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Cyanobacterial mats supplanting coral and spreading coral diseases in tropical reefs, intensified by environmental shifts caused by human-induced pressures, nutrient enrichment, and global climate change, pose grave risks to the survival of coral ecosystems. In this study, we characterized Okeanomitos corallinicola gen. and sp. nov., a newly discovered toxic marine heterocyte-forming cyanobacterium isolated from a coral reef ecosystem of the South China Sea. Phylogenetic analysis, based on the 16S rRNA gene and the secondary structure of the 16S-23S rRNA intergenic region, placed this species in a clade distinct from closely related genera, that is, Sphaerospermopsis stricto sensu, Raphidiopsis, and Amphiheterocytum. The O. corallinicola is a marine benthic species lacking gas vesicles, distinguishing it from other members of the Aphanizomenonaceae family. The genome of O. corallinicola is large and exhibits diverse functional capabilities, potentially contributing to the resilience and adaptability of coral reef ecosystems. In vitro assays revealed that O. corallinicola demonstrates notable cytotoxic activity against various cancer cell lines, suggesting its potential as a source of novel anticancer compounds. Furthermore, the identification of residual saxitoxin biosynthesis function in the genome of O. corallinicola, a marine cyanobacteria, supports the theory that saxitoxin genes in cyanobacteria and dinoflagellates may have been horizontally transferred between them or may have originated from a shared ancestor. Overall, the identification and characterization of O. corallinicola provides valuable contributions to cyanobacterial taxonomy, offering novel perspectives on complex interactions within coral reef ecosystems.
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The marine dinophyte Alexandrium tamiyavanichii is a toxigenic species that produces a group of neurotoxins that is responsible for paralytic shellfish poisoning in humans. Early detection of the species is essential for efficient monitoring. Harmful microalgal monitoring systems have evolved over the years with the advent of environmental DNA (eDNA)-based species detection techniques. In this study, eDNA samples were collected from a large-scale sampling covering the southern South China Sea. The sensitivity and specificity of metabarcoding of the V4 and V9 18S ribosomal DNA barcodes by high-throughput sequencing (HTS) were compared to the species-specific real-time qPCR targeting the A. tamiyavanichii ITS2 region. Environmental samples were screened for A. tamiyavanichii by qPCR (n = 43) and analyzed with metabarcoding (n = 30). Our results revealed a high occupancy profile across samples for both methods; 88% by qPCR, and 80-83% by HTS. When comparing the consistency between the two approaches, only two samples out of 30 were discordant. The V4 and V9 molecular units detected in each sample were positively correlated with the qPCR ITS2 gene copies (V4, rs = 0.67, p < 0.0001; V9, rs = 0.65, p < 0.0001), indicating that metabarcoding could be used as a useful tool for early detection of the species. Our results also revealed that the estimation of A. tamiyavanichii cell abundances based on the HTS read abundances was comparable to that of the qPCR quantification. For long-term monitoring, metabarcoding could serve as a cost-effective screening of detecting not only single HAB species but also simultaneously detecting a multitude of potentially harmful species, which is valuable in informing the subsequent implementation of species-specific monitoring strategies.
Assuntos
Código de Barras de DNA Taxonômico , Dinoflagellida , Monitoramento Ambiental , Reação em Cadeia da Polimerase em Tempo Real , Dinoflagellida/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Código de Barras de DNA Taxonômico/métodos , Monitoramento Ambiental/métodos , DNA Ambiental , China , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Species of the genus Chattonella (Raphidophyceae) are a group of marine protists that are commonly found in coastal waters. Some are known as harmful microalgae that form noxious blooms and cause massive fish mortality in finfish aquaculture. In Malaysia, blooms of Chattonella have been recorded since the 1980s in the Johor Strait. In this study, two strains of Chattonella were established from the strait, and morphological examination revealed characteristics resembling Chattonella subsalsa. The molecular characterization further confirmed the species' identity as C. subsalsa. To precisely detect the cells of C. subsalsa in the environment, a whole-cell fluorescence in-situ hybridisation (FISH) assay was developed. The species-specific oligonucleotide probes were designed in silico based on the nucleotide sequences of the large subunit (LSU) and internal transcribed spacer 2 (ITS2) of the ribosomal DNA (rDNA). The best candidate signature regions in the LSU-rRNA and ITS2-rDNA were selected based on hybridisation efficiency and probe parameters. The probes were synthesised as biotinylated probes and tested by tyramide signal amplification with FISH (FISH-TSA). The results showed the specificity of the probes toward the target cells. FISH-TSA has been proven to be a potential tool in the detection of harmful algae in the environment and could be applied to the harmful algal monitoring program.
