Alle A. Y. Lie

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.

Investigating Microbial Eukaryotic Diversity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of 18S rRNA Genes

ABSTRACT Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.

Changes in the nutrient ratios and phytoplankton community after declines in nutrient concentrations in a semi-enclosed bay in Hong Kong.

Tolo Harbour is a landlocked bay with poor tidal flushings in the northeastern part of Hong Kong. During the 1980s, excessive nutrient loading led to dramatic increase in nutrient concentrations, accompanied by lower N:P ratios, higher algal biomass and shifts in the phytoplankton community. We studied the effects of nutrient loading reduction measures on nutrient concentrations, nutrient ratios and phytoplankton dynamics in Tolo Harbour by comparing data collected before the full implementation of nutrient loading reduction measures (1986-1997) to those after the implementation (1998-2008). Such measures led to declines in nutrient concentrations, changes in N:P and N:Si ratios, lower chlorophyll-a concentrations and fewer algal blooms. Diatoms were the most abundant phytoplankton group in Tolo Harbour both before and after declines in nutrient concentrations. The density of dinoflagellates did not change, but substantial increase in other algal group abundance was recorded.

Estimating Protistan Diversity Using High‐Throughput Sequencing

Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full‐length sequences. A total of 7,432 full‐length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full‐length 18S rRNA gene sequences. Present high‐throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.

Autotrophic and heterotrophic acquisition of carbon and nitrogen by a mixotrophic chrysophyte established through stable isotope analysis

Collectively, phagotrophic algae (mixotrophs) form a functional continuum of nutritional modes between autotrophy and heterotrophy, but the specific physiological benefits of mixotrophic nutrition differ among taxa. Ochromonas spp. are ubiquitous chrysophytes that exhibit high nutritional flexibility, although most species generally fall towards the heterotrophic end of the mixotrophy spectrum. We assessed the sources of carbon and nitrogen in Ochromonas sp. strain BG-1 growing mixotrophically via short-term stable isotope probing. An axenic culture was grown in the presence of either heat-killed bacteria enriched with 15N and 13C, or unlabeled heat-killed bacteria and labeled inorganic substrates (13C-bicarbonate and 15N-ammonium). The alga exhibited high growth rates (up to 2 divisions per day) only until heat-killed bacteria were depleted. NanoSIMS and bulk IRMS isotope analyses revealed that Ochromonas obtained 84–99% of its carbon and 88–95% of its nitrogen from consumed bacteria. The chrysophyte assimilated inorganic 13C-carbon and 15N-nitrogen when bacterial abundances were very low, but autotrophic (photosynthetic) activity was insufficient to support net population growth of the alga. Our use of nanoSIMS represents its first application towards the study of a mixotrophic alga, enabling a better understanding and quantitative assessment of carbon and nutrient acquisition by this species.

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.

Size structure and pigment composition of phytoplankton communities in different hydrographic zones in Hong Kong's coastal seas

The abundance and community composition of phytoplankton are influenced by a suite of interacting environmental factors. Hong Kongs marine environment features a hydrographic gradient from an estuarine zone in the west to a transition zone in the middle and an oceanic zone in the east. Size fractionation combined with high performance liquid chromatography (HPLC) pigment analyses were used to investigate the phytoplankton communities in different hydrographic zones during summer (July–August 2009) and winter (December 2009–January 2010). Clear temporal and spatial variations in environmental parameters occurred among hydrographic zones. Results of principal component analysis (PCA) revealed that the major deviating factors among hydrographic zones were turbidity and salinity in summer and nitrate and phosphate in winter. Phytoplankton abundance showed significant temporal variations, but no zonal variations. Phytoplankton communities in all hydrographic zones were dominated by cells >5 µm in both summer and winter. Chlorophyll a concentrations for most size fractions correlated significantly with temperature. The high concentration of fucoxanthin indicated that the phytoplankton community was dominated by diatoms in both summer and winter, while dinoflagellates, cryptophytes, cyanobacteria and other minor groups occurred sporadically in low abundance. The spatial pattern of phytoplankton in Hong Kongs coastal seas did not reflect the hydrographic zonation, but the phytoplankton in the semi-enclosed Tolo Harbour and Deep Bay were different from those in the other zones.

