Coolia Species Research Articles

First report of epiphytic dinoflagellate Coolia malayensis (Dinophyceae) in the southeastern Pacific Ocean.

Coolia species are epiphytic and benthic dinoflagellates with a cosmopolitan distribution in tropical and subtropical areas. In the austral summer of 2016, during a survey in Bahía Calderilla, a dinoflagellate of the genus Coolia was detected in macroalgae samples, and a clonal culture was established. Subsequently, the cultured cells were observed by scanning electron microscopy (SEM) and identified as C. malayensis based on their morphological characteristics. Phylogenetic analyses based on the LSU rDNA D1/D2 regions confirmed that strain D005-1 corresponded to C. malayensis and clustered with strains isolated from New Zealand, Mexico, and Asia Pacific countries. Although the strain D005-1 culture did not contain yessotoxin (YTX), cooliatoxin, 44-methyl gambierone, or its analogs in detectable amounts by LC-MS/MS, more research is needed to evaluate its toxicity and to determine the possible impact of C. malayensis in northern Chilean waters.

Coolia Species (Dinophyceae) from the Tropical South Atlantic Region: Evidence of Harmfulness of Coolia cf. canariensis Phylogroup II

Benthic dinoflagellates of the Coolia genus have been associated with cytotoxicity and lethal and sublethal effects on marine species. This study aimed to assess the harmful effects of C. cf. canariensis phylogroup II (PII) and C. malayensis strains through bioassays. Experimental exposures (24, 48, and 72 h) of Artemia salina nauplii to Coolia species (330–54,531 cells mL−1) were performed independently. When a concentration-dependent response was achieved, additional experiments were carried out to evaluate the cell-free medium toxicity. The two Coolia species were harmful to Artemia nauplii, inducing significant mortality and sublethal responses. Coolia cf. canariensis PII was the most toxic species, inducing significant lethality at lower concentrations and shorter exposure times, followed by C. malayensis. Only the survival curves achieved after 24 and 48 h of exposure to C. cf. canariensis PII fitted to a concentration–response curve with valid LC50s of 18,064 and 19,968 cells mL−1, respectively. Moreover, extracellular compounds (i.e., culture filtrates) of C. cf. canariensis PII induced significant mortality to nauplii after 48 and 72 h. The toxicity of C. cf. canariensis PII was demonstrated for the first time using bioassays, and it was surprisingly higher than that of the C. malayensis strain, which was previously demonstrated to induce biological activity at the cellular and subcellular levels. Our findings highlight the harmful and lethal effects induced by Coolia cells and the importance of bioassays for toxicity assessments.

Ostreopsis Schmidt and Coolia Meunier (Dinophyceae, Gonyaulacales) from Cook Islands and Niue (South Pacific Ocean), including description of Ostreopsis tairoto sp. nov.

It is important to decipher the diversity and distribution of benthic dinoflagellates, as there are many morphologically indistinct taxa that differ from one another in production of potent toxins. To date, the genus Ostreopsis comprises twelve described species, of which seven are potentially toxic and produce compounds presenting a threat to human and environmental health. In this study, isolates previously identified as “Ostreopsis sp. 3” were sampled from the area where it was first reported, Rarotonga, Cook Islands, and have been taxonomically and phylogenetically characterised as Ostreopsis tairoto sp. nov. Phylogenetically, the species is closely related to “Ostreopsis sp. 8”, O. mascarenensis, “O. sp. 4”, O. fattorussoi, O. rhodesiae and O. cf. siamensis. Previously, it was considered a part of the O. cf. ovata complex but can be distinguished from O. cf. ovata based on the small pores identified on this study, and from O. fattorussoi and O. rhodesiae based on relative lengths of the 2′ plates. No known palytoxin -like compounds were detected in strains investigated in this study. Strains of O. lenticularis, Coolia malayensis and C. tropicalis were also identified and described. This study advances our knowledge of biogeography, distribution, and toxins of Ostreopsis and Coolia species.

