Exploring Endoplasmic Reticulum Stress Pathways in Apostichopus japonicus Under Heat Stress Based on Transcriptome Analysis

doi:10.21203/rs.3.rs-3803431/v1

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Exploring Endoplasmic Reticulum Stress Pathways in Apostichopus japonicus Under Heat Stress Based on Transcriptome Analysis

https://doi.org/10.21203/rs.3.rs-3803431/v1

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Background

High summer temperatures caused by changes in the global environment have significant impacts on marine organisms. The sea cucumber Apostichopus japonicus is a representative marine benthic organism and an economically important shallow-sea aquaculture species in China, and thus it is necessary to explore its response to environmental heat stress.

Results

Endoplasmic reticulum stress is an important regulatory strategy for organisms to respond to changes in the environment, and thus we analyzed the histology, ultrastructure, and transcriptome of the digestive tract of A. japonicus at three temperatures: normal (18°C), summer dormant (25°C), and lethal (32°C). Histological and ultrastructural results showed that heat stress caused morphological changes in the digestive tract of A. japonicus and inflicted alterations and damage to the endoplasmic reticulum of the enterocytes. Totals of 603 and 4615 differentially expressed genes were identified by transcriptome sequencing in the T25-vs-T18 and the T32-vs-T25 group comparisons, respectively. The GO results showed that DEGs in both group comparisons were significantly enriched in protein folding, chaperonin-mediated protein folding, and other protein-related GO terms. Also, KEGG enrichment analysis showed that both groups activated the pathway of protein processing in the endoplasmic reticulum and induced the endoplasmic reticulum stress response. The endoplasmic reticulum molecular chaperones, including BiP, GRP94, and Hsp70, were all up-regulated in expression. At 32°C, in addition to the above endoplasmic reticulum molecular chaperones, the endoplasmic reticulum molecular receptors PERK, TRAF2, and XBP, which are downstream of endoplasmic reticulum stress in the unfolded protein response, were also up-regulated.

Conclusions

This study has characterized the internal molecular regulatory process of A. japonicus under heat stress from the perspective of endoplasmic reticulum stress and provides clues regarding the mechanisms behind homeostatic regulation in invertebrates under stress from the environment.

Apostichopus japonicus

Heat stress

Histomorphology

Transcriptomic sequencing

Endoplasmic reticulum stress

Protein folding and processing

Environmental and global climate changes have led to a substantial increase in the surface temperature of the ocean [1, 2]. In the last century, the Earth’s climate has experienced a temperature increment of about 0.6°C. The net heat uptake of the world's oceans is more than 20 times that of the atmosphere, and average global sea surface temperature by 2100 is projected to increase by 1.5-3°C, a scenario that will have profound effects on ocean phenology, species richness, and aquatic physiology [3–5]. Numerous marine creatures' chances of surviving will likewise be significantly impacted [6–8]. The observed and predicted rate of occurrence and intensity of severe heat waves have notably increased as a result of global warming [9, 10]. Aquaculture animals that are more affected by fluctuating natural conditions and are raised in high densities will be more vulnerable to such extreme heat in summer [11, 12]. In cases of occurrence of extreme heat, the mariculture industry can suffer significant economic losses [13]. As such, it is of increasing attention that research on heat stress in marine aquaculture animals is being carried out.

There are vital cellular organelles responsible for keeping up the normal function of the organism, one such organelle is the endoplasmic reticulum (ER), its primary role is observed in protein and lipid biosynthesis. It regulates protein synthesis, protein aggregation, and protein folding [14–16]. The accretion of misfolded or unfolded proteins happens when the protein folding mechanism is compromised or the ability of ER to fold proteins is impaired, which then causes ER stress and triggers the unfolded protein response (UPR) [17]. Several adverse biotic and abiotic conditions, including salt stress, infections from pathogens, and heat stress may cause ER stress [18–21]. Heat stress can directly or indirectly change or disturb protein homeostasis, causing protein aggregation and denaturation [22]. Protein misfolding caused by heat stress generates ER stress, and cells counteract this by activating the UPR to balance the needs for folding proteins and preserve ER homeostasis [23, 24]. The majority of current research on ER stress focuses on mammals, including mice, porcine animals, and bovines [25–27]. However, little research has been done on the precise ER stress-related pathways caused by heat stress in marine invertebrates.

