Transcriptomes of Rotaria rotatoria under thermal stress

Global warming has raised concerns regarding the potential impact on aquatic biosafety and health. To illuminate the adaptive mechanisms of bdelloid rotifers in response to global warming, the ecological and transcriptomic characteristics of two strains (HX and ZJ) of Rotaria rotatoria were investigated at 25°C and 35°C. Our results showed an obvious genetic divergence between the two geographic populations. Thermal stress significantly reduced the average lifespan of R. rotatoria in both strains, but increased the offspring production in the ZJ strain. Furthermore, the expression levels of genes Hsp70 were significantly upregulated in the HX strain, while GSTo1 and Cu/Zn‐SOD were on the contrary. In the ZJ strain, the expression levels of genes Hsp70, CAT2, and GSTo1 were upregulated under thermal stress. Conversely, a significant decrease in the expression level of the Mn‐SOD gene was observed in the ZJ strain under thermal stress. Transcriptomic profiling analysis revealed a total of 105 and 5288 differentially expressed genes (DEGs) in the HX and ZJ strains under thermal stress, respectively. The PCA results showed clear differences in gene expression pattern between HX and ZJ strains under thermal stress. Interestingly, compared to the HX strain, numerous downregulated DEGs in the ZJ strain were enriched into pathways related to metabolism under thermal stress, suggesting that rotifers from the ZJ strain prioritize resource allocation to reproduction by suppressing costly metabolic processes. This finding is consistent with the life table results. This study provides new insights into the adaptive evolution of aquatic animals in the context of global climate change.

Methods
We collected experimental rotifers from Hexian and Zhanjiang, China, and conducted subsequent experiments following stock culture in the laboratory. 

For transcriptomics experiment, total RNA was extracted from the rotifer samples using Trizol reagent (Invitrogen, CA, USA), following the manufacturer's protocol. The concentration and integrity of the RNA were determined using Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with a RIN (RNA integrity number) ≥ 7.0. Sequencing libraries were obtained following the handbook for the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA). Index codes were added to attribute sequences to each sample. Poly(A) RNA was purified from 5 µg of the total RNA sample using poly-T oligo-attached magnetic beads and two rounds of purification. The mRNA was fragmented into small pieces using divalent cations under elevated temperature conditions. The fragmented RNA was then reverse-transcribed to construct the final cDNA library. The protocol for the mRNASeq sample preparation kit (Illumina, San Diego, USA) was followed, with an average insert size of 300 bp (±50 bp) for the paired-end libraries. Paired-end sequencing was performed using an Illumina Novaseq™ 6000 at LC Sciences (USA), in adherence to the vendor's recommended protocol.