marked differences in fatty acid profiles of some planktonic and benthic marine dinoflagellates from...
TRANSCRIPT
Marked differences in fatty acid profiles of some planktonic and benthic marine
dinoflagellates from Malaysian waters
GIRES USUP*, SITI ZALEHA HAMID, PHENG KOON CHIET, CHENG KOK WAH AND ASMAT AHMAD
Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
G. USUP, S.Z. HAMID, P.K. CHIET, C.K. WAH AND A. AHMAD. 2007. Marked differences in fatty acid profiles of someplanktonic and benthic marine dinoflagellates from Malaysian waters. Phycologia 47: 105–111. DOI: 10.2216/07-55.1
This study was carried out to characterize the fatty acid profiles of some planktonic and benthic marine dinoflagellatesfrom Malaysian waters. Clonal batch cultures of Alexandrium affine, A. leei, A. minutm, A. tamarense, A. tamiyavanichii,Coolia monotis, Prorocentrum emarginatum, P. mexicanum, Ostreopsis ovata and Amphidinium sp. were harvested at lateexponential phase, and total lipid was extracted. Samples were derivatized to produce fatty acid methyl esters (FAMEs).FAMEs were analyzed on a gas chromatograph with flame ionization detection. The total number of fatty acidsdetected in the clones ranged from 10 in the A. tamarense AtPA04 clone to 22 in the C. monotis CmPL01 clone. Fattyacids found in all clones were myristic acid (14 : 0), palmitic acid (16 : 0), stearic acid (18 : 0), linoleic acid (18 : 2v6c)and oleic acid (18 : 1v9c). In all clones only a few fatty acids were dominant. In the Alexandrium clones the dominantfatty acids were 16 : 0, 18 : 0, cis-13,16-docosadienoic acid (22 : 2), 18 : 2v6c and 18 : 1v9c. There was almost completeabsence of omega-3 polyunsaturated fatty acids (PUFA) in the Alexandrium clones. In the benthic species the majorfatty acids were 16 : 0, eicosapentaenoic acid (EPA, 20 : 5v3), docosahexaenoic acid (22 : 6v3), 18 : 2v6c and18 : 1v9c. In the Prorocentrum clones the major fatty acids were 14 : 0, 16 : 0, palmitoleic acid (16 : 1) and EPA. TotalPUFA content in the benthic species were 37%–56%, while in the planktonic species the content was 19%–44%. Thefatty acid profiles could not differentiate between species. However, cluster analysis and principal components analysiswere able to clearly discriminate between the Alexandrium group, Prorocentrum group and benthic species group.
INTRODUCTION
Studies on lipids, sterols and fatty acids of dinoflagellates
are important for several reasons. Economically, some
species may produce well-known or novel fatty acids of
neutraceutical value to humans and reared animals (Cohen
et al. 1995; Mansour 2005; Ward & Singh 2005; Atalah et
al. 2007; Cardozo et al. 2007). For example, the marine
dinoflagellate Crypthecodinium cohnii Biecheler is poten-
tially a very viable source for the omega-3 polyunsaturated
fatty acid (PUFA) docosahexaenoic acid (DHA) (Swaaf et
al. 2003). Ecologically, these analyses can provide insights
into sources of organic material in ecosystems (Marchand
et al. 2005), sources and pathways of organic materials in
food webs (Kirsch et al. 1998; Stevens 2004; Alfaro et al.
2006) and also interaction between different species (Ikawa
2004; Wu et al. 2006). In the case of harmful dinoflagellate
species, some studies have indicated that the presence of
certain lipids, sterols and fatty acids may be responsible for
ichthyotoxic and allelopathic effects (Smolowitz & Shum-
way 1997; Sola et al. 1999; Mooney et al. 2007; Ikawa
2004). Lastly, analysis of these cell constituents may
provide valuable chemotaxonomic biomarkers that might
help delineate taxa (Cho et al. 2001a, b; Zhukova &
Titlyana 2003; Mooney et al. 2007).
