Asellus aquaticus x physiological ethanol concentration

Climate change is one of the most critical challenges of our century. One of its forecasted consequences is the reduction in oxygen availability in aquatic environments and within organisms, potentially leading to ethanol production by these organisms. There is evidence for the presence of very low levels of endogenous ethanol in a wide range of animal species. However, the physiological effects of such low ethanol concentrations in animal systems have been largely overlooked, despite some encouraging preliminary studies.     Type of samples:The biological material used in this study consists of the freshwater isopod Asellus aquaticus, irrespective of age, sex, pigmentation, or reproductive state. Wild populations were collected from a natural pond along the Sentier Nature in Toulouse, France (GPS coordinates: 43.5600, 1.4746) in May 2025.     Variable under investigation:Exposure to a low dose of ethanol.    Experimental conditions:To identify the molecular pathways differentially regulated by a physiological dose of ethanol in whole Asellus aquaticus specimens, we carried out a genome-wide transcriptomic analysis by RNA-seq. Individuals were collected together with surrounding pond water, which was used as the experimental medium. After collection, specimens were acclimated under controlled laboratory conditions for 4 hours. They were then exposed either to 10 mM ethanol for 3 hours or to a control treatment of the same duration. Following incubation, whole specimens were sacrificed and pooled, with each sample containing approximately 75 whole individuals. For each condition (control or 10 mM ethanol), 6 biological replicates were prepared. All samples were subsequently processed for RNA sequencing.    Extraction Protocole: Each pooled sample of whole individuals was homogenized into fine powder using a ball grinder Tissue Lyser II (QIAGEN, Germany) with two consecutive 45 s cycles at 300 Hz. Total RNA was extracted from 1–5 mg of frozen powder using the Arcturus™ PicoPure™ RNA Isolation Kit (KIT0204, Thermo Fisher Scientific) following the manufacturer’s protocol, optimized for Asellus aquaticus. To eliminate genomic DNA contamination, RNA was treated on-column with the RNase-Free DNase Set (79254, QIAGEN, Germany) according to the kit instructions. Library Construction Protocol: Preparation of RNA libraries using the Illumina Truseq Stranded mRNA Kit, and sequencing on 1 line of the Element AVITI high-throughput sequencer. Readings are performed in 2x150bp. List of samples and conditions:  Sample IDlibrary strategyorganismtissuecell line*cell typegenotypetreatmentbatchmoleculesingle or paired-endinstrument modeldescription1-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typecontrol B1polyA RNApaired-endElement AVITIcontrol_bio rep 12-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typeEtOH 10 mMB1polyA RNApaired-endElement AVITIEtOH_bio rep 13-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typecontrol B1polyA RNApaired-endElement AVITIcontrol_bio rep 24-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typeEtOH 10 mMB1polyA RNApaired-endElement AVITIEtOH_bio rep 25-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typecontrol B1polyA RNApaired-endElement AVITIcontrol_bio rep 36-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typeEtOH 10 mMB1polyA RNApaired-endElement AVITIEtOH_bio rep 31b-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typecontrol B2polyA RNApaired-endElement AVITIcontrol_bio rep 42b-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typeEtOH 10 mMB2polyA RNApaired-endElement AVITIEtOH_bio rep 43b-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typecontrol B2polyA RNApaired-endElement AVITIcontrol_bio rep 54b-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typeEtOH 10 mMB2polyA RNApaired-endElement AVITIEtOH_bio rep 55b-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typecontrol B2polyA RNApaired-endElement AVITIcontrol_bio rep 66b-alphaRNA-SeqAsellus aquaticus whole organism wild typewild typewild typeEtOH 10 mMB2polyA RNApaired-endElement AVITIEtOH_bio rep 6