Dsp Toxin and Dinophysis

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Production and excretion of okadaic acid, pectenotoxin-2 and a noveldinophysistoxin from the DSP-causing marine dinoflagellate Dinophysisacuta Effects of light, food availability and growth phase

Lasse Tor Nielsen a,*, Bernd Krock b, Per Juel Hansen a

aMarine Biological Section, University of Copenhagen, Strandpromenaden 5, DK-3000 Helsingr, DenmarkbAlfred-Wegener-Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany

1. Introduction

Diarrhetic shellfish poisoning (DSP) toxins pose a serious threat

to both human health and shellfish industries in many areas of the

world (Reguera et al., 2012). Although first described from two

marine sponges, the primary producers of DSP toxins are species

from the two marine dinoflagellate genera Dinophysis and

Prorocentrum (Yasumoto et al., 1980; Tachibana et al., 1981;

Murakami et al., 1982). In terms of shellfish poisoning and human

health issues, Dinophysis is considered the key player, since DSP

producing Prorocentrum species are benthic, and usually not

readily available for suspension feeding mussels.

The genus Dinophysis contains more than 100 mixotrophic

species and representatives can be found in most oceans and

marine environments of the world (Gomez, 2005). Dinophysis spp.

have long been considered obligate mixotrophs (Jacobson and

Andersen, 1994), but Park et al. (2006) were the first to successfully

grow a Dinophysis species in laboratory culture, by feeding it the

marine ciliate Mesodinium rubrum (= Myrionecta rubra). Prior to

that, all studies on Dinophysis spp. were limited to in situ

populations and single cells picked from natural populations

(e.g. Draisci et al., 1996; Miles et al., 2004; Setala et al., 2005).

The mixotrophic nature of Dinophysis spp. extends beyond

regular mixotrophy, since the genus has recently been shown to

sequester and utilize the chloroplasts of its ciliate prey, M. rubrum

(Park et al., 2007; Wisecaver and Hackett, 2010; Kim et al., 2012).

Therefore, high photosynthetic activity of Dinophysis spp. relies on

continuous food uptake. Starved cells of mixotrophic Dinophysis

species will remain photosynthetically active, although at reduced

rates. This allows mixotrophic Dinophysis spp. to survive without

food for several months, as long as light is available (Kim et al.,

2008; Riisgaard and Hansen, 2009; Nielsen et al., 2012).

Dinophysis spp. produce two groups of DSP toxins: (1) okadaic

acid (OA) and the structurally similar dinophysistoxins (DTXs) and

Harmful Algae 23 (2013) 3445

A R T I C L E I N F O

Article history:

Received 20 September 2012

Received in revised form 21 December 2012

Accepted 25 December 2012

Available online 23 January 2013

Keywords:

Diarrhetic shellfish poisoning (DSP)

Dinophysis acuta

Food availability

Light

Mixotrophy

Okadaic acid (OA)

A B S T R A C T

Diarrhetic shellfish poisoning (DSP) toxinsconstitutea severeeconomic threat to shellfishindustries and

a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in

filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis.

Intraspecific toxin contents ofDinophysis spp. vary a lot, but the regulating factors of toxin content

are stillpoorlyunderstood.Dinophysis spp. have beenshownto sequester anduse chloroplasts from their

ciliateprey, and with this raremodeof nutrition, irradiance and food availabilitycould play a keyrole in

the regulation of toxins contents and production. We investigated toxin contents, production and

excretion of aDinophysisacuta cultureunder different irradiances, food availabilitiesand growthphases.

The newly isolated strain ofD. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel

dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins

were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in

extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found

extracellularly. This was thecaseboth in steady-state exponentialgrowthand in food limited, stationary

growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins.

Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- andextracellularly when starvation reduced growth rates ofD. acuta. Toxin production rates were highest

during exponential growth, but continued at decreased rates when cell division ceased, indicating that

toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of

these new discoveries to shed light on the ecological role of DSP toxins.

2012 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +45 35321992; fax: +45 35321951.

E-mail addresses: [email protected], [email protected] (L.T. Nielsen).

Contents lists available at SciVerse ScienceDirect

Harmful Algae

journ al homepage: www.elsevier .co m/locat e/ha l

1568-9883/$ see front matter 2012 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.hal.2012.12.004
http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004mailto:[email protected]:[email protected]:[email protected]://www.sciencedirect.com/science/journal/15689883http://www.sciencedirect.com/science/journal/15689883http://www.sciencedirect.com/science/journal/15689883http://dx.doi.org/10.1016/j.hal.2012.12.004http://dx.doi.org/10.1016/j.hal.2012.12.004http://www.sciencedirect.com/science/journal/15689883mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.hal.2012.12.004


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(2) pectenotoxins (PTXs) (Yasumoto et al., 1985). OA and DTXs are

free polyether acids that inhibit serine/threonine phosphatase, and

affect the secretion and gene transcription of nerve growth factor

(Pshenichkin and Wise, 1995; Garcia et al., 2003). PTXs are

polyether lactones, but the actual toxicity, and their status as DSP

toxins, is currently under debate. For now, however, the toxin

group remains included in the 160 mg kg1 regulatory threshold

set for all commercial shellfish (EC, 2004; Miles et al., 2004;

Reguera et al., 2012).

