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Monitoring aquatic species by identification of environmental DNA (eDNA) is becoming more common. In order to obtain quantitative datasets for individual species, species-specific quantitative PCR (qPCR) assays are required. Here, we present detailed methodology of qPCR assay design and testing, including in silico, in vitro, and in vivo testing, and comment on the challenges associated with assay design and performance. We use the presented methodology to design assays for three important marine organisms common in the California Current Ecosystem (CCE): humpback whale (Megaptera novaeangliae), shortbelly rockfish (Sebastes jordani), and common murre (Uria aalge). All three assays have excellent sensitivity and high efficiencies ranging from 92% to 99%. However, specificities of the assays varied from species-specific in the case of common murre to the genus-specific shortbelly rockfish assay, to the humpback whale assay which cross-amplified with other two other whale species, including one in a different family. All assays detected their associated targets in complex environmental water samples.

Environmental DNA (eDNA) is the DNA suspended in the environment (e.g., water column), which includes cells, gametes, and other material derived from but not limited to shedding of tissue, scales, mucus, and fecal matter. Amplifying and sequencing marker genes (i.e., metabarcoding) from eDNA can reveal the wide range of taxa present in an ecosystem through analysis of a single water sample. Metabarcoding of eDNA provides higher resolution data than visual surveys, aiding in assessments of ecosystem health. This study conducted eDNA metabarcoding of two molecular markers (cytochrome c oxidase I (COI) and 18S ribosomal RNA (rRNA) genes) to survey eukaryotic diversity across multiple trophic levels in surface water samples collected at three sites along the coral reef tract within the Florida Keys National Marine Sanctuary (FKNMS) during four research cruises in 2015. The 18S rRNA gene sequences recovered 785 genera while the COI gene sequences recovered 115 genera, with only 33 genera shared between the two datasets, emphasizing the complementarity of these marker genes. Community composition for both genetic markers clustered by month of sample collection, suggesting that temporal variation has a larger effect on biodiversity than spatial variability in the FKNMS surface waters. Sequences from both marker genes were dominated by copepods, but each marker recovered distinct phytoplankton groups, with 18S rRNA gene sequences dominated by dinoflagellates and COI sequences dominated by coccolithophores. Although eDNA samples were collected from surface waters, many benthic species such as sponges, crustaceans, and corals were identified. These results show the utility of eDNA metabarcoding for cataloging biodiversity to establish an ecosystem baseline against which future samples can be compared in order to monitor community changes.

Theory suggests that the direct transmission of beneficial endosymbionts (mutualists) from parents to offspring (vertical transmission) in animal hosts is advantageous and evolutionarily stable, yet many host species instead acquire their symbionts from the environment (horizontal acquisition). An outstanding question in marine biology is why some scleractinian corals do not provision their eggs and larvae with the endosymbiotic dinoflagellates that are necessary for a juvenile's ultimate survival. We tested whether the acquisition of photosynthetic endosymbionts (family Symbiodiniaceae) during the planktonic larval stage was advantageous, as is widely assumed, in the ecologically important and threatened Caribbean reef-building coral Orbicella faveolata. Following larval acquisition, similar changes occurred in host energetic lipid use and gene expression regardless of whether their symbionts were photosynthesizing, suggesting the symbionts did not provide the energetic benefit characteristic of the mutualism in adults. Larvae that acquired photosymbionts isolated from conspecific adults on their natal reef exhibited a reduction in swimming, which may interfere with their ability to find suitable settlement substrate, and also a decrease in survival. Larvae exposed to two cultured algal species did not exhibit differences in survival, but decreased their swimming activity in response to one species. We conclude that acquiring photosymbionts during the larval stage confers no advantages and can in fact be disadvantageous to this coral host. The timing of symbiont acquisition appears to be a critical component of a host's life history strategy and overall reproductive fitness, and this timing itself appears to be under selective pressure.

