Toxic Benthic Dinoflagellatesin the West Pacific Regionsfile.iocwestpac.org/HABs/19-22 Dec 2016... · Toxic Benthic Dinoflagellatesin the West Pacific Regions LU Songhui Research

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


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


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


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




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



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



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


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


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


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


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

Toxic Benthic Dinoflagellatesin the West Pacific Regionsfile.iocwestpac.org/HABs/19-22 Dec 2016... · Toxic Benthic Dinoflagellatesin the West Pacific Regions LU Songhui Research

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