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Domoic-acid-production-by-Pseudo-nitzschia-a_2008_Harmful-Algae

Domoic acid production by Pseudo-nitzschia calliantha Lundholm,Moestrup et Hasle (bacillariophyta)isolated from the Black Sea

Sengul Besiktepe a ,*,Larisa Ryabushko b ,Dilek Ediger a ,1,Doruk Yilmaz a ,

Arife Zenginer a ,Vitaly Ryabushko b ,Raisa Lee b

a

Institute of Marine Sciences,Middle East Technical University,P .O.Box 28Erdemli 33731,Mersin,Turkey

b

Institute of Biology of the Southern Seas,National Academy of Sciences of the Ukraine,2Nakhimov Av.,Sevastopol 99011,Ukraine

Received 14May 2007;received in revised form 7September 2007;accepted 28September 2007

Abstract

A species of Pseudo-nitzschia isolated from Sevastopol Bay,Black Sea,was examined for its toxicity.The species was identi?ed as P .calliantha Lundholm,Moestrup et Hasle based on SEM and TEM examination.Domoic acid (DA)was detected in batch culture throughout the growth cycle of P .calliantha .The production of DA by this diatom species was con?rmed by ?uorenylmethoxycarbonyl (FMOC)derivatization and HPLC-?uorescence method.The cellular DA level was higher in the early exponential phase,with the maximum value of 0.95pg DA cell à1.In the stationary phase,the cellular DA levels declined.This is the ?rst record of a DA producing diatom isolated from the Black Sea.#2007Elsevier B.V .All rights reserved.

Keywords:Domoic acid;Harmful algae;Diatom;Pseudo-nitzschia calliantha ;Black Sea

1.Introduction

Production of domoic acid (DA),the toxin responsible for amnesic shell?sh poisoning (ASP),has been reported for several species of the diatom genus Pseudo-nitzschia H.Peragallo .Marine invertebrates,birds and mammals can accumulate considerable amounts of this toxin.Filter feeding bivalves consuming toxin producing Pseudo-nitzschia species can accumulate DA to high concentrations and human consumption of these contaminated bivalves can result in ASP.This event was ?rst recognized in Prince Edward Island,Canada,in 1987with three deaths and over 100people becoming ill after consuming blue mussel Mytilus edulis (Bates et al.,1989).In 1991,the California coast P.australis bloom contaminated anchovies with DA,resulting in the deaths or neurological symptoms of more than 100brown pelican and other marine birds after eating the anchovies (Garrison et al.,1992).Marine mammals deaths were recorded ?rst time due to

the DA poisoning along the California coast (Lefebvre et al.,1999;Scholin et al.,2000).Shell?shery sectors are highly susceptible to impacts of DA toxicity.The high levels of DA in shell?sh have caused the closure of harvesting areas in Oregon and Washington State (Horner and Poster,1993),in the Gulf of https://www.sodocs.net/doc/1018318996.html,wrence (Bates et al.,2002),and in UK waters (Bogan et al.,2007;Campbell et al.,2001;Gallacher et al.,2001).Pseudo-nitzschia species have been commonly observed in the Black Sea (Morozova-V odyanytskaya,1954;Proschkina-Lavrenko,1955;Belogorskaya and Kondratjeva,1965;Bodeanu,1987/1988;Rat’kova,1989;Ryabushko,1991,2003a,b;Mikaelyan et al.,1992;Davidovich and Bates,1998;Bologa et al.,1999;Ryabushko et al.,2000;Moncheva et al.,2001;Vershinin and Kamnev,2001;Uysal,2002;Turkoglu and Koray,2002;Eker-Develi and Kideys,2003;Vershinin et al.,2004,2005).Pseudo-nitzshia pseudodelica-tissima,P .pungens,P .delicatissima ,P .seriata and P.calliantha were reported in Turkish waters of the Black Sea (Turkoglu and Koray,2002;Uysal,2002;Bargu et al.,2002;Eker-Develi and Kideys,2003).High abundance of P .seriata and P .delicatissima were documented along the Bulgarian coasts (Moncheva et al.,2001).Ryabushko (2003a,b)recorded ?ve Pseudo-nitzschia species in the Black Sea and in the Azov Sea;P .delicatissima ,P .fraudulenta ,P.pseudodelicatissima ,P.

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Harmful Algae 7(2008)438–442

*Corresponding author.

E-mail address:sengul@https://www.sodocs.net/doc/1018318996.html,.tr (S.Besiktepe).1

Present address:TUBITAK-Marmara Research Center,Gebze 41470Kocaeli,Turkey.

1568-9883/$–see front matter #2007Elsevier B.V .All rights reserved.doi:10.1016/j.hal.2007.09.004

pungens and P.seriata.Vershinin and Kamnev(2001)cited the blooming of P.pseudodelicatissima and P.seriata(over106 cells Là1)during the winter–spring period in the Black Sea.

