Metagenomic Profiling of a Microbial Assemblage Associated with the California Mussel: A Node in Networks of Carbon and Nitrogen Cycling

Mussels are conspicuous and often abundant members of rocky shores and may constitute an important site for the nitrogen cycle due to their feeding and excretion activities. We used shotgun metagenomics of the microbial community associated with the surface of mussels (Mytilus californianus) on Tatoosh Island in Washington state to test whether there is a nitrogen-based microbial assemblage associated with mussels. Analyses of both tidepool mussels and those on emergent benches revealed a diverse community of Bacteria and Archaea with approximately 31 million bp from 6 mussels in each habitat. Using MG-RAST, between 22.5–25.6% were identifiable using the SEED non-redundant database for proteins. Of those fragments that were identifiable through MG-RAST, the composition was dominated by Cyanobacteria and Alpha- and Gamma-proteobacteria. Microbial composition was highly similar between the tidepool and emergent bench mussels, suggesting similar functions across these different microhabitats. One percent of the proteins identified in each sample were related to nitrogen cycling. When normalized to protein discovery rate, the high diversity and abundance of enzymes related to the nitrogen cycle in mussel-associated microbes is as great or greater than that described for other marine metagenomes. In some instances, the nitrogen-utilizing profile of this assemblage was more concordant with soil metagenomes in the Midwestern U.S. than for open ocean system. Carbon fixation and Calvin cycle enzymes further represented 0.65 and 1.26% of all proteins and their abundance was comparable to a number of open ocean marine metagenomes. In sum, the diversity and abundance of nitrogen and carbon cycle related enzymes in the microbes occupying the shells of Mytilus californianus suggest these mussels provide a node for microbial populations and thus biogeochemical processes

Identifying the plant‐associated microbiome across aquatic and terrestrial environments: the effects of amplification method on taxa discovery

Plants in terrestrial and aquatic environments contain a diverse microbiome. Yet, the chloroplast and mitochondria organelles of the plant eukaryotic cell originate from free‐living cyanobacteria and Rickettsiales. This represents a challenge for sequencing the plant microbiome with universal primers, as ~99% of 16S rRNA sequences may consist of chloroplast and mitochondrial sequences. Peptide nucleic acid clamps offer a potential solution by blocking amplification of host‐associated sequences. We assessed the efficacy of chloroplast and mitochondria‐blocking clamps against a range of microbial taxa from soil, freshwater and marine environments. While we found that the mitochondrial blocking clamps appear to be a robust method for assessing animal‐associated microbiota, Proteobacterial 16S rRNA binds to the chloroplast‐blocking clamp, resulting in a strong sequencing bias against this group. We attribute this bias to a conserved 14‐bp sequence in the Proteobacteria that matches the 17‐bp chloroplast‐blocking clamp sequence. By scanning the Greengenes database, we provide a reference list of nearly 1500 taxa that contain this 14‐bp sequence, including 48 families such as the Rhodobacteraceae, Phyllobacteriaceae, Rhizobiaceae, Kiloniellaceae and Caulobacteraceae. To determine where these taxa are found in nature, we mapped this taxa reference list against the Earth Microbiome Project database. These taxa are abundant in a variety of environments, particularly aquatic and semiaquatic freshwater and marine habitats. To facilitate informed decisions on effective use of organelle‐blocking clamps, we provide a searchable database of microbial taxa in the Greengenes and Silva databases matching various n‐mer oligonucleotides of each PNA sequence.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138887/1/men12645.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138887/2/men12645_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/138887/3/men12645-sup-0001-SupInfo.pd

The microbiome of the canopy-forming kelps, Nereocystis and Macrocystis, from the outer Olympic Coast to the Puget Sound

