| Exopalaemon carinicauda, a shrimp species endemic to China, holds significant economic value due to its desirable meat quality, high protein content, and low fat levels, making it a popular choice among consumers. Its rapid growth, adaptability, and short cultivation cycle further enhance its appeal in aquaculture. However, industrial expansion and rising greenhouse gas emissions have intensified ocean heatwaves, posing a critical challenge to crustacean farming. Temperature fluctuations profoundly affect crustacean physiology, with prolonged high-temperature exposure causing tissue damage, mortality, and economic losses. While prior studies on E. carinicauda have examined temperature impacts on growth and embryonic development, mechanisms underlying its thermal tolerance remain poorly understood. Addressing this gap is vital for developing heat-resilient strains to sustain aquaculture productivity. Heat shock transcription factor 1 (hsf-1) is a key factor in the regulation of the heat shock response, which protects the body from heat stress injury by up-regulating the expression of heat shock proteins and reducing the accumulation of misfolded proteins. Although it has been found that hsf-1 plays an important role in the heat shock response of many species, its role in the heat shock response of E. carinicauda has not been fully studied, and further research is needed to determine whether it has the same function. Therefore, this study was conducted to understand the function of Echsf-1 in high-temperature stress in E. carinicauda. Experiments subjected E. carinicauda to 33℃, a stress threshold identified in preliminary trials. Bioinformatics tools were used to analyze the Echsf-1 gene sequence, and RT-qPCR quantified its expression across healthy tissues (gill, stomach, hepatopancreas, muscle, antennary gland). Hepatopancreas and gill tissues were further evaluated under heat stress. To assess the functional role of Echsf-1, RNA interference (RNAi) was employed via dsRNA injection (4 μg/g), with four experimental groups: normal temperature group (NT), normal temperature dsRNA group (NTD), high temperature group (HT), and high temperature dsRNA group (HTD). Each group included seven replicates (five for sampling, two for survival analysis). Tissues collected at 24 h and 72 h were fixed for histological examination, while hepatopancreas transcriptomes (72 h) were sequenced. Survival rates were monitored at 24 h intervals. The results showed that the amino acid sequence of the E. carinicauda Echsf-1 is closely related to that of the Litopenaeus vannamei, and Echsf-1 was expressed in several tissues of the healthy E. carinicauda, with gill tissues showing the highest level of expression, followed by the stomach and hepatopancreas, suggesting that this transcription factor plays a role in the regulation of genes in a variety of tissues. Under high-temperature stress, Echsf-1 expression levels in hepatopancreas and gills peaked at 48 h and then declined at 72 h. The timing of significant hsf-1 expression was differentiated from that in other species, and species-specificity of the gene was speculated. In RNAi experiments, the E. carinicauda in the high temperature dsRNA group exhibited significantly reduced survival and exacerbated tissue damage, and these results suggest that Echsf-1 is involved in high-temperature stress and improves survival as well as reduces tissue damage under high-temperature stress. The results of transcriptome data showed that in the HT vs NT comparison group, there were 1,240 differentially expressed genes, of which 751 were up-regulated and 489 were down-regulated. In the HTD vs NTD comparison group, there were 482 differentially expressed genes, of which 358 were up-regulated and 124 were down-regulated. KEGG enrichment analysis showed that differentially expressed genes were mainly enriched in immune- and metabolism-related pathways such as antigen processing and presentation, amino sugar and nucleotide sugar metabolism, glycosaminoglycan degradation and sphingolipid metabolism. In the HT vs NT comparison group, hsp70 expression was found to be down-regulated and bip expression was up-regulated in the antigen processing and presentation pathway, leading to increased organismal damage. HSP70 helps misfolded proteins to fold correctly, and its down-regulation leads to the accumulation of misfolded proteins; the up-regulation of bip, which is a molecular chaperone of the endoplasmic reticulum (ER), leads to the over-activation of the unfolded protein response (UPR), which predicts the increase of ER stress, and the result leads to cell death and tissue damage. stress, which results in cell death and tissue damage. Genes associated with heparan sulfate (HS) catabolism in the glycosaminoglycan degradation pathway (e.g., gusb, hspe, and naglu) were generally up-regulated in the HTD vs NTD comparison group, which may allow for the excessive catabolism of HS, resulting in the impaired function of tissue repair. HS plays a critical role in extracellular matrix organization, cell adhesion, tissue development, and repair, and the insufficiency of HS leads to the inability of tissue damage to be effectively repaired, causing death of the organism. In addition, analysis of the HT vs NT and HTD vs NTD comparator groups revealed that phosphoglucose mutase 2 (PGM2) showed opposite expression patterns in these two comparator groups. PGM2 promotes substance reuse, DNA replication, and DNA damage repair. In the HTD vs NTD comparison group, the down-regulation of PGM2 was produced by the knockdown of Echsf-1. The down-regulation of PGM2 leads to an increase in energy expenditure in the organism, and DNA damage could not be repaired in a timely manner, which ultimately led to tissue damage and death of the individuals. This study demonstrates the critical role of Echsf-1 in enhancing thermotolerance by regulating stress-response pathways, reducing protein misfolding, and supporting tissue repair in E. carinicauda. These findings provide a foundation for selective breeding of heat-resistant crustaceans and advance understanding of molecular adaptation to climate-driven thermal stress in aquaculture. |
