightmost kinety commences about half-way down length of cell (Fig. 2g, l; see Gruber et al. 2018). Macronuclear nodules and micronuclei not recognizable in live cells. Cytoplasm colorless, with food vacuoles of various sizes containing ingested ovoidal microalgae (Fig. 2f). Tentaculoids, pin-shaped, about 7μm ×2 μm in size, commencing from outer portions of intermembranellar ridges (Fig. 2f, k). Contractile vacuole, cytopyge, striae, and accessory combs not recognized in live cells. Cell rotates about longitudinal axis while swimming. When disturbed, peduncle contracts and cell proper retracts into lorica with membranelles motionless; on cessation of disturbance, cell proper slowly extends through lorica opening and resumes swimming.

  • Comparison with original and subsequent descriptions

  • This species was first reported as Tintinnopsis baltica var. rotundata by Laackmann (1908) and raised to rank of a species with a new name, T. everta, by Kofoid and Campbell (1929). Our specimens resemble the original description in most lorica features, e.g., 65-95 μm vs. 65-81 μm in length with a posteriorly rounded bowl and a funnel-shaped collar. Hence, we identified our specimens as T. everta, although our population has a larger opening diameter (75-110 μm vs. 50-52 μm) and more annuli (4-6 vs. 3-5), which we consider to be population-dependent variations. Gruber et al. (2018) revealed the cytological features and gave an improved diagnosis of this species. Our population lacks ciliature data, because individuals were insufficiently stained. Nevertheless, our specimens match those of Gruber et al. (2018) in terms of lorica features other than the size of the opening (e.g., lorica campanulate, length 80 μm on average, four annuli on average) and certain cell features (e.g., tentaculoids conspicuous, anterior cilia of left and right ciliary fields extremely elongated).

    Tintinnopsis mulctrellaKofoid and Campbell, 1929 (Fig. 3a-k; Table 1).

    Figure 3.  a-k Drawings (a, b) and photomicrographs (c-k) of Tintinnopsis mulctrella in vivo. a Representative specimen; arrowhead denotes the oblique collar protrusion. b Three collar membranelles, arrowhead denotes the cytoplasmic ridges. c Apical-lateral view of cell, arrowheads mark the cytoplasmic ridges. d Representative specimen; arrowhead denotes the long cilia located in anterior cell portion. e, f Contraction process of same specimen. g Apical view, showing the cytostome. h Details of cell proper, arrowhead shows a long cilium. i Posterior lorica projection. j Empty lorica, arrowhead shows the oblique collar protrusion. k Cell removed from the lorica. CM collar membranelles. Scale bars: 25 μm (a, d, f, g), 40 μm (e)

Description
  • Lorica about 70-85 μm×35-50 μm in size, main part globular, part of collar protrudes obliquely approximately 20 μm above lorica opening (Fig. 3a, d). Lorica opening tilted at an angle of 40° to the long axis, about 25-35 μm in diameter. Posterior end pointed or with an irregular process (Fig. 3a, d, f, i). Hard lorica wall densely agglomerated with mineral and organic particles, about 1-10 μm in diameter causing an irregular outline (Fig. 3a, c-k).

    Cell proper obconical, about 40-60μm ×30-40 μm in size, protrudes almost entirely through lorica opening when fully extended, its posterior end progressively narrows to form a peduncle that is up to 40 μm long and attaches to bottom of lorica (Fig. 3a, b, d). Cilia of collar membranelles 15-20 μm long (Fig. 3a-h); most somatic cilia about 2 μm long, although those in anterior cell portion are about 10 μm long (Fig. 3a, d, h). Macronuclear nodules and micronuclei not recognizable in living cells. Cytoplasm colorless, with food vacuoles of various sizes, which contain ingested ovoidal microalgae (Fig. 3b, e). Tentaculoids not recognized. Cytoplasmic ridges between collar membranelles, about 3-5 μm high (Fig. 3a-c). Accessory combs and cytopyge not recognized. Locomotion by rotating about main cell axis. When disturbed, peduncle contracts and cell proper retracts inside the lorica; on cessation of disturbance, cell proper slowly extends through lorica opening and resumes swimming.

