The morphology of the two marine urostyloid ciliates,
(Cohn, 1866) and
Kahl, 1932, in the family Pseudokeronopsidae, collected from the Yellow Sea, and the East Sea, Korea, respectively, were studied using live observation and protargol impregnation. Additionally, the small subunit ribosomal RNA (SSU rRNA) gene was sequenced. These two species are firstly recorded in Korea. The main diagnostic key is as follows.
: body outline elongate-elliptical, brown-reddish or orange-red in colour
; bicorona of 16-24 frontal cirri; one buccal and two frontoterminal cirri; 7-10 transverse cirri; 5-7 dorsal kineties; two types of cortical granules (one orange- red pigment, mainly grouped around cirri and dorsal bristles, arranged in typical
-pattern; the other, colourless and blood-cell-shaped, and densely distributed); contractile vacuole in the posterior half of the cell on the left side, usually in posterior 1/3-2/5.
: body outline elongate-elliptical, lemon-yellow in colour
; two types of cortical granules (one yellow pigment; the other, blood-cell-shaped, densely distributed); bicorona of 12-18 frontal cirri; 2-3 frontoterminal cirri; two midventral rows comprising 26-35 cirri (consisting of anterior paired cirri, non-paired single cirri, and posterior paired cirri); three dorsal kineties. In addition, the SSU rRNA sequ-ences of the two species were compared with public database of these species and consequently, showed high similarity.
are included in the family Pseudokeronopsidae which was established by Borror and Wicklow (1983).
consists of 10 species and all mem-bers have frontal cirri arranged as a bicorona, which continue posteriorly to two midventral rows and marginal cirri on each side of the body (Borror and Wicklow, 1983; Berger, 2006; Song et al., 2006). Identification of species in the
, however, is somewhat difficult because the diagnostic keys such as body shape, body colour, body size and ciliary pattern either are overlapped or similar among congeners (Song et al., 2006).
Kahl (1932) established the genus
due to the lack of transverse cirri in some species on classification in
Later, Berger (2004) redescribed
by its morphology and morphogenesis, and divided
into the two subgenus,
), thro-ugh the presence of cirrus II/2 in the ordinary position, right of the undulating membranes.
In this study, we described two marine ciliates new to Korea,
, based on live and protargol-impregnated specimens. Moreover, the sequences of the small subunit ribosomal RNA (SSU rRNA) gene from two species were determined and compared with those of known sequences obtained from the NCBI website.
MATERIALS AND METHODS
- Sample collection and identification
The specimens of
were collected from Incheon harbor in the Yellow Sea ( salinity, 28.5‰; temperature, 15℃; 37̊ 26ʻN, 126̊ 35ʻE), Korea, in November 2010, and those of
were collected from Guryongpo, Pohang in the East Sea (salinity, 32.3‰; tempera-ture, 24.1℃; 35̊ 59ʻN, 129̊ 33ʻE), Korea, in September 2008.
After collection and isolation, specimens were maintained in the laboratory, either as pure or raw cultures in Petri dishes and 50 mL tissue culture flasks (Greiner Bio-one, Fricken-hausen, Germany). Autoclaved seawater was supplied with putting rice grains as a substrate for bacterial growth (Jung et al., 2011). The living specimens were observed under a light microscope ( Leica DM2500; Leica Microsystems, Wetzlar, Germany) at 50-1,000 magnification. Protargol impregnation was applied according to Foissner (1991) to reveal the infraciliature.
Terminology and classification are mostly according to Berger (2006) and Lynn (2008).
- DNA sequence determination
A cell (single specimens of each species) was transferred to a 1.5 mL microtube with a minimum volume of water. Geno-mic DNAs were extracted using a RED-Extract-N-Amp Tis-sue PCR kit (Sigma, St. Louis, MO, USA), according to the manufacturer’s protocol. The nearly complete SSU rRNA genes were amplified by polymerase chain reaction (PCR) with the universal eukaryotic primers: New EukA (5ʻ-CTG GTT GAT YCT GCC AGT-3ʻ), modified from Medlin et al. (1988), and LSU rev3 (Sonnenberg et al., 2007) primers. The optimized conditions for this process were as follows: Denaturation at 94℃ for 3 min followed by 35 cycles of denatura-tion at 95℃ for 15 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 4 min, and then a final extension step at 72℃ for 7 min. The PCR products were purified with the QIAquick
PCR Purification kit (Qiagen, Valencia, CA, USA). Three internal primers were used for sequencing: 18S+810 (5ʻ-GCC GGA ATA CAT TAG CAT GG-3ʻ) and 18S-300 (5ʻ-CAT GGT AGT CCA ATA CAC TAC-3ʻ) and 18S+1470(5ʻ-TCT GTG ATG CCC TTA GAT GTC-3ʻ). Sequencing in both directions was conducted by an ABI 3700 Sequencer(Applied Biosystems, Foster City, CA, USA).
