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Fisheries Science ISSN 0919-9268 Fish SciDOI 10.1007/s12562-015-0864-9

Pseudo-nitzschia fukuyoi(Bacillariophyceae), a domoic acid-producing species from Nha Phu Bay,Khanh Hoa Province, Vietnam

Ha Viet Dao, Vy Bao Phan, Sing TungTeng, Hajime Uchida, Chui Pin Leaw, PoTeen Lim, Toshiyuki Suzuki & Ky XuanPham

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Fish SciDOI 10.1007/s12562-015-0864-9

ORIGINAL ARTICLE

Pseudo‑nitzschia fukuyoi (Bacillariophyceae), a domoic acid‑producing species from Nha Phu Bay, Khanh Hoa Province, Vietnam

Ha Viet Dao1 · Vy Bao Phan1 · Sing Tung Teng2 · Hajime Uchida3 · Chui Pin Leaw4 · Po Teen Lim4 · Toshiyuki Suzuki3 · Ky Xuan Pham1

Received: 9 January 2015 / Accepted: 21 February 2015 © Japanese Society of Fisheries Science 2015

potentially toxic Pseudo-nitzschia species were reported in the tropics, including Malaysia [1–5] and Vietnam [6, 7]. We recently documented that one of relatively small-sized species, P. cf. caciantha, from Nha Phu Bay, Khanh Hoa Province, Vietnam, was a DA producer [8]. Furthermore, we suspected that the species was most likely the source of the DA contamination in thorny oyster Spondylus ver-sicolor in the bay. However, DA levels in plankton showed peaks in different seasons, which were around April [8] and August [9]. In the previous studies, during DA peak in plankton in August, DA producing species could not be detected successfully. Therefore, this study aims to investi-gate potential producers of DA in Nha Phu Bay in around August, with emphasis on species of the genus Pseudo-nitzschia using unialgal cultures established form the bay.

Materials and methods

Sampling and algal cultures

On 17 July 2013, 5 l of seawater were collected at a site near Hon Thi Island, Nha Phu Bay (12°38′42″N, 109°22′06″E), Khanh Hoa Province, Vietnam, using a Van Dorn sampler from a 2-m depth. This sample was kept in the dark, without any preservative, and brought back to the laboratory for single-cell isolation.

Single cells of Pseudo-nitzschia species from the water sample were isolated using a fine-drawn Pasteur pipette under an inverted microscope (Nikon TMS-F MFA 20100). Cells were rinsed several times with 0.2 µm filtered seawa-ter before transferring into a 24-multiwell plate containing 1 ml of f/2 medium [10] with pH of 7.8–8.0 at a salinity of 30. Cells that grew successfully were inoculated into 50 ml culture flasks containing 30 ml of f/2 medium, and finally

Abstract Two strains of Pseudo-nitzschia fukuyoi iso-lated from Vietnamese waters produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Species iden-tification was based on detailed morphological observation using a transmission electron microscope and also molec-ular data on large subunit (LSU) and the second internal transcribed spacer (ITS2) with NCBI nucleotide Blast (blastn). Toxin productivity of the two strains was con-firmed and their range were 3.85–4.54 pg/cell by analyses using LC–MS/MS. This is the first report of occurrence of P. fukuyoi in Vietnamese waters, and the first confirmation of productivity of domoic acid of the species.

Keywords Domoic acid · Amnesic shellfish toxin · Pseudo-nitzschia fukuyoi · Vietnam

Introduction

Species of the cosmopolitan genus Pseudo-nitzschia, some of which produce the neurotoxin domoic acid (DA), are often observed in tropical waters. Recently, several

Chemistry and Biochemistry

* Ha Viet Dao daovietha69@gmail.com

1 Institute of Oceanography, Vietnam Academy of Science and Technology, 01 Cau Da, Nhatrang, Vietnam

2 Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan, 94300 Sarawak, Malaysia

3 Tohoku National Fisheries Research Institute, 3-27-5 Shinhama, Shiogama, Miyagi 985-0001, Japan

4 Bachok Marine Research Station, Institute of Ocean and Earth Sciences, University of Malaya, 16310 Bachok, Kelantan, Malaysia

