The prokaryotic and eukaryotic microbiome of Pacific oyster spat is shaped by ocean warming but not acidification.

Pacific oysters (Magallana gigas, a.k.a. Crassostrea gigas), the most widely farmed oysters, are under threat from climate change and emerging pathogens. In part, their resilience may be affected by their microbiome, which, in turn, may be influenced by ocean warming and acidification. To understand these impacts, we exposed early-development Pacific oyster spat to different temperatures (18°C and 24°C) and pCO2 levels (800, 1,600, and 2,800 µatm) in a fully crossed design for 3 weeks. Under all conditions, the microbiome changed over time, with a large decrease in the relative abundance of potentially pathogenic ciliates (Uronema marinum) in all treatments with time. The microbiome composition differed significantly with temperature, but not acidification, indicating that Pacific oyster spat microbiomes can be altered by ocean warming but is resilient to ocean acidification in our experiments. Microbial taxa differed in relative abundance with temperature, implying different adaptive strategies and ecological specializations among microorganisms. Additionally, a small proportion (~0.2% of the total taxa) of the relatively abundant microbial taxa were core constituents (>50% occurrence among samples) across different temperatures, pCO2 levels, or time. Some taxa, including A4b bacteria and members of the family Saprospiraceae in the phyla Chloroflexi (syn. Chloroflexota) and Bacteroidetes (syn. Bacteroidota), respectively, as well as protists in the genera Labyrinthula and Aplanochytrium in the class Labyrinthulomycetes, and Pseudoperkinsus tapetis in the class Ichthyosporea were core constituents across temperatures, pCO2 levels, and time, suggesting that they play an important, albeit unknown, role in maintaining the structural and functional stability of the Pacific oyster spat microbiome in response to ocean warming and acidification. These findings highlight the flexibility of the spat microbiome to environmental changes.IMPORTANCEPacific oysters are the most economically important and widely farmed species of oyster, and their production depends on healthy oyster spat. In turn, spat health and productivity are affected by the associated microbiota; yet, studies have not scrutinized the effects of temperature and pCO2 on the prokaryotic and eukaryotic microbiomes of spat. Here, we show that both the prokaryotic and, for the first time, eukaryotic microbiome of Pacific oyster spat are surprisingly resilient to changes in acidification, but sensitive to ocean warming. The findings have potential implications for oyster survival amid climate change and underscore the need to understand temperature and pCO2 effects on the microbiome and the cascading effects on oyster health and productivity.

Primary Extranodal NK/T-Cell Lymphoma Presenting as Neurolymphomatosis Involving Multiple Cranial Nerves: A Case Report.

Neurolymphomatosis (NL) is a rare condition caused by the lymphomatous or leukemic infiltration of nerves and manifests as neuropathy. Most often, NL is associated with B-lineage non-Hodgkin lymphoma (NHL) and only infrequently occurs in conjunction with T- or NK-lineage NHL. Extranodal NK/T-cell lymphoma (ENKTL)-associated NL is exceedingly unusual, with only 9 cases described in the English language literature, in addition to our case. Diagnosis of NL is challenging, as the entity can mimic neuropathies of more common etiologies, and an adequate biopsy may be difficult to obtain. Timely diagnosis demands a high index of suspicion, especially for patients without a history of hematologic malignancy. We expand upon a unique case of NL exclusively involving cranial nerves and cauda equina nerve roots, as the initial manifestation of ENKTL, and contextualize our findings within the framework of previously reported NK/T-lineage NL cases.

Paraneoplastic neurological syndromes: a single institution 10-year case series.

BACKGROUND: Given its rare incidence, there are few epidemiological case series on paraneoplastic neurologic syndromes (PNS). METHODS: We present a 10-year series compiled in the Section of Neuro-Oncology, Yale Cancer Center between 2002 and 2012. RESULTS: Twenty-five cases met the PNS Euro-network criteria for definitive PNS. Most (64%; 16/25) had no known neoplasm. Cerebrospinal fluid pleocytosis declined logarithmically over time. Neuroimaging abnormalities were seen in 88% of cases (15/17), but with delayed onset. Therapeutic benefit correlated strongly to pre-treatment modified Rankin Scale (mRS) (p < 0.01), but not with time elapsed between syndrome onset to treatment (p = 0.8), first immunotherapy modality (corticosteroids: n = 10; IVIG: n = 10; PLEX: n = 3; p = 0.37), or number of immunotherapy modalities provided (p = 0.17). PNS-related mortality was high (24%; 6/25). Nonetheless, 16% (3/18; 7 living patients censored) survived over 6 times the anticipated median expected by tumor type and stage. CONCLUSIONS: PNS are rare, at an estimated incidence of 3.1 cases per million-person-years. Detection of CSF pleocytosis and MRI abnormalities depend on time of analysis. While PNS-related mortality was high, immunotherapy benefit correlated strongly with pre-treatment mRS and long-term survival is possible.