Several proven and potentially harmful marine dinofla-
gellate species are present in Malaysian waters. These
include the paralytic toxin–producing Pyrodinium baha-
mense Plate var. compressum Bohm, Alexandrium minutum
Halim, A. tamiyavanichii Balech, A. taylori Balech and A.
peruvianum (Balech & Mendiola) Balech and Tangen (Usup
et al. 2002; Lim et al. 2005). Other Alexandrium species
present but nontoxic are A. affine (Inoue & Fukuyo)
Balech, A. leei Balech and A. tamarense (Lebour) Balech. In
addition, there are several potentially harmful benthic
species, including Coolia monotis Meunier, Ostreopsis spp.,
Amphidinium spp. and Gambierdiscus spp. The taxonomy of
some of these species are still problematic. For example,
designation of Alexandrium species is currently based on
sometimes very small differences in morphology. This has
probably led to the creation of duplicate species. The
incorporation of molecular data to supplement morpho-
logical data has had mixed results. In some cases the
molecular data supported morphological delineation, while
in some cases molecular separation followed biogeography
rather than morphology (Scholin et al. 1994; Hansen et al.
2003; Leaw et al. 2005). For these reasons, studies on other
taxonomic markers are actively being pursued. One
potential marker is the fatty acid profile. In bacteriology,
for example, the fatty acid profile is one of the criteria
required for species description. In the case of dinoflagellate
taxonomy, results have been inconclusive. In a recent study
Mooney et al. (2007) suggested that the ratio between
certain fatty acids could potentially differentiate between
species in the gymnodinioid family Kareniaceae. Zhukova
and Titlyanov (2003) proposed that fatty acids are useful
markers for morphophysiological types of zooxanthellae.
Mansour et al. (1999) concluded that while fatty acid
profiles are useful to differentiate between marine micro-
algae classes, they are not useful to differentiate between
species.* Corresponding author ([email protected]).
Phycologia (2008) Volume 47 (1), 105–111 Published 8 January 2008
105
Very few studies have been reported on the fatty acid
profiles of Alexandrium, Prorocentrum and benthic dino-
flagellates despite the importance of these taxa. In this
study we analyzed the fatty acid profiles of some of these
species from Malaysian waters.
MATERIAL AND METHODS
Marine dinoflagellate species used in this study are listed in
Table 1. Clonal cultures were established through single cell
isolations. Cultures were grown in nutrient-replete ES-DK
medium (Kokinos & Anderson 1995) at 26uC under
a 14 : 10-hour light-dark cycle. Cultures for fatty acid
analysis were grown in 250-ml volume in 500-ml flasks.
Cultures for fatty acid analysis were harvested at the end
of the exponential growth phase by sieving through a 10-
mm Nitex mesh. The cell pellet was rinsed briefly with sterile
double-distilled H2O to get rid of excess salt. Fatty acid
extraction was carried out using a modified protocol of
Bligh and Dyer (1959). Briefly, the pellet was extracted in
CHCl3 : MeOH : H2O (1 : 2 : 0.8, v/v/v). The suspension
was centrifuged at 8000 3 g, 4uC for 5 minutes. Chloro-
form and ddH2O were added to the supernatant to final
chloroform/methanol/water ratio of 1 : 1:0.9 (v/v/v). The
chloroform layer was collected and concentrated in a rotary
evaporator under vacuum. Fatty acid methyl esters
(FAME) were produced by heating in MeOH : HCl : H2O
(10 : 1 : 1, v/v/v) at 100uC for 1 hour. FAMEs were
extracted in hexane/chloroform (4 : 1, v/v). FAMEs were
analyzed on a Shimadzu GC-17A gas chromatograph using
flame ionisation detection. Internal standard was hexaco-
sanoic acid (26 : 0) FAME. All clones were analysed in
triplicate.
Relative percentage composition of fatty acids are
presented as the mean 6 standard deviation. Cluster
analysis and principal components analysis of the clones
was carried out using the Multivariate Statistical Package
version 3.13d (Kovach Computing Services).
RESULTS
The total number of fatty acids detected in the clones
ranged from 10 in the A. tamarense AtPA04 clone to 22 in
the C. monotis CmPL01 clone (Table 2). Fatty acids found
in all clones were myristic acid (14 : 0), palmitic acid
(16 : 0), stearic acid (18 : 0), linoleic acid (18 : 2v6c) and
oleic acid (18 : 1v9c).
In all clones only a few fatty acids were dominant. In the
Alexandrium clones the dominant fatty acids were 16 : 0
(14.72%–21.67%), 18 : 0 (8.07%–25.83%), cis-13,16-docosa-
dienoic acid (22 : 2) (1.28%–40.83%), 18 : 2v6c (2.50%–
12.50%) and 18 : 1v9c (16.67%–40.07%); 22 : 2 and
18 : 1v9t were not present in the other genera studied.
