Seafood Toxins - American Chemical Society

21

Ciguatoxigenic Dinoflagellates

from

the C a r i b b e a n S e a

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 28, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch021

DONALD R. TINDALL, ROBERT W. DICKEY, ROLLAND D. CARLSON, and GREGORY MOREY-GAINES Department of Botany, Southern Illinois University, Carbondale, IL 62901 Over 70 separate sites in the British and United States Virgin Islands were surveyed for potential ciguatoxigenic organisms. A culture collection of 65 strains representing 18 species of the most common epiphytic/benthic and planktonic dinoflagellates has been established. To date, nine species have been grown in large-scale culture and screened for toxicity using mice. Five species, namely Gambierdiscus toxicus, Prorocentrum concavum, P. mexicanum, Gymnodinium sanguineum, and Gonyaulax polyedra, produced one or more toxic fractions which killed mice within 48 hours. Results from mouse bioassays and chromatographic treatments of extracts indicate that G. toxicus produces ciguatoxin (and 1 derivative) and maitotoxin; and P. concavum produces a scaritoxin -like toxin, a maitotoxin-like toxin, and at least one (up to 2) very potent, unnamed, fast-acting toxins. A l l of these toxins probably contribute to the ciguatera syndrome in the Caribbean. Ciguatera i s a serious human i n t o x i c a t i o n that results from eating certain t r o p i c a l and subtropical fishes associated with coral reefs and adjacent coastal waters. The disease i s manifested i n humans by a great variety of symptoms (1-4). Those most consistently reported may be generally summarized as follows: (A) moderate to severe gastrointestinal disorders of r e l a t i v e l y short duration; (B) moderate to severe neurological disorders that may persist for days, weeks, or months; and/or (C) i n some cases, death due to respiratory failure. Ciguatera may be caused by over 400 species of marine fishes, including many that are highly prized for food (1_). Banner (_5) provided convincing argument supporting the view that ciguatoxin as defined by Scheuer and coworkers (6) was the p r i n c i p a l toxin causing ciguatera throughout the world. However, a few authors have suggested that the variety of symptoms displayed and the inconsistent response to certain c l i n i c a l treatments by patients 0097-6156/84/0262-0225$06.00/0 © 1984 American Chemical Society

Ragelis; Seafood Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

SEAFOOD TOXINS


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suffering from ciguatera result from there being more than one primary toxin causing the disease (_3» 7). The occurrence of "secondary" toxins (maitotoxin and/or s c a r i t o x i n ) i n association with ciguatoxin i n fishes has been reported (8-14). Randall (15), following an intensive study of the food habits of ciguatoxic fishes, concluded that a l l such fishes were t i e d to the coral reef through the food chain. Furthermore, he speculated that the organism responsible for the production of the toxin was a fine benthic alga or other microorganism. Evidence gathered during the past few years points to certain dinof lagellates that grow i n close association with macroalgae and/or other bottom structures as progenitors of ciguatera toxins. The recently discovered species, Gambierdiscus toxicus (16) has been reported to produce both ciguatoxin and maitotoxin i n the P a c i f i c (17-22). Other species inhabiting the same microhabitats i n the P a c i f i c have been found to produce toxins which also may contribute to the ciguatera syndrome. These include Amphidinium carter!, A. k l e b s i i , Ostreopsis ovata, 0_. siamensis, Prorocentrum concavum (20) and JP. lima (20, 23, 24). Our study of ciguatoxigenic organisms i n the Caribbean was i n i t i a t e d i n May 1978. The primary objectives of this study were: (A) to conduct an extensive search for potentially toxic organisms i n areas of the B r i t i s h and United States V i r g i n Islands known to support toxic fishes and to select s i t e s as permanent stations for subsequent ecological study; (B) to i d e n t i f y , i s o l a t e , and i n i t i a t e cultures of p o t e n t i a l l y toxic microorganisms (with emphasis on species of d i n o f l a g e l l a t e s ) ; (C) to develop large-scale cultures of each species for screening for t o x i c i t y ; (D) to develop procedures for extraction and p u r i f i c a t i o n of toxins from microorganisms; (E) to determine appropriate nerve-muscle preparations for assaying toxins and for determining their s i t e s and modes of action; and (F) to examine d i s t r i b u t i o n , p e r i o d i c i t y , growth, and development of toxic organisms i n the f i e l d and under controlled laboratory conditions. Preliminary results on t o x i c i t y of 3 species of Caribbean dinoflagellates have been reported, namely those on Gambierdiscus toxicus (25), Prorocentrum concavum (as jP. c f . lima) (26), and P_. mexicanum (as ]?_• rhathymum) (27). A more detailed description of the effects of ether-soluble and water-soluble toxins from the same s t r a i n of G_. toxicus on guinea pig i l e a are presented elsewhere i n this volume (28, 29). Results on the growth and development of G. toxicus, P. concavum, and P. mexicanum i n culture also are presented (30). The present paper reports on the development of a culture c o l l e c t i o n of p o t e n t i a l l y toxic dinoflagellates from the V i r g i n Islands, large-scale culture and screening for t o x i c i t y i n nine species, and a more detailed consideration of the toxic properties of Gm toxicus and ?• concavum. Results and Discussion Study area. Over 70 separate s i t e s i n the V i r g i n Islands were surveyed (Figure 1). These s i t e s represented a wide variety of habitat types, namely l i v e and dead coral reefs, open sand bottoms landward and seaward of reefs, protected coves and bays, areas variously exposed to surf, mangrove stands, and salt ponds.



