Removal of Exogenous Stimuli Reveals a Canalization of Circadian Physiology in a Vertically Migrating Copepod.

Diel rhythms are observed across taxa and are important for maintaining synchrony between the environment and organismal physiology. A striking example of this is the diel vertical migration undertaken by zooplankton, some of which, such as the 5 mm-long copepod Pleuromamma xiphias (P. xiphias), migrate hundreds of meters daily between the surface ocean and deeper waters. Some of the molecular pathways that underlie the expressed phenotype at different stages of this migration are entrained by environmental variables (e.g., day length and food availability), while others are regulated by internal clocks. We identified a series of proteomic biomarkers that vary across ocean DVM and applied them to copepods incubated in 24 h of darkness to assess circadian control. The dark-incubated copepods shared some proteomic similarities to the ocean-caught copepods (i.e., increased abundance of carbohydrate metabolism proteins at night). Shipboard-incubated copepods demonstrated a clearer distinction between night and day proteomic profiles, and more proteins were differentially abundant than in the in situ copepods, even in the absence of the photoperiod and other environmental cues. This pattern suggests that there is a canalization of rhythmic diel physiology in P. xiphias that reflects likely circadian clock control over diverse molecular pathways.

Diel metabolic patterns revealed by in situ transcriptome and proteome in a vertically migratory copepod.

Zooplankton undergo a diel vertical migration (DVM) which exposes them to gradients of light, temperature, oxygen, and food availability on a predictable daily schedule. Disentangling the co-varying and potentially synergistic interactions on metabolic rates has proven difficult, despite the importance of this migration for the delivery of metabolic waste products to the distinctly different daytime (deep) and nighttime (surface) habitats. This study examines the transcriptomic and proteomic profiles of the circumglobal migratory copepod, Pleuromamma xiphias, over the diel cycle. The transcriptome showed that 96% of differentially expressed genes were upregulated during the middle of the day - the period often considered to be of lowest zooplankton activity. The changes in protein abundance were more spread out over time, peaking (42% of comparisons) in the early evening. Between 9:00 and 15:00, both the transcriptome and proteome datasets showed increased expression related to chitin synthesis and degradation. Additionally, at 09:00 and 22:00, there were increases in myosin and vitellogenin proteins, potentially linked to the stress of migration and/or reproductive investment. Based on protein abundances detected, there is an inferred switch in broad metabolic processes, shifting from electron transport system in the day to glycolysis and glycogen mobilization in the afternoon/evening. These observations provide evidence of the diel impact of DVM on transcriptomic and proteomic pathways that likely influence metabolic processes and subsequent excretion products, and clarify how this behaviour results in the direct rapid transport of waste metabolites from the surface to the deep ocean.

Proteomic response of early juvenile Pacific oysters (Crassostrea gigas) to temperature.

Pacific oysters (Crassostrea gigas) are a valuable aquaculture product that provides important ecosystem benefits. Among other threats, climate-driven changes in ocean temperature can impact oyster metabolism, survivorship, and immune function. We investigated how elevated temperature impacts larval oysters during settlement (19-33 days post-fertilization), using shotgun proteomics with data-independent acquisition to identify proteins present in the oysters after 2 weeks of exposure to 23 °C or 29 °C. Oysters maintained at elevated temperatures were larger and had a higher settlement rate, with 86% surviving to the end of the experiment; these oysters also had higher abundance trends of proteins related to metabolism and growth. Oysters held at 23 °C were smaller, had a decreased settlement rate, displayed 100% mortality, and had elevated abundance trends of proteins related to immune response. This novel use of proteomics was able to capture characteristic shifts in protein abundance that hint at important differences in the phenotypic response of Pacific oysters to temperature regimes. Additionally, this work has produced a robust proteomic product that will be the basis for future research on bivalve developmental processes.

Coupled microbiome analyses highlights relative functional roles of bacteria in a bivalve hatchery.

