Physical interactions between marine phytoplankton and PET plastics in seawater.

Plastics are the most abundant marine debris globally dispersed in the oceans and its production is rising with documented negative impacts in marine ecosystems. However, the chemical-physical and biological interactions occurring between plastic and planktonic communities of different types of microorganisms are poorly understood. In these respects, it is of paramount importance to understand, on a molecular level on the surface, what happens to plastic fragments when dispersed in the ocean and directly interacting with phytoplankton assemblages. This study presents a computer-aided analysis of electron paramagnetic resonance (EPR) spectra of selected spin probes able to enter the phyoplanktonic cell interface and interact with the plastic surface. Two different marine phytoplankton species were analyzed, such as the diatom Skeletonema marinoi and dinoflagellate Lingulodinium polyedrum, in absence and presence of polyethylene terephthalate (PET) fragments in synthetic seawater (ASPM), in order to in-situ characterize the interactions occurring between the microalgal cells and plastic surfaces. The analysis was performed at increasing incubation times. The cellular growth and adhesion rates of microalgae in batch culture medium and on the plastic fragments were also evaluated. The data agreed with the EPR results, which showed a significant difference in terms of surface properties between the diatom and dinoflagellate species. Low-polar interactions of lipid aggregates with the plastic surface sites were mainly responsible for the cell-plastic adhesion by S. marinoi, which is exponentially growing on the plastic surface over the incubation time.

Devastating farmed abalone mortalities attributed to yessotoxin-producing dinoflagellates.

A large dinoflagellate bloom in Walker Bay (South Africa) in January 2017 impacted 3 land-based abalone farms resulting in the death of several million animals. Satellite-derived images of Chl-a from the Ocean and Land Colour Imager (OLCI) on board the European Space Agency Sentinel-3 A showed bloom initiation in late December 2016 and dispersal in mid-February 2017. The bloom was dominated by two dinoflagellate species identified by light microscopy as Gonyaulax spinifera (Claparède & Lachmann) Diesing, 1866 and Lingulodinium polyedrum (Stein) Dodge, 1989. These morphologically based identifications were confirmed by phylogenetic analysis using partial sequences of the large subunit rDNA of both dinoflagellates. The appearance of yessotoxins (YTX) in abalone clearly coincided with increases in dinoflagellate concentrations. Yessotoxins in both the plankton and abalone were dominated by the two analogues homo-YTX and 45-hydroxy-YTX. The absence of toxins in a clonal culture of L. polyedrum implicated G. spinifera as the likely source of YTX. Toxin concentrations were found to be highest in the gills which showed the most significant pathology, including severe, generalized disruption of the gill epithelium characterized by degeneration and necrosis of epithelial cells accompanied by a modest inflammatory response. Some farms undertook pre-emptive or emergency harvesting to reduce financial losses.

The inhibitory effect of a non-yessotoxin-producing dinoflagellate, Lingulodinium polyedrum (Stein) Dodge, towards Vibrio vulnificus and Staphylococcus aureus

ResumenEl aumento de la resistencia bacteriana a los antibióticos ha causado preocupación a nivel mundial, por lo que se ha promovido la búsqueda de nuevos compuestos. Debido a su abundancia y diversidad, el fitoplancton marino constituye una importante fuente potencial de tales compuestos. La investigación sobre dinoflagelados ha llevado al descubrimiento de inhibidores de crecimiento bacteriano. El dinoflagelado marino Lingulodinium polyedrum causa proliferaciones algales en diferentes regiones del mundo, incluyendo México, y también se sabe que regula el crecimiento de otras especies en las aguas costeras. En este trabajo, se investiga la taxonomía de este dinoflagelado y se caracteriza la capacidad de sus extractos para inhibir el crecimiento de dos bacterias de importancia médica (Vibrio vulnificus y Staphylococcus aureus) en placas de cultivo de agar. La caracterización taxonómica se realizó por PCR y amplificación del gen de ITS, y se confirmó que la especie aislada en la costa del Pacífico de México fue L. polyedrum. Para demostrar el efecto inhibidor de los extractos de L. polyedrum, los cultivos se cosecharon por centrifugación. Los pellets de tres abundancias celulares se extrajeron con agua, metanol, hexano y cloroformo. Los experimentos en V. vulnificus mostraron una inhibición alta del crecimiento para los cuatro extractos, variando entre 77 y 98 %. Sorprendentemente, la inhibición del crecimiento fue menor cuando los extractos se originaron a partir de una mayor abundancia de células L. polyedrum, varía de 0 a 34 %. Para S. aureus, la inhibición del crecimiento también fue alta, pero no estadísticamente diferente para todos los extractos y abundancias de células, con un rango de 62 hasta 99 %. Esto resultados son prometedores para futuras aplicaciones farmacológicas. La cepa mexicana de L. polyedrum no produjo yesotoxinas detectables.

AbstractThe increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins. Rev. Biol. Trop. 64 (2): 805-816. Epub 2016 June 01.

The main nitrate transporter of the dinoflagellate Lingulodinium polyedrum is constitutively expressed and not responsible for daily variations in nitrate uptake rates.