T I N D A L L ET A L .

Ciguatoxigenic

Dinoflagellates

N E C K E R I\

227

*

6

F i g u r e 1. The study area i n the B r i t i s h and U n i t e d S t a t e s V i r g i n I s l a n d s . Dots i n d i c a t e s i t e s surveyed p r i o r t o e s t a b l i s h i n g permanent c o l l e c t i n g s t a t i o n s . Seven permanent s t a t i o n s are i n d i c a t e d by number.



228

SEAFOOD TOXINS

Substrate types characterizing these habitats included surfaces of dead coral, f i n e and coarse sands, organically r i c h mud-sand mixtures, wood and s t e e l surfaces on wrecked ships, a l g a l mats, attached macroalgae, and seagrass beds. Water depths at the various sites ranged from 0.2 to 25 meters. Seven permanent c o l l e c t i n g stations were established (Figure 1): (I) Water Creek i n Hurricane Hole, St. John; (II) Salt Creek, Salt Island; (III) South Creek i n South Sound, V i r g i n Gorda; (IV) Mattie Point i n South Sound, V i r g i n Gorda; (V) Biras Creek i n North Sound, V i r g i n Gorda; (VI) Northwest Beach, Necker Island; and (VII) West End, Anegada. These stations were selected because of their degree of exposure to open seas, unique bottom topography, history of supporting toxic fishes, and/or abundance of d i n o f l a g e l l a t e s and other algae. C o l l e c t i o n and culture of d i n o f l a g e l l a t e s . Large numbers of a l l conspicuous epiphytic, benthic, and planktonic species of dinoflagellates were collected at each station using p l a s t i c bags, syringes, and Van Dorn bottle, respectively. Each c o l l e c t i o n was transported to the f i e l d laboratory i n separate polypropylene bottles. At least 30 c e l l s of each species were i s o l a t e d with micropipetts and inoculated into 10 ml of each of 10 d i f f e r e n t growth media, namely GPM (31), F (32), ES (33), AG (Carolina B i o l o g i c a l Supply Co.), WC (34), ASP and ASP-6 (35), ASP-7 and ASP12 (36), and NS (31). The r e s u l t i n g cultures were maintained i n 30 ml tubes on Plexiglass l i g h t tables which were continuously illuminated from below with four 25 watt cool-white fluorescent lamps. Ambient temperature usually was about 29°C. Over 300 of these crude f i e l d cultures were successfully transported to Southern I l l i n o i s University, Carbondale. After a 10 to 20 day period of acclimation to laboratory conditions, i n d i v i d u a l c e l l s were i s o l a t e d from f i e l d cultures supporting best growth. Careful examination of the f i e l d cultures revealed that best growth of a l l species occurred i n ES enriched seawater. This medium with a 1.5% s o i l extract added was selected as our standard medium for stock cultures. C e l l s were washed several times by s e r i a l transfers through drops of s t e r i l e medium i n Pyrex spot plates. After washing, 30 or more c e l l s of each species were placed i n tubes containing 10 ml of fresh, s t e r i l e medium. Ten ug/ml of germanium dioxide was added to some cultures to eliminate diatom growth. Once unialgal cultures were obtained, they were transferred to 50 ml volumes of medium i n 125 ml f l a s k s . These cultures were used as maintenance stocks and formed the basis of the Southern I l l i n o i s University (SIU) culture c o l l e c t i o n of Caribbean dinoflagellates. To date, we have i s o l a t e d and cultured 65 strains representing 18 of the most common epiphytic/benthic and planktonic species (Table I ) . Several cultures of Gambierdiscus toxicus were i n i t i a t e d by inoculating 5 ml of medium with single c e l l s from various locations. Cultures obtained i n this manner are l i s t e d i n Table I as c l o n a l cultures. A l l cultures were grown at 27°C i n 3200 lux constant cool-white fluorescent illumination and were transferred weekly, using 10% volume inocula. Large-scale culture and harvest of d i n o f l a g e l l a t e s . Large-scale cultures were i n i t i a t e d by f i r s t inoculating 2 l i t e r s of our


21.

TINDALLETAL.

