The potent neurotoxin domoic acid is produced by some species of diatoms in the genus Pseudo-nitzschia, which can lead to amnesic shellfish poisoning (ASP) events in marine food webs. Of the toxin-producing Pseudo-nitzschia, P. australis has caused the most toxic and widespread events, making it a species of concern. Toxic and non-toxic Pseudo-nitzschia species cannot be distinguished easily by light microscopy, necessitating molecular testing. We designed a real-time quantitative PCR assay for rapid and accurate detection of P. australis in environmental DNA (eDNA) samples, targeting the intergenic region between rbcS and psaI in the chloroplast genome, providing species-level specificity and high sensitivity. Experimental determination of rbcS gene copies per cell revealed a limit of detection equivalent to 1 P. australis cell per liter of seawater, facilitating the early detection of this toxic species. This novel qPCR assay was applied to eDNA samples collected from Maine coastal waters between 2021 - 2024 to characterize the nearshore dynamics of P. australis within a regional hub for shellfish aquaculture. Though relatively rare, P. australis peaked in abundance in mid-October when conditions became favorable for this species following convective overturning. The eDNA assay was able to detect a low P. australis signal at a time when 'large' Pseudo-nitzschia spp. cell counts approached 8000 cells l-1 (by microscopy) while qPCR-based estimates of P. australis remained below 10 cells l-1. We demonstrate an eDNA approach for the early detection of HABs, calibration of qPCR assays to cell counts, and provide a new tool for coastal management.

Echinoderm mass mortality events shape marine ecosystems by altering the dynamics among major benthic groups. The sea urchin Diadema antillarum, virtually extirpated in the Caribbean in the early 1980s by an unknown cause, recently experienced another mass mortality beginning in January 2022. We investigated the cause of this mass mortality event through combined molecular biological and veterinary pathologic approaches comparing grossly normal and abnormal animals collected from 23 sites, representing locations that were either affected or unaffected at the time of sampling. Here, we report that a scuticociliate most similar to Philaster apodigitiformis was consistently associated with abnormal urchins at affected sites but was absent from unaffected sites. Experimentally challenging naïve urchins with a Philaster culture isolated from an abnormal, field-collected specimen resulted in gross signs consistent with those of the mortality event. The same ciliate was recovered from treated specimens postmortem, thus fulfilling Koch's postulates for this microorganism. We term this condition D. antillarum scuticociliatosis.

This chapter presents an overview of quantitative real-time PCR (qPCR), with a focus on harmful algae blooms (HABs). It includes an overview of the technology and describes the early development of qPCR assays to detect cryptic species. A review of multispecies and ecotype- or strain-level detection approaches by qPCR is provided. This is followed by examples of qPCR to investigate factors impacting phytoplankton distributions and activities. Approaches to evaluate changes in gene expression are then presented, along with a case study investigating species-specific responses to nitrogen input. Recent advancements in qPCR are discussed, with a case study demonstrating the use of portable qPCR and a comparison to lab-based instruments, followed by a description of droplet digital PCR (ddPCR), a novel advancement in technology that resolves some key issues in the use of qPCR for environmental research and monitoring. The chapter ends with a discussion of efforts to adapt qPCR for community-based science programs, with the potential to enhance our understanding of phytoplankton ecology and improve predictive capabilities for HAB events.

