Cryoprotectant treatment tests on three morphologically diverse marine dinoflagellates and the cryopreservation of Breviolum sp. (Symbiodiniaceae)

Dinoflagellates are among the most diverse group of microalgae. Many dinoflagellate species have been isolated and cultured, and these are used for scientific, industrial, pharmaceutical, and agricultural applications. Maintaining cultures is time-consuming, expensive, and there is a risk of contamination or genetic drift. Cryopreservation offers an efficient means for their long-term preservation. Cryopreservation of larger dinoflagellate species is challenging and to date there has been only limited success. In this study, we explored the effect of cryoprotectant agents (CPAs) and freezing methods on three species: Vulcanodinium rugosum, Alexandrium pacificum and Breviolum sp. A total of 12 CPAs were assessed at concentrations between 5 and 15%, as well as in combination with dimethyl sulfoxide (DMSO) and other non-penetrating CPAs. Two freezing techniques were employed: rapid freezing and controlled-rate freezing. Breviolum sp. was successfully cryopreserved using 15% DMSO. Despite exploring different CPAs and optimizing the freezing techniques, we were unable to successfully cryopreserve V. rugosum and A. pacificum. For Breviolum sp. there was higher cell viability (45.4 ± 2.2%) when using the controlled-rate freezing compared to the rapid freezing technique (10.0 ± 2.8%). This optimized cryopreservation protocol will be of benefit for the cryopreservation of other species from the family Symbiodiniaceae.

A Novel Parasitoid of Marine Dinoflagellates, Pararosarium dinoexitiosum gen. et sp. nov. (Perkinsozoa, Alveolata), Showing Characteristic Beaded Sporocytes

The phylum Perkinsozoa is known as an exclusively parasitic group within alveolates and is widely distributed in various aquatic environments from marine to freshwater environments. Nonetheless, their morphology, life cycle, the identity of the host, and physiological characteristics remain still poorly understood. During intensive sampling along the west coast of Korea in October and November 2017, a new parasitoid, which shares several characteristics with the extant families Perkinsidae and Parviluciferaceae, was discovered and three strains of the new parasitoid were successfully established in cultures. Cross-infection experiments showed that among the examined planktonic groups, only dinoflagellates were susceptible to the new parasitoid, with infections observed in species belonging to eight genera. Even though the new parasitoid shared many morphological and developmental characteristics with other Perkinsozoan parasites, it differed from them by its densely packed trophocyte structure without a large vacuole or hyaline material during the growth stage. These characteristics are common among Parviluciferaceae members. Furthermore, through palintomic extracellular sporogenesis, it produced characteristic interconnected sporocytes resembling a string of beads. Phylogenetic analyses based on the small subunit and large subunit ribosomal DNA sequences revealed that the new parasitoid was distantly related to the family Parviluciferaceae and was more closely related to the families Perkinsidae and Xcellidae. Morphological, ultrastructural, and molecular data on the new parasitoid raised the need to erect a new family, i.e., Pararosariidae, within the phylum Perkinsozoa with Pararosarium dinoexitiosum gen. et sp. nov. as the type species. The isolation and establishment in culture of the new parasitoid outside the family Parviluciferaceae in the present study would contribute to the better understanding of the diversity of Perkinsozoan parasites and provide useful material for comparisons to other parasite species in the further study.

Dietary Exposure Assessment of Paralytic Shellfish Toxins through Shellfish Consumption in Shenzhen Population, China

Paralytic shellfish toxins (PSTs) produced by certain marine dinoflagellates accumulate in filter-feeding marine bivalves. We used LC-MS/MS to detect and quantify 13 PSTs in 188 shellfish samples of 14 species collected from Shenzhen city’s Buji seafood wholesale market from March 2019 to February 2020. Twenty-six of 188 shellfish samples (13.8%) were PST- positive, with highest values in samples of the Noble clam Chlamys nobilis (10/34, 29.4%). Samples originating from Nan’ao island among 11 source sites in China recorded the highest detected rate (7/17, 41.2%). Samples containing PSTs were concentrated in Spring and Winter, with the highest levels in March > December > January. Among PSTs detected, C1 was dominant. Acute dietary exposure assessments for Shenzhen residents were based on P99 consumption data (139.2g/day) and maximum PST concentration for each shellfish species. The outcome for Chlamys nobilis was 2.4 ~ 3.7-fold higher than recommended ARfDs (0.5 ~ 0.7 µg STX eq./kg bw/day). Mean PST concentration (10.9 ~ 134.1 µg STX Eq. /kg), mean shellfish consumption (4.8 g/day) and P99 consumption data were used to assess chronic dietary exposure. The results were lower than the recommended ARfDs. In conclusion, residents in Shenzhen are at risk for acute PST poisoning, while relatively safe from chronic PST exposure.