Pseudo-nitszchia calliantha was recorded for the?rst time in the Turkish waters of the Black Sea by Bargu et al.(2002).The culture isolated from the Karadag,southern coast of the Crimea,was ascribed to P.pseudodelicatissima by Davidovich and Bates(1998)but after re-examination of the culture the species was identi?ed as P.calliantha(Lundholm et al.,2003).

Toxic and non-toxic clones of Pseudo-nitzschia calliantha from different geographical areas have been reported.Domoic acid production of P.calliantha was reported in the Bay of Fundy,Canada,(Martin et al.,1990)and in Danish waters (Lundholm et al.,1997cited in Lundholm et al.,2003),the species was ascribed to P.pseudodelicatissima.The isolates of P.calliantha from Vietnam waters did not show any presence of domoic acid(Lundholm et al.,2003).

The aims of the present study were to isolate P.calliantha from the Black Sea and to test it for the ability to produce DA in the laboratory culture.

2.Material and methods

2.1.Culture

An unialgal culture of P.calliantha was isolated in October 2005from Sevastopol Bay,Black Sea.A stock culture was kept in F/2medium in1L?ask at19?28C in$20ppt salinity water,under an irradiance of65m Einsteins mà2sà1(12h light:12h dark cycle).Irradiance was measured with the LI-COR Spherical quantum sensor(model L1-193SA,USA).

2.2.Microscopy

For studies of the morphology of P.calliantha,the organic material of a subsample of culture was removed by oxidation (Lundholm et al.,2002).A mid-exponential phase culture was ?xed in2%glutaraldehyde.The organic material was removed by adding0.4ml30%H2SO4and2ml saturated KMnO4to a 2ml sample.After24h,the sample was cleared by addition of 2ml saturated aqueous solution of oxalic acid.The sample was then centrifuged at1500rpm for10min and then rinsed with double distilled water.This last step was repeated several times until the removal of oxalic acid.For SEM examination,samples were?ltered on0.45m m pore size,47mm diameter cellulose acetate membrane?lters,and dried in oven at358C for24h. SEM pictures were taken in TUBITAK-Marmara Research Center,Kocaeli,Turkey.For TEM,drops of cleaned material were placed on formvar-coated copper grids,dried and studied in a JEOL,JEM-101L electron microscope in Mersin University,Mersin,Turkey.

2.3.Toxin analysis

Subsamples were taken from the stock culture every2–3 days until the mid-stationary phase.Aliquots around15–70ml (containing total around2?106cells)taken from the culture were?ltered through GF/F(25mm diameter)?lters and kept at à208C until HPLC analysis.The sample was extracted in10% methanol by sonicating for2min at100W.Finally,the extract was centrifuged at4000rpm for10min and?ltered through a 0.2m m disposable acrodisc(25mm surfactant-free cellulose acetate membrane,Nalgene,Hereford,UK)to remove cell debris,and frozen atà208C before analysis.

DA was analyzed using the?uorenylmethoxycarbonyl (FMOC)derivatization and HPLC-?uorescence method(Pock-lington et al.,1990),with the following modi?cations.The chromatographic system consisted of an Agilent1100HPLC equipped with a?uorometric detector(264nm excitation; 313nm emission).Separations were performed on a Vydac RP 18column(250mm?4.6mm i.d.).The mobile phase consisted of acetonitrile and0.1%tri?uoroacetic acid(TFA) and pumped at0.2ml/min.Gradient elution was programmed linearly from30%to40%acetonitrile over10min

and Fig.1.HPLC-?uorescence chromatogram of domoic acid(DA).Domoic acid has a retention time around21min.

S.Besiktepe et al./Harmful Algae7(2008)438–442439

maintained for 10min.Elution was then followed by an increase to 100%acetonitrile over 2min which was maintained for 3min before programming back to initial conditions over 2min.Initial conditions were maintained for a further 12min,resulting in a total cycle time of 39min.The column temperature was 558C.Injections were done by manual injector (Rheodine)equipped with 20m l loop.DACS 1(Marine Analytical Chemistry Standards Program of the NRC,Halifax,NS)was used for instrument calibration solution and dihydrokainic acid (Sigma)was used as an internal standard.DA per cell was calculated by dividing the DA concentration of the aliquots by the cell number.Representative HPLC chromatogramme of DA was given in Fig.1.Domoic acid has a retention time around 21min (Fig.1).3.Results 3.1.Morphology