Currently, two DTXs (DTX-1 and DTX-2) have been described

with full molecular structures from species of Dinophysis. In

addition, other DTXs with yet undisclosed exact molecular

structures have been reported from Irish waters, and the list of

known DTXs could grow within the next years (James et al., 1997;

Draisci et al., 1998). PTX-2 is the primary PTX produced by

Dinophysis spp. but PTX can be found in a variety of forms, with at

least 15 different derivatives presently identified (Miles, 2007;

Anonymous, 2009). Most of these are believed to occur only as

metabolites in shellfish, however (Suzuki et al., 1999). Dinophysis

acuta normally contains OA and PTX-2 as well as either DTX-1 or

DTX-2, but the cellular content of each toxin can vary a lot (Lee

et al., 1989; James et al., 1999; Lindahl et al., 2007; Pizarro et al.,

2008, 2009; Fux et al., 2010).

The ecological role and significance of DSP toxins are currentlylargely unknown (Reguera et al., 2012). Several functions seem

plausible, including food capture, grazer defense, allelopathy and

anti-bacterial deterrent, and some of these have already been

visited (Nagai et al., 1990; Carlsson et al., 1995; Gross, 2003). The

theory of DSP toxins as a defense against grazing is supported by

the finding that some copepods seem to discriminate against D.

acuminata as a food source, whereas another non-discriminating

copepod species experienced reduced survival rates (Carlsson

et al., 1995). The theory of allelopathic effects has also received

backing (Windust et al., 1996), but both ideas remain unproven,

and the ecological role of DSP toxins is still undisclosed.

Many harmful microalgae are mixotrophic (i.e. use particulate

food for growth; e.g. Prymnesium parvum,Alexandrium spp., Karenia

spp., and Karlodinium spp.). In fact, only the non-motile algal groupslike diatoms (e.g. Pseudo-nitzschia spp.) and cyanobacteria (e.g.

Nodularia spumigena) can be considered autotrophs or auxotrophs

(Flynn et al., 2013). Dinophysis spp. are among the very few toxic

microalgae that rely on chloroplasts sequestered from their prey.

Pfisteria spp. maybeanother groupwith similar abilities, butdata on

rates of photosynthesis are still lacking. The available data on

Dinophysis spp. suggest they areobligate mixotrophs. Hence they 1)

cannot live in the long run without food and 2) cannot live in

complete darkness even when supplied excess amounts of food

(Park et al., 2008; Nagai et al., 2008; Nishitani et al., 2008; Kim et al.,

2008; Riisgaard and Hansen, 2009). This raises questions about the

role of food uptake and light for toxin production. It also raises

questions about the possible excretion of DSP toxins and ultimately

about

the

ecological

role

of

DSP

toxins.Here, we investigate the dependence of toxin contents and

production of D. acuta upon irradiance, food availability and

growth phase. Wemeasure both intra- and extracellular levels of

DSP toxins in order to quantify toxin excretion under various

conditions. The aim is to understand Dinophysis spp. toxicity better

and ultimately to unravel the ecological function of DSP toxins.

2. Materials and methods

2.1. Cultures and culturing conditions

Cultures of the cryptophyte Teleaulax amphioxeia (K-0434

(SCCAP)) and the ciliate M. rubrum (MBL-DK2009) were estab-

lished

from

water

samples

from

Helsingr

Harbor

in

2009.

Cultures

of M. rubrum were fed T. amphioxeia at a predator:prey ratio of

1:10 twice a week. During a scientific cruise in the North Atlantic

ca. 100 km south of the Faroe Islands (608240N; 68580W), a non-

clonal culture (DANA-2010) of the DSP producing dinoflagellate D.

acuta was established inJune 2010, by picking and washing several

cells. M. rubrum was added as prey organism twice per week at a

predator:prey ratio of 1:10 to allow mixotrophic growth.

All three species were grown in f/2 medium based on

autoclaved seawater, and with a salinity of 32 1, a dissolved

inorganic carbon (DIC) concentration of 2.3 0.1 mmol L1 and a pH

of 8.0 0.05 (Guillard and Ryther, 1962). They were grown at

15.0 1.0 8C in a temperature controlled room, at an irradiance of

130 mmolphotonsm2 s1 (PAR), controlled by a timer to a light:dark

cycle of 16:8 h. All cultures were non-axenic.

DSP toxins of a D. acuta stock culture were sampled by

transferring 0.5 ml subsamples to spin filters (pore size = 0.45 mm,

VWR, Denmark), and centrifuging these at 400 g for 2 min.

Filtrates were removed, and spin filters were stored at 18 8C until

extraction and analysis.

2.2. Experiment 1 effects of irradiance on growth, photosynthesis

and toxin production

D. acuta was kept well-fed for a minimum of 14 days at fourdifferent irradiances to evaluate the effects of light on photosyn-

thesis, growth rate and toxin content. The four irradiances were 7,

15, 30 and 130 mmol photons m2 s1 (PAR), henceforth termed I7,

I15, I30and I130respectively. All treatments were setup in the same

room, in front of the same light source consisting of Osram cool

white 58 W/640 fluorescent tubes, but with different combina-

tions of neutral density filters in front of I7, I15and I30in order to

achieve the designated irradiances. All four treatments were run in

triplicate 65 ml polystyrene bottles filled to capacity. The ciliate, M.

rubrum, was cultured at I130, but was light acclimated for a week at

the appropriate irradiances before being used as prey for D. acuta.

