| 其他摘要 | The ark shell, Scapharca subcrenata, is an economically important shellfish in China. In recent years, high temperature, frequently combined with the occurrence of Vibrio diseases in summers often leads to massive mortality in mudflat shellfish. Aiming at these problems, this study focused on the selection of heat resistant strain from local populations from Shandong Province and the exploration of the response mechanisms of ark shell to thermal stress. Our results may provide essential basis for molecular assisted breeding of heat-resistant strains of ark shell, and promote healthy and sustainable development of ark shell aquaculture industry in China. (1) Selection of a heat-resistant strains of ark shellIn this study, semi-lethal upper temperature of ark shell was determined as 30.2 °C by temperature tolerance test. The first generation of new heat-resistant strains of ark shell was reproduced from the brook stocks selected by thermal stresses. (2) Comparative transcriptome analyses of ark shell in response to thermal stresses To reveal the underlying molecular mechanisms of heat resistance (32 ℃) in the ark shells, transcriptomic analyses were carried out with haemocytes sampled from ark shells exposed to thermal stress. RNA-seq and quantitative real-time PCR showed that heat shock proteins (HSPs) and apoptosis (TRAF6, GRP78) may be the key differentially expressed genes (DEGs) regulating heat stress. GO and KEGG enrichment analyses showed that the DEGs were mainly associated with apoptosis, NF-kappa B signaling pathway, TNF signaling pathway and RIG-I-like receptor signaling pathway. Noteworthily, the expression of DEGs involved in protein biosynthesis and proteolysis was significantly elevated in ark shells under heat stress. It is speculated that the ark shell may adapt to short-term heat stress by regulating protein metabolism, DNA replication and anti-apoptotic system. However, with the aggravation of high temperature stress, tissue hypoxia and metabolic disorder will cause irreversible damage to the S. subcrenata. (3) Physiological and transcriptional responses to acute and chronic thermal stress in ark shell In order to understand the mechanism of response to acute or chronic stress rise of the ark shell, this study first measured the 96 h median lethal temperature of the ark shell, and then studied their physiological and transcriptional responses to the stress of rapid and slow temperature (32 ℃) rise. A significantly higher cumulative mortality (52% in 96 h) was observed in the acute heating treatment (AHT) group than that (22% in 7 days) in the chronic heating treatment (CHT) group. The apoptosis and necrosis rates of haemocytes were increased significantly in a time-dependent manner under both thermal stress strategies. Activities of antioxidant enzymes (SOD and CAT) increased dramatically in a short time followed by a quick decline and reached to a lower level within 12 h in the AHT group, but maintain relatively high levels over a long period in the CHT group. The contents of MDA were increased significantly firstly and restored to the original later in both acute and chronic thermal stress. Moreover, expression of the genes related to heat shock proteins (HSPs), apoptosis (TRAF6, GRP78, and Casp-3) and antioxidant responses (GST and MRP) could be induced and up-regulated significantly by thermal stress, expression of RGN, MT and PRX was down-regulated dramatically under the two heating treatments. However, the activity of antioxidant enzymes and the expression of most genes could be restored to the initial level in the CHT, so the plasticity of stress resistance was higher. These results suggested that anti-apoptotic system, antioxidant defense system and heat shock proteins could play important roles in thermal tolerance of ark shells. (4) Response mechanism of heat-resistant and wild ark shell strains to thermal stress In order to understand the response mechanism of heat-resistant strain (NTR) and wild strain (WT) to thermal stress, transcriptomic, proteomics and metabolomics association analyses were performed with gills sampled from animals from the NTR and the WT strain after thermal stress (32 ℃). The results showed that compared with the WT that were not exposed to thermal stress, GO and KEGG in differentially expressed genes (DEGs), differentially expressed proteins (DEPs) and differentially expressed metabolites (DEMs) of the WT-H24 were mainly enriched in membrane and its components, NOD-like receptor signaling pathway, cancer pathway and biotin metabolism pathway. With the extension of time, the differences in the WT-H96 were mainly enriched in nucleotide binding, MAPK signaling pathway and apoptosis pathway. However, in the NTR that were not exposed to thermal stress, the differences were mainly enriched in catalytic activity, NOD-like receptor signaling pathway, MAPK signaling pathway and nucleic acid resection repair. The NTR-h24 was mainly enriched in calcium ion binding and PI3K-Akt signaling pathway. The enrichment of DEGs, DEPs and DEMs in the NTR-h96 group was similar to that of the NTR-h24, but the unsaturated fatty acid biosynthesis pathway was gradually enriched with the extension of time. These results indicate that compared with the WT-C, the resistance (NOD-like receptor signaling pathway) and repair pathway (nucleic acid resection repair) have been activated in the NTR-C group, which exhibited a stronger heat resistance to resist external damage when subjected to thermal stress again. However, under the long-term high temperature stress, cellular damage will gradually become apparent in the gills of the ark shell. The results herein may reveal the molecular mechanism of response to high temperature stress of the ark shells and suggest that the method of slow heating stress could be used in the breeding of a heat-resistant strain of S. subcrenata. The results in this study may also lay the groundwork for marker-assisted selection of heat-resistant strains in S. subcrenata. |
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