Ultrastructural examination by SEM and TEM revealed the cultured isolate from the Sevastopol Bay,Black Sea,as Pseudo-nitzschia calliantha .The appearances of the cells are linear shape in valve view and overlapping in colonies.The tapering part of the valve toward the tips is very short (Fig.2A–C),and the eccentric raphe is divided in the middle by a central nodule (Fig.2D).The apical axis ranged from 47to 115m m (commonly 60–90m m),while the transapical axis of valves is between 1.8and 3.6m m (commonly 2.2–3m m).Fibulae are regularly spaced,with 16–17in 10m m (Fig.2D;Fig.3A and B).The central part of the valve has central nodule.Interstriae number 34–37in 10m m (Fig.2D).Striae are composed of a single row of round to square poroids,with 5–6poroids in 1m m (Fig.3A and B).Each poroid is divided into 3–8sectors

(Fig.3A and B).Valvo-copula are 2–3poroids wide and 3–4poroids high (Fig.3C).3.2.DA content

Culture growth was followed for 27days and it remained in exponential growth phase until day 21(Fig.4).The highest cell concentration was recorded as 245,000cell ml à1in stationary phase.Domoic acid levels were highly variable over time within the cells.The maximum cellular DA values were observed during early exponential phase with the average value of 0.7?0.85at day 3,and 0.95pg DA cell à1at day 5.During the mid-exponential phase DA was not detectable,and during late-exponential and stationary phases,cell DA levels ranged from 0.47pg cell à1at day 17to 0.11pg cell à1at day 21(Fig.4

).

Fig.2.Scanning electron micrographs of Pseudo-nitzschia calliantha isolated from Sevastopol Bay,Black Sea.(A)Whole valve.(B–C)Tips of valves.(D)Central part of valve with central

nodule.

Fig. 3.Transmission electron micrographs of Pseudo-nitzschia calliantha isolated from Sevastopol Bay,Black Sea.(A–B)parts of the valves showing the poroid structures.(C)Cingular band.

S.Besiktepe et al./Harmful Algae 7(2008)438–442

440

4.Discussion

The genus Pseudo-nitzschia is a regular component of the marine phytoplankton in the Black Sea waters,with the cell concentrations reaching more than 1–2?106cells L à1in spring in Sevastopol Bay (Ryabushko et al.,2000;Ryabushko,2003a,b ).A red-tide event was observed in summer 1989near the Bulgarian coast due to the bloom of the dino?agellate Noctiluca scintillans together with the diatom Pseudo-nitschia (Ryabushko,1991,2003a,b ).Pseudo-nitzschia pseudodelica-tissima has been found in the planktonic community during the mussel harvesting period in Caucasian coasts of the Black Sea,and DA assays in mussel samples have given a negative result (Vershinin et al.,2005).This study presents the ?rst identi?cation of P .calliantha from Sevastopol Bay,Black Sea,as a DA producer.Domoic acid production has been documented previously in P .calliantha in the Bay of Fundy,Canada,(Martin et al.,1990)and in Danish waters (Lundholm et al.,1997,cited in Lundholm et al.,2003).In both studies,the species was ascribed to P .pseudodelicatissima ,but after re-examination,it has been con?rmed to belong to P .calliantha (Lundholm et al.,2003).Other cultures from Vietnam waters of the same species appear to be non-toxic (Lundholm et al.,2003).We found the toxin level in the cell between non-detectable and 1.3pg DA cell à1using HPLC–FMOC method.These values are comparable with values measured by Martin et al.(1990)who measured cellular DA levels ranging from 0.007to 0.098pg DA cell à1in the Bay of Fundy,Canada.In laboratory batch cultures,high DA production was generally observed in the late exponential or during stationary phase (Bates et al.,1991;Pan et al.,1996a,b;Cusack et al.,2002;Fehling et al.,2004).In our P .calliantha culture,the high DA level was measured in early exponential phase.A similar pattern was reported by Pan et al.(2001)for P .pseudodeli-catissima from Gulf of Mexico.

Detailed examination of the species revealed that the morphology of the species corresponded to published Pseudo-nitzschia calliantha descriptions in Lundholm et al.(2003).However,there are some variations.The cell width of P .calliantha from Sevastopol Bay is 1.8–3.6m m whereas cells described in Lundholm et al.(2003)are 1.4–1.8m m wide.

Instead of 7–10poroid sectors (Lundholm et al.,2003),our culture has 3–8poroid sectors.These differences may result from environmental variabilities that affect the growth rates of the individuals.

Since our study indicates that Pseudo-nitzschia calliantha from the Black Sea is a DA producer,a detailed phytoplankton monitoring program to identify Pseudo-nitzschia species and their ability to produce DA needs to be carried out in the Black Sea.

Acknowledgements

We would like to thank Dr.Y .Fukuyo and his group for con?rming the species as P .calliantha .We are grateful to Drs.Tulin Baykal and Nejat Y?lmaz for providing their TEM facilities and taking the TEM pictures.A special thank you to Dr.H.Orek for carrying out culture irradiance measurements and Dr.Z.Uysal for useful discussions.This study was supported through The Scienti?c and Technical Research Council of Turkey and The Ministry of Ukraine for Education and Science Joint Research Project 104Y053.[TS]References

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