Canopy-forming kelps create vast underwater forests that are among the most productive marine ecosystems. In addition to providing vital habitat for macroscopic organisms, kelps also host an abundant microbial community in their surface mucus layer. In the Salish Sea, two canopy-forming kelps with contrasting life histories co-occur; Macrocystis pyrifera, a perennial species, and Nereocystis luetkeana, an annual species. Kelp-associated microbial communities were sampled along a spatial gradient, including sites from the outer Olympic Coast, Strait of Juan de Fuca, and Puget Sound. We characterized the microbial communities associated with each kelp species and the surrounding seawater using next-generation Illumina sequencing of 16S rRNA genes. Our preliminary results show that canopy-forming kelps host complex microbial symbiont communities that are host-specific and distinct from free-living bacteria in the surrounding seawater. Based on fluorescence microscopy data, up to 25 million bacteria live on just one square centimeter of N. luetkeana tissue. In addition to examining spatial variation in the kelp microbiome, we examined the impact of N. luetkeana and M. pyrifera kelp forests on the surrounding seawater chemistry. We used an enriched carbon stable isotope tracer experiment (13C-bicarbonate) to demonstrate simultaneous carbon fixation and dissolved organic carbon (DOC) production by N. luetkeana from Tatoosh Island, WA. During the daytime, carbon fixed by N. luetkeana is leaked into the surrounding seawater at a rate of 10 μmol DOC per hour per gram of dry mass. This carbon presents an abundant food resource for heterotrophic microbes in the surrounding seawater. Given the immense surface area of kelp forests, the kelp microbiome has potentially far-reaching impacts on kelp forest productivity and coastal nutrient cycling

Successional Dynamics and Seascape-Level Patterns of Microbial Communities on the Canopy-Forming Kelps Nereocystis luetkeana and Macrocystis pyrifera

Canopy-forming kelps create underwater forests that are among the most productive marine ecosystems. On the Pacific coast of North America, two canopy-forming kelps with contrasting life histories co-occur; Macrocystis pyrifera, a perennial species, and Nereocystis luetkeana, an annual species. Kelp blade-associated microbes were sampled from 12 locations across a spatial gradient in Washington, United States, from the outer Pacific Coast to Puget Sound. Microbial communities were characterized using next-generation Illumina sequencing of 16S rRNA genes. At higher taxonomic levels (bacterial phylum and class), canopy-forming kelps hosted remarkably similar microbial communities, but at the amplicon sequence variant level, microbial communities on M. pyrifera and N. luetkeana were host-specific and distinct from free-living bacteria in the surrounding seawater. Microbial communities associated with blades of each kelp species displayed significant geographic variation. The microbiome of N. luetkeana changed along the spatial gradient and was significantly correlated to salinity, with outer Pacific coast sites enriched in Bacteroidetes (family Saprospiraceae) and Gammaproteobacteria (Granulosicoccus sp.), and southern Puget Sound sites enriched in Alphaproteobacteria (family Hyphomonadaceae). We also examined microbial community development and succession on meristematic and apical N. luetkeana blade tissues throughout the summer growing season on Tatoosh Island, WA. Across all dates, microbial communities were less diverse on younger, meristematic blade tissue compared to the older, apical tissues. In addition, phylogenetic relatedness among microbial taxa increased from meristematic to apical blade tissues, suggesting that the addition of microbial taxa to the community was a non-random process that selected for certain phylogenetic groups of microbes. Microbial communities on older, apical tissues displayed significant temporal variation throughout the summer and microbial taxa that were differentially abundant over time displayed clear patterns of community succession. Overall, we report that host species identity, geographic location, and blade tissue age shape the microbial communities on canopy-forming kelps

Exploring B/Ca as a pH proxy in bivalves : relationships between Mytilus californianus B/Ca and environmental data from the northeast Pacific

