Biology subjects

Roberts, S.

Publications and source records attributed to Roberts, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Characterization of Pro-Fibrotic Signaling Pathways using Human Hepatic Organoids

Due to the limitations of available in vitro systems and animal models, we lack a detailed understanding of the pathogenetic mechanisms and have minimal treatment options for liver fibrosis. To overcome this barrier, we engineered a live cell imaging system that identifies collagen producing cells in a human multi-lineage hepatic organoid. This system was adapted for use as a microwell-based platform (i.e., microHOs) where exposure to PDGF or TGF{beta}1 induced the formation of thick collagen fibers. Transcriptomic analysis revealed that TGF{beta}1 exposure converted mesenchymal cells into myofibroblast-like cells that contribute to the development of liver fibrosis. When pro-fibrotic intracellular signaling pathways were examined using pharmacological probes, the anti-fibrotic effect of receptor-specific tyrosine kinase inhibitors was limited to the fibrosis induced by the corresponding growth factor, which indicates that their anti-fibrotic efficacy would be limited to fibrotic diseases that were solely mediated by that growth factor. Transcriptomic and transcription factor activation analyses were used to identify pathways that were jointly activated by PDGF and TGF{beta}1. GSK3{beta} or p38 MAPK inhibitors could prevent TGF{beta}1- or PDGF-induced fibrosis in microHOs because they block intracellular signaling pathways that are commonly utilized by the TGF{beta}1 and PDGF receptors. Hence, these studies identified GSK3{beta} and p38 MAPK inhibitors as potential new broad-spectrum therapies for liver fibrosis, and it is likely that other new therapies could subsequently be identified using this microHO system.

cell biology↗

bioRxiv2023

Genetic modifiers of APOBEC-induced mutagenesis

The cytidine deaminases APOBEC3A and APOBEC3B (A3B) are prominent mutators of human cancer genomes. However, tumor-specific genetic modulators of APOBEC-induced mutagenesis are poorly defined. Here, we utilized a screen to identify 61 gene deletions that increase A3B-induced mutations in yeast. Also, we determined whether each deletion was epistatic with UNG1 loss, which indicated whether the encoded factors participate in the error-free bypass of A3B/Ung1-dependent abasic sites or suppress A3B-catalyzed deamination by protecting against aberrant formation of single stranded DNA (ssDNA). Additionally, we determined that the mutation spectra of A3B-induced mutations revealed genotype-specific patterns of strand-specific ssDNA formation and nucleotide incorporation across APOBEC-induced lesions. Combining these three metrics we were able to establish a multifactorial signature of APOBEC-induced mutations specific to (1) failure to remove H3K56 acetylation, which results in extremely high A3B-induced mutagenesis, (2) defective CTF18-RFC complex function, which results in high levels of A3B induced mutations specifically on the leading strand template that synergistically increase with loss of UNG1, and (3) defective HR-mediated bypass of APOBEC-induced lesions, which were epistatic with Ung1 loss and result from increased Rev1-mediated C-to-G substitutions. We extended these results by analyzing mutation data for human tumors and found BRCA1/2-deficient breast cancer tumors display 3- to 4-fold more APOBEC-induced mutations. Mirroring our results in yeast, for BRCA1/2 deficient tumors Rev1-mediated C-to-G substitutions are solely responsible for increased APOBEC-signature mutations and these mutations occur on the lagging strand during DNA replication. Together these results identify important factors that influence the dynamics of DNA replication and likely the abundance of APOBEC-induced mutation during tumor progression as well as a novel mechanistic role for BRCA1/2 during HR-dependent lesion bypass of APOBEC-induced lesions during cancer cell replication.

genomics↗

bioRxiv2022

Preclinical shortwave infrared tumor screening and resection via pHLIP ICG under ambient lighting conditions