None of the Alexandrium isolates contained docosahexa-
enoic acid (22 : 6v3), while eicosapentaenoic acid (20 : 5v3)
was present only in A. tamiyavanichii. All the Alexandrium
isolates had a PUFA : SFA ratio of , 1, except A.
tamiyavanichii.
In the benthic species the major fatty acids were 16 : 0
(23.43%–29.43%), eicosapentaenoic acid (EPA, 20 : 5v3)
(11.14%–16.86%), docosahexaenoic acid (DHA, 22 : 6v3)
(16.62%–28.80%), 18 : 2v6c (2.23%–10.89%), and
18 : 1v9c (9.18%–21.43%). In all clones except C. monotis
PDB4, the PUFA : SFA ratio was . 1. Lauric acid (12 : 0)
was present in most of the benthic clones but was present
only in P. mexicanum among the planktonic clones.
In the Prorocentrum clones the major fatty acids were
14 : 0 (12.47%–37.85%), 16 : 0 (13.98%–22.75%), palmito-
leic acid (16 : 1) (10.94%–21.85%) and EPA (14.97%–
22.69%). The P. mexicanum and P. emarginatum profiles
were very similar, but P. emarginatum had more variety of
PUFAs. The PUFA : SFA ratio for both species was , 1.
There were some fatty acids that were detected in only
one or two of the species studied. Erucic acid (22 : 1v9) was
found only in A. tamarense AtPA02, and myristoleic acid
(14 : 1) and eicosatrienoic acid (20 : 3v3) were found only
in P. emarginatum and C. monotis CmPL01.
Cluster analysis showed that the fatty acid profile could
not separate the isolates at species or genus level (Fig. 1).
Table 1. The marine dinoflagellate isolates used in this study.
Species Original isolation locality Date of isolation Toxicity
Alexandrium affine AaMS03 Sebatu, Straits of Malacca November 1999 NontoxicAlexandrium minutum AmKB06 Tumpat, South China Sea September 2001 PSP toxin producerAlexandrium leei AlMS01 Sebatu, Straits of Malacca November 1999 NontoxicAlexandrium tamiyavanichii AcMS01 Sebatu, Straits of Malacca November 1998 PSP toxin producerAlexandrium tamarense AtPA01, AtPA02,
AtPA03, AtPA04Penang, Straits of Malacca May 2002 Nontoxic
Prorocentrum mexicanum Tafall PP1E4 Perhentian Island, South China Sea August 2005 UnknownProrocentrum emarginatum Fukuyo PP2D6 Perhentian Island, South China Sea July 2006 UnknownOstreopsis ovata OvPR06 Redang Island, South China Sea July 1997 Unknown; strong
hemolytic activityAmphidinium sp. AsPR01 Redang Island, South China Sea July 1997 UnknownCoolia monotis CmPL01 Langkawi Island, Straits of Malacca September 1997 Unknown; strong
hemolytic activityCoolia monotis CmSA01, CmSA02 Kota Kinabalu, Sabah, South China
SeaOctober 1997 Unknown; strong
hemolytic activityCoolia monotis CmPD03 Port Dickson, Straits of Malacca July 1996 Unknown; strong
hemolytic activityCoolia monotis PP1B6 Perhentian Island, South China Sea July 2006 UnknownCoolia monotis PDB4, PDB5, PDC7 Port Dickson, Straits of Malacca September 2006 Unknown
106 Phycologia, Vol. 47 (1), 2008
There was also no clear separation of toxic from nontoxic
Alexandrium isolates. However, there was a clear separation
of three groups, namely, Alexandrium, Prorocentrum and
the benthic group comprising Coolia, Ostreopsis and
Amphidinium. This group separation was also clearly
defined through principal components analysis (Fig. 2).
DISCUSSION
The fatty acid compositions of the dinoflagellates in this
study were largely similar to others previously reported, but
there were also very significant differences. In all the species
studied the dominant FAs were 16 : 0, 18 : 0 and 18 : 1v9c.