Sea cucumber Apostichopus japonicus as a typical temperate species that naturally distribution in the Russian Far East, Japan, Korea, and the Yellow and Bohai Seas of China [28, 29]. Its potent nutritional and medicinal properties make it one of the most commercially significant species for marine aquaculture in northern China [30, 31]. Of late global warming and intense summer heat have caused widespread deaths of sea cucumbers in many mariculture areas, which has created a barrier to the sea cucumber industry's ability to grow sustainably [32–34]. Furthermore, a great deal of research has demonstrated that sea cucumbers are extremely sensitive to environmental changes, making them perfect model organisms for studying how environmental changes impact marine benthic organisms [35–37]. Studying the mechanisms underlying the sea cucumber aquaculture industry's reaction to ambient temperature stress and the benthic ecosystem's restoration is crucial in light of global warming.

This study examined the effects of varying temperatures on the histological and ultrastructural alterations in A. japonicus’s digestive tract. The transcriptome profile of A. japonicus digestive tract was comprehensively analyzed; the key ER-related functional genes and the pathways during the heat stress response were defined, and the relevant regulatory mechanisms were explored. This research provides insight into the molecular response of A. japonicus to heat stress and provides clues regarding the mechanisms behind homeostatic regulation in invertebrates under stress from the environment.

2.1 Temperature treatment experiment and Sample collection

A. japonicus samples were randomly chosen from active, healthy individuals that were supplied by Yantai Anyuan Aquatic Co. Before the experiments started, the sea cucumbers were regularly cultivated for seven days in seawater with a salinity of 27 ± 0.5 and a temperature of 16 ± 0.5°C to help them adjust to the new surroundings. Every day during this time, the bottom of the pool was cleaned, and one-third of the water was replaced. After the acclimation period, sea cucumbers weighing an average of 116.32 ± 1.91 g were arbitrarily assigned into three separate groups (T18, T25, and T32) for breeding experiments at different temperatures. The 2-kW heating rod was used to increase the temperature of groups T18, T25, and T32 by 2°C/d from 16 ± 0.5°C to 18 ± 0.5°C, 25 ± 0.5°C, and 32 ± 0.5°C. Throughout the experiment, the three groups were maintained at their corresponding temperatures for 12 hours (other factors were consistent except for temperature), and from each group, nine sea cucumbers were chosen for sampling. Each group had three parallel samples lined up, and all of the samples were taken from the middle part of the gastrointestinal system of the sea cucumber. After sampling, the samples were rinsed off with PBS. A portion of each sea cucumber digestive tract was placed in Bouin's solution and 4% Gluta fixative for histological observation, and the remaining portion was promptly put into liquid nitrogen and kept at -80°C to perform transcriptomic sequencing and qRT-PCR analysis.

2.2 Histological observation and transmission electron microscopy

Samples were transferred to 70% alcohol for preservation after 24 hours of fixation in Bouin's solution. After dehydration with an alcohol gradient and immersion in xylene for transparency, a rotary slicer was used to cut the samples into 5 µm slices after they had been fixed in paraffin. The sections were stained with hematoxylin and eosin (H&E). Eventually, the tissue sections were examined under a light microscope.

1% osmium tetroxide was used to fix tissues for 2 hours after they had been treated with 4% Gluta and washed with PBS buffer, followed by gradient alcohol dehydration and incubation with acetone. Following dehydration, the tissues were placed in paraffin and cut with an ultrathin sectioning machine into 80 nm sections.

