Pseudo-nitzschia fukuyoi (Bacillariophyceae), a domoic acidproducing species from Nha Phu Bay, Khanh Hoa Province, Vietnam Ha Viet Dao, Vy Bao Phan, Sing Tung Teng, Hajime Uchida, Chui Pin Leaw, Po Teen Lim, Toshiyuki Suzuki & Ky Xuan Pham Fisheries Science ISSN 0919-9268 Fish Sci DOI 10.1007/s12562-015-0864-9
1 23
Your article is protected by copyright and all rights are held exclusively by Japanese Society of Fisheries Science. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.
1 23
Author's personal copy Fish Sci DOI 10.1007/s12562-015-0864-9
Chemistry and Biochemistry
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
Abstract Two strains of Pseudo-nitzschia fukuyoi isolated from Vietnamese waters produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Species identification was based on detailed morphological observation using a transmission electron microscope and also molecular data on large subunit (LSU) and the second internal transcribed spacer (ITS2) with NCBI nucleotide Blast (blastn). Toxin productivity of the two strains was confirmed 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
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 versicolor 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 investigate potential producers of DA in Nha Phu Bay in around August, with emphasis on species of the genus Pseudonitzschia using unialgal cultures established form the bay.
Introduction
Materials and methods
Species of the cosmopolitan genus Pseudo-nitzschia, some of which produce the neurotoxin domoic acid (DA), are often observed in tropical waters. Recently, several
Sampling and algal cultures
* Ha Viet Dao
[email protected] 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
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 seawater 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
13
Author's personal copy
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 coolwhite 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 morphology observation and molecular sequencing. Cultures at late-stationary phase (day 13) were then collected for DA analysis, as DA production of some Pseudo-nitzschia species 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, Pleasanton, 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 distilled 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 counting 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
13
Fish Sci
D1R-D3Ca [15]. ITS and LSU sequences were obtained using NCBI nucleotide Blast (blastn). The sequences similarities 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 chromatograph (Agilent, Palo Alto, CA, USA) was coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer 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 HypersilBDS-C8 (Keystone Scientific, Bellefonte, PA, USA) maintained at 20 °C. Eluent A was water and B was acetonitrile–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 analysis for toxins was carried out using m/z 312, corresponding to [M + H]+ as the target parent ions in Q1 and particular 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 apices are pointed and have a short tapering (Fig. 1c, d). The eccentric raphe is divided in the middle by a central interspace (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 density of band striae in the valvocopula is 42–43 in 10 µm. The valvocopula contains several striae, each two wide and
Author's personal copy Fish Sci
A
B
C
D
E
F
I
VC
G
C1 C2
C1 VC
J
H
VC
C1
C1 Fig. 1 TEM images of cultured Pseudo-nitzschia fukuyoi from Vietnam: 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)
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 hexagonal (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).
13
13 1.8–2.2 2.7–3.5 1.4–2.0
1.5–2.8 0.9–1.6
1.3–1.8 2.2–2.7 1.5–2.0
84–86 53–75 30–73 30–54 37–79 54–87 33–130 41–98
Lanceolate
Lanceolate, symmetrical Linear and symmetrical
Linear
Linear
Lanceolate, asym- 71–88 metrical
Linear to lanceolate
P. fryxelliana
P. hasleana
P. pseudodelicatissima
P. mannii
P. calliantha
P. circumpora
P. plurisecta
1.7–2.6
17–25
15–19
15–22
17–25
20–25
13–20
(17) 18–25
19–25
15–19
15–19
16–18
16–22
34–45
32–35
34–39
30–40
36–43
31–40
34–40
35–44
28–31
29–32
28–34
30–37
All species showed only one row of poroids between the interstriae and possess a central interspace
P. cuspidata
P. caciantha
P. batesiana