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into 500 ml flasks containing 180 ml of f/2 medium. Two strains were successfully cultured, and coded as Pn 5 and Pn 6. All cultures were maintained at 25 °C under the light intensity of 150 µmol photons m−2 s−1, provided by cool-white fluorescent bulbs with a 12:12 h light:dark cycle, in a temperature-controlled incubator (Sanyo MIR-153). When the cultures reached mid-exponential phase (day 7 after inoculation), 15 ml of each culture was collected for mor-phology observation and molecular sequencing. Cultures at late-stationary phase (day 13) were then collected for DA analysis, as DA production of some Pseudo-nitzschia spe-cies was reported to increase after the phase [11].

Species identification

A 15-ml aliquot of exponentially growing clonal culture was harvested by centrifugation at 4,000×g for 5 min. Cell pellets were acid-washed using 37 % HCl [12], KMnO4 and 10 % oxalic acid [4] to remove the organic material. Cleaned cells were mounted on a Formvar-coated copper grid and dried at 70 °C overnight. The dried samples were then observed using a JEM-1230 transmission electron microscope (TEM). TEM micrographs were taken with an Erlangshen ES500 W camera and morphometrics were obtained using Digital Micrograph software (Gatan, Pleas-anton, CA, USA). Detailed morphological characters were compared with those in previous studies [2–4, 13].

Extraction of domoic acid from the cultures

Cells in 150 ml of culture were collected by filtration onto a GF/F filter, which was then immersed into 10 ml of dis-tilled water (DW), sonicated for 5 min to break the cells, and then centrifuged (10,000×g, 30 min) to obtain the supernatant. The pH of the supernatant was adjusted to 2–3 using 1 M formic acid. Extracts were filtered slowly through Sep-Pak C18 cartridges (Waters Corp., USA). The cartridge column was washed with 10 ml of DW, then DA was eluted with 10 ml of methanol. Cells were enumerated in 1 ml of culture placed into the Sedgewick-Rafter count-ing chamber (repeated three times), then multiplied to total volume of each culture. DA concentration is expressed as ng DA cell−1, by dividing the DA concentration in the extract by the number of cells present in each culture strain (data not shown).

Molecular identification

Genomic DNA of cultures was isolated using DNeasy® Plant Mini Kits (Qiagen, Hilden, Germany). The internal transcript spacer (ITS) and large subunit (LSU) region were amplified using the universal primer pair ITS1/4 [14] and

D1R-D3Ca [15]. ITS and LSU sequences were obtained using NCBI nucleotide Blast (blastn). The sequences simi-larities were used in molecular species identification.

LC–MS/MS analysis

DA was analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). A model 1100 liquid chro-matograph (Agilent, Palo Alto, CA, USA) was coupled to a hybrid triple quadrupole/linear ion trap mass spectrom-eter 3200 Q Trap™ (PE-SCIEX, Thornhill, ON, Canada). LC separation was performed on Quicksilver cartridge columns (50 × 2.1 mm i.d.) packed with 3 µm Hypersil-BDS-C8 (Keystone Scientific, Bellefonte, PA, USA) maintained at 20 °C. Eluent A was water and B was ace-tonitrile–water (95:5), both containing 2 mM ammonium formate and 50 mM formic acid. Linear gradient elution from 5 to 100 % B was performed over 10 min and then held at 100 % B for 10 min, followed by re-equilibration with 5 % B (13 min). The flow rate was 0.2 ml min−1 and the injection volume was 10 µl. MRM LC–MS/MS analy-sis for toxins was carried out using m/z 312, corresponding to [M + H]+ as the target parent ions in Q1 and particu-lar fragment ions of DA at m/z 266 and m/z 91 in Q3. The MRM channel m/z 312 > 266 was used for quantification of DA.

Enhanced product ion (EPI) LC–MS/MS spectra were acquired in positive mode by colliding the Q1-selected precursor ions for [M + H]+ of DA with nitrogen in Q2 operated in radio frequency (rf)—only mode and scanning the linear ion trap, Q3, from m/z 50 to 350. The collision energy was set at −25 V. LC separation of DA was carried out with the same chromatographic conditions as the MRM LC–MS/MS analysis. The injection volume was 10 µl.