2.3 RNA extraction, library construction, and sequencing

The Trizol kit (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA following the manufacturer's instructions [38]. The quality of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and the results were validated using RNase-free agarose gel electrophoresis. After extracting total RNA, eukaryotic mRNA was enriched using oligo(dT) beads. Next, the enriched mRNA was broken up into small fragments using a fragmentation buffer. Then, using an Illumina NEBNext Ultra RNA Library Prep Kit (NEB #7530, New England Biolabs, Ipswich, MA, USA), the mRNA was reverse transcribed into cDNA. After end-repairing and adding a base, the Illumina sequencing adapters were ligated to the purified double-stranded cDNA fragments. The polymerase chain reaction (PCR) was used for amplifying the ligation reaction product after it had been purified using AMPure XP Beads (1.0X). Gene Denovo Biotechnology Co. (Guangzhou, China) used the Illumina Novaseq6000 program to sequence the resultant cDNA library. The Sequence Read Archive (SRA) run accessions in the NCBI are SRR23375701 SRR23375700 SRR23375699 SRR23375698 SRR23375697 SRR23375696 SRR23375695 SRR23375694 SRR23375693 for transcriptome data.

2.4 Data processing and differential expression analysis

The output of the sequencing process was raw reads. Therefore, to obtain high-quality clean reads, we further processed the raw reads using fastp (version 0.18.0) [39]. Using the short read alignment tool Bowtie2 (version 2.2.8), reads were assigned to the ribosomal RNA (rRNA) database [40]. The clean reads that remained following the removal of rRNA-mapped reads were utilized for assembly and gene abundance computations. After which the sequences were matched to the reference genome. HISAT2.2.4 was used to map paired-end clean reads to the reference genome after the reference genome index was created while leaving the remaining parameters at their default settings [41]. StringTie v1.3.1 assembled each sample mapped reads using a reference-based methodology [42, 43]. Using RSEM software, a fragment per kilobase of transcript per million mapped reads (FPKM) value was determined within every transcription area to calculate the abundance and variance in expression [44]. The computed gene expression was utilized to compare the variations in gene expression among samples. Finally, DESeq2 software was employed to perform a differential expression study of RNAs among the two groups [45]. Genes and transcripts exhibiting an absolute fold change of ≥ 2 and a false discovery rate (FDR) ≤ 0.05 were deemed to be differentially expressed genes (DEGs) or transcripts.

2.5 Functional enrichment analysis

Goseq was utilized in Gene Ontology (GO) enrichment analysis, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for a pathway enrichment study [46]. The three ontologies that makeup GO include cellular components are molecular function, and biological process [47]. GO enrichment analysis filters out the DEGs associated with biological functions and generates all of the GO terms that are highly enriched in DEGs relative to the genomic background. KEGG is a publicly availed pathway-related database [48]. When compared to the background of the whole genome, pathway enrichment analysis revealed significantly enriched metabolic or signal transduction pathways in DEGs. FDR correction threshold of FDR ≤ 0.05 was applied to the computed p-value.

2.6 Building networks of protein–protein interactions for DEGs

The top 100 genes in the categories T25-vs-T18 and T32-vs-T25 with the most significant DEGs (with the lowest Q-values) were chosen to create associations between proteins employing the STRING online database (https://cn.string-db.org). The network provides access to key proteins with numerous interactions from the perspective of a network structure.

3.1 The structure of A. japonicus's organization at different temperatures

The mucosa, submucosa, muscular, and serosa layers make up the digestive tract tissue of A. japonicus. Moreover, the mucosa and submucosa usually protrude into the intestinal lumen to form the mucosal folds in a circular pattern. The structural alterations in the layers of A. japonicus's digestive tract tissue exposed to different temperatures were visible using H&E staining (Fig. 1). The findings demonstrated that at 18°C, the morphology of the A. japonicus digestive system was normal, with a relatively regular round shape. Also, the folds in the intestinal lumen were evenly distributed, and the microvilli were stretched out and intact. At 25°C, the mucosal folds began to twist; the microvilli on the folds appeared slightly deficient and disintegrated, and the intestinal lumen was enlarged. As the temperature increased, the digestive tract tissues were significantly damaged at 32°C, as indicated by the severely stretched mucosal layer; the mucosal folds were broken from the base and completely fragmented and degraded, and the intestinal lumen became much larger.

