Next Article in Journal
The Presence of Mycotoxins in Human Amniotic Fluid
Next Article in Special Issue
Critical Review and Conceptual and Quantitative Models for the Transfer and Depuration of Ciguatoxins in Fishes
Previous Article in Journal
Colombian Scorpion Centruroides margaritatus: Purification and Characterization of a Gamma Potassium Toxin with Full-Block Activity on the hERG1 Channel
Previous Article in Special Issue
Acute Toxicity of Gambierone and Quantitative Analysis of Gambierones Produced by Cohabitating Benthic Dinoflagellates
Article

First Report of Domoic Acid Production from Pseudo-nitzschia multistriata in Paracas Bay (Peru)

1
Banco de Germoplasma de Organismos Acuáticos, Instituto del Mar del Perú, Callao 07021, Peru
2
Doctorado en Acuicultura, Programa Cooperativo Universidad de Chile, Pontificia Universidad Católica de Valparaíso, Coquimbo 17811421, Chile
3
Facultad de Ciencias del Mar, Departamento de Acuicultura, Universidad Católica del Norte, Coquimbo 1281, Chile
4
Centro de Investigación y Desarrollo Tecnológico en Algas (CIDTA), Facultad de Ciencias del Mar, Universidad Católica del Norte, Coquimbo 1281, Chile
5
Centro Universitario de Investigaciones Oceanológicas, Universidad de Colima, Manzanillo 28218, Mexico
6
Facultad de Ciencias, Universidad Nacional Agraria La Molina, Avenida La Universidad s/n, La Molina, Lima 15024, Peru
7
Centro de Investigacións Mariñas (Xunta de Galicia), 36620 Vilanova de Arousa, Pontevedra, Spain
*
Authors to whom correspondence should be addressed.
Received: 12 May 2021 / Revised: 3 June 2021 / Accepted: 7 June 2021 / Published: 9 June 2021
(This article belongs to the Special Issue Marine Toxins from Harmful Algae and Seafood Safety)

Abstract

The Peruvian sea is one of the most productive ecosystems in the world. Phytoplankton production provides food for fish, mammals, mollusks and birds. This trophic network is affected by the presence of toxic phytoplankton species. In July 2017, samples of phytoplankton were obtained from Paracas Bay, an important zone for scallop (Argopecten purpuratus) aquaculture in Peru. Morphological analysis revealed the presence of the genus Pseudo-nitzschia, which was isolated and cultivated in laboratory conditions. Subsequently, the monoclonal cultures were observed by scanning electron microscopy (SEM), and identified as P. multistriata, based on both the morphological characteristics, and internal transcribed spacers region (ITS2) sequence phylogenetic analysis. Toxin analysis using liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) revealed the presence of domoic acid (DA) with an estimated amount of 0.004 to 0.010 pg cell−1. This is the first report of DA from the coastal waters of Peru and its detection in P. multistriata indicates that it is a potential risk. Based on our results, routine monitoring of this genus should be considered in order to ensure public health.
Keywords: harmful algae; amnesic shellfish poisoning; ITS2; Argopecten purpuratus; scallop harmful algae; amnesic shellfish poisoning; ITS2; Argopecten purpuratus; scallop

1. Introduction

Marine planktonic diatoms of the genus Pseudo-nitzschia are found in polar, warm and tropical regions; most of their species are cosmopolitan [1,2,3]. Currently, 56 species [4,5] of this genus have been reported, 26 of them producing the neurotoxic compound domoic acid (DA) [6]. Examination of the morphology by optical microscopy is frequently inconclusive, and for this reason scanning and/or transmission electron microscopy is also required. Additionally, molecular analysis of ITS2 [7] of the nuclear encoded ribosomal DNA can identify the species at the molecular level and differentiate between cryptic and pseudo-cryptic species [2,8,9,10,11,12].
The first intoxication in humans by DA was reported at Prince Edward Island, Canada, in 1987. More than 100 people reported becoming ill and at least three people died after eating blue mussels (Mytilus edulis) [13]. Digestive problems and short-term memory loss were the main symptoms of this intoxication, these led to the syndrome being named amnesic shellfish poisoning (ASP) [13,14,15]. Since the report of the first outbreak detected in Canada produced by the diatom Pseudo-nitzschia multiseries, episodes of DA have been recorded in many areas around the world [2,3]. Besides its effects on humans, DA also has severe effects on the trophic transfer between harmful microalgae, filter feeders and mollusks [16,17,18,19,20], spreading to fish [21] and causing mortality of birds and marine mammals [2,22,23,24].
Blooms of Pseudo-nitzschia spp., which produce DA, generate large economic losses due to long periods of closures for recreational and commercial fisheries or marketing of aquaculture products. Recently, these toxic outbreaks have been reported in the United States from 2015 to 2016, affecting the fisheries of the razor clam (Siliqua patula), Dungeness crab (Metacarcinus magister) and rock crabs (Cancer productus, Metacarcinus anthonyi and Romaleon antennarium) [3,24,25,26]. Likewise, in Europe, the DA-producing diatoms of genus Pseudo-nitzschia have led to bans on harvesting the natural populations of the king scallop Pecten maximus, and to the discouragement of aquaculture efforts for this species [27,28], due to its high capability to retain the toxin [29,30].
The coastal upwelling system of Peru constitutes a large part of the Humboldt Current system and is considered one of the most productive regions in the world, fixing 3000–4000 mg C m−2 d−1 [31,32,33,34]. Due to this high productivity, the area is susceptible to harmful algal blooms [2,35,36]. For Peruvian oceanic and coastal areas, the first report is from Hasle [37], who described the presence of Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle and P. australis Frenguelli (as P. pseudo seriata G.R. Hasle) [2,37]. More recently, Tenorio, et al. [38] reported the presence of a non-toxic strain of P. subpacifica (Hasle) Hasle on the central coast of Peru, between San Lorenzo Island, Callao (12°03′S), and Paracas Bay, Ica (13°49′S). In northern Chile, within the framework of the Molluscan Shellfish Safety Program of the National Fisheries and Aquaculture Service (SERNAPESCA), elevated levels of DA have been detected in shellfish from many of the primary scallop Argopecten purpuratus aquaculture sites [39]. The blooms associated with those events have been dominated by diatom Pseudo-nitzschia australis with densities around 1.6 × 106 cell L−1 [40].
In Peru, during the period 2011–2012, intoxication of fur seals (Arctocephalus australis) and sea lions (Otaria byronia) was reported in San Juan de Marcona, Ica (15°20’S), associated with the detection of DA in feces of these marine mammals. During this episode, Pseudo-nitzschia spp. were detected with a maximum of 88,580 cell L−1 in Paracas Bay sampling station (distance of ~155 km to the north) [41]. Unfortunately, there is no additional information about the species that formed the Pseudo-nitzschia assemblage.
Paracas Bay is a traditional aquaculture area of the scallop A. purpuratus, the most important bivalve species in Peru [42]. To date, within the framework of the Molluscan Shellfish Control Program run by the National Fisheries Health Organization of Peru (SANIPES), there is no information about the presence of DA in this bivalve. Nevertheless, the detection of DA in marine mammals in nearby areas indicates that the toxin is a potential risk to aquaculture and suggests that more research is necessary in order to identify different Pseudo-nitzschia species and their capability to produce DA on the coast of Peru.
In 2017, phytoplankton were collected in Paracas Bay to establish monoclonal cultures of Pseudo-nitzschia spp., for their morphological, molecular and toxicological characterization, in order to understand the potentially toxic species of the genus Pseudo-nitzschia in Peruvian waters.

