Production of goniodomin A by the planktonic, chain-forming

dinoflagellate Alexandrium monilatum (Howell)

Balech isolated from the Gulf Coast

of the United States

Michelle H. Hsia a,b, Steve L. Morton c, Laurinda L. Smith c,Kevin R. Beauchesne b, Kevin M. Huncik b, Peter D.R. Moeller b,*

a Marine Biomedicine and Environmental Sciences, Medical University of South Carolina,

Charleston, SC 29425, USAb Toxin Chemistry, National Ocean Service, Hollings Marine Laboratory,

331 Fort Johnson Road, Charleston, SC 29412, USAc Marine Biotoxins Program, National Ocean Service, Hollings Marine Laboratory,

331 Fort Johnson Road, Charleston, SC 29412, USA

Received 8 July 2005; received in revised form 14 August 2005; accepted 22 August 2005

Abstract

The chain-forming dinoflagellate Alexandrium monilatum has been reported to be associated with widespread discolored water

and increased fish mortality in the Mississippi Sound and off the eastern and western coasts of Florida. Previous studies over the last

60–70 years have determined that A. monilatum produces a harmful substance(s) that is predominantly contained in the cell mass as

exhibited by apparent increased toxicity when the organism cytolyses. The current research in our lab corroborated earlier research

demonstrating that A. monilatum produces a lipophilic toxin, unlike its Alexandrium relatives noted for their production of

saxitoxin-like toxins. Using sophisticated chemical, chromatographic, and analytical techniques, we have successfully purified and

identified the molecular structure of the toxin produced by A. monilatum. We utilized a 500 MHz NMR to carry out a number of

experiments (i.e., 1H, 13C, COSY, HSQC, and HMBC) to unambiguously determine the molecular structure of the toxin. In addition,

we report mass analysis of the toxin utilizing electrospray ionization-mass spectrometry (ESI-MS), matrix-assisted laser desorption

ionization-time of flight-mass spectrometry (MALDI-TOF-MS), and Q-TOF mass spectral techniques. The toxin is representative

of a polyether macrolide with an empirical formula of C43H60O12. This toxic compound is shown to be identical to a Japanese

tidepool toxin identified as goniodomin A, which is produced by another Alexandrium species.

Published by Elsevier B.V.

Keywords: Alexandrium spp.; Dinoflagellate; Goniodomin A; Icthyotoxic; Macrolide toxin; Red-tide

www.elsevier.com/locate/hal

Harmful Algae 5 (2006) 290–299

* Corresponding author. Tel.: +1 843 762 8867;

fax: +1 843 762 8737.

E-mail address: Peter.Moeller@noaa.gov (Peter D.R. Moeller).

1568-9883/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.hal.2005.08.004

1. Introduction

The appearance of red, luminescent water and the

mass mortality of fishes in the Offats Bayou near

Galveston, TX, reported by Connell and Cross (1950)

was found to be due to large numbers of the marine

dinoflagellate, Alexandrium spp., formerly known as

Gonylaux spp. (Balech, 1995). Local fisherman of that

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299 291

Fig. 1. Scanning electron micrograph (SEM) of Alexandrium mon-

ilatum cells in chain formation. SEM provided by Steve Morton.

area had noted red water and fish kills 15–20 years prior

to this report. Three years after Connell and Cross’

publication, Howell (1953) reported the occurrence of

red water due to microalgal blooms associated with fish

mortality in Melbourne, FL. He attributed the cause of

the fish mortality to be from the chain-forming

dinoflagellate Alexandrium monilatum (Fig. 1). In

September 1955, Gates and Wilson (1960) again

observed red-colored water and dead fish in Offats

Bayou, TX. This discoloration was found to be caused

by the same organism described by Howell 7 years

earlier. Although summer blooms of A. monilatum are

common in the Gulf of Mexico along the Texas coast,

this species has also been found along Florida’s west

coast (Williams and Ingle, 1972), in the Mississippi

Sound and adjacent Gulf of Mexico (Perry et al., 1979)

as well as the Gulf of Nicoya, Costa Rica (Viquez and

Hargraves, 1995). A. monilatum has now been

implicated in red-tide outbreaks and fish kills for well

over 100 years, yet no structural information of the

toxin responsible for the observed fish kills has been

deduced. This structural information, as well as a

reported appropriate detection method for the toxin(s)

responsible for the massive fish kills, are critical pieces

of information in developing mitigation strategies in

dealing with related human and environmental health

issues.