The transmission electron microscopy results showed that at 18°C, the intestinal cells were structurally intact; the nuclei and cytoplasm were tightly connected; the cells contained more mitochondria and vesicles, and the ER was morphologically normal (Fig. 2A-B). At 25°C, the cytoplasm was shed; a gap began to appear between the cytoplasm and the nucleus, and the ER appeared slightly swollen (Fig. 2C). However, at 32°C, the structure of the nuclei was severely damaged; the separation with the cytoplasm was more pronounced, and the chromatin in the nucleus was shrunken into solid clumps. Moreover, the numbers of mitochondria and vesicles in the cell were reduced, and the ER became grossly swollen and already appeared partially granulated (Fig. 2D).

3.2 Transcriptome sequence and reads mapping

RNA-Seq was used to obtain nine samples (a total of three copies from each group T18, T25, and T32) from the digestive tract of A. japonicus. The results of RNA sequencing are shown in Table S1. Measures of quality control were used to ascertain compliance with sequencing data quality standards. The results from quality control of filtered samples revelead that Q30 values were greater than 90%, and the percentage of GC bases was in the range of 39.88–41.37%. Therefore, the clean reads were carried forward for additional analysis. After HISAT2 software matching, 66.70-099.99% of the total amount of reads were mapped to the genome. The above results suggest that the sequencing results could be used for subsequent analysis (Table S2).

3.3 Transcriptome sequence and reads mapping

Analysis of differentially expressed genes via volcano plots showed that a substantial amount of genes were differentially expressed after heat stress. There were 242 up-regulated expressed genes and 361 down-regulated genes among the DEGs in the T25-vs-T18 group (Fig. 3A). There were 2304 up-regulated expressed and 2311 down-regulated in the T32-vs-T25 group (Fig. 3B). Overall, the amount of DEGs in the T32-vs-T25 group was greater than in the T25-vs-T18 group, and there were more down-regulated genes than up-regulated expressed genes in each comparison group. A cluster heat map analysis of DEGs illustrated that the groups T25 and T18 had fewer differences in DEG expression patterns, while the T32 group differed substantially from the other two groups (Fig. 4).

3.4 GO functional enrichment analysis of DEGs

GO functional enrichment analyses were carried out utilizing DEGs. According to the results of the GO enrichment analysis, a total of 374 DEGs were enriched to 58 GO terms in the T25-vs-T18 group, and 3056 DEGs were enriched to 66 GO terms in the T32-vs-T25 group. The top 20 significantly enriched functional terms of the T25-vs-T18 group included protein folding, chaperone-mediated protein folding, unfolded protein binding, and de novo protein folding (Fig. 5A). The T32-vs-T25 group was significantly enriched in terms such as hydrolase activity, lipid metabolic processes, chaperone-mediated protein folding, and protein folding (Fig. 5B).

3.5 KEGG enrichment analyses for DEGs

In the T25-vs-T18 group, the KEGG enrichment analysis revealed an overall amount of 191 DEGs were annotated to 235 KEGG signaling pathways, among which the 20 most significantly enriched signaling pathways presenting with the lowest Q values were antigen processing and presentation, vitamin digestion and absorption, protein processing in endoplasmic reticulum, and peroxisome (Fig. 6A). There were 1494 differentially expressed genes annotated to 338 KEGG signaling pathways in the T32-vs-T25 group, with significant enrichment in signaling pathways such as protein synthesis and modification in the endoplasmic reticulum, the PPAR signaling pathway, antigen processing and presentation, and peroxisome (Fig. 6B). KEGG signaling pathways enriched and the number of genes annotated in the T32-vs-T25 group were greater as compared to the T25-vs-T18 group. Additionally, the endoplasmic reticulum pathway's protein processing was the top signaling pathway in the T32-vs-T25 group among the significantly enriched signaling pathways as ranked by the Q value.