2. Results

2.1. Morphological Analysis

The isolated strain was morphologically identified as Pseudo-nitzschia multistriata (H. Takano) H. Takano. The cells of strain IMP-BG 440 (Figure 1) had a sigmoid shape in girdle view, and formed stepped chains, with up to four in length and an overlap of 1/6 of the total cell length. The cells were symmetrical and broad lanceolate in valve view. The apical axis ranged from 40 to 48 µm, while the transapical axis of the valves ranged from 3.20 to 4.30 µm. A large central interspace and a central nodule were absent. Valves contained 24 to 28 fibulae and 40 to 42 striae per 10 μm. Striae were formed by 2–3 rows of poroids with a density between 8 to 13 per 1 μm.

2.2. Molecular Analysis

Final alignment yielded 414 characters and comprised 128 ITS2 sequences, including a short sequence (MZ312514, 132 pb) of the strain IMP-BG 440. Phylogenetic analysis of ITS2 sequences using maximum likelihood (ML) and Bayesian inference (BI) showed congruent topologies (Figure 2). Within the genus Pseudo-nitzschia, a well-supported monophyletic clade (BI/ML, 1/100) corresponded to P. multistriata, and 11 strains from Australia, Malaysia, Taiwan, Japan, South Korea, China, Greece, Italy, Spain and Portugal. The clade containing P. multistriata also contained species of Pseudo-nitzschia from France (P. americana) and Malaysia (P. braziliana) with low support (BI, 0.73). This Pseudo-nitzschia clade was positioned within a larger unsupported clade (BI, 0.70) containing Pseudo-nitzschia species from Malaysia (genera type P. pungens), Japan and the USA (P. multiseries). Additionally, the phylogenetic tree shows that P. multistriata is grouped with other species of Pseudo-nitzschia with different levels of support. However, five sequences of Fragilariopsis from Artic, Antarctica and the USA form a supported clade within species of Pseudo-nitzschia (BI/ML, 0.99/64).

2.3. Toxin Analysis

Analysis of extracts (n = 3) of the strain IMP-BG 440 showed that it contained domoic acid (Figure 3). Toxin analysis by LC-HRMS showed a chromatographic peak with a retention time of 6.60 min corresponding to the ion [M+H]+ 312.1449 m/z (mass deviation: 0.64 ppm). The fragmentation mass spectrum of the ion [M+H]+ 312.1449 m/z confirmed the identification of domoic acid (DA) because of its characteristic fragment MS/MS at 294.1332, 266.1384, 248.1277 and 220.1329 m/z (mass deviation in Table S1; Supplementary Materials). The estimated amount was between 0.004 to 0.010 pg cell−1.

3. Discussion

The species of the genus Pseudo-nitzschia are distributed throughout all the coasts of the world [1,2,3]. The present study provides the first report of Pseudo-nitzschia multistriata in Peruvian coastal waters and, as far as we know, in the southeastern Pacific area. The presence of this species has been reported from different geographical locations around the world (Table S2, Supplementary Materials).
The morphological examination of Pseudo-nitzschia cells (Table S2, Supplementary Materials) from the obtained cultures agrees in length of apical axis with the description of strains of P. multistriata from China [43], Tunisia [44], Catalan Coast [45,46], Gulf of Naples [47,48] and the Western Adriatic Sea [49]. Similarly, the width of the transapical axis corresponds to descriptions of cells from Ria de Aveiro, Portugal [50], Tokyo bay [51], New Zealand [52], Mexico [53] and Uruguay [54]. However, the length of the apical axis and the width of the transapical axis of the cell do not match the first description made by Takano [55], given that those were smaller.
The number of fibulae of Paracas strains was close to descriptions of cells from Ria de Aveiro, Portugal [50], Greek coastal waters [56], Gulf Naples, Italy [57] and Gulf of Trieste, Northern Adriatic Sea, Italy [58]. Finally, the striae were formed by 2–3 rows of poroids and their density were similar to those described in cells from Fukukoka Bay, Japan [55] and the Sea of Japan [59].
Phylogenetic analysis of ITS2 sequences support the morphological identification of the strain isolated from Paracas Bay as P. multistriata. This strain from Peru is situated with P. multistriata strains from Australia, Malaysia, South Korea, Taiwan, Japan, China and Europe, forming a well-supported monophyletic clade (BI/ML, 1/100). The phylogenetic tree also shows that P. multistriata is grouped with P. brasiliana and P. americana, within a clade comprising P. pungens (genera type), P. multiseries, P. australis, P. seriata and P. obtuse. Previous phylogenetic analyses of ITS2 sequences had pointed to different relationships of P. multistriata with P. americana, P. brasiliana, and P. pungens. Huang, et al. [60] showed that P. americana is placed on the base of the clade, not clustered with P. multistriata and P. brasiliana. On the contrary, Lim, et al. [61] showed that P. multiseries and P. pungens are placed at the base of the tree. Morphological characteristics have been included by Lim, et al. [61] as representative of some species of the Pseudo-nitzschia clade. Thus, morphological characters, 2–4 rows of poroids, the absence of a central nodule and the lower number of fibulae versus striae in 10 µm, were observed in P. multistriata from Japan [62], matching the description of the strain of P. multistriata (IMP-BG 440) from Peru.
This study confirms P. multistriata as an unequivocal source of domoic acid (DA) on the coast of Peru. The strain IMP-BG 440 tested was able to produce the toxin in culture with a concentration between 0.004 and 0.010 pg cell−1, which is comparable to those reported by Pistocchi, et al. [49] (0.003 pg cell−1) in a strain obtained from the Adriatic Sea that was cultured under similar conditions (16–18 °C; 60–100 μmol photons m−2 s−1). These concentrations were lower than the values reported in Australian strains by Ajani, et al. [63] (1–11 pg cell−1), Rhodes, et al. [64] (1.5 pg cell−1) and in Italian strains registered by Amato, et al. [65] (0.28 pg cell−1), Orsini, et al. [47] (0.69 pg cell−1) and Sarno [48] (0.65 pg cell−1).
The Humboldt Current system (HCS) is considered one of the most productive fishery regions in the world oceans [33,34,66,67]. As mentioned above, due to its high productivity, this upwelling area is susceptible to harmful algal blooms (HABs) [35,36]. In this context, other toxic Pseudo-nitzschia species have been reported in the HCS, specifically on the northern Chilean coast [68,69]. In some cases, DA concentrations have exceeded the regulatory limit (20 mg·kg−1) and the harvesting of scallops (A. purpuratus), from aquaculture sites, has therefore been banned [40]. The DA content in P. multistriata (strain IMP-BG 440) was substantially lower than those reported in P. australis (1.74 pg cell−1) for the southeastern Pacific; however, it was close to the value of P. calliantha (0.01 pg cell−1) [39]. The low content of DA in P. multistriata in Peruvian waters could be one of the reasons that there has not been any detection of this toxin in scallops cultivated in Paracas Bay in the framework of the Molluscan Shellfish Control Program run by SANIPES. A second reason could be the rapid DA depuration of this bivalve in the natural environment as has been demonstrated by Álvarez, et al. [70] in scallops cultivated in Tongoy Bay, Chile. However, the information provided by this work should be taken into consideration in the development of the Molluscan Shellfish Control Program ran by SANIPES [71].
Regarding the intoxication of marine mammals with low levels of DA on the Peruvian coast [41], it is clear that P. multistriata could be involved. However, with the available information we cannot discard the possibility that other species of Pseudo-nitzschia or more toxic strains than the one found in this study could be the principal cause of pinniped intoxication. Finally, more research is needed to find other toxic species, as well as the roll of different environmental variables in the production of DA in different strains of P. multistriata obtained along the Peruvian coast.