Many pathogens can cause cancer, but cancer itself does not normally act as an infectious agent. However, transmissible cancers have been found in a few cases in nature: in Tasmanian devils, dogs, and several bivalve species. The transmissible cancers in dogs and devils are known to spread through direct physical contact, but the exact route of transmission of bivalve transmissible neoplasia (BTN) has not yet been confirmed. It has been hypothesized that cancer cells from bivalves could be released by diseased animals and spread through the water column to infect/engraft into other animals. To test the feasibility of this proposed mechanism of transmission, we tested the ability of BTN cells from the soft-shell clam (Mya arenaria BTN, or MarBTN) to survive in artificial seawater. We found that MarBTN cells are highly sensitive to salinity, with acute toxicity at salinity levels lower than those found in the native marine environment. BTN cells also survive longer at lower temperatures, with 50% of cells surviving greater than 12 days in seawater at 10 °C, and more than 19 days at 4 °C. With one clam donor, living cells were observed for more than eight weeks at 4 °C. We also used qPCR of environmental DNA (eDNA) to detect the presence of MarBTN-specific DNA in the environment. We observed release of MarBTN-specific DNA into the water of laboratory aquaria containing highly MarBTN-diseased clams, and we detected MarBTN-specific DNA in seawater samples collected from MarBTN-endemic areas in Maine, although the copy numbers detected in environmental samples were much lower than those found in aquaria. Overall, these data show that MarBTN cells can survive well in seawater, and they are released into the water by diseased animals. These findings support the hypothesis that BTN is spread from animal-to-animal by free cells through seawater.

Ocean ecosystems are changing, and the climate envelope paradigm predicts a steady shift, approximately poleward, of species ranges. The Gulf of Maine presents a test case of this paradigm, as temperatures have warmed extremely rapidly. Some species have shifted northeastward, matching predictions. Others-namely harmful algal species like Pseudo-nitzschia australis and Karenia mikimotoi-do not appear to have followed climate trajectories, arriving as surprises in the Gulf of Maine. Rare-biosphere dynamics offer one possible ecological lens for understanding and predicting this type of surprise. Rare species in the plankton, possibly more so than southerly ones, may provide management challenges in the future. Improved monitoring and broader coordination of monitoring of the rare biosphere could help develop early warning systems for harmful and toxic algae. A better theoretical understanding of rare biosphere dynamics is also needed. A challenge for the next cohort of ecosystem projections is to predict the newly emerging harmful species of the type that catch us by surprise.

Eastern oyster (Crassostrea virginica) aquaculture is increasingly playing a significant role in the state of Maine’s (USA) coastal economy. Here, we conducted a qPCR-based survey for Haplosporidium nelsoni, Perkinsus marinus, and Perkinsus chesapeaki in C. virginica (n = 1440) from six Maine sites during the summer–fall of 2016 and 2017. In the absence of reported die-offs, our results indicated the continued presence of the three protozoan parasites in the six sites. The highest H. nelsoni qPCR-prevalence corresponded to Jack’s Point and Prentiss Island ( x ¯ = 40 and 48% respectively), both located in the Damariscotta River Estuary. Jack’s Point, Prentiss Island, New Meadows River, and Weskeag River recorded the highest qPCR-prevalence for P. marinus (32–39%). While the P. marinus qPCR-prevalence differed slightly for the years 2016 and 2017, P. chesapeaki qPCR-prevalence in 2016 was markedly lower than 2017 (<20% at all sites versus >60% at all sites for each of the years, respectively). Mean qPCR-prevalence values for P. chesapeaki over the two-year study were ≥40% for samples from Jack’s Point (49%), Prentiss Island (44%), and New Meadows River (40%). This study highlights that large and sustained surveys for parasitic diseases are fundamental for decision making toward the management of the shellfish aquaculture industry, especially for having a baseline in the case that die-offs occur.

