Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression microarray analysis of heat stress in the soil invertebrate Folsomia candida

ABSTRACT: Temperature is an important ecological condition, and sudden temperature changes in soil can induce stress in soil-dwelling invertebrates. Soil animals can move to more favorable habitats and/or adapt physiologically to a stressful environment. Hyperthermic conditions will impact gene expression as one of the first steps. We use a transcriptomics approach to identify the transcripts of which expression changed in response to heat stress in the springtail Folsomia candida using a 5,131 probe microarray. A temperature shift from 20Â°C to 30Â°C for 30 minutes significantly altered the expression of 142 genes, of which 116 were upregulated, and 26 downregulated. Many upregulated genes encoded heat shock proteins (Hsps) or enzymes involved in the synthesis of ATP, such as members of the electron transport chain. Furthermore, genes involved in oxidative stress and anion-transporting ATPases were upregulated. Downregulated were glycoside hydrolases, involved in catalysis of certain disaccharides, which indicate an accumulation of stress-protective disaccharides. The microarray results from this study, which were validated using quantitative RT PCR, reveal a mild response to heat shock in this soil invertebrate, relative to other organisms. This may be due to specific ecological factors during evolution of soil invertebrates, such as the relatively stable temperatures in the soil habitat. This study presents potential candidate genes for future functional studies concerning thermal stress in soil-dwelling invertebrates, like e.g., the investigation of the heat hardening process. Folsomia candida was first acclimated to LUFA 2.2 soil at 20 C for two days. Then animals were either exposed to 30 C for 30 minutes (heat shock treated), or were not heat shocked (reference). For each treatment 4 biological replicates were used, replicate samples consisted of total RNA extracted from ~30 animals exposed in the same jar to either reference or heat shock. Each unique heat shock treated sample was hybridized to a unique reference sample. In total in 4 hybridizations of 8 samples, was performed. The dyes were evenly distributed, which means that for each treatment two samples were labeled with cy3 and the other two with cy5.

ORGANISM(S): Folsomia candida

SUBMITTER: Benjamin Nota

PROVIDER: E-GEOD-17024 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Gene expression microarray analysis of heat stress in the soil invertebrate Folsomia candida.

Nota B B van Straalen N M NM Ylstra B B Roelofs D D

Insect molecular biology 20100114 3

Sudden temperature changes in soil can induce stress in soil-dwelling invertebrates. Hyperthermic conditions have an impact on gene expression as one of the first steps. We use a transcriptomics approach using microarrays to identify expression changes in response to heat in the springtail Folsomia candida. An elevation of temperature (Delta 10 degrees C) altered the expression of 142 genes (116 up-, 26 down-regulated). Many up-regulated genes encoded heat shock proteins, enzymes involved in ATP ...[more]

PMID: 20074298

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Project description:Polycyclic aromatic hydrocarbons are common pollutants in soil, have negative effects on soil ecosystems, and are potentially carcinogenic. The Springtail (Collembola) Folsomia candida is often used as an indicator species for soil toxicity. Here we report a toxicogenomic study that translates the ecological effects of the polycyclic aromatic hydrocarbon phenanthrene in soil to the early transcriptomic responses in Folsomia candida. Microarrays were used to examine two different exposure concentrations of phenanthrene, namely the EC10 (24.95 mg kg-1 soil) and EC50 (45.80 mg kg-1 soil) on reproduction of this springtail, which evoked 405 and 251 differentially expressed transcripts, respectively. Fifty transcripts were differential in response to either concentration. Many transcripts encoding xenobiotic detoxification and biotransformation enzymes (phases I, II, and III) were upregulated in response to either concentration. Furthermore, indications of general and oxidative stress were found in response to phenanthrene. Chitin metabolism appeared to be disrupted particularly at the low concentration, and protein translation appeared suppressed at the high concentration of phenanthrene; most likely in order to reallocate energy budgets for the detoxification process. Finally, an immune response was evoked especially in response to the high effect concentration, which was also described in a previous transcriptomic study using the same effect concentration (EC50) of cadmium. Our study provides new insights in the molecular mode of action of the important polluting class of polycyclic aromatic hydrocarbons in soil animals. Furthermore, we present a fast, sensitive, and specific soil toxicity test which enhances traditional tests and may help to improve current environmental risk assessments and monitoring of potentially polluted sites. Folsomia candida was exposed to phenanthrene spiked soil or untreated (reference/control) soil for 2 days. Two different concentrations of phenanthrene were used, 24.95 and 45.80 mg/kg soil which represent the EC10 and EC50 on reproduction, respectively. For each concentration treatment 4 biological replicates were used, replicate samples consisted of total RNA extracted from ~30 animals exposed in the same jar to either reference or phenanthrene spiked soil. Phenanthrene treated samples were always hybridized to reference samples in an evenly distributed dye-swap manner, which resulted in total in 8 hybridizations of 16 samples.