Morphological and molecular characterization of the genus <italic>Coolia</italic> (Dinophyceae) from Bahía de La Paz, southwest Gulf of California

The genus Coolia A. Meunier 1919 has a global distribution and is a common member of epiphytic dinoflagellate assemblages in neritic ecosystems. Coolia monotis is the type species of the genus and was the only known species for 76 years. Over the past few decades, molecular characterization has unveiled two species complexes that group morphologically very similar species, so their limits are often unclear. To provide new knowledge on the biogeography and species composition of the genus Coolia, 16 strains were isolated from Bahía de La Paz, Gulf of California. The species were identified by applying morphological and molecular approaches. The morphometric characteristics of all isolated Coolia species were consistent with the original taxa descriptions. Phylogenetic analyses (large subunit [LSU] rDNA D1 / D2 and internal transcribed spacer [ITS] 1 / 5.8S / ITS2) revealed a species assemblage comprising Coolia malayensis, C. palmyrensis, C. tropicalis, and the C. cf. canariensis lineage. This is the first report of Coolia palmyrensis and C. cf. canariensis in Mexico and C. tropicalis in the Gulf of California. Our results strengthen the biogeographical understanding of these potentially harmful epiphytic dinoflagellate species.

Temperature variability interacts with mean temperature to influence the predictability of microbial phenotypes.

Despite their relatively high thermal optima (T opt), tropical taxa may be particularly vulnerable to a rising baseline and increased temperature variation because they live in relatively stable temperatures closer to their T opt. We examined how microbial eukaryotes with differing thermal histories responded to temperature fluctuations of different amplitudes (0 control, ±2, ±4°C) around mean temperatures below or above their T opt. Cosmopolitan dinoflagellates were selected based on their distinct thermal traits and included two species of the same genus (tropical and temperate Coolia spp.), and two strains of the same species maintained at different temperatures for >500 generations (tropical Amphidinium massartii control temperature and high temperature, CT and HT, respectively). There was a universal decline in population growth rate under temperature fluctuations, but strains with narrower thermal niche breadth (temperate Coolia and HT) showed ~10% greater reduction in growth. At suboptimal mean temperatures, cells in the cool phase of the fluctuation stopped dividing, fixed less carbon (C) and had enlarged cell volumes that scaled positively with elemental C, N, and P and C:Chlorophyll‐a. However, at a supra‐optimal mean temperature, fixed C was directed away from cell division and novel trait combinations developed, leading to greater phenotypic diversity. At the molecular level, heat‐shock proteins, and chaperones, in addition to transcripts involving genome rearrangements, were upregulated in CT and HT during the warm phase of the supra‐optimal fluctuation (30 ± 4°C), a stress response indicating protection. In contrast, the tropical Coolia species upregulated major energy pathways in the warm phase of its supra‐optimal fluctuation (25 ± 4°C), indicating a broadscale shift in metabolism. Our results demonstrate divergent effects between taxa and that temporal variability in environmental conditions interacts with changes in the thermal mean to mediate microbial responses to global change, with implications for biogeochemical cycling.

Distribution, identification and cytotoxicity of Gambierdiscus (Dinophyceae) in the Atlantic Selvagens Islands (Madeira, Portugal): a ciguatera gateway to Europe

ABSTRACT The emerging threat of ciguatera poisoning (CP) in Europe has been associated with fish captured in the Canary Islands (Spain) and Selvagens Islands (Portugal). The first are heavily populated islands where numerous scientific studies have been carried out. Conversely, the Selvagens Islands are a nature reserve with low human pressure that have been rarely surveyed in terms of the marine benthic microalgae, including the epiphytic ciguatera-causing dinoflagellate species. To investigate the harmful microalgal diversity of the Selvagens Islands, a scientific cruise to these remote islands took place in September, 2018. The Gambierdiscus species composition and distribution, and the associated epiphytic dinoflagellate community, were assessed using artificial substrate devices. Gambierdiscus cells were found in all samples, reaching concentrations of up to 725 cells 100 cm–2. G. australes was the only species identified after morphological and molecular analysis of the retrieved cultures. Species identification was confirmed by molecular characterization based on the LSU D8–D10 region. Nevertheless, phylogenetic studies indicated that some strains diverged from the G. australes clade suggesting genetic differentiation. Toxicity was estimated by neuro-2a cell-based assay in four strains, ranging from 2.46–83 fg of CTX1B eq. cell–1. The epiphytic dinoflagellate community that co-occurred with Gambierdiscus comprised other toxic or potentially toxic dinoflagellates, such as Ostreopsis, Prorocentrum, Amphidinium and Coolia species. Oceanographic and meteorological data were also obtained to characterize the occurrence of Gambierdiscus. This study is the first stage in understanding the role of the Selvagens Islands in the incubation and proliferation of the ciguatera-causing dinoflagellates Gambierdiscus in the NE Atlantic.