4. Conclusions

Pseudo-nitzschia multistriata has been identified from the Peruvian coast based on morphological, phylogenetic and molecular evidence. This is the first report of this species for the Southeast Pacific. The species is confirmed to be a producer of DA which makes it the first known DA producer from Peruvian waters. The presence of toxic P. multistriata is a potential risk for mammals, making it necessary to routinely monitor this species in order to protect public health, as well as the ecosystem of Paracas Bay.

5. Materials and Methods

5.1. Biological Samples and Establishment of Cultures

Phytoplankton samples were obtained periodically in August 2017 in Paracas Bay (13°49′S, 76°17′O) (Figure 4) with temperatures of around 15 to 17 °C and salinity of 35.
Samples were collected using vertical net hauls (20 μm mesh), stored in 250 mL glass bottles and transported to the laboratory in the dark and chilled on ice (10 °C). To establish cultures of the Pseudo-nitzschia species, single chains of Pseudo-nitzschia cells were picked by micropipette and transferred to multi-well culture plates (hydrobios, Germany) filled with 2 mL of L1 culture medium [72] with a salinity of 30. The plates were maintained at 15 °C in a 12:12-h light: dark cycle, with a photon flux of 60 μmol photons m−2 s−1. Established cultures were transferred to borosilicate Erlenmeyer flasks with 150 mL of f/2 medium and grown at 15 °C in a 12:12-h light: dark cycle, with a photon flux of 80 μmol photons m−2 s−1. Mass cultures were grown in borosilicate bottles with 1 L of f/2 medium in triplicate under the above conditions. Two milliliter aliquots of the cultures were preserved with Lugol’s solution for the direct count of the cells. The cell densities in the samples were quantified by the Utermöhl method described by Hasle [73].

5.2. Morphological Analysis

Scanning electron microscopy (SEM) was used to perform detailed morphological analyses of the Pseudo-nitzschia cells. Organic matter was removed from the frustules following the methodology described by Lundholm, et al. [74]. The clean material was retained on a 5.0 μm membrane filter (Isopore Merck KGaA, Darmstadt, Germany), and washed with distilled water to remove salts and preservatives. After being airdried overnight, specimens were gold-coated in a JEOL JFC-1100 (JEOL Ltd., Tokyo, Japan) and observed with a Hitachi SU3500 scanning electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan). Pseudo-nitzschia cells were examined for morphometric characteristics that included width and length of the valve, density of striae, fibulae and poroids.

5.3. Molecular and Phylogenetic Analysis

Molecular identification of the strain of the Pseudo-nitzschia genus was performed by analyzing sequences of the internal transcribed spacer two (ITS2) region. When initial cultures of the strain reached the exponential growth phase, cells were concentrated by successive centrifugations and frozen at −80 °C prior to DNA extraction (24 h). Total genomic DNA was extracted following the cetyltrimethylammonium bromide (CTAB) method [75]. Part of the internal transcribed spacer (ITS) region was amplified by PCR, using ITS1/ITS4 primers (BIOSEARCH TECHNOLOGIES, Petaluma, CA, USA) [76]. The polymerase chain reaction (PCR) conditions for ITS include pre denaturation at 95 °C for 3 min, followed by 39 cycles of 95 °C for 30 s, 45 °C for 30 s and 72 °C for 50 s; and finally, 71 °C for 7 min. The amplicons were visualized on agarose gel (1.2%) (Invitrogen, Carlsbad, USA), purified and sequenced for one strand by Macrogen Inc. (South Korea).
Sequences obtained were checked in BIOEDIT v.7.0.5.3 (Raleigh, NC, USA., 2005) [77] and compared in the GenBank public database using the Basic Local Alignment Search Tool BLASTn. The data block used in the molecular analyses consisted of 128 ITS sequences (Table S3), including the one sequence of Pseudo-nitzschia obtained in this study, sequences of Pseudo-nitzschia and 5 sequences of Fragilariopsis available in the public database and a sequence of Nitzschia longissima as an outgroup. The alignment was constructed using the Muscle algorithm in MEGA7v.7.0.26 (Philadelphia, PA, USA., 2016) [78], checked visually, corrected and trimmed using MEGA7 so that it only contained sequences of the ITS2 region. Final alignment was independently analyzed using maximum likelihood (ML) and Bayesian inference (BI). The best evolutionary models for ML and BI were calculated in jModelTest 2 (Spain, 2012) [79] using the Akaike information criterion (AIC) and the Bayesian information criterion (BIC), respectively. ML analysis was carried out in RAxML v.8.2.X (Karlsruhe, Germany, 2014) [80] using the graphic user interface raxmlGUI v.1.5 5b1 (Frankfurt, Germany, 2012) [81] with the selected model (GTR+I+G) and 1000 bootstrap replications. BI was carried out in MrBayes v.3.2.6 [82] with the selected model (HKY+I+G), two runs of 10 million Markov chain Monte Carlo generations each with 1 cold chain and 3 heated chains, sampling and printing every 1000 generations. The convergence of the runs was checked using Tracer v.1.6.0 (Edinburgh, UK, 2014). A consensus tree was constructed after a burn-in of 25%, and posterior probabilities were estimated.