Results

Morphological analysis

Cell valves are linear and symmetrical (Fig. 1a, b). Cells are 68.8–87.7 µm long and 1.53–2.28 µm wide. Both api-ces are pointed and have a short tapering (Fig. 1c, d). The eccentric raphe is divided in the middle by a central inter-space (Fig. 1e). The fibulae and striae are 16–21 and 33–37 in 10 µm, respectively. Each stria contains one row of round to oval poroids (Fig. 1e). The hymen of the poroids is divided into 2–4 sectors (Fig. 1f, g). The perforations of the poroids are arranged in a hexagonal pattern.

The cingular band contains three girdle bands. The den-sity of band striae in the valvocopula is 42–43 in 10 µm. The valvocopula contains several striae, each two wide and

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two to four high (Fig. 1h–j). Striae of the band II copula contain one row of divided poroids (2–4 sectors) and one longitudinal row of poroids (Fig. 1h, k). Some of the band II copulaeare biseriate, with one row of poroids (Fig. 1j). Each of the band III copulae comprises of two longitudinal rows of poroids (Fig. 1h). The perforation pattern is hex-agonal (Table 1).

Molecular identification

ITS sequences validated the species as P. fukuyoi with high query coverage (99 %; Table 2) and identity (99–100 %; Table 2) by using blastn. The species with the three first hits in blastn were P. fukuyoi, with strain names PnKk36, PnTb55, and PnTb31 (Table 2).

VCC1

C2

C1VC

VC

C1C1

A B C D E

F

G

H

I

J

Fig. 1 TEM images of cultured Pseudo-nitzschia fukuyoi from Viet-nam: a, b valve view, showing linear valves; c, d tapered apices; e central of valve, showing the presence of a central interspace; f detail of the striae and poroids; g detail of poroid sectors; h detail of cingu-

lar bands: alvocopula and copulae (second band and third bands); i detail of valvocopula and first copula; j central sector at valvocopula; k detail of second band (copula). Scale bars 20 μm (a); 10 μm (b); 2 μm (c, d); 1 μm (e); 0.5 μm (f); 0.2 μm (h–k); 0.1 μm (g)

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Tabl

e 1

Com

pari

son

of m

orph

omet

ric

data

of

cultu

red

Pse

udo-

nitz

schi

a fu

kuyo

i fro

m V

ietn

am w

ith it

s cl

osel

y re

late

d sp

ecie

s in

the

P. p

seud

odel

icat

issi

ma

com

plex

; mea

n ±

SD

are

sho

wn

in

pare

nthe

ses;

n, n

umbe

r of

cel

ls m

easu

red

All

spec

ies

show

ed o

nly

one

row

of

poro

ids

betw

een

the

inte

rstr

iae

and

poss

ess

a ce

ntra

l int

ersp

ace

Spec

ies

Val

veL

engt

h (µ

m )

Wid

th (

µm )

Fibu

lae

Stri

aeSe

ctor

s in

hy

men

Poro

ids

in

1 µ

mB

and

stri

ae in

10

µm

Val

voco

pula

pa

ttern

Cop

ula

I pa

ttern

Ref

eren

ce

Pse

udo-

nitz

schi

a fu

kuyo

i

Com

plet

ely

linea

r, sy

mm

etri

cal

68.8

–87.

71.

53–2

.28

16–2

133

–37

(0–1

) 2–

4 (5

–6)

4–7

42–4

32

× (

2) 3

–41–

3 ×

1–2

Thi

s st

udy

76.9

5 ±

5.3

01.

95 ±

0.2

118

.94

± 1

.26

34.3

3 ±

0.9

12.

31 ±

1.0

35.

40 ±

0.7

642

.29

± 0

.49

n =

65

n =

16

n =

20

n =

19

n =

19

n =

94

n =

64

n =

8

P. fu

kuyo

iL

inea

r to

lanc

eo-

late

, sym

met

ri-

cal

74–8

11.

5–1.

917

–19

32–3

42–

3 (4

)5–

639

–47

2 ×

3-4

Lim

et a

l. [3

]

P. a

bren

sis

Lin

ear

to la

nceo

-la

te, s

ymm

etri

-ca

l

66.5

–74.

