Oysters and eelgrass: potential partners in a high pCO2 ocean.

Climate change is affecting the health and physiology of marine organisms and altering species interactions. Ocean acidification (OA) threatens calcifying organisms such as the Pacific oyster, Crassostrea gigas. In contrast, seagrasses, such as the eelgrass Zostera marina, can benefit from the increase in available carbon for photosynthesis found at a lower seawater pH. Seagrasses can remove dissolved inorganic carbon from OA environments, creating local daytime pH refugia. Pacific oysters may improve the health of eelgrass by filtering out pathogens such as Labyrinthula zosterae (LZ), which causes eelgrass wasting disease (EWD). We examined how co-culture of eelgrass ramets and juvenile oysters affected the health and growth of eelgrass and the mass of oysters under different pCO2 exposures. In Phase I, each species was cultured alone or in co-culture at 12°C across ambient, medium, and high pCO2 conditions, (656, 1,158 and 1,606 µatm pCO2 , respectively). Under high pCO2 , eelgrass grew faster and had less severe EWD (contracted in the field prior to the experiment). Co-culture with oysters also reduced the severity of EWD. While the presence of eelgrass decreased daytime pCO2 , this reduction was not substantial enough to ameliorate the negative impact of high pCO2 on oyster mass. In Phase II, eelgrass alone or oysters and eelgrass in co-culture were held at 15°C under ambient and high pCO2 conditions, (488 and 2,013 µatm pCO2 , respectively). Half of the replicates were challenged with cultured LZ. Concentrations of defensive compounds in eelgrass (total phenolics and tannins), were altered by LZ exposure and pCO2 treatments. Greater pathogen loads and increased EWD severity were detected in LZ exposed eelgrass ramets; EWD severity was reduced at high relative to low pCO2 . Oyster presence did not influence pathogen load or EWD severity; high LZ concentrations in experimental treatments may have masked the effect of this treatment. Collectively, these results indicate that, when exposed to natural concentrations of LZ under high pCO2 conditions, eelgrass can benefit from co-culture with oysters. Further experimentation is necessary to quantify how oysters may benefit from co-culture with eelgrass, examine these interactions in the field and quantify context-dependency.

A case study characterizing animal fecal sources in surface water using a mitochondrial DNA marker.

Water quality impairment by fecal waste in coastal watersheds is a public health issue. The present study provided evidence for the use of a mitochondrial (mtDNA) marker to detect animal fecal sources in surface water. The accurate identification of fecal pollution is based on the notion that fecal microorganisms preferentially inhabit a host animal's gut environment. In contrast, mtDNA host-specific markers are inherent to eukaryotic host cells, which offers the advantage by detecting DNA from the host rather than its fecal bacteria. The present study focused on sampling water presumably from non-point sources (NPS), which can increase bacterial and nitrogen concentrations to receiving water bodies. Stream sampling sites located within the Piscataqua River Watershed (PRW), New Hampshire, USA, were sampled from a range of sites that experienced nitrogen inputs such as sewer and septic systems and suburban runoff. Three mitochondrial (mtDNA) gene marker assays (human, bovine, and canine) were tested from surface water. Nineteen sites were sampled during an 18-month period. Analyses of the combined single and multiplex assay results showed that the proportion of occurrence was highest for bovine (15.6%; n = 77) compared to canine (5.6%; n = 70) and human (5.7%; n = 107) mtDNA gene markers. For the human mtDNA marker, there was a statistically significant relationship between presence vs. absence and land use (Fisher's test p = 0.0031). This result was evident particularly for rural suburban septic, which showed the highest proportion of presence (19.2%) compared to the urban sewered (3.3%), suburban sewered (0%), and agricultural (0%) as well as forested septic (0%) sites. Although further testing across varied land use is needed, our study provides evidence for using the mtDNA marker in large watersheds.

Metagenomics to characterize sediment microbial biodiversity associated with fishing exposure within the Stellwagen Bank National Marine Sanctuary.