None of the isolates contain the common dinoflagellate
PUFA octadecapentaenoic acid (18 : 5v3) or the very long
chain PUFA 28 : 7v6 and 28 : 8v3. It has previously been
suggested that a biomarker for fatty acids of dinoflagellate
origin is docosahexaenoic acid or DHA (22 : 6v3) and
eicosapentaenoic acid or EPA (20 : 5v3) (Joseph 1975;
Parrish et al. 2000). The major surprise in this study was the
complete absence of 22 : 6v3 in all the Alexandrium species
studied. In fact, there was almost complete absence of all
omega-3 fatty acids in all the Alexandrium. It is not known
whether this is peculiar to these Malaysian isolates or true
of tropical Alexandrium in general. In a previous study Cho
et al. (2001a) detected high levels of DHA in some A.
tamarense isolates from South Korea. Wen and Chen
(2003) proposed that in microalgae, EPA and DHA are
produced in microalgae through a pathway as follows:
acetyl-CoA R oleic acid (18 : 1v9) R linoleic acid
(18 : 2v6c) R a-linoleic acid (18 : 3v3) R R R EPA
(20 : 5v3) R R R DHA (22 : 6v3). The Alexandrium
isolates from Malaysia all produced a high proportion of
18 : 1v9, as much as 40% in A. affine and a significant
content of 18 : 2v6c. However, the isolates did not contain
18 : 3v3, presumably resulting in no EPA and DHA being
produced. The results suggest that these Alexandrium
isolates could be unique among marine dinoflagellates
because they lack the enzyme D15 desaturase for the
Fig. 1. Clustering of the dinoflagellates based on fatty acid composition.
Fig. 2. Distribution of the dinoflagellate clones studied based on principal components analysis. Axes are principal components 1 and 2.
Usup et al.: Fatty acids in some tropical planktonic and benthic dinoflagellates 107
Table
2.
Fatt
yaci
dco
mp
osi
tio
ns
of
the
din
ofl
agel
late
s.V
alu
esare
giv
enas
mea
n6
s(n
53).
Fatt
yaci
d
Ale
xandri
um
Pro
roce
ntr
um
mex
icanum
Pro
rocen
trum
em
arg
inatu
maff
ine
min
utu
mle
eita
miy
avanic
hii
tam
are
nse
tam
are
nse
tam
are
nse
tam
are
nse
AaM
S03
Am
KB
06
AlM
S01
AcM
S01
AtP
A01
AtP
A02
AtP
A03
AtP
A04
PP
1E
4P
P2D
612
:0
1.1
16
1.4
714
:0
0.5
56
0.0
72.2
26
0.1
30.5
66
0.1
02.2
26
0.2
61.5
96
0.2
31.1
86
0.0
80.8
36
0.1
11.6
76
0.3
037.8
56
2.2
712.4
76
2.1
215
:0
0.5
66
0.1
00.6
06
0.1
10.7
56
0.0
416
:0
18.8
96
3.4
015.8
36
2.3
721.6
76
4.1
116.3
96
2.1
320.0
06
0.2
218.8
96
1.7
018.5
