[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 08/2015; 10(7). DOI:10.1371/journal.pone.0133053 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A single-stranded DNA (ssDNA) virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in a Forbes sea star displaying symptoms of sea star wasting disease (SSWD). The AfaCV genome organization is typical of circular Rep-encoding ssDNA (CRESS-DNA) viruses and is similar to that of members of the family Circoviridae. PCR-based surveys indicate that AfaCV is not clearly associated with SSWD, whereas the sea star-associated densovirus (SSaDV), recently implicated in SSWD in the Pacific, was prevalent in symptomatic specimens. AfaCV represents the first CRESS-DNA virus detected in echinoderms, adding to the growing diversity of these viruses recently recovered from invertebrates.
Archives of Virology 06/2015; 160(9). DOI:10.1007/s00705-015-2503-2 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sea star wasting disease (SSWD) describes a suite of symptoms reported in asteroids of the North American Pacific Coast. We performed a metatranscriptomic survey of asymptomatic and symptomatic sunflower star (Pycnopodia helianthoides) body wall tissues to understand holobiont gene expression in tissues affected by SSWD. Metatranscriptomes were highly variable between replicate libraries, and most differentially expressed genes represented either transcripts of associated microorganisms (particularly Pseudomonas and Vibrio relatives) or low-level echinoderm transcripts of unknown function. However, the pattern of annotated host functional genes reflects enhanced apoptotic and tissue degradation processes and decreased energy metabolism, while signalling of death-related proteins was greater in asymptomatic and symptomatic tissues. Our results suggest that the body wall tissues of SSWD-affected asteroids may undergo structural changes during disease progression, and that they are stimulated to undergo autocatalytic cell death processes.
PLoS ONE 05/2015; 10(5):e0128150. DOI:10.1371/journal.pone.0128150 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intense annual spring phytoplankton blooms and thermohaline stratification lead to anoxia in Chesapeake Bay bottom waters. Once oxygen becomes depleted in the system, microbial communities use energetically favorable alternative electron acceptors for respiration. The extent to which changes in respiration are reflected in community gene expression have only recently been investigated. Metatranscriptomes prepared from near-bottom water plankton over a four month time series in central Chesapeake Bay demonstrated changes consistent with terminal electron acceptor availability. The frequency of respiration-related genes in metatranscriptomes was examined by BLASTx against curated databases of genes intimately and exclusively involved in specific electron acceptor utilization pathways. The relative expression of genes involved in denitrification and dissimilatory nitrate reduction to ammonium were coincident with changes in nitrate, nitrite and ammonium concentrations. Dissimilatory iron and manganese reduction transcript ratios increase during anoxic conditions and corresponded with the highest soluble reactive phosphate and manganese concentrations. The sulfide concentration peaked in late July and early August and also matched dissimilatory sulfate reduction transcript ratios. We show that rather than abrupt transitions between terminal electron acceptors, there seems to be substantial overlap in time and space of these various anaerobic respiratory processes in Chesapeake Bay.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Populations of at least 20 asteroid species on the Northeast Pacific Coast have recently experienced an extensive outbreak of sea-star (asteroid) wasting disease (SSWD). The disease leads to behavioral changes, lesions, loss of turgor, limb autotomy, and death charac-terized by rapid degradation ("melting"). Here, we present evidence from experimental challenge studies and field observations that link the mass mortalities to a densovirus (Parvoviridae). Virus-sized ma-terial (i.e., <0.2 μm) from symptomatic tissues that was inoculated into asymptomatic asteroids consistently resulted in SSWD signs whereas animals receiving heat-killed (i.e., control) virus-sized inoc-ulum remained asymptomatic. Viral metagenomic investigations revealed the sea star-associated densovirus (SSaDV) as the most likely candidate virus associated with tissues from symptomatic asteroids. Quantification of SSaDV during transmission trials indi-cated that progression of SSWD paralleled increased SSaDV load. In field surveys, SSaDV loads were more abundant in symptomatic than in asymptomatic asteroids. SSaDV could be detected in plank-ton, sediments and in nonasteroid echinoderms, providing a possible mechanism for viral spread. SSaDV was detected in museum speci-mens of asteroids from 1942, suggesting that it has been present on the North American Pacific Coast for at least 72 y. SSaDV is therefore the most promising candidate disease agent responsible for asteroid mass mortality. virus | Asteroidea | disease | densovirus | wasting
Proceedings of the National Academy of Sciences 11/2014; 111(48). DOI:10.1073/pnas.1416625111/-/DCSupplemental · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background/Question/Methods
Skin microbial communities provide protection against pathogens, and regulate physiological processes and immune responses. We hypothesized that ontogenetic and seasonal shifts can alter the balance between “protective” and “harmful” bacteria (also known as dysbiosis), leading to increased rates of infection to pathogens. To test our hypothesis, we examined the transition of skin microbial communities between periods of immunesupression, either caused by development or environmental stress, in declining amphibians affected by the pathogenic fungus Batrachochytrium dendrobatidis. We used automated ribosomal intergenic spacer analyses (ARISA) and Illumina MiSeq sequencing to quantify microbial diversity and composition in Eleutherodactylus coqui and Lithobates yavapaiensis, two anurans with well-characterized infection dynamics but different life histories.