The same was done with the cryptophyte T. amphioxeia. Initial cell

concentrations at the experimental setup were 200 and

2500 cells ml

1 of D. acuta and M. rubrum, respectively.Every 24 days, 3 ml subsamples were removed from each flask

for enumerations of D. acuta, M. rubrum and T. amphioxeia. 1 ml

SedgewickRafter sedimentation chambers were used, and cells

were counted on an Olympus CK2 inverted microscope at 40

400. A minimum of 200 cells were counted, unless cell

concentrations were below 200 ml1 at which point a maximum

1 ml was inspected. After each sampling, cell concentrations of D.

acuta and M. rubrum were adjusted to 200 and 2000 cells ml1

respectively, by adding f/2 medium and light acclimated, well fed

M. rubrum.

On the final day, 1 ml subsamples were removed from each

triplicate bottle for determination of photosynthetic activity. 80 D.

acuta cells were picked from each subsample under a stereo

microscope,

and

photosynthetic

activities

were

determinedexactly like presented earlier, including 14C addition (as HCO3

)

to both light and dark samples, 3 h incubations and checks of

specific activity (Nielsen et al., 2012).

Toxin samples were also taken from each flask on the final day of

the first experiment. Subsamples of 0.5 ml were transferred to spin

filters, and these were centrifuged at 400 g for 2 min. Filtrates

were removed, and spin filters stored at 18 8C until extraction and

analysis. This toxin extraction method has previously been shown

not to affect intracellular toxins quotas of Dinophysis acuminata at

centrifugal forces up to 12,800 g (Nielsen et al., 2012).

For growth rates and toxin contents, averages of the last three

values were defined as the well-fed, light acclimated values

(hereafter termed steady-state), and these were used for

comparisons

between

irradiances.

L.T. Nielsen et al./Harmful Algae 23 (2013) 3445 35


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2.3. Experiment 2 toxin content, production and excretion as a

function of growth phase

Based on results from the first experiment, the two irradiances

I15 and I130were selected for the set of subsequent experiments.

Triplicate 65 ml polystyrene bottles of each of the two irradiances

were set up with 200 D. acuta cells ml1 and 2500 M. rubrum

cells ml1 initially. Under both treatments, D. acuta was kept well

fed for the first 14 days to ensure light acclimation. This was done

by sampling, enumerating and diluting every few days, parallel to

the first experiment. After the first 14 days, food additions were

stopped, and bottles were instead diluted with f/2 medium only, in

order to ensure optimal conditions of nutrients, pH, etc. Dilutions

with f/2 were stopped once division rates of D. acuta declined due

to starvation.

In this set of experiments, DSP toxins were sampled in three

different ways. Firstly, standard spin filter samples of 0.5 ml were

taken on all samplings following the method described for the first

experiment. Secondly, on four occasions during the experiments,

100 D. acuta cells from each bottle were picked and washed with

micropipettes under the stereoscope. After washing, cells were

picked and transferred to spin filters that were subsequently

treated like the regular spin filter samples. Thirdly, on five

occasions during the experiments, a 10 ml subsample from eachbottle was transferred to a glass scintillation vial, which was then

stored at 18 8C until further analysis. Since D. acuta cells were not

removed from these samples prior to freezing, these samples were

a measure of total toxin (intra- and extracellular).

As in the first experiment, averages of the last three values from

the well-fed period were defined as steady-state values repre-

senting well-fed and light acclimated conditions, and these values

were used for comparisons.

2.4. Toxin extraction and analysis

For extraction of spin filter samples, 100 ml methanol (100%)

was added, and after 1 h incubation, these were centrifuged at

800 g for 2 min. The extract was then transferred to a 2 ml glassHPLC vial with a 250 ml glass insert. Extractions from the 10 ml

water samples taken in the second experiment were achieved with

solid phase extraction (SPE). 1 ml LC-18 SPE cartridges (Sigma

Aldrich1, Germany) were pre-conditioned with 100% methanol,

and then flushed with distilled water to remove excess methanol.

All 10 ml of each sample were then slowly (1 ml min1) passed

through an SPE cartridge. The cartridge was washed three times

with 1 ml distilled water, after which DSP toxins were eluded with

1 ml 100% methanol directly into a 2 ml glass HPLC vial.

