African Journal of Marine Science 2006, 28(2): 185–191Printed in South Africa — All rights reserved

Copyright © NISC Pty LtdAFRICAN JOURNAL OF

MARINE SCIENCEEISSN 1814–2338

The taxonomy and growth of a Crypthecodinium species (Dinophyceae)isolated from a brackish-water fish aquarium

MW Parrow1*, M Elbrächter2, MK Krause3, JM Burkholder1, NJ Deamer1, N Htyte3 and EH Allen1

1 Center for Applied Aquatic Ecology, North Carolina State University, 620 Hutton Street, Suite 104, Raleigh, NC 27606, USA;current address: Department of Biology, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte,NC 28223, USA2 Deutsches Zentrum für Marine Biodiversitätsforschung, Forschungsinstitut Senckenberg, Wattenmeerstation Sylt,Hafenstrasse 43, D-25992 List/Sylt, Germany3 Department of Biology, 114 Hofstra University, Hempstead, NY 11549, USA* Corresponding author, e-mail: mwparrow@email.uncc.edu

Introduction

Biecheler (1938, 1952) described the heterotrophic dinofla-

gellate Crypthecodinium setense from the Mediterranean

coast of France using silver staining and light microscopy to

determine the thecal plate tabulation. Biecheler (1952)

placed C. setense in a new family, the Crypthecodiniaceae,

and considered the possibility that it might be identical to

the previously described Gyrodinium cohnii (Seligo) Schiller,

if the same plate tabulation could be proven in the latter.

Chatton (1952) regarded these dinoflagellates as conspe-

cific, and introduced the combination Crypthecodiniumcohnii (Seligo) Chatton, but did not demonstrate a shared

thecal structure.

Since then, heterotrophic dinoflagellates roughly similar

in size and shape have been called C. cohnii and used

extensively in research on dinoflagellate physiology, ultra-

structure and genetics (Beam and Himes 1984, Barlow and

Triemer 1986, Perret et al. 1993, Bhaud et al. 1994, 2000, Lam

et al. 2001). Sexual compatibility experiments and genetic

analyses of numerous isolates have suggested that the

present morphospecies conception of C. cohnii may encom-

pass several biological species (Beam and Himes 1977,

1982, Beam et al. 1993).

Ucko et al. (1989, 1997) reported a C. cohnii-like dino-

flagellate occurring as a deleterious contaminant in commer-

cial pond cultures of Porphyridium sp., a unicellular red alga

mass-cultured for production of valuable biochemicals. The

Crypthecodinium dinoflagellate caused reduced yield and

collapse of the Porphyridium cultures by feeding on the red

algal cells (Ucko et al. 1989). Therefore, although not known

to be toxic, Crypthecodinium may be considered a poten-

tially harmful alga because it can negatively affect some

commercial mass-algal culture operations, resulting in finan-

cial loss.

A relatively small heterotrophic dinoflagellate found in

abundance in a brackish-water fish aquarium at North

Carolina State University, USA, was suspected of being a

Crypthecodinium species, based on observations using light

microscopy. During similar culture experiments (Parrow etal. 2005), a clonal isolate of this dinoflagellate was estab-

lished and has been cultivated for more than 1.5 years using

a Chinook salmon Oncorhynchus tshawytscha cell line as

the food source. The objectives of this study were to (1)

evaluate the taxonomy of this dinoflagellate by examining

its morphology and small sub-unit (18S) rDNA sequence;