1.1. Previous findings

In contrast to other red-tide algal toxins, such as

saxitoxin, and the well-characterized toxins produced

by K. brevis and P. parvum (Sasner, 1973), little was

known about the physical or chemical nature of the

toxins produced by or associated with A. monilatum.

Neither was there a clear understanding of the resultant

physiological effects produced by them. Although little

or no molecular structural information has been

reported to date, early attempts at characterizing the

toxin did yield useful preliminary chemical data.

Upon observing and collecting an A. monilatum

bloom on the Texas coast, Gates and Wilson (1960)

performed tests to determine the effects of A. monilatum

cultures on mullet (Mugil cephalus). Their tests indicated

that disrupting the dinoflagellates by heating or freezing

resulted in increased fish mortality. This was due to the

release of the toxic substance within the cells. Fish

exposed to the cultures exhibited distress by frenzied

activity, followed by the loss of equilibrium. The fish

often turned upside down on their side and exhibited

slowed opercular movement (Gates and Wilson, 1960).

In this study, the authors reported that A. monilatum

appeared to have a paralyzing effect on mullet.

Confirming the findings of Gates and Wilson (1960), a

subsequent study conducted by Aldrich et al. (1967)

concluded that A. monilatum produced a toxic, lipophilic

compound that was contained primarily in the cell mass

and was released by autolysis or other cell disruption

mechanisms. The authors of this report speculated that a

cell-bound toxin was being produced and released as the

algal cells were disrupted by autolytic processes

accompanying or following cell death.

Still other research groups reported that the

lipophilic toxin derived from A. monilatum appeared

to be related to the polycyclic, polyether toxins, such as

brevetoxin and ciguatoxin (Sasner, 1973). Interestingly,

in his report, Sasner pointed out that the A. monilatum

toxin(s) activity paralleled that of the toxins produced

by Prymnesium parvum and K. brevis, due to its

hemolytic character. These comparisons were based in

part on earlier findings by Sievers (1969) who had

reported on the toxin activity of A. monilatum and K.

brevis, as compared and tested against fish, crustaceans,

annelids and mollusks. His experiments determined that

the fish were very sensitive to K. brevis and A.

monilatum, while the crustaceans appeared resistant to

both. The annelids and mollusks, on the other hand,

were more sensitive to A. monilatum than to K. brevis.

These data indicated that although K. brevis and A.

monilatum toxins may have similar chemical properties,

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299292

the mechanism of action by which these two toxins have

attributed, appears to be quite different (Sievers, 1969).

Earlier studies established that the main active toxic

compound(s) was not appreciably soluble in water

bearing a highly lipophilic nature. The toxin(s) appeared

to be primarily contained within healthy cells rather than

actively exuded into the environment. It became the goal

of this study to investigate and establish the molecular

nature of the toxic principles of A. monilatum. We report

the isolation and first unambiguous structural character-

ization of the toxin produced by A. monilatum.

2. Materials and methods

2.1. Isolation, identification and mass culturing of

A. monilatum

Samples from a ‘‘red-tide’’ of A. monilatum off

Gulfport, MS were collected by Dr. John Rogers (USEPA

Gulf Breeze, FL) and shipped to the NOAA Marine

Biotoxins Laboratory (Charleston, SC). Single cells were

isolated using the micro-pipette method of Guillard and

Morton (2003). Isolates were grown in modified L-1

medium (Guillard and Morton, 2003) at a salinity of

34 psu at 27 8C with 40 mE/(cm2 s) on a 16-h light:8-h

dark cycle. Seven isolates (AM01-07) were grown and

placed into the Marine Biotoxins Culture Collection.

All isolates were identified using both light micro-

scopy and scanning electron microscopy (SEM). For

light microscopy, an Olympus BX51 fitted with a

magnafire digital color camera was used. Plate

tabulation and visualization was achieved using 0.1%

Caulfour white fluorescent stain. For SEM, cells were

fixed with 1% gluteraldehyde and desalted using a

gradient of seawater to freshwater. Preparations were

dehydrated using a series of acetone and a series of

hexomethyldisilizane (HMDS). The samples were

coated with approximately 150 A of gold using a

Denton sputter-edge coater. SEM samples were imaged

on an environmental JEOL 5600LV.