Using ARISA, we found similar responses in both species where immune-suppressed hosts (juveniles and winter-collected frogs) exhibited increased operational taxonomic unit (OTU) richness, diversity, and evenness. In addition, skin microbial community structure overlapped very little between ages in E. coqui and seasons in L. yavapaiensis, suggesting drastic shifts driven by the addition or loss of specific OTUs or by changes in their relative abundances. Juvenile E. coqui frogs lost eight unique OTUs not present in adults, whereas adult L. yavapaiensis gained 13 unique OTUs from summer to winter. By amplifying the V4 region of 16S ribosomal RNA, we found significant differences in core microbiota for E. coqui. Acinetobacter johnsonii formed part of the core microbiome of juveniles, whereas Pseudomonadaceae dominated in adults. Unfortunately, due to insufficient template material, we were only able to sequence four winter-collected L. yavapaiensis, precluding phylotype analyses between seasons. Our findings indicate that immune suppression during susceptible states is accompanied by the addition of new microbial taxa which causes skin dysbioses in amphibians. Identifying whether these microbial community changes promote health or disease requires further functional investigation, particularly if future conservation strategies are to include the use of probiotics in successful disease mitigation.
[Show abstract][Hide abstract] ABSTRACT: Annual intense spring phytoplankton blooms and thermohaline stratification lead to anoxia in Chesapeake Bay bottom waters. Once oxygen becomes depleted in the system, microbial communities utilize energetically favorable alternative electron acceptors for respiration. The extent to which changes in respiration are reflected in community gene expression have only recently been investigated. The relative transcript abundance of respiration-related genes in bottom water metatranscriptomes was examined by BLASTx against curated databases of genes intimately and exclusively involved in specific electron acceptor utilization pathways. The relative expression of transcripts involved in denitrification and dissimilatory nitrate reduction to ammonium were coincident with changes in NO3-, NO2- and NH4+ concentrations. Dissimilatory iron and manganese reduction transcript ratios increased during anoxic conditions and corresponded with the highest soluble reactive PO43- and Mn(II) concentrations. The sulfide concentration peaked in late July and early August and also matched dissimilatory sulfate reduction transcript ratios. This study provides evidence that anaerobic respiratory transcript ratios correspond to the environmental availability of the terminal electron acceptor of highest potential energy yield in Chesapeake Bay.
[Show abstract][Hide abstract] ABSTRACT: Cyanobacteria are biogeochemically significant constituents of coral reef ecosystems, however, little is known about biotic and abiotic factors influencing the abundance and composition of cyanobacterial communities in fringing coral reef waters. To understand patterns of cyanobacterial biogeography in relation to coastal environmental factors, we examined the diversity of planktonic and benthic cyanobacteria at 12 sites along the west coast of Hawaii's Big Island. We found distinct cyanobacterial communities in sediments compared to the water column. In both sediments and water, community structure was strongly related to overall biomass (chlorophyll a concentration), though both these communities corresponded to different sets of biotic/abiotic variables. To examine the influence of freshwater input on planktonic cyanobacterial communities, we conducted a mesocosm experiment where seawater was amended with freshwater from two sources representing high- and low-human population influence. Planktonic cyanobacterial abundance decreased over time in mesocosms, though chlorophyll a concentration significantly increased with time, indicating cyanobacteria were likely outcompeted by other phytoplankton in incubations. Our results show that cyanobacterial community structure may be affected by runoff from terrestrial habitats, but that the composition of cyanobacterial communities inhabiting these locations are also structured by factors not measured in this study.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: A sea star wasting disase epizootic was first noted in June 2013 and continues to present (February 2014) and involves 12 species of sea stars (Asteroidea) on the Pacific Coast. This is leading to localized mass mortality events in some species and locations. The dominant species affected are Pycnopodia helianthoides, Evasterias troschelli and Pisaster ochraceus. Mortalities and signs of wasting were first reported in Pisaster ochraceus on the outer coast of Washington State, followed by mass mortality of Pycnopodia helianthoides north of Vancouver BC. By mid September, Pisaster ochraceus were affected in Central California and a mass mortality event involving 12 species of stars occurred in November in Central California. Localized mass mortality events continue in Washington State, British Columbia, Central and southern California waters. Diversity of viruses and bacteria in diseased and healthy sea stars are being investigated in the Hewson lab using metagenomics, community fingerprinting of bacteria and quantification of constituent genotypes. Histology and preliminary bacterial metagenomics allows us to rule out fungi, protozoans, larger parasites and ricketsial bacteria as causative. Infection experiments are underway to test different fractions.
