Primary Microbial Succession in the Anchialine Ecosystem.

When new land is created, initial microbial colonization lays the foundation for further ecological succession of plant and animal communities. Primary microbial succession of new aquatic habitats formed during volcanic activity has received little attention. The anchialine ecosystem, which includes coastal ponds in young lava flows, offers an opportunity to examine this process. Here, we characterized microbial communities of anchialine habitats in Hawaii that were created during volcanic eruptions in 2018. Benthic samples from three habitats were collected ∼2 years after their formation and at later time points spanning ∼1 year. Sequence profiling (16S and 18S) of prokaryotic and eukaryotic communities was used to test whether communities were similar to those from older, established anchialine habitats, and if community structure changed over time. Results show that microbial communities from the new habitats were unlike any from established anchialine microbial communities, having higher proportions of Planctomycetota and Chloroflexi but lower proportions of green algae. Each new habitat also harbored its own unique community relative to other habitats. While community composition in each habitat underwent statistically significant changes over time, they remained distinctive from established anchialine habitats. New habitats also had highly elevated temperatures compared to other habitats. These results suggest that idiosyncratic microbial consortia form during early succession of Hawaiian anchialine habitats. Future monitoring will reveal whether the early communities described here remain stable after temperatures decline and macro-organisms become more abundant, or if microbial communities will continue to change and eventually resemble those of established habitats. This work is a key first step in examining primary volcanic succession in aquatic habitats and suggests young anchialine habitats may warrant special conservation status.

River Flow Impacts Bacterial and Archaeal Community Structure in Surface Sediments in the Northern Gulf of Mexico.

Meiobenthic community structure in the northern Gulf of Mexico has been shown to be driven by geographical differences due to inshore-offshore gradients and location relative to river discharge. Samples collected along three transects spanning Mobile Bay, Alabama, showed significant differences in meiobenthic communities east of the bay compared to those sampled from the west. In contrast, analysis of bacterial and archaeal communities from the same sediment samples shows that the inshore-offshore gradient has minimal impact on their community structure. Significant differences in community structure were observed for Bacteria and Archaea between the east and west samples, but there was no difference in richness or diversity. Grouped by sediment type, higher richness was observed in silty samples compared to sandy samples. Significant differences were also observed among sediment types for community structure with bacteria communities in silty samples having more anaerobic sulfate reducers compared to aerobic heterotrophs, which had higher abundances in sandy sediments. This is likely due to increased organic matter in the silty sediments from the overlying river leading to low oxygen habitats. Most archaeal sequences represented poorly characterized high-level taxa, limiting interpretation of their distributions. Overlap between groups based on transect and sediment characteristics made determining which factor is more important in structuring bacterial and archaeal communities difficult. However, both factors are driven by discharge from the Mobile River. Although inshore-offshore gradients do not affect Bacteria or Archaea to the same extent as the meiobenthic communities, all three groups are strongly affected by sediment characteristics.

Spatial and temporal variation of intertidal nematodes in the northern Gulf of Mexico after the Deepwater Horizon oil spill.

Nematodes are an abundant and diverse interstitial component of sedimentary habitats that have been reported to serve as important bioindicators. Though the 2010 Deepwater Horizon (DWH) disaster occurred 60 km offshore in the Gulf of Mexico (GOM) at a depth of 1525 m, oil rose to the surface and washed ashore, subjecting large segments of coastline in the northern GOM to contamination. Previous metabarcoding work shows intertidal nematode communities were negatively affected by the oil spill. Here we examine the subsequent recovery of nematode community structure at five sites along the Alabama coast over a two-year period. The latter part of the study (July 2011-July 2012) also included an examination of nematode vertical distribution in intertidal sediments. Results showed nematode composition within this region was more influenced by sample locality than time and depth. The five sampling sites were characterized by distinct nematode assemblages that varied by sampling dates. Nematode diversity decreased four months after the oil spill but increased after one year, returning to previous levels at all sites except Bayfront Park (BP). There was no significant difference among nematode assemblages in reference to vertical distribution. Although the composition of nematode assemblages changed, the feeding guilds they represented were not significantly different even though some variation was noted. Data from morphological observations integrated with metabarcoding data indicated similar spatial variation in nematode distribution patterns, indicating the potential of using these faster approaches to examine overall disturbance impact trends within communities. Heterogeneity of microhabitats in the intertidal zone indicates that future sampling and fine-scale studies of nematodes are needed to examine such anthropogenic effects.