An unidentified heterotrophic dinoflagellate found

growing in abundance in a brackish-water fish aquar-

ium was isolated and serially cultivated using a fish

cell line as the food source. Prominent characteristics

of this dinoflagellate included a cingulum that did not

fully encircle the motile cell, cell division in non-motile

cysts, and a theca composed of thin but structured

plates. Morphological analysis of flagellate cells by

scanning electron microscopy revealed a Kofoid thecal

plate tabulation of 4’, 4a, 4’’, ‘X’, 5 or 6c, ?s, 5’’’, 1p, 1’’’’,

most consistent with the original description of Cryp-thecodinium setense Biecheler. This Crypthecodinium

species exhibited a high maximum division rate (3.2

divisions day–1) and cell yield (>106 cells ml–1) when fed

cultured fish cells. Small sub-unit rDNA phylogenetic

analyses supported relatedness with a previously

studied Crypthecodinium-like dinoflagellate, but a

significant difference in aligned gene sequences was

found. This study provides the first clear demonstra-

tion of the plate tabulation of a Crypthecodinium spe-

cies since the original description over 60 years ago,

allowing the original morphological conception of

Crypthecodinium to be linked with molecular phylo-

genetic information.

Keywords: Crypthecodinium, dinoflagellate, phylogeny, small sub-unit rDNA, taxonomy, theca

Parrow, Elbrächter, Krause, Burkholder, Deamer, Htyte and Allen186

(2) describe its general mode of feeding and reproduction;

and (3) document its high rate of proliferation when culti-

vated on fish cells.

Material and Methods

The fish cell line (ATCC CRL-1681, Manassas, Virginia,

USA) was serially cultivated in Eagle’s minimum essential

medium (MEM) with 10% fetal bovine serum (FBS) in poly-

styrene cell culture flasks following the procedures of

Parrow et al. (2005). Prior to inoculation with dinoflagel-

lates, the MEM + FBS medium overlaying the adherent fish

cell layer was removed and replaced with filter-sterilised

(0.22µm pore size) artificial saltwater medium (ASM)

composed of (in g l–1) NaCl (8.1), MgSO4 (1.6), MgCl2 (1.2),

CaCl2 (0.4), KCl (0.3), NaHCO3 (0.1) and glucose (1.0) in

deionised water. The dinoflagellate culture was initiated by

treating a 20ml sample taken from the aquarium with peni-

cillin (50IU ml–1) and streptomycin (0.1mg ml–1) for 24h,

before isolating a single motile dinoflagellate cell from this

population using a sterile glass micropipette. The cell was

washed by micropipette through eight drops of sterile ASM

under aseptic conditions, and deposited into a culture flask

containing adherent fish cells overlaid with ASM. After the

dinoflagellate had proliferated into a cultured population, a

subsample (0.1ml) was serially transferred every seven

days into a newly prepared flask of fish cells. Observations

and photographs of living cells were made using light

microscopy in culture flask or Petri dish subcultures.

For scanning electron microscopy (SEM), culture aliquots

were treated with a 40% reduction in salinity for 20min and

then fixed in 1% OsO4, 2% glutaraldehyde and 0.1mol

sodium cacodylate (final concentration) at 4°C for 20min.

Fixed cells were filtered onto polycarbonate filters (3µm

pore size), rinsed in 0.1mol sodium cacodylate, dehydrated

through a graded ethanol concentration series (30%, 50%,

70%, 90%, 100%; 15min per step), critical point dried using

CO2, sputter coated with 25nm Au/Pd and viewed at 15kV

on a JEOL 5900LV SEM.

DNA was isolated from dinoflagellate samples using a

Qiagen Plant Mini Kit, and universal dinoflagellate 18S

primers (Saldarriaga et al. 2001) were used for sequence

amplification. Gel-purified 18S rDNA fragments were ampli-

fied by PCR, isolated and sequenced in both directions using

Amersham Thermo SequenaseTM. ClustalW was used for

multiple sequence alignment, and MEGA Version 3.1 was

used for phylogenetic analyses (Kumar et al. 2004). A mini-

mum evolution/maximum likelihood-distance consensus

phylogenetic tree was constructed with 1 500 bootstrap

replicates, based on alignment of sequence data from 41

additional taxa obtained from GenBank covering major

dinoflagellate groups (Table 1). Parameters were estimated

by a Kimura 2-parameter (Gamma) model and calculated with

the Close-Neighbour-Interchange algorithm. Toxoplasmagondii Nicolle et Manceaux was used as an outgroup.

