Mucus-Trap-Assisted Feeding Is a Common Strategy of the Small Mixoplanktonic Prorocentrum pervagatum and P. cordatum (Prorocentrales, Dinophyceae).

Prorocentrum comprises a diverse group of bloom-forming dinophytes with a worldwide distribution. Although photosynthetic, mixoplanktonic phagotrophy has also been described. Recently, the small P. cf. balticum was shown to use a remarkable feeding strategy by crafting globular mucus traps to capture and immobilize potential prey. Here we present evidence showing that two additional related species, the recently described P. pervagatum and the cosmopolitan bloom-forming P. cordatum, also produce large (80-120 µm) mucus traps supporting their mixoplanktonic activity. Prey are captured within the traps either through passive entanglement upon contact with the outside surface, or through active water movement created by rotating Prorocentrum cells eddying particles to the inside surface where trapped live prey cells became immobilized. Entrapment in mucus assisted deployment into the prey of a peduncle extruded from the apical area of the Prorocentrum cell. Phagotrophy by P. pervagatum supported faster growth compared to unfed controls and time series quantification of food vacuoles revealed ingestion rates of ca. 10-12 Teleaulax prey cells day-1. Model calculations show clear advantages of deploying a mucus trap for increasing prey encounter rates. This study demonstrates that the large size and immobilization properties of mucus traps successfully increase the availability of prey for small Prorocentrum species, whose peduncle feeding mode impedes consumption of actively moving prey, and that this strategy is common among certain clades of small planktonic Prorocentrum species.

Response of planktonic microbial assemblages to disturbance in an urban sub-tropical estuary.

Microbes are sensitive indicators of estuarine processes because they respond rapidly to dynamic disturbance events. As most of the world's population lives in urban areas and climate change-related disturbance events are becoming more frequent, estuaries bounded by cities are experiencing increasing stressors, at the same time that their ecosystem services are required more than ever. Here, using a multidisciplinary approach, we determined the response of planktonic microbial assemblages in response to seasonality and a rainfall disturbance in an urban estuary bounded by Australia's largest city, Sydney. We used molecular barcoding (16S, 18S V4 rRNA) and microscopy-based identification to compare microbial assemblages at locations with differing characteristics and urbanisation histories. Across 142 samples, we identified 8,496 unique free-living bacterial zOTUs, 8,175 unique particle associated bacterial zOTUs, and 1,920 unique microbial eukaryotic zOTUs. Using microscopy, we identified only the top <10% abundant, larger eukaryotic taxa (>10 µm), however quantification was possible. The site with the greater history of anthropogenic impact showed a more even community of associated bacteria and eukaryotes, and a significant increase in dissolved inorganic nitrogen following rainfall, when compared to the more buffered site. This coincided with a reduced proportional abundance of Actinomarina and Synechococcus spp., a change in SAR 11 clades, and an increase in the eukaryotic microbial groups Dinophyceae, Mediophyceae and Bathyoccocaceae, including a temporary dominance of the harmful algal bloom dinoflagellate Prorocentrum cordatum (syn. P. minimum). Finally, a validated hydrodynamic model of the estuary supported these results, showing that the more highly urbanised and upstream location consistently experienced a higher magnitude of salinity reduction in response to rainfall events during the study period. The best abiotic variables to explain community dissimilarities between locations were TDP, PN, modelled temperature and salinity (r = 0.73) for the free living bacteria, TP for the associated bacteria (r = 0.43), and modelled temperature (r = 0.28) for the microbial eukaryotic communities. Overall, these results show that a minor disturbance such as a brief rainfall event can significantly shift the microbial assemblage of an anthropogenically impacted area within an urban estuary to a greater degree than a seasonal change, but may result in a lesser response to the same disturbance at a buffered, more oceanic influenced location. Fine scale research into the factors driving the response of microbial communities in urban estuaries to climate related disturbances will be necessary to understand and implement changes to maintain future estuarine ecosystem services.

Diverse RNA Viruses Associated with Diatom, Eustigmatophyte, Dinoflagellate, and Rhodophyte Microalgae Cultures.