  • Comparison with original description

  • Tintinnopsis mulctrella is a rare species that previously has been reported only once, i.e., by Kofoid and Campbell (1929) who described it as having a "milk pitcher-like" lorica. The specimens from Qingdao match the original population in the lorica size (length 70-85 μm vs. 77 μm), bowl width (30-40 µm vs. about 35 µm, inferred from the original illustration), and especially the oblique collar protrusion, which is unique to this species. Thus, we identified our Qingdao population as T. mulctrella, although it differs from the type population in smaller opening diameter (25-35 μm vs. 44-77 μm), slightly bigger ratio of length to opening diameter (2.4-3.2 vs. 1-2.26), and shorter collar protrusion (15-25 μm vs. 35-40 μm, inferred from the original illustration) (Kofoid and Campbell 1929).

    Family Eutintinnidae Bachy et al., 2012

    Genus Eutintinnus Kofoid and Campbell, 1939

    Eutintinnus inflatusMarshall, 1969 (Fig. 4a-h; Table 1)

    Figure 4.  a-h Drawings (a) and photomicrographs (b-h) of Euintinnus inflatus in vivo. a Lateral view of representative specimen. b, d Different views of a representative individual, arrowheads show the asymmetric notches in the lorica (lateral view). c, e Lateral view of another individual, arrowhead shows a notch in the lorica. f A wider lorica. g Cell proper, arrowhead shows the somatic cilia. h Peduncle. P peduncle. Scale bars: 50 μm (a-e), 30 μm (f)

Description
  • Lorica goblet-like, about 95-125 μm×30-45 μm in size. Anterior opening about 30-35 μm across with inconspicuous trumpet-shaped rim, posterior opening not or slightly flared, about 5-15 μm across (Fig. 4a-f). Middle portion slightly swollen. Posterior portion narrowed at an angle of about 15-30° to main axis. Widest portion of bowl about 30-45 μm across, narrowest portion about 10-15 μm (Fig. 4a-f, h). Two asymmetric notches were visible only when viewed from lateral aspect near widest portion of bowl, about 15-30 μm long and 5-10 μm depth (Fig. 4d, e).

    Cell proper obconical, about 30-55μm ×20-40 μm in vivo when fully extended, narrowing posteriorly to form a peduncle (about 40 μm long) that attaches to posterior 1/5 of inner lorica wall (Fig. 4a-f, h). Cytoplasm colorless, full of food vacuoles of various sizes containing ingested ovoidal microalgae (Fig. 4a-g). Cilia of collar membranelles about 15-25 μm long (Fig. 4a-f); no elongated somatic cilia were observed in living cells; lateral ciliary field with densely arranged cilia, about 1-2 μm long (Fig. 4g). Macronuclear nodules, micronuclei, tentaculoids, striae, accessory combs, contractile vacuole, cytopyge, and extrusomes not recognized. Locomotion by swimming slowly while rotating about main cell axis.

  • Comparison of lorica features with original description

  • Eutintinnus inflatus was first discovered by Silva (1953) from coastal waters of southern Europe and recorded as an unidentified form E. sp., Marshall (1969) named it E. inflatus but failed to supply a detailed redescription. The present population closely matches the original specimens in terms of its lorica length (95-125 μm vs. 100 μm), diameter of posterior opening (5-15 μm vs. 14 μm), and the bowl slightly wider than anterior opening. However, it differs from the original population in having a wider anterior opening (30-35 µm vs. 21 µm), which is assumed to reflect intraspecific polymorphism. Neither of the previous reports mention the asymmetric notches on the bowl. This feature was, however, conspicuous in all specimens of our study so we do not consider it to be an artifact but possibly a population-dependent character.

  • Comparison with congeners

  • Eutintinnus angustatus (Daday 1887) Kofoid and Campbell 1939 has similar lorica size, swollen bowl, and narrowed posterior end to but differs from E. inflatus in having a longer lorica (135-144 µm vs. 95-125 µm), a wider anterior opening (42-48 µm vs. 30-35 µm), anterior portion of the bowl straight-walled (vs. with slightly curving walls), and the commencement of the narrowing of the posterior portion bowl about 4/5 (vs. 2/3) down length of lorica (Daday 1887).

    Family Ptychocylididae Kofoid and Campbell, 1929

    GenusFavellaJörgensen, 1924

    Favella ehrenbergii(Claparède and Lachmann, 1858) Jörgensen, 1924 (Fig. 5a-m; Table 1).