Toxin samples from the first experiment were only analyzed in

the positive ionization mode, whereas all toxin samples from the

second experiment were analyzed in duplicates: once in positive

ionization mode to detect PTXs, and once in negative ionization

mode

to

detect

OA

and

DTXs.Separation of toxins was achieved on an Agilent (Waldbronn,

Germany) model 1100 liquid chromatograph (LC). The LC

equipment included a solvent reservoir, in-line degasser

(G1379A), binary pump (G1311A), refrigerated autosampler

(G1329A/G1330B), and temperature-controlled column oven

(G1316A). After injection of 5 ml of sample, separation of lipophilic

toxins was performed by reverse-phase chromatography on a C8

column (50 2 mm) packed with 3 mm Hypersil BDS 120 A

(Phenomenex, Aschaffenburg, Germany) and maintained at

20 8C. The flow rate was 0.2 mL min1 and gradient elution was

performed with two eluants, where eluant A was water and eluent

B was acetonitrile/water (95:5, v/v), both containing 2.0 mmol l1

ammonium formate and 50 mmol l1 formic acid. Initial condi-

tions

were

12

min

column

equilibration

with

5%

B,

followed

by

a

linear gradient to 100% B within 10 min and isocratic elution until

15 min with 100% B. The programme was then returned to initial

conditions for 18 min (total run time: 30 min).

For okadaic acid (OA) and dinophysistoxins (DTXs) large

volume injection of 50 ml was used. After injection the sample

was first flushed for 1 min by the initial eluent composition via a

six-port valve onto a cartridge (Oasis HLB 5 mm, 2.1 20 mm;

Water, Eschborn, Germany) for removal of sample solvent, and the

analytes were then backflushed by valve switch onto the analytical

column and chromatographed as described above.

Detection of toxins was performed on an ABI-SCIEX-4000 Q

Trap (Applied Biosystems, Darmstadt, Germany), triple quadrupole

mass spectrometer equipped with a TurboSpray1 interface. PTX-2

was detected in the positive ionization mode by selected reaction

monitoring (SRM) experiments with the transition m/z876 > 213

using the following parameters: curtain gas: 10 psi, CAD gas:

medium, ion spray voltage: 5500 V, temperature: ambient,

nebulizer gas: 10 psi, auxiliary gas: off, interface heater: on,

declustering potential: 50 V, entrance potential: 10 V, collision

energy: 55 V, exit potential: 15 V. OA and DTXs were detected in

the negative ionization mode with the transitions m/z803 > 255

and m/z 803 > 113 for OA and DTX-2 and m/z817 > 255 and m/z

817 > 113 for DTX-1 using the following parameters: curtain gas:

10 psi, CAD gas: high, ion spray voltage: 4500 V, temperature:500 8C, nebulizer gas: 30 psi, auxiliary gas: 30 psi interface heater:

off, declustering potential: 120 V, entrance potential: 10 V,

collision energy: 60 V, exit potential: 2 V. Dwell times of 100

200 ms were used for each transition.

Due to the finding of a novel DTX-1 isomer, collision induced

dissociation (CID) spectra of DTX-1 and the new compound were

recorded in the enhanced product ion (EPI) mode of m/z 836 (mass

range m/z100830) in the positive and m/z 803 (mass range m/z

100820) in the negative mode. Mass spectrometric parameters

were as in the respective SRM experiments.

2.5. Calculations and statistical analysis

In both sets of experiments, toxin production rates, Rtoxwerecalculated as:

Rtox T2 T1N t2 t1

where T1 and T2 are toxin contents per ml culture at two

subsequent samplings taken on days t1 and t2, and N is the ln-

average of the cell concentration per ml calculated as:

N N2 N1lnN2=N1

Total toxin values from SPE samples were used for this in the

second

experiment.

However,

calculations

of

toxin

productionrates require two consecutive measurements, and since only one

SPE measurement was taken during the well-fed period, rates from

this period could only be obtained by assuming that the steady-

state condition gave similar toxin contents at day 11 and 14. Thus,

we set the toxin contents (per cell) on day 11 to mirror those found

in the only steady-state measurement from day 14. This

assumption seems justified, since D. acuta had grown well for

several samplings up until this point. At the same time, carry-over

effects from the stock culture were minimal, since dilutions had

left only 4 and 2% of the original stock culture in the two

treatments I15 and I130, respectively.

One-way ANOVA was used for all statistical comparisons, with a

predefined a of 0.05 and a Tukey test for pairwise comparisons.

Analyses

were

done

using

the

software

SigmaStat

3.5.

L.T. Nielsen et al./Harmful Algae 23 (2013) 344536


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treatment wasnot statistically significantlydifferent from I15 and I130,

but all other treatment combinations were (p < 0.02, one-way

ANOVA, n = 3).

3.3. Experiment 2 toxin content, production and excretion as a

function of growth phase

3.3.1. Growth characteristics

When well-fed, D. acuta concentrations increased betweensamplings from averages of 159 20 and 167 4 to 262 33 and

359 8 cells ml1 in the two treatments I15 and I130 respectively.

Comparably, M. rubrum concentrations decreased from averages

of 2306 100 and 2313 65 cells ml1 in the two treatments to

695 244 and 913 81 cells ml1 between samplings (Fig. 6). After

the final feeding at day 11, the two treatments developed somewhat

differently. In I15, M. rubrumdecreased to 231 220 cells ml1 onday

17, andwas allbutgone fourdays later. Simultaneously, D. acutagrew

unaffectedly until day 21, where after growth continued at a

decreasing rate until day 55 (maximum 690 103 cells ml1). In

the I130 treatment, M. rubrumdecreased comparably, but D. acutaonlygrew unaffectedly for six days (until day 17), and only showed

positive growth until day 28.