Spesies genus Chattonella (Raphidophyceae) ialah sekumpulan protista marin yang biasa ditemui di perairan laut pantai. Sesetengahnya dikenali sebagai mikroalga berbahaya yang membentuk ledakan alga berbahaya dan menyebabkan kematian ikan secara besar-besaran dalam akuakultur ikan sirip. Di Malaysia, ledakan alga Chattonella telah direkodkan sejak tahun 1980-an di Selat Johor. Dalam kajian ini, dua strain Chattonella telah didirikan dari selat, dan pemeriksaan morfologi mendedahkan ciri-ciri yang menyerupai Chattonella subsalsa. Pencirian molekul seterusnya mengesahkan identiti spesies sebagai C. subsalsa. Untuk mengesan dengan tepat sel-sel C. subsalsa di dalam persekitaran, ujian penghibridan in-situ berpendarfluor (FISH) ke atas sel keseluruhan telah dibangunkan. Prob oligonukleotida spesies telah direka secara spesifik secara siliko berdasarkan jujukan nukleotida subunit besar (LSU) dan spacer transkripsi dalaman 2 (ITS2) gen DNA ribosom (rDNA). Calon terbaik kawasan tanda dalam LSU-rRNA dan ITS2-rDNA telah dipilih berdasarkan kecekapan penghibridan dan parameter prob. Prob telah disintesis sebagai prob biotinilasi dan diuji dengan penguatan isyarat tyramide dengan FISH (FISH-TSA). Keputusan menunjukkan kekhususan prob ke atas sel sasaran. FISH-TSA telah terbukti sebagai alat yang berpotensi dalam pengesanan alga berbahaya di alam sekitar dan boleh digunakan untuk program pemantauan alga berbahaya.
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A large-scale sampling was undertaken during a research cruise across the South China Sea in August 2016, covering an area of about 100,000 km2 to investigate the molecular diversity and distributions of micro-eukaryotic protists, with a focus on the potentially harmful microalgal (HAB) species along the east coast of Peninsular Malaysia. Environmental DNAs from 30 stations were extracted and DNA metabarcoding targeting the V4 and V9 markers in the 18S rDNA was performed. Many protistan molecular units, including previously unreported HAB taxa, were discovered for the first time in the water. Our findings also revealed interesting spatial distribution patterns, with a marked signal of compositional turnover between latitudinal regimes of water masses, where dinophytes and diatom compositions were among the most strongly enhanced at the fronts, leading to distinct niches. Our results further confirmed the widespread distribution of HAB species, such as the toxigenic Alexandrium tamiyavaichii and Pseudo-nitzschia species, and the fish-killing Margalefidinium polykrikoides and Karlodinium veneficum. The molecular information obtained from this study provides an updated HAB species inventory and a toolset that could facilitate existing HAB monitoring schemes in the region to better inform management decisions.