There is a critical need to improve optical imaging that will lead to its widespread acceptance for routine clinical procedures. Shortwave infrared (SWIR, 900-1700nm) imaging has demonstrated clear advantages over visible and near-infrared imaging (reduced autofluorescence with improved contrast, resolution, and sensitivity at tissue depth). Here we show that the previously reported compound, pH low insertion peptide (pHLIP) conjugated to indocyanine green (ICG, pHLIP ICG) currently in clinical trials, serves as an excellent candidate for SWIR imaging protocols. SWIRs increased sensitivity enabled preclinical tumor screening and resection at exposure times as low as 0.1 ms with acceptable signal-to-noise and contrast-to-noise ratios. Imaging was performed under ambient lighting conditions, and SWIRs sensitivity enabled an extended surgical resection window up to 96 hrs post injection in an orthotopic breast cancer mouse model. This work provides a direct precedent for the clinical translation of SWIR pHLIP ICG imaging for cancer resection. One Sentence SummarySWIR imaging under ambient lighting is highly sensitive to pHLIP ICG, a cancer targeting fluorescent agent currently under clinical investigation.

cancer biology↗

bioRxiv2022

The native mussel Mytilus chilensis genome reveals adaptative molecular signatures facing the marine environment.

The blue mussel Mytilus chilensis is a key socioeconomic species inhabiting the southern coast of Chile. This endemic marine mussel supports a booming aquaculture industry, which entirely relies on artificially collected seeds from natural beds that are translocated to a diverse physical-chemical ocean conditions for farming. Furthermore, mussel production is threatened by a broad range of microorganisms, pollution, and environmental stressors that eventually impact its survival and growth. Herein, understanding the genomic basis of the local adaption is pivotal to developing sustainable shellfish aquaculture. We present a high-quality reference genome of M. chilensis, which is the first chromosome-level genome for a Mytilidae member in South America. The assembled genome size was 1.93 Gb, with a contig N50 of 134 Mb. Through Hi-C proximity ligation, 11,868 contigs were clustered, ordered, and assembled into 14 chromosomes in congruence with the karyological evidence. The M. chilensis genome comprises 34,530 genes and 4,795 non-coding RNAs. A total of 57% of the genome contains repetitive sequences with predominancy of LTR-retrotransposons and unknown elements. Comparative genome analysis was conducted among M. chilensis and M. coruscus, revealing genic rearrangements distributed into the whole genome. Notably, Steamer-like elements associated with horizontal transmissible cancer were explored in reference genomes, suggesting putative phylogenetic relationships at the chromosome level in Bivalvia. Genome expression analysis was also conducted, showing putative genomic differences between two ecologically different mussel populations. Collectively, the evidence suggests that local genome adaptation can be analyzed to develop sustainable mussel production. The genome of M. chilensis provides pivotal molecular knowledge for the Mytilus complex evolution and will help to understand how climate change can impact mussel biology.

genomics↗

bioRxiv2022

CSL-Tox: An open-source analytical framework for the comparison of short-term and long-term toxicity end points and exploring the opportunities for decreasing in-vivo studies conducted for drug development programs.

In-vivo toxicity assessment is an important step prior to clinical development and is still the main source of data for overall risk assessment of a new molecular entity (NCE). All in-vivo studies are performed according to regulatory requirements and many efforts have been exerted to minimize these studies in accordance with the (Replacement, Reduction and Refinement) 3Rs principle. Many aspects of in-vivo toxicology packages can be optimized to reduce animal use, including the number of studies performed as well as study durations, which is the main focus of this analysis. We performed a statistical comparison of adverse findings observed in 116 short-term versus 78 long-term studies in order to explore the possibility of using only short-term studies as a prediction tool for the longer-term effects. Annotation of treatment related findings was one of the challenges faced during this work. A specific focus was therefore put on the summary and conclusion sections of the reports since they contain expert assessments on whether the findings were considered adverse or were attributed to other reasons. Our analysis showed a general good concordance between short-term and long-term toxicity findings for large molecules and the majority of small molecules. Less concordance was seen for certain "target organ systems findings. While this work supports the minimization of in-vivo study durations, a larger-scale effort would be needed to provide more evidence. We therefore present the steps performed in this study as an open-source R workflow (CSL-Tox) and we provide the dataset used in the work to allow researchers to reproduce such analysis and to promote large-scale application of this study.

