Avery O. Tatters

Short- and long-term conditioning of a temperate marine diatom community to acidification and warming

Ocean acidification and greenhouse warming will interactively influence competitive success of key phytoplankton groups such as diatoms, but how long-term responses to global change will affect community structure is unknown. We incubated a mixed natural diatom community from coastal New Zealand waters in a short-term (two-week) incubation experiment using a factorial matrix of warming and/or elevated pCO2 and measured effects on community structure. We then isolated the dominant diatoms in clonal cultures and conditioned them for 1 year under the same temperature and pCO2 conditions from which they were isolated, in order to allow for extended selection or acclimation by these abiotic environmental change factors in the absence of interspecific interactions. These conditioned isolates were then recombined into ‘artificial’ communities modelled after the original natural assemblage and allowed to compete under conditions identical to those in the short-term natural community experiment. In general, the resulting structure of both the unconditioned natural community and conditioned ‘artificial’ community experiments was similar, despite differences such as the loss of two species in the latter. pCO2 and temperature had both individual and interactive effects on community structure, but temperature was more influential, as warming significantly reduced species richness. In this case, our short-term manipulative experiment with a mixed natural assemblage spanning weeks served as a reasonable proxy to predict the effects of global change forcing on diatom community structure after the component species were conditioned in isolation over an extended timescale. Future studies will be required to assess whether or not this is also the case for other types of algal communities from other marine regimes.

High CO2 and silicate limitation synergistically increase the toxicity of Pseudo-nitzschia fraudulenta.

Anthropogenic CO2 is progressively acidifying the ocean, but the responses of harmful algal bloom species that produce toxins that can bioaccumulate remain virtually unknown. The neurotoxin domoic acid is produced by the globally-distributed diatom genus Pseudo-nitzschia. This toxin is responsible for amnesic shellfish poisoning, which can result in illness or death in humans and regularly causes mass mortalities of marine mammals and birds. Domoic acid production by Pseudo-nitzschia cells is known to be regulated by nutrient availability, but potential interactions with increasing seawater CO2 concentrations are poorly understood. Here we present experiments measuring domoic acid production by acclimatized cultures of Pseudo-nitzschia fraudulenta that demonstrate a strong synergism between projected future CO2 levels (765 ppm) and silicate-limited growth, which greatly increases cellular toxicity relative to growth under modern atmospheric (360 ppm) or pre-industrial (200 ppm) CO2 conditions. Cellular Si∶C ratios decrease with increasing CO2, in a trend opposite to that seen for domoic acid production. The coastal California upwelling system where this species was isolated currently exhibits rapidly increasing levels of anthropogenic acidification, as well as widespread episodic silicate limitation of diatom growth. Our results suggest that the current ecosystem and human health impacts of toxic Pseudo-nitzschia blooms could be greatly exacerbated by future ocean acidification and ‘carbon fertilization’ of the coastal ocean.

SHORT- VERSUS LONG-TERM RESPONSES TO CHANGING CO2 IN A COASTAL DINOFLAGELLATE BLOOM: IMPLICATIONS FOR INTERSPECIFIC COMPETITIVE INTERACTIONS AND COMMUNITY STRUCTURE

Increasing pCO2 (partial pressure of CO2) in an “acidified” ocean will affect phytoplankton community structure, but manipulation experiments with assemblages briefly acclimated to simulated future conditions may not accurately predict the long‐term evolutionary shifts that could affect inter‐specific competitive success. We assessed community structure changes in a natural mixed dinoflagellate bloom incubated at three pCO2 levels (230, 433, and 765 ppm) in a short‐term experiment (2 weeks). The four dominant species were then isolated from each treatment into clonal cultures, and maintained at all three pCO2 levels for approximately 1 year. Periodically (4, 8, and 12 months), these pCO2‐conditioned clones were recombined into artificial communities, and allowed to compete at their conditioning pCO2 level or at higher and lower levels. The dominant species in these artificial communities of CO2‐conditioned clones differed from those in the original short‐term experiment, but individual species relative abundance trends across pCO2 treatments were often similar. Specific growth rates showed no strong evidence for fitness increases attributable to conditioning pCO2 level. Although pCO2 significantly structured our experimental communities, conditioning time and biotic interactions like mixotrophy also had major roles in determining competitive outcomes. New methods of carrying out extended mixed species experiments are needed to accurately predict future long‐term phytoplankton community responses to changing pCO2.

Structure and relative potency of several karlotoxins from Karlodinium veneficum.