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Biogeosciences 8 (2011): 2567-2579, doi:10.5194/bg-8-2567-2011.A distinct gap in our ability to understand changes in coastal biology that may be associated with recent ocean acidification is the paucity of directly measured ocean environmental parameters at coastal sites in recent decades. Thus, many researchers have turned to sclerochronological reconstructions of water chemistry to document the historical seawater environment. In this study, we explore the relationships between B/Ca and pH to test the feasibility of B/Ca measured on the ion probe as a pH proxy in the California mussel, Mytilus californianus. Heterogeneity in a range of ion microprobe standards is assessed, leading to reproducible B/Ca ratios at the 5% level. The B/Ca data exhibit large excursions during winter months, which are particularly pronounced during the severe winters of 2004–2005 and 2005–2006. Furthermore, B/Ca ratios are offset in different parts of the skeleton that calcified at the same time. We compare the M. californianus B/Ca record to directly measured environmental data during mussel growth from the period of 1999–2009 to examine whether seawater chemistry or temperature plays a role in controlling shell B/Ca. A suite of growth rate models based on measured temperature are compared to the B/Ca data to optimise the potential fit of B/Ca to pH. Despite sampling conditions that were well-suited to testing a pH control on B/Ca, including a close proximity to an environmental record, a distinct change in pH at the sampling locale, and a growth model designed to optimise the correlations between seawater pH and shell B/Ca, we do not see a strong correlations between pH and shell B/Ca (maximum coefficient of determination, r2, of 0.207). Instead, our data indicate a strong biological control on B/Ca as observed in some other carbonate-forming organisms.Financial support was provided by USGSWHOI Co-operative agreement and NSF-ANT award number 0902957 to L. F. Robinson, a SeaDoc Society grant to C. A. Pfister and J. T. Wootton, and a NASA Planetary Biology Internship to S. J. McCoy with L. F. Robinson and D. M. Glover

Microbial associations with macrobiota in coastal ecosystems : patterns and implications for nitrogen cycling

Author Posting. © Ecological Society of America, 2016. This article is posted here by permission of Ecological Society of America for personal use, not for redistribution. The definitive version was published in Frontiers in Ecology and the Environment 14 (2016): 200-208, doi:10.1002/fee.1262.In addition to their important effects on nitrogen (N) cycling via excretion and assimilation (by macrofauna and macroflora, respectively), many macrobiota also host or facilitate microbial taxa responsible for N transformations. Interest in this topic is expanding, especially as it applies to coastal marine systems where N is a limiting nutrient. Our understanding of the diversity of microbes associated with coastal marine macrofauna (invertebrate and vertebrate animals) and macrophytes (seaweeds and marine plants) is improving, and recent studies indicate that the collection of microbes living in direct association with macrobiota (the microbiome) may directly contribute to N cycling. Here, we review the roles that macrobiota play in coastal N cycling, review current knowledge of macrobial–microbial associations in terms of N processing, and suggest implications for coastal ecosystem function as animals are harvested and as foundational habitat is lost or degraded. Given the biodiversity of microbial associates of macrobiota, we advocate for more research into the functional consequences of these associations for the coastal N cycle.University of Chicago-Marine Biological Laboratories (MBL

Magnitude and predictability of ph fluctuations shape plastic responses to ocean acidification

16 pages, 5 figures, supplemental material https://www.journals.uchicago.edu/doi/suppl/10.1086/712930.-- Data and Code Availability: All raw data and referenced supplemental files in this article have been deposited in the Dryad Digital Repository (https://doi.org/10.5061/dryad.tqjq2bvxc; Bitter et al. 2020). All code associated with statistical analyses and figure generation for this article are publicly available at GitHub (https://github.com/MarkCBitter/pHFluctuation_Plasticity) and Zenodo (https://doi.org/10.5281/zenodo.4306829; Bitter 2020)Phenotypic plasticity is expected to facilitate the persistence of natural populations as global change progresses. The attributes of fluctuating environments that favor the evolution of plasticity have received extensive theoretical investigation, yet empirical validation of these findings is still in its infancy. Here, we combine high-resolution environmental data with a laboratory-based experiment to explore the influence of habitat pH fluctuation dynamics on the plasticity of gene expression in two populations of the Mediterranean mussel, Mytilus galloprovincialis. We linked differences in the magnitude and predictability of pH fluctuations in two habitats to population-specific gene expression profiles in ambient and stressful pH treatments. Our results demonstrate population-based differentiation in gene expression plasticity, whereby mussels native to a habitat exhibiting a large magnitude of pH fluctuations with low predictability display reduced phenotypic plasticity between experimentally imposed pH treatments. This work validates recent theoretical findings on evolution in fluctuating environments, suggesting that the predictability of fluctuating selection pressures may play a predominant role in shaping the phenotypic variation observed across natural populationsM.C.B. was supported by a National Science Foundation Graduate Research Fellowship Program grant (1746045) and a Department of Education grant (P200A150101). L.K. was supported by a National Science Foundation grant (OCE-1521597), which provided research support for this work. This research was also supported in part by the University of Chicago’s France and Chicago Collaborating in the Sciences program to C.A.P. and M.C.B.With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)Peer reviewe