BACKGROUND: Microbial communities are ubiquitous throughout ecosystems and are commensal with hosts across taxonomic boundaries. Environmental and species-specific microbiomes are instrumental in maintaining ecosystem and host health, respectively. The introduction of pathogenic microbes that shift microbiome community structure can lead to illness and death. Understanding the dynamics of microbiomes across a diversity of environments and hosts will help us to better understand which taxa forecast survival and which forecast mortality events. RESULTS: We characterized the bacterial community microbiome in the water of a commercial shellfish hatchery in Washington state, USA, where the hatchery has been plagued by recurring and unexplained larval mortality events. By applying the complementary methods of metagenomics and metaproteomics we were able to more fully characterize the bacterial taxa in the hatchery at high (pH 8.2) and low (pH 7.1) pH that were metabolically active versus present but not contributing metabolically. There were shifts in the taxonomy and functional profile of the microbiome between pH and over time. Based on detected metagenomic reads and metaproteomic peptide spectral matches, some taxa were more metabolically active than expected based on presence alone (Deltaproteobacteria, Alphaproteobacteria) and some were less metabolically active than expected (e.g., Betaproteobacteria, Cytophagia). There was little correlation between potential and realized metabolic function based on Gene Ontology analysis of detected genes and peptides. CONCLUSION: The complementary methods of metagenomics and metaproteomics contribute to a more full characterization of bacterial taxa that are potentially active versus truly metabolically active and thus impact water quality and inter-trophic relationships.

Physiological and molecular responses of lobe coral indicate nearshore adaptations to anthropogenic stressors.

Corals in nearshore marine environments are increasingly exposed to reduced water quality, which is the primary local threat to Hawaiian coral reefs. It is unclear if corals surviving in such conditions have adapted to withstand sedimentation, pollutants, and other environmental stressors. Lobe coral populations from Maunalua Bay, Hawaii showed clear genetic differentiation between the 'polluted, high-stress' nearshore site and the 'less polluted, lower-stress' offshore site. To understand the driving force of the observed genetic partitioning, reciprocal transplant and common-garden experiments were conducted to assess phenotypic differences between these two populations. Physiological responses differed significantly between the populations, revealing more stress-resilient traits in the nearshore corals. Changes in protein profiles highlighted the inherent differences in the cellular metabolic processes and activities between the two; nearshore corals did not significantly alter their proteome between the sites, while offshore corals responded to nearshore transplantation with increased abundances of proteins associated with detoxification, antioxidant defense, and regulation of cellular metabolic processes. The response differences across multiple phenotypes between the populations suggest local adaptation of nearshore corals to reduced water quality. Our results provide insight into coral's adaptive potential and its underlying processes, and reveal potential protein biomarkers that could be used to predict resiliency.

Temporal proteomic profiling reveals insight into critical developmental processes and temperature-influenced physiological response differences in a bivalve mollusc.

BACKGROUND: Protein expression patterns underlie physiological processes and phenotypic differences including those occurring during early development. The Pacific oyster (Crassostrea gigas) undergoes a major phenotypic change in early development from free-swimming larval form to sessile benthic dweller while proliferating in environments with broad temperature ranges. Despite the economic and ecological importance of the species, physiological processes occurring throughout metamorphosis and the impact of temperature on these processes have not yet been mapped out. RESULTS: Towards this, we comprehensively characterized protein abundance patterns for 7978 proteins throughout metamorphosis in the Pacific oyster at different temperature regimes. We used a multi-statistical approach including principal component analysis, ANOVA-simultaneous component analysis, and hierarchical clustering coupled with functional enrichment analysis to characterize these data. We identified distinct sets of proteins with time-dependent abundances generally not affected by temperature. Over 12 days, adhesion and calcification related proteins acutely decreased, organogenesis and extracellular matrix related proteins gradually decreased, proteins related to signaling showed sinusoidal abundance patterns, and proteins related to metabolic and growth processes gradually increased. Contrastingly, different sets of proteins showed temperature-dependent abundance patterns with proteins related to immune response showing lower abundance and catabolic pro-growth processes showing higher abundance in animals reared at 29 °C relative to 23 °C. CONCLUSION: Although time was a stronger driver than temperature of metamorphic proteome changes, temperature-induced proteome differences led to pro-growth physiology corresponding to larger oyster size at 29 °C, and to altered specific metamorphic processes and possible pathogen presence at 23 °C. These findings offer high resolution insight into why oysters may experience high mortality rates during this life transition in both field and culture settings. The proteome resource generated by this study provides data-driven guidance for future work on developmental changes in molluscs. Furthermore, the analytical approach taken here provides a foundation for effective shotgun proteomic analyses across a variety of taxa.