5.4. Sample Preparation and Toxin Analysis

A 1 L sample from the culture of Pseudo-nitzschia spp. (densities ranged from 267,170 to 305,691 cell mL−1) was taken in the stationary phase of growth. The sample was concentrated by centrifugation at 4000 g for 10 min with a centrifuge (Hettich Rotina 420R, Germany). The obtained pellets were mixed with 10 mL of aqueous methanol (Merck KGaA, Darmstadt, Germany) (50%, v/v) and the cells disrupted with a Branson Ultrasonic 250 (Danbury, CT, USA). The extract was clarified by centrifugation at 10,000 g for 20 min (Centurion K2015R, Centurion Scientific Ltd., Stoughton, West Sussex, UK). A one-milliliter aliquot was filtered through 0.22 μm Clarinert nylon syringe filters (13 mm diameter) (Bonna-Agela technologies, Torrance, CA, USA) and stored in an autosampler vial at −20 °C until analysis. The presence of DA (cellular content) in the extracts was determined following the method described by de la Iglesia, et al. [83] with modifications. The instrumental analysis was developed using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Sunnyvale, CA, USA). A reversed-phase HPLC column Kinetex C18 (50 mm × 2.1 mm; 2.6 µm) with an Ultra Guard column C18 from Phenomenex (Torrance, CA, USA) was used. The flow rate was set to 0.28 mL min−1, and the injection volume was 10 µL. Mobile phases A and B were water (Milli-Q) and MeOH, respectively, both containing 0.1% formic acid. The following gradient was used to achieve the chromatographic separation: 100% phase A, held for the first 0.5 min. Afterwards, separation was carried out at 12.5% B up to 3 min, decreased to 3% B over 7 min and then returned to the initial conditions over 2 min. The total analysis run time was 12 min.
The detection of DA was carried out by a high-resolution mass spectrometer Q Exactive Focus equipped with an electrospray interphase HESI II (Thermo Fisher Scientific, Sunnyvale, CA, USA). The interface was operated in positive ionization mode with a spray voltage of 3.5 kV. The temperature of the ion transfer tube and the HESI II vaporizer were set at 250 °C. Nitrogen (>99.98%) was employed as a sheath gas and auxiliary gas at pressures of 20 and 10 arbitrary units, respectively. Data were acquired in selected ion monitoring (SIM) and data-dependent (ddMS2) acquisition modes (for quantification and confirmation, respectively). In SIM mode, the mass was set to 312.1404 m/z, the scan mass range was set at m/z 100–1000 with a mass resolution of 70,000, the automatic gain control (AGC) was set at 5 × 104 and the maximum injection time (IT) 3000 ms. For dds2 the mass resolution was set at 70,000, AGC at 5 × 104 and IT 3000 ms. In both cases, the isolation windows were 2 m/z. DA was quantified by external calibration, using a DA-certified reference solution (CRM-DA-g) (NRC, CNRC, Canada). Limits of detection were calculated as the average concentration of DA producing a signal-to-noise ratio (S/N) of 3 and corresponding to 0.5 ng mL−1, while the limit of quantification of the method was 2 ng mL−1.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/toxins13060408/s1, Table S1: Accurate mass and mass deviation (ppm) of domoic acid and its main fragments. Table S2: Comparison of morphometric data between Peruvian strain of Pseudo-nitzschia multistriata with strains obtained from different locations around the world, Table S3: List of sequences of Pseudo-nitzschia, Fragilariopsis and Nitzschia strains included in the molecular analysis. Species, locality, strain code and GenBank accession numbers for the ITS2 gene marker.