The sequencing fragments of the SSU rRNA gene were combined via BioEdit (Hall, 1999) and were aligned using Clustal X 1.81 (Jeanmougin et al., 1998). Mega 4.0 (Tamura et al., 2007) was used to calculate genetic distance by applying the Kimura two-parameter distance method (Kimura, 1980).
Korean name: 1*뚱뚱이홍색위각모충 (신칭)
Cohn, 1866: 288, 300.
Holosticha (Keronopsis) rubra
(Cohn, 1866) Kahl, 1932: 573.
(Cohn, 1866) nov. comb. Wirns-berger et al., 1987: 79, fig. 9, Tables .1-3
sensu Shi and Xu, 2003: 23-30.
Hu, Warren and Suzuki, 2004: 351-368.
(Cohn, 1866) Wirnsberger et al.,
Phylum Ciliophora Doflein, 1901
Class Spirotrichea Bütschli, 1889
Order Urostylida Jankowski, 1979
Family Pseudokeronopsidae Borror and Wicklow, 1983
GenusPseudokeronopsisBorror and Wicklow, 1983
1*Pseudokeronopsis carnea(Cohn, 1866)
Wirnsberger et al., 1987 (Table 1, Figs.1 A-D, 2)
Morphometric characterization ofPseudokeronopsiscarneaAll data, including the Korean population (first line) and the Chinese population(second line), are based on protargol-impregnated specimens.The data of the Chinese population is cited from Song et al. (2006). Min, minimum; Max, maximum; Mean, arithmetic mean; SD, standard devia-tion; SE, standard error of the mean; CV, coefficient of variation in % n, number of individuals examined; MVR, midventral row.
Morphometric characterization of Pseudokeronopsiscarnea All data, including the Korean population (first line) and the Chinese population(second line), are based on protargol-impregnated specimens.The data of the Chinese population is cited from Song et al. (2006). Min, minimum; Max, maximum; Mean, arithmetic mean; SD, standard devia-tion; SE, standard error of the mean; CV, coefficient of variation in % n, number of individuals examined; MVR, midventral row.
Morphology and infraciliature of Pseudokeronopsis carnea and Uroleptopsis citrina from live specimens (A B E F) and afterprotargol impregnation (C D G H). A-D Pseudokeronopsis carnea: A Ventral view of live specimen arrowhead in (A) denotes CV;B Two types of granules; infraciliature of the ventral (C) and dorsal (D) sides. E-H Uroleptopsis citrina: E Ventral view of livespecimen; F Two types of granules; infraciliature of the ventral (G) and dorsal (H) sides. AZM adoral zone of membranelles; BCbuccal cirri; CV contractile vacuole; DK dorsal kineties; EM endoral membrane; FTC frontoterminal cirri; LMR left marginal row;Ma macronuclei; MVR midventral row; PM paroral membrane; RMR; right marginal row; TC transverse cirri. Scale bars=100 μm.
Morphology and infraciliature of Pseudokeronopsis carnea from live specimens (A-C E-G) and after protargol impregnation(D H-L). A B Ventral views of live specimen; C Dorsal views of live specimen arrow marks a contractile vacuole; E Cortical granulesaround dorsal kineties; Arrows in (F G) indicate ring-shaped hollow structures arrowheads show cortical granules; D H-K Ventraland (L) dorsal views of protargol-impregnated specimen; D General ciliature of the specimen; H Frontal (bicorona) arrow indicatesthe buccal cirrus; I Two frontoterminal cirri arrows indicate the cirri near the distal end of adoral zone; J Two midventral rows; KDenotes transverse ventral cirri; Arrows in (L) show the dorsal kineties. Scale bars=100 μm.
