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

WESTPAC HAB Workshop 2016, Nha Trong Toxic Benthic Dinoflagellates in the West Pacific Regions LU Songhui Research Center for Harmful Algae and Marine Biology, Jinan University, Guangzhou, China

01 Benthic Dinos in China 02 Taxonomy and Diversity Outline 03 Distribution and Toxicity 04 Perspectives

01 Benthic Dinos in China

Introduction Humans are affected by CFP after eating reef fish containing the naturally occurring toxins known collectively as ciguatoxins(CTXs). Other toxic species of different genera often occur associated in a benthic dinoflagellate assemblage, such as Ostreopsis , Prorocentrum , Amphidinium , and Coolia . Gambierdiscus Ostreopsis Prorocentrum Amphidinium Coolia

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

Ciguatoxins in wild fish from southern China 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

Gambierdiscus from China 3 species has been identified : G. caribaeus G. pacificus G. australes Zhang & Lu, 2016. Phycol. Res

Species1 Gambierdiscus caribaeus scale ： a – d: 10 μm scale ： a – f: 10 μm 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; Zhang & Lu, 2016. Phycol. Res

Species 2 Gambierdiscus pacificus scale ： a – b: 10 μm ； c: 5μm ； d: 1μm scale ： a – f: 10 μm 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; Zhang & Lu, 2016. Phycol. Res

Species 3 Gambierdiscus australes scale ： a – : 10 μm scale ： a – d: 10 μm 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; Zhang & Lu, 2016. Phycol. Res

Prorocentrum 7 Species hes been identified: P . lima P . rhathymum P. concavum P. fukuyoi P. emarginatum The latter 5 species was first P. maculosum reported in the Chinese waters P. panamense 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.

5 Morphotypes of Prorocentrum lima 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 Prorocentrum lima morphotype 1 Zhang & Lu, 2015. Phycologia

P. lima 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 Prorocentrum lima morphotype 2 Zhang & Lu, 2015. Phycologia

P. lima Prorocentrum lima morphotype 3 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 Zhang & Lu, 2015. Phycologia

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 Prorocentrum lima morphotype 4 Zhang & Lu, 2015. Phycologia

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 Prorocentrum lima morphotype 5 Zhang & Lu, 2015. Phycologia

Phylogenetic analyses of P . lima and other Prorocentrum species P. lima LSU (D1-D3) rDNA P. lima ITS Zhang & Lu, 2015. Phycologia

Ostreopsis along Chinese seas 48 sampling sites Ostreopsis isolated from 19 sites 130 monoclonal strains established Dalian Changdao, Penglai Qingdao Shenzhen Weizhou Island Hainan Island Paracel Islands

Morphology of Ostreopsis in China

Morphology of Ostreopsis in China

O. cf. siamensis Mediterranean/Atlantic Phylogenetic analyses O. cf. ovata Pacific MediterraneanAtlanti c Maximum likelihood phylogenetic tree of the genus Ostreopsis inferred from LSU ribosomal gene sequences Ostreopsis sp. Pacific/Atlantic O. cf. lenticularis Pacific Ostreopsis sp.5 Pacific

O. cf. ovata Pacific Atlantic Phylogenetic analyses Mediterranean Maximum likelihood phylogenetic tree of the genus Ostreopsis inferred from ITS-5.8S ribosomal gene sequences O. cf. siamensis O. cf. fattorussoi Atlantic/Mediterranean Ostreopsis sp. Pacific

PLTXs extraction 130 monoclonal strains established

Mouse bioassay

Hemolysis neutralization assay E: erythrocyte 1.0 S: sample E+PBS O: ouabain E+S E+S+O .8 E+O absorbance (abs) .6 .4 V U .2 0.0 0 60 120 180 240 Time (minutes)

LCMS

LCHRMS Intens. 400 300 200 100 0 0 2 4 6 8 10 12 14 Time [min] T1 -20160802_BC2_01_279.d: EIC 1315.7000±0.1 +All MS m/z =1315.7162

Strain PLTXs amount （ pg/cell ） WZD G2 0.369 PLG7 OVTX-a （ m/z 1315.7162 ） WZD G6 0.195 OVTX-g （ m/z 1307.7552 ） WZD 111 0.200 WZD111 OVTX-c (m/z 1345.7319) XPD28 2.679 OVTX-d/e (m/z 1323.7461) OVTX-g ZZD2 0.154 OVTX-f (m/z 1329.7094) ZZD13 0.249 ZZD17 0.247 ZZD44 0.203 PLG7 0.156 PLG39 0.418

02 Taxonomy and Diversity

Gambierdiscus 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. 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)

Prorocentrum At least nine benthic species of the genus Prorocentrum have been shown to produce the toxins okadaic acid (OA) and its analogues ( Hoppenrath et al. , 2013). 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)

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)

03 Distribution and Toxicity

Distribution of Gambierdiscus species 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. Litaker et al., 2010

Geographical distribution of P. lima (Nagahama et al. , 2011; Zhang et al ., 2015; Luo et al ., 2016)

Geographical distribution of the genus Ostreopsis Redrawn and updated from Rhodes (2010) by A. Zingone.(GEOHAB 2012)

Distribution of Gambierdiscus in WEST Pacific Species Location in WESTPAC References Japan, Vietnam Fukuyo, 1981; Faust, 1996; Roeder et al. 2010. G. toxicus Japan, Malayisa Faust, 1996; Leaw et al ., 2011. G. belizeanus Malaysia, China Mohammad-Noor et al., 2007; Zhang et al ., 2016. G. pacificus Japan, China Zhang et al. , 2016; Kibler et al. , 2012. G. australes - G. polynesiensis ‐ Korea, Thailand, China G. caribaeus Zhang et al. , 2016; Jeong et al. , 2012; Tawong et al. , 2016; Tawong et al. , 2015. - - G. carolinianus Guam, the Philipines Litaker et al. , 2010, Azanza, 2016. G. carpenteri - - G. excentricus Japan Nishimura et al. , 2014 G. scabrosus - - G. silvae - - G. balechii - - G. cheloniae