3.6 Construction of protein interaction networks using the DEGs with significant differences

The top 100 genes with the most significant DEGs (with the least Q-values) in the T25-vs-T18 and T32-vs-T25 comparisons were selected. After removing the genes that were not annotated to the gene bank and duplicated genes, 47 and 52 genes remained in the groups T25-vs-T18 and T32-vs-T25, respectively. Protein–protein interaction networks were built using the protein sequences (Fig. 7). In a network of protein interactions, there are complex interactions among the proteins close to the center of the network to the other proteins. In the T25-vs-T18 group, the proteins with larger numbers of interactions close to the center of the network were Akt1, Hsp90ab1, and Pdia6. In the T32-vs-T25 comparison, the proteins with higher numbers of interactions were Akt1, Hspa5, and Dnajb1. Moreover, ER molecular chaperone proteins Pdia6 and Bip (Hspa5) had more links in the groups T25-vs-T18 and T32-vs-T25, respectively. This result also confirmed that the significance of ER stress-related proteins in A. japonicus's heat stress response.

4.1 Temperature influences the morphology and internal structural composition of the digestive tract in A. japonicus

Temperature is a powerful abiotic component that influences the daily functions of marine mollusks, and it can directly or indirectly modify physiological and metabolic processes [49]. As an intertidal species, A. japonicus is particularly susceptible to environmental temperature changes compared to other marine organisms [50, 51]. Some research has revealed that the ideal temperature range for A. japonicus growth is between 12–18°C [52, 53], and water temperatures above 25°C will trigger A. japonicus will enter into the stage of aestivation [34, 54]. During this stage, the primary physiological characteristics of A. japonicus include reduced feeding activity, degradation and atrophy of the digestive tract, and a reduced metabolism [55, 56]. However, in recent years, the extreme weather in summer has led to water temperatures of 30°C or more, and the survival strategy of aestivation for A. japonicus has thus become ineffective [57]. When extremely high temperatures reach 32°C, there will be mass mortality of A. japonicus. As such, this study focused on the changes in physiological activities of A. japonicus at lethal temperature and aestivation temperature, two representative temperature phases.

Compared to terrestrial organisms, the digestive tract of marine organisms are relatively open organs that are susceptible to variations from such factors as temperature or others in the environment, and damage to tissue structure is one of the effects. Zhao et al. assessed the impact of heat stress on the digestive tract histology of oysters and found that the intestinal microvilli were shortened and detached, and the amount of goblet cells multiplied with the increase in temperature [58]. The digestive tract in A. japonicus is a vital organ, it is not only used in food digestion and nutrient absorption but in immune homeostasis as well. Due to its sensitivity to environmental changes, studies on the heat stress reaction of A. japonicus have focused on digestive tract tissues [59]. For example, Xu et al. conducted histological and ultrastructural studies on the digestive tract of A. japonicus under heat stress and found that ultrastructural damage was intensified with increasing heat stress [33]. In our study, compared with the normal temperature, the microvilli on the folds in the digestive tract of A. japonicus appeared slightly damaged; gaps between the cytoplasm and the nucleus began to appear with a slight swelling of the ER at 25°C. This was consistent with the physiological phenomenon of reduced feeding and degradation of the digestive tract during the aestivation period. At 32°C, the mucosal folds in the digestive tract of A. japonicus were broken from the base; the gap between the cytoplasm and the nucleus was enlarged, and the ER was severely swollen. The results suggest that 32°C had seriously threatened the life of A. japonicus, and various organs such as the digestive tract had undergone severe structural damage.

4.2 RNA-Seq reveals that heat stress alters gene expression in A. japonicus

The organism's reaction to heat stress is a quick and intricate process that may involve the differential expression of many genes [57, 60]. Using this stress response system, the organism responds to the change in the environmental temperature. Many researchers have investigated the response mechanisms of organisms to heat stress through RNA-Seq. Huang et al. analyzed the transcriptome of rainbow trout head kidney tissue under heat stress conditions which identified 443 DEGs and found that many genes involved in maintaining homeostasis or adapting to stress and stimuli were highly induced in response to high temperature [61]. To identify possible molecular and cellular mechanisms of heat stress in a scleractinian coral, Onyango et al. obtained 1362 DEGs from RNA-Seq and observed that the heat stress response in the coral constitutes a reaction of protection [62]. In our analysis, comparisons T25-vs-T18 and T32-vs-T25 contained 603 and 4615 DEGs, respectively. The results revealed that the amount of DEGs in T32-vs-T25 group were higher compared to T25-vs-T18 group. We speculate that the degree of environmental change was more dramatic from 25°C to 32°C compared with 18°C to 25°C. When responding to such environmental temperature changes, the metabolism and other activities of A. japonicus are increased, and thus gene expression is altered at the transcriptional level to respond to the stress.