The karlotoxins are a family of amphidinol-like compounds that play roles in avoiding predation and in prey capture for the toxic dinoflagellate Karlodinium veneficum. The first member of the toxin group to be reported was KmTx 1 (1), and here we report an additional five new members of this family (3-7) from the same strain. Of these additional compounds, KmTx 3 (3) differs from KmTx 1 (1) in having one less methylene group in the saturated portion of its lipophilic arm. In addition, 64-E-chloro-KmTx 3 (4) and 10-O-sulfo-KmTx 3 (5) were identified. Likewise, 65-E-chloro-KmTx 1 (6) and 10-O-sulfo-KmTx 1 (7) were also isolated. Comparison of the hemolytic activities of the newly isolated compounds to that of KmTx 1 shows that potency correlates positively with the length of the lipophilic arm and is disrupted by sulfonation of the polyol arm.

Long-Term Conditioning to Elevated pCO2 and Warming Influences the Fatty and Amino Acid Composition of the Diatom Cylindrotheca fusiformis

The unabated rise in anthropogenic CO₂ emissions is predicted to strongly influence the ocean’s environment, increasing the mean sea-surface temperature by 4°C and causing a pH decline of 0.3 units by the year 2100. These changes are likely to affect the nutritional value of marine food sources since temperature and CO₂ can influence the fatty (FA) and amino acid (AA) composition of marine primary producers. Here, essential amino (EA) and polyunsaturated fatty (PUFA) acids are of particular importance due to their nutritional value to higher trophic levels. In order to determine the interactive effects of CO₂ and temperature on the nutritional quality of a primary producer, we analyzed the relative PUFA and EA composition of the diatom Cylindrotheca fusiformis cultured under a factorial matrix of 2 temperatures (14 and 19°C) and 3 partial pressures of CO₂ (180, 380, 750 μatm) for >250 generations. Our results show a decay of ~3% and ~6% in PUFA and EA content in algae kept at a pCO₂ of 750 μatm (high) compared to the 380 μatm (intermediate) CO₂ treatments at 14°C. Cultures kept at 19°C displayed a ~3% lower PUFA content under high compared to intermediate pCO₂, while EA did not show differences between treatments. Algae grown at a pCO₂ of 180 μatm (low) had a lower PUFA and AA content in relation to those at intermediate and high CO₂ levels at 14°C, but there were no differences in EA at 19°C for any CO₂ treatment. This study is the first to report adverse effects of warming and acidification on the EA of a primary producer, and corroborates previous observations of negative effects of these stressors on PUFA. Considering that only ~20% of essential biomolecules such as PUFA (and possibly EA) are incorporated into new biomass at the next trophic level, the potential impacts of adverse effects of ocean warming and acidification at the base of the food web may be amplified towards higher trophic levels, which rely on them as source of essential biomolecules.

Microcystin Prevalence throughout Lentic Waterbodies in Coastal Southern California

Toxin producing cyanobacterial blooms have increased globally in recent decades in both frequency and intensity. Despite the recognition of this growing risk, the extent and magnitude of cyanobacterial blooms and cyanotoxin prevalence is poorly characterized in the heavily populated region of southern California. Recent assessments of lentic waterbodies (depressional wetlands, lakes, reservoirs and coastal lagoons) determined the prevalence of microcystins and, in some cases, additional cyanotoxins. Microcystins were present in all waterbody types surveyed although toxin concentrations were generally low across most habitats, as only a small number of sites exceeded California’s recreational health thresholds for acute toxicity. Results from passive samplers (Solid Phase Adsorption Toxin Tracking (SPATT)) indicated microcystins were prevalent throughout lentic waterbodies and that traditional discrete samples underestimated the presence of microcystins. Multiple cyanotoxins were detected simultaneously in some systems, indicating multiple stressors, the risk of which is uncertain since health thresholds are based on exposures to single toxins. Anatoxin-a was detected for the first time from lakes in southern California. The persistence of detectable microcystins across years and seasons indicates a low-level, chronic risk through both direct and indirect exposure. The influence of toxic cyanobacterial blooms is a more complex stressor than presently recognized and should be included in water quality monitoring programs.

Single-cell transcriptomics of small microbial eukaryotes: limitations and potential

Single-cell transcriptomics is an emerging research tool that has huge untapped potential in the study of microbial eukaryotes. Its application has been tested in microbial eukaryotes 50 μm or larger, and it generated transcriptomes similar to those obtained from culture-based RNA-seq. However, microbial eukaryotes have a wide range of sizes and can be as small as 1 μm. Single-cell RNA-seq was tested in two smaller protists (8 and 15 μm). Transcript recovery rate was much lower and randomness in observed gene expression levels was much higher in single-cell transcriptomes than those derived from bulk cultures of cells. We found that the reason of such observation is that the smaller organisms had much lower mRNA copy numbers. We discuss the application of single-cell RNA-seq in studying smaller microbial eukaryotes in the context of these limitations.