Author Contributions

Conceptualization, C.T., G.Á., E.U., S.Q.-S. and J.B.; methodology, C.T., G.Á., E.U., S.Q.-S., M.A. and N.A.; software, C.T., S.Q.-S., N.A., M.P.-A., M.A. and G.Á.; investigation, C.T., G.Á., S.Q.-S., M.P.-A., N.A., M.A., F.Á., J.B. and E.U.; formal analysis, C.T., G.Á., S.Q.-S., E.U. and J.B.; visualization, G.Á., S.Q.-S., E.U. and J.B.; writing—original draft preparation, C.T., G.Á., M.A., E.U., S.Q.-S., E.U. and J.B.; writing—review and editing, G.Á., S.Q.-S. and J.B.; visualization, C.T., G.Á., M.A. and J.B.; supervision, G.Á., S.Q.-S., E.U. and J.B.; funding acquisition, G.Á. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the “ANID + FONDEF/PRIMER CONCURSO INVESTIGACIÓN TECNOLÓGICA TEMATICO ENSISTEMAS PESQUERO ACUICOLAS FRENTE A FLORECIMIENTOS ALGALES NOCIVOS FANS IDeA DEL FONDO DE FOMENTO AL DESARROLLO CIENTÍFICO Y TECNOLÓGICO, FONDEF/ANID 2017, IT17F10002” developed within the framework of a cooperation agreement between the Consellería do Mar, Xunta de Galicia, Spain and the Universidad Católica del Norte, Chile and the Budget National Program N°094 (PpR094): “Management and Development of Aquaculture”—PRODUCE, PERU, through the project “Technological Development in Aquaculture”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We acknowledge the project the FONDEQUIP150109-Chile.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Hasle, G.R. Are most of the domoic acid-producing species of the diatom genus Pseudo-nitzschia cosmopolites? Harmful Algae 2002, 1, 137–146. [Google Scholar] [CrossRef]
  2. Trainer, V.L.; Bates, S.S.; Lundholm, N.; Thessen, A.E.; Cochlan, W.P.; Adams, N.G.; Trick, C.G. Pseudo-nitzschia physiological ecology, phylogeny, toxicity, monitoring and impacts on ecosystem health. Harmful Algae 2012, 14, 271–300. [Google Scholar] [CrossRef]
  3. Bates, S.S.; Hubbard, K.A.; Lundholm, N.; Montresor, M.; Leaw, C.P. Pseudo-nitzschia, Nitzschia, and domoic acid: New research since 2011. Harmful Algae 2018, 79, 3–43. [Google Scholar] [CrossRef]
  4. Chen, X.M.; Pang, J.X.; Huang, C.X.; Lundholm, N.; Teng, S.T.; Li, A.; Li, Y. Two New and Nontoxigenic Pseudo-nitzschia species (Bacillariophyceae) from Chinese Southeast Coastal Waters. J. Phycol. 2021, 57, 335–344. [Google Scholar] [CrossRef]
  5. Guiry, M.D.; Guiry, G.M. AlgaeBase. Available online: http://www.algaebase.org (accessed on 1 March 2021).
  6. Lundholm, N. Bacillariophyceae, in IOC-UNESCO Taxonomic Reference List of Harmful Micro Algae. Available online: http://www.marinespecies.org/hab (accessed on 25 February 2021).
  7. Coleman, A.W. ITS2 is a double-edged tool for eukaryote evolutionary comparisons. Trends Genet. 2003, 19, 370–375. [Google Scholar] [CrossRef]
  8. Quijano-Scheggia, S.I.; Garcés, E.; Lundholm, N.; Moestrup, Ø.; Andree, K.; Camp, J. Morphology, physiology, molecular phylogeny and sexual compatibility of the cryptic Pseudo-nitzschia delicatissima complex (Bacillariophyta), including the description of P. arenysensis sp. nov. Phycologia 2009, 48, 492–509. [Google Scholar] [CrossRef]
  9. Quijano-Scheggia, S.I.; Olivos-Ortiz, A.; Garcia-Mendoza, E.; Sánchez-Bravo, Y.; Sosa-Avalos, R.; Salas Marias, N.; Lim, H.C. Phylogenetic relationships of Pseudo-nitzschia subpacifica (Bacillariophyceae) from the Mexican Pacific, and its production of domoic acid in culture. PLoS ONE 2020, 15, e0231902. [Google Scholar] [CrossRef] [PubMed]
  10. Lundholm, N.; Moestrup, Ø.; Hasle, G.R.; Hoef-Emden, K. A study of the Pseudonitzschia pseudodelicatissima/cuspidata complex (Bacillariophyceae): What is P. pseudodelicatissima? J. Phycol. 2003, 39, 797–813. [Google Scholar] [CrossRef]
  11. Lundholm, N.; Bates, S.S.; Baugh, K.A.; Bill, B.D.; Connell, L.B.; Léger, C.; Trainer, V.L. Cryptic and pseudo-cryptic diversity in diatoms—with descriptions of pseudo-nitzschia hasleana sp. nov. and p. fryxelliana sp. nov. 1. J. Phycol. 2012, 48, 436–454. [Google Scholar] [CrossRef]
  12. Amato, A.; Kooistra, W.H.; Ghiron, J.H.L.; Mann, D.G.; Pröschold, T.; Montresor, M. Reproductive isolation among sympatric cryptic species in marine diatoms. Protist 2007, 158, 193–207. [Google Scholar] [CrossRef]
  13. Bates, S.; Bird, C.J.; Freitas, A.d.; Foxall, R.; Gilgan, M.; Hanic, L.A.; Johnson, G.R.; Mc Culloch, A.; Odense, P.; Pocklington, R.; et al. Pennate diatom Nitzschia pungens as the primary source of domoic acid, a toxin in shellfish from eastern Prince Edward Island, Canada. Can. J. Fish. Aquat. Sci. 1989, 46, 1203–1215. [Google Scholar] [CrossRef]
  14. Perl, T.M.; Bédard, L.; Kosatsky, T.; Hockin, J.C.; Todd, E.C.; Remis, R.S. An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid. N Engl. J. Med. 1990, 322, 1775–1780. [Google Scholar] [CrossRef]
  15. Wright, J.; Boyd, R.; Freitas, A.d.; Falk, M.; Foxall, R.; Jamieson, W.; Laycock, M.; McCulloch, A.; McInnes, A.; Odense, P. Identification of domoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Can. J. Chem. 1989, 67, 481–490. [Google Scholar] [CrossRef]
  16. Bates, S. Toxic phytoplankton on the Canadian east coast: Implications for aquaculture. Bull. Aquacult. Assoc. Can. 1997, 97, 9–18. [Google Scholar]
  17. La Barre, S.; Bates, S.S.; Quilliam, M.A. Domoic acid. In Outstanding Marine Molecules: Chemistry, Biology, Analysis; La Barre, S., Kornprobst, J.M., Eds.; Wiley-VCH Verlag GmbH & KgaA: Weinheim, Germany, 2014; pp. 189–216. [Google Scholar]
  18. Goldberg, J.D. Domoic Acid in the Benthic Food Web of Monterey Bay, California. Master’s Thesis, University Monterey Bay, Seaside, CA, USA, 2003. [Google Scholar]
  19. Zabaglo, K.; Chrapusta, E.; Bober, B.; Kaminski, A.; Adamski, M.; Bialczyk, J. Environmental roles and biological activity of domoic acid: A review. Algal Res. 2016, 13, 94–101. [Google Scholar] [CrossRef]