96
1.6
714.7
26
2.5
113.9
86
1.9
522.7
56
2.5
017
:0
0.4
96
0.0
32.6
06
0.3
32.1
46
0.1
718
:0
8.0
76
1.1
319.4
46
3.6
916.6
76
1.6
516.6
76
2.5
025.8
36
1.2
916.3
96
0.8
119.4
06
2.3
316.6
76
0.8
31.6
16
0.2
81.0
06
0.0
620
:0
0.5
66
0.0
30.3
76
0.0
60.8
36
0.0
90.8
36
0.0
80.2
86
0.0
60.4
66
0.0
40.5
86
0.0
91.3
96
0.2
721
:0
1.6
16
0.2
822
:0
0.2
86
0.0
40.3
76
0.0
40.5
16
0.0
61.3
96
0.1
30.4
86
0.0
50.4
26
0.0
30.2
86
0.0
40.2
76
0.0
223
:0
1.3
96
0.0
91.7
26
0.1
80.5
56
0.4
50.2
86
0.0
30.4
56
0.0
70.6
06
0.1
11.1
16
0.1
90.2
96
0.0
524
:0
0.2
86
0.0
42.2
16
0.1
31.3
96
0.2
23.6
76
0.4
71.5
66
0.2
814
:1
0.8
16
0.0
515
:1
16
:1
0.5
66
0.0
80.5
66
0.0
910.9
46
0.7
621.8
56
4.3
717
:1
2.4
86
0.3
218
:1v
9t
1.1
16
0.1
30.3
76
0.0
32.5
06
0.3
50.5
66
0.0
60.6
36
0.0
80.8
36
0.0
95.2
86
0.6
36.1
16
0.5
518
:1v
9c
40.0
76
2.8
021.6
76
3.4
724.4
46
4.8
816.6
76
3.3
322.9
96
2.3
338.3
36
2.2
919.1
76
0.9
622.2
26
1.5
56.6
16
0.3
32.7
76
0.4
918
:2v
6t
0.5
26
0.1
10.5
96
0.1
00.3
96
0.0
218
:2v
6c
12.5
06
1.5
05.5
66
0.5
64.1
46
0.2
12.7
86
0.2
25.0
06
0.8
58.3
36
1.3
32.7
76
0.4
42.5
06
0.3
21.7
76
0.0
80.7
36
0.1
318
:3v
30.6
06
0.1
12.4
16
0.2
418
:3v
65.2
86
1.0
02.5
06
0.3
21.9
46
0.1
21.1
16
0.0
61.5
06
0.1
12.7
86
0.3
31.9
46
0.2
72.7
86
0.1
40.7
86
0.0
420
:1v
98.0
66
1.5
30.4
66
0.0
90.3
66
0.0
220
:3v
32.5
86
0.4
920
:3v
620
:4v
620
:5v
32.7
86
0.3
014.9
76
1.6
422.6
96
1.3
622
:1v
90.5
96
0.1
122
:2
1.2
86
0.1
928.8
96
3.7
522.2
26
3.5
540.8
36
4.0
819.4
46
3.1
17.5
06
1.0
530.0
06
4.8
030.8
36
5.5
422
:6v
33.4
86
0.4
52.1
26
0.3
824
:1v
90.2
86
0.0
20.5
66
0.0
5T
ota
lS
FA
30.0
238.3
244.1
738.0
550.0
739.6
740.8
435.5
661.4
241.2
3T
ota
lM
UF
A49.8
022.0
426.9
417.2
323.9
941.3
324.4
528.3
318.3
627.4
6T
ota
lP
UF
A20.1
839.7
328.8
944.7
225.9
419.0
034.7
136.1
120.2
231.3
1P
UF
A:
SF
A0.7
:1
1:
10.7
:1
1.2
:1
0.5
:1
0.5
:1
0.8
:1
1:
10.3
:1
0.8
:1
108 Phycologia, Vol. 47 (1), 2008
Table
2.
Co
nti
nu
ed.
Fatt
yaci
dC
ooli
am
onoti
sO
stre
opsi
sova
taO
vP
R06
Am
phid
iniu
msp
AsP
R01
PD
B4
PD
B5
PD
C7
Cm
PD
02
Cm
PD
03
Cm
PL
01
Cm
SA
01
Cm
SA
02
PP
1B
612
:0
0.1
66
0.0
30.4
56
0.0
20.8
26
0.1
10.1
36
0.0
20.8
16
0.1
51.8
76
0.3
314
:0
1.8
86
0.3
13.0
16
0.4
83.4
66
0.4
82.9
26
0.1
43.2
46
0.5
88.8
86
0.6
22.7
56
0.2
82.4
76
0.5
42.2
16
0.3
52.8
46
0.3
71.5
96
0.1
915
:0
0.1
86
0.0
316
:0
29.4
36
2.0
628.2
56
2.5
429.0