Larval Geoduck (Panopea generosa) Proteomic Response to Ciliates.

The innate immune response is active in invertebrate larvae from early development. Induction of immune response pathways may occur as part of the natural progression of larval development, but an up-regulation of pathways can also occur in response to a pathogen. Here, we took advantage of a protozoan ciliate infestation of a larval geoduck clam culture in a commercial hatchery to investigate the molecular underpinnings of the innate immune response of the larvae to the pathogen. Larval proteomes were analyzed on days 4-10 post-fertilization; ciliates were present on days 8 and 10 post-fertilization. Through comparisons with larval cultures that did not encounter ciliates, proteins implicated in the response to ciliate presence were identified using mass spectrometry-based proteomics. Ciliate response proteins included many associated with ribosomal synthesis and protein translation, suggesting the importance of protein synthesis during the larval immune response. There was also an increased abundance of proteins typically associated with the stress and immune responses during ciliate exposure, such as heat shock proteins, glutathione metabolism, and the reactive oxygen species response. These findings provide a basic understanding of the bivalve molecular response to a mortality-inducing ciliate and improved characterization of the ontogenetic development of the innate immune response.

Dynamic response in the larval geoduck (Panopea generosa) proteome to elevated pCO2.

Pacific geoducks (Panopea generosa) are clams found along the northeast Pacific coast where they are important components of coastal and estuarine ecosystems and a major aquaculture product. The Pacific coastline, however, is also experiencing rapidly changing ocean habitat, including significant reductions in pH. To better understand the physiological impact of ocean acidification on geoduck clams, we characterized for the first time the proteomic profile of this bivalve during larval development and compared it to that of larvae exposed to low pH conditions. Geoduck larvae were reared at pH 7.5 (ambient) or pH 7.1 in a commercial shellfish hatchery from day 6 to day 19 postfertilization and sampled at six time points for an in-depth proteomics analysis using high-resolution data-dependent analysis. Larvae reared at low pH were smaller than those reared at ambient pH, especially in the prodissoconch II phase of development, and displayed a delay in their competency for settlement. Proteomic profiles revealed that metabolic, cell cycle, and protein turnover pathways differed between the two pH and suggested that differing phenotypic outcomes between pH 7.5 and 7.1 are likely due to environmental disruptions to the timing of physiological events. In summary, ocean acidification results in elevated energetic demand on geoduck larvae, resulting in delayed development and disruptions to normal molecular developmental pathways, such as carbohydrate metabolism, cell growth, and protein synthesis.

Metaproteomics reveal that rapid perturbations in organic matter prioritize functional restructuring over taxonomy in western Arctic Ocean microbiomes.

We examined metaproteome profiles from two Arctic microbiomes during 10-day shipboard incubations to directly track early functional and taxonomic responses to a simulated algal bloom and an oligotrophic control. Using a novel peptide-based enrichment analysis, significant changes (p-value < 0.01) in biological and molecular functions associated with carbon and nitrogen recycling were observed. Within the first day under both organic matter conditions, Bering Strait surface microbiomes increased protein synthesis, carbohydrate degradation, and cellular redox processes while decreasing C1 metabolism. Taxonomic assignments revealed that the core microbiome collectively responded to algal substrates by assimilating carbon before select taxa utilize and metabolize nitrogen intracellularly. Incubations of Chukchi Sea bottom water microbiomes showed similar, but delayed functional responses to identical treatments. Although 24 functional terms were shared between experimental treatments, the timing, and degree of the remaining responses were highly variable, showing that organic matter perturbation directs community functionality prior to alterations to the taxonomic distribution at the microbiome class level. The dynamic responses of these two oceanic microbial communities have important implications for timing and magnitude of responses to organic perturbations within the Arctic Ocean and how community-level functions may forecast biogeochemical gradients in oceans.

Growth phase proteomics of the heterotrophic marine bacterium Ruegeria pomeroyi.