Microbes in marine sediments constitute a large percentage of the global marine ecosystem and function to maintain a healthy food web. In continental shelf habitats such as the Gulf of Maine (GoM), relatively little is known of the microbial community abundance, biodiversity, and natural product potential. This report is the first to provide a time-series assessment (2017-2020) of the sediment microbial structure in areas open and closed to fishing within the Stellwagen Bank National Marine Sanctuary (SBNMS). A whole metagenome sequencing (WMS) approach was used to characterize the sediment microbial community. Taxonomic abundance was calculated across seven geographic sites with 14 individual sediment samples collected during the summer and fall seasons. Bioinformatics analyses identified more than 5900 different species across multiple years. Non-metric multidimensional scaling methods and generalized linear models demonstrated that species richness was inversely associated with fishing exposure levels and varied by year. Additionally, the discovery of 12 unique biosynthetic gene clusters (BGCs) collected across sites confirmed the potential for medically relevant natural product discovery in the SBNMS. This study provides a practical assessment of how fishing exposure and temporal trends may affect microbial community structure in a coastal marine sanctuary.

Whole metagenomic sequencing to characterize the sediment microbial community within the Stellwagen Bank National Marine Sanctuary and preliminary biosynthetic gene cluster screening of Streptomyces scabrisporus.

Understanding the marine sediment microbial community structure is of increasing importance to microbiologists since little is known of the diverse taxonomy that exists within this environment. Quantifying microbial species distribution patterns within marine sanctuaries is necessary to address conservation requirements. The objectives of this study were to characterize the relative abundance and biodiversity of metagenome samples of the sediment microbial community in the Stellwagen Bank National Marine Sanctuary (SBNMS). Related to the need for a comprehensive assessment of the microbial habitat within marine sanctuaries is the increased threat of antibiotic-resistant pathogens, coupled with multi-resistant bacterial strains. This has necessitated a renewed search for bioactive compounds in marine benthic habitat. An additional aim was to initiate quantification of biosynthetic gene clusters in species that have potential for natural product and drug discovery relevant to human health. Surficial sediment from 18 samples was collected in the summer and fall of 2017 from three benthic sites in the SBNMS. Microbial DNA was extracted from samples, and sequencing libraries were prepared for taxonomic analysis. Whole metagenome sequencing (WMGS) in combination with a bioinformatics pipeline was employed to delineate the taxa of bacteria present in each sample. Among all sampling sites, biodiversity was higher for summer compared to fall for class (p = 0.0013; F = 4.5) and genus (p = 0.0219; F = 4.4). Actinobacteria was the fifth most abundant class in both seasons (7.81%). Streptomyces was observed to be the fourth most abundant genus in both seasons with significantly higher prevalence in summer compared to fall samples. In summer, site 3 had the highest percentage of Streptomyces (1.71%) compared to sites 2 (1.62%) and 1 (1.37%). The results enabled preliminary quantification of the sequenced hits from the SBNMS sites with the highest potential for harboring secondary metabolite biosynthetic gene clusters for Streptomyces scabrisporus strain (NF3) genomic regions. This study is one of the first to use a whole metagenomics approach to characterize sediment microbial biodiversity in partnership with the SBNMS and demonstrates the potential for future ecological and biomedical research.

Metabarcoding assessment of prokaryotic and eukaryotic taxa in sediments from Stellwagen Bank National Marine Sanctuary.

Stellwagen Bank National Marine Sanctuary (SBNMS) in the Gulf of Maine is a historic fishing ground renowned for remarkable productivity. Biodiversity conservation is a key management priority for SBNMS and yet data on the diversity of microorganisms, both prokaryotic and eukaryotic, is lacking. This study utilized next generation sequencing to characterize sedimentary communities within SBNMS at three sites over two seasons. Targeting 16S and 18S small subunit (SSU) rRNA genes and fungal Internal Transcribed Spacer (ITS) rDNA sequences, samples contained high diversity at all taxonomic levels and identified 127 phyla, including 115 not previously represented in the SBNMS Management Plan and Environmental Assessment. A majority of the diversity was bacterial, with 59 phyla, but also represented were nine Archaea, 18 Animalia, 14 Chromista, eight Protozoa, two Plantae, and 17 Fungi phyla. Samples from different sites and seasons were dominated by the same high abundance organisms but displayed considerable variation in rare taxa. The levels of biodiversity seen on this small spatial scale suggest that benthic communities of this area support a diverse array of micro- and macro-organisms, and provide a baseline for future studies to assess changes in community structure in response to rapid warming in the Gulf of Maine.

Analytical validation of a novel multiplex test for detection of advanced adenoma and colorectal cancer in symptomatic patients.