Ten liter batch cultures of strain AM01 were grown

using 15 L Bellco m spinner flasks, fitted with a Teflon

impeller. Growth rates (Guillard, 1973) were calculated

by removing 1 mL of culture every 3 days. The number of

cells were counted using a Palmer–Maloney Counting

Chamber. All cultures were harvested at late log growth

phase, approximately 18 days after inoculation.

2.2. Cell harvest and extraction

A. monilatum cells were filtered from growth media

using a 25 mm sieve. Cells were lyophilized and horn

sonicated in 100 mL of ethyl acetate with a Branson

Sonifier 450 Horn Sonicator at full power for 15 min to

both disrupt the outer theca and ensure maximum

release of toxin from the cellular debris. An equal

volume of water was added after horn sonication for

solvent–solvent partitioning. Both layers were col-

lected, concentrated and tested for cytotoxicity using

GH4C1 rat pituitary cells and icthyotoxicity using

Sheepshead minnow. The layer with the greatest

toxicity was determined and taken on for further

purification.

2.3. GH4C1 rat pituitary cytotoxicity bioassay

(Mosmann, 1983)

GH4C1 rat pituitary cells were plated onto a 96-

well plate and allowed to incubate for 6 h to allow for

adherence to the plate. Dried fractions to be tested

were solubilized in 100 mL methanol of and sonicated

to ensure maximum solubility prior to carrying out

the bioassay. The assay was carried out by adding

4 mL (or smaller amount) of the methanol-solubilized

fractions to individual wells in the 96-well plate;

4 mL or less of pure methanol has been previously

tested to be safe to these cells (i.e., a negative

control). The plate was then allowed to incubate for a

minimum of 18 h; 15 mL of 3-(4,5-cimethylthiazol-2-

yl)-2,5-diphenyl tetrazolium bromide (MTT) dye was

added after incubation and allowed to incubate for

another 4 h before adding 100 mL of SDS to each

well. The plate was allowed to incubate for another

4 h prior to reading the assay.

2.4. Preparative thin layer chromatography (TLC)

The crude, concentrated ethyl acetate extract of A.

monilatum was loaded onto a Whatman Silica Gel

60A preparative TLC glass-backed plate and then

developed in a mixture of 7:3 ethyl acetate:benzene.

All colored and non-colored bands were scraped from

the plate, eluted with ethyl acetate, concentrated and

tested for cytotoxicity. These bioassays used metha-

nol as a carrier against the GH4C1 rat pituitary cells

and an MTT dye as determining the endpoint

(Mosmann, 1983). This procedure both established

the Rf value of the toxic compound and effectively

separated the toxin from contaminating pigments. A

modified molybidic acid stain (Stahl, 1965; H2SO4

added as modification-internal communication, Moel-

ler) was used in addition to cytotoxicity testing in

order to parallel activity of colored and non-colored

bands.

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299 293

Fig. 2. (a) ESI-MS of toxic extract; 791.2 indicates mass of extract in positive ion mode. (b) MALDI-TOF of toxic extract; 791.23 indicates mass of

extract in positive ion mode.

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299294

Fig. 3. (a) Proton NMR spectra of toxic extract in CDCl3. (b) Carbon NMR spectra (top) and APT NMR spectra (bottom) of toxic extract in CDCl3.

2.5. Mass spectrometry

Mass spectrometric detection of the A. monilatum

toxic extract was carried out using a number of mass

spectrometric techniques. Using a SL trap electrospray

ionization-mass spectrometry (ESI-MS) with an HPLC

interface, an ABI Voyager matrix-assisted laser

desorption ionization-time of flight-mass spectrometry

(MALDI-TOF-MS) with 2,5-dihydrobenzoic acid (2,5-

DHB) as a matrix and an ABI Voyager MALDI-TOF/

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299 295

TOF, purified toxic extract was tested in efforts to first,

fingerprint active fractions; secondly, to provide a non-

bioassay method to analytically detect and follow the

toxin derived from cellular extract(s); thirdly, to develop

an analytical technique to quantify the toxin where

necessary.