3.3.2. DSP toxins

The three DSP toxins PTX-2, okadaic acid (OA) and DTX-1b

found in experiment 1 also dominated the toxin profile of D. acuta

during experiment 2.

3.3.2.1. Spin filters. Based on spin filter samples, steady-state

(well-fed, light acclimated) contents of PTX-2, OA and DTX-1b

were 58.8 12.4, 3.0 0.4 and 7.6 1.4 pg cell1 in the I15treatment, and 52.6 1.1, 2.7 0.2 and 6.5 0.4 pg cell1 in the

I130 treatment respectively (Fig. 7). All three toxins accumulated

under

both

irradiances

when

D.

acuta

was

starved,

and

correspondingvalues on the final day of the experiments were 97.2 10.6,

15.4 4.3 and 33.2 10.2 pg cell1 for I15 and 130.5 17.1,

12.9 3.0 and 29.5 8.5 pg cell1 for I130.

3.3.2.2. Intracellular toxinfrompicked cells. From samples of picked

cells, intracellular contents of PTX-2, OA and DTX-1b in the steady-

state phase were 43.7 7.5, 1.7 0.5 and 4.0 1.3 pg cell1 under

I15 and 32.8 1.4, 2.8 0.3 and 6.0 0.9 pg cell1 under I130,

respectively (Fig. 7). When cultures were starved, intracellular toxin

values increased in a similar way as with the spin filter samples. On

the final day of the experiment, values of PTX-2, OA and DTX-1b had

thus increased to 67.6 0.6, 7.2 1.0 and 16.0 2.3 pg cell1 under

I15 and 84.2 3.1, 8.2 0.3 and 17.7 0.8 pg cell1 under I130,

respectively.

465

-H2O

447/ 461-H2O429 / 443337

-H2O

319 / 305 -H

2O

301/ 287

HO

O O

OHO

337 319 / 305 301 / 287

1

31

A

HO

O

OOH

OO

OOH

OH

31

35

29

12

22

335/ 321255 / 255 563 / 577B

HOO

O

O O

O

OHO

1

3112

O

OHO O

OOH

OH

35

2922

Fig. 3. Fragmentation scheme of DTX-1 in (A) the positive mode and (B) the negative mode. Fragments of DTX-1/compound 1, respectively, observed in the spectra are printed

in bold.

A

Cellconcentr

ation(cellsml-1)

0

200

400

600

800

D. acuta

B

Day

0 2 4 6 8 10 12 14 1601000

2000

3000

4000

M. rubrum

Fig. 4. Steady-state growth of Dinophysis acuta (A) and Mesodinium rubrum (B) in the

first experiment. Example of experimental design and dilution scheme at the

irradiance treatment I30. Symbols and error bars represent means SD (n = 3).



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3.3.2.3. Total toxin via SPE. By comparison, the total amounts of

PTX-2, OA and DTX-1b found in the steady-state phase were

125.2 30.0, 16.3 3.0 and 37.9 8.0 pg cell1 respectively under

I15, and the corresponding values for I130were 93.9 7.4, 10.1 0.5

and 22.6 1.1 pg cell1 (Fig. 7). As with the other two types of toxin

measurements, most values of total toxin also increased when cells

were starved. The only exception was the PTX-2 value at I15, which

didnot increasenoticeablyduring starvation. Thus, on thefinal day of

the experiment, values of PTX-2, OA and DTX-1b were 134.1 14.9,

33.6 5.2 and 78.0 11.6 pg cell1, respectively in the I15 treatment,

and 182.0 31.1, 51.8 14.3 and 115.4 33.0 pg cell1 under I130.

3.3.3. Intracellular vs. total toxin content

When in steady-state, intracellular PTX-2 from picked cells of

I15 and I130 each accounted for 35.7 7.0 and35.0 1.0% of the total

amount of PTX-2 found in the corresponding whole-water SPE

samples (Fig. 8C and D). When starved, the intracellular proportions

initially increased markedly to 72.9 8.0 and 74.3 14.8% respec-

tively, but these later decreased again somewhat, and ended at

53.2 7.7 and 43.0 8.3% on the final day of the experiment.

In terms of intracellular versus extracellular toxin pools, OA and

DTX-1b almost perfectly mirrored each other at both irradiances

(Fig. 8C and D). Initially, when well-fed, 10.7 2.6 and10.4 2.3%of

OA and DTX-1b respectively were found intracellularly at I15. Thus,90% of both OA andDTX-1b were found extracellularly in the growth

medium. With corresponding values of 27.3 2.6 and 26.5 3.9%,

statistically significantly more was found intracellularly at I130 (OA:

p = 0.001, DTX-1b: p = 0.003, one-way ANOVA, n = 3). At I15, the

intracellular proportion increased a little during starvation, especially

toward the end of the experiment, where intracellular proportions of

OA and DTX-1b were 21.6 2.4 and 20.6 1.5%, respectively. At I130,

the intracellular proportions ofOA andDTX-1bwereunaffected for the

first 38 days of starvation. At the last measurement, on day 66, the

intracellular proportions decreased somewhat to 16.8 5.0 and

16.4 5.5% for OA and DTX-1b, respectively.