Morphology and molecular phylogeny of three species of Coolia (Dinophyceae) from Hainan Island, South China Sea

Marine benthic dinoflagellates in the genus Coolia were reported in both tropical and temperate regions. We conducted a morphological and phylogenetic characterization of Coolia species from Hainan Island, South China Sea. The morphologies of three Coolia species were similar to those of the original descriptions of Coolia canariensis, C. tropicalis, and C. malayensis. In phylogenetic analyses based on the LSU rDNA and ITS regions (ITS1-5.8S-ITS2), the Hainan strains of C. canariensis, C. tropicalis, and C. malayensis clustered within the clades of these species with other isolates from different areas. No differences (p-distance) in LSU rDNA sequences were found between the Hainan C. malayensis strains and the strains from New Zealand, Florida of USA, Malaysia, Japan, Dominican Republic and Guangxi and Hong Kong of China. For C. canariensis and C. tropicalis, no differences (p-distance) in the LSU rDNA sequences were found between the Hainan strains (D1C2 and DS5F4, respectively) and the Australian strains (NQAIF252 and NQAIF90, respectively). Our study reveals the morphological and genetic diversity of Coolia species from Hainan Island, South China Sea, which provides a detailed understanding of Coolia species of this area.

Diversity and Toxicity of the Genus Coolia Meunier in Brazil, and Detection of 44-methyl Gambierone in Coolia tropicalis

Coolia is a genus of marine benthic dinoflagellates which is widely distributed in tropical and temperate zones. Toxicity has been reported in selected Coolia species, although the identity of causative compounds is still controversial. In this study, we investigated the taxonomical and toxicological aspects of Coolia species from Brazil. Since light- and electron microscopy-based morphology was not enough to distinguish small-celled species, ITS and LSU D1-D3 phylogenetic analyses were used for species definition. Cultures of Coolia palmyrensis and Coolia santacroce were established from samples collected along the northeastern Brazilian coast, the first record of both species in South Atlantic waters. Cultures of Coolia malayensis and Coolia tropicalis were also established and exhibited acute in vivo toxicity to adults of Artemia salina, while C. palmyrensis and C. santacroce were non-toxic. The presence of 30 yessotoxin analogues, 7 metabolites of Coolia and 44 Gambierdiscus metabolites was screened in 14 strains of Coolia. 44-methyl gambierone (formerly referred to as MTX3) and a new isomer of this compound were detected only in C. tropicalis, using both low- and high-resolution LC-MS/MS. To our knowledge, this is the first report of gambierone analogues in dinoflagellates other than Gambierdiscus; the role of C. tropicalis in ciguatera poisoning thus deserves to be considered in further investigations.

Transcriptomic investigation into polyketide toxin synthesis in Ostreopsis (Dinophyceae) species.

In marine ecosystems, dinoflagellates can become highly abundant and even dominant at times, despite their comparatively slow growth. Their ecological success may be related to their production of complex toxic polyketide compounds. Ostreopsis species produce potent palytoxin-like compounds (PLTX), which are associated with human skin and eye irritations, and illnesses through the consumption of contaminated seafood. To investigate the genetic basis of PLTX-like compounds, we sequenced and annotated transcriptomes from two PLTX-producing Ostreopsis species; O. cf. ovata, O. cf. siamensis, one non-PLTX producing species, O. rhodesae and compared them to a close phylogenetic relative and non-PLTX producer, Coolia malayensis. We found no clear differences in the presence or diversity of ketosynthase and ketoreductase transcripts between PLTX producing and non-producing Ostreopsis and Coolia species, as both groups contained >90 and > 10 phylogenetically diverse ketosynthase and ketoreductase transcripts, respectively. We report for the first-time type I single-, multi-domain polyketide synthases (PKSs) and hybrid non-ribosomal peptide synthase/PKS transcripts from all species. The long multi-modular PKSs were insufficient by themselves to synthesize the large complex polyether backbone of PLTX-like compounds. This implies that numerous PKS domains, including both single and multi-, work together on the biosynthesis of PLTX-like and other related polyketide compounds.