1987, Song et al., 2006: 271-287, figs.1 A-G, 2, 3, 9C, Tables .1-3
One population was obtained from In-cheon harbor on November 2, 2010.
slender shape, 190-255×55-70 ㎛, usually 225×61.3 ㎛ (
A); anterior end bluntly rounded (
B); posterior end inconspicuously narrowed; both anterior and posterior ends round; dorsoventrally flattened. Contractile vacuole located on the left side usually in posterior 1/3-2/5 (
C, arrow); reddish cortex due to underlying reddish-brown or orange-red in colour cortical granules, which are around both dorsal kine-ties and cirri (
E; F, G, arrowhead); cortical granules colourless, blood cell shaped, scattered throughout the cell body (
F, G, arrow).
The adoral zone of membranelles distinct, approximately 1/3 of the cell length, and composed of about 69 membran-elles(
D, H). Bicorona of frontal cirri slightly enlarged,composed of about 8-12 cirral pairs, extending as a midven-tral complex consecutively. One buccal cirrus near the paro-ral membrane (
H, arrow), whereas two frontoterminal cirri behind the distal end of the adoral zone (
I, arrows); midventral complex distinctly separated rows (
J), com-posed of 30-46 cirral pairs, terminating near transverse cirri; both posterior ends of marginal cirral rows not overlapped; 7-10 transverse cirri located between both posterior ends of the left and right marginal cirral rows (
K). Almost no gap found between the midventral rows and the transverse cirri; from five to seven dorsal kineties (
North Sea, German, Denmark, Mediterranean,Yugoslavia, China and Korea (this study).
Cohn (1866) published
without any illustration. As the former species is almost iden-tical to
, he classified it as a variety of
The derivation of the name was not given in the original description of
The meaning of c
in Latin is “fleshy.” In 1882, Kent transferred
to the genus
. Kahl (1932) classified
as a subgenus of
Then, Kahl named it
Holosticha (Keronopsis) rubra
Even after several taxono-mists recorded this species, they were considered it
in confusion. Entz (1884) con-sidered
as a transitional form between
The neotype of
was fixed by Wirns-berger et al. (1987) and until now, a Chinese population of
has been redescribed solely (Song et al., 2006).
Eight species among the genus
live in marine habitats. Because
species are somewhat difficult to classify and identify among congeners, the colour as main diagnostic key is the critical factor disting-uishing
from the other congeners (Hu and Song, 2001). The orange-red colour of cortical granules is essen-tial for identifying
; brick-red and yellow,
). Moreover, with the exception of the colour of the cortical granules, the ciliary pattern and position of the contractile vacuole support species separation (Song et al., 2002; Berger, 2006). Like the name suggests, this species has the most plump body shape among the congeners. Alth-ough the anterior end is bluntly rounded, the posterior end is inconspicuously narrowed. This species can be separated from the other congeners by having: more cirral pairs in both the bicorona and the midventral rows; more transverse cirri; more dorsal kineties; a contractile vacuole in the posterior half of the cell, usually in the posterior 1/3-2/5; more conspi-cuous pigment granules, always dark red or orange-red. The number of adoral membranelles in this organism is also con-spicuously more than that of other congeners. In addition, the adoral zone of membranelles is relatively long compared to body length (ratio, 1 : 3), and almost no gap exists bet-ween the midventral rows and the transverse cirri.
The Korean population,
, has a few differences from the Chinese population of
(Song et al., 2006) as follows: (1) dorsal kineties (5-7 vs. 7-8); and (2) transverse cirri (on average 8 vs. 8.6). In addition, we ascertained that the sequence was successfully amplified on the partial region of the SSU rRNA gene and the amplified se-quence length is 1,756 bp (GenBank accession no: JN714476) and shows 99.89% similarity with the Chinese population (GenBank accession no: AY881633).
Uroleptopsis citrina Kahl, 1932 (Table 2, Figs.1 E-H, 3)
Kahl, 1932: 543, fig.87 ; Kahl, 1933:107, fig. 1612.; Kudo, 1950: 672; Borror, 1972: 11; Berger, 2004: 99-121, 5-28, 35-42, table 1.