Phylogenomics of Lophotrochozoa with Consideration of Systematic Error.

Phylogenomic studies have improved understanding of deep metazoan phylogeny and show promise for resolving incongruences among analyses based on limited numbers of loci. One region of the animal tree that has been especially difficult to resolve, even with phylogenomic approaches, is relationships within Lophotrochozoa (the animal clade that includes molluscs, annelids, and flatworms among others). Lack of resolution in phylogenomic analyses could be due to insufficient phylogenetic signal, limitations in taxon and/or gene sampling, or systematic error. Here, we investigated why lophotrochozoan phylogeny has been such a difficult question to answer by identifying and reducing sources of systematic error. We supplemented existing data with 32 new transcriptomes spanning the diversity of Lophotrochozoa and constructed a new set of Lophotrochozoa-specific core orthologs. Of these, 638 orthologous groups (OGs) passed strict screening for paralogy using a tree-based approach. In order to reduce possible sources of systematic error, we calculated branch-length heterogeneity, evolutionary rate, percent missing data, compositional bias, and saturation for each OG and analyzed increasingly stricter subsets of only the most stringent (best) OGs for these five variables. Principal component analysis of the values for each factor examined for each OG revealed that compositional heterogeneity and average patristic distance contributed most to the variance observed along the first principal component while branch-length heterogeneity and, to a lesser extent, saturation contributed most to the variance observed along the second. Missing data did not strongly contribute to either. Additional sensitivity analyses examined effects of removing taxa with heterogeneous branch lengths, large amounts of missing data, and compositional heterogeneity. Although our analyses do not unambiguously resolve lophotrochozoan phylogeny, we advance the field by reducing the list of viable hypotheses. Moreover, our systematic approach for dissection of phylogenomic data can be applied to explore sources of incongruence and poor support in any phylogenomic data set. [Annelida; Brachiopoda; Bryozoa; Entoprocta; Mollusca; Nemertea; Phoronida; Platyzoa; Polyzoa; Spiralia; Trochozoa.].

Genetic assessment of meiobenthic community composition and spatial distribution in coastal sediments along northern Gulf of Mexico.

Meiobenthic (meiofauna and micro-eukaryotes) organisms are important contributors to ecosystem functioning in aquatic environments through their roles in nutrient transport, sediment stability, and food web interactions. Despite their ecological importance, information pertaining to variation of these communities at various spatial and temporal scales is not widely known. Many studies in the Gulf of Mexico (GOM) have focused either on deep sea or continental shelf areas, while little attention has been paid to bays and coastal regions. Herein, we take a holistic approach by using high-throughput sequencing approaches to examine spatial variation in meiobenthic communities within Alabama bays and the coastal northern GOM region. Sediment samples were collected along three transects (Mississippi Sound: MS, FOCAL: FT, and Orange Beach: OB) from September 2010 to April 2012 and community composition was determined by metabarcoding the V9 hypervariable region of the nuclear18S rRNA gene. Results showed that Stramenopiles (diatoms), annelids, arthropods (copepods), and nematodes were the dominate groups within samples, while there was presence of other phyla throughout the dataset. Location played a larger role than time sampled in community composition. However, samples were collected over a short temporal scale. Samples clustered in reference to transect, with the most eastern transect (OB) having a distinct community composition in comparison to the other two transects (MS and FT). Communities also differed in reference to region (Bay versus Shelf). Bulk density and percent inorganic carbon were the only measured environmental factors that were correlated with community composition.

Metabarcoding reveals environmental factors influencing spatio-temporal variation in pelagic micro-eukaryotes.