Results and Discussion

The flagellate cells of this Crypthecodinium species were

8–24µm long, colourless, appeared superficially gymnodinoid

under light microscopy, and were approximately ovoid in

ventral or dorsal view and slightly compressed dorsoven-

trally. The cingulum did not fully encircle the cell, exhibiting

a leftward descending spiral that terminated near the right

lateral side after traversing roughly two-thirds of the cell

circumference (Figure 1a, b). The longitudinal flagellum

was approximately 1.5–2X the cell length (Figure 1a).

Investigation by SEM revealed that the flagellate cells

had thin thecae composed of structured plates. The thecal

plate tabulation was consistent with a Kofoid formula of 4’,

4a, 4”, ‘X’, 5 or 6c, ?s, 5’’’, 1p, 1’’’’ (Figure 2). No distinct apical

pore complex was discerned. The sulcal plates were not

resolved clearly enough to permit description and it was

uncertain whether the number of cingular plates was five or

six. The observed thecal plates were arranged in a pattern

most consistent with the original C. setense description

Table 1: List of species used in the phylogenetic analysis, with

GenBank accession numbers

Species Accession number

Alexandrium catenella AB088284

A. fundyense U09048

A. minutum U27499

Amylax diacantha AY443013

Amyloodinium ocellatum AF080096

Ceratium furca AJ276699

C. fusus AF022153

C. hirundinella AY443014

C. tenue AF022192

Ceratocorys horrida AF022154

Crypthecodinium cohnii M64245

Fragilidium subglobosum AF033869

Gloeodinium viscum L13716

Gonyaulax spinifera AF022155

Gymnodinium beii U37367

G. catenatum AF022193

G. fuscum AF022194

Gyrodinium instriatum AY443015

G. uncatenum AF274263

Hemidinium nasutum AY443016

Karenia brevis AF172714

K. mikimotoi AF022195

Karlodinium micrum AY245692

Lessardia elongata AF521100

Lingulodinium polyedrum AY421788

Noctiluca scintillans AF022200

Peridinium polonicum AY443017

Pfiesteria piscicida AF077055

P. shumwayae AY245694

Polarella glacialis AF099183

Prorocentrum micans AY585526

P. minimum Y16238

Protoceratium reticulatum AF274273

Protoperidinium conicum AY443020

P. pellucidum AY443022

Pyrocystis noctiluca AF022156

Scrippsiella sweeneyae AF274276

S. trochoidea AJ415515

Symbiodinium microadriaticum M88521

Thecadinium dragescoi AY238479

Toxoplasma gondii U00458

African Journal of Marine Science 2006, 28(2): 185–191 187

Figure 1: Scanning electron micrographs of the Crypthecodinium species including (a) a flagellate cell (ventral view) showing characteristic

morphology and (b) a flagellate cell (right lateral view) showing the terminus of the cingulum and transverse flagellum (arrow). Scale bars = 1µm

Figure 2: Scanning electron micrographs showing (a–b) ventral/antapical, (c–d) dorsal/apical and (e–f) dorsal/antapical views of the thecal

plate arrangement of the Crypthecodinium species. The Kofoid plate labels proposed by Fensome et al. (1993) are shown rather than the

original plate labels of Biecheler (1938). Scale bars = 1µm

Parrow, Elbrächter, Krause, Burkholder, Deamer, Htyte and Allen188

(Biecheler 1938; Figure 3), including the asymmetrical first

apical (1’) and the large ‘X’ plate to the right of the sulcus

extending from the epicone to the hypocone. The 1’ and 3’

plates differed from the original description, however, by

joining at a point rather than sharing a suture and having

one less side each (Figures 2d and 4). Possible taxonomic

implications of this morphological difference are uncertain,

because this type of plate variation can occur within some

dinoflagellate species (Elbrächter and Meyer 2001).