pharmacology and toxicology↗

bioRxiv2022

Dynamic DNA methylation contributes to carryover effects and beneficial acclimatization in geoduck clams

Given the rapidly changing global climate, there is a growing need to understand the acclimatory basis of animal response to environmental change. To investigate the role of DNA methylation in environmental acclimatization, we generated a reference genome and surveyed the physiology and DNA methylomes of juvenile geoduck clams, Panopea generosa, under differing seawater pH regimes. Juveniles were initially exposed to one of three seawater pH conditions, followed by ambient common-garden conditions, then a second reciprocal exposure to ambient pH or pH 7.4. Within 10 days of the initial low pH exposure, juvenile clams showed decreased shell size relative to ambient pH with corresponding differential DNA methylation. Following four months of ambient common-garden conditions, juveniles initially exposed to low pH compensatorily grew larger, with DNA methylation indicative of these phenotypic differences, demonstrating epigenetic carryover effects persisted months after initial exposure. Functional enrichment analysis of differentially methylated genes revealed regulation of signal transduction through widespread changes in the Wnt signaling pathways that influence cell growth, proliferation, tissue and skeletal formation, and cytoskeletal change. After 10 days of secondary exposure to pH 7.4, naive juvenile clams were more sensitive to low pH compared to those initially exposed, showing reduced growth and having nearly a 2-fold greater change in DNA methylation. Collectively, this new genomic resource and coordinated phenotypic and methylomic response support that epigenetic mechanisms underlie acclimatization to provide beneficial phenotypes.

genomics↗

bioRxiv2021

Variability in mosquito community composition associated with estuarine wetlands in Northern NSW, Australia

The Northern Rivers region of NSW, Australia, is well documented as being impacted by nuisance-biting mosquitoes and mosquito-borne disease. Mosquitoes of greatest concern are those associated with estuarine and brackish water habitats associated with coastal wetlands and understanding the spatial variability in abundance and diversity will assist the assessment of risk and inform surveillance and control programs. Adult mosquito populations were sampled, using carbon dioxide baited traps, at four locations within the Richmond River estuary at Ballina, NSW, Australia, during January and February 2021. Concomitant sampling of habitats for immature mosquitoes was also undertaken. A total of 16,467 mosquitoes was collected at all sites across two sampling periods with the most abundant mosquitoes, Verrallina funerea, Aedes vigilax, and Culex sitiens, those typically associated with estuarine environments. Culex annulirostris, a mosquito associated with freshwater habitats, and Aedes notoscriptus, a mosquito associated with water-holding containers, were also commonly collected. The mosquito communities differed, in relative abundance and species richness, between the four locations. The result highlighted the need for authorities to understand the variability in productivity of potential mosquito habitats, beyond those determinants associated with vegetation communities alone, when assessing suitable locations of mosquito surveillance and integrated mosquito management.

ecology↗

bioRxiv2021

Non-invasive Imaging of Sense of Smell by Tracking the Voltage-Gated Sodium Channel NaV1.7

The sense of smell (olfaction) is one of the most important senses for animals including humans. Despite significant advances in the understanding mechanism of olfaction, currently, there are no objective non-invasive methods that can identify loss of smell. Covid-19-related loss of smell has highlighted the need to develop methods that can identify loss of olfaction. Voltage-gated sodium channel 1.7 (NaV1.7) plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. We have identified several conditions such as chronic inflammation and viral infections such as Covid-19 that lead to loss of smell correlate with downregulation of NaV1.7 expression at transcript and protein levels in the olfactory epithelium. Leveraging this knowledge, we have developed a novel fluorescent probe Tsp1a-IR800 that targets NaV1.7. Using fluorescence imaging we can objectively measure the loss of sense of smell in live animals non-invasively. We also demonstrate that our non-invasive method is semiquantitative because the loss of fluorescence intensity correlates with the level of smell loss. Our results indicate, that our probe Tsp1a-IR800, can objectively diagnose anosmia in animal and human subjects using infrared fluorescence. We believe this method to non-invasively diagnose loss of smell objectively is a significant advancement in relation to current methods that rely on highly subjective behavioral studies and can aid in studying olfaction loss and the development of therapeutic interventions.