The heterotrophic marine bacterium, Ruegeria pomeroyi, was experimentally cultured under environmentally realistic carbon conditions and with a tracer-level addition of 13C-labeled leucine to track bacterial protein biosynthesis through growth phases. A combination of methods allowed observation of real-time bacterial protein production to understand metabolic priorities through the different growth phases. Over 2000 proteins were identified in each experimental culture from exponential and stationary growth phases. Within two hours of the 13C-labeled leucine addition, R. pomeroyi significantly assimilated the newly encountered substrate into new proteins. This dataset provides a fundamental baseline for understanding growth phase differences in molecular physiology of a cosmopolitan marine bacterium.

Pacific geoduck (Panopea generosa) resilience to natural pH variation.

Pacific geoduck aquaculture is a growing industry, however, little is known about how geoduck respond to varying environmental conditions, or how the industry will fare under projected climate conditions. To understand how geoduck production may be impacted by low pH associated with ocean acidification, multi-faceted environmental heterogeneity needs to be included to understand species and community responses. In this study, eelgrass habitats and environmental heterogeneity across four estuarine bays were leveraged to examine low pH effects on geoduck under different natural regimes, using targeted proteomics to assess physiology. Juvenile geoduck were deployed in eelgrass and adjacent unvegetated habitats for 30 days while pH, temperature, dissolved oxygen, and salinity were monitored. Across the four bays, pH was lower in unvegetated habitats compared to eelgrass habitats. However this did not impact geoduck growth, survival, or proteomic abundance patterns in gill tissue. Temperature and dissolved oxygen differences across all locations corresponded to differences in growth and targeted protein abundance patterns. Specifically, three protein abundance levels (trifunctional-enzyme ß-subunit, puromycin-sensitive aminopeptidase, and heat shock protein 90-α) and shell growth positively correlated with dissolved oxygen variability and inversely correlated with mean temperature. These results demonstrate that geoduck may be resilient to low pH in a natural setting, but other abiotic factors (i.e. temperature, dissolved oxygen variability) may have a greater influence on geoduck physiology. In addition this study contributes to the understanding of how eelgrass patches influences water chemistry.

Variations in Copepod Proteome and Respiration Rate in Association with Diel Vertical Migration and Circadian Cycle.

The diel vertical migration of zooplankton is a process during which individuals spend the night in surface waters and retreat to depth during the daytime, with substantial implications for carbon transport and the ecology of midwater ecosystems. The physiological consequences of this daily pattern have, however, been poorly studied beyond investigations of speed and the energetic cost of swimming. Many other processes are likely influenced, such as fuel use, energetic trade-offs, underlying diel (circadian) rhythms, and antioxidant responses. Using a new reference transcriptome, proteomic analyses were applied to compare the physiological state of a migratory copepod, Pleuromamma xiphias, immediately after arriving to the surface at night and six hours later. Oxygen consumption was monitored semi-continuously to explore underlying cyclical patterns in metabolic rate under dark-dark conditions. The proteomic analysis suggests a distinct shift in physiology that reflects migratory exertion and changes in metabolism. These proteomic analyses are supported by the respiration experiments, which show an underlying cycle in metabolic rate, with a peak at dawn. This project generates molecular tools (transcriptome and proteome) that will allow for more detailed understanding of the underlying physiological processes that influence and are influenced by diel vertical migration. Further, these studies suggest that P. xiphias is a tractable model for continuing investigations of circadian and diel vertical migration influences on plankton physiology. Previous studies did not account for this cyclic pattern of respiration and may therefore have unrepresented respiratory carbon fluxes from copepods by about 24%.

MS analysis of a dilution series of bacteria:phytoplankton to improve detection of low abundance bacterial peptides.