Ciguatoxigenic

229

Dinoflagellates


Table I. Southern I l l i n o i s University (SIU) Culture C o l l e c t i o n of Dinoflagellates From the V i r g i n Islands. A l l Stock Cultures are Maintained i n ES Enriched Natural Seawater Containing 1.5% S o i l Extract. The Letter c Next to Strain Number Denotes Clonal Culture. Species

SIU Strain Number

Origin

Amphidinium elegans

547

Ceratium furca

682

Cochlodinium polykrikoides Coolia monotis C. monotis E n s i c u l i f e r a sp. nov. Gambierdiscus toxicus

283,489 263 390 415 711, 756c, 763c , 772c 350, 453, 467, 566, 570 661, 774c, 775c , 777c 509 619, 623, 740c, 741c , 742c, 744c 747c 783 403 780, 781 278 373, 374, 497 499 437 533 376, 379, 473 474 665 702, 705 364 662 700 722 228, 262, 273, 276 86, 404, 724 344 556, 557, 558, 582, 587, 685

South Sound, V i r g i n Gorda (VG) Hurricane Hole, St. John (SJ) South Sound, VG Hurricane Hole, SJ South Sound, VG South Sound, VG Hurricane Hole, SJ

G. toxicus G. toxicus G. toxicus G. toxicus

Gonyaulax diacantha Gonyaulax g r i n d l e y i G. g r i n d l e y i Gonyaulax polyedra Gymnodinium sanguineum G. sanguineum G. sanguineum Gyrodinium fissum Gyrodinium sp. Ostreopsis sp. Prorocentrum concavum P. concavum Prorocentrum lima Prorocentrum mexicanum P. mexicanum S c r i p p s i e l l a subsalsa S. subsalsa S c r i p p s i e l l a trochoidea

South Sound, VG Biras Creek, VG West End, Anegada St. Thomas Lagoon

Hurricane Hole, SJ Hurricane Hole, SJ Drake s Channe1 Hurricane Hole, SJ South Sound, VG 1

Biras Creek, VG Hurricane Hole, SJ South Sound, VG Biras Creek, VG St. Thomas Lagoon Salt Island Biras Creek, VG South Sound, VG Hurricane Hole, VG Salt Island Hurricane Hole, SJ Biras Creek, VG South Sound, VG


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standard medium with c e l l s from dense stock cultures. After approximately 20 ddys of growth, the 2 - l i t e r cultures were each used to inoculate 16 to 18 l i t e r s of medium i n 20 l i t e r carboys. These cultures were aerated to prevent CO2 depletion and to provide gentle aggitation. Light and temperature were maintained at 4300 lux and 27°C, respectively. In some cases, 1 or 2 l i t e r s of the large cultures were used to inoculate new large-scale cultures. Cultures were harvested by continuous flow centrifugation. The resulting moist c e l l p e l l e t s were removed from the centrifuge chamber and weighed. These products were recorded as y i e l d i n grams fresh weight (g f.w.). Nine species have been growth i n large-scale culture. Total volume of cultures and y i e l d for each species are included i n Table I I . Growth rates of selected species i n largescale culture are reported elsewhere i n this volume (30). Table I I . Production of Cells and Crude Extracts of Nine Species of Dinoflagellates i n Large-Scale Culture. Ages of Cultures Ranged From 20 to 36 Days. Species (SIU Strain Number) Gambierdiscus toxicus (350) Prorocentrum concavum (364) Prorocentrum mexicanum (276) Coolia monotis (263) Amphidinium elegans (547) Scrippsiella subsalsa (344) Gonyaulax g r i n d l e y i (403) Gonyaulax polyedra (278) Gymnodinium sanguineum (373)

Culture Volume (1)

Yield (g f.w. )

ESAF

Crude Extracts WSAF ESAP (mg)

WSAP

712.4

192.6

1214.3

1129.5

308.9

629.6

581.0

191.1

1418.0

481.6

734.7

265.9

466.5

29.5

210.6

141.7

69.9

171.2

32.0

10.3

87.7

81.5

22.7

0.0

32.0

5.6

66.3

35.6

17.7

1.8

72.0

46.5

123.0

0.0

33.7

0.0

54.0

11.7

72.3

4.8

15.8

0.4

16.0

4.7

27.2

1.6

4.6

0.1

16.0

3.2

33.6

1.2

12.1

0.8

Extraction and p a r t i a l p u r i f i c a t i o n . The c e l l pellets derived from large-scale cultures were subjected to our standard extraction and p a r t i t i o n i n g procedure for i s o l a t i n g ciguatera toxins (Figure 2). The f i n a l cold acetone treatment of d i e t h y l ether and water soluble fractions usually resulted i n four products: (A) ether soluble acetone f i l t r a t e (ESAF), (B) ether soluble acetone precipitate (ESAP), (C) water soluble acetone f i l t r a t e (WSAF), and (D) water soluble acetone precipitate (WSAP). These acetone soluble and i n soluble products were not p u r i f i e d further for this study; however, chromatographic procedures which we have u t i l i z e d for such p u r i f i c a tion are presented i n this volume (28, 29). The amounts of these fractions obtained from large-scale cultures are shown i n Table I I .


21.

TINDALLETAL.

Ciguatoxigenic

231

Dinoflagellates

L A R G E SCALE CULTURE

I Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 28, 2018 | https://pubs.acs.org Publication Date: September 19, 1984 | doi: 10.1021/bk-1984-0262.ch021

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Seafood Toxins - American Chemical Society

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