Detection of microalgal resting cysts in European coastal sediments using a PCR-based assay

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Detection of microalgal resting cysts in European coastal sediments using aPCR-based assay

Antonella Penna a,�, Cecilia Battocchi a, Esther Garces b, Silvia Angl�es b, Emellina Cucchiari c,Cecilia Totti c, Anke Kremp d, Cecilia Satta e, Maria Grazia Giacobbe f, Isabel Bravo g, Mauro Bastianini h

a Department of Biomolecular Sciences, University of Urbino, Viale Trieste 296, 61100 Pesaro, Italyb Institut de Ci�encies del Mar, CSIC, Passeig Marıtim de la Barceloneta 37-43, 08003 Barcelona, Spainc Dipartimento di Scienze del Mare, Universit�a Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italyd Tvarminne Zoological Station, University of Helsinki, 10900 Hanko, Finlande Dip. Botanica ed Ecologia Vegetale, University of Sassari, 07100 Sassari, Italyf Istituto per l’Ambiente Marino Costiero, CNR, Spianata S. Raineri 86, 98122 Messina, Italyg Instituto Espanol de Oceanografıa, Apdo. 1552, 36200 Vigo, Spainh CNR-ISMAR, Istituto di Scienze Marine, Castello 1364/A, 30122 Venezia, Italy

a r t i c l e i n f o

Available online 23 September 2009

Keywords:

HAB species

Mediterranean Sea

PCR

Ribosomal genes

Resting stages

Sediments

a b s t r a c t

A PCR-based assay was developed and applied to sediment and sediment trap samples for the detection

of different cysts belonging to dinoflagellates and raphidophytes in European coastal areas.

Oligonucleotide primers were designed based on the ITS-5.8S and LSU ribosomal gene sequences.

The specificity and sensitivity of the PCR assay were assessed using genomic DNA from clonal cultures,

plasmid copy number of cloned target sequences, as well as from sediment samples. Qualitative PCR

determinations of different cysts in sediment and sediment trap samples were compared to taxonomic

examinations by light microscopy. This molecular methodology permitted a fast and specific detection

of target cysts in sediment samples. We also detected dinoflagellate and raphidophyte cysts at

concentrations not detectable by microscopic methods or that are difficult to identify. The results given

by molecular and microscopic methods were comparable. However, higher values of positive detection

for target cysts were obtained by PCR than with microscopy. Some taxa were detected in 100% of the

samples using PCR, while others were only found in 10% of the samples. The data obtained in this study

showed that the PCR-based method is a valid tool for cyst identification in marine sediments.

& 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Harmful Algal Blooms (HABs) are recurring events in Europeancoastal waters (Giacobbe et al., 2007; Smayda, 2007). Many HABspecies are responsible for these events and the majority aredinoflagellates (IOC Taxonomic reference list, http://www.bi.ku.dk/ioc/). Most HAB dinoflagellates display heteromorphic lifecycles, including motile planktonic stages, as well as immotilebenthic resting cysts. All stages of the life cycle, and especially thedormant cysts, have a large impact on bloom dynamics. Thebloom development of cyst-forming HAB species may also bedependent on the presence of cyst beds seeding local blooms(Garces et al., 2002; Steidinger and Garces, 2006). While greateffort has been dedicated to the study of the planktonic stages ofthese microorganisms, we still have limited knowledge of theirother life stages, especially benthic stages. In some dinoflagellates

and raphidophytes, the resting stages with their resistant wallspermits the species to tolerate environmental conditions andtherefore to expand their geographical distribution (Amorim et al.,2001; McGillicuddy et al., 2003; De Boer et al., 2004; Edvardsenand Imai, 2006).

Knowledge of the cyst bed composition and location in bottomsediments can provide information on the long-term presence of aspecies in an area, as well as enabling prediction of subsequentblooms. Mapping the cyst distribution of HAB species alsoprovides information on cyst transport to new areas, dependingon oceanic currents. Moreover, cyst distribution reflects thesedimentary dynamics and the location of blooms in the overlyingsurface waters (Angl�es et al., 2010). Further, cyst assemblage dataprovide an indication of the potential plankton diversity reservoirin a locality. All this information is necessary to understand andpredict potential HAB development and expansion. Therefore,monitoring the distribution of cyst densities in coastal areas priorto an outbreak is important for localizing hot spots for blooms andto minimize the damage caused by harmful blooms (Bravo et al.,2006).

ARTICLE IN PRESS

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/dsr2

Deep-Sea Research II

0967-0645/$ - see front matter & 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.dsr2.2009.09.010

� Corresponding author.

E-mail address: [email protected] (A. Penna).

Deep-Sea Research II 57 (2010) 288–300

https://www.researchgate.net/publication/222717228_Recurrent_high-biomass_blooms_of_Alexandrium_taylorii_Dinophyceae_a_HAB_species_expanding_in_the_Mediterranean_Sea?el=1_x_8&enrichId=rgreq-5b3247a4373fc4123056998a33b03841-XXX&enrichSource=Y292ZXJQYWdlOzIyMzY0Njg4OTtBUzo5NzMwOTE3NDAwOTg2MUAxNDAwMjExNzIzNTU4

https://www.researchgate.net/publication/223582418_Reflections_on_the_ballast_water_dispersal-harmful_algal_bloom_paradigm_Harmful_Algae_6601-622?el=1_x_8&enrichId=rgreq-5b3247a4373fc4123056998a33b03841-XXX&enrichSource=Y292ZXJQYWdlOzIyMzY0Njg4OTtBUzo5NzMwOTE3NDAwOTg2MUAxNDAwMjExNzIzNTU4

https://www.researchgate.net/publication/226142732_Importance_of_Life_Cycles_in_the_Ecology_of_Harmful_Microalgae?el=1_x_8&enrichId=rgreq-5b3247a4373fc4123056998a33b03841-XXX&enrichSource=Y292ZXJQYWdlOzIyMzY0Njg4OTtBUzo5NzMwOTE3NDAwOTg2MUAxNDAwMjExNzIzNTU4

https://www.researchgate.net/publication/223854085_Alexandrium_minutum_resting_cyst_distribution_dynamics_in_a_confined_site?el=1_x_8&enrichId=rgreq-5b3247a4373fc4123056998a33b03841-XXX&enrichSource=Y292ZXJQYWdlOzIyMzY0Njg4OTtBUzo5NzMwOTE3NDAwOTg2MUAxNDAwMjExNzIzNTU4

Author's personal copyARTICLE IN PRESS

Studies on resting cyst diversity and distribution have beenhampered by difficulties in the reliable identification of species-specific resting stages, since dinoflagellate and raphidophyte cystsdo not always have species-specific morphological features.Traditional microscopic methods, which are commonly employedfor monitoring of HAB species, often do not allow identification,unless the corresponding vegetative form can be providedthrough in vitro germination of the respective cysts. Moleculartechniques and in particular, PCR techniques based on theamplification of targeted ribosomal genes, have been developedfor the rapid and accurate detection and quantification ofvegetative cells of HAB species both in culture and field samples(Galluzzi et al., 2004; Coyne et al., 2005; Godhe et al., 2007; Pennaet al., 2007). The PCR detection technique can be applied tosamples from sediment cores and sediment traps containing avariety of cysts. Such molecular-based assays can accurately andrapidly identify a variety of specific taxa in the sediments,overcoming the problem of taxonomic identification by micro-scopy (Bolch, 2001; Bowers et al., 2006). However, to date, only afew dinoflagellates have been identified in sediment samplesusing the PCR approach (Godhe et al., 2002; Kamikawa et al.,2007). The success of the PCR-based approach in sediments isstrictly dependent on the inhibitory substances contained in thesediment material (Saito et al., 2002). Different strategies areapplied to overcome the problem of PCR inhibition caused bycontaminating substances: the use of commercial nucleic acidextraction kits that remove inhibitors and facilitate purification;the use of thermostable DNA polymerase; the dilution of templateDNA prior to PCR assay and the use of BSA (bovine serumalbumin) in the PCR assay.

In this study, a PCR-based assay was developed and applied tosediment and sediment trap samples for the detection of restingcysts from several taxa. The specificity and sensitivity of the PCRassay were assessed both in clonal microalgal culture and fieldsediment samples that were collected from several Europeancoastal areas. Oligonucleotide primers specific to several HABdinoflagellate species, namely Alexandrium spp., Lingulodinium

polyedrum, Protoceratium reticulatum, Gymnodinium catenatum,G. nolleri, and the raphidophyte Fibrocapsa japonica were applied inthe PCR-based assay to identify the corresponding cyst morpho-types in sediment and sediment trap samples from different areasof the Mediterranean, Baltic Sea, and the East Atlantic coast. Theassay was based on the LSU, 5.8S rRNA genes and ITS regions astarget regions for the taxa specific primers and was validated onthe genomic DNA of the clonal cultures and preserved cystsamples. A protocol for species-specific PCR detection of cystswas developed based on the use of a nucleic acid purification kit.The specificity and sensitivity of each assay was determined andcomparisons of the qualitative determinations of the PCR analysisand optical microscopy were made. Data on the detection of theresting cyst stage of the different HA species are shown.

2. Methods

2.1. Study sites

Surface sediment samples were collected during the years2006 and 2007 from 38 sampling stations located at 11 differentsites (Table 1) in the Mediterranean and Baltic Sea and the EastAtlantic (Fig. 1). In the Mediterranean Sea, the sampling sites weredistributed in four regional Seas: the North West Adriatic, Ionian,Tyrrhenian, and Catalan Sea. The sampling sites in the Baltic Seaand East Atlantic Ocean were located in the coastal NorthernBaltic Sea and the western coast of Spain, respectively. Stationswere located in areas where dinoflagellate or raphidophyte

blooms occur, and in sediment accumulation areas, as indicatedby deposition maps, e.g. for the Arenys harbor (Garces et al., 2004;Angl�es et al., 2010) and Ria de Vigo (Bravo and Anderson, 1994).