neuroscience↗

bioRxiv2021

Characterization of the gene repertoire and environmentally driven expression patterns in Tanner crab (Chionoecetes bairdi)

Tanner crab (Chionoecetes bairdi) are an economically important species that is threatened by ocean warming and Bitter Crab Disease, which is caused by an endoparasitic dinoflagellate, Hematodinium. Little is known about disease transmission or its link to host mortality, or how ocean warming will affect pathogenicity or host susceptibility. To provide a transcriptomic resource for the Tanner crab, we generated a suite of RNA-seq libraries encompassing pooled hemolymph samples from crab displaying differing infection statuses and maintained at different temperatures (ambient (7.5{degrees}C), elevated (10{degrees}C), or decreased (4{degrees}C)). After assembling a transcriptome and performing a multifactor differential gene expression analysis, we found genes influenced by temperature in relation to infection, and detected some of those genes over time at the individual level using RNAseq data from one crab. Biological processes associated with those genes include lipid storage, transcription, response to oxidative stress, cell adhesion, and morphogenesis. Alteration in lipid storage and transcription provide insight into how temperature impacts energy allocation in Hematodinium infected crabs. Alteration in expression patterns in genes associated with morphogenesis could suggest hemocytes were changing morphology and/or type in response to temperature. This project provides insight into how Hematodinium infection could influence crab physiology as oceans warm.

physiology↗

bioRxiv2021

The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Cellular differentiation, which is necessary for HPV life cycle completion disrupts CTCF-dependent chromatin looping of HPV18 episomes inducing enhanced activity of the HPV18 early promoter P105 and increased viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species in these cultures, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells (HPV18-{Delta}CTCF) identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF of HPV18 promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate importance of CTCF-dependent transcription regulation at multiple stages of the HPV life cycle. IMPORTANCEOncogenic human papillomavirus (HPV) infection is the cause of a subset of epithelial cancers of the uterine cervix, other anogenital areas and the oropharynx. HPV infection is established in the basal cells of epithelia where a restricted programme of viral gene expression is required for replication and maintenance of the viral episome. Completion of the HPV life cycle is dependent on the maturation (differentiation) of infected cells which induces enhanced viral gene expression and induction of capsid production. We previously reported that the host cell transcriptional regulator, CTCF, is hijacked by HPV to control viral gene expression. In this study, we use long-read mRNA sequencing to quantitatively map the variety and abundance of HPV transcripts produced in early and late stages of the HPV life cycle and to dissect the function of CTCF in controlling HPV gene expression and transcript processing.

microbiology↗

bioRxiv2021

The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Finally, the Shu complex exhibits increased affinity for 3meC-containing DNA. Thus, our work identifies a novel mechanism for coping with alkylation adducts.

genetics↗

bioRxiv2021

DNA METHYLATION PROFILING OF A CNIDARIAN-ALGAL SYMBIOSIS USING NANOPORE SEQUENCING

Symbiosis with protists is common among cnidarians such as corals and sea anemones, and is associated with homeostatic and phenotypic changes in the host that could have epigenetic underpinnings, such as methylation of CpG dinucleotides. We leveraged the sensitivity to base modifications of nanopore sequencing to probe the effect of symbiosis with the chlorophyte Elliptochloris marina on methylation in the sea anemone Anthopleura elegantissima. We first validated the approach by comparison of nanopore-derived methylation levels with CpG depletion analysis of a published transcriptome, finding that high methylation levels are associated with CpG depletion as expected. Next, using reads generated exclusively from aposymbiotic anemones, a largely complete draft genome comprising 243 Mb was assembled. Reads from aposymbiotic and symbiotic sea anemones were then mapped to this genome and assessed for methylation using the program Nanopolish, which detects signal disruptions from base modifications as they pass through the nanopore. Based on assessment of 452,841 CpGs for which there was adequate read coverage (approximately 8% of the CpGs in the genome), symbiosis with E. marina was, surprisingly, associated with only subtle changes in the host methylome. However, we did identify one extended genomic region with consistently higher methylation among symbiotic individuals. The region was associated with a DNA polymerase zeta that is noted for its role in translesion synthesis, which opens interesting questions about the biology of this symbiosis. Our study highlights the power and relative simplicity of nanopore sequencing for studies of nucleic acid base modifications in non-model species.