Assuntos
Diatomáceas , Dinoflagellida , Microalgas , Animais , Proliferação Nociva de Algas , Microalgas/genética , Dinoflagellida/genética , Diatomáceas/genética , China , ÁguaRESUMO
The scleractinian coral-associated symbiotic algae Symbiodiniaceae plays an important role in bleaching tolerance and coral resilience. In this study, coral-associated Symbiodiniaceae communities of 14 reef sites of Perhentian and Redang Islands Marine Parks (Malaysia, South China Sea) were characterized using the high-throughput next-generation amplicon sequencing on the ITS2 rDNA marker to inventory the Symbiodiniaceae diversity from a healthy tropical reef system and to generate a baseline for future studies. A total of 64 coral-Symbiodiniaceae associations were characterized in 18 genera (10 families) of scleractinian corals using the SymPortal analytical framework. The results revealed the predominance of Symbiodiniaceae genera Cladocopium (average 82%) and Durusdinium (18%), while Symbiodinium, Breviolum, Fugacium, and Gerakladium were found as minor groups (<0.01%). Of the 39 Cladocopium and Durusdinium major ITS2 sequences, 14 were considered dominant/sub-dominant, with C3u as the predominant type (63.3%), followed by D1 (15%), C27 (10.1%), and C15 (6.9%). A total of 19 and 13 Cladocopium and Durusdinium ITS2-type profiles were detected across the coral species, respectively. Symbiodiniaceae diversity and richness recorded in this study were higher when compared to other reefs in the proximity. With the increasing coral-Symbiodiniaceae associations archived, the database would provide a baseline to assess the changes of Symbiodiniaceae communities in the coral hosts and to explore the potential adaptive roles of this coral-algal association.
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Antozoários , Dinoflagellida , Animais , Antozoários/genética , China , Recifes de Corais , Dinoflagellida/genética , Sequenciamento de Nucleotídeos em Larga Escala , SimbioseRESUMO
Harmful algal blooms (HABs) were not new to the tropical semi-enclosed Johor Strait, with incident records that could trace back to the 1980s. HAB monitoring in the area, often, is reactive, focusing only on HAB taxa previously causing problems but neglecting potential emerging HABs. To develop datasets on HABs that can better inform and improve management practices, monitoring should expand to sample whole microphytoplankton communities. In this study, microphytoplankton community structure across the Strait was investigated. Abundances of microphytoplankton and a suite of in situ water parameters of temperatures, salinity, pH, dissolved oxygen levels, macronutrients, and chlorophyll-a contents were collected at ten sites across the Strait at monthly intervals from January 2017 to December 2018. A total of 48 genera (51 taxa) microphytoplankton were identified microscopically. Diatom was the most diverse group (32 genera), followed by dinophyte (15 genera). Bloom-forming species included diatoms Chaetoceros, Coscinodiscus, Eucampia, Pseudo-nitzschia, Rhizosolenia, Skeletonema, Thalassiosira, and dinophytes Blixaea quinquecornis and Scrippsiella. Diatom taxa that exhibit high in situ growth rates were predominant in the low-nutrient marine-influenced environment. Bloom-forming taxa including HAB taxa were found dominant in the environment with high nutrient levels and mesohaline, salinity-stratified conditions. This study provides valuable baseline data that could assist in monitoring and prediction of HABs in the Johor Strait and could be of reference to other similar tropical coastal systems.
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Diatomáceas , Dinoflagellida , Microalgas , Proliferação Nociva de Algas , MalásiaRESUMO
Coastal eutrophication is one of the pivotal factors driving occurrence of harmful algal blooms (HABs), whose underlying mechanism remained unclear. To better understand the nutrient regime triggering HABs and their formation process, the phytoplankton composition and its response to varying nitrogen (N) and phosphorus (P), physio-chemical parameters in water and sediment in Johor Strait in March 2019 were analyzed. Surface and sub-surface HABs were observed with the main causative species of Skeletonema, Chaetoceros and Karlodinium. The ecophysiological responses of Skeletonema to the low ambient N/P ratio such as secreting alkaline phosphatase, regulating cell morphology (volume; surface area/volume ratio) might play an important role in dominating the community. Anaerobic sediment iron-bound P release and simultaneous N removal by denitrification and anammox, shaped the stoichiometry of N and P in water column. The decrease of N/P ratio might shift the phytoplankton community into the dominance of HABs causative diatoms and dinoflagellates.