Effect of light and prey availability on gene expression of the mixotrophic chrysophyte, Ochromonas sp.

BackgroundOchromonas is a genus of mixotrophic chrysophytes that is found ubiquitously in many aquatic environments. Species in this genus can be important consumers of bacteria but vary in their ability to perform photosynthesis. We studied the effect of light and bacteria on growth and gene expression of a predominantly phagotrophic Ochromonas species. Axenic cultures of Ochromonas sp. were fed with heat-killed bacteria (HKB) and grown in constant light or darkness. RNA was extracted from cultures in the light or in the dark with HKB present (Light + HKB; Dark + HKB), and in the light after HKB were depleted (Light + depleted HKB).ResultsThere were no significant differences in the growth or bacterial ingestion rates between algae grown in light or dark conditions. The availability of light led to a differential expression of only 8% of genes in the transcriptome. A number of genes associated with photosynthesis, phagotrophy, and tetrapyrrole synthesis was upregulated in the Light + HKB treatment compared to Dark + HKB. Conversely, the comparison between the Light + HKB and Light + depleted HKB treatments revealed that the presence of HKB led to differential expression of 59% of genes, including the majority of genes involved in major carbon and nitrogen metabolic pathways. Genes coding for unidirectional enzymes for the utilization of glucose were upregulated in the presence of HKB, implying increased glycolytic activities during phagotrophy. Algae without HKB upregulated their expression of genes coding for ammonium transporters, implying uptake of inorganic nitrogen from the culture medium when prey were unavailable.ConclusionsTranscriptomic results agreed with previous observations that light had minimal effect on the population growth of Ochromonas sp. However, light led to the upregulation of a number of phototrophy- and phagotrophy-related genes, while the availability of bacterial prey led to prominent changes in major carbon and nitrogen metabolic pathways. Our study demonstrated the potential of transcriptomic approaches to improve our understanding of the trophic physiologies of complex mixotrophs, and revealed responses in Ochromonas sp. not apparent from traditional culture studies.

Multiple Stressors at the Land-Sea Interface: Cyanotoxins at the Land-Sea Interface in the Southern California Bight

Blooms of toxic cyanobacteria in freshwater ecosystems have received considerable attention in recent years, but their occurrence and potential importance at the land-sea interface has not been widely recognized. Here we present the results of a survey of discrete samples conducted in more than fifty brackish water sites along the coastline of southern California. Our objectives were to characterize cyanobacterial community composition and determine if specific groups of cyanotoxins (anatoxins, cylindrospermopsins, microcystins, nodularins, and saxitoxins) were present. We report the identification of numerous potentially harmful taxa and the co-occurrence of multiple toxins, previously undocumented, at several locations. Our findings reveal a potential health concern based on the range of organisms present and the widespread prevalence of recognized toxic compounds. Our results raise concerns for recreation, harvesting of finfish and shellfish, and wildlife and desalination operations, highlighting the need for assessments and implementation of monitoring programs. Such programs appear to be particularly necessary in regions susceptible to urban influence.

Understanding the blob bloom: Warming increases toxicity and abundance of the harmful bloom diatom Pseudo-nitzschia in California coastal waters

The toxic diatom genus Pseudo-nitzschia produces environmentally damaging harmful algal blooms (HABs) along the U.S. west coast and elsewhere, and a recent ocean warming event coincided with toxic blooms of record extent. This study examined the effects of temperature on growth, domoic acid toxin production, and competitive dominance of two Pseudo-nitzschia species from Southern California. Growth rates of cultured P. australis were maximal at 23°C (∼0.8d-1), similar to the maximum temperature recorded during the 2014-2015 warming anomaly, and decreased to ∼0.1 d-1 by 30°C. In contrast, cellular domoic acid concentrations only became detectable at 23°C, and increased to maximum levels at 30°C. In two incubation experiments using natural Southern California phytoplankton communities, warming also increased the relative abundance of another potentially toxic local species, P. delicatissima. These results suggest that both the toxicity and the competitive success of particular Pseudo-nitzschia spp. can be positively correlated with temperature, and therefore there is a need to determine whether harmful blooms of this diatom genus may be increasingly prevalent in a warmer future coastal ocean.