2.2. Cyst sample collection, purification, and microscopic

morphotype identification

Vertical sediment samples up to 8–10 cm in depth werecollected using a gravity corer (7.2 cm internal diameter). Threereplicate samples were taken per station. Sediment samples ofArenys de Mar (Catalan Sea) and Olbia (Tyrrhenian Sea) harborswere collected and processed as indicated in Satta et al. (2010).Sediment samples (7.5 ml) were extracted from the undisturbed3-cm surface layer in the sample core container using a syringeand were transferred into a 50-ml conical tube. In addition,sediment samples of 10 ml were collected from the bottom 3 cm ofthe sediment surface layer by a SCUBA diver using a plasticsyringe. Samples were preserved by adding Lugol’s solution(Andersen and Throndsen, 2003) in filtered seawater and storingthem in the dark at 4 1C until analysis. Three samples were takenfrom sediment traps, collected at the Arenys harbor (Catalan Sea)and Baiona site (East Atlantic coast); the sampling of sedimenttraps was carried out following the method of Garces et al. (2004).Settled material was collected from the traps every 4 days andtrap subsamples of 48 ml were fixed with Lugol’s solution andkept in the dark at 4 1C until analysis.

Subsamples of 2.5 ml of suspended sediments were sonicated(Bandelin, Germany) to disaggregate cysts from sediment parti-cles, sieved on steel membrane (Endecotts, UK) using sizefractionation of 100- and 10-mm membranes and transferred intofiltered seawater. The density gradient method was applied tosediment subsamples to separate cysts from detrital material(Amorim et al., 2001; Bravo et al., 2006). Samples were then usedboth for microscopic examination (2 ml subsamples) and mole-cular analysis (5-ml subsamples). The resting cysts were identifiedand counted under an inverted microscope at 200� and 400�magnification by scanning the entire Utermohl sedimentationchamber. Different morphotypes of dinoflagellate and raphido-phyte cysts were classified according to shape, color, wallthickness, and size. Cyst abundance was expressed as the cystnumber per volume of wet sediment sample and total cystnumber processed for molecular analysis.

2.3. Genomic DNA extraction of sediment and trap samples

Sediment samples (5 ml), obtained as above, were centrifugedat 4000 rpm for 10 min at room temperature; the supernatant wasgently discarded and 20 ml of filtered seawater were added;samples were again centrifuged at 4000 rpm for 10 min at roomtemperature. This washing step was repeated twice; then, the cystpellet was resuspended in 1 ml of sterile MilliQ water andcentrifuged at 10,000 rpm for 5 min at room temperature; thesupernatant was discarded and the pellet was frozen at �80 1Cuntil nucleic acid extraction. The pellet was thawed at +65 1C for15 min, and re-frozen at �80 1C for 15 min; this freeze–thaw stepwas repeated twice. After the last thawing, the pellet wassonicated for 20 min in an ultrasonic bath and then added to atube containing beads and lysis buffer of the UltraClean Soil DNaKit (MoBio Lab In., Solana Beach, CA). DNA extraction andpurification were carried out according to the manufacturer’sinstructions. Purified genomic DNA was quantified on an agarosegel using serially diluted Lambda DNA Marker (MBI Fermentas,Germany) and a gel-documentation apparatus (Bio-Rad, Hercules,CA, USA).

A. Penna et al. / Deep-Sea Research II 57 (2010) 288–300 289


Ta

ble

1S

ed

ime

nt

sam

ple

sco

lle

cte

da

lon

gse

ve

ral

coa

sta

la

rea

sin

the

Me

dit

err

an

ea

n,

Ba

ltic

Se

aa

nd

Ea

ste

rnA

tla

nti

c,cy

sta

bu

nd

an

cep

er

sam

ple

an

dp

roce

sse

dcy

sta

bu

nd

an

cein

the

mo

lecu

lar

ba

sed

ass

ay.

Ge

og

rap

hic

al

are

aLo

cali

tie

sa

nd

sam

pli

ng

sta

tio

ns

Sta

tio

nco

ord

ina

tes

Sa

mp

lin

gp

eri

od

Sta

tio

n

nu

mb

er

Sa

mp

lin

gty

pe

Cy

sta

bu

nd

an

ce

(ml�

1�

we

tse

dim

en

t)

To

tal

pro

cess

ed

cyst

ab

un

da

nce

NW

Ad

ria

tic

Se

a,

Ita

lyV

en

ezi

a,

C1

04

51

150

0000

N,

121

450

6000

EJa

nu

ary

20

06

1S

ed

ime

nt

core

42

84

28

6

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,S

ou

thA

431

300

0900

N,

131

410

0000

EJu

ly2

00

62

Se

dim

en

tco

re1

26

18

90

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,S

ou

thC

431

300

0000

N,

131

410

0700

EJu

ly2

00

63

Se

dim

en

tco

re9

41

41

5

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,S

ou

thB

431

300

0500

N,

131

410

0300

EJu

ly2

00

64

Se

dim

en

tco

re7

71

10

5

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,S

ou

th-o

ut

CD

431

300

0000

N,

131

410

0700

ED

ece

mb

er

20

06

5S

ed

ime

nt

core

12

01

80

5

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,N

ort

h-o

ut

AB

431

300

0900

N,

131

410

0000

ED

ece

mb

er

20

06

6S

ed

ime

nt

core

59

89

0

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,S

ou

th-i

nA

B4

31

300

0500

N,

131

410

0300

ED

ece

mb

er

20

06

7S

ed

ime

nt

core

78

11

75

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,N

ort

hC

431

410

0400

N,

131

230

0000

ED

ece

mb

er

20

06

8S

ed

ime

nt

core

30

64

59

0

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,N

ort

h-i

nA

BL

431

420

0100

N,

131

220

0000

ED

ece

mb

er

20

06

9S

ed

ime

nt

core

17

02

55

5

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,N

ort

h-o

ut

AB

L4

31

430

0400

N,

131

190

0000

ED

ece

mb

er

20

06

10

Se

dim

en

tco

re2

47

37

00

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,N

ort

hB

431

430

0400

N,

131

190

0000

EJu

ly2

00

61

1S

ed

ime

nt

core

51

27

68

0

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,N

ort

hA

431

420

0100

N,

131

220

0000

EJu

ly,

20

06

12

Se

dim

en

tco

re4

70

70

50

NW

Ad

ria

tic

Se

a,

Ita

lyA

nco

na

,N

ort

h-o

ut

DE

R4

31

410

0400

N,

131

230

0000

EJu

ly2

00

61

3S

ed

ime

nt

core

38

65

79

0

NT

yrr

he

nia

nS

ea

,It

aly

Olb

iaA

401

550

4500

N,

91

300

2500

EM

ay

20

07

14

Se

dim

en

tco

re1

21

37

5

NT

yrr

he

nia

nS

ea

,It

aly

Olb

iaB

401

550

4500

N,

91

300

4500

EM

ay

20

07

15

Se

dim

en

tco

re5

91

77

NT

yrr

he

nia

nS

ea

,It

aly

Olb

iaG

401

550

1500

N,

91

300

1400

EM

ay

20

07

16

Se

dim

en

tco

re9

72

91

ST

yrr

he

nia

nS

ea

,It

aly

Vu

lca

no

381

250

0700

N,

141

570

1100

EM

ay

20

06

17

Se

dim

en

tco

re7

10

0

Ion

ian

Se

a,

Ita

lyS

t.1

C,

Sir

acu

sa3

71

030

5100

N,

1511

70

0100

EM

arc

h2

00

71

8S

ed

ime

nt

core

15

93

65

Ion

ian

Se

a,

Ita

lyS

t.2

C,

Sir

acu

sa3

71

030

2100

N,

1511

70

4000

EM

arc

h2

00

71

9S

ed

ime

nt

core

13

42

81

Ion

ian

Se

a,

Ita

lyS

t.E

,S

ira

cusa

371

030

5100

N,

151

170

01

EM

arc

h2

00

72

0S

ed

ime

nt

core

13

Ion

ian

Se

a,

Ita

lyA

qu

acu

lt.

B,

Sir

acu

sa3

71

020

0900

N,

151

170

20

EM

arc

h2

00

72

1S

ed

ime

nt

core

56

17

5

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,S

t.1

54

11

340

6900

N,

21

330

6800

EA

pri

l2

00

62

2T

rap

sam

ple

36

18

07

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,S

t.1

14

11

340

6900

N,

21

330

6800

EA

pri

l2

00

62

3T

rap

sam

ple

38

18

95

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,S

t.2

3-1

411

340

7300

N,

21

330

3400

EJu

ne

20

06

24

Se

dim

en

tco

re1

85

73

71

4

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,S

t.1

2+

St.