2.6. Liquid chromatography–mass spectrometry

(LC–MS)

Toxin identification and preparative toxin production

was augmented by using preparative LC–MS in the

reverse phase. Preparative toxin production was carried

out using an Agilent 1100 Purification Series LC/MSD

system equipped with a 9.4 cm � 50 mm, 5 mm, Zorbax

SBC18 column operating under APCI–MS conditions.

The following conditions were set for all LC–MS

experiments: drying gas rate at 5 L/min, gas temperature

at 300 8C, vaporizer at 325 8C, and corona voltage at

4000 V. Filtered A. monilatum toxic extract obtained

from preparative TLC was chromatographed using mass

based detection. Fractions of the appropriate mass were

collected using single ion monitoring in positive ion

mode. The Agilent system operated at a flow rate of

5 mL/min, using an injection volume of 500 mL. The first

purification procedure involved a gradient from 1:1

H2O:MeOH to 5:9.5 H2O:MeOH over 25 min. The

desired compound was collected, concentrated and dried

in preparation for the second LC–MS method. The

second purification LC–MS method followed the same

operations as above, but eluted the compound with an

isocratic method of 1:1 water:acetonitrile over 26 min.

The active compound was collected, concentrated and

dried in preparation for the third and final purification

step. This operation involved a step gradient using

acetonitrile and water over 30 min. The first 5 min

involved 9:1 water:acetonitrile, followed by 5 min of 3:1

water:acetonitrile, 5 min of 6.5:3.5 water:acetonitrile and

then 15 min at 1:1 water:acetonitrile.

2.7. Nuclear magnetic resonance (NMR)

Molecular structural analysis of the toxic compound

was carried out using a 500 MHz Bruker DMX NMR

equipped with a 5 mm triple nucleus gradient probe.

Purified sample (approximately 25 mg) was brought up in

CDCl3 as the lock solvent. The following NMR

experimental experiments were carried out to establish

molecular structure: 1H, 13C, APT, COSY (Neuhaus

et al., 1985), HSQC (Keeler and Neuhaus, 1985), HMBC

(Keeler and Neuhaus, 1985), and NOESY (Bax and

Davis, 1985a,b).

2.8. Accurate mass analysis

Accurate mass detection of the A. monilatum toxic

extract was performed using direct infusion of the

sample into a Waters Micromass Quadrupole Q-TOF

mass spectrometer operating with electrospray ioniza-

tion in positive ion mode. KBr and NH4 were added in

order to test for adduct formation. Chemical formulae

were calculated utilizing the elemental composition

tools within the Waters Masslynx software.

3. Results

3.1. Purification using preparative thin layer

chromatography

This method efficiently separated the toxic extract

from naturally occurring pigments also produced by

A. monilatum. This was an important step since co-

occurring peridinin and xanthophylls are cytotoxic in

high concentrations. Under this elution scheme, the

toxic component yielded an Rf value of 0.9, indicative of

its high lipophilicity. This upper-most band tested

positive against the GH4C1 rat pituitary cell line

cytotoxicity bioassay. A modified molybdic acid stain

was developed to highlight and detect both the active

and non-active non-colored/non-chromophoric bands in

efforts to develop an analytical technique to circumvent

the use of sample expensive bioassays. Non-colored

bands were clearly visible using this procedure. The

toxin itself was colorless and in pure state yielded no

UV chromophore under Agilent PDA detection

between 210 and 280 nm. It did, however, co-elute

with several green chlorophylls under the conditions

described.

The active compound(s) was eluted from the

preparative TLC plate and taken for further purification

and characterization on MS and LC–MS. The pre-

parative TLC method separated the active material from

most other organic soluble molecular components,

providing a large scale, time-efficient process for the

purification of the active toxin from the toxic pigments

produced by the dinoflagellate, A. monilatum.

3.2. Mass spectrometry of the toxic extract

The mass of the toxic extract (Fig. 2a and b) resulted in

a m/z for the toxic extract of 791.2 in positive ion mode on

both ESI-MS and MALDI-TOF-MS. The apparent mass

of 790 amu was used as an analytical tag for further mass

purification using LC–MS. With this MS method in hand,

it was possible to eliminate the need for bioassay

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299296

Table 1

Comparison of goniodomin A NMR data from Alexandrium monila-

tum and Alexandrium pseudogoniaulax

Alexandrium monilatum Alexandrium pseudogoniaulax

(Murukami et al., 1988)