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Diatomáceas , Dinoflagellida , Anaerobiose , Eutrofização , Malásia , Nitrogênio/análise , Nutrientes , Fósforo/análise , FitoplânctonRESUMO
Coastal ecosystems are often subjected to anthropogenic disturbances that lead to water quality deterioration and an increase in harmful algal bloom (HAB) events. Using the next-generation molecular tool of 18S rDNA metabarcoding, we examined the community assemblages of HAB species in the Johor Strait, Malaysia between May 2018 and September 2019, covering 19 stations across the strait. The molecular operational taxonomic units (OTUs) of HAB taxa retrieved from the dataset (n = 194) revealed a much higher number of HAB taxa (26 OTUs) than before, with 12 taxa belong to new records in the strait. As revealed in the findings of this study, the diversity and community structure of HAB taxa varied significantly over time and space. The most common and abundant HAB taxa in the strait (frequency of occurrence >70%) comprised Heterosigma akashiwo, Fibrocapsa japonica, Pseudo-nitzschia pungens, Dinophysis spp., Gymnodinium catenatum, Alexandrium leei, and A. tamiyavanichii. Also, our results demonstrated that the HAB community assemblages in the strait were dependent on the interplay of environmental variables that influence by the monsoonal effects. Different HAB taxa, constitute various functional types, occupied and prevailed in different environmental niches across space and time, leading to diverse community assemblages and population density. This study adds to the current understandings of HAB dynamics and provides a robust overview of temporal-spatial changes in HAB community assemblages along the environmental gradients in a tropical eutrophic coastal ecosystem.
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Dinoflagellida , Microalgas , Ecossistema , Proliferação Nociva de Algas , FitoplânctonRESUMO
The Johor Strait has experienced rapid development of various human activities and served as the main marine aquaculture area for the two countries that bordered the strait. Several fish kill incidents in 2014 and 2015 have been confirmed, attributed to the algal blooms of ichthyotoxic dinoflagellates; however, the cause of fish kill events after 2016 was not clarified and the causative organisms remained unknown. To clarify the potential cause of fish kills along the Johor Strait, a 1-year field investigation was conducted with monthly sampling between May 2018 and April 2019. Monthly vertical profiles of physical water parameters (temperature, salinity, and dissolved oxygen levels) were measured in situ at different depths (subsurface, 1 m, 5 m, and 8 m) depending on the ambient depth of the water column at the sampling stations. The spatial-temporal variability of macronutrients and chlorophyll a content was analyzed. Our results showed that high chlorophyll a concentration (up to 48.8 µg/L) and high biomass blooms of Skeletonema, Chaetoceros, Rhizosolenia, and Thalassiosira were observed seasonally at the inner part of the strait. A hypoxic to anoxic dead zone, with the dissolved oxygen levels ranging from 0.19 to 1.7 mg/L, was identified in the inner Johor Strait, covering an estimated area of 10.3 km2. The occurrence of high biomass diatom blooms and formation of the hypoxic-anoxic zone along the inner part Johor Strait were likely the culprits of some fish kill incidents after 2016.
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Diatomáceas , Dinoflagellida , Animais , Biomassa , Clorofila A , EutrofizaçãoRESUMO
The marine dinoflagellate Alexandrium minutum is known to produce saxitoxins that cause paralytic shellfish poisoning in human worldwide through consumption of the contaminated shellfish mollusks. Despite numerous studies on the growth physiology and saxitoxin production of this species, the knowledge on the molecular basis of nutrient uptakes in relation to toxin production in this species is limited. In this study, relative expressions of the high-affinity transporter genes of nitrate, ammonium, and phosphate (AmNrt2, AmAmt1 and AmPiPT1) and the assimilation genes, nitrate reductase (AmNas), glutamine synthase (AmGSIII) and carbamoyl phosphate synthase (AmCPSII) from A. minutum were studied in batch clonal culture condition with two nitrogen sources (nitrate: NO3- or ammonium: NH4+) under different N:P ratios (high-P: N:P of 14 and 16, and low-P: N:P of 155). The expression of AmAmt1 was suppressed in excess NH4+-grown condition but was not observed in AmNrt2 and AmNas. Expressions of AmAmt1, AmNrt2, AmNas, AmGSIII, AmCPSII, and AmPiPT1 were high in P-deficient condition, showing that A. minutum is likely to take up nutrients for growth under P-stress condition. Conversely, relative expression of AmCPSII was incongruent with cell growth, but was well correlated with toxin quota, suggesting that the gene might involve in arginine metabolism and related toxin production pathway. The expression of AmGSIII is found coincided with higher toxin production and is believed to involve in mechanism to detoxify the cells from excess ammonium stress. The gene regulation observed in this study has provided better insights into the ecophysiology of A. minutum in relation to its adaptive strategies in unfavorable environments.