17

411

340

7200

N,

21

330

7000

E4

11

340

6000

N,

21

330

4400

E

Jun

e2

00

62

5S

ed

ime

nt

core

26

56

10

64

0

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,S

t.8

+S

t.1

34

11

340

6900

N,

21

330

4200

E4

11

340

6600

N,

21

330

7000

E

Jun

e2

00

62

6S

ed

ime

nt

core

25

91

10

36

4

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,2

411

340

7300

N,

21

330

3400

EO

cto

be

r2

00

62

7S

ed

ime

nt

core

24

15

48

30

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,1

/34

11

340

7300

N,

21

330

3400

EO

cto

be

r2

00

62

8S

ed

ime

nt

core

23

45

93

80

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,2

-4

411

340

7300

N,

21

330

3400

ED

ece

mb

er

20

06

29

Se

dim

en

tco

re2

83

71

13

48

NC

ata

lan

Se

a,

Sp

ain

Are

ny

s,1

/5/6

411

340

7300

N,

21

330

3400

ED

ece

mb

er

20

06

30

Se

dim

en

tco

re3

13

21

87

92

Ea

stA

tla

nti

c,S

pa

inB

aio

na

24

21

070

1200

N,

81

500

1500

EA

ug

ust

20

06

31

Tra

psa

mp

le1

14

05

70

0

Ea

stA

tla

nti

c,S

pa

inR

iad

eV

igo

a4

21

130

0800

N,

81

510

1400

EM

arc

h2

00

63

2S

ed

ime

nt

core

15

18

0

Ea

stA

tla

nti

c,S

pa

inR

iad

eV

igo

b4

21

130

0800

N,

81

510

1400

EM

arc

h2

00

63

3S

ed

ime

nt

core

11

41

36

8

Ea

stA

tla

nti

c,S

pa

inB

aio

na

28

421

070

1200

N,

81

500

1500

EJu

ly2

00

63

4T

rap

sam

ple

65

53

60

0

Ea

stA

tla

nti

c,S

pa

inB

aio

na

64

21

070

1200

N,

81

500

1500

ED

ece

mb

er

20

06

35

Se

dim

en

tco

re2

80

14

00

Ba

ltic

Se

a,

Sw

ed

en

Him

me

rsfj

ard

en

581

590

0700

N,

171

430

6000

EM

arc

h2

00

73

6S

ed

ime

nt

core

43

43

90

60

Ba

ltic

Se

a,

Fin

lan

dS

torf

jard

en

591

510

31

400

N,

2311

30

00

100

EM

ay

,2

00

73

7S

ed

ime

nt

core

88

01

79

21

35

Ba

ltic

Se

a,

Fin

lan

dLa

ng

ska

r5

91

470

33

400

N,

231;

200

28

400

EM

ay

20

07

38

Se

dim

en

tco

re6

34

22

85

39

0

A. Penna et al. / Deep-Sea Research II 57 (2010) 288–300290


2.4. Microalgal cultures and genomic DNA extraction

Clonal cultures were used for taxon-specific PCR-based assays(Table 2). Cultures were kept in F/2 or L1 media (https://ccmp.bigelow.org/) at 2071 1C in a 12:12 h (light:dark) photoperiod.Illumination was provided by photon irradiance of 100mmolm–2 s–1. Culture subsamples containing 100,000 cells of eachtarget species were used for the genomic DNA extraction using aDNeasy Plant Kit (Qiagen, Valencia, CA, USA) according to Pennaet al. (2005).

2.5. PCR amplification detection assay

Genus (Alexandrium) and species-specific (Alexandrium minu-

tum, A. taylori, A. tamarense, A. catenella, L. polyedrum,P. reticulatum, F. japonica) primers were designed in the 5.8SrDNA-ITS regions and/or were derived from Penna et al. (2007).The primers for the genus Scrippsiella and species G. catenatum,G. nolleri and Scrippsiella hangoei were designed in the LSU and5.8S rDNA-ITS regions by using OLIGO software ver. 6.65 andBioEdit software ver. 7.0.5 (Table 3). The BLAST (Basic LocalAlignment Search Tool) analysis of alignment nucleotidesequences (Altschul et al., 1990) were as follows: primerG. catenatum: F0 ATTGCAGAATTCCGTGAA, total alignment length:18 nt, overall identity: 100% (18/18 nt). Primer G. nolleri

F0 TTTCAGCGATGGATGTCT, total alignment length: 18 nt, overallidentity: 100% (18/18 nt). Primer Scrippsiella sp. F0 CTGAAA

GGAAAGCGAATGGAG, total alignment length: 22 nt, overallidentity: 63.64% (14/22 nt). Primer S. hangoei F0 TTCGTTCGGAAGTGGTTT, total alignment length: 18 nt, overall identity:100% (18/18 nt). The LSU and 5.8S rDNA-ITS sequences forG. catenatum VGO-744 (AM998536), G. nolleri VGO-663(AM998534), G. nolleri 922I (AM998535), Scrippsiella rotunda

CBA-4 (AM998538), and S. trochoidea VGO-S3V (AM998537)were obtained in this study; the 5.8S rDNA-ITS sequences forS. hangoei were obtained from GenBank. Amplification fromsediment and sediment trap samples for the detection ofdifferent cyst taxa was performed in an Applied Biosystems DNAThermo Cycler 2720 (Foster City, CA, USA). PCR amplificationswere carried out directly using genus- or species-specific primersor by following two steps as follows: an initial PCR usingeukaryotic primers targeting the ITS-5.8S or LSU ribosomalgenes; then, a second (i.e. nested) PCR reaction, using genus-and species-specific primers on amplified products of ribosomalgenes (Fig. 2).

2.6. Direct PCR-based assay

Direct PCR using genus- or species-specific primers was asfollows: reaction tubes contained a 50mL mixture of 200–400mMof each dNTP; 0.2–0.4mM of each primer; 3.0–6.0 mM MgCl2; 1�Reaction Buffer (Eppendorf, Germany); 0.5–1� Enhancer Buffer(Eppendorf, Germany); 0.2–1.5mg/ul BSA (Table 4); 0.5 U of TaqPolymerase (Eppendorf, Germany) and 0.5–1.0 ng of sediment

Fig. 1. Locations of the sampling stations in the Mediterranean Sea, East Atlantic and Baltic Sea (see Table 1 for locality names).

Table 2Clonal cultures used together to test the specificity of genus and species specific PCR identification assay.

Species Strain Collection site Source

Alexandrium minutum VGO663 Tyrrhenian Sea, Olbia, Italy Fraga S.

Chaetoceros socialis CCMP 204 Catalan Sea, Marseille, France Berland B.

Fibrocapsa japonica CBA-1 Adriatic Sea, Ancona, Italy Totti C.

Gymnodinium catenatum GC12V East Atlantic, Vigo, Spain Figueroa R.I.

Gymnodinium nolleri 922I Kattegat, Denmark Ellegard M.

Scrippsiella hangoei SHTV-1 Baltic Sea, Tvarmine, Finland Kremp, A.

Scrippsiella rotunda CBA-4 Adriatic Sea, Pesaro, Italy Ingarao C.

Scrippsiella trochoidea VGOS3V East Atlantic, Vigo, Spain Bravo I.

Prymnesium parvum CCMP708 North Atlantic, Scotland, UK Droop M.

Skeletonema marinoi CBA-2 Adriatic Sea, Pesaro, Italy Penna A.

Ostreopsis ovata CNR-D1 Ligurian Sea, Genova, Italy Giacobbe M.G.

Pseudo-nitzschia spp. CBA-4 Adriatic Sea, Pesaro, Italy Capellacci S.



Table 3Oligonucleotide primers targeting the ITS-5.8S rDNA and LSU regions of different phytoplankton genera and species used in this study.

Target taxa Forward Primer Reverse primer Forward primer sequence (50- 30) -

Reverse primer sequence (50- 30) ’

Amplification

size (bp)

G + C % Primer positions

Alexandrium spp.a 5.8S-50 5.8S-30 F0-GCAADGAATGTCTTAGCTCAA 135 38.0 5.8S (50-30)

R0-GCAMACCTTCAAGMATATCCC 42.8 5.8S (30’50)

A. catenellaa ITS1c 5.8S-30 F0-AGCATGATTTGTTTTTCAAGC 226 33.3 ITS1 (50-30)


A. minutuma ITS1m 5.8S-30 F0-CATGCTGCTGTGTTGATGACC 212 52.3 ITS1 (50-30)


A. tamarensea 5.8S-50 ITS2t F0- TGTTACTTGTACCTTTGGGA 134 40.0 5.8S (50-30)

R0- ACAACACCCAGGTTCAAT 44.4 ITS2 (30’50)

A. tayloria ITS1t 5.8S-30 F0-TGGTGTTTGAATGCGGTTGT 297 45.0 ITS1 (50-30)


F. japonicaa Fibrocapsa F Fibrocapsa R F0-GCAGAGTCCAGCGAGTCATCA 180 57.1 5.8S (50-30)

R0-TAATATCCCAGACCACGCCAGA 50.0 ITS2 (30’50)

G. catenatumb Gymno-cat F Gymno-cat R F0-ATTGCAGAATTCCGTGAA 290 38.9 5.8S (50-30)

R0-GATCGATGCGAATGAAAC 44.4 ITS2 (30’50)

G. nollerib Gymno-nolleri F Gymno-nolleri R F0-TTTCAGCGATGGATGTCT 350 44.4 5.8S (50-30)

R0-TGAAGGCACGATTGACAC 50.0 ITS2 (30’50)

L. polyedruma L. Poly GF L. Poly GR F0-ATGTGTTCTCATCGGATGTTG 383 45.5 ITS1 (50-30)

R0-CACAGTACCGCTGCCACTTAAA 50.0 ITS2 (30’50)

P. reticulatuma P. ret F P. ret. R F0-TGCTGATTGCCATCTATCTT 382 40.0 ITS1 (50-30)

R0-CAGAAGCGCGTTAAACAG 50.0 ITS2 (30’50)

Scrippsiella sp.b Scripp F Scripp R F0-CTGAAAGGAAAGCGAATGGAG 221 47.6 LSU (D1-D2)

R0-CCGCAAATGAGTTCCAACAAG 47.6 LSU (D1-D2)

S. hangoeib Scripp-hangoei F Scripp-hangoei F F0-TTCGTTCGGAAGTGGTTT 274 44.4 ITS1 (50-30)

R0-CCCGAGAGCACCTTAACA 55.6 ITS2 (30’50)

Degenerate code D = A/G/T.

Degenerate code M = A/C.

Degenerate code Y = C/T.

Degenerate code R = A/G.

a From Penna et al. (2007).b designed in this study.