genomics↗

bioRxiv2020

RRM2 is a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma

Neuroblastoma is a pediatric tumor originating from the sympathetic nervous system responsible for 10-15 percent of all childhood cancer deaths. Half of all neuroblastoma patients present with high-risk disease at diagnosis. Despite intensive multi-modal therapies nearly 50 percent of high-risk cases relapse and die of their disease. In contrast to the overall paucity of mutations, high-risk neuroblastoma nearly invariably present with recurrent somatic segmental chromosome copy number variants. For several focal aberrations (e.g. MYCN and LIN28B amplification), the direct role in tumor formation has been established. However, for recurrent aberrations, such as chromosome 2p and 17q gains, the identification of genes contributing to tumor initiation or progression has been challenging due to the scarcity of small segmental gains or amplifications. In this study, we identified and functionally evaluated the ribonucleotide reductase regulatory subunit 2 (RRM2) as a top-ranked 2p putative co-driver and therapeutic target in high-risk neuroblastoma enforcing replicative stress resistance. In vitro knock down and pharmacological RRM2 inhibition highlight RRM2 dependency in neuroblastoma cells, further supported by the finding that co-overexpression of RRM2 in a d{beta}h-MYCN transgenic zebrafish line increased tumor penetrance with 80% and accelerated tumor formation. Given the critical role of RRM2 in replication fork progression and regulation of RRM2 through ATR/CHK1 signaling, we tested combined RRM2 and ATR/CHK1 small molecule inhibition with triapine and BAY1895344/prexasertib respectively, and observed strong synergism, in particular for combined RRM2 and CHK1 inhibition. Transcriptome analysis following combinatorial drugging revealed HEXIM1 as one of the strongest upregulated genes. Using programmable DNA binding of dCas9 with a promiscuous biotin ligase, RRM2 promotor bound proteins were identified including HEXIM1 and NurRD complex members, supporting a cooperative role for HEXIM1 upregulation together with CHK1 inhibition in further attenuating RRM2 expression levels. We evaluated the impact of combined RRM2/CHK1 inhibition in vivo, with treatment of a murine xenograft model showing rapid and complete tumor regression, without tumor regrowth upon treatment arrest. In conclusion, we identified RRM2 as a novel dependency gene in neuroblastoma and promising target for synergistic drug combinations with small compounds targeting DNA checkpoint regulators.

cancer biology↗

bioRxiv2020

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress which is known to induce genomic instability causing chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea induced replicative stress versus normal growth conditions in an equally-mixed cell population of isogenic euploid and 17q+ hESCs. We could show that 17q+ hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

cancer biology↗

bioRxiv2020

Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

immunology↗

bioRxiv2020

A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity In Mice

A self-transcribing and replicating RNA (STARR) based vaccine (LUNAR(R)-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolong SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell mediated immunity produced a strong viral antigen specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for IFN-{gamma} and IL-4 positive CD4+ T helper lymphocytes as well as anti-spike glycoprotein IgG2a/IgG1 ratios supported a strong Th1 dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 g and 10 g doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of Lunar-COV19 as a single dose vaccine.

immunology↗

bioRxiv2020

Ocean acidification induces subtle shifts in gene expression and DNA methylation in mantle tissue of the Eastern oyster (Crassostrea virginica)