In this study, the number of GO terms enriched in the T32-vs-T25 group was substantially when compared with T25-vs-T18 group. The results reflect the changes in gene expression that become more pronounced with increased heat stress. Interestingly, GO terms correlated with protein folding were significantly enriched in the two comparison groups T25-vs-T18 and T32-vs-T25. These terms included protein folding, chaperone-mediated protein folding, and unfolded protein binding. Studies have shown that heat stress, heavy metal ions, or ultraviolet light stress can cause injury to proteins in the cell and disrupt the balance of protein groups [63]. When protein disorder occurs, transcription of genes is needed to adjust the expression pattern to regulate protein folding and repair [64]. Molecular chaperones play an important role in the quality control of proteins, a process necessary to ensure the correct folding and refolding of selected proteins and control the abnormal folding of isomers [65–67]. Mao et al. performed a transcriptomic analysis of gill tissues from three species of scallops under heat stress and also found a significant enrichment of many GO terms associated with protein folding [68]. Therefore, we speculate that heat stress disrupts the protein homeostasis of the intestinal cells of A. japonicus and to adapt to this temperature change, protein folding-related genes are transcribed to regulate the expression of related proteins, thereby participating in the process of other proteins obtaining their correct functional structure and arrangement.

4.3 Heat stress activated ER stress in A. japonicus

The KEGG enrichment analysis showed that there were 191 DEGs annotated into 235 KEGG signaling pathways in the T25-vs-T18 group, while 1494 DEGs were annotated into 338 KEGG signaling pathways in the T32-vs-T25 group. The amount of pathways enriched in the T32-vs-T25 group was greater than that in the T25-vs-T18 group, consistent with the GO enrichment results. This implies that more pathways need to be activated and more genes need to be transcribed in A. japonicus to deal with higher environmental temperature. In addition, we also found that the protein processing in the endoplasmic reticulum pathway was significantly enriched in both comparison groups T25-vs-T18 and T32-vs-T25.

After exposure to adverse stresses, the ER may become dysfunctional and can induce the ER stress response [16]. Therefore, the ER has a complex network of pathways for processing cellular secretions as well as membrane protein synthesis and ER-dependent adaptive responses to stress [69]. A. japonicus's digestive tract cells may be actively responding to ER stress, as suggested by the substantial enrichment of numerous GO terms associated with protein folding in our study and the KEGG analysis of protein processing in the endoplasmic reticulum pathway. To preserve protein homeostasis, this response must recognize the misfolded proteins and specifically direct their refolding or degradation by activating the ER pathway and up-regulating associated genes. We also selected the part of the genes with the most significant DEGs in the T25-vs-T18 and T32-vs-T25 comparison groups to construct protein–protein interaction networks. We found that the ER molecular chaperone proteins Bip and Pdia6 appeared to have more links in the two groups, also indicating the important role of the response mechanism. By combining the results, we hypothesize that A. japonicus may benefit from the ER stress response as a protective and compensatory mechanism for A. japonicus under heat stress and that the genes participating in the ER stress response are crucial when it is exposed to heat stress.

4.4 The key DEGs involved in the ER stress response by the digestive tract of A. japonicus

In our study, we have suggested that the DEGs involved in ER stress were key genes for A. japonicus to respond to changes in ambient temperature and that they interact to alleviate the stress. At 25°C, ER stress occurred in A. japonicus’s digestive tract, and the genes involved included ER molecular chaperones such as BiP, Grp94, and PDI. When the temperature was increased to 32°C, ER molecular chaperones were not adequate to deal with the stress at that temperature, and thus the ER molecule receptors PERK, TRAF2, and XBP in the UPR downstream of endoplasmic reticulum stress were activated.

HSPs, including HSP40, HSP70, and HSP90, which are strongly conserved proteins, are frequently used as biomarkers of environmental stress [70, 71]. It has been demonstrated that HSPs serve as crucial "helpers" by assisting in the correct folding, assembly, or disassembly of proteins and protein complexes as well as the correct refolding of aberrant or damaged proteins [72, 73]. This has been supported in several studies of heat stress in marine animals, including A. japonicus [35, 60, 74–76].