Ocean Sciences Meeting, Honolulu, Hawaii USA; 02/2014
[Show abstract][Hide abstract] ABSTRACT: Our goal is to strengthen the foundations of metaproteomics as a microbial community analysis tool that links the functional identity of actively expressed gene products with host phylogeny. We used shotgun metaproteomics to survey waters in six disparate aquatic habitats (Cayuga Lake, NY; Oneida Lake, NY; Gulf of Maine; Chesapeake Bay, MD; Gulf of Mexico; and the South Pacific). Peptide pools prepared from filter-gathered microbial biomass, analyzed by nano-liquid chromatography-mass spectrometry (MS/MS) generating 9,693 ± 1,073 mass spectra identified 326 ± 107 bacterial proteins per sample. Distribution of proteobacterial (Alpha and Beta) and cyanobacterial (Prochlorococcus and Synechococcus spp.) protein hosts across all six samples was consistent with the previously published biogeography for these microorganisms. Marine samples were enriched in transport proteins (TRAP-type for dicarboxylates and ATP binding cassette (ABC)-type for amino acids and carbohydrates) compared with the freshwater samples. We were able to match in situ expression of many key proteins catalyzing C-, N-, and S-cycle processes with their bacterial hosts across all six habitats. Pelagibacter was identified as the host of ABC-type sugar-, organic polyanion-, and glycine betaine-transport proteins; this extends previously published studies of Pelagibacter's in situ biogeochemical role in marine C- and N-metabolism. Proteins matched to Ruegeria confirmed these organism's role in marine waters oxidizing both carbon monoxide and sulfide. By documenting both processes expressed in situ and the identity of host cells, metaproteomics tested several existing hypotheses about ecophysiological processes and provided fodder for new ones.
[Show abstract][Hide abstract] ABSTRACT: To assess the effects of seasonal oxygen transition on microbial metabolism, we measured spatiotemporal changes in total dissolved inorganic carbon, respiratory products, and geochemical constituents in the mesohaline region of Chesapeake Bay from May to October 2010. Vertical redox zonation was examined, and a spatial transect survey was also conducted from the southern to northern limit of the mesohaline region in July providing an alternative approach for assessing the temporal dynamics of oxygen transition. The transitions from oxic to hypoxic to anoxic and back to oxic were illustrated by the pattern of nitrogen redox species. Respiration, measured from changes in total dissolved inorganic carbon (∆DIC) and dissolved oxygen (∆DO) during incubations, had an average respiratory quotient (∆DIC/∆DO) of 1.04 ± 0.06 under oxic conditions and 1.58 ± 0.48 under hypoxic conditions. The difference in the respiratory quotients suggested that oxygen-based respiration measurements would underestimate community respiration rates in hypoxic conditions. In the present study, we observed within the surface-mixed layer three- to sevenfold differences in temporal and vertical variation of community respiration, while net respiration across oxyclines and anaerobic respiration in deep waters had lower rates and variability. In some anoxic samples, there was a net decrease in dissolved inorganic carbon that was exacerbated with experimental augmentation of terminal electron acceptors. Potential carbon fixation rates of chemoautotrophs within and below oxyclines were estimated and ranged from 0.63 to 116.9 mg C m−2 day−1 depending on growth conditions. These results indicate that anaerobic metabolism during the seasonal anoxic transition and at oxic/anoxic interface can play an important role in the estuarine carbon cycle.
[Show abstract][Hide abstract] ABSTRACT: Echinoderms are important constituents of marine ecosystems, where they may influence the recruitment success of benthic flora and fauna, and are important consumers of detritus and plant materials. There are currently no described viruses of echinoderms. We used a viral metagenomic approach to examine viral consortia within three urchins - Colobocentrotus atratus, Tripneustes gratilla, and Echinometra mathaei - which are common constituents of reef communities in the Hawaiian archipelago. Metagenomic libraries revealed the presence of bacteriophage and densoviruses (Parvoviridae) in tissues of all three urchins. Densoviruses are typically known to infect terrestrial and aquatic arthropods. Urchin-associated densoviruses were detected by qPCR in all tissues tested, and were also detected in filtered suspended matter (> 0.2µm) from plankton and in sediments at several locations near to where urchins were collected for metagenomic analysis. This is the first report of echinoderm-associated viruses, which extends the known host range of parvoviruses.
