Understanding the risks of co-exposures in a changing world: a case study of dual monitoring of the biotoxin domoic acid and Vibrio spp. in Pacific oyster.

Assessing the co-occurrence of multiple health risk factors in coastal ecosystems is challenging due to the complexity of multi-factor interactions and limited availability of simultaneously collected data. Understanding co-occurrence is particularly important for risk factors that may be associated with, or occur in similar environmental conditions. In marine ecosystems, the co-occurrence of harmful algal bloom toxins and bacterial pathogens within the genus Vibrio may impact both ecosystem and human health. This study examined the co-occurrence of Vibrio spp. and domoic acid (DA) produced by the harmful algae Pseudo-nitzschia by (1) analyzing existing California Department of Public Health monitoring data for V. parahaemolyticus and DA in oysters; and (2) conducting a 1-year seasonal monitoring of these risk factors across two Southern California embayments. Existing public health monitoring efforts in the state were robust for individual risk factors; however, it was difficult to evaluate the co-occurrence of these risk factors in oysters due to low number of co-monitoring instances between 2015 and 2020. Seasonal co-monitoring of DA and Vibrio spp. (V. vulnificus or V. parahaemolyticus) at two embayments revealed the co-occurrence of these health risk factors in 35% of sampled oysters in most seasons. Interestingly, both the overall detection frequency and co-occurrence of these risk factors were considerably less frequent in water samples. These findings may in part suggest the slow depuration of Vibrio spp. and DA in oysters as residual levels may be retained. This study expanded our understanding of the simultaneous presence of DA and Vibrio spp. in bivalves and demonstrates the feasibility of co-monitoring different risk factors from the same sample. Individual programs monitoring for different risk factors from the same sample matrix may consider combining efforts to reduce cost, streamline the process, and better understand the prevalence of co-occurring health risk factors.

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

BACKGROUND: Ochromonas 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). RESULTS: There 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. CONCLUSIONS: Transcriptomic 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.

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.

Investigating microbial eukaryotic diversity from a global census: insights from a comparison of pyrotag and full-length sequences of 18S rRNA genes.

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.

The Effect of pH and Salinity on the Toxicity and Growth of the Golden Alga, Prymnesium parvum.

Bioassays using cultures of the toxic haptophyte Prymnesium parvum and the ciliate Cyclidium sp. as prey were conducted to test the effect of pH (range = 6.5 - 8.5), salinity (range = 1.50 - 7.50‰), and a combination of pH and salinity on the toxicity of P. parvum. pH had a significant effect on P. parvum toxicity. Toxicity was rapidly (within 24 hr) induced by increasing pH of the medium, or reduced by lowering pH. Conversely, lowering salinity reduced toxicity, albeit less effectively compared to pH, and P. parvum cells remained toxic at the lowest values tested (1.50‰ at pH 7.5). An additional effect between pH and salinity was also observed: low salinity combined with low pH led to not only decreased toxicity, but also resulted in lower P. parvum growth rates. Such effects of pH and salinity on P. parvum growth and toxicity provide insight into the environmental factors supporting community dominance and toxic blooms of the alga.

Co-occurring dissolved algal toxins observed at multiple coastal sites in southern California via solid phase adsorption toxin tracking.

Algal toxins (domoic acid, saxitoxin, okadaic acid) were monitored at seven locations off southern California using Solid Phase Adsorption Toxin Tracking. At least two types of toxins were found at all locations, with co-occurrence of two and three toxins in 12% and 10% of samples, respectively. This study expands our limited understanding of the simultaneous presence of multiple algal toxins along the coast and raises questions regarding the potential health ramifications of such co-occurrences.

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.

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.

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.

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

BACKGROUND: Predatory 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. RESULTS: P. 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. CONCLUSIONS: The 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.

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.