3.3.4. Toxin ratios

Average ratios of PTX-2: OA from the steady-state period were20.9 5.5 and 20.4 1.0 for I15and I130, respectively (Fig. 8E and F).

Corresponding values forPTX-2:DTX-1b were 8.1 2.2 and 8.2 0.3,

and for DTX-1b: OA they were 2.6 0.1 and 2.5 0.1. The DTX-1b:

OA ratios were remarkably stable throughout the experiment,

regardless of irradiance and starvation. The ratios of PTX-2 to OA

and to DTX-1b decreased, on the other hand, under both irradiances

when D. acuta was starved. Thus, PTX-2: OA ratios ended at 6.6 1.5

and 10.3 1.5 at I15 and I130 respectively, and the corresponding

ratios of PTX-2: DTX-1b were 3.1 0.7 and 4.6 0.8.

3.3.5. Rates of toxin production

Total toxin production rates in steady-state were 21.8 6.4 pg

PTX-2 cell1 d1, 2.9 1.0 pg OA cell1 d1 and 6.7 2.1 pg DTX-

1b

cell1

d1

for

I15 and

28.9

0.9

pg

PTX-2

cell1

d1

,

3.1

0.2

pgOA cell1 d1 and 7.0 0.4 pg DTX-1b cell1 d1 for I130 (Fig. 9). All

these rates declined markedly when growth rates declined, and the

corresponding values on the final day were 1.5 0.9, 0.2 0.2 and

0.4 0.5 pg PTX-2/OA/DTX-1b cell1 d1 for I15 and 3.0 0.5,

0.1 0.3 and 0.3 0.8 pg PTX-2/OA/DTX-1b cell1 d1 for I130.

4. Discussion

4.1. Structure of compound 1 and the toxin profile of Dinophysis

acuta

Under collision induced dissociation (CID) conditions in the

positive and negative ionization mode, compound 1 showed the

same

fragmentation patterns

as

DTX-1, except

for a

14Da

C

PTX-2

content(pgcell-1)

0

20

40

60

80

100

120

140

B

Photosyn

thesis(pgC

cell-1h

-1)

0

20

40

60

80

100

120

140

160

180

A

(d-1)

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

D

PAR (mol photons m-2

s-1

)

0 20 40 60 80 100 120 140PTX-2production(pgcell-1d-1)

0

5

10

15

20

25

Fig. 5. Steady-state values of growth rate (A), photosynthetic activity (B), PTX-2

content (C) and daily PTX-2 production rate (D) in Dinophysis acuta as functions of

treatment irradiance. For A, C and D, mean values of the last three measurements

during steady-state growth were used. Only one measurement was made for B. A, B

and D were statistically significantly affected by irradiance, whereas C was not.

Symbols and error bars represent means SD (n = 3).



7/12

enlarged C1C22 fragment and a 14 Da smaller C23C35

fragment (Figs. 1 and 2). This is strong evidence that in

comparison to DTX-1, compound 1 carries an additional methyl

(or methylene) group at C1C22, and a methyl (or methylene)

group less in the part C23C35. Carey et al. (2012) established a

fragmentationpatternof DTX-1 forthe negative ionizationmode,

which basically gave the same fragments as in the positive mode

(Fig. 3B). The fact that DTX-1 and compound 1 showed the exact

same differences in the positive and the negative ionization

mode strengthen our argument that compound 1 is closely

related to DTX-1.

Interestingly, both DTX-1 and compound 1 formed the

fragments m/z 151, 191 and 255, the latter originating from acleavage between C11 and C12 (Fig. 3B). This is strong evidence

that both molecules share an identical structure from C1 to C11.

Taking into account that OA, which was also produced by this

strain of D. acuta, does not possess a methyl group at C35, the

information at hand strongly suggest the structure of compound

1 to be 1222-methyl OA. However, the exact locations of the

missing methyl (or methylene) group between C26 and C35 and

the additional methyl (or methylene) group between C12 and

C22 cannot be unambiguously assigned by mass spectrometry

alone. For an unambiguous structural elucidation of compound

1, approximately 500 mg of pure substance is required for

nuclear magnetic resonance (NMR) spectroscopy, and this was

unfortunately not possible to achieve within this work. Even

though the

complete structure of

compound

1

remainsunresolved, we demonstrate that there is a yet undescribed

dinophysistoxins in this strain of D. acuta, and that the

molecular structure seems to be that of a DTX-1 isomer. The

high similarity of the spectra and the partly identical mass

fragments may have caused compound 1 to be misidentified as

DTX-1 in the past.

D. acuta normally contains only OA, PTX-2 and either DTX-1 or

DTX-2; only on a single occasion has it been found with both DTX-1

and DTX-2 (Johansen, 2008). Two novel DTX-2 isomers have

previously been reported in D. acuta, but with precise chemical

structures yet to be determined (James et al., 1997; Draisci et al.,

1998). Together with our report on a new DTX-1 isomer, this

demonstrates that DTXs D. acuta are not limited to the standard

DTX-1

and

DTX-2

molecules.

Future

work

should

be

meticulous

with the identification of DTXs, and more information on novel

toxins is required, not least on their toxicity.