Project description:Field-selected tolerance to heavy metals has been reported for Orchesella cincta (Arthropoda: Collembola) populations occurring at metal-contaminated mining sites. This tolerance is correlated with heritable increase of metal excretion efficiency; less pronounce cadmium induced growth reduction and, over-expression of the metallothionein gene. We applied transcriptomics to determine differential gene expression caused by this abiotic stress in reference and cadmium tolerant populations. Many cDNAs responded to cadmium exposure in a reference population. Significantly fewer clones were cadmium responsive in tolerant animals. Analysis of variance revealed transcripts that interact between cadmium exposure and population. Hierarchical clustering of these clones revealed two major groups. The first one contained cDNAs that were up regulated by cadmium in the reference culture, but non-responsive or down regulated in tolerant animals. This cluster was also characterized by elevated constitutive expression in the tolerant population. Gene ontology analysis revealed that these cDNAs were involved in structural integrity of the cuticle, anti-microbial defense, calcium-channel blocking, neurotransmitter transport, chromatin remodeling and, endoplasmatic vesicle activity. The second group consisted of cDNAs down regulated in reference animals but not responding or slightly up regulated in tolerant animals. Their functions involved carbohydrate metabolic processes, Ca2+ dependent stress signaling, proteolysis and digestion. The reference population showed a strong signature of stress-induced genome-wide perturbation of gene expression, whereas the tolerant animals maintained normal gene expression upon cadmium exposure. We confirmed the micro-evolutionary processes occurring in soil arthropod populations and suggest a major contribution of gene regulation to the evolution of a stress-adapted phenotype. Orchesella cincta (Collembola) from the laboratory population (LC) at the department of Animal Ecology, Vrije Universiteit Amsterdam were taken as the reference group. This population originated from a pine forest from the reference area (Roggebotzand, the Netherlands, latitude N = 52 º 34’17’’, longitude E = 5 º 47’56’’) that contains on average < 0.5 μg cadmium per gram litter and humus. Tolerant animals were collected from randomly selected litter samples at two areas of the abandoned, but still heavily polluted lead/zinc mining site of Plombières (Belgium, latitude N = 50˚44’03’’, longitude E = 5˚58’02’’): the locations contain an average cadmium concentration between 10 and 52 μg.g-1 soil (Lock et al. 2003; Sterenborg 2003; Van Straalen et al. 1987). To diminish putative environmental effects from the field the animals were reared in a climate room (20°C, 75% humidity, LD 12 h:12 h) in PVC jars on a moist plaster of Paris layer feeding on algae present on twigs for at least three generations before experimental treatment. Animals were exposed to cadmium (nominal concentration of 112,4 μg cadmium.g-1 dry algae) for 3 days, directly after molting, via algal food. Control groups received the same treatment except that water was spiked into the algal food instead of cadmium solution. The cadmium exposure concentration is two times the no observed effect concentration (NOEC) for reproduction in chronically exposed animals. Total RNA was extracted from times 12 pooled animals per treatment using SV Total RNA Isolation system (Promega) and quantified on a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies). Total RNA was visualized on a 1.5% agarose gel to verify its integrity. Each experimental group (pool of 12 animals) was replicated 8 times. Dyes were swapped between biological replicates: 4 Cy-3 labeled replicates and 4 Cy-5 lebeled replicates per experimental group Microarray experiment was designed in a closed loop Lab culture clean vs Cd, Lab culture Cd vs Tolerant culture Cd, Tolerant culture Cd vs Tolerant culture clean, Tolerant culture clean vs lab culture clean

Dataset Information

Gene expression microarray analysis of heat stress in the soil invertebrate Folsomia candida

Publications

Gene expression microarray analysis of heat stress in the soil invertebrate Folsomia candida.

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