Transcriptional responses of the marine green alga Micromonas pusilla and an infecting prasinovirus under different phosphate conditions.

Prasinophytes are widespread marine algae for which responses to nutrient limitation and viral infection are not well understood. We studied the picoprasinophyte, Micromonas pusilla, grown under phosphate-replete (0.65 ± 0.07 d-1 ) and 10-fold lower (low)-phosphate (0.11 ± 0.04 d-1 ) conditions, and infected by the phycodnavirus MpV-SP1. Expression of 17% of Micromonas genes in uninfected cells differed by >1.5-fold (q < 0.01) between nutrient conditions, with genes for P-metabolism and the uniquely-enriched Sel1-like repeat (SLR) family having higher relative transcript abundances, while phospholipid-synthesis genes were lower in low-P than P-replete. Approximately 70% (P-replete) and 30% (low-P) of cells were lysed 24 h post-infection, and expression of ≤5.8% of host genes changed relative to uninfected treatments. Host genes for CAZymes and glycolysis were activated by infection, supporting importance in viral production, which was significantly lower in slower growing (low-P) hosts. All MpV-SP1 genes were expressed, and our analyses suggest responses to differing host-phosphate backgrounds involve few viral genes, while the temporal program of infection involves many more, and is largely independent of host-phosphate background. Our study (i) identifies genes previously unassociated with nutrient acclimation or viral infection, (ii) provides insights into the temporal program of prasinovirus gene expression by hosts and (iii) establishes cell biological aspects of an ecologically important host-prasinovirus system that differ from other marine algal-virus systems.

An amplicon panel for high-throughput and low-cost genotyping of Pacific oyster.

Maintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea (Magallana) gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia, Canada. The use of the panel for parentage applications was evaluated using multiple generations of oysters from a breeding program on Vancouver Island, Canada (n = 181) and families selected for Ostreid herpesvirus-1 resistance from the Molluscan Broodstock Program in Oregon, USA (n = 136). Population characterization was evaluated using wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 190). Technical replicates showed high genotype concordance (97.5%; n = 68 replicates). Parentage analysis found suspected pedigree and sample handling errors, demonstrating the panel's value for quality control in breeding programs. Suspected null alleles were identified and found to be largely population dependent, suggesting population-specific variation impacting target amplification. Null alleles were identified using existing data without the need for pedigree information, and once they were removed, assignment rates increased to 93.0% and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output, amplitools, is also provided.

Mortality by ribosomal sequencing (MoRS) provides a window into taxon-specific cell lysis.

Microbes are by far the dominant biomass in the world's oceans and drive biogeochemical cycles that are critical to life on Earth. The composition of marine microbial communities is highly dynamic, spatially and temporally, with consequent effects on their functional roles. In part, these changes in composition result from viral lysis, which is taxon-specific and estimated to account for about half of marine microbial mortality. Here, we show that extracellular ribosomal RNA (rRNAext) is produced by viral lysis, and that specific lysed populations can be identified by sequencing rRNAext recovered from seawater samples. In ten seawater samples collected at five depths between the surface and 265 m during and following a phytoplankton bloom, lysis was detected in about 15% of 16,946 prokaryotic taxa, identified from amplicon sequence variants (ASVs), with lysis occurring in up to 34% of taxa within a water sample. The ratio of rRNAext to cellular rRNA (rRNAcell) was used as an index of taxon-specific lysis, and revealed that higher relative lysis was most commonly associated with copiotrophic bacteria that were in relatively low abundance, such as those in the genera Escherichia and Shigella spp., as well as members of the Bacteriodetes; whereas, relatively low lysis was more common in taxa that are often relatively abundant, such as members of the Pelagibacterales (i.e., SAR11 clade), cyanobacteria in the genus Synechococcus, and members of the phylum Thaumarchaeota (synonym, Nitrososphaerota) that comprised about 13-15% of the 16 S rRNA gene sequences below 30 m. These results provide an explanation for the long-standing conundrum of why highly productive bacteria that are readily isolated from seawater are often in very low abundance. The ability to estimate taxon-specific cell lysis will help explore the distribution and abundance of microbial populations in nature.