Microorganisms and burrowing animals exert a pronounced impact on the cycling of redox sensitive metals in coastal sediments. We hypothesized that the iron biogeochemical cycle and associated sedimentary microbial community will respond to seasonal changes in a bioturbated intertidal mudflat. In this study, we monitored the spatiotemporal dynamics of porewater and highly reactive solid phase iron with the corresponding prokaryotic and eukaryotic sedimentary microbial communities over one annual cycle from November 2015 to November 2016. Continuous and seasonally variable pools of both porewater Fe(II) and highly reactive iron (FeHR) were observed throughout the seasons with significant increases of Fe(II) and FeHR in response to increased sediment temperature in summer months. Maximum concentrations of Fe(II) and FeHR were predominantly confined to the upper 5 cm of sediment throughout the year. Iron-oxidizing and-reducing microorganisms were present and stable temporally, and exhibited a depth-dependent stratification likely due to availability of Fe(II) and FeHR pools, respectively. Zetaproteobacteria, presumptive lithotrophic iron-oxidizing bacteria, were present at abundances around 0.5-1% in the top 5 cm of sediment with decline abundance with depth. As a whole the microbial community was relatively stable across the seasons, and showed strongest separation with depth, probably driven by changes in oxygen availability and organic matter. The Deltaproteobacteria, principally taxa known to be associated with sulfur and iron cycling, were prevalent, especially at >5 cm depth. Gammaproteobacteria and Bacteroidetes were also abundant, with putatively aerobic members especially prevalent in the cm of the sediment. The relative abundance of diatoms, estimated from abundance of 18S rRNA gene counts, showed evidence of a seasonal signal possibly tied to spring and fall blooms. Overall, analysis of phytoplankton found significant abundance at depth, likely due to the feeding and bio-mixing activity of marine worms. Macro-, and meiofauna, consistent with expected taxa, were detected throughout the year via 18S gene counts, and showed some seasonal variations that may influence sedimentary iron transformations by active microbial grazing. In summary, this analysis revealed relatively consistent temporal and spatial trends in iron geochemistry and microbial and macrobial community composition, while also indicating a complex dynamic of microbial and macrobial interactions are responsible for maintaining these processes.

Microbial eukaryotes have important roles in marine food webs, but their diversity and activities in hydrothermal vent ecosystems are poorly characterized. In this study, we analyzed microbial eukaryotic communities associated with bacterial (Beggiatoa) mats in the 2,000 m deep-sea Guaymas Basin hydrothermal vent system using 18S rRNA gene high-throughput sequencing of the V4 region. We detected 6,954 distinct Operational Taxonomic Units (OTUs) across various mat systems. Of the sequences that aligned with known protistan phylotypes, most were affiliated with alveolates (especially dinoflagellates and ciliates) and cercozoans. OTU richness and community structure differed among sediment habitats (e.g. different mat types and cold sediments away from mats). Additionally, full-length 18S rRNA genes amplified and cloned from single cells revealed the identities of some of the most commonly encountered, active ciliates in this hydrothermal vent ecosystem. Observations and experiments were also conducted to demonstrate that ciliates were trophically active and ingesting fluorescent bacteria or Beggiatoa trichomes. Our work suggests that the active and diverse protistan community at the Guaymas Basin hydrothermal vent ecosystem likely consumes substantial amounts of bacterial biomass, and that the different habitats, often defined by distances of just a few 10s of cm, select for particular assemblages and levels of diversity.

Shellfish are known as a potential source of Toxoplasma gondii (responsible for toxoplasmosis), and Cryptosporidium parvum, which is one of the major causes of gastroenteritis in the world. Here we performed a comprehensive qPCR-based monthly survey for T. gondii and C. parvum during 2016 and 2017 in oysters (Crassostrea virginica) (n = 1440) from all six sites along the coast of Maine (USA). Pooled samples (mantle, gills, and rectum) from individual oysters were used for DNA extraction and qPCR. Our study resulted in detections of qPCR positives oysters for T. gondii and C. parvum at each of the six sites sampled (in 31% and 10% of total oysters, respectively). The prevalence of T. gondii was low in 2016, and in September 2017 several sites peaked in prevalence with 100% of the samples testing positive. The prevalence of C. parvum was very low except in one estuarine location (Jack's Point) in June 2016 (58%), and in October of 2016, when both prevalence and density of C. parvum at most of the sampling sites were among the highest values detected. Statistical analysis of environmental data did not identify clear drivers of retention, but there were some notable statistically significant patterns including current direction and nitrate along with the T. gondii prevalence. The major C. parvum retention event (in October 2016) corresponded with the month of highest dissolved oxygen measurements as well as a shift in the current direction revealed by nearby instrumentation. This study may guide future research to locate any contributing parasite reservoirs and evaluate the potential risk to human consumption.

