ABSTRACT: The goals of these studies are to explore the mechanisms that may account for evolutionary divergence of adaptive osmotic physiologies among taxa that occupy different osmotic niches. In a common-garden environment, we track genome expression responses to hyper-osmotic (brackish water) challenge during a time-course of acclimation, and contrast these responses between species. We seek to identify mechanisms that facilitate osmotic acclimation that are evolutionarily conserved between basal and derived physiologies, and also to identify mechanisms that are uniquely derived in alternate osmotic environments.
Project description:The goals of these studies are to explore the mechanisms that enable extreme physiological plasticity and that may account for evolutionary divergence of adaptive osmotic physiologies among taxa that occupy different osmotic niches. In a common-garden environment, we track physiological and genome expression responses to hypo-osmotic (freshwater) challenge during a time-course of acclimation, and contrast these responses within and between species. We seek to identify mechanisms that facilitate osmotic acclimation that are evolutionarily conserved between basal and derived physiologies, and identify mechanisms that are uniquely derived to enable the extreme osmotic plasticity exhibited by F. heteroclitus. Importantly, previous studies using a comparable experimental design have identified physiological changes and genome expression responses that are adaptive for populations of F. heteroclitus that live in fresh water. As such, this enables us to test whether mechanisms of adaptive micro-evolutionary divergence across osmotic gradients within F. heteroclitus are shared with the mechanisms that account for patterns of macro-evolutionary divergence between F. heteroclitus and F. majalis that we identify in this study. That is, are the targets of micro-evolutionary fine-tuning the same or different as the targets of macro-evolutionary divergence across osmotic boundaries? Population comparisons include between populations from Chesapeake Bay (CB), coastal Virginia (VA), and coastal Georgia (GA).
Project description:In this study we characterize the gill transcriptome changes that coincide with the arrival of contaminating oil in field-collected Gulf killifish Fundulus grandis. Gill transcription was contrasted before and after the arrival of oil, and between oil impacted and reference sites. Animals were sampled from field sites at four times. The oil impacted site is Grand Terre Island Louisiana (GT) and the two reference sites are Bay St. Louis Mississippi (BSL) and Bayou La Batre Alabama (BLB). The first timepoint (05/01/2010 to 05/09/2010) was before the arrival of contaminating oil, the second and third timepoints (06/28/2010 to 06/29/2010, and 08/30/2010 to 09/01/2010) were after the arrival of contaminating oil, and the fourth timepoint 08/28/2011) was over a year after the arrival of contaminating oil.
Project description:In this study we characterize the gill transcriptome changes in Gulf killifish (Fundulus grandis) that coincide with controlled laboratory-based exposure to various concentrations of experimentally-weathered south Louisiana crude oil. Gill transcription was contrasted between doses and across timepoints following dosing.
Project description:This is a common-garden experiment comparing the transcriptional response to hypo-osmotic acclimation among multiple populations of the killifish Fundulus heteroclitus. Original data are in the archive: E-MTAB-524.additional.zip
Project description:The invasive marine mussel Mytilus galloprovincialis has displaced the native congener Mytilus trossulus from central and southern California, but the native species remains dominant at more northerly sites that have high levels of freshwater input. To determine the extent to which interspecific differences in physiological tolerance to low salinity might explain limits to the invasive species’ biogeography, we used an oligonucleotide microarray to compare the transcriptional responses of these two species to an acute decrease in salinity. Among 6,777 genes on the microarray, 117 genes showed significant changes that were similar between species, and 12 genes showed significant species-specific responses to salinity stress. Osmoregulation and cell cycle control were important aspects of the shared transcriptomic response to salinity stress, whereas the genes with species-specific expression patterns were involved in mRNA splicing, polyamine synthesis, exocytosis, translation, cell adhesion, and cell signaling. Forty-five genes that changed expression significantly during salinity stress also changed expression during heat stress, but the direction of change in expression was typically opposite for the two forms of stress. These results (i) provide insights into the role of changes in gene expression in establishing physiological tolerance to acute decreases in salinity, and (ii) indicate that transcriptomic differences between M. galloprovincialis and M. trossulus in response to salinity stress are subtle and involve only a minor fraction of the overall suite of gene regulatory responses. Two species (Mytilus galloprovincialis, Mytilus trossulus), hypo-osmotic shock for four hours (850 mOs/kg), one control group (1000 mOs/kg) sampled at the end of the treatment exposure (850 mOs/kg), one control group (1000 mOs/kg) sampled at the beginning. Biological replicates: 6 in each treatment group, 6 in each control group. Heterologous and homologous hybridization to a microarray constructed from Mytilus californianus and Mytilus galloprovincialis sequences. A reference design that used separate pools of reference RNA for each species was employed. Reference amplified RNA (aRNA) was created for each species by pooling RNA before and after amplification. The reference pool was made up of RNA from six different samples: two base-line control samples from the beginning of the experiment, two treatment samples from the end of the four-hour hypo-osmotic exposure, and two time-control samples from the end of the four-hour exposure time. To accurately compare the transcriptomes of Mytilus galloprovincialis and M. trossulus, we chose to develop a common microarray format that could be used for both species. This microarray design consisted of probe sequences generated from the out-group species, M. californianus. M. trossulus and M. galloprovincialis are approximately 7.5 million years divergent from M. californianus, yet only 3.5 million years divergent from each other (Seed, 1992). Therefore, heterologous hybridization to the microarray allowed us to compare transcriptional responses of M. galloprovincialis and M. trossulus without the inherent sequence biases that would result from a microarray that was designed from sequences of either M. galloprovincialis or M. trossulus. A limited number of sequences (556) from ESTs from M. galloprovincialis that matched M. californianus ESTs were included on the microarray to test for the effects of sequence mismatches. Only probes that performed well for both M. galloprovincialis and M. trossulus were used in our analyses. In order to determine significant changes in expression, we conducted a two-way ANOVA, in which salinity and species were modeled as fixed effects, and focused on genes that were significant for the salinity effect or the species x temperature interaction. We ignored the species term from the ANOVA as this effect could have highlighted genes that differentially bound to probes on the microarray due to differences in sequence homology, thus not reflecting true differences in gene expression. In accordance with statistical convention, all genes with a significant species x temperature interaction were deemed not to have significant temperature effects, even if the temperature term from the ANOVA had a low P-value. All genes with FDR-corrected (Benjamini and Hochberg, 1995) P-values less than 0.05 were considered significant. Analyses were conducted in the R statistical programming environment (R Development Core Team, 2009).