Environmental DNA (eDNA) metabarcoding is a new approach for assessing marine biodiversity that may overcome challenges of traditional monitoring and complement both existing surveys and biodiversity assessments. There are limited eDNA studies that evaluate vertebrate biodiversity in the marine environment or compare patterns of biodiversity with traditional methods. This study uses eDNA metabarcoding of the mitochondrial 12S rRNA genes present in seawater samples to characterize vertebrate biodiversity and distribution within National Marine Sanctuaries located in the California Current upwelling ecosystem. The epipelagic community in the study region has been monitored using traditional (mid-water trawl and marine mammal) survey methods since 1983. During 2016 and 2017, we concurrently sampled the epipelagic community using traditional survey methods and water for eDNA analysis to assess agreement among the methods. We collected replicate eDNA samples from 25 stations at depths of 10, 40, and 80 m, resulting in 131 small volume (1 L) environmental water samples to examine eDNA sequences. Across the eDNA and traditional survey methods, 80 taxa were identified. Taxa identified by eDNA partially overlapped with taxa through trawl and marine mammal surveys, but more taxa were identified by eDNA. Diversity and distribution patterns of marine vertebrates inferred from eDNA sequences reflected known spatial distribution patterns in species occurrence and community structure (e.g., cross-shelf and alongshore patterns). During both years, we identified fishery taxa Sebastes (rockfish), Merluccius (hake), Citharichthys (sanddab), and Engraulis (anchovy) across the majority of the stations using eDNA metabarcoding. The marine vertebrate assemblage identified by eDNA in 2016 was statistically different from the 2017 assemblage and more marine mammals were identified in 2017 than in 2016. Differences in assemblages identified by eDNA were coincident with different oceanographic conditions (e.g., upwelling and stratification). In 2016, weak upwelling and warmer than average conditions were measured, and vertebrate assemblages were not different among ecological regions [Point Reyes, Pescadero, and Monterey Bay]. While in 2017, average upwelling conditions returned, vertebrate assemblages differed at each region. This study illustrates that eDNA provides a new baseline for vertebrate assessments that can both augment traditional biomonitoring surveys and aid our understanding of changes in biodiversity.

Environmental DNA (eDNA) analysis allows the simultaneous examination of organisms across multiple trophic levels and domains of life, providing critical information about the complex biotic interactions related to ecosystem change. Here we used multilocus amplicon sequencing of eDNA to survey biodiversity from an eighteen-month (2015–2016) time-series of seawater samples from Monterey Bay, California. The resulting dataset encompasses 663 taxonomic groups (at Family or higher taxonomic rank) ranging from microorganisms to mammals. We inferred changes in the composition of communities, revealing putative interactions among taxa and identifying correlations between these communities and environmental properties over time. Community network analysis provided evidence of expected predator-prey relationships, trophic linkages, and seasonal shifts across all domains of life. We conclude that eDNA-based analyses can provide detailed information about marine ecosystem dynamics and identify sensitive biological indicators that can suggest ecosystem changes and inform conservation strategies., Increasingly, eDNA is being used to infer ecological interactions. Here the authors sample eDNA over 18 months in a marine environment and use co-occurrence network analyses to infer potential interactions among organisms from microbes to mammals, testing how they change over time in response to oceanographic factors.

Metabarcoding of marine environmental DNA (eDNA), the DNA suspended in seawater originating from tissue, cells, or extracellular DNA, offers the opportunity to survey the biological composition of communities across multiple trophic levels from a non-invasive seawater sample. Here we compare results of eDNA metabarcoding of multiple trophic levels from the same seawater samples collected from a kelp forest in Monterey Bay, California in order to establish methods for future cross-trophic level eDNA analysis. Triplicate 1 L water samples were filtered using five different 47 mm diameter membrane filters (PVDF, PES, GFF, PCTE, and NC) and DNA was extracted from triplicates of each filter-type using three widely-used extraction methods (the DNeasy Blood and Tissue kit, the MoBio PowerWater DNA Isolation kit, and standard phenol/chloroform methods) resulting in 45 individual eDNA samples prepared with 15 workflow combinations. Each DNA extract was amplified using PCR primers for the 16S rRNA gene (microorganisms; Bacteria and Archaea), 18S rRNA gene (phytoplankton), and the 12S rRNA gene (vertebrates) genes, and PCR products were sequenced on an Illumina MiSeq platform. The richness and community composition of microbial, phytoplankton, and vertebrate OTUs were not significantly different between any of the 0.2 μm pore-size filter types extracted with the DNeasy or MoBio kits. However, phenol/chloroform extraction resulted in significantly different community structures. This study provides insight into multiple choices for extraction and filtration methods to use eDNA metabarcoding for biodiversity assessment of multiple trophic levels from a single sample. We recommend any combination of either DNeasy or MoBio with PES, PCTE, PVDF, or NC filters for a cross trophic level comparison.

