BioRisk 35 97-1 10 (2009) A peer-reviewed open-access journal

doi: 10.3897 /biorisk.3.16 & BioRisk

www.pensoftonline.net/biorisk Biodiversity & Ecosystem Risk Assessment

Biodiversity of free-living flagellates
in Kuwait’s intertidal sediments

Maria Saburova'”, Faiza Al-Yamani’, Igor Polikarpov’

| Institute of Biology of the Southern Seas, Sevastopol, Ukraine 2 Mariculture and Fisheries Department,
Kuwait Institute for Scientific Research, Kuwait

Corresponding author: Maria Saburova (msaburova@gmail.com)

Academic editors: L.J. Musselman, FE Krupp | Received 14 March 2009 | Accepted 15 December 2009 | Published 28 December 2009

Citation: Saburova M, Al-Yamani F, Polikarpov I (2009) Biodiversity of free-living flagellates in Kuwait's intertidal sedi-
ments. In: Krupp FE Musselman LJ, Kotb MMA, Weidig I (Eds) Environment, Biodiversity and Conservation in the Mid-
dle East. Proceedings of the First Middle Eastern Biodiversity Congress, Aqaba, Jordan, 20-23 October 2008. BioRisk 3:
97-110. doi: 10.3897/biorisk.3.16

Abstract

Taxonomic data of free-living benthic flagellates in Kuwait's intertidal sediments are summarized. A full
list of the species composition is presented, including distribution on different sediment types, species
occurrence and light micrographs for each taxon identified. A total of 67 flagellate species were identified,
representing six classes. Most of them are reported from Kuwait for the first time. The most abundant and

diverse species were sand-dwelling dinoflagellates (43 taxa).

Keywords

Benthic flagellates, intertidal sediments, Kuwait

Introduction

The marine coast of Kuwait, which extends for about 170 km, is composed of a variety
of coastal habitats (Al-Yamani et al. 2004). Kuwait’s coast may conveniently be divided
into two primary regions, which reflect the position of Kuwait’s offshore energy zones.
In the north, suspended material from the Shatt al-Arab delta has settled to form ex-
tensive intertidal mud and sand flats within the rather protected low-energy zone of
Kuwait Bay. Intertidal sediments grade from mud in the north and west of the Bay,
where limited salt marshes may occur, to medium or fine sand beaches at the entrance
of the Bay. On the more exposed open coast south of Kuwait Bay in high-energy ar-

Copyright M. Saburova, F Al-Yamani, |. Polikarpov. This is an open access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

98 Maria Saburova, Faiza Al-Yamani & Igor Polikarpov / BioRisk 3: 97-110 (2009)

eas, medium and coarse sand beaches extend down to the Saudi Arabian border and
beyond. Within these two broad categories, however, there are several other shoreline
types, which can grade into one another. Thus, all stages of mud, sandy mud, muddy
sand, sand, sandy-rocky flats, or rocky shore can be found along the coastline of Ku-
wait, often in close proximity of each other (Jones 1986a, b).

This unique and very productive component of Kuwait’s marine ecosystem is poor-
ly studied in terms of its microbenthic community. Previous investigations of marine
protists in some parts of Kuwait's coastline focused on their general productivity and/
or diversity especially for the diatom/cyanobacteria component of the microalgal com-
munity (e.g. Hendey 1970, Clayton 1986, Jones 1986b, Hoffman 1996, Al-Yamani
et al. 2004, Al-Zaidan et al. 2006), whereas information on taxonomy and ecology of
benthic flagellates in Kuwait’s coastal zone is totally lacking. Many of the benthic flag-
ellates are abundant in intertidal substrata and their contributions to benthic and shal-
low marine ecosystems may be significant. Several species from the benthic flagellated
community are known to be potentially toxic; therefore, the study of their biology and
potential toxic reactions in shallow waters is important.

The aim of this study was to document the biodiversity of benthic flagellates along
the Kuwaiti coastline. Additionally, their abundance and distribution on different sedi-
ment types are reported.

