Life cycle, toxinological features, and genetic characterization of the Harmful Algal Bloom producer dinoflagellate Protoceratium reticulatum from the austral coast of Chile (~44º–53º S)
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1
Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Subida a Radio Faro 50, 36390, Vigo , Spain
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2
Instituto de Fomento Pesquero (IFOP), Enrique Abello 0552, Punta Arenas, Chile
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3
Instituto de Investigaciones Marinas (IIM-CSIC), Eduardo Cabello 6, 36208, Vigo, Spain
Introduction
The frequency of harmful algal blooms (HABs) produced by marine dinoflagellates has increased worldwide over the last several decades, with serious negative impacts on public health and on the economies of the affected areas. Protoceratium reticulatum is considered an important species among HAB species due to the lipophilic toxin production of the yessotoxins (YTXs) type and its cosmopolitan distribution in the coastal environments. Although YTXs have not been directly linked to human intoxications, the presence of YTXs in shellfish samples produces false-positives in mouse bioassay tests creating serious problems in toxin detection methods. The knowledge of life strategy and the basic features of the HAB-involved species in order to understand their behavior is, so far, the only way of fighting against the impact of toxic microalgae.
Methodology
Protoceratium reticulatum strains were isolated from Queulat (strains PRAY1-B11, PRAY2, PRAY3, and PRAY4) and Otway (strain PRENM) Sounds, in Aysén (~44º S) and Magallanes (~53º S) regions, respectively, during the regular monitoring of red tides from southern Chile in 2010 and 2013. Cultures were maintained in L1-Si medium adjusted to a salinity of 32, at a temperature of 15 ºC, and with a photoperiod of 12:12 h light:dark (photon flux approximately 100 μmol m–2 s–1). For the genetic study total DNA was extracted using the Chelex resin method. The D1-D2 region of the large subunit (LSU) rRNA gene was performed using the primer pair D1R/D2C as described by Lenaers et al. (1989). Morphological studies and observations of cell nuclei were performed by Calcofluor white and Sybr Green staining, respectively. Sexual stages (gametes, planozygotes, and resting cysts) were promoted by crossing compatible strains in phosphate-depleted medium (L1-P). For the of toxin study cultures were filtered and filters stored at -20 ºC. Subsequently the filters were extracted twice with 100% methanol for YTXs toxin analysis. YTX and their analogues were identified by LC–HRMS according to Gerssen et al. 2009.
Results
The phylogenetic analysis showed that the sequences from the strains PRENM, PRAY1-B11, and PRAY3 were identical to most of the other sequences of P. reticulatum retrieved from Genbank database. The Maximum Likelihood tree revealed that strains were part of a monophyletic clade denominated “Clade A” by Akselman et al. 2015.
The toxin analysis showed that P. reticulatum cells clearly produced YTX and 45-OH-YTX analogue, but also suspected of NoroxoYTX-enone and NoroxoHomoYTX-enone (Table 1). Mass spectrometric analysis showed that the toxin profiles of all strains were very similar and that the major compound was YTX. Total YTX per cell was variable among strains ranging from 8.7 pg YTX cell-1 in PRAY1-B11 to 22.8 pg YTX cell-1 in PRENM.
Morphological characteristics of the most common mobile stages observed during P. reticulatum life cycle study showed the same morphology already described in the literature (e.g., Hansen et al. 1996) with plate formula: Po, 4', 0a, 6'', 6C, 5''', 0p, 2'''' (Fig. 1A–D). The cell fission suture appeared consistent with a typical gonyaulacoid desmoschisis (Figs. 1E–H). Vegetative cells (30.6–40.8 µm long, 21.4–35.5 µm wide) were solitary, showed numerous golden-brown colour chloroplasts, and a nucleus lobulated in U-shaped located in the posterior dorsal part of the cell (Figs. 2A–C). The mating type experiment indicated that P. reticulatum follows a complex heterothallic mating type behavior because clonal strains were not successful for resting cyst formation. Gametes (23.7–30.5 µm long, 19.5–26.4 µm wide) were smaller than vegetative cells; they had a pale colour, and were seen in couple after the day 3 of the crossing (Figs. 2D,E). Conjugation of gametes resulted in a planozygote with two trailing longitudinal flagella (Fig. 2F). Planozygotes (39.9–51.9 µm long, 33.3–42.6 µm wide) were larger than vegetative cells, dark colour, with a lot of granules, and a large U-shaped nucleus (Figs. 2F–H). They were found four days after the initial date of the crossing experiment. The first appearance of resting cysts (39.9–51.9 µm diameter) occurred on day 7 after sexual induction. First, cysts formed a transparent outer membrane shaped balloon, and then processes appeared on the cell wall (Fig. 3A). A double cell wall (1.3–3.9 µm thickness) became thicker with time (Figs. 3B,C). Resting cysts were mainly spherical, although sometimes maintained planozygote shape, showed many reserve granules, one orange accumulation body, and many hollow processes of variable length (7.3–16.0 µm long) with capitate distal ends (Fig. 3C). Its nucleus was ring-shaped (Fig. 3D).