    Figure 5.  a-m Photomicrographs of Favella ehrenbergii in vivo (a-h) and after protargol staining (i-m). a Fully extended cell, double arrowhead indicates an unidentified parasite, arrowhead marks posterior projection. b-d Different contracted forms of a same individual, arrowheads show the spiraled annulus. e Another individual with a bulge on the peduncle (arrowhead), arrow indicates an unidentified parasite. f Cell proper. g Collar membranelles (arrowhead) and buccal membranelle within the buccal cavity. h Alveolar structure of lorica in ×1000 magnification. i, j Ventral views of different specimens. k Dorsal view, arrowhead indicates the first dorsal kinety. l Apical view. m Endoral membrane. n Kinetal map of a morphostatic specimen. CM collar membranelles, DK dorsal kinety, DK2 the second dorsal kinety, EM endoral membrane, LA lateral ciliary field, LF left ciliary field, Ma macronuclear nodule, P parasite, RF right ciliary field, VK ventral kinety. Scale bars: 50 μm

    Lorica hyaline, 230-300 μm long, bowl mostly cylindrical rounded posteriorly and with a slender spine-like projection, mono-laminar alveoli on lorica surface (Fig. 5a-e, h). Opening is widest portion of lorica, 70-105 μm in diameter; spiraled annulus about 8 μm below opening (Fig. 5a-e). Posterior spine-like projection about 30-60 μm long and 15-30 μm wide (Fig. 5a-e).

    Cell proper obconical, measuring 70-110μm ×70-105 μm in size in vivo, posterior region narrowing gradually to form a peduncle that is 90-150 μm long, occasionally with one or two bulges, and is attached to bottom of lorica (Fig. 5a-g). Two irregular macronuclear nodules, each about 30-60μm ×15-25 μm in size after protargol staining were recognized (Fig. 5i-k). Micronuclei, tentaculoids, striae, accessory comb, cytopyge, contractile vacuole, and capsules not recognized. Cytoplasm colorless, with numerous lipid droplets and food vacuoles of various sizes containing ingested ovoid microalgae, sometimes diatoms (Fig. 5a-g). Different parasites usually attached to the cell proper or peduncle, spheroidal or arch shape, ca. 30-80 μm in diameter (Fig. 5a-f). Cell movement occurs slowly by rotating about the cell main axis, twitching back when meeting obstacles. When disturbed, cell proper retracts quickly into lorica due to contraction of peduncle and posterior portion; membranelles bend towards centre of peristomial field.

    Somatic ciliature comprises a right, left, and lateral ciliary field as well as a ventral and two dorsal kineties (Fig. 5i-k). Length of kineties and numbers of kinetids usually highly variable. Kinetids of each ciliary row ostensibly connected by an argyrophilic fiber. Ventral kinety about 29-47 μm long, composed of 33-46 monokinetids (Fig. 5i, j). Right ciliary field commences about 8 μm posteriorly to collar membranelles, composed of 40-58 kineties, neighboring kineties about 3 μm apart, each of which consists of about ten monokinetids and one anterior dikinetid (Fig. 5i, j). Left ciliary field commences at same level as right field, composed of 27-40 kineties, each kinety comprises about eight monokinetids and one anterior dikinetid; cilia about 3 μm long after protargol staining (Fig. 5k). Each basal body in left and right ciliary fields bears a cilium; anterior cilium in each dikinetid about 15 μm long after protargol staining; all other cilia are about 3 μm long after protargol staining (Fig. 5i-k). Lateral ciliary field lies between ventral kinety and left ciliary field commences at same level as right and left fields, composed of 7-14 monokinetidal kineties; monokinetids in anterior portion packed significantly densely than those in posterior portion (Fig. 5j). Two dorsal kineties commence about 10 μm posteriorly to collar membranelles, 58-80 μm and 62-90 μm in length, composed of 72-85 and 80-92 monokinetids, respectively (Fig. 5k). Posterior kinety absent.

    Oral apparatus occupies anterior portion of cell proper. Collar zone closed, composed of 16 membranelles with cilia about 30-40 μm long, five of which notably extend into buccal cavity; bases of collar membranelles about 40 μm wide; kinetal structures of membranelles could not be recognized (Fig. 5f, g, m, l). Single buccal membranelle located within buccal cavity (Fig. 5g). Endoral membrane encircles oral cavity, composed of a single row of basal bodies (Fig. 5l, m). Argyrophilic fibers associated with oral apparatus insufficiently impregnated to be observed in protargol preparations.

  • Comparison with original and subsequent descriptions

  • Favella ehrenbergii is a neritic form and has been described many times. It was originally described under the name Tintinnus ehrenbergii (Claparède and Lachmann 1858). Jörgensen (1924) transferred it to the newly established genus Favella, and Kofoid and Campbell (1929) designated F. ehrenbergii as the type species of this genus. The Jiaozhou Bay population closely resembles the original description in terms of its lorica shape and the presence of a spine-like posterior projection, although it has a larger size (230-300 μm vs. about 190 μm in length). Laval-Peuto (1981) revealed polymorphism of the lorica during the life cycle in cultures of F. ehrenbergii. However, we did not observe significant polymorphism of the lorica in the present study. Kim et al. (2010) first revealed the ciliature of F. ehrenbergii and improved the species diagnosis. The Jiaozhou Bay population matches well with the emended diagnosis of Kim et al. (2010) in terms of both its lorica features and the pattern of its ciliature.