Carbon # Carbon Hydrogen Carbon Hydrogen

1 168.5 168.1

2 76.6 4.53 76.2 4.309

3 140.3 139.7

4 112.7 4.82 112.1 4.779

4.98 5.002

5 41.1 2.26 40.7 2.331

2.76 2.82

6 70.6 3.83 70.2 4.133

7 80.7 3.55 80.2 3.683

8 73.5 4.81 73.1 5.133

9 149.3 148.6

10 110.8 4.96 110.4 4.933

5.05 5.177

11 34.1 2.63 33.6 2.537

12 20.4 1.35 20 1.412

1.35 1.412

1.35 1.412

13 44.6 1.70 44.1 1.73

2.19 2.095

14 100.6 101

15 150.6 150.1

16 107.8 4.95 107.5 4.675

5.03 4.933

17 27.92 1.82 27.5 1.948

1.92 1.948

18 25.7 1.47 25.4 1.17

2.04 1.33

19 76.9 3.68 76 3.686

20 76.5 3.86 76.5 3.818

21 27.95 2.34 27.5 1.55

2.34 1.55

22 123.6 5.81 123.2 5.659

23 129.6 5.97 129.1 6.277

24 76.8 4.14 76.3 4.39

25 81.8 3.86 81.4 4.03

26 29.9 1.29 29.9 2.226

1.29 2.226

27 31.8 1.78 31.4 1.55

2.34 2.14

28 79.7 5.04 79.1 5.203

29 147.2 147

30 114.3 5.16 113.4 5.039

5.16 5.039

31 81.8 3.86 81 4.073

32 73.2 3.68 72.8 3.93

Table 1 (Continued )

Alexandrium monilatum Alexandrium pseudogoniaulax

(Murukami et al., 1988)

Carbon # Carbon Hydrogen Carbon Hydrogen

33 32.1 1.94 31.8 2.14

2.75 2.976

34 135.4 6.16 135.1 6.453

35 123.8 5.59 123.3 5.866

36 74 5.71 73.5 5.962

37 98 97.5

38 41.3 1.25 40.9 1.329

39 13.2 0.93 12.7 0.98

0.93 0.98

0.93 0.98

40 31.3 1.70 30.8 1.664

41 20.4 0.93 20 0.74

0.93 0.74

0.93 0.74

42 34.3 1.35 34.2 1.17

1.53 1.17

43 61.1 3.70 60.6 3.56

3.95 3.902

Carbon count corresponds to Fig. 5.

cytotoxicity tests to monitor for the toxin. This technique

is amenable to all MS equipment in any lab.

3.3. Liquid chromatography–mass spectrometry of

the toxic extract

Three sequential purification experiments carried out

on the LC/MS preparative system resulted in highly

purified material suitable for structural analysis. In the

first purification method, the desired compound eluted

at 20–21 min. The second purification method eluted

the active compound in 9.5–10.5 min. The third step

eluted the active compound(s) at 13, 20–21, 26–27 min.

These three compounds, all exhibiting the same

nominal mass, were collected separately based on their

respective elution times. The 26–27 min fraction was

the major compound of interest and was taken on for

total structural analysis using NMR.

3.4. Nuclear magnetic resonance of the toxic

extract

The LC–MS purified sample resulted in a colorless

amorphous solid. Structural elucidation of this highly

toxic substance was accomplished by detailed data

analyses of a number of NMR experiments. The 1H,

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299 297

Fig. 4. (a) Accurate mass analysis of toxic extract; 807.3716 indicates the potassium adduct in positive ion mode. (b) Accurate mass analysis of toxic

extract; 786.4421 represents the ammonium adduct in positive ion mode.

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299298

13C, and APT (Fig. 3a and b) gave valuable information

regarding preliminary structural analysis and compound

class. These experiments clearly defined a polycyclic/

polyether type of molecule. Subsequent two-dimen-

sional NMR experiments provided the necessary

molecular connectivity information to establish the

molecular structure of the targeted toxin. 13C and HSQC

spectra revealed 43 signals representing 3 methyls, 14

methylenes, 19 methines, and 7 quaternary carbons, and

the existence of 7 double bonds in the molecule; 13C

chemical shift data are listed in Table 1.

This carbon and associated hydrogen count resulted

in some early confusion as the NMR data did not appear

to fit the observed mass at 790 amu. This issue was

resolved through the use of accurate mass analysis,

which provided information regarding the toxic

extract’s mass and empirical formula (see below).

