Real-time quantitative PCR analysis of the effect of non-lethal heat shock in two strains of rotifer Brachionus sp.
Description
Temperature plays an important role in the occurrence and performance of organisms in aquatic ecosystems and is also one of the main environmental factors affecting species’ survival and growth in aquaculture. As an important species for aquaculture sustainability, the rotifer Brachionus sp. benefits from inducible phenotypic traits that allow the organisms to cope with environmental stress. The exposure to high temperature has shown to increase production of heat shock proteins (HSPs) and histone modifications in several organisms, resulting in induced thermotolerance. This study aimed to evaluate the potential of non-lethal heat shock (NLHS) to induce thermotolerance in two strains of B. koreanus (MRS10 and IBA3) and pinpoint some of the molecular mechanisms involved in the process. For this, neonates were exposed to a 30 min NLHS, after which triplicates of heat shocked (NLHS treatment) and non-heat shocked (Reference treatment) organisms were placed under control conditions for 8 h. Sampling was done at 1 h, 4 h and 8 h, in each repliclate, to analyse gene expression of heat shock proteins and epigenetic-related genes. This was done for both rotifer strains studied. Results showed that, overall, induced thermotolerance was concomitant with a typical heat shock response, with up-regulation of HSP genes, after 1 and 4 h of recovery, especially seen for HSP40 and HSP90 for both strains. The exposure to a single NLHS did not promote significant alterations in the gene expression of several epigenetic markers.
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Total RNA was extracted using TRIzol® reagent and, using iScript™ cDNA Synthesis Kit, total RNA was converted into cDNA. Amplification reactions were then performed in triplicates for all samples, for the 9 target genes coding for heat shock proteins and epigenetic proteins involved in histone modifications, plus 2 housekeeping genes. Reaction conditions consisted of one initial cycle of 30 s at 95 °C (activation step), and 40 cycles of a combined denaturation (5 s at 95 °C) and annealing (30 s at 60 °C) step. Melting curves were generated by an additional cycle at 65 ºC for 5 s, followed by increasing steps of 0.5 °C, and a final cycle for 5 s at 95 ºC. Expression values of the target genes were normalized by the expression of two housekeeping genes (HK), 18S ribosomal RNA (18S rRNA) and elongation factor 1 alpha (EF1α). The relative gene expression (normalized data) of all genes was calculated using the equation: Gene expression ratio = (EGOI)^ΔCt GOI / (EHK)^ΔCt HK, where E is the efficiency of the primer for each gene of interest (GOI) and housekeeping genes (HK) and ΔCT is the difference between the minimum CT of each treatment and the CT of each replicate from the same treatment. The geometric mean (GeoMean) between the two HK genes was used. Fold change was calculated using the equation: Relative Fold Change (RFC) = (EGOI)^ΔCt GOI / GeoMean[(EHK)^ΔCt HK], where ΔCT is the difference between the mean CT of Reference treatment and the CT of each sample of the NLHS treatment. Fold change results were log2 normalized.