Metabarcoding of environmental DNA (eDNA) samples holds new promise to increase our ability to measure changes in biodiversity and community composition over time. It can allow the characterization of large groups of organisms where traditional sampling may be impractical or not cost-effective. However, it is still unclear how best to compare and combine this information with morphological counts in order to inform policies and biodiversity metrics that are based on traditional sampling results. Under the Marine Biodiversity Observation Network (MBON) initiative, multiple taxonomic marker genes (16S rRNA, 18S rRNA, mitochondrial cytochrome c oxidase subunit I (COI), and 12S rRNA) have been used concurrently to examine the phylogenetic diversity of samples across trophic levels from microbes to vertebrates. Marker genes and their amplification primers target a different (and sometimes overlapping) group of organisms. Just as with traditional sampling methods, each have biases towards detecting certain organisms over others. Though eDNA metabarcoding often detects many more species than can be identified through microscopic or macroscopic net tow counts, processing and relating sequence data to traditional counts and biodiversity measures is an ongoing challenge. For samples collected within the MBON project, an analysis pipeline has been adapted to standardize sequence analysis of each marker gene. The pipeline processes reads from quality control and trimming through clustering of sequences into Operational Taxonomic Units (OTUs). Taxonomic identification of OTUs uses publically available sequence databases. Finally, the results of the analysis pipeline are combined into a Biological Observation Matrix (BIOM) file with metadata pertaining to the biological sample, PCR processing, and bioinformatic analysis. BIOM files can be used in downstream analysis to analyze biodiversity patterns within the samples. Monterey Bay in California, USA, is a hot spot of biodiversity and productivity fed by nutrient-rich upwelling water along the coast. A local time-series of samples has been collected by the Monterey Bay Aquarium Research Institute at coastal stations within the bay, providing several decades of contextual environmental data. Samples taken from this time series are ideal for testing the ability of eDNA sequencing to show variability in taxonomic groups over time. For metabarcoding analysis, samples were chosen representing different seasons corresponding to spring (early) upwelling, summer (late) upwelling, fall oceanic regime, and a winter (Davidson) regime from the years 2013-2016. Samples were analyzed across four taxonomic marker genes: two small-subunit ribosomal RNA genes targeting prokaryotic (16S rRNA) and eukaryotic (18S rRNA) organisms and two mitochondrial genes targeting eukaryotes (cytochrome c oxidase subunit I gene (COI)) and vertebrates (mitochondrial small-subunit ribosomal RNA gene (12S)). In order to combine data from multiple markers, species occupancy modeling was used to determine the probability that an OTU is truly present in a sample (as described in Kelly et al. 2017 and Lahoz-Monfort et al. 2015). Many taxonomic groups show seasonal trends in species abundance and diversity in Monterey Bay. Together this work illustrates the rewards and challenges of applying multiple genetic markers to eDNA sequencing analysis of an environmental time series.

Given the rapid rise of environmental DNA (eDNA) surveys in ecology and environmental science, it is important to be able to compare the results of these surveys to traditional methods of measuring biodiversity. Here we compare samples from a traditional method (a manual tow-net) to companion eDNA samples sequenced at three different genetic loci. We find only partial taxonomic overlap among the resulting datasets, with each reflecting a portion of the larger suite of taxa present in the sampled nearshore marine environment. In the larger context of eDNA sequencing surveys, our results suggest that primer amplification bias drives much of the taxonomic bias in eDNA detection, and that the baseline probability of detecting any given taxon with a broad-spectrum primer set is likely to be low. Whether catching fish with different nets or using different PCR primer sets, multiple data types can provide complementary views of a common ecosystem. However, it remains difficult to cross-validate eDNA sequencing techniques in the field, either for presence/absence or for abundance, particularly for primer sets that target very wide taxonomic ranges. Finally, our results highlight the breadth of diversity in a single habitat, and although eDNA does capture a richer sample of the community than traditional methods of sampling, a large number of eDNA primer sets focusing on different subsets of the biota would be necessary to survey any ecological community in a reasonably comprehensive way.