66
1.4
526.9
46
4.8
423.4
36
3.2
825.3
76
2.0
327.3
76
6.0
223.7
36
2.2
723.6
36
3.5
428.9
86
4.3
527.8
96
4.1
817
:0
0.7
26
0.1
10.6
06
0.1
10.5
76
0.0
50.5
36
0.0
61.2
26
0.0
71.1
26
0.1
50.3
86
0.0
90.7
96
0.0
91.0
46
0.1
21.1
76
0.0
91.0
96
0.1
218
:0
12.3
56
1.3
53.5
76
0.4
24.0
16
0.3
63.3
26
0.5
62.6
86
0.3
71.9
96
0.2
23.0
06
0.6
33.1
66
0.6
94.0
16
0.4
82.8
66
0.2
02.8
96
0.3
820
:0
0.3
36
0.0
50.3
46
0.0
50.1
96
0.0
221
:0
22
:0
1.0
06
0.2
423
:0
0.1
76
0.0
20.3
46
0.0
30.4
26
0.0
20.1
36
0.0
10.0
96
0.0
20.1
16
0.0
30.5
36
0.0
70.2
76
0.0
30.3
36
0.0
41.3
06
0.2
224
:0
0.4
86
0.0
90.3
46
0.0
20.3
66
0.0
70.5
46
0.0
41.0
96
0.1
50.3
26
0.0
30.3
36
0.0
40.6
86
0.0
60.4
86
0.0
814
:1
0.1
46
0.0
215
:1
0.2
36
0.0
40.1
46
0.0
316
:1
0.2
16
0.0
30.2
26
0.0
41.9
06
0.2
25.3
26
1.0
20.1
86
0.0
217
:1
0.2
36
0.0
20.4
86
0.0
918
:1v
9t
18
:1v
9c
17.1
26
2.7
310.6
36
0.5
312.1
46
2.1
814.1
56
1.2
719.8
36
1.3
99.3
36
1.3
19.1
86
2.0
221.4
36
3.4
311.7
26
0.9
410.4
76
2.3
121.1
66
4.0
218
:2v
6t
18
:2v
6c
7.4
36
1.4
17.1
76
1.3
67.3
46
0.6
67.3
06
0.5
85.6
76
0.6
25.6
76
0.9
66.5
56
1.4
45.2
16
0.2
610.8
96
0.9
87.0
86
0.7
82.2
36
0.1
818
:3v
30.4
86
0.0
81.0
16
0.1
61.1
16
0.0
90.9
86
0.1
10.2
36
0.0
40.8
96
0.1
71.1
66
0.1
518
:3v
61.3
16
0.0
70.4
96
0.0
30.9
06
0.1
40.4
06
0.0
30.9
66
0.1
10.4
66
0.0
70.3
26
0.0
61.7
46
0.2
12.1
46
0.1
70.6
06
0.1
320
:1n
-920
:3v
30.1
26
0.0
220
:3v
60.3
36
0.0
30.6
16
0.0
30.4
26
0.0
50.7
26
0.0
40.2
56
0.0
40.4
56
0.0
60.6
36
0.0
50.6
46
0.0
320
:4v
60.4
36
0.0
30.9
76
0.0
60.3
16
0.0
50.1
66
0.0
30.4
76
0.0
40.4
06
0.0
51.5
26
0.3
320
:5v
311.1
46
1.4
416.7
66
2.6
814.8
06
1.9
216.5
76
1.3
217.5
96
2.4
616.4
96
1.9
813.8
96
1.6
716.6
76
3.1
612.8
16
1.1
516.2
96
1.9
516.8
66
3.7
122
:1v
922
:2
22
:6v
316.6
26
1.3
227.1
86
2.1
725.7
26
3.8
525.6
66
1.5
419.8
76
2.7
821.4
26
4.2
835.2
76
6.3
521.9
56
1.0
928.8
06
2.9
327.6
96
3.8
820.6
06
1.8
524
:1v
9T
ota
lS
FA
145.3
635.9
338.2
534.9
531.5
839.0
433.9
331.0
131.8
437.4
737.6
3T
ota
lM
UF
A2
17.3
310.8
512.1
414.1
522.1
915.4
19.3
621.4
311.7
210.4
721.1
6T
ota
lP
UF
A3
37.3
153.2
249.6
150.9
046.2
345.5
556.7
147.5
656.4
452.0
641.2
1P
UF
A:
SF
A0.8
2:
11.5
:1
1.3
:1
1.5
:1
1.5
:1
1.2
:1
1.7
:1
1.5
:1
1.8
:1
1.4
:1
1.1
:1
1S
FA
,sa
tura
ted
fatt
yaci
d.
2M
UF
A,
mo
no
un
satu
rate
dfa
tty
aci
d.
3P
UF
A,
po
lyu
nsa
tura
ted
fatt
yaci
d.