Broadcast spawners release their gametes into the water column for ‘chance’ fertilization events. However, detection of such events in near real-time is extremely difficult, but needed to improve fisheries and conservation management practices. It is practically impossible to distinguish the gametes of many species by microscopy; therefore, DNA-based techniques are preferable to detect and quantify gametes from field-collected plankton samples. We developed a quantitative PCR (qPCR) approach to detect and quantify broadcast spawning events in marine environmental DNA (eDNA) samples. We applied this approach to a commercially valuable broadcast spawning bivalve species, the sea scallop Placopecten magellanicus. Our approach includes (1) sequencing the internal transcribed spacer (ITS) region, (2) developing a novel species-specific probe and primer set, (3) testing the probe and primer set on a dilution series of sea scallop sperm to quantify the relationship between gamete abundance and DNA copy number, and (4) conducting dockside field tests of our method on plankton samples adjacent to naturally spawning sea scallops. Quantitative PCR revealed a clear relationship between DNA copy number and P. magellanicus sperm cell abundance, indicating that this method is reliable for detecting sperm release by male scallops during spawning events. Plankton samples collected during the scallop spawning season revealed spikes of scallop eDNA in both the <20 μm (sperm) and >20 μm (possible eggs) particle size-fractions. This method holds great potential to provide more efficient estimates of the timing, magnitude, and spatial scale of reproductive events than conventional methods for a wide range of broadcast spawners.

High-throughput sequencing of ocean biomes has revealed vast eukaryotic microbial diversity, a significant proportion of which remains uncharacterized. Here we use a temporal approach to understanding eukaryotic diversity at the Scripps Pier, La Jolla, California, USA, via high-throughput amplicon sequencing of the 18S rRNA gene, the abundances of both Synechococcus and Synechococcus grazers, and traditional oceanographic parameters. We also exploit our ability to track operational taxonomic units (OTUs) temporally to evaluate the ability of 18S sequence-based OTU assignments to meaningfully reflect ecological dynamics. The eukaryotic community is highly dynamic in terms of both species richness and composition, although proportional representation of higher-order taxa remains fairly consistent over time. Synechococcus abundance fluctuates throughout the year. OTUs unique to dates of Synechococcus blooms and crashes or enriched in Synechococcus addition incubation experiments suggest that the prasinophyte Tetraselmis sp. and Gymnodinium-like dinoflagellates are likely Synechococcus grazers under certain conditions, and may play an important role in their population fluctuations.

Long-term time series data show that ocean acidification is occurring in the subtropical oceans. As a component of an in situ mesocosm experiment carried out off Gran Canaria in the subtropical North Atlantic, we examined the influence of ocean acidification on the net production of dimethylsulfide (DMS). Over 23 days under oligotrophic conditions, time-integrated DMS concentrations showed an inverse relationship of −0.21 ± 0.02 nmol DMS nmol−1 H+ across the gradient of H+ concentration of 8.8–23.3 nmol l−1, equivalent to a range of pCO2 of 400–1,252 atm. Proportionally similar decreases in the concentrations of both dissolved and particulate dimethylsulfoniopropionate (DMSP) were observed in relation to increasing H+ concentration between the mesocosms. The reduced net production of DMSP with increased acidity appeared to result from a decrease in abundance of a DMSP-rich nanophytoplankton population. A 35S-DMSP tracer approach was used to determine rates of dissolved DMSP catabolism, including DMS production, across the mesocosm treatments. Over a phase of increasing DMS concentrations during the experiment, the specific rates of DMS production were significantly reduced at elevated H+ concentration. These rates were closely correlated to the rates of net DMS production indicating that transformation of dissolved DMSP to DMS by bacteria was a major component of DMS production. It was not possible to resolve whether catabolism of DMSP was directly influenced by H+ concentrations or was an indirect response in the bacterial community composition associated with reduced DMSP availability. There is a pressing need to understand how subtropical planktonic communities respond to the predicted gradual prolonged ocean acidification, as alterations in the emission of DMS from the vast subtropical oceans could influence atmospheric chemistry and potentially climate, over a large proportion of the Earth's surface.