3.5. Accurate mass analysis of analytically pure

compound

Addition of KBr to the purified sample resulted in a

potassium adduct with a m/z of 807.3716 in positive ion

mode (Fig. 4a). Confirmation of the potassium adduct

was established via the addition of an NH4 to the

purified compound. With the addition of ammonium, a

m/z of 786.4421 in positive ion mode was obtained,

clearly demonstrating adduct formation (Fig. 4b).

Purified samples subjected to accurate mass analysis

Fig. 5. Toxic compound isolated from Alexandrium monilatum:

goniodomin A.

demonstrated that the mass of 791.0 found from

previous ESI-MS and MALDI-TOF-MS experiments

was a sodium adduct with an accurate mass of 791.3974

in positive ion mode. The actual non-adduct molecular

ion was also observed at m/z 768. The best-fit formula

for the accurate mass that fit the NMR data were

C43H60O12. This formula defines 14 degrees of unsa-

turation, as corroborated in the NMR spectra. These

14 degrees of unsaturation are defined as 4 exocyclic

methylenes, 2 double bonds, 7 rings and 1 ester ca-

rbonyl (Fig. 5).

4. Discussion

By utilizing the combined NMR experiments, it was

possible to fully assign and structurally characterize a

polyether macrolide compound from A. monilatum. This

compound had a rare characteristic boasting of four

exocyclic methylenes. The structure is somewhat re-

miniscent of the spirolides, toxic compounds produced

by the dinoflagellate, Alexandrium ostenfeldii (Paulsen)

(Balech and Tangen, 1985). Established unambiguously

using high-field NMR and mass spectrometry, we now

report that A. monilatum produces the toxin goniodomin

A (Fig. 5), a toxin reportedly produced by the Japanese

rock pool dinoflagellate, Alexandrium pseudogoniaulax.

The NMR data obtained from the cultures are identical to

the macrolide toxin reported for this organism (Supple-

mentary material, Murukami et al., 1988). A comparison

of the 13C NMR signals is listed in Table 1. Pharma-

cological research carried out on this substance indicated

that goniodomin A has extreme effects on the reorganiza-

tion of the cytoskeleton. Abe et al. (2002) found that this

compound inhibited angiogenic properties of endothelial

cells partly by inhibition of actin reorganization. The

findings from Furukawa et al. (1993) suggested that

goniodomin A causes a conformational change resulting

in an enhancement of actomyosin ATPase activity at

concentration < 10�6 M, but conformational changes at

concentrations > 10�6 M may lead to a loss of ATPase

activity in rabbit skeletal muscle. This modulation

differed according to the type of cardiac myosin (Yasuda

et al., 1998), and the activation of skeletal actomyosin

ATPase was sensitive to the troponin/tropomyosin co-

mplex (Matsunaga et al., 1999). Other findings also

indicated that goniodomin A inhibited cell division in the

fertilized sea urchin (Murukami et al., 1988). In addition,

this compound demonstrates antifungal activity against

Candida albicans and Mortierella ramannianus (Mur-

ukami et al., 1988).

Previous reports by Clemons et al. (1980) and Bass

et al. (1983) have found evidence of a hemolytic

M.H. Hsia et al. / Harmful Algae 5 (2006) 290–299 299

principle associated with A. monilatum, but no toxin

was identified at the time. We did not test goniodomin A

against a hemolytic assay for this report. However, with

pure compound in hand, we plan on subsequent research

to establish whether or not this toxin exhibits hemolytic

activity.

5. Conclusions

We have found that A. monilatum produces the toxic

compound, goniodomin A. This toxin is identical to the

active compound produced by the Japanese rock pool

organism Alexandrium pseudogoniaulax. NMR data

chemical shifts match identically with those found by

Murukami et al. (1988). In the process of this research,

we have also developed highly sensitive mass spectro-

metric methods that are currently being optimized to

detect goniodomin A in marine matrices, such as

shellfish and mammal tissue. Other ongoing research

will focus on the algal–bacteria relationships as related

to toxin production.

Acknowledgement

This work was supported by NOAA/NOS. [SES]

Appendix A. Supplementary data

Supplementary data associated with this article can

be found, in the online version, at 10.1016/j.hal.2005.

08.004.

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