Bacterial diet and weak cadmium stress affect the survivability of Caenorhabditis elegans and its resistance to severe stress.

Stress may have negative or positive effects in dependence of its intensity (hormesis). We studied this phenomenon in Caenorhabditis elegans by applying weak or severe abiotic (cadmium, CdCl2) and/or biotic stress (different bacterial diets) during cultivation/breeding of the worms and determining their developmental speed or survival and performing transcriptome profiling and RT-qPCR analyses to explore the genetic basis of the detected phenotypic differences. To specify weak or severe stress, developmental speed was measured at different cadmium concentrations, and survival assays were carried out on different bacterial species as feed for the worms. These studies showed that 0.1 µmol/L or 10 mmol/L of CdCl2 were weak or severe abiotic stressors, and that E. coli HT115 or Chitinophaga arvensicola feeding can be considered as weak or severe biotic stress. Extensive phenotypic studies on wild type (WT) and different signaling mutants (e.g., kgb-1Δ and pmk-1Δ) and genetic studies on WT revealed, inter alia, the following results. WT worms bred on E. coli OP50, which is a known cause of high lipid levels in the worms, showed high resistance to severe abiotic stress and elevated gene expression for protein biosynthesis. WT worms bred under weak biotic stress (E. coli HT115 feeding which causes lower lipid levels) showed an elevated resistance to severe biotic stress, elevated gene expression for the innate immune response and signaling but reduced gene expression for protein biosynthesis. WT worms bred under weak biotic and abiotic stress (E. coli HT115 feeding plus 0.1 µmol/L of CdCl2) showed high resistance to severe biotic stress, elevated expression of DAF-16 target genes (e.g., genes for small heat shock proteins) but further reduced gene expression for protein biosynthesis. WT worms bred under weak biotic but higher abiotic stress (E. coli HT115 feeding plus 10 µmol/L of CdCl2) showed re-intensified gene expression for the innate immune response, signaling, and protein biosynthesis, which, however, did not caused a higher resistance to severe biotic stress. E. coli OP50 feeding as well as weak abiotic and biotic stress during incubations also improved the age-specific survival probability of adult WT worms. Thus, this study showed that a bacterial diet resulting in higher levels of energy resources in the worms (E. coli OP50 feeding) or weak abiotic and biotic stress promote the resistance to severe abiotic or biotic stress and the age-specific survival probability of WT.

PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans.

The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.

Detoxification and sensing mechanisms are of similar importance for Cd resistance in Caenorhabditis elegans.

The present study employed mass spectrometry (ICP-MS) to measure the internal cadmium concentrations (Cdint) in Caenorhabditis elegans to determine Cd uptake from a Cd-containing environment as well as Cd release under Cd-free conditions. To analyze the functional role of several ATP binding cassette (ABC) transporters (e.g., HMT-1 and MRP-1) and phytochelatin synthase (PCS), we compared wild-type (WT) and different mutant strains of C. elegans. As a pre-test on selected mutant strains, several time-resolved experiments were performed to determine the survival rate and avoidance behavior of C. elegans under Cd stress, which confirmed the already known Cd sensitivity of the deletion mutants mrp-1Δ, pcs-1Δ, and hmt-1Δ. In addition, these experiments revealed flight reactions under Cd stress to be almost completely absent in mrp-1Δ mutants. The ICP-MS studies showed Cd uptake to be significantly higher in mrp-1Δ and WT than in hmt-1Δ. As Cd is ingested with food, food refusal due to very early Cd stress and its perception was likely the reason for the reduced Cd uptake of hmt-1Δ. Cd release (detoxification) was found to be maximal in mrp-1Δ, minimal in hmt-1Δ, and intermediate in WT. High mortality under Cd stress, food refusal, and minimal Cd release in the case of hmt-1Δ suggest a vital importance of the HMT-1/PCS-1 detoxification system for the survival of C. elegans under Cd stress. High mortality under Cd stress, absence of an avoidance behavior, missing food refusal, and maximal Cd release in the case of mrp-1Δ indicate that MRP-1 is less important for Cd detoxification under severe stress, but is probably important for Cd perception. Accordingly, our results suggest that the survival of WT under Cd stress (or possibly other forms of metal stress) primarily depends on the function of the HMT-1/PCS-1 detoxification system and the presence of a sensing mechanism to control the uptake of Cd (or other metals), which keeps internal Cd (or metal) concentrations under control, to some extent, for the timely mobilization of protection and repair systems.

Adjustments of serine proteases of Daphnia pulex in response to temperature changes.

Elevated temperatures considerably challenge aquatic invertebrates, and enhanced energy metabolism and protein turnover require adjustments of digestion. In Daphnia, the serine proteases chymotrypsin and trypsin represent the major proteolytic enzymes. Daphnia pulex acclimated to different temperature conditions or subjected to acute heat stress showed increased expression level of serine proteases with rising temperatures. Transcripts of trypsin isoforms were always present in higher amounts than observed for chymotrypsin. Additionally, trypsin isoform transcripts were induced by elevated temperatures to a larger extent. Correspondingly, trypsin activity dominated in cold-acclimated animals. However, the enzymatic activity of chymotrypsin increased at elevated temperatures, whereas trypsin activity slightly decreased, resulting in a shift to dominating chymotrypsin activity in warm-acclimated animals. Zymograms revealed eight bands with proteolytic activity in the range of 20 to 86 kDa. The single bands were assigned to trypsin or chymotrypsin activity applying specific inhibitors or from casein cleavage products identified by mass spectrometric analysis. The total amount of proteolytic activity was elevated with acclimation temperature increase and showed a transient decrease under acute heat stress. The contribution of the different isoforms to protein digestion indicated induction of chymotrypsin with increasing acclimation temperature. For trypsin, the share of one isoform decreased with elevated temperature, while another isoform was enhanced. Thus differential expression of serine proteases was observed in response to chronic and acute temperature changes. The observed phenotypic plasticity adjusts the set of active proteases to the altered needs of protein metabolism optimizing protein digestion for the temperature conditions experienced in the habitat.