4.2. Effects of light ongrowth,photosynthesis and PTX-2 content and

production

Irradiance significantly affected the growth rates of D. acuta,

even when prey was supplied in excess. The growth rate at the I7treatment was reduced to 18% of that obtained at I130. Irradiance

also affected the photosynthetic rate of D. acuta, and the rate of

photosynthesis at I7was reduced to 22% of that obtained at I130.

Similar effects of irradiance on growth and photosynthesis have

been found in other Dinophysis species, indicating that they arefunctionally identical (Kim et al., 2008; Riisgaard and Hansen,

2009; Nielsen et al., 2012). The fact that the growth rate was so

tightly coupled to the rate of photosynthesis indicates that food

ingestion cannot substitute for phototrophically derived carbon.

Irradiance had only marginal effects on the cellular contents of

PTX-2 in D. acuta. If anything, the cellular PTX-2 content decreased

with irradiance. On the other hand, with growth rates significantly

affected by irradiance and PTX-2 contents more or less stable,

cellular PTX-2 production rates were obviously higher at increased

irradiances. This was also the case for D. acuminata, although this

species showed slightly increased PTX-2 contents at higher

irradiances (Nielsen et al., 2012). Contrary, Tong et al. (2011)

found that irradiance had no effect on rates of toxin production in

D.

acuminata, but

they

applied

only

irradiances

from

65

to300 mmol photons m2 s1 that were all growth saturating.

Together, these result suggest, that PTX-2 production is linked

somehow to growth rate. In fact, in the current dataset, there was

an almost perfect linear relationship between growth rates and

PTX-2 production rates of the four well-fed irradiance treatments,

I7I130(r2 = 0.99). The influence of irradiance on toxin production

seems, thus, to owe to the influence held via effects on growth

rates. Beyond that, PTX-2 production appears unaffected by

irradiance.

4.3. Effects of growth phase on toxin content and production

Production of all three DSP toxins continued as growth rates

declined

due

to

starvation,

resulting

in

significant

accumulation

of

0 10 20 30 40 50 60 7001000

2000

3000

130 mol

0

200

400

600

800

Time (d)

0 10 20 30 40 50 60 7001000

2000

3000

M. rubrum

15 mol

Cellconcentra

tions(cellsml-1)

0

200

400

600

800

D. acuta

Fig. 6. Cell concentrations of Dinophysis acuta (top) and its prey Mesodinium rubrum (bottom) at the two irradiance treatments I15and I130used in the second experiment.

Flasks were diluted with f/2 growth medium every 24 days until Day 21. Mesodinium rubrum was added every 24 days until day 14. Symbols and error bars represent

means SD (n = 3).



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these toxins in D. acuta (Figs. 7 and 9). This is parallel to what has

been observed before, and it is now evident that senescent

populations of Dinophysis spp. contain more toxin than those in

exponential growth (Tong et al., 2011; Nagai et al., 2011; Nielsen

et

al.,

2012). This

has

also

been

observed

in

field

populations

of

D. acuta (Pizarro et al., 2008, 2009). The fact that toxin production

is not coupled directly to the growth rate when growth rates

decline due to starvation seems in direct contrast to the conclusion

reached in Section 4.2, and the correlation appears to only hold

true

under

food

replete

conditions.

The

obvious

interpretation

PTX-2(pgcell-1)

0

50

100

150

200

250

OA(pgcell-1)

0

10

20

30

40

50

60

70

SPE (total)

Spin filters

Picked cells

Time (d)

0 10 20 30 40 50 60 70

DTX-1b(pgcell-1)

0

20

40

60

80

100

120

140

160

Time (d)

0 10 20 30 40 50 60 70

A B

C D

E F

130 mol15 mol

(d-1)

0,0

0,1

0,2

0,3

0,4

0,5

G H

Fig. 7. Toxin contents of Dinophysis acuta at irradiance treatments I15 (left) and I130 (right) during the second experiment. PTX-2 (C and D), okadaic acid (E and F) and the novel

toxin DTX-1b (G and H) in the three different sample types total toxin (dashed), spin filters (solid) and picked cells (dotted). Growth rates of D. acuta (A and B) for

references. Dashed, vertical lines indicate when growth rates of D. acuta started to decline. Symbols and error bars represent means SD (n = 3).



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Time (d)

0 10 20 30 40 50 60 70

PTX-2:OA

PTX-2:DTX-1b

DTX-1b:OA

130 mol

Time (d)

0 10 20 30 40 50 60 70

Toxinratios

0

10

20

30

40

Cellular:total

0,0

0,2

0,4

0,6

0,8

1,0PTX-2

OA

DTX-1b

15 mol

(d-1)

0,0

0,1

0,2

0,3

0,4

0,5A B

C D

E F

Fig. 8. Toxin contents of Dinophysis acuta at irradiance treatments I15(left) and I130(right) during the second experiment. Ratios of cellular: total toxin (C and D) and ratios

between the three toxins PTX-2, okadaic acid (OA) and the novel toxin DTX-1b (E and F). Growth rates of D. acuta (A and B) for references. Dashed, vertical lines indicate when

growth rates of D. acuta started to decline. Symbols and error bars represent means SD (n = 3).