Unicellular microalgae are of immense ecological importance with growing commercial potential in industries such as renewable energy, food, and pharmacology. Viral infections can have a profound impact on the growth and evolution of their hosts. However, very little is known of the diversity within, and the effect of, unicellular microalgal RNA viruses. In addition, identifying RNA viruses in these organisms that could have originated more than a billion years ago constitutes a robust data set to dissect molecular events and address fundamental questions in virus evolution. We assessed the diversity of RNA viruses in eight microalgal cultures, including representatives from the diatom, eustigmatophyte, dinoflagellate, red algae, and euglenid groups. Using metatranscriptomic sequencing combined with bioinformatic approaches optimized to detect highly divergent RNA viruses, we identified 10 RNA virus sequences, with nine constituting new viral species. Most of the newly identified RNA viruses belonged to the double-stranded Totiviridae, Endornaviridae, and Partitiviridae, greatly expanding the reported host range for these families. Two new species belonging to the single-stranded RNA viral clade Marnaviridae, commonly associated with microalgal hosts, were also identified. This study highlights that a substantial diversity of RNA viruses likely exists undetected within the unicellular microalgae. It also highlights the necessity for RNA viral characterization and for investigation of the effects of viral infections on microalgal physiology, biology, and growth, considering their environmental and industrial roles. IMPORTANCE Our knowledge of the diversity of RNA viruses infecting microbial algae-the microalgae-is minimal. However, describing the RNA viruses infecting these organisms is of primary importance at both the ecological and economic scales because of the fundamental roles these organisms play in aquatic environments and their growing value across a range of industrial fields. Using metatranscriptomic sequencing, we aimed to reveal the RNA viruses present in cultures of eight microalgae species belonging to the diatom, dinoflagellate, eustigmatophyte, rhodophyte, and euglena major clades of algae. Accordingly, we identified 10 new divergent RNA virus species belonging to RNA virus families as diverse as the double-stranded Totiviridae, Endornaviridae, and Partitiviridae and the single-stranded Marnaviridae. By expanding the known diversity of RNA viruses infecting unicellular eukaryotes, this study contributes to a better understanding of the early evolution of the virosphere and will inform the use of microalgae in industrial applications.

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

Despite their relatively high thermal optima (Topt ), tropical taxa may be particularly vulnerable to a rising baseline and increased temperature variation because they live in relatively stable temperatures closer to their Topt . 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 Topt . 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.

Predictability of thermal fluctuations influences functional traits of a cosmopolitan marine diatom.

Evolutionary theory predicts that organismal plasticity should evolve in environments that fluctuate regularly. However, in environments that fluctuate less predictably, plasticity may be constrained because environmental cues become less reliable for expressing the optimum phenotype. Here, we examine how the predictability of +5°C temperature fluctuations impacts the phenotype of the marine diatom Thalassiosira pseudonana. Thermal regimes were informed by temperatures experienced by microbes in an ocean simulation and featured regular or irregular temporal sequences of fluctuations that induced mild physiological stress. Physiological traits (growth, cell size, complexity and pigmentation) were quantified at the individual cell level using flow cytometry. Changes in cellular complexity emerged as the first impact of predictability after only 8-11 days, followed by deleterious impacts on growth on days 13-16. Specifically, cells with a history of irregular fluctuation exposure exhibited a 50% reduction in growth compared with the stable reference environment, while growth was 3-18 times higher when fluctuations were regular. We observed no evidence of heat hardening (increasingly positive growth) with recurrent fluctuations. This study demonstrates that unpredictable temperature fluctuations impact this cosmopolitan diatom under ecologically relevant time frames, suggesting shifts in environmental stochasticity under a changing climate could have widespread consequences among ocean primary producers.

Mucospheres produced by a mixotrophic protist impact ocean carbon cycling.