Methods

We selected 14 localities along Kuwait’s coast (Fig. 1) for our investigation. Each
study site stretched for about 100 m and consisted of one or more of the following
substrates: mud, sandy mud, muddy sand, or sand. In total, we collected 127 sedi-
ment samples from different intertidal heights during 2005 to 2007. Using plastic
tube corers, we sampled the top 3 to 4 cm of sandy sediment, or 3 to 4 mm of
muddy sediment. These samples were transported to the laboratory where flagellates
were separated from the sediment using Uhlig’s (1964) frozen seawater method and a
110 um mesh filter. Flagellates were collected in a Petri dish beneath the filter and ex-
amined alive with a Leica DMIL inverted microscope at x35 to x200 magnification.
For detailed observations, flagellates were isolated by micropipette and examined with
a Leica DMLM microscope or with a Carl Zeiss Axiovert 200M microscope using
transmitted-light with a bright field and phase contrast at x400 to x1000 magnifica-
tion. Flagellate plate patterns were made using Calcofluor White M2R (Fritz and
Triemer 1985). The cells were examined on an epifluorescent (violate excitation ca
430 nm, blue emission ca 490 nm) Axiovert 200M microscope. We examined both
the dorsal and ventral sides of each flagellate, and obtained micrographs using either
Axiovert 200M microscope equipped with an AxioCam HRc digital camera or a Leica
DMLM microscope with a Leica DFC 320 digital camera. Cell size was measured
by light microscopy using a calibrated ocular micrometer, and some morphometric
measurements were obtained from micrographs using Carl Zeiss Axio Visiton 3.0

Biodiversity of free-living flagellates in Kuwaits intertidal sediments 99

Figure I. Area of investigation. A Arabian Gulf, with inset showing the greater region in which the
sampling area was located B Map of Kuwait showing sampling sites (sites located within low-energy and

high-energy zones are labeled with black dots and black squares, respectively).

software. All observations presented here are based on light microscope investigations
of freshly-collected living cells.

We used the taxonomic classification scheme of Throndsen (1997), which was a
partially modified classification of Christensen (1962, 1966). The recent taxonomic
position of Protaspis is presented in accordance with Ad et al. (2005). Species names
are listed in alphabetical order within each class (Table 1).

Results and discussion

Flagellated inhabitants of Kuwait's intertidal sediments were very diverse and mostly
composed. of sand-dwelling dinoflagellates, euglenids and cryptomonads. We identi-
fied a total of 67 flagellate species (Table 1, Figs 2-7); most of them being reported

from Kuwait from the first time.

100 Maria Saburova, Faiza Al-Yamani & Igor Polikarpov / BioRisk 3: 97-110 (2009)

Table I. List of species encountered during the survey of free-living flagellates associated with intertidal
sediments along the coastline of Kuwait, 2005-2007. Abbreviations for substrate are as follows: ‘M’ —
mud, ‘MS’ — muddy sand, ‘S’ — sand, ‘P’ — small saline ponds, ‘MA’ — macroalgae; categories of occurrence
are: ‘C’ — common, ‘F’ — frequent, ‘R’ — rare; light microphotographs: figure numbers are followed by the

numbers of the photographs.

ww | dea “ae
Taxon 5 E =

Aion ce
Class Dinophyceae
Adenoides eludens (Herdman) Balech 1956 S 23
Amphidiniella sp. S 2:4, 5
Amphidiniopsis arenaria Hoppenrath 2000 S Cc 2218
Amphidiniopsis dentata Hoppenrath 2000 5 2:6-8
Amphidiniopsis swedmarkii (Balech) Dodge 1982 MS, S 2:9-11
Amphidinium carterae Hulbert 1957 MS, S C avi
Amphidinium corpulentum Kofoid et Swezy 1921 S R Si eA0
Amphidinium corrugatum Larsen et Patterson 1990 S R 3:6-8
Amphidinium gibbosum (Maranda et Shimizu) Fla Jorgensen et Murray 2004 S R 3:4
Amphidinium glabrum Hoppenrath et Okolodkov 2000 5 R 3:95:10
Amphidinium herdmanii Kofoid et Swezy 1921 S c SA ee)
Amphidinium incoloratum Campbell 1973 S C oD
Amphidinium mootonorum Murray et Patterson 2002 S Cc Olds. 16
Amphidinium operculatum Claparéde et Lachmann 1859 S € eo
Amphidinium poecilochroum Larsen 1985 S R 4:1
Amphidinium psittacus Larsen 1985 MS, S F 4:2
Amphidinium scissum Kofoid et Swezy 1921 MS, S e 3:11-14
Amphidinium semilunatum Herdman 1924 MS, S 3:18
Amphidinium steinii Lemmermann 1910 C 3:3,