Fig. 2. Schematic representation of the PCR assays based on the use of genus and species-specific or eukaryotic primers. Direct or nested PCR assays were used for the PCR

amplification of target taxa cyst in sediment and trap samples.



template DNA. PCR conditions were as follows: an initialdenaturation step of 10 min at 95 1C, 40 cycles of 30 s at 95 1C,30 s at 58 1C, and 30 s at 72 1C, and a final extension step of 7 minat 72 1C.

2.7. Nested PCR-based assay

For the nested PCR eukaryotic-specific primers targeting theITS-5.8S rDNA (Adachi et al., 1994) and LSU rDNA (Scholin et al.,1994) primers were used in the first PCR. Reaction tubes containeda 25mL mixture of: 200 mM of each dNTP; 2 pmol of each primer;4 mM MgCl2; 1�Reaction Buffer (Eppendorf, Germany); 0.5 U ofTaq Polymerase (Eppendorf, Germany) and 0.5–1.0 ng of sedimenttemplate DNA. PCR conditions were as follows: an initialdenaturation step of 10 min at 95 1C, 40 cycles of 30 s at 95 1C,30 s at 55 1C and 30 s at 72 1C, and a final extension step of 7 min at72 1C. The PCR-amplified products, which were derived from thePCR assay using universal eukaryotic primers and were not directlyvisualized on agarose gel, were used as template in nested PCRamplification with genus- or species-specific primers using 1mL ofthe first PCR-amplified product; the PCR conditions were asdescribed above for the direct PCR amplification with the exceptionof 35 cycles rather than 40.

2.8. Sequencing

The 5.8S gene and ITS regions of genomic DNA from microalgalspecies were amplified using ITSA/ITSB primers (Adachi et al.,1994). The PCR-amplified products were purified using the QiagenPurification Gel Extraction Kit (Qiagen, CA, California) according tothe manufacturer’s instructions and were then directly sequenced.PCR-amplified products were sequenced on an ABI PRISM 310Genetic Analyser (Applied Biosystems, USA) using the dyeterminator method according to the manufacturer’s instructions(ABI PRISM Big Dye Terminator Cycle Sequencing Ready ReactionKit, Applied Biosystems). Genus- and species-specific PCR-ampli-fied products obtained from sediment samples, such as those fromStation 23 (Arenys) for Alexandrium, Station 11 (Ancona, North B)for A. minutum, Station 8 (Ancona, North C) for A. tamarense,Station 27 (Arenys) for Scrippsiella, Station 18 (Siracusa) forL. polyedrum, Station 12 (Ancona, North A) for P. reticulatum,Station 9 (Ancona, North-in ABL) for F. japonica, Station 33 (Ria deVigo) for G. catenatum and Station 31 (Baiona) for G. nolleri, wereexcised from the gel, purified using QIAquick Gel extraction Kit(Qiagen) and directly sequenced.

2.9. Specificity and sensitivity of the PCR assay

The specificity and sensitivity of the PCR reactions had alreadybeen assessed for Alexandrium, A. minutum, A. tamarense,L. polyedrum, P. reticulatum and F. japonica in Penna et al. (2007).The specificity of the Scrippsiella spp., S. hangoei, G. catenatum andG. nolleri oligonucleotide primers were tested by amplifying targetDNA of the various microalgal genera and species from both clonalcultures of target species and sediment samples with designedspecies-specific primers. The Scrippsiella spp., S. hangoei, G. catena-

tum and G. nolleri genomic DNA was amplified with specific primersin the presence of non-target mixed genomic DNA obtained fromdifferent clonal strains; 1 ng of genomic DNA was used for eachbackground species. The 5.8S rDNA-ITS regions of Scrippsiella spp.,S. hangoei, G. catenatum and G. nolleri were amplified and cloned intothe Pcr 2.1 vector following the manufacturer’s instructions(Invitrogen, Carlsbad, CA, USA). Plasmids containing the target ITS-5.8S and LSU rDNA sequences and non-target mixed genomic DNAwere used as a positive and negative control, respectively.

To assess the sensitivity of the PCR assay, 100, 10 and 1 pg ofeach genomic DNA of Scrippsiella spp., S. hangoei, G. catenatum andG. nolleri were spiked into 10, 5 and 1 ng of the mixed cystpopulation DNA, providing background sediment DNA in themixture, which was then subjected to the PCR assays. Thesensitivity assays were done in triplicate for each amount ofgenomic DNA and taxon examined. The background sedimentsample was checked for the absence of the target taxa (Scrippsiella

spp., S. hangoei, G. catenatum and G. nolleri) by microscopy.Assessment of sensitivity was also performed on a retroviralcloned sequence in an external (non-microalgal) plasmid DNA(Casabianca et al., 1998); this non-microalgal plasmid DNA wasspiked as 104, 103, 102 and 101 copies into 10, 5 and 1 ng of mixedcyst population DNA. The PCR products were resolved on a 1.8%(w/v) agarose, 0.5X TBE buffer gel and were visualized by standardethidium bromide staining under UV light.

3. Results

3.1. Sample collection and cyst content identification by microscopy

A total of 38 sediment and sediment trap samples werecollected during the study in several coastal localities of theMediterranean and Baltic Sea and the north-western coast of Spain.A relatively high diversity of cyst taxa was found, with a total of 32cyst morphotypes identified by microscopy (Table 5). Dinoflagellate

Table 4Different concentrations of dNTPs, primers, MgCl2, Enhancer and BSA specific for each microalgal taxon used in the PCR based assay.

Taxa dNTPs (mM) Primers (mM) MgCl2 (mM) Enhancera BSA (mg/ml)

Alexandrium spp. 0.2 0.4 6.0 No added 0.2

Alexandrium catenella 0.2 0.4 6.0 0.5 0.2

Alexandrium minutum 0.2 0.4 6.0 1.0� 1.0

Alexandrium tamarense 0.4 0.4 6.0 No added 1.0

Alexandrium taylori 0.4 0.4 6.0 0.5 0.4

Gymnodinium catenatum 0.4 0.4 6.0 1x 1.5

Gymnodinium nolleri 0.4 0.4 6.0 No added 0.4

Fibrocapsa japonica 0.4 0.2 3.0 No added 0.2

Lingulodinium polyedrum 0.4 0.2 3.0 0.5 No added

Protoceratium reticulatum 0.4 0.4 3.0 No added 0.2

Scrippsiella spp. 0.1 0.4 6.0 0.5 0.2

Scrippsiella hangoei 0.4 0.4 3.0 No added 0.4

The Taq Master PCR enhancer is a buffer additive that improves thermostability (enzyme half-life) and processing of Taq DNA polymerase by stabilizing the enzyme during

PCR.

a The TaqMaster PCR enhancer is a buffer furnished with the Taq Polymerase (Eppendorf, Germany).



species belonging to Peridiniales and Gonyaulacales dominated theassemblages at all stations. In particular, Protoperidinium andScrippsiella species within the Peridiniales were dominant; butcysts of potentially toxic dinoflagellate species, such as Alexandrium

spp., A. minutum, and Gymnodinium spinifera, were also recorded.Gymnodinium cysts were also found, but were not identified at thespecies level with the exception of G. catenatum, G. nolleri andGymnodinium impudicum. Further, F. japonica cysts were identifiedin sediment samples from the northern Adriatic Sea.

3.2. Specificity and sensitivity of the PCR-based assay

PCR amplifications using primers designed in the highlyvariable ITS regions and conserved 5.8S and LSU (D1/D2)

ribosomal genes of one dinoflagellate genus and three speciesgave amplified fragments of different base pair lengths. PCR-amplified products for the genus Scrippsiella were 221 bp long andfor the three species, S. hangoei, G. catenatum and G. nolleri, rangedfrom 274 to 350 bp in length. The specificity of the genus- andspecies-specific primers was assessed both by multiple alignmenton the silico platform BLAST and by PCR amplification of thegenomic DNA from each genus and species in the presence ofmixed non-target taxa clonal strain DNA together with targetspecies clonal strains. The target genus- and species-specificprimers showed high specificity in all selected PCR-based assaysand no other detectable bands were observed; non-targetgenomic DNA used as background DNA templates was notamplified. The specificity of the PCR-based assay for the otherdinoflagellate genera and species, and the raphidophyte F.

japonica, was assessed in an earlier study by Penna et al. (2007).The sensitivity and the absence of inhibitors of the PCR-based

assay using specific primers were assessed on the plasmid targetcloned sequence of a murine retroviral complex. The PCR assay onplasmid DNA was sensitive enough to detect ten copies of thecloned sequence in the presence of 1 ng of sediment DNA asbackground. The sensitivity of the PCR assay was also assessedusing genomic DNA as a template. The sensitivity of thePCR-based assay carried out on genomic DNA corresponded tothe specific PCR amplification of 1 pg. The presence of 1 ng ofbackground genomic DNA from the sediment sample, containingmixed dinoflagellate resting cysts, did not have an effect on thesensitivity of any of the species-specific PCR-based assays. Assaysusing genomic DNA were inhibited by Z5 ng of the backgroundDNA from the sediment sample (data not shown).