Coral diseases impact reefs globally. Although we continue to describe diseases, little is known about the etiology or progression of even the most common cases. To examine a spectrum of coral health and determine factors of disease progression we examined Orbicella faveolata exhibiting signs of Yellow Band Disease (YBD), a widespread condition in the Caribbean. We used a novel combined approach to assess three members of the coral holobiont: the coral-host, associated Symbiodinium algae, and bacteria. We profiled three conditions: (1) healthy-appearing colonies (HH), (2) healthy-appearing tissue on diseased colonies (HD), and (3) diseased lesion (DD). Restriction fragment length polymorphism analysis revealed health state-specific diversity in Symbiodinium clade associations. 16S ribosomal RNA gene microarrays (PhyloChips) and O. faveolata complimentary DNA microarrays revealed the bacterial community structure and host transcriptional response, respectively. A distinct bacterial community structure marked each health state. Diseased samples were associated with two to three times more bacterial diversity. HD samples had the highest bacterial richness, which included components associated with HH and DD, as well as additional unique families. The host transcriptome under YBD revealed a reduced cellular expression of defense- and metabolism-related processes, while the neighboring HD condition exhibited an intermediate expression profile. Although HD tissue appeared visibly healthy, the microbial communities and gene expression profiles were distinct. HD should be regarded as an additional (intermediate) state of disease, which is important for understanding the progression of YBD.

In the last two decades, genetic and genomic studies have revealed the astonishing diversity and ubiquity of microorganisms. Emergence and expansion of the human microbiome project has reshaped our thinking about how microbes control host health—not only as pathogens, but also as symbionts. In coral reef environments, scientists have begun to examine the role that microorganisms play in coral life history. Herein, we review the current literature on coral-microbe interactions within the context of their role in evolution, development, and ecology. We ask the following questions, first posed by McFall-Ngai et al. (2013) in their review of animal evolution, with specific attention to how coral-microbial interactions may be affected under future environmental conditions: (1) How do corals and their microbiome affect each other's genomes? (2) How does coral development depend on microbial partners? (3) How is homeostasis maintained between corals and their microbial symbionts? (4) How can ecological approaches deepen our understanding of the multiple levels of coral-microbial interactions? Elucidating the role that microorganisms play in the structure and function of the holobiont is essential for understanding how corals maintain homeostasis and acclimate to changing environmental conditions., Singapore-MIT Alliance for Research and Technology. Center for Environmental Sensing and Modeling, Massachusetts Institute of Technology. Department of Civil and Environmental Engineering, Massachusetts Institute of Technology. Presidential Fellowship

Echinoderms, positioned taxonomically at the base of deuterostomes, provide an important system for the study of the evolution of the immune system. However, there is little known about the cellular components and genes associated with echinoderm immunity. The 2013-2014 sea star wasting disease outbreak is an emergent, rapidly spreading disease, which has led to large population declines of asteroids in the North American Pacific. While evidence suggests that the signs of this disease, twisting arms and lesions, may be attributed to a viral infection, the host response to infection is still poorly understood. In order to examine transcriptional responses of the sea star Pycnopodia helianthoides to sea star wasting disease, we injected a viral sized fraction (0.2 μm) homogenate prepared from symptomatic P. helianthoides into apparently healthy stars. Nine days following injection, when all stars were displaying signs of the disease, specimens were sacrificed and coelomocytes were extracted for RNA-seq analyses. A number of immune genes, including those involved in Toll signaling pathways, complement cascade, melanization response, and arachidonic acid metabolism, were differentially expressed. Furthermore, genes involved in nervous system processes and tissue remodeling were also differentially expressed, pointing to transcriptional changes underlying the signs of sea star wasting disease. The genomic resources presented here not only increase understanding of host response to sea star wasting disease, but also provide greater insight into the mechanisms underlying immune function in echinoderms.