Epibenthic Harmful Marine Dinoflagellates from Fuerteventura (Canary Islands), with Special Reference to the Ciguatoxin-Producing Gambierdiscus

The relationship between the ciguatoxin-producer benthic dinoflagellate Gambierdiscus and other epibenthic dinoflagellates in the Canary Islands was examined in macrophyte samples obtained from two locations of Fuerteventura Island in September 2016. The genera examined included Coolia, Gambierdiscus, Ostreopsis, Prorocentrum, Scrippsiella, Sinophysis, and Vulcanodinium. Distinct assemblages among these benthic dinoflagellates and preferential macroalgal communities were observed. Vulcanodinium showed the highest cell concentrations (81.6 × 103 cells gr−1 wet weight macrophyte), followed by Ostreopsis (25.2 × 103 cells gr−1 wet weight macrophyte). These two species were most represented at a station (Playitas) characterized by turfy Rhodophytes. In turn, Gambierdiscus (3.8 × 103 cells gr−1 wet weight macrophyte) and Sinophysis (2.6 × 103 cells gr−1 wet weight macrophyte) were mostly found in a second station (Cotillo) dominated by Rhodophytes and Phaeophytes. The influence of macrophyte’s thallus architecture on the abundance of dinoflagellates was observed. Filamentous morphotypes followed by macroalgae arranged in entangled clumps presented more richness of epiphytic dinoflagellates. Morphometric analysis was applied to Gambierdiscus specimens. By large, G. excentricus was the most abundant species and G. australes occupied the second place. The toxigenic potential of some of the genera/species distributed in the benthic habitats of the Canary coasts, together with the already known presence of ciguatera in the region, merits future studies on possible transmission of their toxins in the marine food chain.

Comparison of Five TRIzol-Based Protein Preparation Methods for 2-DE Production From Challenging Marine Dinoflagellate Samples: A Case Study on Two Benthic Prorocentrum Species

Two-dimensional gel electrophoresis (2-DE) is a major element of conventional gel-based proteomics, which resolves complex protein mixtures. Protein extraction with the removal of interfering substances from the sample remains the key to producing high-quality 2-DE profiles. Marine dinoflagellates contain large endogenous amounts of salts, nucleic acids, polysaccharides, phenolic compounds, pigments, and other interfering compounds. These substances are detrimental to the quality of gel images. Protein preparation using TRIzol reagent is a promising method for producing high-quality 2-DE profiles for dinoflagellate samples. In addition to its remarkable performance, the TRIzol method’s several advantages have made it a popular and widely used method in the field of 2-DE sample preparation. Nonetheless, the quality of 2-DE of samples from certain dinoflagellate species is not as high as previously reported when the same TRIzol protocol is applied. Therefore, modifications to the original TRIzol method are required to remove interfering substances from those challenging dinoflagellate samples. In this study, the original TRIzol method and four modified methods, namely the aliquot TRIzol method, re-TRIzol method, TRIzol method with a commercial clean-up kit, and TRIzol method with trichloroacetic acid/acetone precipitation, were compared. Performance of these five methods in terms of protein yield, background signal, and resolution and number of protein spots was investigated on samples from two benthic Prorocentrum species: P. lima and P. hoffmannianum. Our results demonstrated that high-quality 2-DE could be achieved from P. lima samples prepared using both the original TRIzol method and the TRIzol method with a commercial clean-up kit. However, the original TRIzol method failed to produce high-quality 2-DE profiles for P. hoffmannianum samples. Among the four modified TRIzol methods, only the TRIzol method with a commercial clean-up kit could yield substantially improved high-quality 2-DE profiles for P. hoffmannianum samples. This combination of the conventional TRIzol method with a commercial clean-up kit potentially represents a promising protein extraction methodology for obtaining high-quality 2-DE profiles for difficult dinoflagellate samples.