3.3. Detection of dinoflagellate and raphidophyte resting cysts in

coastal sediments

PCR amplification was performed on several Lugol-fixedsediment and sediment trap samples collected during the years2006 and 2007 along the coastal areas of the Mediterranean Sea,north-western coast of Spain and Baltic Sea to detect the presenceof either dinoflagellate resting cysts belonging to the generaAlexandrium and Scrippsiella and species A. minutum, A. tamarense,

A. catenella, A. taylori, G. catenatum, G. nolleri, L. polyedrum,

P. reticulatum, and S. hangoei or raphidophyte resting cystsbelonging to the species F. japonica (Table 6). These samplescontained mixed cyst communities including the targetmorphotypes, with total cyst abundances ranging from 3.0�101

to 7.9�105. The amount of target taxa in the analyzed samplesranged from 1.0�101 to 2.3�105 of cysts. Several direct PCRamplifications, using genus- and species-specific primers onsediment samples, which contained low numbers of target cysts,resulted in faint bands or the absence of bands on agarose gel.Thus, nested PCR amplification reactions were performed onsediment and trap samples using genus- and species-specificprimers. Nested PCR amplification reactions with eukaryotic-specific primers targeting the 5.8S-ITS and LSU rDNA regions first,and then amplifying the genus- or species-specific regions, alwaysgave positive PCR products from the genomic DNA in sedimentsamples. PCR products of the expected size for each genus andspecies were detectable in the samples containing the target cystmorphotype, thus confirming the ability to amplify the targetgenomic DNA by direct or nested PCR-based assay. The PCR assaydetected the presence of the resting stages belonging to differentdinoflagellate taxa even if target cysts were not observed inthe sediment and sediment trap samples by microscopyexaminations. The PCR assays were positive for the presence ofcysts of the genera Alexandrium and Scrippsiella and the species

Table 5List of cyst morphotypes from sediment and sediment trap samples of the

Mediterranean and Baltic Sea and East Atlantic coast sampling stations identified

by microscopy.

Taxa Adriatic

Sea

Ionian

Sea

Tyrrhenian

Sea

Catalan

Sea

Baltic

Sea

DinophyceaeGymnodinialesPolykrikos spp. + +

Polykrikos kofoidii +

Polykrikos schwartzii + +

Gymnodinium spp. + + +

G. catenatum

G. nolleri +

G. impudicum + +

GonyaulacalesAlexandrium spp. + + + + +

Alexandrium minutum + + + + +

Alexandrium

pseudogoniaulax

+

Alexandrium ostenfeldii +

Gonyaulax sp. + + + +

G. spinifera + + +

Woloszynskia halophila +

Lingulodinium

polyedrum

+ + +

Protoceratium

reticulatum

+

Peridiniella catenata +

PeridinialesPentapharsodinium

dalei

+

Pentapharsodinium

tyrrhenicum

+ +

Protoperidinium spp. + + + +

Protoperidinium cfr.

claudicans

+ + +

Protoperidinium

conicum

+ + +

Protoperidinium

compressum

+ +


divaricatum

+

Protoperidinium granii +


oblongum

+ +

Scrippsiella spp. + + + + +

Scrippsiella hangoei +

Scrippsiella lachrymosa + +

Scrippsiella trochoidea + + + +

Undetermined

dinoflagellates

+ + +

RaphidophyceaeFibrocapsa japonica +Total cyst type 18 15 12 14 12



Ta

ble

6.

PC

Rb

ase

da

ssay

on

the

ITS

-5.8

Sa

nd

LSU

rDN

Aa

nd

mic

rosc

op

ya

na

lysi

so

fv

ari

ou

sp

hy

top

lan

kto

ng

en

era

an

dsp

eci

es

pro

cess

ed

cyst

nu

mb

er

fro

mse

dim

en

ta

nd

tra

psa

mp

les

coll

ect

ed

du

rin

gth

ep

eri

od

20

06

-20

07

alo

ng

sev

era

l

coa

sta

la

rea

so

fth

eM

ed

ite

rra

ne

an

,B

alt

icS

ea

an

dE

ast

Atl

an

tic.

Sta

tio

nn

um

be

ra

nd

reg

ion

al

Se

a

Ale

xan

dri

um

spp

.A

.m

inu

tum

A.

tam

are

nse

Scri

pp

siel

lasp

p.

Ge

nu

ssp

eci

fic

PC

R

ass

ay

Pro

cess

ed

cyst

nu

mb

er

Sp

eci

es

spe

cifi

cP

CR

ass

ay

Pro

cess

ed

cyst

nu

mb

er

Sp

eci

es

spe

cifi

cP

CR

ass

ay

Pro

cess

ed

cyst

nu

mb

er

Ge

nu

ssp

eci

fic

PC

R

ass

ay

Pro

cess

ed

cyst

nu

mb

er

St.

1,

NW

Ad

ria

tic

+3

93

+3

93

�a

n.i

.+

74

St.

2,

NW

Ad

ria

tic

+a

22

5+

an

.i.

�a

n.i

.+

18

0

St.

3,

NW

Ad

ria

tic

+3

50

+n

.i.

�a

n.i

.+

26

0

St.

4,

NW

Ad

ria

tic

+a

26

5+

an

.i.

�a

n.i

.+

21

0

St.

5,

NW

Ad

ria

tic

+2

70

+n

.i.

�a

n.i

.+

30

0

St.

6,

NW

Ad

ria

tic

+a

65

+a

n.i

.�

an

.i.

+2

30

St.

7,

NW

Ad

ria

tic

+a

14

0+

an

.i.

�a

n.i

.+

18

0

St.

8,

NW

Ad

ria

tic

+7

15

+n

.i.

+n

.i.

+9

65

St.

9,

NW

Ad

ria

tic

+a

44

5+

an

.i.

�n

.i.

+5

80

St.

10

,N

WA

dri

ati

c+

61

2�

n.i

.+

an

.i.

+9

17

St.

11

,N

WA

dri

ati

c+

10

10

+n

.i.

�n

.i.

+1

88

2

St.

12

,N

WA

dri

ati

c+

89

0+

n.i

.�

n.i

.+

15

15

St.

13

,N

WA

dri

ati

c+

82

5�

n.i

.+

n.i

.+

80

5

St.

14

,N

Ty

rrh

en

ian

�a

3�

a3

�a

n.i

.+

a1

41

St.

15

,N

Ty

rrh

en

ian

�a

n.i

.�

an

.i.

�a

n.i

.+

a8

7

St.

16

,N

Ty

rrh

en

ian

+n

.i.

+n

.i.

�n

.i.

+a

59

St.

17

,S

Ty

rrh

en

ian

+n

.i.

�n

.i.

+n

.i.

n.a

.n

.i.

St.

18

,Io

nia

n+

a2

3+

a2

3�

an

.i.

+a

45

St.

19

,Io

nia

n+

a9

8+

a9

8�

an

.i.

+a

58

St.

20

,Io

nia

n�

a1

�a

n.i

.�

an

.i.

�a

n.i

.

St.

21

,Io

nia

n�

a6

�a

n.i

.�

an

.i.

+7

7

St.

22

,C

ata

lan

+2

3,6

84

+2

3,6

84

�a

n.i

.+

14

,21

1

St.

23

,C

ata

lan

+2

0,4

55

+2

0,4

55

�a

n.i

.+

15

,68

2

St.

24

,C

ata

lan

+8

25

+8

25

�a

n.i

.�

a7

12

St.

25

,C

ata

lan

+8

59

+8

59

�a

n.i

.+

12

03

St.

26

,C

ata

lan

+9

67

+9

67

�a

n.i

.+

11

18

St.

27

,C

ata

lan

+7

44

+7

44

�a

n.i

.+

12

86

St.

28

,C

ata

lan

+4

94

+4

94

�a

n.i

.+

a1

22

9

St.

29

,C

ata

lan

+a

32

6+

a3

26

�a

n.i

.�

a1

25

1

St.

30

,C

ata

lan

+4

37

+4

37

�a

n.i

.+

14

29

St.

31

,E

ast

Atl

an

tic

+5

70

0+

57

00

�a

n.i

.+

an

.i.

St.

32

,E

ast

Atl

an

tic

+n

.i.

+a

n.i

.�

an

.i.

+a

n.i

.

St.

33

,E

ast

Atl

an

tic

+n

.i.

+a

n.i

.�

an

.i.

+a

n.i

.

St.

34

,E

ast

Atl

an

tic

+3

60

0+

36

00

�a

n.i

.+

n.i

.

St.

35

,E

ast

Atl

an

tic

+a

n.i

.+

an

.i.

�a

n.i

.+

n.i

.

St.

36

,B

alt

icS

ea

+1

30

0+

30

0�

an

.i.

+7

83

0

St.

37

,B

alt

icS

ea

+6

50

�n

.i.

�a

n.i

.+

29

70

St.

38

,B

alt

icS

ea

+1

75

0�

n.i

.�

an

.i.

+5

82

0



Sta

tio

nn

um

be

r

an

dre

gio

na

lS

ea

L.p

oly

edru

mP.

reti

cula

tum

F.ja

po

nic

aG

.ca

ten

atu

mG

.n

oll

eri

Spec

ies

spec

ific

PC

R

ass

ay

Pro

cess

edcy

st

nu

mb

er

Spec

ies

spec

ific

PC

R

ass

ay

Pro

cess

ed

cyst

nu

mb

er

Sp

eci

es

spe

cifi

cP

CR

ass

ay

Pro

cess

ed

cyst

nu

mb

er

Sp

eci

es

spe

cifi

cP

CR

ass

ay

Pro

cess

edcy

st

nu

mb

er

Spec

ies

spec

ific

PC

R

ass

ay

Pro

cess

edcy

st

nu

mb

er

St.

1,

NW

Ad

ria

tic

+6

07

+n

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

2,

NW

Ad

ria

tic

+a

10

0�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

3,

NW

Ad

ria

tic

+a

80

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

4,

NW

Ad

ria

tic

+a

15

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

5,

NW

Ad

ria

tic

+a

70

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

6,

NW

Ad

ria

tic

+a

50

�a

5.0

�a

n.i

.�

an

.i.

�a

n.i

.

St.

7,

NW

Ad

ria

tic

+a

75

+a

10

�a

n.i

.�

an

.i.

�a

n.i

.

St.

8,

NW

Ad

ria

tic

+4

15

+a

60

+a

85

�a

n.i

.�

an

.i.

St.

9,

NW

Ad

ria

tic

+2

20

+a

40

+a

80

�a

n.i

.�

an

.i.

St.