Morphology and molecular phylogeny of Coolia tropicalis, Coolia malayensis and a new lineage of the Coolia canariensis species complex (Dinophyceae) isolated from Brazil

ABSTRACT The morphology and molecular phylogenetic relationships of Coolia species isolated from Brazil, including C. malayensis, C. tropicalis and a new lineage of the Coolia canariensis complex, are presented. Morphology was investigated by light and scanning electron microscopy. No significant morphological differences between the Brazilian strains and other geographically distant strains of C. malayensis and C. tropicalis were observed. In LSU and ITS rDNA phylogenies C. malayensis and C. tropicalis sequences from Brazil branched within the clade of these species with strains isolated from elsewhere. Phylogenetic relationships (LSU rDNA) within the C. canariensis species complex support the existence of three cryptic species. No morphological difference was discernible among species in this complex. Additional molecular, morphological and toxin data from globally distributed isolates should be acquired to strengthen our knowledge of the C. canariensis species complex.

Molecular phylogeny and toxicity of harmful benthic dinoflagellates Coolia (Ostreopsidaceae, Dinophyceae) in a sub-tropical marine ecosystem: The first record from Hong Kong

Coolia are marine benthic dinoflagellates which are globally distributed and potentially toxic. This study provides the first investigation of species diversity and toxicity assessment of Coolia in Hong Kong waters. Fifty-one strains of four Coolia species, including C. malayensis, C. canariensis, C. tropicalis, and C. palmyrensis, were isolated from twelve sub-tidal habitats, and identified phylogenetically using 28S rDNA sequences. Exposure experiments (48-hour) demonstrated that the algal lysates extracted from the four Coolia species exhibited different toxic effects on the lethality and abnormality of two invertebrate larvae, i.e., brine shrimp Artemia franciscana and sea urchin Heliocidaris crassispina. Heliocidaris crassispina was more sensitive to the toxic effects of Coolia species than A. franciscana. Toxicity tests from both larvae revealed that C. malayensis was generally more toxic, and caused higher mortality rates when compared with the other three species. The emerging threat of harmful benthic dinoflagellates to marine environments and sensitive biota is discussed.

Morphology, phylogeny and novel chemical compounds from Coolia malayensis (Dinophyceae) from Okinawa, Japan

Marine benthic dinoflagellates within the genus Coolia have been reported to produce natural products, some of which are known to be toxic (i.e., cooliatoxin). To date, five species of Coolia have been reported in tropical and temperate waters around the world; however, very few studies have combined detailed morphological and molecular data with chemical analyses. In this study, a clonal culture of Coolia malayensis was isolated and mass cultivated from a coral reef on the island of Okinawa, Japan. Analysis of the thecal plate morphology and molecular phylogeny from 28S rDNA strongly supported the close relationship between this new isolate of C. malayensis from Okinawa and other isolates of C. malayensis from around the world. Following methanol extraction of 250L of mass culture, chemical analyses using NanoLiquid chromatography mass spectrometry revealed the mass profiles of water-soluble and ethyl acetate-soluble parts. High-resolution mass spectrometry derived the molecular formulas of three novel disulphated polyether analogs of yessotoxin (C56H78O18S2 1102.4 (Compound 1), C57H80O18S2 1116.4 (Compound 2), and C57H78O19S2 1130.4 (Compound 3)); two potential homologous compounds (Compounds 4 and 5) were also observed on the high-resolution mass, albeit with low signal intensity. The five compounds in the C. malayensis from Okinawa are composed of less oxygen, compared to cooliatoxin and other analogs of yessotoxin, suggesting the metabolites produced by C. malayensis are unique to those previously reported from other strains of Coolia.

Morphology and distribution of the three epiphytic dinoflagellate species Coolia monotis, C. tropicalis, and C. canariensis (Ostreopsidaceae, Gonyaulacales, Dinophyceae) from Vietnamese coastal waters

Three epiphytic dinoflagellate species of the genus Coolia isolated from Vietnamese coastal waters were identified and described. Among the three Coolia species, Coolia monotis, C. tropicalis and C. canariensis, the latter two are recorded for the first time in Vietnam. The morphology of these species is illustrated by light and scanning electron micrographs. The heavily areolated thecal surface of C. areolata and the apical quadrangular 3′ plate of C. malayensis have been reported in original descriptions. The two species C. areolata and C. malayensis were not found in this study.