The coordinated transcriptional up-regulation of resident ER chaperones like Grp78, Grp94, and PDIs is a well-known characteristic of the ER stress response [77, 78]. In addition to being extremely selective in its chaperone role, Grp94 is essential for protein folding and preserving the integrity of the ER [79, 80].Additionally, earlier research has demonstrated that Grp94 possesses ATPase activity, which is required for Grp94 to bind to client proteins [81, 82]. An oxidoreductase and isomerase, PDIA6 catalyzes the oxidoreduction and isomerization of disulfide bonds [83, 84]. It is normally located in the ER and acts as a molecular chaperone to prevent the aggregation of misfolded and unfolded proteins in response to different adverse environmental [85, 86]. Researchers studying razor clams and oysters under heat stress and have obtained similar results [87, 88].

BiP is a member of the HSP70s and is homologous to Grp94 and HSP90, both of which are related to the newly synthesized immunoglobulin light chain and heavy chain and two ER stress proteins [89, 90]. These genes provide protein quality control for the cytoplasm, ER, and mitochondria, and all have similar mechanisms of action. BiP that functions as a companion that promotes Grp94's shutdown. The BiP nucleotide-binding domain’s interaction with the Grp94 intermediate domain is in control of the shutdown acceleration of Grp94. Hsp70 is also involved in the accelerated shutdown of Hsp90 [91].

Under ER stress, PERK serves as the primary sensor of the UPR [92]. It is a branch of the unfolded protein response, is in charge of preventing protein translation during the initial phases of the ER stress response in order to reduce the amount of unfolded proteins in the body [93, 94]. Moreover, research has demonstrated that PERK-related pathway gene expression was up-regulated in largemouth bass exposed to heat stress, consistent with the above studies [95]. Under ER stress, cells activate an UPR via PERK, reversible dissociation of BiP from the PERK lumen domain is facilitated by disturbances in protein folding. The absence of BiP is associated with the formation of activated PERK polymer-mass complexes, while overexpression of BiP attenuates PERK activation [96]. According to a prior study, BiP may act as a UPR sensor, sensing the unfolded protein through a typical binding substrate, and PERK signals the interaction [97]. PDIs prevent 26S proteasome degradation of activated PERK and thus help maintain PERK signaling as a mandatory regulator of the PERK pathway [98].

4.5 The regulatory process of ER stress and Unfolded Protein Response at different temperatures

Based on our experimental data, we hypothesized the pathways involved in the ER stress and unfolded protein response of the digestive tract in A. japonicus at 25°C and 32°C and considered how the key DEGs functioning at these two temperatures are regulated (Fig. 8).

Protein folding function was disrupted at 25°C due to the elevated ambient temperature, and unfolded proteins accumulated in the reticulum lumen of A. japonicus's digestive tract. The ER, like a "baroreceptor," receives signals to activate signaling pathways such as the UPR to help cells quickly return to a normal physiological state; this is the endoplasmic reticulum stress response. HSPs, especially molecular chaperone proteins such as Bip, Grp94, and ERP57, are upregulated to handle unfolded proteins, clearing and degrading excess substrate proteins from the endoplasmic reticulum cavity to maintain normal cell function and adapt to the rising environmental temperatures.

As the temperature increased, at 32°C the level of environmental temperature stress became severe, disrupting normal physiological functions of cells, increasing the number of denatured proteins, and disrupting intracellular protein function and activity. Receptors for ER molecules in the UPR downstream of ER stress exhibited significant differential expression. The significantly up-regulated DEGs such as PERK, TRAF2, and S2P indicated that all three ER transmembrane proteins dissociated with Bip, and a part of the proteins in each of the three branches responded. The heat induced A. japonicus's digestive tract triggered the three UPR signaling pathways: the IRE1 signaling pathway, the ATF6 signaling pathway, and the PERK signaling pathway. PERK dissociates from Bip, which phosphorylates its own protein, thereby responding to heat stress. IRE1 is also dissociated, not only shearing XBP-1 to generate active XBP-1s but also recruiting TRAF2 to alleviate the stress experienced by activating the associated kinase. ATF6 becomes dissociated and is transferred from the membrane of the ER to that of Golgi apparatus as vesicles. The protease S2P cleaves ATF6 transported to the Golgi apparatus to activate the genes downstream of the ER stress-responsive progenitor genes to induce the transcription of additional genes. When the environmental temperature stress continues or intensifies and the endoplasmic reticulum stress is too strong or persists for too long, cells will eventually induce autophagy or apoptosis as a form of protection for normal body function.