11.

7–2.

516

–22

30–3

71–

44–

636

–38

2 ×

2–4

Ori

ve e

t al.

[16]

P. lu

ndho

lmia

eL

ance

olat

e, s

ym-

met

rica

l63

–73

1.7–

2.3

16–1

828

–34

1–2

(3)

4–6

35-4

01–

2 ×

2-3

Lim

et a

l. [3

]

P. b

ates

iana

Lan

ceol

ate,

sym

-m

etri

cal

84–8

61.

8–2.

215

–19

29–3

22–

35–

640

–43

2 ×

3–4

Lim

et a

l. [3

]

P. c

acia

ntha

Lan

ceol

ate,

asy

m-

met

rica

l53

–75

2.7–

3.5

15–1

928

–31

4–5

3.5–

533

–38

2 ×

3–5

Lun

dhol

m

et a

l. [1

7]

P. c

uspi

data

Lan

ceol

ate

30–7

31.

4–2.

019

–25

35–4

42

4–6

47–5

31

× 2

Lun

dhol

m

et a

l. [1

7]

P. fr

yxel

lian

aL

ance

olat

e30

–54

2.1–

2.5

(17)

18–

2534

–40

(1)

2–3

5–6

(7)

41–5

02

× 1

–3L

undh

olm

et

al.

[12]

P. h

asle

ana

Lan

ceol

ate,

sym

-m

etri

cal

37–7

91.

5–2.

813

–20

31–4

02–

65–

637

–46

(47)

2 ×

3–6

Lun

dhol

m

et a

l. [1

2]

P. p

seud

odel

i-ca

tiss

ima

Lin

ear

and

sym

-m

etri

cal

54–8

70.

9–1.

620

–25

36–4

32

5–6

48–5

5Sp

lit p

oroi

dL

undh

olm

et

al.

[17]

P. m

anni

iL

inea

r33

–130

1.7–

2.6

17–2

530

–40

2–7

4–6

46–4

72

× 3

–4A

mat

o et

al.

[18]

P. c

alli

anth

aL

inea

r41

–98

1.3–

1.8

15–2

234

–39

7–10

4–6

42–4

82–

3 ×

4–5

(6)

Lun

dhol

m

et a

l. [1

7]

P. c

ircu

mpo

raL

ance

olat

e, a

sym

-m

etri

cal

71–8

82.

2–2.

715

–19

32–3

5>

71–

440

–42

2 ×

4L

im e

t al.

[2]

P. p

luri

sect

aL

inea

r to

lanc

eo-

late

56.3

–59.

71.

5–2.

017

–25

34–4

53–

104–

745

–48.

52

× 3

–5O

rive

et a

l. [1

6]

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LSU sequences were also examined with blastn. The LSU sequences showed 100 % coverage with 99 % iden-tity and 0.0 E values. The first three hits were P. fukuyoi (PnTb72, PnTb39, and PnTb25).

Domoic acid production in cultured clones

Results from the LC–MS/MS analysis showed detectable DA in both cultured strain extracts. Cellular DA concentra-tion was calculated as 3.85 pg/cell in Pn 5 and 4.54 pg/cell in Pn 6. These culture extracts showed a peak at the same retention time as that of the DA standard, with the pseudo-molecular ion [M + H]+ (m/z 312) in both m/z 312 > 266 and 266 > 91 (Fig. 2a, b). Fragmentation patterns of daugh-ter ions characteristic of DA (m/z 266, 248, 161), which are

identical for DA (Fig. 2c), were observed consistently in the culture extract (Fig. 2d).

Discussion

The Pseudo-nitzschia fukuyoi strains in this study are almost identical morphologically to the original descrip-tion of P. fukuyoi from Malaysian waters [3]. Some minor differences, however, are noted. Our P. fukuyoi strain from Vietnamese waters is linear in valve view; this dif-fers slightly from the original description, which is linear to lanceolate. It also has a wider range in width (1.53–2.28 µm) compared to the original description (1.5–1.9 µm) [3]. Also, our P. fukuyoi has a wider range in density of striae in 10 µm (33–37), comparing to the original descrip-tion (32–34). Otherwise, P. fukuyoi in this study shows the same morphological data, i.e., apical valve length, density of fibulae and poroids, and structure and band striae pattern of the valvocopula.