10

,N

WA

dri

ati

c+

39

5+

a5

2+

a1

65

�a

n.i

.�

an

.i.

St.

11

,N

WA

dri

ati

c+

a1

75

+a

37

+3

05

�a

n.i

.�

an

.i.

St.

12

,N

WA

dri

ati

c+

48

0+

a3

0+

25

5�

an

.i.

�a

n.i

.

St.

13

,N

WA

dri

ati

c+

20

0+

a5

0+

a1

20

�a

n.i

.�

an

.i.

St.

14

,N

Ty

rrh

en

ian

�n

.i.

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

15

,N

Ty

rrh

en

ian

�n

.i.

�a

n.i

.�

an

.i.

+a

n.i

.�

an

.i.

St.

16

,N

Ty

rrh

en

ian

�n

.i.

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

17

,S

Ty

rrh

en

ian

n.a

.n

.in

.a.

n.i

.n

.a.

n.i

n.a

.n

.in

.a.

n.i

St.

18

,Io

nia

n+

21

6�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

19

,Io

nia

n+

a5

8�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

20

,Io

nia

n+

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

21

,Io

nia

n+

a5

8�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

22

,C

ata

lan

�n

.i.

+n

.i.

�n

.i.

�a

n.i

.�

an

.i.

St.

23

,C

ata

lan

�n

.i.

�a

n.i

.�

n.i

.�

an

.i.

�a

n.i

.

St.

24

,C

ata

lan

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

25

,C

ata

lan

+a

10

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

26

,C

ata

lan

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

27

,C

ata

lan

�a

7�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

28

,C

ata

lan

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

29

,C

ata

lan

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

30

,C

ata

lan

+a

11

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

St.

31

,E

ast

Atl

an

tic

�a

n.i

.�

an

.i.

�a

n.i

.+

an

.i.

+a

n.i

.

St.

32

,E

ast

Atl

an

tic

�a

n.i

.+

an

.i.

�a

n.i

.+

18

0�

an

.i.

St.

33

,E

ast

Atl

an

tic

�a

n.i

.+

an

.i.

�a

n.i

.+

13

68

�a

n.i

.

St.

34

,E

ast

Atl

an

tic

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�n

.i.

St.

35

,E

ast

Atl

an

tic

+a

n.i

.�

an

.i.

�a

n.i

.+

14

00

�n

.i.

St.

36

,B

alt

icS

ea

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

37

,B

alt

icS

ea

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

St.

38

,B

alt

icS

ea

�a

n.i

.�

an

.i.

�a

n.i

.�

an

.i.

�a

n.i

.

Th

eP

CR

de

tect

ion

so

fth

esp

eci

es

A.c

ate

nel

la,A

.ta

ylo

ria

nd

S.h

an

go

eiin

the

sed

ime

nts

we

ren

ot

sho

wn

.A.c

ate

nel

laa

nd

A.t

ayl

ori

wer

en

ot

pre

sen

tin

the

sed

ime

nt

sam

ple

s.S.

ha

ng

oei

wa

sp

rese

nt

on

lyin

the

Ba

ltic

Se

ase

dim

en

t

sam

ple

s.

+=

po

siti

ve

am

pli

fica

tio

n;�

=n

eg

ati

ve

am

pli

fica

tio

n.

n.i

.=n

ot

ide

nti

fie

d;

n.a

.=n

ot

ava

ila

ble

.

aN

est

ed

on

the

ITS

-5.8

S,

LS

UrD

NA

.



A. minutum, A. tamarense, G. catenatum, G. nolleri, L. polyedrum,P. reticulatum, and S. hangoei (data not shown). The resting cysts ofspecies A. catenella and A. taylori were never detected in thesediment samples examined by PCR assay and microscopy (datanot shown).

The positive detection of the two genera and eight speciesobtained by PCR assay and microscopy were compared (Fig. 3). ForF. japonica, 12/12 samples were positive for both methods; incontrast, the species-specific identification of A. tamarense,G. catenatum and G. nolleri was confirmed by the molecularmethod only. The number of positive detections obtained byPCR assay was higher than microscopy determinations in 14samples for A. minutum, 5 samples for Alexandrium spp. andScrippsiella spp., 4 samples for P. reticulatum, and 2 samples forL. polyedrum.

Furthermore, both methods revealed negative detections oftarget taxa cysts in the field samples, when the total number ofprocessed samples was considered (Fig. 3). These values werehigher for Gymnodinium species and F. japonica, while lowervalues of negative detections were found for the Gonyaulacalesand Peridiniales groups. False negatives were also obtained by thePCR assay in some cases, in which cysts were positively identifiedby microscopy.

4. Discussion

4.1. Molecular techniques

Molecular methodologies are highly specific, sensitive, andrapid techniques for the diagnostic identification of microbialeukaryotes in aquatic environments (Galluzzi et al., 2005, 2008;Gescher et al., 2008). Moreover, gene amplification techniques(PCR methods) with taxon-specific oligonucleotide primers andprobes have been extensively developed and have shown greatpotential with regard to the identification and enumeration ofmany harmful dinoflagellate species (Dyhrman et al., 2006;Handy et al., 2006). The PCR technique has mostly been utilizedfor the identification of vegetative cells in the water column ratherthan other life cycle stages, such as the resting stages insediments, with the exception of Godhe et al (2002) and Erdneret al. (2010).

The goal of this study was to evaluate the utility of the PCR-based technique for the identification of dinoflagellate andraphidophyte cysts in coastal sediment samples compared tomicroscopy determinations. Furthermore, this study proposesproviding an assessment of the PCR method used, since theapplication of the PCR method to sediments is problematic due toinhibitory contaminants and the low concentrations of targetcysts. Based on the results obtained in this study, the techniqueproduced higher detection efficiency than the microscopicmethod, as shown by the higher positive percentage identifica-tions of the target harmful cysts in sediments and sediment traps.

4.2. PCR-based analysis

The PCR-based assay using new and tested oligonucleotideprimers was developed to identify several dinoflagellate taxa andthe raphidophyte F. japonica in sediments and sediment traps. ThePCR assay for genus- and species-specific cyst detection wasperformed in mixed culture samples and then validated in cystsediment samples using different concentrations of PCR compo-nents for each assessed target genus- or species-specific primers.It was found that BSA can co-precipitate the inhibitor compoundsand that the TaqMaster Enhancer buffer makes the Taq DNAPolymerase less sensitive to exogenous PCR-inhibiting contami-nants. The ribosomal genes were useful in designing genus-specific Scrippsiella and species-specific G. catenatum, G. nolleri,and S. hangoei primers; the highly variable ITS regions with moreconserved rDNA genes permits discrimination at the inter-specieslevel in sediment samples containing various cyst populations, asdemonstrated for other genera and species in seawater samples inour previous study (Penna et al., 2007).

4.3. Sensitivity of the PCR assay

The sensitivity of the PCR assay was assessed by spikingplasmid DNA with a known amount of sediment genomic DNA asbackground. The sensitivity of the PCR for amplifying the lowestamount of target DNA was determined by the amplification of tencopies of plasmid DNA/ng background sediment DNA.

Furthermore, the PCR assay amplified 1 pg of genomic DNAfrom cultured Scrippsiella and Gymnodinium species in thepresence of 1 ng of mixed cyst population DNA as background,

Fig. 3. Comparison of the total sample number processed both by PCR and microscopy methods for the detection of different taxa in sediment and trap samples collected at

different regional Seas of the Mediterranean and Baltic Sea and the East Atlantic coast during the years 2006 and 2007. Analyses of positive and negative PCR amplifications

compared with the corresponding positive and negative microscopic examinations of the sediment samples are shown.



while the taxon-specific PCR-based assay was inhibited in thepresence of 10 and 5 ng of sediment genomic DNA as background.It is likely that the presence of some inhibitory substances,which are not completely eliminated from sediments duringextraction and purification using the commercial kit, cannegatively affect the PCR reaction. Inhibitor substances, such ashumic acids, polyphenols, polysaccharides and metals, andnuclease activity, are the major concern when extracting genomicDNA from marine sediments (Stults et al., 2001). In fact, the co-precipitation of compounds that inhibit PCR confuses themolecular analyses of field samples by producing false nega-tive results (Tebbe and Vahjen, 1993). The most common strategyused to overcome PCR inhibition by contaminants is dilution to alower concentration of the template DNA. In this study, weapplied a total DNA extraction and purifying procedures using acommercial kit to eliminate the potential inhibitors of the PCRreaction.

4.4. Specificity of PCR assay

The PCR-based assay was effective for the qualitative detectionof the target cysts in sediment and sediment trap samples. TotalDNA extraction was accomplished with a commercial kit that waseffective in obtaining the total quantity of DNA visible on agarosegel, which was then quantifiable for subsequent PCR reactions.The commercial kit was also successful in removing majorinhibitors, since the direct PCR amplification using either taxon-specific or eukaryotic-specific primers targeting the LSU or 5.8SrDNA and ITS was positive. Nevertheless, absence of PCRamplifications were also obtained as direct PCR using genus-and species-specific primers or eukaryotic primers often resultedin very faint bands or no observed bands at all, thus yieldingapparent false negatives. A nested taxon-specific PCR amplifica-tion of the first amplified fragment using eukaryotic-specificprimers, always gave a positive PCR reaction. The lack ofPCR-amplified fragments was observed in those sediment samplescontaining a low fraction of target taxa cysts (n=7) (Fig. 3). Directand nested PCR-based amplifications with isolated single cystshave been achieved by minimizing cyst DNA loss by the Bolch(2001) and Ki and Han (2007) methods. However, a suitablemethod for the rapid and specific detection of dinoflagellate cystsin numerous sediment samples during monitoring activity isrequired. The single cyst PCR technique requires the manipulationand isolation of the single cysts from the sediment samples undera microscope, and a single isolated cyst may be sufficient to carryout only a few PCR amplification reactions.