  20. Bates, S.S.; Garrison, D.L.; Horner, R.A. Bloom dynamics and physiology of domoic-acid-producing Pseudo-nitzschia species. Nato Asi Ser. G Ecol. Sci. 1998, 41, 267–292. [Google Scholar]
  21. Lefebvre, K.A.; Frame, E.R.; Kendrick, P.S. Domoic acid and fish behavior: A review. Harmful Algae 2012, 13, 126–130. [Google Scholar] [CrossRef]
  22. D’Agostino, V.C.; Degrati, M.; Sastre, V.; Santinelli, N.; Krock, B.; Krohn, T.; Dans, S.L.; Hoffmeyer, M.S. Domoic acid in a marine pelagic food web: Exposure of southern right whales Eubalaena australis to domoic acid on the Peninsula Valdes calving ground, Argentina. Harmful Algae 2017, 68, 248–257. [Google Scholar] [CrossRef]
  23. Di Liberto, T. This summer’s West Coast algal bloom was unusual. What would Usual Look Like? 30 September 2015. [Google Scholar]
  24. McCabe, R.M.; Hickey, B.M.; Kudela, R.M.; Lefebvre, K.A.; Adams, N.G.; Bill, B.D.; Gulland, F.M.; Thomson, R.E.; Cochlan, W.P.; Trainer, V.L. An unprecedented coastwide toxic algal bloom linked to anomalous ocean conditions. Geophys. Res. Lett. 2016, 43, 10366–310376. [Google Scholar] [CrossRef]
  25. Ritzman, J.; Brodbeck, A.; Brostrom, S.; McGrew, S.; Dreyer, S.; Klinger, T.; Moore, S.K. Economic and sociocultural impacts of fisheries closures in two fishing-dependent communities following the massive 2015 US West Coast harmful algal bloom. Harmful Algae 2018, 80, 35–45. [Google Scholar] [CrossRef]
  26. Du, X.; Peterson, W.; Fisher, J.; Hunter, M.; Peterson, J. Initiation and development of a toxic and persistent Pseudo-nitzschia bloom off the Oregon coast in spring/summer 2015. PLoS ONE 2016, 11, e0163977. [Google Scholar] [CrossRef]
  27. Arévalo, F.; Bermúdez de la Puente, M.; Salgado, C. Seguimiento de biotoxinas marinas en las Rías Gallegas: Control y evolución durante los años 1995–1996. V Reunión Ibérica de Fitoplancton Tóxico y Biotoxinas. ANFACO-CECOPESCA, Vigo 1997, 90–101. [Google Scholar]
  28. Blanco, J.; Acosta, C.; De La Puente, M.B.; Salgado, C. Depuration and anatomical distribution of the amnesic shellfish poisoning (ASP) toxin domoic acid in the king scallop Pecten maximus. Aquat. Toxicol. 2002, 60, 111–121. [Google Scholar] [CrossRef]
  29. Mauriz, A.; Blanco, J. Distribution and linkage of domoic acid (amnesic shellfish poisoning toxins) in subcellular fractions of the digestive gland of the scallop Pecten maximus. Toxicon 2010, 55, 606–611. [Google Scholar] [CrossRef] [PubMed]
  30. Blanco, J.; Mauríz, A.; Álvarez, G. Distribution of Domoic Acid in the Digestive Gland of the King Scallop Pecten maximus. Toxins 2020, 12, 371. [Google Scholar] [CrossRef] [PubMed]
  31. Calienes, R.; Guillén, O.; Lostaunau, N. Variabilidad espacio-temporal de clorofila, producción primaria y nutrientes frente a la costa peruana. Boletin Instituto del Mar del Peru 1985, 10, 1–44. [Google Scholar]
  32. Graco, M.; Ledesma, J.; Flores, G.; Giron, M. Nutrients, oxygen and biogeochemical processes in the Humboldt upwelling current system off Peru. Rev. Peru. Biol 2007, 14, 117–128. [Google Scholar]
  33. Echevin, V.; Gévaudan, M.; Espinoza-Morriberón, D.; Tam, J.; Aumont, O.; Gutierrez, D.; Colas, F. Physical and biogeochemical impacts of RCP8. 5 scenario in the Peru upwelling system. Biogeosciences 2020, 17, 3317–3341. [Google Scholar] [CrossRef]
  34. Bakun, A.; Weeks, S.J. The marine ecosystem off Peru: What are the secrets of its fishery productivity and what might its future hold? Prog. Oceanogr. 2008, 79, 290–299. [Google Scholar] [CrossRef]
  35. Pitcher, G.C.; Jiménez, A.B.; Kudela, R.M.; Reguera, B. Harmful algal blooms in eastern boundary upwelling systems. Oceanography 2010, 30, 22. [Google Scholar] [CrossRef]
  36. Trainer, V.L.; Pitcher, G.C.; Reguera, B.; Smayda, T.J. The distribution and impacts of harmful algal bloom species in eastern boundary upwelling systems. Prog. Oceanogr. 2010, 85, 33–52. [Google Scholar] [CrossRef]
  37. Hasle, G.R. Nitzschia and Fragilariopsis species studied in the light and electron microscopes. II. The group Pseudonitzschia. Skr. Nor. Vidensk-Akad. I. Mat.-Nat. Kl. Ny Ser. 1965, 18, 1–45. [Google Scholar]
  38. Tenorio, C.; Uribe, E.; Gil-Kodaka, P.; Blanco, J.; Álvarez, G. Morphological and toxicological studies ofPseudo-nitzschiaspecies from the central coast of Peru. Diatom Res. 2016, 31, 331–338. [Google Scholar] [CrossRef]
  39. Álvarez, G.; Uribe, E.; Quijano-Scheggia, S.; López-Rivera, A.; Mariño, C.; Blanco, J. Domoic acid production by Pseudo-nitzschia australis and Pseudo-nitzschia calliantha isolated from North Chile. Harmful Algae 2009, 8, 938–945. [Google Scholar] [CrossRef]
  40. Díaz, P.A.; Álvarez, A.; Varela, D.; Pérez-Santos, I.; Díaz, M.; Molinet, C.; Seguel, M.; Aguilera-Belmonte, A.; Guzmán, L.; Uribe, E. Impacts of harmful algal blooms on the aquaculture industry: Chile as a case study. Perspect. Phycol 2019. [Google Scholar] [CrossRef]
  41. Fire, S.E.; Adkesson, M.J.; Wang, Z.; Jankowski, G.; Cárdenas-Alayza, S.; Broadwater, M. Peruvian fur seals (Arctocephalus australis ssp.) and South American sea lions (Otaria byronia) in Peru are exposed to the harmful algal toxins domoic acid and okadaic acid. Mar. Mammal Sci. 2017, 33, 630–644. [Google Scholar] [CrossRef]
  42. Kluger, L.C.; Taylor, M.H.; Wolff, M.; Stotz, W.; Mendo, J. From an open-access fishery to a regulated aquaculture business: The case of the most important Latin American bay scallop (Argopecten purpuratus). Rev. Aquac. 2019, 11, 187–203. [Google Scholar] [CrossRef]
  43. Lü, S.; Li, Y.; Lundholm, N.; Ma, Y.; Ho, K. Diversity, taxonomy and biogeographical distribution of the genus Pseudo-nitzschia (Bacillariophyceae) in Guangdong coastal waters, South China Sea. Nova Hedwig. 2012, 95, 123–152. [Google Scholar] [CrossRef]