Complete Genome Sequence of Vibrio natriegens Strain PWH3a.

Vibrio natriegens strain PWH3a, isolated from the Texas Gulf Coast, is used as a model organism in marine microbiology. Here, we report the complete genome sequence of strain PWH3a, which has two circular chromosomes, 4,650 coding sequences, 34 rRNA, 4 noncoding RNA (ncRNA), 131 tRNA genes, and one Mu-like prophage sequence.

Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS).

BACKGROUND: The microbiome affects the health of plants and animals, including humans, and has many biological, ecological, and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. RESULTS: To overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that > 96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. CONCLUSION: CCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Video Abstract.

Relative genomic impacts of translocation history, hatchery practices, and farm selection in Pacific oyster Crassostrea gigas throughout the Northern Hemisphere.

Pacific oyster Crassostrea gigas, endemic to coastal Asia, has been translocated globally throughout the past century, resulting in self-sustaining introduced populations (naturalized). Oyster aquaculture industries in many parts of the world depend on commercially available seed (hatchery-farmed) or naturalized/wild oysters to move onto a farm (naturalized-farmed). It is therefore important to understand genetic variation among populations and farm types. Here, we genotype naturalized/wild populations from France, Japan, China, and most extensively in coastal British Columbia, Canada. We also genotype cultured populations from throughout the Northern Hemisphere to compare with naturalized populations. In total, 16,942 markers were identified using double-digest RAD-sequencing in 182 naturalized, 112 hatchery-farmed, and 72 naturalized-farmed oysters (n = 366). Consistent with previous studies, very low genetic differentiation was observed around Vancouver Island (mean F ST = 0.0019) and low differentiation between countries in the Japan-Canada-France historical translocation lineage (France-Canada F ST = 0.0024; Japan-Canada F ST = 0.0060). Chinese populations were more differentiated (China-Japan F ST = 0.0241). Hatchery-propagated populations had higher interindividual relatedness suggesting family structure. Within-population inbreeding was not detected on farms, but nucleotide diversity and polymorphism rate were lower in one farm population. Moving oysters from nature onto farms did not result in strong within-generation selection. Private alleles at substantial frequency were identified in several hatchery populations grown in BC, suggesting nonlocal origins. Tests of selection identified outlier loci consistent with selective differences associated with domestication, in some cases consistently identified in multiple farms. Top outlier candidates were nearby genes involved in calcium signaling and calmodulin activity. Implications of potential introgression from hatchery-farmed oysters depend on whether naturalized populations are valued as a locally adapted resource or as an introduced, invasive species. Given the value of the industry in BC and the challenges the industry faces (e.g., climate change, crop losses, biotic stressors), this remains an important question.

The marine viromes of four oceanic regions.

Viruses are the most common biological entities in the marine environment. There has not been a global survey of these viruses, and consequently, it is not known what types of viruses are in Earth's oceans or how they are distributed. Metagenomic analyses of 184 viral assemblages collected over a decade and representing 68 sites in four major oceanic regions showed that most of the viral sequences were not similar to those in the current databases. There was a distinct "marine-ness" quality to the viral assemblages. Global diversity was very high, presumably several hundred thousand of species, and regional richness varied on a North-South latitudinal gradient. The marine regions had different assemblages of viruses. Cyanophages and a newly discovered clade of single-stranded DNA phages dominated the Sargasso Sea sample, whereas prophage-like sequences were most common in the Arctic. However most viral species were found to be widespread. With a majority of shared species between oceanic regions, most of the differences between viral assemblages seemed to be explained by variation in the occurrence of the most common viral species and not by exclusion of different viral genomes. These results support the idea that viruses are widely dispersed and that local environmental conditions enrich for certain viral types through selective pressure.

Incidence and Outcomes of Intraoperative Descemet Membrane Perforations During Deep Anterior Lamellar Keratoplasty.