Amphidinium testudo Herdman 1924 Sal

S

S
Amphidinium sp. 1 S 4:3-5
Amphidinium sp. 2 S R 4:6, 7
Bysmatrum teres Murray, Hoppenrath, Larsen et Patterson 2006 S 4:14-16
Coolia cf. areolata Ten-Hage, Turquet, Quod et Couté 2000 S

R 2:16
Coolia monotis Meunier 1919 MA, S R 2:14, 15

Gymnodinium venator Fle Jorgensen et Murray 2004 S 4:10, 11
Gyrodinium estuariale Hulburt 1957 M-S 4:8
Gyrodinium sp. S R 4:9
Herdmania litoralis (Dodge) Hoppenrath 2000 S C 4:18, 19
Heterocapsa cf. psammophila Tamura, Iwataki et Horiguchi 2005 S c 5:6-8
Heterocapsa sp. S C 4:12, 13
Katodinium asymmetricum (Massart) Loeblich 1965 MS, S F 5:5
Katodinium glandula (Herdman) Loeblich 1965 5 c 5:1-4
Oxyrrhis marina Dujardin 1841 M-S ‘é 4:20, 21
Peridinium quinquecorne Abé 1927 S R 4:17
Prorocentrum concavum Fukuyo 1981 MA, S R 5:9-11
Prorocentrum fukuyoi Murray et Nagahama 2007 5 © S285 19
Prorocentrum lima (Ehrenberg) Dodge 1975 MA, S F 5:12, 13

TR | 5:14-17

=
oa
WN
rs)

Prorocentrum rhathymum Loeblich III, Sherley et Schmidt 1979

Biodiversity of free-living flagellates in Kuwaits intertidal sediments 101

g a.
Taxon re = i}
D eo) a
Roscoffia minor Horiguchi et Kubo 1997 S 5:20, 21
Sinophysis ebriolum (Herdman) Balech 1956 S
Sinophysis stenosoma Hoppenrath 2000 S
Thecadinium ovatum Yoshimatsu, Toriumi et Dodge 2004 S 2:17, 18
Class Cryptophyceae
Platychilomonas psammobia Larsen et Patterson 1990 S 6:1, 2
Rhodomonas salina (Wislouch) Hill et Wetherbee 1989 MS, S 6:3-5
Class Chlorophyceae
Dunaliella salina (Dunal) Teodoresco 1905 P 3 6:6, 7
Class Prasinophyceae
Pyramimonas cf. octopus Moestrup e¢ Kristiansen 1987 S F 6:8, 9
Class Euglenophyceae
Anisonema acinus Dujardin 1841 MS, S C 6:12, 13
Chasmostoma nieuportense Massart 1920 M, MS R 7:3
Dinema litorale Skuja 1939 S F 6:10, 11
Dinema validum Larsen et Patterson 1990 M, MS F 6:14-16
Eutreptia pertyi Pringsheim 1953 S 7:1
Eutreptiella sp. S ee
Heteronema exaratum Larsen et Patterson 1990 MS R 6:17, 18
Heteronema larseni Lee et Patterson 2000 S 6:19
Heteronema ovale Kahl 1928 5 6:20
Notosolenus ostium Larsen et Patterson 1990 MS, S C 7:9
Petalomonas minor Larsen et Patterson 1990 M, MS
Ploeotia heracleum Larsen et Patterson 1990 S
Ploeotia cf. oblonga Larsen et Patterson 1990 S
Ploeotia pseudoanisonema Larsen et Patterson 1990 M, MS
Ploeotia sp. S
Urceolus sabulosus Stokes 1886 M, MS
Cercozoa
Protaspis grandis Hoppenrath et Leander 2006 S
Protaspis maior Skuja 1939 S
Protaspis obliqua Larsen et Patterson 1990 MS, S
Protaspis sp. S

The diversity of the flagellated group was mainly due to sand-dwelling dinoflagel-
lates and euglenids. Amphidinium, with 17 species, was among the most abundant and
diverse sand-dwelling dinoflagellate genera. Within this genus, the large, unarmored
A, scissum was widely distributed throughout the year and may be the most abundant
on Kuwait's intertidal sand flats. This species was occasionally found in other habitats.