Journal of General Virology 12/2013; 95(Pt_3). DOI:10.1099/vir.0.060780-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circular replication initiator protein (rep)-encoding ssDNA (CRESS-DNA) viruses have been widely reported in viral metagenomic surveys and in association with invertebrate zooplankton in freshwater and marine habitats. However, there have been no systematic or quantitative studies of their distribution in marine waters. We investigated the distribution of CRESS-DNA viruses in net plankton (>64 mu m) collected from geographically widespread locations, using quantitative PCR that targets viral genotypes previously recovered from soil, freshwater and estuarine free-living viruses, and viruses associated with arthropod tissues. We detected CRESS-DNA viruses in most net plankton samples except for a sample containing only the cyanobacterium Trichodesmium. Soil and freshwater plankton CRESS-DNA viruses were detected only at sites with substantial freshwater and runoff effects, while 2 CRESS-DNA viruses recovered from plankton of the Chesapeake Bay were detected in most net plankton tested. CRESS-DNA viruses recovered from marine copepods, the freshwater cladoceran Daphnia and the freshwater amphipod Diporeia were primarily detected in habitats where similar hosts were observed in zooplankton counts. Our data suggest that CRESS-DNA viruses previously recovered from invertebrate tissues and from virioplankton may be widely distributed in plankton >64 mu m, providing evidence for a zooplankton origin of this viral group.
[Show abstract][Hide abstract] ABSTRACT: We used metatranscriptomics to study the gene transcription patterns of microbial plankton (0.2 - 64 μm) at a mesohaline station in Chesapeake Bay under transitions from oxic to anoxic waters in spring, and anoxic to oxic waters in autumn. Samples were collected from surface [i.e. above pycnocline] waters (3m) and waters beneath the pycnocline (16 - 22 m) in both 2010 and 2011. Metatranscriptome profiles based on function and potential phylogeny were different between 2010 and 2011, and strongly variable in 2011. This difference in variability corresponded with a highly variable ratio of eukaryotic to bacterial sequences (0.3 - 5.5), reflecting transient algal blooms in 2011 that were absent in 2010. The similarity between metatranscriptomes changed at a lower rate during the transition from oxic to anoxic waters than after the return to oxic conditions. Transcripts related to photosynthesis and low-affinity cytochrome oxidases were significantly higher in shallow than in deep waters, while in deep water genes involved in anaerobic metabolism, particularly sulfate reduction, succinyl to propionyl CoA conversion, and menaquinone synthesis were enriched relative to shallow waters. Expected transitions in metabolism between oxic and anoxic deep waters were reflected in elevated anaerobic respiratory reductases and utilization of propenediol and acetoin. The percentage of archaeal transcripts increased in both years in late summer (0.1 - 4.4 % of all transcripts in 2010 and from 0.1 to 6.2 % in 2011). Denitrification-related genes were expressed in a predicted pattern during the oxic-anoxic transition. Overall, our data suggest that Chesapeake Bay microbial assemblages express gene suites differently in shallow and deep waters, and that differences in deep waters reflect variable redox states.
[Show abstract][Hide abstract] ABSTRACT: The benthic amphipod Diporeia is an ecologically and biogeochemically important constituent of deep freshwater lakes in North America. The proliferation of dreissenid mussels in the mid-1990s coincided with a sharp decrease in Diporeia populations in several Laurentian Great Lakes; however the ultimate cause and mechanisms of their decline are still unknown. Here we examined the composition of DNA viruses associated with Diporeia collected from populations of Lake Michigan that had declined and stable populations in Lake Superior and Owasco Lake (Finger Lake in central New York State). Viral metagenomic libraries from Owasco Lake and Lake Superior were comprised primarily of bacteriophages, which may infect bacteria within the amphipod microbiome. In contrast, the metagenomic library from Lake Michigan contained well-represented ssDNA circular viral genomes. The prevalence and viral load of one putative Type V ssDNA circular virus (LM29173) that recruited almost 30% of total viral sequence reads in the Lake Michigan library was analyzed by quantitative PCR. The prevalence of LM29173 was over two orders of magnitude greater in Lake Michigan compared to the other two lakes. Although further research is necessary to establish the pathology and epidemiological extent of viral-Diporeia interactions, our data suggest that viruses may be numerically significant constituents of the Diporeia microbiome, and if pathogenic some of these viruses may be a stressor of Great Lakes Diporeia populations. Our data further indicate that special attention should be given to the circovirus that was prevalent in the declining Michigan population but uncommon in the other two lakes.
