Toxic Benthic Dinoflagellates in the West Pacific Regions
LU SonghuiResearch Center for Harmful Algae and Marine Biology, Jinan University, Guangzhou,
China
WESTPAC HAB Workshop 2016, Nha Trong
Outline
01 Benthic Dinos in China
02 Taxonomy and Diversity
03 Distribution and Toxicity
04 Perspectives
Benthic Dinos in China01
Humans are affected by CFP after eating reef fish containing the naturally occurring toxins known collectively as ciguatoxins(CTXs).
Gambierdiscus
Other toxic species of different genera often occur
associated in a benthic dinoflagellate assemblage, such as
Ostreopsis, Prorocentrum, Amphidinium, and Coolia .
Ostreopsis Prorocentrum Amphidinium Coolia
Introduction
IntroductionHabitats
A : Sandy beach, Sanya, Hainan Island. B: Seabed, Lingshui, Hainan Island. C: Sandy sediment, Sanya, Hainan Island.D: Sea grass and macroalgae, Hainan Island. E: Sand, Hainan Island. F: Coral reefs, Malaysia (BHAB workshop, 2012) G: Padina sp. and Sargassum sp.
Algal Biotoxins in China
Dolah et al., 2000; GEOHAB 2012
CFP Incidents and Number of People Affected in Hong Kong
Wong et al. Toxicon 46 (2005) 563–571
The mouse bioassay showed that ciguatoxic fish existed in all six sampling sites, and 47.8% of the samples were confirmed to be contaminated with CTXs. According to HPLC–MS/MS, no P-CTX-1 was identified in any of the samples that the mouse bioassay identified as positive.
Ni Wu, et al. 2015. Marine and Freshwater Research
Ciguatoxins in wild fish from southern China
Gambierdiscus from China
3 species has been identified:
G. caribaeusG. pacificusG. australes
Zhang & Lu, 2016. Phycol. Res
Gambierdiscus caribaeus
D: 73.88–98.36 (88.17±6.83) μm; W: 70.85–94.63 (85.15±6.59) μm; D/W: 1.01–1.13 (1.04±0.03); L: 48.11–58.01 (53.75±4.15) μm;
Species1
Zhang & Lu, 2016. Phycol. Res
scale:a–d: 10 μmscale:a–f: 10 μm
Gambierdiscus pacificus
D: 57.64–78.44 (65.46±5.59) μm; W: 54.48–71.96 (60.77±4.73) μm; D/W: 0.98–1.25(1.08±0.04); L: 38.08–50.73 (44.40±3.17) μm;
Species 2
scale:a–b: 10 μm;c: 5μm;d: 1μm
scale:a–f: 10 μm
Zhang & Lu, 2016. Phycol. Res
Gambierdiscus australes
D: 70.71–92.22 (80.58±4.51) μm; W: 63.47–92.81 (77.60±5.68) μm; D/W: 0.88–1.13 (1.04±0.04); L: 33.03–56.99 (45.21±5.24) μm;
Species 3
Zhang & Lu, 2016. Phycol. Res
scale:a–d: 10 μmscale:a–: 10 μm
Prorocentrum
7 Species hes been identified:
P. lima
P. rhathymum
P. concavum
P. fukuyoi
P. emarginatum
P. maculosum
P. panamense
The latter 5 species was first reported in the Chinese waters
Luo, Zhang and Lu et al. 2016. Algal Res.
The Toxin Profile of the Chinese Strains of Prorocentrum Species
Luo, Zhang and Lu et al. 2016. Algal Res.
Luo, Zhang and Lu et al. 2016. Algal Res.
Luo, Zhang and Lu et al. 2016. Algal Res.