56.3–59.7
1.7–2.3
63–73
P. lundholmiae
2.1–2.5
1.7–2.5
66.5–74.1
P. abrensis
P. fukuyoi
n = 19 32–34
34.33 ± 0.91
n = 19 17–19
18.94 ± 1.26
n = 20 1.5–1.9
n = 16 74–81
n = 65 Linear to lanceolate, symmetrical Linear to lanceolate, symmetrical Lanceolate, symmetrical Lanceolate, symmetrical Lanceolate, asymmetrical Lanceolate
33–37
16–21
Completely linear, 68.8–87.7 1.53–2.28 symmetrical 76.95 ± 5.30 1.95 ± 0.21
Pseudonitzschia fukuyoi
Striae
Fibulae
Length (µm ) Width (µm )
Valve
Species
3–10
>7
7–10
2–7
2
2–6
(1) 2–3
2
4–5
2–3
1–2 (3)
1–4
n = 94 2–3 (4)
(0–1) 2–4 (5–6) 2.31 ± 1.03
Sectors in hymen
4–7
1–4
4–6
4–6
5–6
5–6
5–6 (7)
4–6
3.5–5
5–6
4–6
4–6
n = 64 5–6
5.40 ± 0.76
4–7
Poroids in 1 µm
45–48.5
40–42
42–48
46–47
48–55
37–46 (47)
41–50
47–53
33–38
40–43
35-40
36–38
n = 8 39–47
42.29 ± 0.49
42–43
Band striae in 10 µm
2 × 3–5
2 × 4
2–3 × 4–5 (6)
2 × 3–4
Split poroid
2 × 3–6
2 × 1–3
1 × 2
2 × 3–5
2 × 3–4
1–2 × 2-3
2 × 2–4
2 × 3-4
2 × (2) 3–4
Valvocopula pattern
Reference
Orive et al. [16]
Amato et al. [18] Lundholm et al. [17] Lim et al. [2]
Lim et al. [3] Lim et al. [3] Lundholm et al. [17] Lundholm et al. [17] Lundholm et al. [12] Lundholm et al. [12] Lundholm et al. [17]
Orive et al. [16]
Lim et al. [3]
1–3 × 1–2 This study
Copula I pattern
Table 1 Comparison of morphometric data of cultured Pseudo-nitzschia fukuyoi from Vietnam with its closely related species in the P. pseudodelicatissima complex; mean ± SD are shown in parentheses; n, number of cells measured
Author's personal copy
Fish Sci
Author's personal copy Fish Sci Table 2 GenBank blast hits for the ITS and LSU sequences of Pseudo-nitzschia fukuyoi in this study (E value = 0.0) Pn 5/Pn 6
Pn 5/Pn 6
Accession number
Query cover- Identity % Reference age %
ITS sequences PnKk36 KC147515 PnTb55 KC147520 PnTb31 KC147517 LSU sequences PnTb72 KC147537 PnTb39 KC147536 PnTb25
KC147535
99
100
Lim et al. [3]
99 99
99 99
Lim et al. [3] Lim et al. [3]
100 100
99 99
Lim et al. [3] Lim et al. [3]
100
99
Lim et al. [3]
LSU sequences were also examined with blastn. The LSU sequences showed 100 % coverage with 99 % identity and 0.0 E values. The first three hits were P. fukuyoi (PnTb72, PnTb39, and PnTb25). Domoic acid production in cultured clones
Intensity, cps
Results from the LC–MS/MS analysis showed detectable DA in both cultured strain extracts. Cellular DA concentration 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 pseudomolecular ion [M + H]+ (m/z 312) in both m/z 312 > 266 and 266 > 91 (Fig. 2a, b). Fragmentation patterns of daughter ions characteristic of DA (m/z 266, 248, 161), which are 4736 4500 4000 3500 3000 2500 2000 2000 1500 1000 500 0
Discussion The Pseudo-nitzschia fukuyoi strains in this study are almost identical morphologically to the original description 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 differs 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 description (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 documentation of this species from Vietnamese waters [6, 7].
8.94 min
(a)
m/z 312>266
m/z 312>91
Intensity, cps
Strain
identical for DA (Fig. 2c), were observed consistently in the culture extract (Fig. 2d).
3.7e4 3.5e4
312.1
(c)
266.3
3.0e4 161.2
2.5e4 1.5e4
220.0
1.0e4 5000.0 1
2
3
4
5
6
7
8
9
60
10 11 12 13 14 15 16 17 18 19
80
100 120 140 160 180 200 220 240 260 280 300 320
Time, min.
3.0e4
8.92 min
(b)
m/z 312>266
2.5e4 2.0e4 1.5e4
m/z 312>91
1.0e4 5000.0 0
1
m/z, amu
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19
Time, min.
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-
Intensity, cps
Intensity, cps
3.4e4
248.3
2.0e4
2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4
266.2
(d)
312.1 161.0 220.1
60
80
248.3
100 120 140 160 180 200 220 240 260 280 300 320
m/z, amu
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
13
Author's personal copy
Therefore, this is the first record of P. fukuyoi in Vietnamese 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 originating 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 toxigenic 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-producing 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 identified as P. fukuyoi, while no other potential toxic Pseudonitzschia 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 elucidated yet, but it is inevitable to clarify toxin accumulation process and system in the bay.