PURPOSE: To report the incidence and outcomes of intraoperative Descemet membrane (DM) perforations during deep anterior lamellar keratoplasty (DALK). DESIGN: Retrospective, consecutive, interventional case series. METHODS: A retrospective audit of all DALK cases performed from 2004 to 2015 in a tertiary center, with and without intraoperative DM perforations. We excluded cases with preexisting corneal perforations before surgery. RESULTS: There were a total of 540 eyes, of which 101 (18.7%) had intraoperative DM perforations. These included 79 eyes (78.2%) with microperforations and 15 eyes (14.9%) with macroperforation. The most common steps at which DM perforation occurred intraoperatively were during deep lamellar dissection (32 cases; 31.7%), air injection (27 cases; 26.7%), and suturing (21 cases; 20.8%). Management of the DM perforations included a combination of intracameral air tamponade (49 cases; 48.5%), stromal patching (10 cases; 9.9%), fibrin glue (8 cases; 7.9%), and suturing of the defect (1 case; 1.1%). There were 2 eyes (2/540; 0.37%) that were converted to penetrating keratoplasty (PK). There were no significant differences in the postoperative unaided or best-corrected visual acuity, or in the numbers of patients with postoperative graft failure, graft rejection, or subsequent surgery at postoperative years 1 and 3. CONCLUSIONS: DALK cases with DM perforations intraoperatively are often able to be managed without conversion to PK. Cases with DM perforations intraoperatively have equivalent visual acuity outcomes compared to those without DM perforations, and did not have any increased risk of graft failure, rejection, or subsequent surgery at postoperative years 1 and 3.

Cerebral aspergillosis within new tumour site presents as incidental new brain lesion in patient receiving temozolomide for glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an aggressive tumour that can lead to lymphopaenia. Its standard treatment involves temozolomide (TMZ) chemotherapy with radiation, often with addition of corticosteroids for symptomatic management. Although TMZ is also immunosuppressive, patients receiving TMZ rarely develop disseminated opportunistic infections. Here, we report the case of a patient with GBM receiving TMZ, radiotherapy and corticosteroids, who develops an incidental new brain lesion that is found to be disseminated Aspergillus within a new GBM tumour site. The patient received successful early treatment of her central nervous system aspergillosis. This case illustrates the profound immunosuppressive potential of GBM in conjunction with TMZ and corticosteroids, which can lead to high-morbidity opportunistic infections concurrently with tumour progression. Future research is needed to elucidate GBM, TMZ and corticosteroids' compound immune effects and guide management that strikes a balance between treating high-morbidity infections and continuing with immunosuppressive chemotherapy.

Isolation and Purification of Viruses Infecting Cyanobacteria Using a Liquid Bioassay Approach.

The following protocol describes the isolation and purification of viruses infecting cyanobacteria using a liquid bioassay approach. Viruses infecting cyanobacteria are also known as cyanophages. This protocol was written specifically for the isolation of cyanophages infecting freshwater cyanobacteria particularly, cyanobacteria that cannot be cultured on solid media. The use of a clonal cyanobacterial culture is recommended for the isolation of viruses. Growth conditions (i.e., media, light cycle and temperature) should be modified based on the host of interest.

Variation in the Genetic Repertoire of Viruses Infecting Micromonas pusilla Reflects Horizontal Gene Transfer and Links to Their Environmental Distribution.

Prasinophytes, a group of eukaryotic phytoplankton, has a global distribution and is infected by large double-stranded DNA viruses (prasinoviruses) in the family Phycodnaviridae. This study examines the genetic repertoire, phylogeny, and environmental distribution of phycodnaviruses infecting Micromonas pusilla, other prasinophytes and chlorophytes. Based on comparisons among the genomes of viruses infecting M. pusilla and other phycodnaviruses, as well as the genome from a host isolate of M. pusilla, viruses infecting M. pusilla (MpVs) share a limited set of core genes, but vary strongly in their flexible pan-genome that includes numerous metabolic genes, such as those associated with amino acid synthesis and sugar manipulation. Surprisingly, few of these presumably host-derived genes are shared with M. pusilla, but rather have their closest non-viral homologue in bacteria and other eukaryotes, indicating horizontal gene transfer. A comparative analysis of full-length DNA polymerase (DNApol) genes from prasinoviruses with their overall gene content, demonstrated that the phylogeny of DNApol gene fragments reflects the gene content of the viruses; hence, environmental DNApol gene sequences from prasinoviruses can be used to infer their overall genetic repertoire. Thus, the distribution of virus ecotypes across environmental samples based on DNApol sequences implies substantial underlying differences in gene content that reflect local environmental conditions. Moreover, the high diversity observed in the genetic repertoire of prasinoviruses has been driven by horizontal gene transfer throughout their evolutionary history, resulting in a broad suite of functional capabilities and a high diversity of prasinovirus ecotypes.