A limited number of benthic dinoflagellates are potentially harmful, as they are
capable of producing toxins, which may result in an intoxication of the marine en-
vironment. Among taxa of sand-dwelling dinoflagellates recorded in Kuwait were
eight species that must be considered potentially harmful. Prorocentrum concavum

102 Maria Saburova, Faiza Al-Yamani & Igor Polikarpov / BioRisk 3: 97-110 (2009)

Figure 2. Light micrographs of the genera Adenoides, Amphidiniella, Amphidiniopsis, Coolia, Thecadin-
ium and Sinophysis. \-3 Adenoides eludens 4,5 Amphidiniella sp. 6-8 Amphidiniopsis dentata 9-11 Am-
phidiniopsis swedmarkii 12, 13 Amphidiniopsis arenaria 14, 15 Coolia monotis 16 Coolia cf. areolata \7,
18 Thecadinium ovatum \9 Sinophysis ebriolum 20 Sinophysis stenosoma. Photos 2, 3, 10, 11, 15: epifluo-
rescence. Scale bar = 10 um for all photos.

Biodiversity of free-living flagellates in Kuwaits intertidal sediments 103

19 20 21

Figure 3. Light micrographs of the genus Amphidinium. | Amphidinium carterae 2 Amphidinium oper-
culatum 3 Amphidinium steinii 4 Amphidinium gibbosum § Amphidinium incoloratum 6-8 Amphidinium
corrugatum 9, 10 Amphidinium glabrum 11-14 Amphidinium scissum 15,16 Amphidinium mootonorum
17 Amphidinium testudo 18 Amphidinium semilunatum 19, 20 Amphidinium corpulentum 2\, 22 Am-
phidinium herdmanii. Scale bar = 10 um for all photos.

104 Maria Saburova, Faiza Al-Yamani & Igor Polikarpov / BioRisk 3: 97-110 (2009)

| al 20
Figure 4. Light micrographs of the genera Amphidinium, Gyrodinium, Gymnodinium, Heterocapsa, Bys-
matrum, Peridinium, Herdmania and Oxyrrhis. \ Amphidinium poecilochroum 2. Amphidinium psittacus
3-5 Amphidinium sp. 1 6, 7 Amphidinium sp. 2 8 Gyrodinium estuariale 9 Gyrodinium sp. 10, 11 Gym-
nodinium venator 12, 13 Heterocapsa sp. 14—16 Bysmatrum teres \7 Peridinium quinquecorne 18, 19
Herdmania litoralis 20,21 Oxyrrhis marina. Photo 16: epifluorescence. Scale bar = 10 pm for all photos.

Biodiversity of free-living flagellates in Kuwaits intertidal sediments 105

Figure 5. Light micrographs of the genera Katodinium, Heterocapsa, Prorocentrum and Roscoffia. \-4
Katodinium glandula § Katodinium asymmetricum 6-8 Heterocapsa cf. psammophila 9=—\\ Prorocentrum
concavum \2, 13 Prorocentrum lima \4-17 Prorocentrum rhathymum \8,\9 Prorocentrum fukuyoi 20, 21
Roscoffia minor. Photos 7, 8, 21: epifluorescence. Scale bar = 10 pm for all photos.

106 Maria Saburova, Faiza Al-Yamani & Igor Polikarpov / BioRisk 3: 97-110 (2009)

Figure 6. Light micrographs of the genera Platychilomonas, Rhodomonas, Dunaliella, Pyramimonas, Din-
ema, Anisonema and Heteronema. \,2. Platychilomonas psammobia 3-5 Rhodomonas salina 6,7 Dunaliella
salina 8,9 Pyramimonas cf. octopus 10, 11 Dinema litorale 12,13 Anisonema acinus 1\4=16 Dinema validum
17,18 Heteronema exaratum \9 Heteronema larseni 20 Heteronema ovale. Scale bar = 10 um for all photos.