The ITS and LSU sequences of P. fukuyoi show very high query coverage (99–100 %) and identity (99–100 %) in blastn. Further validation of the identity of P. fukuyoi is that it shares the same genetic information as the P. fukuyoi reported by Lim et al. [3]. Therefore, the species in this study is designated as P. fukuyoi.

Some species of Pseudo-nitzschia are cosmopolitan and are often observed in plankton samples of tropical waters. Pseudo-nitzschia fukuyoi was first reported from the Strait of Malacca, Malaysia [3], but there has been no docu-mentation of this species from Vietnamese waters [6, 7].

Table 2 GenBank blast hits for the ITS and LSU sequences of Pseudo-nitzschia fukuyoi in this study (E value = 0.0)

Strain Pn 5/Pn 6 Pn 5/Pn 6

Accession number

Query cover-age %

Identity % Reference

ITS sequences

PnKk36 KC147515 99 100 Lim et al. [3]

PnTb55 KC147520 99 99 Lim et al. [3]

PnTb31 KC147517 99 99 Lim et al. [3]

LSU sequences

PnTb72 KC147537 100 99 Lim et al. [3]

PnTb39 KC147536 100 99 Lim et al. [3]

PnTb25 KC147535 100 99 Lim et al. [3]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(a)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(b)

80 100 120 140 160 180 200 220 240 260 280 300 320 60

(c)

161.2

220.0

248.3

266.3

312.1

80 100 120 140 160 180 200 220 240 260 280 300 320 60

(d)

161.0

220.1 248.3

266.2

312.1

1.0e5 8.0e4 6.0e4 4.0e4 2.0e4

1.2e5 1.4e5 1.6e5 1.8e5 2.0e5 2.2e5

Inte

nsity

, cps

Inte

nsity

, cps

In

tens

ity, c

ps

Inte

nsity

, cps

2000 1500 1000 500

0

2000 2500 3000 3500

4500 4000

4736

3.4e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4

5000.0 0

8.94 min

8.92 min

Time, min.

Time, min.

m/z, amu

m/z, amu

3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4

5000.0

3.7e4 m/z 312>266

m/z 312>91

m/z 312>266

m/z 312>91

Fig. 2 LC–MS/MS chromatographs of domoic acid in cultured Pseudo-nitzschia fukuyoi from Vietnam: a elution pattern of domoic acid standard in LC, scanned by (M + H)+ 312 m/z; b elution pat-

tern of cultured P. fukuyoi from Vietnam in LC, scanned by (M + H)+ 312 m/z; c fragmentation pattern of DA standard; d fragmentation pattern of cultured P. fukuyoi from Vietnam

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Therefore, this is the first record of P. fukuyoi in Vietnam-ese waters.

To date, 17 species of Pseudo-nitzschia have been shown to be toxigenic [5, 8, 19, 20]. In this study, LC–MS/MS analysis confirmed the presence of DA in cultured P. fukuyoi from Vietnamese waters. This is in contrast to the results from monoclonal cultures strains of this species from Malaysian waters, in which no toxin was detected [3]. This study is, therefore, the first report of DA production by P. fukuyoi in Vietnamese waters and as a novel producer of DA in the world. The results are robust because the identity of DA was confirmed by LC–MS/MS. This is the third toxigenic Pseudo-nitzschia species reported recently in Southeast Asian waters, after P. kodamae from Malaysia and P. cf. caciantha from Vietnam. Some studies reported that strains of the same species originating from different areas may be toxic or nontoxic. For example, P. multiseries from Prince Edward Island, Canada, was reported as highly toxic, while no toxin was detected in cultured strains origi-nating from France and Brazil (reviewed in [21]). This is also the case for P. australis; strains from the United States showed high toxin levels, but no toxin was detected in strains from Spain and Australia [21]. Investigation of the toxin production ability of P. fukuyoi strains originating from other tropical waters is required.