The Genome of the Beluga Whale (Delphinapterus leucas).

The beluga whale is a cetacean that inhabits arctic and subarctic regions, and is the only living member of the genus Delphinapterus. The genome of the beluga whale was determined using DNA sequencing approaches that employed both microfluidic partitioning library and non-partitioned library construction. The former allowed for the construction of a highly contiguous assembly with a scaffold N50 length of over 19 Mbp and total reconstruction of 2.32 Gbp. To aid our understanding of the functional elements, transcriptome data was also derived from brain, duodenum, heart, lung, spleen, and liver tissue. Assembled sequence and all of the underlying sequence data are available at the National Center for Biotechnology Information (NCBI) under the Bioproject accession number PRJNA360851A.

Metagenomic Analysis Suggests Modern Freshwater Microbialites Harbor a Distinct Core Microbial Community.

Modern microbialites are complex microbial communities that interface with abiotic factors to form carbonate-rich organosedimentary structures whose ancestors provide the earliest evidence of life. Past studies primarily on marine microbialites have inventoried diverse taxa and metabolic pathways, but it is unclear which of these are members of the microbialite community and which are introduced from adjacent environments. Here we control for these factors by sampling the surrounding water and nearby sediment, in addition to the microbialites and use a metagenomics approach to interrogate the microbial community. Our findings suggest that the Pavilion Lake microbialite community profile, metabolic potential and pathway distributions are distinct from those in the neighboring sediments and water. Based on RefSeq classification, members of the Proteobacteria (e.g., alpha and delta classes) were the dominant taxa in the microbialites, and possessed novel functional guilds associated with the metabolism of heavy metals, antibiotic resistance, primary alcohol biosynthesis and urea metabolism; the latter may help drive biomineralization. Urea metabolism within Pavilion Lake microbialites is a feature not previously associated in other microbialites. The microbialite communities were also significantly enriched for cyanobacteria and acidobacteria, which likely play an important role in biomineralization. Additional findings suggest that Pavilion Lake microbialites are under viral selection as genes associated with viral infection (e.g CRISPR-Cas, phage shock and phage excision) are abundant within the microbialite metagenomes. The morphology of Pavilion Lake microbialites changes dramatically with depth; yet, metagenomic data did not vary significantly by morphology or depth, indicating that microbialite morphology is altered by other factors, perhaps transcriptional differences or abiotic conditions. This work provides a comprehensive metagenomic perspective of the interactions and differences between microbialites and their surrounding environment, and reveals the distinct nature of these complex communities.

Genetic richness of vibriophages isolated in a coastal environment.

The purpose of this study was to characterize Vibrio parahaemolyticus viruses (VpVs) isolated from different environments within and adjacent to the Strait of Georgia, and to examine the relative influences of distance and environment on host-range and genetic richness. Nearly all seawater enrichment cultures (29/31) generated isolates, implying that VpVs were widespread in the virioplankton, yet at low abundances (< 1 l(-1)). Viruses were not detected in sediments (n = 99). Fourteen of the 16 viruses characterized were siphoviruses, with genome sizes ranging from approximately 45-106 kb, and half were capable of infecting other Vibrio species. The VpVs infected bacteria isolated from oysters and sediments fairly well (55% and 46% of the host-virus combinations, respectively), but were unable to infect many of the bacteria isolated from the water column (< 13% of 112 combinations). When compared with VpVs from oysters, it was clear that the major determinant of phenotypic (host-range) and genetic richness (by the DP-RAPD assay) was not geography, but the source environment from which the VpVs originated. Therefore, the VpV population within the Strait of Georgia is a highly diverse mixture of phenotypes and genotypes.