Mixotrophic protists (unicellular eukaryotes) that engage in both phototrophy (photosynthesis) and phago-heterotrophy (engulfment of particles)-are predicted to contribute substantially to energy fluxes and marine biogeochemical cycles. However, their impact remains largely unquantified. Here we describe the sophisticated foraging strategy of a widespread mixotrophic dinoflagellate, involving the production of carbon-rich 'mucospheres' that attract, capture, and immobilise microbial prey facilitating their consumption. We provide a detailed characterisation of this previously undescribed behaviour and reveal that it represents an overlooked, yet quantitatively significant mechanism for oceanic carbon fluxes. Following feeding, the mucospheres laden with surplus prey are discarded and sink, contributing an estimated 0.17-1.24 mg m-2 d-1 of particulate organic carbon, or 0.02-0.15 Gt to the biological pump annually, which represents 0.1-0.7% of the estimated total export from the euphotic zone. These findings demonstrate how the complex foraging behaviour of a single species of mixotrophic protist can disproportionally contribute to the vertical flux of carbon in the ocean.

Phenotypic trait variability as an indication of adaptive capacity in a cosmopolitan marine diatom.

Determining the adaptive capacity of marine phytoplankton is important in predicting changes in phytoplankton responses to ocean warming. Phytoplankton may consist of high levels of standing phenotypic and genetic variability, the basis of rapid evolution; however, few studies have quantified trait variability within and amongst closely related diatom species. Using 35 clonal cultures of the ubiquitous marine diatom Leptocylindrus isolated from six locations, spanning 2000 km of the south-eastern Australian coastline, we found evidence of significant intraspecific morphological and metabolic trait variability, which for 8 of 9 traits (growth rate, biovolume, C:N, silica deposition, silica incorporation rate, chl-a, and photosynthetic efficiency under dark adapted, growth irradiance, and high-light adaptation) were greater within a species than between species. Moreover, only two traits revealed a latitudinal trend with strains isolated from lower latitudes showing significantly higher silicification rates and protein:lipid content compared to their higher latitude counterparts. These data mirror recent studies on diatom intraspecific genetic diversity, which has found comparable levels of genetic diversity at a single site to those thousands of kilometres apart, and provide evidence of a functional role of diatom diversity that will allow for rapid adaptation via ecological selection on standing variation in response to changing conditions.

The role of 44-methylgambierone in ciguatera fish poisoning: Acute toxicity, production by marine microalgae and its potential as a biomarker for Gambierdiscus spp.

Ciguatera fish poisoning (CFP) is prevalent around the tropical and sub-tropical latitudes of the world and impacts many Pacific island communities intrinsically linked to the reef system for sustenance and trade. While the genus Gambierdiscus has been linked with CFP, it is commonly found on tropical reef systems in microalgal assemblages with other genera of toxin-producing, epiphytic and/or benthic dinoflagellates - Amphidinium, Coolia, Fukuyoa, Ostreopsis and Prorocentrum. Identifying a biomarker compound that can be used for the early detection of Gambierdiscus blooms, specifically in a mixed microalgal community, is paramount in enabling the development of management and mitigation strategies. Following on from the recent structural elucidation of 44-methylgambierone, its potential to contribute to CFP intoxication events and applicability as a biomarker compound for Gambierdiscus spp. was investigated. The acute toxicity of this secondary metabolite was determined by intraperitoneal injection using mice, which showed it to be of low toxicity, with an LD50 between 20 and 38 mg kg-1. The production of 44-methylgambierone by 252 marine microalgal isolates consisting of 90 species from 32 genera across seven classes, was assessed by liquid chromatography-tandem mass spectrometry. It was discovered that the production of this secondary metabolite was ubiquitous to the eight Gambierdiscus species tested, however not all isolates of G. carpenteri, and some species/isolates of Coolia and Fukuyoa.

Characterisation of Two Toxic Gambierdiscus spp. (Gonyaulacales, Dinophyceae) from the Great Barrier Reef (Australia): G. lewisii sp. nov. and G. holmesii sp. nov.