  44. Sahraoui, I.; Grami, B.; Bates, S.S.; Bouchouicha, D.; Chikhaoui, M.A.; Mabrouk, H.H.; Hlaili, A.S. Response of potentially toxic Pseudo-nitzschia (Bacillariophyceae) populations and domoic acid to environmental conditions in a eutrophied, SW Mediterranean coastal lagoon (Tunisia). Estuar. Coast. Shelf Sci. 2012, 102, 95–104. [Google Scholar] [CrossRef]
  45. Quijano-Scheggia, S.; Garcés, E.; Andree, K.B.; De la Iglesia, P.; Diogène, J.; Fortuño, J.M.; Camp, J. Pseudo-nitzschia species on the Catalan coast: Characterization and contribution to the current knowledge of the distribution of this genus in the Mediterranean Sea. Sci. Mar. 2010, 74, 395–410. [Google Scholar] [CrossRef]
  46. Quijano-Sheggia, S.; Garcés, E.; Sampedro, N.; Van Lenning, K.; Flo Arcas, E.; Andree, K.; Fortuño Alós, J.M.; Camp, J. Identification and characterisation of the dominant Pseudo-nitzschia species (Bacillariophyceae) along the NE Spanish coast (Catalonia, NW Mediterranean). Sci. Mar. 2008, 72, 343–359. [Google Scholar]
  47. Orsini, L.; Sarno, D.; Procaccini, G.; Poletti, R.; Dahlmann, J.; Montresor, M. Toxic Pseudo-nitzschia multistriata (Bacillariophyceae) from the Gulf of Naples: Morphology, toxin analysis and phylogenetic relationships with other Pseudo-nitzschia species. Eur. J. Phycol. 2002, 37, 247–257. [Google Scholar] [CrossRef]
  48. Sarno, D. Production of domoic acid in another species of Pseudo-nitzschia: P. multistriata in the Gulf of Naples (Mediterranean Sea). Harmful Algal News 2000, 21, 5. [Google Scholar]
  49. Pistocchi, R.; Guerrini, F.; Pezzolesi, L.; Riccardi, M.; Vanucci, S.; Ciminiello, P.; Dell’Aversano, C.; Forino, M.; Fattorusso, E.; Tartaglione, L. Toxin levels and profiles in microalgae from the North-Western Adriatic Sea—15 years of studies on cultured species. Mar. Drugs 2012, 10, 140–162. [Google Scholar] [CrossRef]
  50. Churro, C.I.; Carreira, C.C.; Rodrigues, F.J.; Craveiro, S.C.; Calado, A.J.; Casteleyn, G.; Lundholm, N. Diversity and abundance of potentially toxic Pseudo-nitzschia Peragallo in Aveiro coastal lagoon, Portugal and description of a new variety, P. pungens var. aveirensis var. nov. Diatom Res. 2009, 24, 35–62. [Google Scholar] [CrossRef]
  51. Yap-Dejeto, L.G.; Omura, T.; Nagahama, Y.; Fukuyo, Y. Observations of eleven Pseudo nitzschia species in Tokyo Bay, Japan. La mer 2010, 48, 1–16. [Google Scholar]
  52. Rhodes, L.L.; Adamson, J.; Scholin, C. Pseudo-nitzschia multistriata(Bacillariophyceae) in New Zealand. New Zealand J. Mar. Freshw. Res. 2000, 34, 463–467. [Google Scholar] [CrossRef]
  53. Rivera-Vilarelle, M.; Quijano-Scheggia, S.; Olivos-Ortiz, A.; Gaviño-Rodríguez, J.H.; Castro-Ochoa, F.; Reyes-Herrera, A. The genus Pseudo-nitzschia (Bacillariophyceae) in Manzanillo and Santiago Bays, Colima, Mexico. Bot. Mar. 2013, 56, 357–373. [Google Scholar] [CrossRef]
  54. Méndez, S.M.; Ferrario, M.; Cefarelli, A.O. Description of toxigenic species of the genus Pseudo-nitzschia in coastal waters of Uruguay: Morphology and distribution. Harmful Algae 2012, 19, 53–60. [Google Scholar] [CrossRef]
  55. Takano, H. Marine diatom Nitzschia multistriata sp. nov. common at inlets of southern Japan. Diatom 1993, 8, 39–41. [Google Scholar]
  56. Moschandreou, K.K.; Baxevanis, A.D.; Katikou, P.; Papaefthimiou, D.; Nikolaidis, G.; Abatzopoulos, T.J. Inter- and intra-specific diversity of Pseudo-nitzschia (Bacillariophyceae) in the northeastern Mediterranean. Eur. J. Phycol. 2012, 47, 321–339. [Google Scholar] [CrossRef]
  57. D’Alelio, D.; Amato, A.; Kooistra, W.H.; Procaccini, G.; Casotti, R.; Montresor, M. Internal transcribed spacer polymorphism in Pseudo-nitzschia multistriata (Bacillariophyceae) in the Gulf of Naples: Recent divergence or intraspecific hybridization? Protist 2009, 160, 9–20. [Google Scholar] [CrossRef]
  58. Dermastia, T.T.; Cerino, F.; Stanković, D.; Francé, J.; Ramšak, A.; Tušek, M.Ž.; Beran, A.; Natali, V.; Cabrini, M.; Mozetič, P. Ecological time series and integrative taxonomy unveil seasonality and diversity of the toxic diatom Pseudo-nitzschia H. Peragallo in the northern Adriatic Sea. Harmful Algae 2020, 93, 101773. [Google Scholar] [CrossRef] [PubMed]
  59. Stonik, I.V.; Orlova, T.Y.; Lundholm, N. Diversity of Pseudo-nitzschia H. Peragallo from the western North Pacific. Diatom Res. 2011, 26, 121–134. [Google Scholar] [CrossRef]
  60. Huang, C.X.; Dong, H.C.; Lundholm, N.; Teng, S.T.; Zheng, G.C.; Tan, Z.J.; Lim, P.T.; Li, Y. Species composition and toxicity of the genus Pseudo-nitzschia in Taiwan Strait, including P. chiniana sp. nov. and P. qiana sp. nov. Harmful Algae 2019, 84, 195–209. [Google Scholar] [CrossRef]
  61. Lim, H.C.; Tan, S.N.; Teng, S.T.; Lundholm, N.; Orive, E.; David, H.; Quijano-Scheggia, S.; Leong, S.C.Y.; Wolf, M.; Bates, S.S.; et al. Phylogeny and species delineation in the marine diatom Pseudo-nitzschia (Bacillariophyta) using cox1, LSU, and ITS2 rRNA genes: A perspective in character evolution. J. Phycol. 2018, 54, 234–248. [Google Scholar] [CrossRef] [PubMed]
  62. Stonik, I.; Orlova, T.Y.; Propp, L.; Demchenko, N.; Skriptsova, A. An autumn bloom of diatoms of the genus Pseudo-nitzschia H. Peragallo, 1900 in Amursky Bay, the Sea of Japan. Russ. J. Mar. Biol. 2012, 38, 211–217. [Google Scholar] [CrossRef]
  63. Ajani, P.; Murray, S.; Hallegraeff, G.; Lundholm, N.; Gillings, M.; Brett, S.; Armand, L. The diatom genus Pseudo-nitzschia (Bacillariophyceae) in New South Wales, A ustralia: Morphotaxonomy, molecular phylogeny, toxicity, and distribution. J. Phycol. 2013, 49, 765–785. [Google Scholar] [CrossRef]
  64. Rhodes, L.; Jiang, W.; Knight, B.; Adamson, J.; Smith, K.; Langi, V.; Edgar, M. The genus Pseudo-nitzschia (Bacillariophyceae) in New Zealand: Analysis of the last decade’s monitoring data. New Zealand J. Mar. Freshw. Res. 2013, 47, 490–503. [Google Scholar] [CrossRef]