Project description:Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater. Saltwater acclimation requires salmonids to make various adjustments in color, shape, size, metabolism, catabolism, and osmotic and ion regulation. The molecular mechanisms underlying this transition are largely unknown. The present study acclimated coho salmon (Oncorhynchus kisutch) to four different salinities (<0.5, 8, 16, and 32 ppth) and assessed gene expression through microarray analysis of gill, liver and olfactory tissues. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory tissues are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1,616. Gills had 1,074 differentially expressed genes and olfactory tissue had 924. The difference in the number of differentially expressed genes may be due to the higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy to fuel other metabolic and osmoregulatory tissues like gills. Differentially expressed genes were tissue and salinity treatment dependent. There were no genes differentially expressed in all salinity treatments and all three tissues. Five genes were targeted for microarray confirmation by qPCR and included CCAAT/enhancer binding protein ? (CEBPB), calpain 1 (CAPN1), proto-oncogene, serine/threonine kinase (Pim1), aldolase B, fructose-bisphosphate (aldob), and complement component 3 (c3). qPCR expression profiles of these genes matched array outputs. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significant. Most terms were tissue dependent. For liver, oxygen binding and transport terms were highlighted, suggesting possible impacts on metabolism. For gills, muscle and cytoskeleton related terms were emphasized and for olfactory tissues, immune response related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosenors and signal transduction cascades. Overall this study suggests that Pacific salmonids share many salinity acclimation molecular mechanisms with other species, with a few new genes identified, and that although the three tissues shared certain underlying mechanism, many of the differentially expressed genes were tissue-specific. To assess how salinity acclimation in coho salmon (Oncorhynchus kisutch) impacted gene expression in gills, liver, and olfactory tissues.
Project description:In this study we characterize the liver transcriptome changes in Gulf killifish (Fundulus grandis) that coincide with controlled laboratory-based exposure to various concentrations of experimentally-weathered south Louisiana crude oil. Liver transcription was contrasted between doses and across timepoints following dosing.
Project description:The goal of this study was to use microarrays to identify genes differentially regulated under conditions of formaldehyde stress relative to two other stress conditions (oxidative, osmotic) in an effort to identify genes that might be involved in a formaldehyde-specific stress response, rather than a general stress response, in the model methylotroph Methylobacterium extorquens AM1. Two color experiment, three treatments, three biological replicates per treatment, and two technical (dye swap) replicates per biological replicate: formaldehyde-stressed vs. unstressed cells; oxidative-stressed vs. unstressed cells; and osmotic-stressed vs. unstressed cells.
Project description:The angio-suppressive effect of 20(R)-ginsenoside Rg3 (Rg3-R) has been previously demonstrated, and microRNAs (miRNAs) are a vital group of small non-coding RNAs that function as post-transcriptional modulator of gene expression. Thus, using human umbilical vein endothelial cells (HUVEC) as model, we compared the microRNA (miRNA) expression profile of vascular endothelial growth factor (VEGF)-induced cells with the profile of the cell co-treated with VEGF and Rg3-R. Among the screened 553 human miRNAs, 6 up-regulated (miR-520h, miR-487b, miR-197, miR-524*, miR-342 and miR-219) and 3 down-regulated (miR-23a, miR-489 and miR-377) miRNAs were detected in Rg3-R treated vascular endothelial growth factor (VEGF)-induced HUVECs compared to VEGF alone. Real time RT-PCR was subsequently performed to verify the miRNA microarray result. Two condition experiment: VEGF-induced HUVEC and VEGF-induced HUVEC treated with Rg3-R. Three independent microarray experiments, with triplicate per microarray.
Project description:Male Wistar rats (6 weeks, 250g), (Charles River, Sulzfeld, Germany) were equipped with micro osmotic pumps model 2001 from Alzet (Cupertino, CA) containing 400mM PETN dissolved in DMSO or the solvent and infusion was maintained for four days at 1?l/h (10.5?g/kg/min). For direct comparison, rats were also infused with 450mM NTG (6.6?g/kg/min in ethanol or the solvent as a control). <br>After 4d rats were sacrificed by exsanguinations under Isofluran anesthesia (5% inhalant in room air), the heart was rapidly excised, placed in ice-cold Krebs-HEPES-solution (composition in g/l: 5.78 NaCl, 0.35 KCl, 0.37 CaCl2, 0.30 MgSO4, 2.1 NaHCO3, 0.14 K2HPO4, 5.21 HEPES and 2.0 D-glucose) and dissected.<br>Total RNA was isolated from the hearts. Direct labeled cDNA was synthezised and hybridized to Rat OpArray Microarray (total genom expression profiling).