13
Fish Sci Acknowledgments This study was funded by project 106.992010.22—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 manuscript, 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 (Bacillariophyceae) 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, Lundholm N, Kotaki Y, Lim PT (2012) Morphology and molecular characterization of Pseudo-nitzschia (Bacillariophyceae) from Malaysian Borneo, including the new species Pseudonitzschiacircumpora 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. batesiana sp. nov., P. lundholmiae sp. nov., and P. fukuyoi sp. nov. (Bacillariophyceae) from the Strait of Malacca, Malaysia. J Phycol 49(5):902–916 4. Teng ST, Leaw CP, Lim HC, Lim PT (2013) The genus Pseudonitzschia (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 toxigenic 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: Potentially 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 7. Doan NH, Tin NT, Anh NTM, Lam NN (2013) Species composition and cell density variation of diatoms Pseudo-nitzschia spp. in coastal waters of Khanh Hoa, Vietnam. In: Bui HL et al (eds) Proceedings of the international conference on “Bien Dong 2012”, 12–14 September, 2012 (in Vietnamese with English abstract), pp 159–268 8. Dao VH, Lim PT, Ky PX, Takata Y, Teng ST, Omura T, Fukuyo Y, Kodama M (2014) Diatom Pseudo-nitzschia cf. caciantha (Bacillariophyceae), the most likely source of domoic acid contamination in the thorny oyster Spondylusversicolor Schreibers 1793 in Nha Phu Bay, Khanh Hoa Province, Vietnam. Asian Fish Sci 27:16–29 9. DaoVH, Takata Y, Omura T, Sato S, Fukuyo Y, Kodama M (2009) Seasonal variation of domoic acid in Spondylus versicolor in association with that in plankton samples in Nha Phu Bay, Khanh Hoa, Vietnam. Fish Sci 75:507–512 10. Guillard RRL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH (eds) Culture of marine invertebrate animals. Plenum Press, New York, pp 26–60 11. Bates SS, de Freltas ASW, Milley JE, Pocklington R, Quilliam MA, Smith JC, Worms J (1991) Controls on domoic acid production by the diatom Nitzschiapungens f. multiseries in culture nutrients and irradiance. Can J Fish Aquat Sci 48:1136–1144 12. Lundholm N, Bates SS, Baugh KA, Bill BD, Connell LB, Léger C, Trainer VL (2012) Cryptic and pseudo-cryptic diversity in diatoms-with descriptions of Pseudo-nitzschiahasleana sp. nov. and P. fryxelliana sp. nov. J Phycol 48(2):436–454 13. Bargu S, Koray T, Lundholm N (2002) First report of Pseudonitzschia calliantha Lundholm, Moestrup and Hasle 2003, a new
Author's personal copy Fish Sci
14.
15.
16.
17.
potentially toxic species from Turkish Coast. J Fish Aqua Sci 19(3–4):479–483 White T, Bruns T, Lee S, Taylor J, Innis M, Gelfand D, Shinsky J, White T (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA et al (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 315–322 Scholin CA, Herzog M, Sogin M, Anderson DM (1994) Identification of group-and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae). II. Sequence analysis of a fragment of the LSU rRNA gene. J Phycol 30(6):999–1011 Orive E, Pérez-Aicua L, David H, García-Etxebarria K, LazaMartínez A, Seoane S, Miguel I (2013) The genus Pseudonitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov. J Phycol 49:1192–1206 Lundholm N, Moestrup Ø, Hasle GR, Hoef-Emden K (2003) A study of the Pseudo-nitzschia pseudodelicatissima/cuspidata complex (Bacillariophyceae): what is P. pseudodelicatissima? J Phycol 39:797–813
18. Amato A, Montresor M (2008) Morphology, phylogeny, and sexual cycle of Pseudo-nitzschia mannii sp. nov. (Bacillariophyceae): a pseudocryptic species within the P. pseudodelicatissima complex. Phycologia 47:487–497 19. Lelong AL, Hégaret HH, Soudant P, Bates SS (2012) Pseudonitzschia (Bacillariophyceae) species, domoic acid and amnesic shellfish poisoning: revisiting previous paradigms. Phycologia 51:168–216 20. Fernandes LF, Hubbard KA, Richlen M, Smith J, Bates SS, Ehrman J, Léger C, Mafra LL Jr, Kulis D, Quilliam M, Erdner D, Libera K, McCauley L, Anderson D (2014) Diversity and toxicity of the diatom Pseudo-nitzschia Peragallo in the Gulf of Maine, Northwestern Atlantic Ocean. Deep Sea Res II 103:139–162 21. Trainer VL, Bates SS, Lundholm N, Thessen AE, Cochlan WP, Adams NG, Trick CG (2012) Pseudo-nitzschia physiological ecology, phylogeny, toxicity, monitoring and impacts on ecosystem health. Harmful Algae 14:271–300
13