Additionally, a particular cell type with a different morphology from the rest of those cells of the P. reticulatum life cycle was also identified. This type of cell (35.2–57.7 µm long, 29.6–47.7 µm wide) showed a strong dorsoventral compression (Fig. 4A), it was seen with and without reticulation in their cellulosic plates (Figs. 4B,C), and with one or two trailing longitudinal flagella, which is indicative of vegetative and zygote cells, respectively. Its nucleus was flattened (Fig. 4D). These flat cells were rarely swimmers, especially were immobile at the bottom of cultures. This cell type was recorded in all strains studied, and was mainly observed in the stationary phase of cultures.
Main conclusions
-This study shows for the first time different sexual stages of the Protoceratium reticulatum life cycle.
-An unknown flat morphotype was identified during the life cycle, which needs to be studied in detail.
-The presence of YTX and 45-OH-YTX analogue is confirmed for P. reticulatum strains from southern Chile.
Acknowledgements
This work is a contribution of Unidad Asociada “Microalgas Nocivas” (CSIC-IEO) and was carried out at the Instituto Español de Oceanografía (IEO) in Vigo and was financially supported by the CCVIEO project and CICAN-2013-40671-R (Ministry of Economy and Competitiveness). P. Salgado is a researcher at the IFOP, which provides financial support for his doctoral stay.
References
Akselman, R., Krock, B., Alpermann, T.J., Tillmann, U., Borel, C.M., Almandoz, G.O., Ferrario, M.E. 2015. Protoceratium reticulatum (Dinophyceae) in the austral Southwestern Atlantic and the first report on YTX-production in shelf waters of Argentina. Harmful Algae 45: 40–52
Gerssen, A., Mulder, P., McElhinney, M.A., de Boer, J. 2009. Liquid chromatography–tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions. J. Chromatogr. A 1216: 1421–1430
Hansen, G., Moestrup, Ø, Roberts, K.R. 1996. Light and electron microscopical observations on Protoceratium reticulatum (Dinophyceae). Arch. Protistenkd. 147: 381–391
Lenaers, G., Maroteaux, L., Michot, B., Herzog, M., 1989. Dinoflagellates in evolution. A molecular phylogenetic analysis of large subunit ribosomal RNA. J. Mol. Evol. 29, 40–51
Keywords:
life cycle,
Resting cysts,
Protoceratium reticulatum,
Flat cell,
Yessotoxin,
Harmful Algal Bloom
Conference:
XIX Iberian Symposium on Marine Biology Studies, Porto, Portugal, 5 Sep - 9 Sep, 2016.
Presentation Type:
Poster Presentation
Topic:
1. ECOLOGY, BIODIVERSITY AND VULNERABLE ECOSYSTEMS
Citation:
Salgado
P,
Riobó
P,
Rodríguez
F and
Bravo
I
(2016). Life cycle, toxinological features, and genetic characterization of the Harmful Algal Bloom producer dinoflagellate Protoceratium reticulatum from the austral coast of Chile (~44º–53º S).
Front. Mar. Sci.
Conference Abstract:
XIX Iberian Symposium on Marine Biology Studies.
doi: 10.3389/conf.FMARS.2016.05.00174
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Received:
26 Apr 2016;
Published Online:
03 Sep 2016.
*
Correspondence:
PhD. Pablo Salgado, Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Subida a Radio Faro 50, 36390, Vigo, Vigo, Spain, pablo.salgado@ifop.cl