A tale of two mixotrophic chrysophytes: Insights into the metabolisms of two Ochromonas species (Chrysophyceae) through a comparison of gene expression

Ochromonas spp. strains CCMP1393 and BG-1 are phagotrophic phytoflagellates with different nutritional strategies. Strain CCMP1393 is an obligate phototroph while strain BG-1 readily grows in continuous darkness in the presence of bacterial prey. Growth and gene expression of strain CCMP1393 were investigated under conditions allowing phagotrophic, mixotrophic, or phototrophic nutrition. The availability of light and bacterial prey led to the differential expression of 42% or 45–59% of all genes, respectively. Data from strain CCMP1393 were compared to those from a study conducted previously on strain BG-1, and revealed notable differences in carbon and nitrogen metabolism between the 2 congeners under similar environmental conditions. Strain BG-1 utilized bacterial carbon and amino acids through glycolysis and the tricarboxylic acid cycle, while downregulating light harvesting and carbon fixation in the Calvin cycle when both light and bacteria were available. In contrast, the upregulation of genes related to photosynthesis, light harvesting, chlorophyll synthesis, and carbon fixation in the presence of light and prey for strain CCMP1393 implied that this species is more phototrophic than strain BG-1, and that phagotrophy may have enhanced phototrophy. Cellular chlorophyll a content was also significantly higher in strain CCMP1393 supplied with bacteria compared to those without prey. Our results thus point to very different physiological strategies for mixotrophic nutrition in these closely related chrysophyte species.

The seasonal distribution, diel vertical distribution and feeding behavior of Paraeuchaeta concinna in the shallow subtropical coastal waters of eastern Hong Kong

BackgroundPredatory copepods of the family Euchaetidae are widely distributed in polar, temperate, subtropical and tropical oceans. Paraeuchaeta concinna is the most abundant Euchaetidae in the subtropical coastal seas of Hong Kong and southern China. However, compared to Euchaetidae species in temperate and polar regions, relatively little information is available on the ecology of P. concinna and other Euchaetidae species in the subtropical oceans. This paper provides information on the seasonal abundance of P. concinna in the coastal seas of eastern Hong Kong. The diel vertical distribution of P. concinna, feeding behavior, and predation impact on mesozooplankton in eastern Hong Kong were also investigated.ResultsP. concinna is most abundant in winter and spring. Their abundance decreases shoreward, and densities are generally higher in the open waters of eastern Hong Kong than in the inner parts of Mirs Bay and Tolo Harbour. P. concinna exhibits both diel vertical migration and diel feeding rhythms in Mirs Bay. P. concinna females show strong preference for the copepods of the genera Acrocalanus, Paracalanus, and Parvocalanus, and remove ~4% of their standing stocks daily.ConclusionsThe low abundance of P. concinna during most of the year suggests it is not indigenous to coastal seas of eastern Hong Kong. P. concinna performs diel vertical migration, most likely as a strategy to avoid visual predation. Gut content analysis showed that Acrocalanus, Paracalanus, and Parvocalanus are highly preferred prey of P. concinna. A daily predation impact of ~4% of the standing stocks of Acrocalanus, Paracalanus, and Parvocalanus suggests that P. concinna may play an important role in regulating the populations of these small copepods in Mirs Bay, especially during winter and spring.

Single-Cell View of Carbon and Nitrogen Acquisition in the Mixotrophic Alga Prymnesium parvum (Haptophyta) Inferred From Stable Isotope Tracers and NanoSIMS

Nutritional modes of unicellular eukaryotes range from pure photoautotrophy of some phytoplankton to pure heterotrophy of species typically called protozoa. Between these two extremes lies a functional continuum of nutrient and energy acquisition modes termed mixotrophy. Prymnesium parvum is an ecologically important mixotrophic haptophyte alga that can produce toxins and form ecosystem disruptive blooms that result in fish kills and changes in planktonic food web structure. We investigated carbon and nitrogen acquisition strategies of single cells of P. parvum using a combined experimental-imaging approach employing labeling of live cells with stable isotope tracers (13C and 15N) followed by measurement of cellular isotopic ratios using nanometer-scale secondary ion mass spectrometry (NanoSIMS). With this method, we were able to quantify the relative contributions of photosynthesis and heterotrophy to the nutrition of the alga. Our results suggest that P. parvum relies on predation primarily for nitrogen, while most carbon for cellular building blocks is obtained from inorganic sources. Our analysis further revealed that nitrogen assimilation can vary up to an order of magnitude among individual cells, a finding that would be difficult to determine using other methods. These results help to improve our understanding of mixotrophy across the enormous diversity of eukaryotes, one cell and one species at a time.