Materials and methods

    Sample collection and environmental factor measurement

  • All samples were collected from the surface water (0-2 m water depth) in coastal waters of the Yellow Sea at Qingdao, China using a plankton net (mesh size 25 μm) (Fig. 1). Tintinnopsis mulctrella and T. cf. radix were found near Zhanqiao Pier (36° 03′ 36′′ N, 120° 18′ 54′′ E) in September, 2018 (Site 2 in Fig. 6). Tintinnopsis everta, Eutintinnus inflatus, and Favella ehrenbergii were found in Jiaozhou Bay (36° 07′ 46′′ N, 120° 15′ 18′′ E) in November, 2018 (Site 1 in Fig. 6). Water temperature, salinity, pH and dissolved oxygen concentration (DO) were measured in situ using a water quality-measuring instrument (YSI Professional Plus, America). Concentrations of nitrate nitrogen (NO3-N), nitrite nitrogen (NO2-N), ammonium nitrogen (NH4-N), and phosphate phosphorus (PO4-P) were measured with a water quality analyser (YSI-9500, America). Chlorophyll-a concentration was measured on a 10-AU field fluorimeter (Turner Design, America), calibrated with purified Chlorophyll-a standard (Sigma). A list of the environmental factors measured is given in Table 2.

    Figure 6.  a-d Sampling sites and surrounding areas. a Map of China with sampling area (yellow circle). b Map of Jiaozhou Bay and surrounding area, red circle 1 depicts the location of (c), red circle 2 depicts the location of (d). c, d Part of Jiaozhou Bay (c) and coastal area around Zhanqiao Pier, Qingdao (d)

    Si Locations T(℃) S(psu) pH DO(mg/L) Chl-a(μg/L) NO2-N(mg/L) NO3-N(mg/L) NH4-N(mg/L) PO4-P(mg/L)
    1 36°07′46"N
    120° 15′ 18"E
    18.3 31.85 8.2 4.88 0.673 0.021 0.415 0.07 0.51
    2 36° 03′ 36"N
    120° 18′ 52"E
    21.7 32.06 8.2 5.14 0.452 0.046 0.528 0.18 0.16

    Table 2.  Environmental factors of the sampling sites

  • Observation and identification

  • The water subsamples were transferred into Petri dishes of 200 mm in diameter with about 5 mm water depth. The tintinnines were isolated by micropipettes under a stereo microscope (Guiguang XTL-200, China) at a×45 magnification. Cells were observed with bright-field and differential interference contrast microscope in vivo. Lorica measurements were calculated from the photomicrographs of live cells taken at×100-400 magnifications, and estimated to accuracy of±5 μm. To examine the infraciliature and nuclear apparatus, specimens were fixed in Bouin's solution, the cell proper was removed from its lorica using an eyebrow brush after fixation, and stained using the protargol method (Song et al. 1999). The ciliary patterns of only two of the five species, i.e., Tintinnopsis cf. radix and Favella ehrenbergii, were sufficiently stained to reveal the infraciliature. The protargol powder was manually synthesized following Pan et al. (2013). Measurements of protargol-stained specimens were performed at a×1000 magnification. Drawings were made at a ×1000 magnification with the aid of a camera lucida. Identifications were based on original species descriptions (Brandt 1907; Claparède and Lachmann 1858; Imhof 1886; Kofoid and Campbell 1929; Silva 1953). Terminology and classification follow Agatha and Riedel-Lorjé (2006) and Adl et al. (2019), respectively.

    Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos: 41776133, 31801955, 31702009). We thank Prof. Weibo Song, Ocean University of China for his comments on the manuscript. We are also grateful to the editor and anonymous reviewers for their constructive suggestions.

    Author contributions XH conceived and guided the study. YB and RW conducted sampling and performed laboratory work. XH, YB, and RW identified the species. YB drafted the manuscript, and TS, WS, and XH made further revisions. All authors read and approved the final version of manuscript.

Compliance with ethical standards
  • Conflict of interest The authors declare no conflicts of interests.

    Animal and human rights statement No animal and human rights are involved in this article.

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