Marine environments harbour a vast diversity of micro-eukaryotic organisms (protists and other small eukaryotes) that play important roles in structuring marine ecosystems. However, micro-eukaryote diversity is not well understood. Likewise, knowledge is limited regarding micro-eukaryote spatial and seasonal distribution, especially over long temporal scales. Given the importance of this group for mobilizing energy from lower trophic levels near the base of the food chain to larger organisms, assessing community stability, diversity and resilience is important to understand ecosystem health. Herein, we use a metabarcoding approach to examine pelagic micro-eukaryote communities over a 2.5-year time series. Bimonthly surface sampling (July 2009 to December 2011) was conducted at four locations within Mobile Bay (Bay) and along the Alabama continental shelf (Shelf). Alpha-diversity only showed significant differences in Shelf sites, with the greatest differences observed between summer and winter. Beta-diversity showed significant differences in community composition in relation to season and the Bay was dominated by diatoms, while the Shelf was characterized by dinoflagellates and copepods. The northern Gulf of Mexico is heavily influenced by the Mobile River Basin, which brings low-salinity nutrient-rich water mostly during winter and spring. Community composition was correlated with salinity, temperature and dissolved silicate. However, species interactions (e.g. predation and parasitism) may also contribute to the observed variation, especially on the Shelf, which warrants further exploration. Metabarcoding revealed clear patterns in surface pelagic micro-eukaryote communities that were consistent over multiple years, demonstrating how these techniques could be greatly beneficial to ecological monitoring and management over temporal scales.

Biogeochemical and Microbial Variation across 5500 km of Antarctic Surface Sediment Implicates Organic Matter as a Driver of Benthic Community Structure.

Western Antarctica, one of the fastest warming locations on Earth, is a unique environment that is underexplored with regards to biodiversity. Although pelagic microbial communities in the Southern Ocean and coastal Antarctic waters have been well-studied, there are fewer investigations of benthic communities and most have a focused geographic range. We sampled surface sediment from 24 sites across a 5500 km region of Western Antarctica (covering the Ross Sea to the Weddell Sea) to examine relationships between microbial communities and sediment geochemistry. Sequencing of the 16S and 18S rRNA genes showed microbial communities in sediments from the Antarctic Peninsula (AP) and Western Antarctica (WA), including the Ross, Amundsen, and Bellingshausen Seas, could be distinguished by correlations with organic matter concentrations and stable isotope fractionation (total organic carbon; TOC, total nitrogen; TN, and δ(13)C). Overall, samples from the AP were higher in nutrient content (TOC, TN, and NH4 (+)) and communities in these samples had higher relative abundances of operational taxonomic units (OTUs) classified as the diatom, Chaetoceros, a marine cercozoan, and four OTUs classified as Flammeovirgaceae or Flavobacteria. As these OTUs were strongly correlated with TOC, the data suggests the diatoms could be a source of organic matter and the Bacteroidetes and cercozoan are grazers that consume the organic matter. Additionally, samples from WA have lower nutrients and were dominated by Thaumarchaeota, which could be related to their known ability to thrive as lithotrophs. This study documents the largest analysis of benthic microbial communities to date in the Southern Ocean, representing almost half the continental shoreline of Antarctica, and documents trophic interactions and coupling of pelagic and benthic communities. Our results indicate potential modifications in carbon sequestration processes related to change in community composition, identifying a prospective mechanism that links climate change to carbon availability.

Meiofaunal community analysis by high-throughput sequencing: comparison of extraction, quality filtering, and clustering methods.

Using molecular tools to examine community composition of meiofauna, animals 45µm to 1mm in size living between sediment grains in aquatic environments, is relatively new in comparison to bacterial and archaeal microbial studies. Although high-throughput molecular approaches are starting to be applied to these ccommunities, effectiveness of different approaches for nucleic acid extraction from meiofauna is poorly known and bioinformatic pipelines vary between studies. Given this situation, there is a need for protocols to be developed that promote consistency in sample collection and processing, sequence quality filtering, and Operational Taxonomic Unit (OTU) clustering methods. Herein, we assess different approaches used for DNA extraction (DNA extracted directly from sediment versus elutriated material retained on a 45µm sieve) as well as how different quality filtering methods of sequences and OTU clustering algorithms impact genetic assessment of meiofauna community composition. DNA extracted directly from sediment resulted in higher presence of non-metazoan eukaryotic taxa; in contrast, an elutriation (resuspension with decanting) approach increased meiofauna abundance and enriched metazoan OTUs. In regards to bioinformatics analyses, the number of overall OTUs varied by clustering algorithm, primarily due to the applied method of sequence quality filtering. However, alpha and beta diversity analyses showed similar trends regardless of bioinformatics pipeline utilized. Based on our results, we recommend studies of meiofauna communities first elutriate samples prior to DNA extraction and include multiple biological replicates to account for variation in community-level composition. The quality filtering method should be carefully considered as this step accounted for large discrepancy in the number of OTUs inferred.

