Toxic Benthic Dinoflagellates in the West Pacific Regions LU - - PowerPoint PPT Presentation

Toxic Benthic Dinoflagellates in the West Pacific Regions

LU Songhui

Research 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 China

01

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

Introduction

Habitats

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. caribaeus
• G. pacificus
• G. 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 μm scale：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 μm scale：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 2 Zhang & 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 5 Zhang & 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 sites Ostreopsis isolated from 19 sites 130 monoclonal strains established

Dalian Qingdao Changdao, Penglai Shenzhen Weizhou Island Hainan Island Paracel 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. siamensis

Mediterranean/Atlantic

• O. cf. ovata

Pacific MediterraneanAtlanti c Ostreopsis sp.5 Pacific Ostreopsis sp. Pacific/Atlantic

• O. cf. lenticularis Pacific

Maximum likelihood phylogenetic tree of the genus Ostreopsis inferred from ITS-5.8S ribosomal gene sequences

Phylogenetic analyses

• O. cf. ovata

Pacific Atlantic Mediterranean Ostreopsis sp. Pacific

• O. cf. fattorussoi

Atlantic/Mediterranean

• O. cf. siamensis

PLTXs extraction

130 monoclonal strains established

Mouse bioassay

Hemolysis neutralization assay

E: erythrocyte S: sample O: ouabain

Time (minutes)

60 120 180 240

U V absorbance (abs)

0.0 .2 .4 .6 .8 1.0

E+PBS E+S E+S+O E+O

LCMS

LCHRMS

m/z =1315.7162

2 4 6 8 10 12 14 Time [min] 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 PLG7 OVTX-a（m/z 1315.7162） OVTX-g（m/z 1307.7552） WZD111 OVTX-c (m/z 1345.7319) OVTX-d/e (m/z 1323.7461) OVTX-g OVTX-f (m/z 1329.7094)

Taxonomy and Diversity

02

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

• f the genus Prorocentrum

have been shown to produce the toxins okadaic acid (OA) and its analogues (Hoppenrath et 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 Toxicity

03

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 Pacific

Species 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 Pacific

Species 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 Ostreopsis based on LSU D1/D2 rDNA sequences

Genetic Diversity

Efimova et al. 2014

• O. siamensis
• O. ovata

Cells X 105

I: Totti et al, HA 2010 B: Graneli & Vidyarathna, HA 2010 H: Parsons & Preskitt, HA 2007 S: Vila et al, AME 2001 J: Adachi et al, 2010, 14th HAB Conf NZ: Shears & Ross, HA 2009

Ostreopsis cells / gr FW seaweeds

2 4 6 8 24 25 26 27 28 29 30

Cells . 103 . ml -1 Temperature (°C)

Maximum biomass

Japanese Mediterranean

Graneli, per commin.

Toxicity

Temperature (°C)

Mediterranean Japanese

1 6 4 3 2

24 25 26 27 28 29 30

ng SE . cell-1

5 Graneli, per commin.

1000 2000 3000 4000 5000 6000 7000 8000

Brazilian Mediterranean Japanese

Nr of O. ovata cells causing 100% hemolysis at 24 °C

Low toxic High toxic 100 cells

Graneli, per commin.

Temperature (°C)

Carbohydrate production in O. ovata

Declining phase Glucose eq (ng /cell)

16 12 8 4

20 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-Noor et 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