Assigning links between microbial activity and biogeochemical cycles in the ocean is a primary objective for ecologists and oceanographers. Bacteria represent a small ecosystem component by mass, but act as the nexus for both nutrient transformation and organic matter recycling. There are limited methods to explore the full suite of active bacterial proteins largely responsible for degradation. Mass spectrometry (MS)-based proteomics now has the potential to document bacterial physiology within these complex systems. Global proteome profiling using MS, known as data dependent acquisition (DDA), is limited by the stochastic nature of ion selection, decreasing the detection of low abundance peptides. The suitability of MS-based proteomics methods in revealing bacterial signatures outnumbered by phytoplankton proteins was explored using a dilution series of pure bacteria (Ruegeria pomeroyi) and diatoms (Thalassiosira pseudonana). Two common acquisition strategies were utilized: DDA and selected reaction monitoring (SRM). SRM improved detection of bacterial peptides at low bacterial cellular abundance that were undetectable with DDA from a wide range of physiological processes (e.g. amino acid synthesis, lipid metabolism, and transport). We demonstrate the benefits and drawbacks of two different proteomic approaches for investigating species-specific physiological processes across relative abundances of bacteria that vary by orders of magnitude.

Differential impacts of individual and combined exposures of deoxynivalenol and zearalenone on the HepaRG human hepatic cell proteome.

Numerous surveys have highlighted the natural co-occurrence of deoxynivalenol (DON) and zearalenone (ZEA) mycotoxins in food and feed. Nevertheless, data regarding cellular mechanisms involved in response to their individual and simultaneous exposures are lacking. In this study, in order to analyze how low mycotoxin doses could impact cellular physiology and homeostasis, proteomic profiles of proliferating human hepatic cells (HepaRG) exposed for 1h and 24h to low DON and ZEA cytotoxicity levels (0.2 and 20µM respectively), alone or in combination, were analyzed by LC-MS/MS. Proteome analyses of mycotoxin-treated cells identified 4000 proteins with about 1.4% and 3.7% of these proteins exhibiting a significantly modified abundance compared to controls after 1h or 24h, respectively. Analysis of the Gene Ontology biological process annotations showed that cell cycle, proliferation and/or development as well as on DNA metabolic processes were affected for most treatments. Overall, different proteins, and thus biological processes, were impacted depending on the considered mycotoxin and exposure duration. Finally, despite the important proteome changes observed following 24h exposure to both mycotoxins, only the uptake of ZEA by the cells was suggested by the mycotoxin quantification in cell supernatants. BIOLOGICAL SIGNIFICANCE: This study investigated the proteomic changes that occurred after DON and ZEA (individually and in combination) short exposures at low cytotoxicity levels in proliferating HepaRG cells using LC-MS/MS. The obtained results showed that the cellular response is time- and mycotoxin or mixture-dependent. In particular, after 1h exposure, the DON+ZEA combination led to more proteomic changes than DON or ZEA alone, whereas the opposite was observed after 24h. In addition, the significant cellular response to stress induced by ZEA after 24h exposure seemed to be reduced when combined with DON. Thus, these results supported a possible mitigation by the hepatocytes when exposed to the mycotoxin mixture for a long duration. These findings represent an essential step to further explore adaptive cell response to mycotoxin exposure using with more complex incubation kinetics and combining different "omics" tools. Moreover, as mycotoxin quantification in cell supernatants showed different behaviors for DON and ZEA, this also raises the question about how mycotoxins actually trigger the cell response.

MetaGOmics: A Web-Based Tool for Peptide-Centric Functional and Taxonomic Analysis of Metaproteomics Data.

Metaproteomics is the characterization of all proteins being expressed by a community of organisms in a complex biological sample at a single point in time. Applications of metaproteomics range from the comparative analysis of environmental samples (such as ocean water and soil) to microbiome data from multicellular organisms (such as the human gut). Metaproteomics research is often focused on the quantitative functional makeup of the metaproteome and which organisms are making those proteins. That is: What are the functions of the currently expressed proteins? How much of the metaproteome is associated with those functions? And, which microorganisms are expressing the proteins that perform those functions? However, traditional protein-centric functional analysis is greatly complicated by the large size, redundancy, and lack of biological annotations for the protein sequences in the database used to search the data. To help address these issues, we have developed an algorithm and web application (dubbed "MetaGOmics") that automates the quantitative functional (using Gene Ontology) and taxonomic analysis of metaproteomics data and subsequent visualization of the results. MetaGOmics is designed to overcome the shortcomings of traditional proteomics analysis when used with metaproteomics data. It is easy to use, requires minimal input, and fully automates most steps of the analysis-including comparing the functional makeup between samples. MetaGOmics is freely available at https://www.yeastrc.org/metagomics/.