Prorocentrum lima morphotype 1
L: 33.51–44.42 (37.63±2.05)
μm; W:27.29–36.45
(30.29±1.69 ) μm ;
L:W: 1.14–1.36 (1.24±0.37);
V-pores: 52–84;
M-pores: 42–77;
scale:
a, b, c: 10 μm;
d, e, g,h: 5 μm;
f: 2 μm; i: 1 μm
5 Morphotypes of Prorocentrum lima
Zhang & Lu, 2015. Phycologia
L: 31.97–39.21 (36.10±1.46)
μm;
W: 28.28–32.85 (30.23±1.11)
μm;
L:W: 1.10–1.29 (1.19±0.04);
V-pores: 45–74 ;
M-pores: 39–51;
scale:
a, b, c: 10 μm;
d, e, g, h: 5 μm;
f: 2 μm; i: 1 μm
P. lima
Prorocentrum lima morphotype 2Zhang & Lu, 2015. Phycologia
L: 36.82–39.96 (38.32 ± 0.76) μm; W: 25.60–28.85 (27.39 ± 0.74)μm; L:W: 1.33–1.45 (1.40 ± 0.03); V-pores: 50–72 ; M-pores: 48–60;
标尺:a, b, c: 10 μm; d, e: 5 μm; f: 2 μm; g: 1 μm
P. lima
Prorocentrum lima morphotype 3
Zhang & Lu, 2015. Phycologia
L: 37.50–48.05 (41.79±1.53) μm; W: 27.31–38.23
(30.28±1.45) μm;
L:W: 1.23–1.51
(1.38±0.046);
V-pores: 52–72 ;
M-pores: 54–80 ;
scale:
a, b, c: 10 μm;
d, e, g,: 5 μm;
f, h: 2 μm; i: 1 μm
P. lima
Prorocentrum lima morphotype 4
Zhang & Lu, 2015. Phycologia
L: 39.01–44.19 (41.57±1.26)
μm;
W: 30.74–34.89 (33.09±1.01)
μm;
L:W: 1.25–1.33 (1.25±0.02);
V-pores: 71–94 ;
M-pores: 49–69;
scale:
a, b, c: 10 μm;
d, e, g,: 5 μm;
f, h, i: 1 μm
P. lima
Prorocentrum lima morphotype 5Zhang & Lu, 2015. Phycologia
P. lima LSU (D1-D3) rDNA P. lima ITS
Phylogenetic analyses of P. lima and other Prorocentrum species
Zhang & Lu, 2015. Phycologia
48 sampling sitesOstreopsis isolated from 19 sites130 monoclonal strains established
Dalian
QingdaoChangdao, Penglai
ShenzhenWeizhou IslandHainan IslandParacel Islands
Ostreopsis along Chinese seas
Morphology of Ostreopsis in China
Morphology of Ostreopsis in China
Phylogenetic analyses
Maximum likelihood phylogenetic tree of the genus Ostreopsis inferred from LSU ribosomal gene
sequences
O. cf. siamensisMediterranean/Atlantic
O. cf. ovataPacificMediterraneanAtlantic
Ostreopsis sp.5Pacific
Ostreopsis sp.Pacific/AtlanticO. cf. lenticularis Pacific
Maximum likelihood phylogenetictree of the genus Ostreopsis inferred from ITS-5.8S ribosomal gene sequences
Phylogenetic analyses
O. cf. ovataPacificAtlanticMediterranean
Ostreopsis sp.Pacific
O. cf. fattorussoiAtlantic/Mediterranean
O. cf. siamensis
PLTXs extraction
130 monoclonal strains established
Mouse bioassay
Hemolysis neutralization assay
E: erythrocyteS: sampleO: ouabain
Time (minutes)0 60 120 180 240
UV
abs
orba
nce
(abs
)
0.0
.2
.4
.6
.8
1.0E+PBSE+SE+S+OE+O
LCMS
LCHRMS
m/z =1315.7162
0 2 4 6 8 10 12 14 Time [min]0
100
200
300
400
Intens.
T1 -20160802_BC2_01_279.d: EIC 1315.7000±0.1 +All MS
Strain PLTXs amount(pg/cell)
WZD G2 0.369
WZD G6 0.195
WZD 111 0.200
XPD28 2.679
ZZD2 0.154
ZZD13 0.249
ZZD17 0.247
ZZD44 0.203
PLG7 0.156
PLG39 0.418
PLG7OVTX-a(m/z 1315.7162)OVTX-g(m/z 1307.7552)
WZD111OVTX-c (m/z 1345.7319) OVTX-d/e (m/z 1323.7461) OVTX-g OVTX-f (m/z 1329.7094)
Taxonomy and Diversity02
Gambierdiscus
Gambierdiscus species(Litaker et al., 2009; Fraga et al., 2011; Fraga & Rodríguez, 2014; Nishimura et al., 2014; Fraga et al., 2016; Smith et al., 2016)
1. Totally 11 species has been described.
2. G. yasumotoi and G. ruetzleri has been moved to the genus Fukuyoa(Gómez et al., 2015).3. Two new species, G. balechii Fraga, Rodríguez et Bravo (Fraga et al., 2016) and G. cheloniae Smith, Rhodes & Murray (Smith et al., 2016) have been described in 2016.
