Project description:As a result of increasing thermal fluctuations and mean temperature values, organisms will experience conditions beyond their physiological limits. In situ adaptation to thermal regimes is mediated via directional selection and phenotypic plasticity. The latter involves physiological and morphological adjustments realized by underlying molecular mechanisms. Understanding species' adaptive capacities requires investigating these adjustive processes. Yet, acclimation through phenotypic plasticity remains largely unexplored, especially at the molecular level; For example, whether cold-adapted species inhabiting freshwater spring ecosystems have evolved adaptive mechanisms to cope with warming of freshwater habitats has, to our knowledge, never been investigated. This work reports a comprehensive proteomics study of the stenotopic species Crunoecia irrorata (Curtis, 1834) (Trichoptera: Lepistomatidae) acclimated to 10, 15 and 20 °C for 168 h. A liquid chromatography tandem mass spectrometry (LC-MS/MS)-based shotgun proteomics approach identified molecular mechanisms underlying acclimation. We constructed a homology-based database by combining genomic and transcriptomic data from related species and quantified 1356 proteins, of which 186 were differentially expressed between temperature treatments. Through functional annotation, we identified candidate proteins facilitating, among others, trehalose accumulation, tracheal system alteration, and heat shock protein regulation, then discuss concomitant ecophysiological implications. These results provide new insights into the mechanisms of adaptive responses to warming of species inhabiting freshwater ecosystems sensitive to climate change. Further, identified candidate proteins will aid in developing targeted experiments to understand their compensatory physiology. To our knowledge, this is the first study utilizing this approach to investigate the nature of phenotypic plasticity of aquatic macroinvertebrates.

Project description:Digestive Gland Samples: A manila clam oligo microarray platform (GPL10900) was used to profile gene expression in digestive gland of R. philippinarum. Total RNA was extracted from three (3) independent biological replicates of digestive gland for each sampling site, each consisting of tissue pools of five (5) animals. Statistical analysis with SAM (Significance Analysis of Microarray) identified1,127 probes differentially expressed. Gills Samples: A manila clam oligo microarray platform (GPL10900) was used to profile gene expression in gills of R. philippinarum. Total RNA was extracted from three (3) independent biological replicates of gills for each sampling site, each consisting of tissue pools of five (5) animals. Statistical analysis with SAM (Significance Analysis of Microarray) identified1,127 probes differentially expressed. Digestive Gland Samples: In this study, we analyzed six (6) samples, three (3) pools of digestive gland of Manila clam sampled in Marghera and three(3) pools of digestive gland of Manila clam sampled in Alberoni. Gene expression profiling was performed using the Agilent-019810 Ruditapes philippinarum Oligo Microarray platform (GPL10900) based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal. Gills Samples: In this study, we analyzed six (6) samples, three (3) pools of gills of Manila clam sampled in Marghera and three(3) pools of gills of Manila clam sampled in Alberoni. Gene expression profiling was performed using the Agilent-019810 Ruditapes philippinarum Oligo Microarray platform (GPL10900) based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

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