The Diversity of Coolia spp. (Dinophyceae Ostreopsidaceae) in the Central Great Barrier Reef Region

BackgroundDinoflagellates are important primary producers, crucial in marine food webs. Toxic strains, however, are the main causative agents of non-bacterial seafood poisoning, a major concern for public health worldwide. Despite their importance, taxonomic uncertainty within many genera of dinoflagellates is still high. The genus Coolia includes potentially harmful species and the diversity within the genus is just starting to become apparent.Methodology/Principal FindingsIn the current study, cultures were established from strains of Coolia spp. isolated from the central Great Barrier Reef (GBR). Cultures were identified based on thecal plate morphology and analyses of sequences (18S, ITS and 28S) from the nuclear rRNA operon. We report that the central GBR harbors a high diversity of Coolia species, including two species known to be capable of toxin production (C. tropicalis and C. malayensis), as well as the non-toxic C. canariensis. The strain of C. canariensis isolated from the GBR may in fact be a cryptic species, closely related but nevertheless phylogenetically distinct from the strain on which the holotype of C. canariensis was based. We also found evidence of the occurrence of a cryptic species morphologically very similar to both C. malayensis and C. monotis. The consequences of taxonomic confusion within the genus are discussed.Conclusion/SignificanceThe central GBR region harbors a previously unreported high diversity of Coolia spp., including two species known to potentially produce toxins. The presence of a cryptic species of unknown toxicity highlights the importance of cryptic diversity within dinoflagellates.

Autecology and phylogeny of Coolia tropicalis and Coolia malayensis (Dinophyceae), with emphasis on taxonomy of C. tropicalis based on light microscopy, scanning electron microscopy and LSU rDNA(1).

Coolia is a widespread and ecologically important genus of benthic marine dinoflagellates found in tropical regions. Historically, there has been taxonomic confusion about the taxonomy and toxicity of this group. The goal of this study was to resolve morphological questions concerning Coolia tropicalis and determine the taxonomic identity of the Australian Coolia isolate which has been reported to produce cooliatoxins. To accomplish this, the morphology of tropical strains from Belize (the type locality of C.tropicalis), Malaysia, Indonesia, and Australia were examined and compared to published reports. The morphological analysis showed that C.tropicalis differs from the original description in that it has a slightly larger size (35-47μm long by 30-45μm wide versus 23-40μm long by 25-39μm wide), and the shape of fourth apical plate, and the length of Po plate (7.4-12μm versus 7μm). Based on both morphology and phylogenetic analysis using LSU D1- D3 rDNA sequences, the clones of C.tropicalis from Malaysia, Indonesia, and Belize were found to form a monophyletic clade within the genus. The strain producing cooliatoxin was found to be C.tropicalis, not Coolia monotis as originally assumed. To explore the factors influencing the growth of Coolia species, the growth rates of C.tropicalis and Coolia malayensis were determined at different temperatures and salinities. Both species tolerated a wide range of temperatures, but cannot survive at temperatures <20°C or >35°C. C.monotis, the dominant species reported in the literature, probably does not produce toxins.

MORPHOLOGY AND MOLECULAR CHARACTERIZATION OF A NEW SPECIES OF THECATE BENTHIC DINOFLAGELLATE, COOLIA MALAYENSIS SP. NOV. (DINOPHYCEAE)1

Coolia Merunier is an important component of benthic dinoflagellate assemblages in tropical and subtropical seas. In this study, detailed morphological observation of Coolia species from Malaysians waters was carried out using light and electron microscopy in parallel with molecular characterization of nuclearencoded partial LSU rDNA, and internal transcribed spacer (ITS) regions.

COOLIA CANARIENSIS SP. NOV. (DINOPHYCEAE), A NEW NONTOXIC EPIPHYTIC BENTHIC DINOFLAGELLATE FROM THE CANARY ISLANDS(1).

A new photosynthetic dinoflagellate species, Coolia canariensis S. Fraga sp. nov., is described based on samples taken from tidal ponds on the rocky shore of the Canary Islands, northeast Atlantic Ocean. Its morphology was studied by LM and SEM. It is almost spherical and has a thick smooth theca with many scattered pores. Plate 1' is the biggest of the epithecal plates, and 7″ is twice as wide as it is long. Phylogeny inferred from the D1/D2 regions of the LSU nuclear rDNA of three strains of C.canariensis and several strains of other Coolia species, C.monotis, C.sp., showed that C.canariensis strains clustered in a well-supported clade distinct from the other species. No toxins were detected using mouse bioassay, liquid chromatography with Fluorescence detection (LC-FLD) or liquid chromatography-mass spectrometry (LC-MS). Its pigment composition is of the peridinin type of dinoflagellates. Together with this new species, many other strains of C.monotis from the Atlantic Ocean and Mediterranean Sea have been analyzed for toxin presence, and no evidence of toxin production related to yessotoxins (YTXs) was found, as was previously suggested for C.monotis from Australia.