Reconstruction of cyclooxygenase evolution in animals suggests variable, lineage-specific duplications, and homologs with low sequence identity.

Cyclooxygenase (COX) enzymatically converts arachidonic acid into prostaglandin G/H in animals and has importance during pregnancy, digestion, and other physiological functions in mammals. COX genes have mainly been described from vertebrates, where gene duplications are common, but few studies have examined COX in invertebrates. Given the increasing ease in generating genomic data, as well as recent, although incomplete descriptions of potential COX sequences in Mollusca, Crustacea, and Insecta, assessing COX evolution across Metazoa is now possible. Here, we recover 40 putative COX orthologs by searching publicly available genomic resources as well as ~250 novel invertebrate transcriptomic datasets. Results suggest the common ancestor of Cnidaria and Bilateria possessed a COX homolog similar to those of vertebrates, although such homologs were not found in poriferan and ctenophore genomes. COX was found in most crustaceans and the majority of molluscs examined, but only specific taxa/lineages within Cnidaria and Annelida. For example, all octocorallians appear to have COX, while no COX homologs were found in hexacorallian datasets. Most species examined had a single homolog, although species-specific COX duplications were found in members of Annelida, Mollusca, and Cnidaria. Additionally, COX genes were not found in Hemichordata, Echinodermata, or Platyhelminthes, and the few previously described COX genes in Insecta lacked appreciable sequence homology (although structural analyses suggest these may still be functional COX enzymes). This analysis provides a benchmark for identifying COX homologs in future genomic and transcriptomic datasets, and identifies lineages for future studies of COX.

High-throughput sequencing characterizes intertidal meiofaunal communities in northern Gulf of Mexico (Dauphin Island and Mobile Bay, Alabama).

Meiofauna are important components of food webs and for nutrient exchange between the benthos and water column. Recent studies have focused on these communities in the Gulf of Mexico due to potential impacts of the Deepwater Horizon Oil Spill (DWHOS). In particular, intertidal meiofaunal communities from Mobile Bay and Dauphin Island, Alabama, were previously shown to shift from predominately metazoan taxa prior to DWHOS to a fungal-dominated community after the spill. However, knowledge of variability within these communities remains unknown. Herein, we used Illumina high-throughput amplicon sequencing to examine variation throughout a year for the same locations for which the organismal shift was noted. Sediment samples were collected bi-monthly for a year (July 2011-July 2012) from which the meiofaunal community was examined by sequencing the eukaryotic hypervariable V9 region of the 18S rRNA gene. Results showed that the presence of fungal taxa was limited within these communities, suggesting that previously reported acute impacts of the DWHOS on meiofauna were apparently short term. However, these meiofaunal communities show shifts in proportions of metazoan taxa compared to pre-spill samples. Whether this change is due to prolonged impacts of the spill or variation in community composition is unclear. Taxonomic variation within and between sampled locations throughout the study was observed, suggesting potential yearly variation in communities. Continued sampling over a longer timeframe will provide a more complete understanding of seasonality and variation within these communities. Such a baseline is required to assess future anthropogenic impacts.

A role for PKC-epsilon in Fc gammaR-mediated phagocytosis by RAW 264.7 cells.

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC- epsilon in Fc gamma receptor (Fc gammaR)-dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG-opsonized beads. PKC- epsilon, but not PKC-delta, concentrated around the beads. PKC- epsilon accumulation was transient; apparent as a "flash" on target ingestion. Similarly, endogenous PKC- epsilon was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC- epsilon, but not PKC-alpha, PKC-delta, or PKC-gamma enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC- epsilon expressors was twice that of cells expressing GFP PKC-delta. Expression of the regulatory domain ( epsilon RD) and the first variable region ( epsilon V1) of PKC- epsilon inhibited uptake, whereas the corresponding PKC-delta region had no effect. Actin polymerization was enhanced on expression of GFP PKC- epsilon and epsilon RD, but decreased in cells expressing epsilon V1, suggesting that the epsilon RD and epsilon V1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC- epsilon in Fc gammaR-mediated phagocytosis that is independent of its effects on actin assembly.