4.5. Molecular PCR and microscopic determinations of sediment

samples

PCR detection of the target species was compared with themicroscopic analyses of the same sediment and sediment trapsamples. The molecular technique provided a higher positivedetection rate of target cysts than microscopy. Target cysts weredetected by PCR in sediment samples presenting mixed restingstages at low concentrations or concentrations below the detec-tion limit of the microscopy method, and when it was not possibleto recognize the target cyst taxonomically. Based on thePCR-based assay higher rates of positive detection were observedfor the genera Alexandrium and Scrippsiella, and speciesA. minutum, A. tamarense, G. catenatum, G. nolleri, L. polyedrum

and P. reticulatum due to a widespread distribution. Theraphidophyte F. japonica cysts were identified equally in sedimentsamples by the two methods. PCR analysis detected 100% ofA. tamarense, G. nolleri and G. catenatum cysts in sedimentsamples. This is a relevant finding as the different morphotypecysts of Gymnodinium spp. and Alexandrium spp. are quite difficultto recognize and distinguish (Bolch et al., 1999; Bravo et al., 2006).

Furthermore, when PCR and microscopy methods were appliedto sediments and seawater for the detection of target taxa (restingor vegetative stages), the PCR method produced a higher numberof positive detections compared to the positive microscopydeterminations both in sediment and seawater substrates (Fig. 4).

The PCR-based assay detected the presence of A. minutum andA. tamarense in the Mediterranean Sea, where the two specieswere retrieved from both sediments and seawaters. The PCR assayalso detected cysts of Alexandrium spp. in all three stationssampled in the Baltic Sea. The PCR assay also confirmed thepresence of A. minutum cysts, which were identified by micro-scopy in Himmerfjarden sediments from the Swedish easterncoast. This is the first record of this species in the lower salinewaters in the northern areas of the Baltic Sea, implying thatA. minutum might be expanding and potentially cause harmfulalgal blooms in this area. Further, in the present study, S. hangoei

and Gymnodinium sp. cysts were detected in the Baltic sedimentsamples by the PCR detection assay, confirming the tentativeidentification of the S. hangoei cyst morphotype in the sedimentsamples. While Woloszinskia halophila and Gymnodinium sp. formcharacteristic cysts, which can be recognized by microscopy(Kremp et al., 2005), S. hangoei cysts lack distinctive features andthe morphotype might easily be confused with other smooth,transparent and colorless cyst types. In the sediment samples ofthe Baltic Sea, overall cyst numbers were approximately ten timeshigher than in the sediment samples from the East Atlantic and

y = 1.270x + 4.111

60

80

100

120

140

y = 0.925x + 3.282

0

20

40

60

0Samples positives by microscopy

20 40 60 80 100 120 140

R2 = 0.945

R2 = 0.947

Fig. 4. Number of samples that showed positive detection with both methods (PCR-based assay and microscopy) for different dinoflagellate and raphidophyte cysts (’) and

vegetative cells (E), in sediments and seawater samples, respectively. The diagonal line (1:1) indicates equal detection of both methods in sediments and seawater

samples. Sediment samples were collected in European coastal waters during 2006 and 2007. Positive PCR and microscopy determinations on seawater samples were

derived from Penna et al. (2007).



the Mediterranean Sea. Such high total cyst concentrations aredue to the dominance of W. halophila, which encysts in largenumbers after the spring bloom, resulting in exceptionally highcyst fluxes in the sediments (Kremp and Heiskanen, 1999). Thegenus Scrippsiella was also highly abundant among the cyst-forming dinoflagellates in the examined areas; Scrippsiella cellsare commonly found in coastal waters of these regions (Montresoret al., 1994). The life strategy of this genus includes a shortdormancy period with rapid turnover rates in cyst/vegetative cells,which may explain the formation of the bloom and its abundancealong with its long-term presence in a widespread area.

The identification of Gymnodinium cysts was quite difficult dueto the many almost indistinguishable morphotypes, some ofwhich correspond to the same species (Matsuoka and Fukuyo,2003). Within the genus Gymnodinium, G. catenatum is a toxicspecies producing a reticulated cyst, which makes it easier todistinguish from other cysts, but it is difficult to differentiate fromG. nolleri cysts (Bolch et al., 1999). This species is reported to berecurrent on the western Atlantic coast of Spain (Bravo andAnderson, 1994) and in the Alboran Sea in the Mediterranean Sea(Bravo et al., 1990). Moreover, it has appeared sporadically in someregions of the western Mediterranean Sea and the cysts weredetected during occasional surveys in southern western Mediter-ranean sediments (Calbet et al., 2002). In this study, G. catenatum

cysts were found in the Spanish Atlantic sediments by microscopyand PCR assay, and also by the PCR method in one locality of theTyrrhenian Sea where the cysts had never been detected bymicroscopy.

With regards to the raphidophyte species, reports on F. japonica

cysts are found in the in vitro study of De Boer et al. (2004), andthe identification of the cyst morphotype is limited to marinesediments (Yoshimatsu, 1987). In the Mediterranean Sea,F. japonica blooms have only been registered in the Tyrrhenianand north-western Adriatic Seas (Cucchiari et al., 2008). In thislatter coastal area, F. japonica produces abundant blooms duringthe summer period (Totti C., pers. comm.). In the present study,cysts of F. japonica were detected in the north-western Adriaticsediment samples by both methods, proving for the first time thepresence of F. japonica resting stages in the area where this speciescauses blooms. Moreover, the taxonomic identification of cystmorphotype by microscopy was confirmed by molecular assay.The cysts of the toxic species L. polyedrum and P. reticulatum werefound at almost all sampling stations, since they are widespreadin European coastal seawaters.

5. Conclusions

In this study the specificity and sensitivity of the PCR-basedtechnique for the detection of target cysts in marine sediment andsediment trap samples was demonstrated. The PCR methodpermitted higher detection efficiency than the microscopic method,illustrated by the higher positive percentage identification of theharmful target cysts in sediments. Knowledge of species composi-tion is important to understand bloom events in the coastal areas;it is also crucial to have information on the presence of novel andpotentially introduced taxa and to confirm the recurrent events of aspecies. In the future, the PCR method could be used for mappingthe distribution of target species cysts in coastal sediments,particularly given its high specificity and sensitivity.

Acknowledgments

We thank S. Casabianca and A. Casabianca for molecularanalysis assistance and suggestions; S. Fraga for culture strains;

S. Capellacci for technical assistance. Thanks to the two anon-ymous reviewers who made an effort in improving the paper. Thisstudy was financed by the EU funded Research Project SEED(GOCE-CT-2005-003875). E. Garces was sustained by a Ramon yCajal contract from the Spanish Ministry of Science and Education.

References

Adachi, M., Sako, Y., Ispida, Y., 1994. Restriction fragment length polymorphism ofribosomal DNA internal transcribed spacer and 5.8S regions in JapaneseAlexandrium species (Dinophyceae). Journal of Phycology 30, 857–863.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic localalignment search tool. Journal of Molecular Biology 215, 403–410.

Amorim, A., Dale, B., Godinho, R., Brotas, V., 2001. Gymnodinium catenatum-likecysts (Dinophyceae) in recent sediments from the coast of Portugal. Phycologia40 (6), 572–582.

Andersen, P., Throndsen, J., 2003. Estimating cell number. In: Hallegraeff, G.M.,Anderson, D.M., Cembella, A.D. (Eds.), Manual on Harmful Marine Microalgae.UNESCO, Paris, pp. 99–129.

Angl�es, S., Garces, E., Jordi, A., Basterretxea, G., Palanques, A., 2010. Alexandriumminutum resting cyst distribution dynamics in a confined site. Deep-SeaResearch Part II 57 (3–4), 210–221.

Bolch, C.J., 2001. PCR protocol for genetic identification of dinoflagellates directlyfrom single cysts and plankton cells. Phycologia 40 (2), 162–167.

Bolch, C.J.S., Negri, A.P., Hallegraeff, G.M., 1999. Gymnodinium microreticulatum sp.nov. (Dinophyceae): a naked, microreticulate cyst-producing dinoflagellate,distinct from Gymnodinium catenatum and Gymnodinium nolleri. Phycologia 38(4), 301–313.

Bowers, H.A., Trice, M.T., Magnien, R.E., Goshorn, D.M., Michael, B., Schaefer, E.F.,Rublee, P.A., Oldach, D.W., 2006. Detection of Pfiesteria spp. by PCR in surfacesediments collected from Chesapeake Bay tributaries (Maryland). HarmfulAlgae 5 (4), 342–351.

Bravo, I., Garces, E., Diogene, J., Fraga, S., Sampedro, N., Figueroa, R.I., 2006.Resting cysts of the toxigenic dinoflagellate genus Alexandrium in recentsediments from the Western Mediterranean coast, including first descriptionof cysts of A. kutnerae and A. peruvianum. European Journal of Phycology 41 (4),293–302.

Bravo, I., Anderson, D.M., 1994. The effects of temperature, growth mediumand darkness on excystment and growth of the toxic dinoflagellate Gymno-dinium catenatum from northwest Spain. Journal of Plankton Research 16 (5),513–525.

Bravo, I., Reguera, B., Martınez, A., Fraga, S., 1990. First report of Gymnodiniumcatenatum in the Spanish Mediterranean coast. In: Graneli, E., Anderson, D.M.,Edler, L., Sundstrom, B. (Eds.), Toxic Marine Phytoplankton. Elsevier, New York,pp. 449–452.

Calbet, A., Broglio, E., Saiz, E., Alcaraz, M., 2002. Low grazing impact ofmesozooplankton on the microbial communities of the Alboran Sea: a possiblecase of inhibitory effects by the toxic dinoflagellate Gymnodinium catenatum.Aquatic Microbial Ecology 26 (3), 235–246.