Biodiversity of free-living flagellates in Kuwait’ intertidal sediments

»
.
'
rd
Ne
%
a
.
,
a

*

Figure 7. Light micrographs of the genera Eutreptia, Eutreptiella, Chasmostoma, Ploeotia, Notosolenus,
Petalomonas, Urceolus and Protaspis. | Eutreptia pertyi 2 Eutreptiella sp. 3 Chasmostoma nieuportense 4
Ploeotia heracleum § Ploeotia cf. oblonga 6, 7 Ploeotia pseudoanisonema 8 Ploeotia sp. 9 Notosolenus ostium

10, 11 Petalomonas minor 12-16 Urceolus sabulosus 11 Protaspis grandis \8 Protaspis maior \9 Protaspis
obliqua 20, 21 Protaspis sp. Scale bar = 10 pm for all photos.

107

108 Maria Saburova, Faiza Al-Yamani & Igor Polikarpov / BioRisk 3: 97-110 (2009)

has been reported to produce three diol esters of okadaic acid and ichthyotoxin (Yas-
umoto et al. 1987, Hu et al. 1993). Prorocentrum lima may produce okaidic acid and
dinophysistoxins, which may cause diarrhetic shellfish poisoning (Murakami et al.
1982, Tindall et al. 1984, Torigoe et al. 1988, Lee et al. 1989, Hu et al. 1993). Pro-
rocentrum rhathymum may produce water-soluble fast-acting toxins and hemolytic
effects (Nakajima et al. 1981, Tindall et al. 1989). In the summer of 1999, a bloom
of P rhathymum (reported as P mexicanum) caused a massive fish kill in Kuwait Bay
(Al-Yamani et al. 2004). Coolia monotis is known to produce cooliatoxin, which
is presumably related to yessotoxin (Holmes et al. 1995). It may be involved in
ciguatera (Tindall and Morton 1998). Haemolysins, compounds toxic to fish, have
been isolated from Amphidinium carterae (Yasumoto et al. 1987). Amphidinium gib-
bosum may produce cytotoxic metabolites, the most potent of which (caribenolide
I) had anti-tumor effects (Bauer et al. 1994, Bauer et al. 1995a, b, Maranda and
Shimizu 1996). Haemolytic and antifungal properties (amphidinols) are reported
from A. operculatum; it may also be toxic to fish (Yasumoto et al. 1987). Peridinium
quinquecorne can cause anoxia and fish kills, if occurring with very high cell densi-
ties (Fukuyo et al. 1990). These potentially harmful dinoflagellates were present in
Kuwait’s benthic microalgal community during 2005 to 2007, but never occurred
in great numbers.

Euglenids comprised auto- and heterotrophic species. Among them, Anisonema
acinus was the most common and widespread species in Kuwait’s muddy and sandy
sediments. Petalomonas minor and Urceolus sabulosus were mainly recorded in intertidal
mudflats of Kuwait Bay, where they can be quite abundant.

The highest species diversity of flagellates in Kuwait’s soft sediments was as-
sociated with the southern intertidal sand flats. Together with benthic diatoms and
cyanobacteria, autotrophic flagellates are likely to be among the most important
contributors to primary productivity in the intertidal zone. Heterotrophic species
may play an important role in intertidal food webs, consuming even large diatoms
and other flagellates.

Acknowledgements

We are particularly grateful to Dr Jacob Larsen (IOC-Science and Communication
Centre on Harmful Algae, Botanical Institute, University of Copenhagen, Denmark)
and to Dr Mona Hoppenrath (Senckenberg Research Institute, Germany) for their
thoughtful and creative comments. We also would like to express our thanks to Dr
Won Je Lee (Kyungam University, Korea) and Dr Denis V. Tikhonenkov (Institute
for Biology of Inland Waters, Russia) for their critical reviews of the manuscript. The
assistance of Alan Lennox (Kuwait Institute for Scientific Research, Kuwait) is also
highly appreciated. We thank Dr James Bishop (Kuwait Institute for Scientific Re-
search, Kuwait) for correcting the English. We are grateful to the Kuwait Institute for
Scientific Research for funding this research project.

Biodiversity of free-living flagellates in Kuwaits intertidal sediments 109

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