One population was obtained from Guryongpo, Pohang in September 2008.
slender shape, 118-165× 45-55 ㎛, usually 130.2× 50 ㎛ (
A, B); body shape elon-gate-elliptical; both anterior and posterior ends round and dorsoventrally flattened. Contractile vacuole difficult to recognize, located on the left side of usually slightly squeezed cells. Body colour is lemon-yellow due to cortical granules,
Morphology and infraciliature of Uroleptopsis citrina from live specimens (A B E F) and after protargol impregnation (C DG-I). A B Ventral views of live specimen; C Dorsal and (D G-I) ventral views of protargol-impregnated specimen; C Arrows markthe invariable three dorsal kineties; C D Arrowheads point to gap in adoral zone; D General ciliature of the specimen; E F Arrowand arrowheads indicate the two kinds of granules respectively; G Anterior pairs and single cirri (arrow mark) on the midventralcomplex; H Indicates macronucleus; I Frontal cirri (bicorona); arrows show two frontoterminal cirri. Scale bars=100 μm.
which are around both dorsal kineties and cirri; cortical gra-nules colourless, blood cell shaped, scattered throughout the cell body (
The adoral zone of membranelles distinct; about 1/3 of cell length, and composed of about 40 membranelles (
D, I), left anterior corner a minute process causing a break (
C, D, arrowhead). Bicorona of frontal cirri slightly enlarged, composed of about 6-9 cirral pairs, extending as a midventral complex consecutively (
I). Midventral complex distin-ctly separated rows, composed of 26-35 cirri containing anterior, single cirri (
G, arrow) in middle portion, posterior portion. Two or three frontoterminal cirri behind the distal end of the adoral zone (
I, arrows); invariably three dorsal kineties (
C, arrows); of particular interest, there is no buccal cirrus and transverse cirri.
Adriatic Sea, and Korea (this study).
. Kahl (1932) established the genus
and described firstly
. Later, Berger (2004) rede-scribed
of the Adriatic Sea by its morphology and morphogenesis.
has a gap in the adoral zone and lacks transverse cirri. The loss of the transverse cirri is the main diagnostic character to separate
from other Pseudokeronopsidae species. This species has conspicuous differences from the congener
Morphometric characterization ofUroleptopsis citrinaAll data, including the Korean population (first line) and the Adriatic Seapopulation (second line), are based on protargol-impregnated specimens. The Data of the Adriatic Sea population is cited from Berger et al. (2004). Min, minimum; Max, maximum; Mean, arithmetic mean; SD, standarddeviation; SE, standard error of the mean; CV, coefficient of variation in%; n, number of individuals examined; MVC, midventral complex.
Morphometric characterization of Uroleptopsis citrina All data, including the Korean population (first line) and the Adriatic Seapopulation (second line), are based on protargol-impregnated specimens. The Data of the Adriatic Sea population is cited from Berger et al. (2004). Min, minimum; Max, maximum; Mean, arithmetic mean; SD, standarddeviation; SE, standard error of the mean; CV, coefficient of variation in%; n, number of individuals examined; MVC, midventral complex.
lows: presence of a buccal cirrus and the pattern of the mid-ventral complex. Circumstantially,
lacks a buccal cirrus in the ordinary position, right of the paroral, whereas is present in
. Also, the anterior and posterior portion of the midventral complex in this species primarily consist of ordinary midventral pairs; the middle portion is compos-ed only of the right cirri of the cirral pairs, whereas the an-terior portion of the midventral complex in
is com-posed of paired cirri, and the middle and posterior portion consist of non-paired cirri (Mihailowitsch and Wilbert, 1990). Yellow cortical granules and ring-shaped structures are under-neath the cell surface. Consequently,
is transferred to the subgenus
by Berger (2004).
is a little different from
in that the cell colour is yellow. However,
has one buccal cirrus in the ordinary position, 2-4 transverse cirri, 3-4 dorsal kineties, and lacks a break in the adoral zone (Song et al., 2004).
The Korean population,
, has a few differences from the Adriatic Sea population of
(Berger, 2004) as follows: (1) left marginal cirri (26-41 vs. 28-49); (2) right marginal cirri (29-53 vs. 34-63); and (3) single midventral cirri (on average 8 vs. 11). Additionally, we ascertained that the sequence was successfully amplified on the partial region of the SSU rRNA gene and the amplified sequence length is 1,754 bp (GenBank accession no: JN714477) and shows 99.88% similarity with that of Chinese population (GenBank accession no: GU437211). Unfortunately, no Adriatic Sea population sequence is available in GenBank.
This study was supported by the Invasive Species Manage-ment Program in Marine Ecosystem, Korean Ministry of Land, Transport & Maritime Affairs of Korean Govern-ment, and also funded by the National Fisheries Research & Development Institute (NFRDI) of Korea and Polar Acade-mic Program (PAP), KOPRI.
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