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Técnicas de Cultura Celular por Lotes/métodos , Dinoflagellida/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Compostos de Amônio/metabolismo , Dinoflagellida/metabolismo , Regulação da Expressão Gênica , Nitratos/metabolismo , Fosfatos/metabolismo , Saxitoxina/genética , Estresse FisiológicoRESUMO
Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0-1.6 pg cell-1) and P. caipirignum (3.1 pg cell-1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.
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Dinoflagellida , DNA Ribossômico , Ilhas , Malásia , FilogeniaRESUMO
In 2015, a remarkably high density bloom of Alexandrium minutum occurred in Sungai Geting, a semi-enclosed lagoon situated in the northeast of Peninsular Malaysia, causing severe discoloration and contaminated the benthic clams (Polymesoda). Plankton and water samples were collected to investigate the mechanisms of bloom development of this toxic species. Analysis of bloom samples using flow cytometry indicated that the bloom was initiated by the process of active excystment, as planomycetes (>4C cells) were observed in the early stage of the bloom. Increase in planozygotes (2C cells) was evident during the middle stage of the bloom, coinciding with an abrupt decrease in salinity and increase of temperature. The bloom was sustained through the combination of binary division of vegetative cells, division of planozygotes, and cyst germination through continuous excystment. Nutrient depletion followed by precipitation subsequently caused the bloom to terminate. This study provides the first continuous record of in situ life-cycle stages of a natural bloom population of A. minutum through a complete bloom cycle. The event has provided a fundamental understanding of the pelagic life-cycle stages of this tropical dinoflagellate, and demonstrated a unique bloom development characteristic shared among toxic Alexandrium species in coastal embayments.
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Dinoflagellida/crescimento & desenvolvimento , Eutrofização , Povo Asiático , Divisão Celular , Humanos , Malásia , Salinidade , Estações do Ano , TemperaturaRESUMO
Saxitoxins (STXs) constitute a family of potent sodium channel blocking toxins, causative agents of paralytic shellfish poisoning (PSP), and are produced by several species of marine dinoflagellates and cyanobacteria. Two STX-core genes, sxtA and sxtG, have been well elucidated in Alexandrium but the expression of these genes under various nutritional modes in tropical species remains unclear. This study investigates the physiological responses of a tropical Pacific strain of Alexandrium minutum growing with nitrate or ammonium, and with various nitrogen to phosphorus (N:P) supply ratios. The transcriptional responses of the sxt genes were observed. Likewise, a putative sxtI encoding O-carbamoyltransferase (herein designated as AmsxtI) was recovered from the transcriptomic data, and its expression was investigated. The results revealed that the cellular toxin quota (Qt) was higher in P-depleted, nitrate-grown cultures. With cultures at similar N:P (<16), cells grown with excess ammonium showed a higher Qt than those grown with nitrate. sxtA1 was not expressed under any culture conditions, suggesting that this gene might not be involved in STX biosynthesis by this strain. Conversely, sxtA4 and sxtG showed positive correlations with Qt, and were up-regulated in P-depleted, nitrate-grown cultures and with excess ambient ammonium. On the other hand, AmsxtI was expressed only when induced by P-depletion, suggesting that this gene may play an important role in P-recycling metabolism, while simultaneously enhancing toxin production.