The Comparative Transcriptome Study Of Two Species Of Hypotrichida Ciliates Under Salinity Stress

The salinity variation between different environments is one of the most challenging obstacles for marine and terrestrial organisms.For those living at the intersection of sea and land,they have evolved mechanisms to resist the impact of salinity changes on their normal physiological activities.These mechanisms involve multiple biochemical pathways such as energy flow and protein metabolism,requiring multiple organs to work together.However,for single-celled organisms,maintaining osmotic balance inside and outside the cell becomes a major challenge when the salinity of the environment changes.To maintain the stability of intracellular water content,organisms need to evolve adaptive mechanisms,such as synthesizing acid proteins or organic solutes to resist salinity changes and maintain their normal life activities.While the mechanisms in single-celled algae and some salt-tolerant yeast have been deeply studied,the molecular mechanisms of osmotic strategies in protist groups（especially ciliates）are still unknown.In studies and investigations related to the population and community of ciliated protozoa in nearshore waters,we observed that marine planktonic ciliates are widely distributed in different salinity gradients.Meanwhile,due to the changes in salinity gradients,the composition of ciliate populations and communities also undergoes significant changes.For ciliates inhabiting brackish water environments such as estuaries and coastal wetlands,they are believed to be the result of adaptive evolution of seawater species to the decreased salinity in their living environment.In our analysis of these ciliate populations,we found that species that can resist changes in habitat salinity generally have a thick pellicle,which we speculate provides protection for ciliates transitioning from freshwater to seawater.In this study,we used the fresh-water species Gastrostyla setifera as experimental material for salinity acclimation and the marine species Euplotes vannus as the experimental material for low-salt acclimation,compared to the original population,and analyzed their transcriptome using comparative transcriptomics.This paper established the transcriptome information of G.setifera and studied the transcriptional response levels of the two ciliates under salinity stress.The main conclusions are as follows:（1）The first deep sequencing and assembly of a gene library for RNA-seq data from G.setifera was conducted,resulting in 486,767 single-gene sequences of G.setifera after splicing and removing r RNA,mitochondrial,and bacterial contaminants.The SPOCS method was used to detect orthologs between G.setifera and two model species,Tetrahymena thermophila and Paramecium tetraurelia,as well as four other closely related ciliates.Overlapping homologous genes were found at a high degree of coincidence,demonstrating the integrity of the obtained G.setifera gene library.（2）After growing to mid-exponential stage under 3 psu salinity,11,690 differentially expressed genes（log₂Fold Change>2,p-value<0.05）were screened from the transcriptome of G.setifera,including 7,662 down-regulated genes and 4,028 up-regulated genes.The screened differentially expressed genes were annotated in the KEGG pathway database and GO database It was found that most of the differentially expressed genes were enriched to ion channel proteins,protein kinases,protein phosphorylases,tricarboxylic acid cycle and other pathways under salinity stress.This indicates that salinity stress has a significant effect on energy metabolism,cell growth,transcription and differentiation of G.setifera,and that G.setifera adapts to the increase of external salinity by transporting ions outward and activating protein functions.The transcriptome expression of the above physiological pathways was reduced or decreased to adapt to environmental salinity changes and maintain the basic survival state.（3）Transcriptome analysis of E.vannus living in 30 psu and 15 psu salinity revealed an overall upward trend in gene expression after low salinity stress.Differential gene expression screening identified 21,860 differentially expressed genes,including 3,177 genes that were down-regulated and 18,683 genes that were up-regulated.Annotation of these genes with KEGG pathways and GO databases showed that most of the up-regulated genes were annotated to catalytic activity,transferase activity,DNA binding,metabolic pathways,and the tricarboxylic acid cycle.These findings suggest that as the salinity of the living environment decreases,the gene expression levels and physiological and biochemical pathways of the fan-shaped amoeba show higher levels of expression,which may reflect its adaptation strategy to low salt environments.In addition,the comparative transcriptomic studies also revealed that the ciliates in the subclass hypotrichida have a high degree of flexibility in adapting to changes in salinity.They can adjust their gene expression levels to respond to changes in salinity,which allows them to survive and thrive in different salinity environments.Overall,the comparative transcriptomic studies of ciliates in the subclass hypotrichida provide insights into the molecular mechanisms underlying their adaptation to changes in salinity.These findings have important implications for understanding the ecology and evolution of these ciliates,as well as for the development of strategies to manage their populations in aquatic ecosystems.