A novel 18S rDNA sequence was obtained from clonal

culture of this dinoflagellate (1 736 nucleotides; GenBank

accession number: DQ322643). Phylogenetic analysis

indicated relatedness between the 18S rDNA sequence of

this dinoflagellate and that of the sole published sequence

for an unspecified strain referred to as C. cohnii by

Gajadhar et al. (1991) (Figure 5). The aligned sequences

shared only 87% of base pairs, however, indicating that

these taxa were not identical. Branching order in the

phylogenetic tree was not well resolved, but there was

support for placement of Crypthecodinium in a major

clade that links dinoflagellate species currently assigned

to the order Gonyaulacales. The position of Cryptheco-dinium in a common gonyaulacoid lineage has been both

supported (Saldarriaga et al. 2001, 2004) and not suppor-

ted (Murray et al. 2005) in previous molecular phyloge-

netic analyses. This phylogenetic analysis did not indicate

close relatedness between Crypthecodinium and a fresh-

water dinoflagellate considered to be Hemidinium nasu-tum Stein, which also has thin thecal plates and a cingu-

lum that does not completely encircle the cell (Stein 1883,

Woloszyñska 1925). Crypthecodinium and Hemidiniumhave reported differences in tabulation pattern, and the

morphological characteristic of a cingulum that does not

fully encircle the cell may have arisen independently in

these taxa.

In this culture system, the dinoflagellates fed on fish cell

cytoplasm using an extensible feeding tube (Figure 6a),

and underwent cell division(s) within non-motile spherical

cysts (Figure 6b) that each produced 2–8 flagellate

offspring cells. Final yields of >106 dinoflagellate cells ml–1

occurred when this Crypthecodinium was cultivated

with fish cells as food (Figure 7), with a corresponding

maximum growth of 3.2 divisions day–1. Most present

Crypthecodinium-like strains are known for their ability to

grow on dissolved compounds in liquid and solid media

(Tuttle and Loeblich 1975, Beam and Himes 1977, 1982,

1984). However, Crypthecodinium has also been described

as a phagotroph that feeds on other protists (Biecheler

1952, Ucko et al. 1997). Feeding by Crypthecodinium on

fish cells in this study may have been a consequence of

the cultivation system rather than an indication of the natu-

ral prey preference of the dinoflagellate. Although this

Crypthecodinium strain might be capable of feeding on

living fish epithelium, no unusual fish deaths occurred in

the aquarium from which the dinoflagellate was isolated.

This Crypthecodinium strain can also feed on some chloro-

phyte and cryptophyte microalgae (MWP, unpublished

data), and is considered therefore to feed naturally on

various organisms.

Figure 3: The reported thecal plate pattern of Crypthecodinium setense: (a) ventral, (b) dorsal, (c) apical and (d) antapical; modified from

Biecheler (1938) and Fensome et al. (1993), using Kofoid plate labels adapted from Fensome et al. (1993)

Figure 4: Illustration of the observed apical plate arrangement of

the Crypthecodinium species

African Journal of Marine Science 2006, 28(2): 185–191 189

The dinoflagellate examined here appeared very similar

to that described by Ucko et al. (1997) of the Elat, Israel

Crypthecodinium strain feeding on Porphyridium. That

strain, like the present one, fits best with Biecheler’s (1938,

1952) description of C. setense (Ucko et al. 1997). Unfor-

tunately, the plate tabulation of the Elat Crypthecodiniumstrain was not completely resolved before the strain was

lost (ME, unpublished data). Excepting perhaps a partial

description by Javornický (1962), this is the first published

documentation of the Crypthecodinium plate tabulation

since Biecheler (1938), and the first to do so by SEM. There-

fore, it is uncertain how closely other dinoflagellate strains

regarded as Crypthecodinium relate to the taxon studied

here. Further morphological and genetic studies are

needed to clarify the taxonomy of dinoflagellates presently

regarded as Crypthecodinium.

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Figure 5: Representative phylogeny of 41 dinoflagellate taxa inferred from 18S rDNA sequence homologies with Toxoplasma gondii used as

an outgroup. Bootstrap values >50% are indicated. The asterisk denotes the Crypthecodinium species investigated here

Parrow, Elbrächter, Krause, Burkholder, Deamer, Htyte and Allen190

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