Early evidence suggests that DNA methylation can mediate phenotypic responses of marine calcifying species to ocean acidification (OA). Few studies, however, have explicitly studied DNA methylation in calcifying tissues through time. Here, we examined the phenotypic and molecular responses in the extrapallial fluid and mantle (fluid and tissue at the calcification site) in the Eastern oyster (Crassostrea virginica) exposed to experimental OA over 80 days. Oysters were reared under three experimental pCO2 treatments ( control, 580 atm; moderate OA, 1000 uatm; high OA, 2800 atm) and sampled at 6 time points (24 hours - 80 days). We found that high OA initially induced changes in the pH of the extrapallial fluid (pHEPF) relative to the external seawater, but the magnitude of this difference was highest at 9 days and diminished over time. Calcification rates were significantly lower in the high OA treatment compared to the other treatments. To explore how oysters regulate their extrapallial fluid, gene expression and DNA methylation were examined in the mantle-edge tissue of oysters from day 9 and 80 in the control and high OA treatments. Mantle tissue mounted a significant global molecular response (both in the transcriptome and methylome) to OA that shifted through time. Although we did not find individual genes that were significantly differentially expressed to OA, the pHEPF was correlated with the eigengene expression of several co-expressed gene clusters. A small number of OA-induced differentially methylated loci were discovered, which corresponded with a weak association between OA-induced changes in genome-wide gene body DNA methylation and gene expression. Gene body methylation, however, was not significantly correlated with the eigengene expression of pHEPF correlated gene clusters. These results suggest that in C. virginica, OA induces a subtle response in a large number of genes, but also indicates that plasticity at the molecular level may be limited. Our study highlights the need to re-assess the plasticity of tissue-specific molecular responses in marine calcifiers, as well as the role of DNA methylation and gene expression in mediating physiological and biomineralization responses to OA.

molecular biology↗

bioRxiv2020

Effects of winter conditions on Olympia oyster reproduction and larval yield

For ectothermic marine invertebrates living in temperate regions, impacts of ocean warming will vary considerably by season. In many species, reproductive and metabolic processes are tightly linked to the seasonal change from winter to spring, yet we know little about how these processes will shift as winters become milder. This study examined latent effects of winter warming on spring reproduction in the Olympia oyster, Ostrea lurida. Adults were collected in autumn from central Puget Sound, WA, USA, and exposed to two winter temperatures (7{degrees}C, 10{degrees}C) in the presence of food limited (5k algal cells/mL) and food abundant (50k algal cells/mL) environments. Following treatments, adults exposed to elevated winter temperature contained larger oocytes regardless of feeding regime, and those also fed abundant food contained more developed sperm. Adults then spawned in common conditions, and larvae were reared through settlement to assess carryover effects of winter conditions on larval viability. Adults previously exposed to elevated winter temperature (10{degrees}C) produced larger larvae, particularly if they were also fed high food levels. More developed gametes and larger larvae suggest that gametogenesis occurred at low levels throughout the winter, possibly resulting in increased maternal provisioning to influence larval size. Interestingly, winter temperature did not impact larval survival, or the timing or magnitude of larval production. In the wild, more developed gametes and larger larvae following milder winters could greatly impact recruitment patterns, possibly benefitting O. lurida populations. In the hatchery setting, larval production and survival is not contingent upon winter conditions, and larval survival does not correlate with oocyte and larval size. Our results suggest that O. lurida reproduction is resilient to winter warming. Furthermore, as global temperature continues to rise, winter conditions should not be overlooked when examining reproductive cycles of O. lurida and other temperate marine invertebrates with similar reproductive cycles. Highlights of the manuscriptO_LIElevated winter temperature resulted in more developed O. lurida sperm, larger oocytes, and larger larvae. C_LIO_LIIn experimental settings, O. lurida oocyte and larval size upon release did not predict larval survival, but in the wild where rearing conditions are more challenging, winter warming could benefit wild populations by increasing recruitment. C_LIO_LIWinter temperature did not affect larval production timing or magnitude, indicating that O. lurida reproductive capacity is relatively resilient to increasing winter temperatures. C_LI

physiology↗