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Phenotypic plasticity of temperature induced Daphnia gene expression; experiment 2

ABSTRACT: This study measured the differential expression of genes from a single clonal Daphnia pulex isolate obtained from an eutrophic flooded opencast mine near Gräfenhain (Saxony, Germany) and has been kept in the laboratory since 2002. The daphniids (50 adult animals per batch) were raised in 3-L glass beakers under a 16 h:8 h L:D photoperiod. Three-quarter of the culture medium (M4) was renewed once a week, and the animals were equally fed ad libitum with algae (Desmodesmus subspicatus; SAG 53.80, Göttingen, Germany). Long-term-acclimated animals were kept at three different temperatures (i.e., 10 ± 0.5°C, 20 ± 0.3°C, and 24 ± 0.3°C) under normoxia (Po2 ≥ 20 kPa) for at least twelve weeks. Time-resolved experiments (acute heat stress) were carried out with long-term 20°C acclimated animals, which were exposed for three different time intervals (2, 4, and 8 h) to either 30 ± 0.2°C (test conditions) or 20 ± 0.3°C (control conditions). For this, 50 animals each were rapidly transferred with a sieve (mesh size: 1.2 mm) from 20°C-medium to a set of beakers containing 30°C-medium. As control, 50 animals each were transferred from 20°C-medium to another set of beakers with 20°C-medium. Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments. Animals were not fed during short-term exposures. During experiments, all animals were in good physical condition. After thermal acclimation or heat exposure, animals were transferred into 1.5-ml microcentrifuge tubes and shock-frozen in liquid nitrogen after removing adhering water with a tissue paper. Samples were short-term stored at -80°C. For all experimental conditions, four independent replicates (50 animals each) were analyzed. This GEO record is for the acute heat stress temperature contrasts between animals conditioned to 20 degrees Celsius (condition 1) versus heat shock to 30 degrees Celsius (condition 2) exposed for 2 hours (treatment 1), 4 hours (treatment 2), and 8 hours (treatment 3).

ORGANISM(S): Daphnia pulex

PROVIDER: GSE91030 | GEO | 2017/09/18

SECONDARY ACCESSION(S): PRJNA357527

REPOSITORIES: GEO

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Project description:This study measured the differential expression of genes from a single clonal Daphnia pulex isolate obtained from an eutrophic flooded opencast mine near Gräfenhain (Saxony, Germany) and has been kept in the laboratory since 2002. The daphniids (50 adult animals per batch) were raised in 3-L glass beakers under a 16 h:8 h L:D photoperiod. Three-quarter of the culture medium (M4) was renewed once a week, and the animals were equally fed ad libitum with algae (Desmodesmus subspicatus; SAG 53.80, Göttingen, Germany). Long-term-acclimated animals were kept at three different temperatures (i.e., 10 ± 0.5°C, 20 ± 0.3°C, and 24 ± 0.3°C) under normoxia (Po2 ≥ 20 kPa) for at least twelve weeks. Time-resolved experiments (acute heat stress) were carried out with long-term 20°C acclimated animals, which were exposed for three different time intervals (2, 4, and 8 h) to either 30 ± 0.2°C (test conditions) or 20 ± 0.3°C (control conditions). For this, 50 animals each were rapidly transferred with a sieve (mesh size: 1.2 mm) from 20°C-medium to a set of beakers containing 30°C-medium. As control, 50 animals each were transferred from 20°C-medium to another set of beakers with 20°C-medium. Only adult female animals with a body length of 2−2.5 mm (between the base of the apical spine and the anterior part of the head) and carrying parthenogenetic eggs and embryos were used for experiments. Animals were not fed during short-term exposures. During experiments, all animals were in good physical condition. After thermal acclimation or heat exposure, animals were transferred into 1.5-ml microcentrifuge tubes and shock-frozen in liquid nitrogen after removing adhering water with a tissue paper. Samples were short-term stored at -80°C. For all experimental conditions, four independent replicates (50 animals each) were analyzed. This GEO record is for the contrasts between animals conditioned to 10 degrees Celsius (condition 1) versus 20 degrees Celsius (condition 2), 10 degrees Celsius (condition 1) versus 24 degrees Celsius (condition 3), and 20 degrees Celsius (condition 2) versus 24 degrees Celsius (condition 3)

Project description:We probed the mechanism of cross-regulation of osmotic and heat stress responses by characterizing the effects of high osmolarity (0.3M vs. 0.0M NaCl) and temperature (43oC vs. 30oC) on the transcriptome of Escherichia coli K12 using E. coli Genome 2 Array (Affymetrix, Inc.). Independent array hybridizations were carried out for 3 biological replicates (independent cultures). Total RNA was extracted using a hot phenol-chloroform method. cDNA synthesis, fragmentation and labeling, and washing and scanning of E. coli GeneChip Arrays were performed according to the instructions of the manufacturer (Affymetrix Technical Manual, Affymetrix, Inc., USA). Labeled cDNA was hybridized to E. coli Genome 2 Array (Affymetrix, Inc.). Independent array hybridizations were carried out for 3 biological replicates (independent cultures) of each condition. A number of genes in the SoxRS and OxyR oxidative stress regulons were up-regulated by high osmolarity, high temperature, and/or by the combination of both stresses. This result could account for cross-protection of osmotic stress against oxidative stress. The trehalose biosynthetic genes were induced by both stresses, in accord with the proposed protective role of this disaccharide against thermal and oxidative damage. Experiment Overall Design: E. coli K12 strain NCM3722 cultures were grown with aeration in K medium containing 10 or 20 mM glucose. To impose osmotic stress, the osmolarity of the medium was increased to 0.64 osm by supplementation with 0.3 M NaCl. Because E. coli is a methionine auxotroph above 42oC the K medium cultures were supplemented with 0.5 mM methionine. Glycine betaine (GB) was added to the high osmolarity media to a final concentration of 1 mM. Cells taken from a single colony on LB agar were inoculated into 1ml liquid LB and grown to saturation at 37oC, then 0.05 ml were inoculated into i) K medium, 0.5 mM methionine, 10 mM glucose; ii) K medium, 0.5 mM methionine, 10 mM glucose, 0.3 M NaCl; and iii) K medium, 0.5 mM methionine, 10 mM glucose, 0.3 M NaCl, 1 mM GB. The cultures were grown to saturation at 30oC and 43oC, and sub-cultured once into the same medium and grown at the same temperatures as before. Exponentially growing cells form these cultures were then inoculated into Erlenmeyer flasks (starting OD600nm 0.05) containing 40 ml of the same medium, and incubated in the same salt and temperature conditions as used previously, except that the glucose concentration was increased to 20 mM. When the cells reached OD600nm of 0.4-0.5 (~3 doublings), 25 ml were added to a 2.5 ml mixture of cold 95% ethanol and 5% phenol to preserve RNA. The cells were cooled briefly on ice, immediately harvested by centrifugation (4oC), and the pellet was frozen on dry ice. The six different growth conditions used were: C1 - 30oC, no NaCl; C2: 30oC, 0.3 M NaCl; C3: 30oC, 0.3 M NaCl, 1 mM GB; C4: 43oC, no NaCl; C5: 43oC, 0.3 M NaCl; C6: 43oC, 0.3 M NaCl, 1 mM GB.

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Phenotypic plasticity of temperature induced Daphnia gene expression; experiment 2

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