Dinoflagellates are unicellular eukaryotes capable of forming spectacular harmful algal blooms (HABs). Eutrophication of coastal waters by fertilizer runoff, nitrate in particular, has contributed to recent increases in the frequency, magnitude and geographic extent of HABs. Although physiological nitrate uptake and assimilation in dinoflagellates have often been measured in the field and in the laboratory, no molecular components involved in nitrate transport have yet been reported. This study reports the first identification and characterization of dinoflagellate nitrate transporters, found in the transcriptome of the bloom-forming Lingulodinium polyedrum. Of the 23 putative transporters found by BLAST searches, only members of the nitrate transporter 2 (NRT2) family contained all key amino acids known to be essential for nitrate transport. The dinoflagellate NRT2 sequences have 12 predicted transmembrane domains, as do the NRT2 sequences of bacteria, plants and fungi. The NRT2 sequences in Lingulodinium appear to have two different evolutionary origins, as determined by phylogenetic analyses. The most expressed transcript of all putative nitrate transporters was determined by RNA-Seq to be LpNRT2.1. An antibody raised against this transporter showed that the same amount of protein was found at different times over the light dark cycle and with different sources of N. Finally, global nitrate uptake was assessed using a 15N tracer, which showed that the process was not under circadian-control as previously suggested, but simply light-regulated.

Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch-activated ion channels.

Dinoflagellate bioluminescence serves as a whole-cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage-sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 µM Gadolinium III (Gd(3+) ), a nonspecific blocker of stretch-activated and some voltage-gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 µM nifedipine, an inhibitor of L-type voltage-gated Ca(2+) channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol-stimulated bioluminescence was inhibited by Gd(3+) but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch-activated and voltage-gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch-activated component at the plasma membrane.

Is yessotoxin the main phycotoxin in Croatian waters?

With the aim of investigating whether yessotoxin (YTX) is responsible for diarrhetic shellfish poisoning (DSP) events in Croatian waters, three different methods were combined: a modified mouse bioassay (MBA) that discriminates YTX from other DSP toxins, the enzyme-linked immunosorbent assay method (ELISA) and liquid chromatography-mass spectrometry (LC-MS/MS). Among 453 samples of mussels and seawater analyzed in 2007, 10 samples were DSP positive. Results obtained by the modified MBA method revealed that most of the samples were positive for YTX, with the exception of samples from Lim Bay (LB 1) The ELISA method also identified the presence of YTX in these samples. DSP toxin profiles showed the presence of okadaic acid (OA) in three, and YTX in four out of nine samples that were analyzed by LC-MS/MS. The phytoplankton community structure pattern revealed Lingulodinium polyedrum (Stein) Dodge, which was present in the water prior to and/or during toxicity events at low concentrations (80 to 1440 cells L(-1)), as a potential YTX producing species. It is proposed that L. polyedrum cells accumulated in mussels and the subsequently observed toxicity may be related to metabolism after ingestion, resulting in carboxy YTX as the major analog in the mussel.

Yessotoxins, a group of marine polyether toxins: an overview.

Yessotoxin (YTX) is a marine polyether toxin that was first isolated in 1986 from the scallop Patinopecten yessoensis. Subsequently, it was reported that YTX is produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. YTXs have been associated with diarrhetic shellfish poisoning (DSP) because they are often simultaneously extracted with DSP toxins, and give positive results when tested in the conventional mouse bioassay for DSP toxins. However, recent evidence suggests that YTXs should be excluded from the DSP toxins group, because unlike okadaic acid (OA) and dinophyisistoxin-1 (DTX-1), YTXs do not cause either diarrhea or inhibition of protein phosphatases. In spite of the increasing number of molecular studies focused on the toxicity of YTX, the precise mechanism of action is currently unknown. Since the discovery of YTX, almost forty new analogues isolated from both mussels and dinoflagellates have been characterized by NMR or LC-MS/MS techniques. These studies indicate a wide variability in the profile and the relative abundance of YTXs in both, bivalves and dinoflagellates. This review covers current knowledge on the origin, producer organisms and vectors, chemical structures, metabolism, biosynthetic origin, toxicological properties, potential risks to human health and advances in detection methods of YTXs.

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES(1).

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high-resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two-step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1-5.8S-ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.

Marea roja producida por Lingulodinium polyedrum (Peridiniales, Dinophyceae) en Bahía Culebra, Golfo de Papagayo, Costa Rica / Red tide bloom produced by Lingulodinium polyedrum (Peridiniales, Dinophyceae) in Bahía Culebra, Papagayo Gulf, Costa Rica

This is the first record of the dinoflagellate Lingulodinium polyedrum in a red tide bloom in the North Pacific coast of Costa Rica. The sample was collected on April 2000 at Culebra Bay, Gulf of Papagayo, from a patch of aproximatly 2000 m2, which produced a red discoloration of the water and a peculiar strong odor. This species produces spherical hypnocysts that may remain for decades when dark or anoxic conditions are present; L. polyedrum had been associated with the production of paralyzing toxins such as saxitoxins and yessotoxins. A second smaller patch was observed close Panama beach, into the bay, where we found seven puffer fish (Diodontidae) and two lobsters dead in the sand. It is important to develop a monitoring program to identify seasonal behavior of this species and ameliorate its impact on coastal human communities.