This study has comprehensively explored the morphological changes and molecular functional mechanisms of the A. japonicus digestive tract under heat stress conditions from histological, ultrastructural, and transcriptome sequencing perspectives. The ultrastructural results showed that heat stress caused alterations and damage to the ER of A. japonicus intestinal cells, and the transcriptomic sequencing results also indicated that high temperature caused significant enrichment of differentially expressed genes of ER-associated pathways, leading to ER stress. A. japonicus initiated varying degrees of endoplasmic reticulum stress in response to environmental temperature stress. The response restores the protein homeostasis of the endoplasmic reticulum by treating unfolded or misfolded proteins, reducing or alleviating the burden and damage to the endoplasmic reticulum, and restoring normal life activities. This study has characterized the internal molecular regulatory process of A. japonicus under heat stress from the perspective of endoplasmic reticulum stress, providing insights for future research on the response of A. japonicus to environmental stress and contributing to the protection and utilization of A. japonicus resources under global environmental change.

A. japonicus: Apostichopus japonicus

ER: Endoplasmic Reticulum

UPR: Unfolded Protein Response

H&E: Hematoxylin and Eosin

FPKM: Fragment per kilobase of transcript per million mapped reads

FDR: False Discovery Rate

DEGs: Differentially Expressed Genes

GO: Gene Ontology

KEGG: Kyoto Encyclopedia of Genes and Genomes

Conflict of Interest

The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Availability of data and materials

The sequencing data that support the findings of this study are openly available in the NCBI Sequence Read Archive (SRA) under BioProject Accession No. PRJNA932476.

Author Contributions

GS, JY designed and supervised the study. YY, LL, SL, RX, AZ and JZ prepared the samples. YY and SZ analyzed all sequencing data. YY, GS and JY wrote the manuscript. All authors have read and approved the final manuscript.

Funding

This work was supported by the Key Technology Research and Development Program of Shandong Province (2023LZGC019, 2023CXGC010410, 2022TZXD005), Natural Science Foundation of Shandong Province (ZR2021MC023), Agriculture Industry System of Shandong Province (SDAIT-22-02), and Taishan Industry Leading Talents, China (tscy20190114).

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No competing interests reported.

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Exploring Endoplasmic Reticulum Stress Pathways in Apostichopus japonicus Under Heat Stress Based on Transcriptome Analysis

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Abstract

Background

Results

Conclusions

Figures

1. Background

2. Materials and Methods

2.1 Temperature treatment experiment and Sample collection

2.2 Histological observation and transmission electron microscopy

2.3 RNA extraction, library construction, and sequencing

2.4 Data processing and differential expression analysis

2.5 Functional enrichment analysis

2.6 Building networks of protein–protein interactions for DEGs

3. Results

3.1 The structure of A. japonicus's organization at different temperatures

3.2 Transcriptome sequence and reads mapping

3.3 Transcriptome sequence and reads mapping

3.4 GO functional enrichment analysis of DEGs

3.5 KEGG enrichment analyses for DEGs

3.6 Construction of protein interaction networks using the DEGs with significant differences

4 Discussion

4.1 Temperature influences the morphology and internal structural composition of the digestive tract in A. japonicus

4.2 RNA-Seq reveals that heat stress alters gene expression in A. japonicus

4.3 Heat stress activated ER stress in A. japonicus

4.4 The key DEGs involved in the ER stress response by the digestive tract of A. japonicus

4.5 The regulatory process of ER stress and Unfolded Protein Response at different temperatures

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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