Drawings of the species in right thecal view showing cell shape, periflagellar area shape, part of the ornamentation, and the pore pattern. (by Hoppenrath et al., 2013)
Prorocentrum
At least nine benthic species of the genus Prorocentrumhave been shown to produce the toxins okadaic acid (OA) and its analogues (Hoppenrathet al., 2013).
Ostreopsis (10 species)
Line drawings of the 10 described Ostreopsis
species in epithecal (upper) and hypothecal
(lower) view. (a) O. siamensis after Schmidt
(1901); (b-d) O. siamensis, O. lenticularis and
O. ovata, respectively, after Steidinger and
Tangen (1996); (e) O. mascarenensis, after
Quod (1994); (f) O. heptagona, after Norris et
al. (1985); (g) O. labens, redrawn from Faust
and Morton (1985); (h-j) O. belizeana, O.
marina, and O. caribbeanus, respectively,
after Faust (1999). From Penna et al. (2005)
(GEOHAB 2012). (k) O. fattorussoi Accoroni
et al., 2016
Coolia (7 species)
Coolia species
(Meunier 1919; Faust 1995; Ten-Hage et al. 2000; Fraga et al. 2008; Leaw et al., 2010; Karafas et al., 2015)
Distribution and Toxicity03
A) found only in the Atlantic, B)
found only in the Pacific or C)
found in both the Atlantic and
Pacific. Solid or dashed line(s)
are used to associate specific
species with the location
(indicated by the filled circles)
where they were collected.
Distribution of Gambierdiscus species
Litaker et al., 2010
Geographical distribution of P. lima
(Nagahama et al., 2011; Zhang et al., 2015; Luo et al., 2016)
Redrawn and updated from Rhodes (2010) by A. Zingone.(GEOHAB 2012)
Geographical distribution of the genus Ostreopsis
Distribution of Gambierdiscus in WEST PacificSpecies Location in WESTPAC References
G. toxicus Japan, Vietnam Fukuyo, 1981; Faust, 1996; Roeder et al. 2010.
G. belizeanus Japan, Malayisa Faust, 1996; Leaw et al., 2011.
G. pacificus Malaysia, China Mohammad-Noor et al., 2007; Zhang et al., 2016.
G. australes Japan, China Zhang et al., 2016; Kibler et al., 2012.
G. polynesiensis - ‐
G. caribaeus Korea, Thailand, China Zhang et al., 2016; Jeong et al., 2012; Tawong et al., 2016;
Tawong et al., 2015.
G. carolinianus - -
G. carpenteri Guam, the Philipines Litaker et al., 2010, Azanza, 2016.
G. excentricus - -
G. scabrosus Japan Nishimura et al., 2014
G. silvae - -
G. balechii - -
G. cheloniae - -
Distribution of toxic Prorocentrum species in WEST Pacific
Species Location in WESTPAC References
P. lima Japan, Malaysia, China, Russia Fukuyo, 1981; Faust, 1996; Ono,1999; Grzebyk et al., 1998; Mohammad-Noor et al., 2007; Konovalova & Selina, 2010; Selina & Levchenko, 2011; Zhang et al., 2015; Luo et al., 2016
P. belizeanum - -P. borbonicum - -P. concavum Japan, Malaysia, China Fukuyo, 1981; Mohammad-Noor et al., 2007; Luo et al.,
2016
P. faustiae Malaysia Mohammad-Noor et al., 2007
P. hoffmannianum - -
P. levis - -
P. “maculosum” China Luo et al., 2016
P. rhathymum Japan, Malaysia, China Fukuyo, 1981; Mohammad-Noor et al., 2007; Luo et al., 2016
Distribution of Ostreopsis in WEST PacificSpecies Location in WESTPAC References
O. siamensis Japan, Thailand, Russia,Vietnam
Schmidt, 1901; Fukuyo, 1981; Taniyama et al., 2003;
Sagara, 2008; Adachi et al., 2008; Sato et al., 2011; Chu Van 2002; Selina & Orlova 2010.
O. lenticularis Japan, Vietnam, Philippines, Indonesia; Malaysia, China
Faust, 1996; Chu Van 2002; Leaw et al., 2001;
Sidabutar et al., 2000; Pacsidio & Dimaano 2004;
Mohammad-Noor et al., 2007; Zhang et al., unpubl.