Casabianca, A., Vallanti, G., Magnani, M., 1998. Competitive PCR for quantificationof BM5d proviral DNA in mice with AIDS. Journal of Clinical Microbiology 36,2371–2374.

Coyne, K.J., Handy, S.M., Demir, E., Whereat, E.B., Hutchins, D.A., Portune, K.J.,Doblin, M.A., Cary, S.C., 2005. Improved quantitative real-time PCR assays forenumeration of harmful algal species in field samples using an exogenous DNAreference standard. Limnology and Oceanography: Methods 3, 381–391.

Cucchiari, E., Guerrini, F., Penna, A., Totti, C., Pistocchi, R., 2008. Effect of salinity,temperature, organic and inorganic nutrients on growth of cultured Fibrocapsajaponica (Raphidophyceae) from the northern Adriatic Sea. Harmful Algae 7,405–414.

De Boer, M.K., Van Rijssel, M., Vrieling, E.G., 2004. Morphology of Fibrocapsajaponica cysts formed under laboratory conditions. In: Steidinger, K.A.,Landsberg, J.H., Tomas, C.R., Vargo, G.A. (Eds.), Harmful Algae 2002. UNESCO,Paris, pp. 455–457.

Dyhrman, S.T., Erdner, D., La Du, J., Galac, M., Anderson, D.M., 2006. Molecularquantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 5 (3), 242–250.

Erdner, D.L., Percy, L., Lewis, J., Anderson, D.M., 2010. A quantitative real-timePCR assay for the identification and enumeration of Alexandrium cysts inmarine sediments. Deep-Sea Research Part II 57 (3–4), 279–287.

Edvardsen, B., Imai, I., 2006. The ecology of harmful flagellates within Prymne-siophyceae and Raphidophyceae. In: Graneli, E., Turner, J.T. (Eds.), Ecology ofHarmful Algae. Springer-Verlag, Berlin, pp. 67–79.

Galluzzi, L., Bertozzini, E., Penna, A., Perini, F., Pigalarga, A., Graneli, E., Magnani, M.,2008. Detection and quantification of Prymnesium parvum (Haptophyceae) byreal-time PCR. Letters in Applied Microbiology 46, 261–266.

Galluzzi, L., Penna, A., Bertozzini, E., Vila, M., Garces, E., Giacobbe, M.G., Prioli, S.,Magnani, M., 2005. Development of a qualitative PCR method for theAlexandrium (Dinophyceae) detection in contaminated mussels (Mytilusgalloprovincialis). Harmful Algae 4 (6), 973–983.

Galluzzi, L., Penna, A., Bertozzini, E., Vila, M., Garces, E., Magnani, M., 2004.Development of a real-time PCR assay for rapid detection and quantification of



Alexandrium minutum (a dinoflagellate). Applied and Environmental Micro-biology 70, 1199–1206.

Garces, E., Bravo, I., Vila, M., Figueroa, R.I., Maso, M., Sampedro, N., 2004.Relationship between vegetative cells and cyst production during Alexandriumminutum bloom in Arenys de Mar harbour (NW Mediterranean). Journal ofPlankton Research 26 (6), 637–645.

Garces, E., Zingone, A., Montresor, M., Reguera, B., Dale, B., 2002. Lifehab: LifeHistories of Microalgal Species Causing Harmful Blooms. Office for the OfficialPublications of the European Communities, Luxembourg, pp. 1-189.

Gescher, C., Metfies, K., Medlin, L.K., 2008. The ALEX CHIP-development of a DNAchip for identification and monitoring of Alexandrium. Harmful Algae 7 (4),485–494.

Giacobbe, M.G., Penna, A., Gangemi, E., Maso, M., Garces, E., Fraga, S., Bravo, I.,Azzaro, F., Decembrini, F., Penna, N., 2007. Recurrent high-biomass blooms ofAlexandrium taylorii (Dinophyceae), a HAB species expanding in the Mediter-ranean Sea. Hydrobiologia 580, 125–133.

Godhe, A., Cusack, C., Pedersen, J., Andersen, P., Anderson, D.M., Bresnan, E.,Cembella, A., Dahl, E., Diercks, S., Elbrachter, M., Edler, L., Galluzzi, L., Gescher,C., Gladstone, M., Karlson, B., Kulis, D., LeGresley, M., Lindahl, O., Marin, R.,McDermott, G., Medlin, L.K., Naustvoll, L.J., Penna, A., T +obe, K., 2007.Intercalibration of classical and molecular techniques for identification ofAlexandrium fundyense (Dinophyceae) and estimation of cell densities.Harmful Algae 6 (1), 56–72.

Godhe, A., Rehnstam-Holm, A-S., Karunasagar, I., Karunasagar, I., 2002. PCRdetection of dinoflagellate cysts in field sediment samples from tropic andtemperate environments. Harmful Algae 1 (4), 361–373.

Handy, S.M., Hutchins, D.A., Cary, S.C., Coyne, K., 2006. Simultaneous enume-ration of multiple raphidophyte species by quantitative real-time PCR:capabilities and limitations. Limnology and Oceanography: methods 4,193–204.

Kamikawa, R., Nagai, S., Hosoi-Tanabe, S., Itakura, S., Yamaguchi, M., Uchida, Y.,Baba, T., Sako, Y., 2007. Application of real-time PCR assay for detection andquantification of Alexandrium tamarense and Alexandrium catenella cysts frommarine sediments. Harmful Algae 6 (3), 413–420.

Ki, J.S., Han, M.S., 2007. Rapid molecular identification of the harmful freshwaterdinoflagellate Peridinium in various life stages using genus-specific single-cellPCR. Journal of Applied Phycology 19, 467–470.

Kremp, A., Elbrachter, M., Schweikert, M., Wolny, J., Gottschling, M., 2005.Woloszynskia halophila (Biecheler) comb. nov.—a bloom forming cold-waterdinoflagellate co-occurring with Scrippsiella hangoei (Dinophyceae) in theBaltic Sea. Journal of Phycology 41 (3), 629–642.

Kremp, A., Heiskanen, A.S., 1999. Sexuality and cyst formation of the spring-bloomdinoflagellate Scrippsiella hangoei in the coastal northern Baltic Sea. MarineBiology 134 (4), 771–777.

Matsuoka, K., Fukuyo, Y., 2003. Taxonomy of cysts. In: Hallegraeff, G.M., Anderson,D.M., Cembella, A.D. (Eds.), Manual on Harmful Marine Microalgae. UNESCO,Paris, pp. 563–592.

McGillicuddy, D.J., Signell, R.P., Stock, C.A., Keafer, B.A., Keller, M.D., Hetland, R.D.,Anderson, D.M., 2003. A mechanism for offshore initiation of harmfulalgal blooms in the coastal Gulf of Maine. Journal of Plankton Research 25,1131–1138.

Montresor, M., Montesarchio, E., Marino, D., Zingone, A., 1994. Calcareousdinoflagellate cysts in marine sediments of the Gulf of Naples (MediterraneanSea). Review of Paleobotany and Palynology 84, 45–56.

Penna, A., Bertozzini, E., Battocchi, C., Galluzzi, L., Giacobbe, M.G., Vila, M., Garces,E., Lugli�e, A., Magnani, M., 2007. Monitoring of HAB species in theMediterranean Sea through molecular methods. Journal of Plankton Research29 (1), 19–38.

Penna, A., Garces, E., Vila, M., Giacobbe, M.G., Fraga, S., Lugli�e, A., Bravo, I.,Bertozzini, E., Vernesi, C., 2005. Alexandrium catenella (Dinophyceae), a toxicribotype expanding in the NW Mediterranean Sea. Marine Biology 148 (1), 13–23.

Saito, K., Drgon, T., Robledo, J., Krupatkita, D., Vasta, G., 2002. Characterization ofthe rRNA locus of Pfiesteria piscicida and development of standard andquantitative PCR-based detection. Applied and Environmental Microbiology 68(11), 5394–5407.

Satta, C., Angles, S., Garces, E., Luglie, A., Padedda, B., Sechi, N., 2010. Dinoflagellatecysts in recent sediments from two semi-enclosed areas of the WesternMediterranean Sea subject to high human impact. Deep Sea Research II 57(3–4), 256–267.

Scholin, C.A., Herzog, M., Sogin, M., Anderson, D.M., 1994. Identification of group-and strain-specific genetic markers from globally distributed Alexandrium(Dinophyceae). II. Sequence analysis of fragments of the LSU rRNA gene.Journal of Phycology 30, 999–1011.

Smayda, T.J., 2007. Reflections on the ballast water dispersal—harmful algal bloomparadigm. Harmful Algae 6 (4), 601–622.

Steidinger, K.A., Garces, E., 2006. Importance of life cycles in the ecology of harmfulmicroalgae. In: Graneli, E., Turner, J.T. (Eds.), Ecology of Harmful Algae.Springer-Verlag, Berlin, pp. 37–49.

Stults, J.R., Snoeyenbos-West, O., Methe, B., Lovley, D.R., Chandler, D.P., 2001.Application of the 50 fluorogenic exonuclease assay (Taqman) for quantitativeribosomal DNA and rRNA analysis in sediments. Applied EnvironmentalMicrobiology 67, 2781–2789.

Tebbe, C.C., Vahjen, W., 1993. Interference of humic acids and DNA extracted directlyfrom soil in detection and transformation of recombinant DNA from bacteriaand a yeast. Applied and Environmental Microbiology 59, 2657–2665.

Yoshimatsu, S., 1987. The cysts of Fibrocapsa japonica (Raphidophyceae) found inbottom sediment in Harima Nada, eastern Inland Sea of Japan. Bulletin ofPlankton Society Japan 34, 25–31.


Documents

Detection of microalgal resting cysts in European coastal sediments using a PCR-based assay