Early detection of colorectal cancer (CRC) is key to reducing associated mortality. Despite the importance of early detection, approximately 40% of individuals in the United States between the ages of 50-75 have never been screened for CRC. The low compliance with colonoscopy and fecal-based screening may be addressed with a non-invasive alternative such as a blood-based test. We describe here the analytical validation of a multiplexed blood-based assay that measures the plasma concentrations of 15 proteins to assess advanced adenoma (AA) and CRC risk in symptomatic patients. The test was developed on an electrochemiluminescent immunoassay platform employing four multi-marker panels, to be implemented in the clinic as a laboratory developed test (LDT). Under the Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) regulations, a United States-based clinical laboratory utilizing an LDT must establish performance characteristics relating to analytical validity prior to releasing patient test results. This report describes a series of studies demonstrating the precision, accuracy, analytical sensitivity, and analytical specificity for each of the 15 assays, as required by CLIA/CAP. In addition, the report describes studies characterizing each of the assays' dynamic range, parallelism, tolerance to common interfering substances, spike recovery, and stability to sample freeze-thaw cycles. Upon completion of the analytical characterization, a clinical accuracy study was performed to evaluate concordance of AA and CRC classifier model calls using the analytical method intended for use in the clinic. Of 434 symptomatic patient samples tested, the percent agreement with original CRC and AA calls was 87% and 92% respectively. All studies followed CLSI guidelines and met the regulatory requirements for implementation of a new LDT. The results provide the analytical evidence to support the implementation of the novel multi-marker test as a clinical test for evaluating CRC and AA risk in symptomatic individuals.

Revelation of the IFNα, IL-10, IL-8 and IL-1ß as promising biomarkers reflecting immuno-pathological mechanisms in porcine Huntington's disease model.

Studies on Huntington's disease (HD) demonstrated altered immune response in HD gene carriers. Using multiplexing immunoassay, we simultaneously investigated seven cytokines in secretomes of microglia and blood monocytes, cerebrospinal fluid (CSF) and serum collected from transgenic HD minipigs at pre-symptomatic disease stage. Decline in IFNα and IL-10 was observed in CSF and secretome of microglia whilst elevated IL-8 and IL-1ß levels were secreted by microglia. Additionally, IL-8 was increased in serum. The proportion of mutant huntingtin in microglia may have causative impact on cytokine production. IFNα, IL-10, IL-8 and IL-1ß represent promising biomarkers reflecting immuno-pathological mechanisms in porcine HD model.

Use of a High-Density Protein Microarray to Identify Autoantibodies in Subjects with Type 2 Diabetes Mellitus and an HLA Background Associated with Reduced Insulin Secretion.

New biomarkers for type 2 diabetes mellitus (T2DM) may aid diagnosis, drug development or clinical treatment. Evidence is increasing for the adaptive immune system's role in T2DM and suggests the presence of unidentified autoantibodies. While high-density protein microarrays have emerged as a useful technology to identify possible novel autoantigens in autoimmune diseases, its application in T2DM has lagged. In Pima Indians, the HLA haplotype (HLA-DRB1*02) is protective against T2DM and, when studied when they have normal glucose tolerance, subjects with this HLA haplotype have higher insulin secretion compared to those without the protective haplotype. Possible autoantibody biomarkers were identified using microarrays containing 9480 proteins in plasma from Pima Indians with T2DM without the protective haplotype (n = 7) compared with those with normal glucose regulation (NGR) with the protective haplotype (n = 11). A subsequent validation phase involving 45 cases and 45 controls, matched by age, sex and specimen storage time, evaluated 77 proteins. Eleven autoantigens had higher antibody signals among T2DM subjects with the lower insulin-secretion HLA background compared with NGR subjects with the higher insulin-secretion HLA background (p<0.05, adjusted for multiple comparisons). PPARG2 and UBE2M had lowest p-values (adjusted p = 0.023) while PPARG2 and RGS17 had highest case-to-control antibody signal ratios (1.7). A multi-protein classifier involving the 11 autoantigens had sensitivity, specificity, and area under the receiver operating characteristics curve of 0.73, 0.80, and 0.83 (95% CI 0.74-0.91, p = 3.4x10-8), respectively. This study identified 11 novel autoantigens which were associated with T2DM and an HLA background associated with reduced insulin secretion. While further studies are needed to distinguish whether these antibodies are associated with insulin secretion via the HLA background, T2DM more broadly, or a combination of the two, this study may aid the search for autoantibody biomarkers by narrowing the list of protein targets.