Ciguatera fish poisoning (CFP) is a human illness caused via consumption of seafood contaminated with neurotoxins produced by some species from the epiphytic dinoflagellate genus Gambierdiscus. In this study, we describe two new species of Gambierdiscus isolated from Heron Island in the Southern Great Barrier Reef, Queensland, Australia. These new species were analysed using light microscopy, scanning electron microscopy, and phylogenetic analyses of nuclear encoded ribosomal ITS, SSU as well as D1-D3 and D8-D10 of the LSU gene regions. Gambierdiscus lewisii sp. nov. (Po, 3', 0a, 7″, 6c,? s, 5‴, 0p, 2'‴) is distinguished by its strong reticulate-foveate ornamentation and is genetically distinct from its sister species, G. pacificus. Gambierdiscus holmesii sp. nov. (Po, 3', 0a, 7″, 6c, 6s?, 5‴, 0p, 2'‴) is morphologically distinct from the genetically similar species G. silvae because of a strongly ventrally displaced apical pore complex and a characteristic fold at the anterior edge of the sulcus. Both G. lewisii and G. holmesii produce putative Maitotoxin-(44-Methylgambierone) and compounds which show ciguatoxin and maitotoxin-like activities. Identification of two new Gambierdiscus species will enable us to more accurately assess the risk of CFP in Australia and internationally.

Live cell analysis at sea reveals divergent thermal performance between photosynthetic ocean microbial eukaryote populations.

Experimentation at sea provides insight into which traits of ocean microbes are linked to performance in situ. Here we show distinct patterns in thermal tolerance of microbial phototrophs from adjacent water masses sampled in the south-west Pacific Ocean, determined using a fluorescent marker for reactive oxygen species (ROS). ROS content of pico-eukaryotes was assessed after 1, 5 and 25 h of incubation along a temperature gradient (15.6-32.1 °C). Pico-eukaryotes from the East Australian Current (EAC) had relatively constant ROS and showed greatest mortality after 25 h at 7 °C below ambient, whereas those from the Tasman Sea had elevated ROS in both warm and cool temperature extremes and greatest mortality at temperatures 6-10 °C above ambient, interpreted as the outcome of thermal stress. Tracking of water masses within an oceanographic circulation model showed populations had distinct thermal histories, with EAC pico-eukaryotes experiencing higher average temperatures for at least 1 week prior to sampling. While acclimatization and community assembly could both influence biological responses, this study clearly demonstrates that phenotypic divergence occurs along planktonic drift trajectories.

First description of the environmental niche of the epibenthic dinoflagellate species Coolia palmyrensis, C. malayensis, and C. tropicalis (Dinophyceae) from Eastern Australia.

Environmental variables such as temperature, salinity, and irradiance are significant drivers of microalgal growth and distribution. Therefore, understanding how these variables influence fitness of potentially toxic microalgal species is particularly important. In this study, strains of the potentially harmful epibenthic dinoflagellate species Coolia palmyrensis, C. malayensis, and C. tropicalis were isolated from coastal shallow water habitats on the east coast of Australia and identified using the D1-D3 region of the large subunit (LSU) ribosomal DNA (rDNA). To determine the environmental niche of each taxon, growth was measured across a gradient of temperature (15-30°C), salinity (20-38), and irradiance (10-200 µmol photons · m-2  · s-1 ). Specific growth rates of Coolia tropicalis were highest under warm temperatures (27°C), low salinities (ca. 23), and intermediate irradiance levels (150 µmol photons · m-2  · s-1 ), while C. malayensis showed the highest growth at moderate temperatures (24°C) and irradiance levels (150 µmol photons · m-2  · s-1 ) and growth rates were consistent across the range of salinity levels tested (20-38). Coolia palmyrensis had the highest growth rate of all species tested and favored moderate temperatures (24°C), oceanic salinity (35), and high irradiance (>200 µmol photons · m-2  · s-1 ). This is the first study to characterize the environmental niche of species from the benthic harmful algal bloom genus Coolia and provides important information to help define species distributions and inform risk management.

Toxicology of Gambierdiscus spp. (Dinophyceae) from Tropical and Temperate Australian Waters.