The cellular DA levels of this diatom (3.85–4.54 pg/cell) are lower than the maximum levels of some other toxi-genic Pseudo-nitzschia species, e.g., P. multiseries (67 pg/cell), P. australis (37 pg/cell), P. seriata (33.6 pg DA/cell) (reviewed in [21]), and P. kodamae (42.5 pg DA/cell) [5]. Nevertheless, toxin levels are higher than other DA-pro-ducing Pseudo-nitzschiaspecies; for example, higher than in two other phylogenetically closely related species in the P. pseudodelicatissima complex, P. pseudodelicatissima (0.0078 pg/cell) and P. cuspidata (0.031 pg/cell) (reviewed in [21]). In this study, both single clones isolated were iden-tified as P. fukuyoi, while no other potential toxic Pseudo-nitzschia species including toxic P. cf. caciantha [8] could be isolated from the same plankton sample. From the result of toxin production in the cultures, this species is more toxic than P. cf. caciantha from the same area (Nha Phu Bay, Khanh Hoa Province, Vietnam), although toxin production in P. cf. caciantha could not be quantitative at that time [8]. Further studies are required to determine cellular DA levels at different stages of the growth curve of this species.

Based on toxin productivity analyses described above, responsible species for toxin contamination in thorny oyster in Nha Phu Bay could be not only P. cf. caciantha reported before [8], but also P. fukuyoi endemic in the bay. Seasonal blooming pattern of the two species in relation to changing pattern of the toxin in shellfish has not been elu-cidated yet, but it is inevitable to clarify toxin accumulation process and system in the bay.

Acknowledgments This study was funded by project 106.99-2010.22—NAFOSTED, Vietnam. The authors wish to express the deep thank to Dr. Stephen S. Bates (Fisheries and Oceans Canada, Gulf Fisheries Centre, Canada) for the help to improve the manu-script, both in scientific ideas and English correction.

References

1. Lim HC, Su SNP, Mohamed-Ali H, Kotaki Y, Leaw CP, Lim PT (2010) Toxicity of diatom Pseudo-nitzschia (Bacillariophy-ceae) analyzed using high performance liquid chromatography (HPLC). J Sci Technol Trop 6:S116–S119

2. Lim HC, Leaw CP, Su SNP, Teng ST, Usup G, Noor NM, Lun-dholm N, Kotaki Y, Lim PT (2012) Morphology and molecu-lar characterization of Pseudo-nitzschia (Bacillariophyceae) from Malaysian Borneo, including the new species Pseudo-nitzschiacircumpora sp. nov. J Phycol 48(5):1232–1247

3. Lim HC, Teng ST, Leaw CP, Lim PT (2013) Three novel species in the Pseudo-nitzschia pseudo delicatissima complex: P. bate-siana sp. nov., P. lundholmiae sp. nov., and P. fukuyoi sp. nov. (Bacillariophyceae) from the Strait of Malacca, Malaysia. J Phy-col 49(5):902–916

4. Teng ST, Leaw CP, Lim HC, Lim PT (2013) The genus Pseudo-nitzschia (Bacillariophyceae) in Malaysia, including new records and a keyto species inferred from morphology-based phylogeny. Bot Mar 56:375–398

5. Teng ST, Lim HC, Lim PT, Dao VH, Bates SS, Leaw CP (2014) Pseudo-nitzschiakodamae sp. nov. (Bacillariophyceae), a toxi-genic species from the Strait of Malacca, Malaysia. Harmful Algae 34:17–28. doi:10.1016/j.hal.2014.02.005

6. Skov J, Ton TP, Do TBL (2004) Bacillariophyceae. In: Poten-tially toxic microalgae of Vietnamese waters. In: Larsen J, Nguyen NL (eds) Opera Botanica, vol 140. Council for Nordic Publications in Botany, Copenhagen, pp 23–51

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Pseudo-nitzschia fukuyoi (Bacillariophyceae), a domoic acid-producing species from Nha Phu Bay, Khanh Hoa Province, VietnamAbstract IntroductionMaterials and methodsSampling and algal culturesSpecies identificationExtraction of domoic acid from the culturesMolecular identificationLC–MSMS analysis

ResultsMorphological analysisMolecular identificationDomoic acid production in cultured clones

DiscussionAcknowledgments References