Mortality of native and invasive ladybirds co-infected by ectoparasitic and entomopathogenic fungi

Harmonia axyridis is an invasive alien ladybird in North America and Europe. Studies show that multiple natural enemies are using Ha. axyridis as a new host. However, thus far, no research has been undertaken to study the effects of simultaneous infection by multiple natural enemies on Ha. axyridis. We hypothesized that high thallus densities of the ectoparasitic fungus Hesperomyces virescens on a ladybird weaken the host's defenses, thereby making it more susceptible to infection by other natural enemies. We examined mortality of the North American-native Olla nu-nigrum and Ha. axyridis co-infected with He. virescens and an entomopathogenic fungus-either Beauveria bassiana or Metarhizium brunneum. Laboratory assays revealed that He. virescens- infected O. nu-nigrum individuals are more susceptible to entomopathogenic fungi, but Ha. axyridis does not suffer the same effects. This is in line with the enemy release hypothesis, which predicts that invasive alien species in new geographic areas experience reduced regulatory effects from natural enemies compared to native species. Considering our results, we can ask how He. virescens affects survival when confronted by other pathogens that previously had little impact on Ha. axyridis

Spatial organization of the kelp microbiome at micron scales

Background: Elucidating the spatial structure of host-associated microbial communities is essential for understanding taxon-taxon interactions within the microbiota and between microbiota and host. Macroalgae are colonized by complex microbial communities, suggesting intimate symbioses that likely play key roles in both macroalgal and bacterial biology, yet little is known about the spatial organization of microbes associated with macroalgae. Canopy-forming kelp are ecologically significant, fixing teragrams of carbon per year in coastal kelp forest ecosystems. We characterized the micron-scale spatial organization of bacterial communities on blades of the kelp Nereocystis luetkeana using fluorescence in situ hybridization and spectral imaging with a probe set combining phylum-, class-, and genus-level probes to localize and identify > 90% of the microbial community. Results: We show that kelp blades host a dense microbial biofilm composed of disparate microbial taxa in close contact with one another. The biofilm is spatially differentiated, with clustered cells of the dominant symbiont Granulosicoccus sp. (Gammaproteobacteria) close to the kelp surface and filamentous Bacteroidetes and Alphaproteobacteria relatively more abundant near the biofilm-seawater interface. A community rich in Bacteroidetes colonized the interior of kelp tissues. Microbial cell density increased markedly along the length of the kelp blade, from sparse microbial colonization of newly produced tissues at the meristematic base of the blade to an abundant microbial biofilm on older tissues at the blade tip. Kelp from a declining population hosted fewer microbial cells compared to kelp from a stable population. Conclusions: Imaging revealed close association, at micrometer scales, of different microbial taxa with one another and with the host. This spatial organization creates the conditions necessary for metabolic exchange among microbes and between host and microbiota, such as provisioning of organic carbon to the microbiota and impacts of microbial nitrogen metabolisms on host kelp. The biofilm coating the surface of the kelp blade is well-positioned to mediate interactions between the host and surrounding organisms and to modulate the chemistry of the surrounding water column. The high density of microbial cells on kelp blades (10(5)-10(7) cells/cm(2)), combined with the immense surface area of kelp forests, indicates that biogeochemical functions of the kelp microbiome may play an important role in coastal ecosystems