Ciguatera Fish Poisoning (CFP) is a human illness caused by the consumption of marine fish contaminated with ciguatoxins (CTX) and possibly maitotoxins (MTX), produced by species from the benthic dinoflagellate genus Gambierdiscus. Here, we describe the identity and toxicology of Gambierdiscus spp. isolated from the tropical and temperate waters of eastern Australia. Based on newly cultured strains, we found that four Gambierdiscus species were present at the tropical location, including G. carpenteri, G. lapillus and two others which were not genetically identical to other currently described species within the genus, and may represent new species. Only G. carpenteri was identified from the temperate location. Using LC-MS/MS analysis we did not find any characterized microalgal CTXs (P-CTX-3B, P-CTX-3C, P-CTX-4A and P-CTX-4B) or MTX-1; however, putative maitotoxin-3 (MTX-3) was detected in all species except for the temperate population of G. carpenteri. Using the Ca2+ influx SH-SY5Y cell Fluorescent Imaging Plate Reader (FLIPR) bioassay we found CTX-like activity in extracts of the unidentified Gambierdiscus strains and trace level activity in strains of G. lapillus. While no detectable CTX-like activity was observed in tropical or temperate strains of G. carpenteri, all species showed strong maitotoxin-like activity. This study, which represents the most comprehensive analyses of the toxicology of Gambierdiscus strains isolated from Australia to date, suggests that CFP in this region may be caused by currently undescribed ciguatoxins and maitotoxins.

Ciguatoxins and Maitotoxins in Extracts of Sixteen Gambierdiscus Isolates and One Fukuyoa Isolate from the South Pacific and Their Toxicity to Mice by Intraperitoneal and Oral Administration.

Ciguatoxins (CTXs), and possibly maitotoxins (MTXs), are responsible for Ciguatera Fish Poisoning, an important health problem for consumers of reef fish (such as inhabitants of islands in the South Pacific Ocean). The habitational range of the Gambierdiscus species is expanding, and new species are being discovered. In order to provide information on the potential health risk of the Gambierdiscus species, and one Fukuyoa species (found in the Cook Islands, the Kermadec Islands, mainland New Zealand, and New South Wales, Australia), 17 microalgae isolates were collected from these areas. Unialgal cultures were grown and extracts of the culture isolates were analysed for CTXs and MTXs by liquid chromatography tandem mass spectrometry (LC-MS/MS), and their toxicity to mice was determined by intraperitoneal and oral administration. An isolate of G. carpenteri contained neither CTXs nor MTXs, while 15 other isolates (including G. australes, G. cheloniae, G. pacificus, G.honu, and F. paulensis) contained only MTX-1 and/or MTX-3. An isolate of G. polynesiensis contained both CTXs and MTX-3. All the extracts were toxic to mice by intraperitoneal injection, but those containing only MTX-1 and/or -3 were much less toxic by oral administration. The extract of G. polynesiensis was highly toxic by both routes of administration.

Long-term perspective on the relationship between phytoplankton and nutrient concentrations in a southeastern Australian estuary.

Sixteen years (1997-2013) of physicochemical, nutrient and phytoplankton biomass (Chlorophyll-a (Chl-a)) data and a decade (2003-2013) of phytoplankton composition and abundance data were analyzed to assess how the algal community in a temperate southeastern Australian estuary has responded to decreased chronic point source nitrogen loading following effluent treatment upgrade works in 2003. Nitrogen concentrations were significantly lower (P<0.05) following enhanced effluent treatment and Chl-a levels decreased (P<0.05) during the warmer months. Temperature and nutrient concentrations significantly influenced temporal changes of Chl-a (explaining 55% of variability), while salinity, temperature, pH and nutrient concentrations influenced phytoplankton abundance and composition (25% explained). Harmful Algal Bloom (HAB) dynamics differed between sites likely influenced by physical attributes of the estuary. This study demonstrates that enhanced effluent treatment can significantly decrease chronic point source nitrogen loading and that Chl-a concentrations can be lowered during the warmer months when the risk of blooms and HABs is greatest.