O. ovata Japan, Vietnam, Indonesia;Thailand, Korea, Singapore, Malaysia, Russia, China
Fukuyo, 1981; Faust, 1996; Holmes et al., 1998; Sidabutar et al. 2000; Leaw et al., 2001;
Mohammad-Noor et al., 2007; Adachi et al., 2008; Sato et al., 2011; Selina et al., 2014;
Tawong et al., 2014; Kang et al., 2013; Chu Van 2002; Zhang et al., unpubl.
O. heptagona - -
O. mascaranensis - -
O. labens Japan, Malaysia Faust & Morton, 1995; Faust, 1996; Mohammad-Noor et al., 2007
O. marina Vietnam Larsen & Nguyen-Ngoc 2004
O. belizeana - -
O. caribbeanus - -
O. fattorussoi -- -
Brissard et al.2015
GEOHAB, 2012
Ostreopsis genus distribution (based on Rhodes, 2010 and updated to March 2015)
Cioccio, 2015
Summary of the toxins identified in Ostreopsis spp.
Cioccio, 2015
Maximum likelihood phylogeny of Ostreopsis inferred from ITS sequence
Sato et al. 2011
phylogenetic tree based on LSU rDNA D8/D10 sequences of various Ostreopsis strains
Tawong et al. 2014
Phylogeny of Ostreopsisbased on LSU D1/D2 rDNAsequences
Genetic Diversity
Efimova et al. 2014
O. siamensisO. ovata
Cells X 105
I: Totti et al, HA 2010B: Graneli & Vidyarathna, HA 2010H: Parsons & Preskitt, HA 2007S: Vila et al, AME 2001J: Adachi et al, 2010, 14th HAB ConfNZ: Shears & Ross, HA 2009
Ostreopsis cells / gr FW seaweeds
0
2
4
6
8
24 25 26 27 28 29 30
Cel
ls.
103
. ml -1
Temperature (°C)
Maximum biomass
Japanese
Mediterranean
Graneli, per commin.
Toxicity
Temperature (°C)
Mediterranean
Japanese1
6
4
3
2
024 25 26 27 28 29 30
ngSE
. cel
l-1
5
Graneli, per commin.
0
1000
2000
3000
4000
5000
6000
7000
8000
Brazilian Mediterranean Japanese
Nr of O. ovata cells causing 100% hemolysisat 24 °C
Low toxic
High toxic
100 cells
Graneli, per commin.
Temperature (°C)
Carbohydrate production in O. ovata
Declining phase
Glu
cose
eq
(ng
/cel
l)
16
12
8
4
020 24 25 26 27 28 29 30
Mediterranean
Japanese
Graneli, per commin.
Distribution of Coolia in WEST Pacific
Species Location in WESTPAC References
C. monotis Japan, China, Vietnam Fukuyo, 1981; Liang et al. 2009; Ho & Nguyen, 2014
C. tropicalis Japan, Malaysia, Vietnam, Thailand, Indonesia, China
Faust, 1996; Mohammad-Noor et al., 2013; Ho & Nguyen, 2014; Tawong et al., 2015; Zhang et al., unpubl.
C. areolata - -
C. canariensis Korea, Vietnam, China Jeong et al., 2012; Ho & Nguyen, 2014; Zhang et al., unpubl.
C. malayensis Malaysia, Japan, Thailand, Korea,China
Leaw et al., 2010; Jeong et al., 2012; Mohammad-Nooret al., 2013; Tawong et al., 2015; Wakeman et al., 2015; Leaw et al., 2016; Zhang et al., unpubl.
C. santacroce - -
C. palmyrensis - -
Species/Strain Host/Origin References
A. gibbosum (S1-36-5) Free-swimming, US Virgin Islands Bauer et al. 1995
Amphidinium sp. HYA024 Benthic, Japan Tsuda et al. 2007; Kobayashi 2008; Oguchi et al. 2008.
A. carterae Bahamas Meng et al. 2010
A. carterae CAWD57 Benthic, New Zealand Echigoya et al. 2005
A. klebsii NIES613 Surface of seaweed, Japan Morsy et al. 2005, 2006; Paul et al. 1995, 1997; Satake et al. 1991
Amphidinium sp. China Huang et al. 2004
A. carterae Surface of seaweed, Taiwan Huang et al. 2009
A. carterae CAWD152 Surface of seaweed Halimeda sp., in Cook Islands Rhodes et al. 2010
Known toxic species of Amphidinium (Hoppenrath et al. 2014)
1.Further biodiversity and taxonomy study of Benthic dinoflagellates in the WESTPAC region.
2.Distribution map of toxic species in the WESTPAC region.
3.Factors regulating the toxin production