  65. Amato, A.; Lüdeking, A.; Kooistra, W.H. Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy). Toxicon 2010, 55, 157–161. [Google Scholar] [CrossRef]
  66. Oyarzún, D.; Brierley, C.M. The future of coastal upwelling in the Humboldt current from model projections. Clim. Dyn. 2019, 52, 599–615. [Google Scholar] [CrossRef]
  67. Brink, K.; Halpern, D.; Huyer, A.; Smith, R. The physical environment of the Peruvian upwelling system. Prog. Oceanogr. 1983, 12, 285–305. [Google Scholar] [CrossRef]
  68. Suárez-Isla, B.A.; López, A.; Hernández, C.; Clement, A.; Guzmán, L. Impacto económico de las floraciones de microalgas nocivas en Chile y datos recientes sobre la ocurrencia de veneno amnésico de los mariscos. Floraciones Algales Nocivas en el Cono Sur Americano, Inst. Esp. Oceanogr. Madrid, España. In Floraciones Algales Nocivas en el Cono Sur Americano; Sar, E.A., Ferrario, M.E., Reguera, B., Eds.; Instituto Español de Oceanografía: Madrid, Spain, 2002; pp. 257–268. [Google Scholar]
  69. Lopez-Rivera, A.; Pinto, M.; Insinilla, A.; Suarez Isla, B.; Uribe, E.; Alvarez, G.; Lehane, M.; Furey, A.; James, K.J. The occurrence of domoic acid linked to a toxic diatom bloom in a new potential vector: The tunicate Pyura chilensis (piure). Toxicon 2009, 54, 754–762. [Google Scholar] [CrossRef]
  70. Álvarez, G.; Rengel, J.; Araya, M.; Álvarez, F.; Pino, R.; Uribe, E.; Díaz, P.A.; Rossignoli, A.E.; López-Rivera, A.; Blanco, J. Rapid Domoic Acid Depuration in the Scallop Argopecten purpuratus and Its Transfer from the Digestive Gland to Other Organs. Toxins 2020, 12, 698. [Google Scholar] [CrossRef]
  71. National. Fisheries Health Organization of Perú-Organismo Nacional de Sanidad Pesquera(SANIPES). Available online: https://www.sanipes.gob.pe/web/index.php/es/fitoplancton (accessed on 15 April 2021).
  72. Guillard, R.R.L.; Hargraves, P.E. Stichochrysis immobilis is a diatom, not a chrysophyte. Phycologia 1993, 32, 234–236. [Google Scholar] [CrossRef]
  73. Hasle, G.R. The Inverted-Microscope Method; Sournia, A., Ed.; Phytoplankton Manual. Monographs on Oceanic Methodology; 1978; pp. 88–96. [Google Scholar]
  74. Lundholm, N.; Hasle, G.R.; Fryxell, G.A.; Hargraves, P.E. Morphology, phylogeny and taxonomy of species within the Pseudo-nitzschia americana complex (Bacillariophyceae) with descriptions of two new species, Pseudo-nitzschia brasiliana and Pseudo-nitzschia linea. Phycologia 2002, 41, 480–497. [Google Scholar] [CrossRef]
  75. Doyle, J.J.; Doyle, J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987, 19, 11–15. [Google Scholar]
  76. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pcr Protoc. A Guide Methods Appl. 1990, 18, 315–322. [Google Scholar]
  77. Hall, T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 1999, 41, 95–98. [Google Scholar]
  78. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed]
  79. Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. jModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 2012, 9, 772. [Google Scholar] [CrossRef]
  80. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef] [PubMed]
  81. Silvestro, D.; Michalak, I. raxmlGUI: A graphical front-end for RAxML. Org. Divers. Evol. 2012, 12, 335–337. [Google Scholar] [CrossRef]
  82. Ronquist, F.; Teslenko, M.; Van Der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012, 61, 539–542. [Google Scholar] [CrossRef] [PubMed]
  83. de la Iglesia, P.; Gimenez, G.; Diogene, J. Determination of dissolved domoic acid in seawater with reversed-phase extraction disks and rapid resolution liquid chromatography tandem mass spectrometry with head-column trapping. J. Chromatogr. A 2008, 1215, 116–124. [Google Scholar] [CrossRef]
Figure 1. Pseudo-nitzschia multistriata (MEB), (A) whole valve; (B) valve end (2 µm); (C) mid valve, no central interspace (5 µm).
Figure 1. Pseudo-nitzschia multistriata (MEB), (A) whole valve; (B) valve end (2 µm); (C) mid valve, no central interspace (5 µm).
Toxins 13 00408 g001
Figure 2. Pseudo-nitzschia Bayesian tree based on ITS2 sequences. Numbers above lines represent BI posterior probabilities/ML bootstrap values. “-“ indicates a different phylogeny structure for ML analysis. Boldface indicates the studied strain as P. multistriata. Phylogenetic ITS2 trees (BI and ML) showed six general groups. The taxon P. multistriata is included in one of these groups comprising also P. americana + P. brasiliana, P. pungens + P. multiseries, P. australis + P. seriata + P. cf. obtusa, Fragilariopsis nana + F. cylindricus + Fragilariopsis sp. and P. subfraudulenta + P. fraudulenta.
Figure 2. Pseudo-nitzschia Bayesian tree based on ITS2 sequences. Numbers above lines represent BI posterior probabilities/ML bootstrap values. “-“ indicates a different phylogeny structure for ML analysis. Boldface indicates the studied strain as P. multistriata. Phylogenetic ITS2 trees (BI and ML) showed six general groups. The taxon P. multistriata is included in one of these groups comprising also P. americana + P. brasiliana, P. pungens + P. multiseries, P. australis + P. seriata + P. cf. obtusa, Fragilariopsis nana + F. cylindricus + Fragilariopsis sp. and P. subfraudulenta + P. fraudulenta.
Toxins 13 00408 g002
Figure 3. SIM chromatogram (upper panel) and mass spectrum (lower panel) of DA in Pseudo-nitzschia multistriata culture.
Figure 3. SIM chromatogram (upper panel) and mass spectrum (lower panel) of DA in Pseudo-nitzschia multistriata culture.
Toxins 13 00408 g003
Figure 4. Location of the sampling station Paracas Bay, Peru.
Figure 4. Location of the sampling station Paracas Bay, Peru.
Toxins 13 00408 g004
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Back to TopTop