Integrating Discovery-driven Proteomics and Selected Reaction Monitoring To Develop a Noninvasive Assay for Geoduck Reproductive Maturation.

Geoduck clams (Panopea generosa) are an increasingly important fishery and aquaculture product along the eastern Pacific coast from Baja California, Mexico, to Alaska. These long-lived clams are highly fecund, although sustainable hatchery production of genetically diverse larvae is hindered by the lack of sexual dimorphism, resulting in asynchronous spawning of broodstock, unequal sex ratios, and low numbers of breeders. The development of assays of gonad physiology could indicate sex and maturation stage as well as be used to assess the status of natural populations. Proteomic profiles were determined for three reproductive maturation stages in both male and female clams using data-dependent acquisition (DDA) of gonad proteins. Gonad proteomes became increasingly divergent between males and females as maturation progressed. The DDA data were used to develop targets analyzed with selected reaction monitoring (SRM) in gonad tissue as well as hemolymph. The SRM assay yielded a suite of indicator peptides that can be used as an efficient assay to determine geoduck gonad maturation status. Application of SRM in hemolymph samples demonstrates that this procedure could effectively be used to assess reproductive status in marine mollusks in a nonlethal manner.

Human-mediated evolution in a threatened species? Juvenile life-history changes in Snake River salmon.

Evaluations of human impacts on Earth's ecosystems often ignore evolutionary changes in response to altered selective regimes. Freshwater habitats for Snake River fall Chinook salmon (SRFCS), a threatened species in the US, have been dramatically changed by hydropower development and other watershed modifications. Associated biological changes include a shift in juvenile life history: Historically essentially 100% of juveniles migrated to sea as subyearlings, but a substantial fraction have migrated as yearlings in recent years. In contemplating future management actions for this species should major Snake River dams ever be removed (as many have proposed), it will be important to understand whether evolution is at least partially responsible for this life-history change. We hypothesized that if this trait is genetically based, parents who migrated to sea as subyearlings should produce faster-growing offspring that would be more likely to reach a size threshold to migrate to sea in their first year. We tested this with phenotypic data for over 2,600 juvenile SRFCS that were genetically matched to parents of hatchery and natural origin. Three lines of evidence supported our hypothesis: (i) the animal model estimated substantial heritability for juvenile growth rate for three consecutive cohorts; (ii) linear modeling showed an association between juvenile life history of parents and offspring growth rate; and (iii) faster-growing juveniles migrated at greater speeds, as expected if they were more likely to be heading to sea. Surprisingly, we also found that parents reared a full year in a hatchery produced the fastest growing offspring of all-apparently an example of cross-generational plasticity associated with artificial propagation. We suggest that SRFCS is an example of a potentially large class of species that can be considered to be "anthro-evolutionary"-signifying those whose evolutionary trajectories have been profoundly shaped by altered selective regimes in human-dominated landscapes.

An Alignment-Free "Metapeptide" Strategy for Metaproteomic Characterization of Microbiome Samples Using Shotgun Metagenomic Sequencing.

In principle, tandem mass spectrometry can be used to detect and quantify the peptides present in a microbiome sample, enabling functional and taxonomic insight into microbiome metabolic activity. However, the phylogenetic diversity constituting a particular microbiome is often unknown, and many of the organisms present may not have assembled genomes. In ocean microbiome samples, with particularly diverse and uncultured bacterial communities, it is difficult to construct protein databases that contain the bulk of the peptides in the sample without losing detection sensitivity due to the overwhelming number of candidate peptides for each tandem mass spectrum. We describe a method for deriving "metapeptides" (short amino acid sequences that may be represented in multiple organisms) from shotgun metagenomic sequencing of microbiome samples. In two ocean microbiome samples, we constructed site-specific metapeptide databases to detect more than one and a half times as many peptides as by searching against predicted genes from an assembled metagenome and roughly three times as many peptides as by searching against the NCBI environmental proteome database. The increased peptide yield has the potential to enrich the taxonomic and functional characterization of sample metaproteomes.