Shellfish aquaculture (in particular oyster cultivation) has the potential to play a significant role in refreshing the coastal economy in the state of Maine (USA). Although ocean warming and acidification are often listed as the primary manifestations of climate change in the marine environment, the issue of expanding geographic ranges of parasites and pathogens is an equally serious threat to shellfish populations. Protozoan parasites of the genera Bonamia, Haplosporidium, and Perkinsus, the bacterial pathogen responsible for Roseovarius oyster disease, and the disease condition is known as disseminated neoplasia are currently recognized as some of the significant threats to natural and farmed bivalve populations. We have analyzed the peer-reviewed literature for reports of these pathogens/conditions in Maine. Most reports focus on oysters from the Damariscotta River Estuary and are the result of directed studies into the biology of a particular pathogen rather than the result of intensive monitoring programs. The sampling effort could be interpreted in several ways including; the pathogens/conditions not impacting the annual harvest to any great extent due to limited distribution of the causative agent, the oysters developing a tolerance to the diseases, or just a lack of resources directed at studying this topic. With the shellfish aquaculture industry expected to grow in the next several decades, we recommend that a rigorous and sustained survey of parasitic diseases and believe that such an effort is fundamental to the success, resilience, and well-being of Maine's shellfish aquaculture industry.

Biogeochemistry in marine plankton communities is strongly influenced by the activities of microbial species. Understanding the composition and dynamics of these assemblages is essential for modeling emergent community-level processes, yet few studies have examined all of the biological assemblages present in the plankton, and benchmark data of this sort from time-series studies are rare. Abundance and biomass of the entire microbial assemblage and mesozooplankton ( > 200 mu m) were determined vertically, monthly and seasonally over a 3-year period at a coastal time-series station in the San Pedro Basin off the southwestern coast of the USA. All compartments of the planktonic community were enumerated (viruses in the femtoplankton size range [0.02-0.2 mu m], bacteria + archaea and cyanobacteria in the picoplankton size range [0.2-2.0 mu m], phototrophic and heterotrophic protists in the nanoplanktonic [2-20 mu m] and microplanktonic [20-200 mu m] size ranges, and mesozooplankton [ > 200 mu m]. Carbon biomass of each category was estimated using standard conversion factors. Plankton abundances varied over seven orders of magnitude across all categories, and total carbon biomass averaged approximately 60 mu g C l(-1) in surface waters of the 890 m water column over the study period. Bacteria + archaea comprised the single largest component of biomass ( > 1/3 of the total), with the sum of phototrophic protistan biomass making up a similar proportion. Temporal variability at this subtropical station was not dramatic. Monthly depth-specific and depth-integrated biomass varied 2-fold at the station, while seasonal variances were generally < 50%. This study provides benchmark information for investigating long-term environmental forcing on the composition and dynamics of the microbes that dominate food web structure and function at this coastal observatory.

The Dirichlet-multinomial mixture model (DMM) and its extensions provide powerful new tools for interpreting the ecological dynamics underlying taxon abundance data. However, like many complex models, how effectively they capture the many features of empirical data is not well understood. In this work, we expand the DMM to an infinite mixture model (iDMM) and use posterior predictive distributions (PPDs) to explore the performance in three case studies, including two amplicon metagenomic time series. We avoid concentrating on fluctuations within individual taxa and instead focus on consortial-level dynamics, using straight-forward methods for visualizing this perspective. In each study, the iDMM appears to perform well in organizing the data as a framework for biological interpretation. Using the PPDs, we also observe several exceptions where the data appear to significantly depart from the model in ways that give useful ecological insight. We summarize the conclusions as a set of considerations for field researchers: problems with samples and taxa; relevant scales of ecological fluctuation; additional niches as outgroups; and possible violations of niche neutrality.

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.

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.