A

Time (d)

0 10 20 30 40 50 60 70

Toxin

production

(p

g

cell-1

d-1)

-5

0

5

10

15

20

25

30

PTX-2

OA

DTX-1b

B

Time (d)

0 10 20 30 40 50 60 70

Fig. 9. Rates of PTX-2, OA and DTX-1b production in Dinophysis acuta at irradiance treatments I15(left) and I130(right) during the second experiment. Dashed, vertical lines

indicate when growth rates of D. acuta started to decline. Production rates were calculated from measures of total toxin (SPE samples). Symbols and error bars represent

means SD (n = 3).



10/12

would be that both growth and toxin production are limited by

irradiance, but that only growth, and not toxin production, is

limited by heterotrophic intake of ciliate prey.

4.4. Intra- and extracellular toxin pools

The spin filters used for the standard toxin analysis in both

experiments had a pore diameter of 0.45 mm, and thus sampled

particles of roughly this size and larger. However, this could

potentially encompass several toxin pools including intracellular

toxin from D. acuta and from bacteria as well as extracellular toxins

bound to organic matter particles, e.g. cell debris. In addition, spin

filters omitted toxin from the 0.45 mm) and (3) the

dissolved organic matter (DOM) fraction (0.45 mm) was much smaller,

generally at the same level at that found intracellularly in D. acuta.

In steady-state, exponential growth, the DOM pool also contained

the larger part of the extracellular PTX-2. This changed when cell

growth ceased, and here the POM pool was often as large as the

DOM pool. The awareness that all DSP toxins are excreted, but to

completely

different

levels,

and

that

growth

phase

affects

the

excretion of the different toxins differently could lead to a better

understanding of the biological role of the different DSP toxins.

For OA and DTX-1b, starvation caused both intra- and

extracellular levels to increase, resulting in fairly stable ratios

between intra- and extracellular pools of toxin. PTX-2, on the other

hand, only accumulated intracellularly, and the proportion of toxin

found extracellularly thus decreased as growth ceased. At the same

time, the ratios of PTX-2 to OA and PTX-2 to DTX-1b continuously

decreased during the stationary growth phase (Fig. 8E and F). PTX-

2 to OA/DTX-1 ratios also decreased markedly during stationary

growth in the experiment by (Tong et al., 2011). Hence, it would

seem that PTX-2 production and excretion are regulated differ-

ently than OA and DTX-1b, and this may relate to the function of

these different toxins.

4.5. Potential functions of DSP toxins

The results presented here could help reveal the ecological role

of DSP toxins. With the finding that the majority of OA and DTX-1b

was found outside D. acuta cells, the idea that these compounds

play an extracellular role seems increasingly likely.

4.5.1. Allelopathy

Observations of swimming and feeding behavior in mixedM.rubrum and Dinophysis spp. cultures have previously led to the

suggestion that some form of allelopathic interaction occurs

(Nagai et al., 2008; Nishitani et al., 2008). Also, free OA has been

shown to have allelopathic effects on other, potentially compet-

ing, microalgae (Windust et al.,1996). However, concentrations of

free OA 1 mmol l1 were needed to induce a 10% inhibition of

growth in the competing organisms. Evenby optimistically using

the highest possible toxin contents achieved in this experiment

(150 pg OA + DTX-1b cell1), this corresponds to >5 million -

cells l1 a concentration far beyond those found in natural

blooms. It is worth mentioning that this could potentially be

different for the other OA producing genus, Prorocentrum: its

benthic habitat could facilitate local hotspots of high OA

concentrations in the vicinity of Prorocentrum spp. populations,due to stagnant water and high local cell densities. Besides,

competing microalgae, such as those studied by (Windust et al.,

1996), may not even be the intended target organisms of

allelopathiceffects of OAand DTXs.Okadaic acid hasbeen shown

to affectfungi, andthese, or even a third group of organisms, could

be the primary intended targets (Nagai et al., 1990). For now, OA

and DTXs as allelochemicals remains an unproven, largely

unexplored, but still plausible theory.

4.5.2. Grazer defense

DSP toxins have also been suggested to work as grazer

repellents (Carlsson et al., 1995), and some copepod species have

been shown to select against Dinophysis spp. in mixed phyto-

plankton

assemblages

(Carlsson

et

al.,

1995;

Kozlowsky-Suzukiet al., 2006; Setala et al., 2009). But food selectivity is common in

copepods, and may result from sizes or food quality rather than

toxicity. This was exemplified in a study involving Dinophysis

norvegica by (Jansen et al., 2006), who showed that only one out of

three copepods grazed on D. norvegica, but that the same copepod

species was also the only one to graze Ceratium furca, a similar-

sized non-DSP containing dinoflagellate. The study by Carlsson

et al. (1995) is, to our knowledge, so far the only study to show

direct negative effects of Dinophysis spp. consumption on the

copepod grazer. Negative effects on e.g. survival or egg production,

or behavioral changes such as those found when copepods are fed

other toxic algae (Huntley et al., 1986; Sykes and Huntley, 1987),

needs to be demonstrated if the theory of DSP toxins as grazer

repellents

is

to

gain

favor.



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http://www.recmar-galicia.org/abstract.asp?Idpro=3


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