Journal of Great Lakes Research 09/2013; 39(3):499–506. DOI:10.1016/j.jglr.2013.06.006 · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used a metagenomic approach to identify viruses that may be involved in the ecology of Daphnia spp. in Oneida and Cayuga lakes (upstate New York). We identified several highly represented, putative eukaryotic, circular, single-stranded deoxyribonucleic acid (DNA) viral genomes. Among these, we discovered a genotype similar in both sequence and genomic architecture to a virus previously reported from a hyperthermal lake that shares characteristics of both single-stranded ribonucleic acid (RNA) and single-stranded DNA viruses. We used quantitative polymerase chain reactions to study the prevalence and viral load of both positive-sense and negative-sense strands of the Daphnia mendotae-associated (Cladocera) hybrid virus (DMClaHV) over a summer season in Oneida and Cayuga lakes. DMClaHV had high prevalence within Daphnia populations, where viral load and the proportion of virus-positive individuals were higher preceding host population decline. DMClaHV viral load was different between two species of Daphnia (D. mendotae and D. retrocurva), and the dynamics between viruses and their hosts varied between the two lakes. We detected DMClaHV in eggs (ephippia) retrieved from Oneida Lake sediments with an estimated age of 30 yr. Using transmission electron microscopy, we observed small (20 nm diameter) virus-like particles in Daphnia that were well away from gut tissues and not associated with intracellular parasites. Because Daphnia plays a critical role in many lake ecosystems, DMClaHV may have important effects on herbivory and thus carbon flow through the lake ecosystem.
Limnology and Oceanography 09/2013; 58(5):1605-1620. DOI:10.4319/lo.2013.58.5.1605 · 3.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Annual spring phytoplankton blooms ultimately lead to anoxic stratification in Chesapeake Bay bottom waters, which in turn triggers shifts in microbial respiration to inorganic ions and metals. The extent to which these changes in respiration are reflected in host community gene expression are not known. Plankton metatranscriptomes generated from near-bottom water samples taken during a four month time series in the central Chesapeake Bay demonstrate changes consistent with terminal electron acceptor availability. The relative expression of genes involved in nitrogen processes (denitrification, DNRA and anammox) follow changes in nitrate, nitrite and ammonium concentrations. Iron and manganese reduction gene expression increase during anoxic conditions and show a relationship with the highest soluble reactive phosphorus concentration. Sulfide concentration peaks in late July and early August also match sulfur respiration gene expression. This study provides metatranscriptomic evidence for anaerobic respiration pathways correlated to chemical profiles of highest potential energy yield for bacterioplankton in Chesapeake Bay.
[Show abstract][Hide abstract] ABSTRACT: As dominant members of marine mesozooplankton communities, copepods play critical roles in oceanic food webs and biogeochemical cycling. Despite the ecological significance of copepods, little is known regarding the causes of copepod mortality, and up to 35% of total copepod mortality cannot be accounted for by predation alone. Viruses have been established as ecologically important infectious agents in the oceans; however, viral infection has not been investigated in mesozooplankton communities. Here we used molecular and microscopic techniques to document viral infection in natural populations of the calanoid copepods Acartia tonsa (Dana) and Labidocera aestiva (Wheeler) in Tampa Bay, FL. Viral metagenomics revealed previously undocumented viruses in each species, named Acartia tonsa copepod circo-like virus (AtCopCV) and Labidocera aestiva copepod circo-like virus (LaCopCV). LaCopCV was found to be extremely prevalent and abundant in L. aestiva populations, with up to 100% prevalence in some samples and average viral loads of 1.13 × 10(5) copies per individual. LaCopCV transcription was also detected in the majority of L. aestiva individuals, indicating viral activity. AtCopCV was sporadically detected in A. tonsa populations year-round, suggesting temporal variability in viral infection dynamics. Finally, virus-like particles of unknown identity were observed in the connective tissues of A. tonsa and L. aestiva by transmission electron microscopy, demonstrating that viruses were actively proliferating in copepod connective tissue as opposed to infecting gut contents, parasites, or symbionts. Taken together, these results provide strong independent lines of evidence for active viral infection in dominant copepod species, indicating that viruses may significantly influence mesozooplankton ecology.
Proceedings of the National Academy of Sciences 01/2013; 110(4). DOI:10.1073/pnas.1216595110 · 9.67 Impact Factor