Usup et al.: Fatty acids in some tropical planktonic and benthic dinoflagellates 109
conversion of 18 : 2v6c to 18 : 3v3. Ecologically, EPA and
DHA are essential fatty acids to grazers, so the absence of
these fatty acids would presumably lower the nutritional
value of Alexandrium to zooplankters and would be
selectively not grazed upon in the presence of more
nutritious prey. On the other hand, all the Alexandrium
species contained 22 : 2 and 18 : 1v9t but not the other
species studied. We also did not come across these two FAs
in previous reports on other dinoflagellates. Perhaps these
two FAs could be used as biomarkers to separate
Alexandrium from other dinoflagellate species.
In contrast to Alexandrium, omega-3 FAs were very high
in Prorocentrum, Coolia, Ostreopsis and Amphidinium. EPA
content reached 20% in P. emarginatum, while DHA
reached 35% in C. monotis CmSA01. The EPA percentages
in these isolates were comparable to those reported for
several other microalgal classes (reviewed in Wen & Chen
2003), while the DHA proportions were higher and
comparable to those found in C. cohnii. The benthic
dinoflagellates also contained high levels of oleic acid
(18 : 1v9c), a precursor for EPA and DHA. The 18 : 3v3
content was low in most of the benthic dinoflagellate
isolates and was not detected in C. monotis clones PDC7
and CmPD02 as well as O. ovata and Amphidinium spp.
This suggested that the fatty acid is preferably converted to
EPA and DHA in the cells. Results of this study suggest
that these benthic dinoflagellates and Prorocentrum spp.
could be viable commercial sources for essential FAs either
for human consumption or aquaculture feeds since they
could be easily cultured in large volumes. However, these
species would not be suitable for single-cell oils because
they most probably contain other compounds toxic to
humans.
Results of this study clearly indicate that the fatty acid
profile is not applicable as a chemotaxonomic marker at
species or genus level. It is as yet unclear if fatty acid profile
would be useful as a supporting taxonomic criterion as
applied in bacteriology. This will be clearer as more data
become available. In a recent study Mooney et al. (2007)
suggested that the ratio of 28 : 7v6 to 28 : 8v3 may be
useful as a taxonomic marker at species level for the family
Kareniaceae. It is likely that the fatty acids produced by
a particular isolate will remain invariable since this will be
determined by the genetic makeup of the isolate. This may
remain stable even after prolonged culturing in the
laboratory. For example, the C. monotis isolates that were
used in this study have been in laboratory culture for
periods of 1–10 years, but the fatty acid profiles were fairly
consistent across all clones. The relative proportions
between each fatty acid, however, might change under
different physiological and growth conditions (Swaaf et al.
2003; Wen & Chen 2003; Zhukova & Titlyanov 2003, 2006).
The same situation is known to occur in the case of
paralytic poisoning toxin production (e.g. Usup et al. 1994).
Thus, the use of ratios between particular fatty acids to
delineate taxa has to be applied with caution.
In this study both cluster and principal components
analyses showed that the FA profile did have some
discriminatory value since Alexandrium, Prorocentrum and
the benthic dinoflagellates were separated into clearly
distinct groups, although Prorocentrum might have clus-
tered together with Coolia and Ostreopsis if more isolates
were analysed. The basis for separation of the benthic and
planktonic dinoflagellates observed in this study was not
clear. One possibility was that it could be related to the
utilization of the fatty acids in buoyancy regulation.
However, there was no clear excess of long carbon chain
FAs in the benthic dinoflagellates compared to the
planktonic species. One clear difference between the
planktonic and benthic species was that in the former
group the ratio of polyunsaturated to saturated fatty acids
was , 1, while in the latter it was . 1. Perhaps the
differences between these three groups have a phylogenetic
basis. Zhukova and Titlyanov (2006) suggested that light-
dependent changes in fatty acid composition in zooxan-
thellae are probably due to correlation of activity of
photosystems with processes of production and desatura-
tion of fatty acids. Thus, the differences between the
planktonic and benthic dinoflagellates observed in this
study, especially with regard to the PUFA : SFA ratios,
could be the result of genetic adaptations to the different
light levels where these species are typically found. More
data are required in order to determine if this pattern
applies to most marine dinoflagellates.
ACKNOWLEDGEMENTS
This study was funded by the Malaysia government
through research grants IRPA 09-02-02-EA0079 and 02-
01-02-SF0203.
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Received 13 July 2007; accepted 9 October 2007
Associate editor: John Beardall
Usup et al.: Fatty acids in some tropical planktonic and benthic dinoflagellates 111