The Intestinal Microbiota Involves in the Deterioration of Live Sea Cucumber During Storage

ABSTRACT This study was designed to explore the relationship between the deterioration of sea cucumbers and intestinal flora’s diversity change during preservation using 16S rRNA gene sequencing. We show that the body wall of sea cucumber develops white spots on day 4, and the epidermis dissolves on day 7. The distinctive OTUs remain relatively low during four days of storage but increase on day 7. The potential gut microbiota biomarkers are Psychrobacter-cibarius, Ralstonia-pickettii, and Clostridiales during the deterioration of sea cucumber. The result of mixed storage of fresh and deteriorated sea cucumbers validates the influence of microbiota on healthy sea cucumbers. GRAPHICAL ABSTRACT


Introduction
The sea cucumber (Apostichopus japonicus) is an echinoderm species belonging to the Holothuroidea order and Stichopodidae family (Purcell et al. 2013). It is considered an expensive delicacy with high nutrition (Hossain et al. 2020;Li et al. 2019). In recent years, the output of sea cucumbers in China has exceeded 170,000 tons and boasts extensive market demand (Bureau of Fisheries in Ministry of Agriculture 2020; Su et al. 2018). Live sea cucumbers decompose at the tissue level due to environmental factors during storage and transportation. Decomposition manifests as epidermal damage and increases mortality (Tonna et al. 2016). The physiological stress, mechanical abrasion, and pathogenic microorganisms during the capture, processing, and transportation adversely affect the survival of sea cucumbers (Zamora and Jeffs 2015).
Pathogenic microorganism infection can cause epidermal ruptures of sea cucumbers and the acceleration of death (Li et al. 2010;Liu et al. 2010;Yang et al. 2016). The microorganisms that cause oral swelling, skin ulcers, and many fatal lesions during the cultivation of sea cucumbers mainly belong to V. splendidus, Shewanella sp., Pseudoalteromonas tetraodonis, and Serratia odorifera biogroup I (Li et al. 2010). Moreover, it has been reported that V. splendidus can destroy the immune function of sea cucumbers and cause skin ulceration syndrome (Lv et al. 2019). These studies explained that the reproduction and growth of pathogenic microorganisms have a substantial negative impact on the health of sea cucumbers in breeding.
Nonetheless, the effect of environmental microbial change on the natural deterioration process during sea cucumber transportation remains unknown. It is necessary to systematically and comprehensively study the role of environmental microorganisms on the deterioration procedure of sea cucumbers during storage. The flora composition in the transport seawater changes rapidly and is extremely unstable. Therefore, the current study uses sea cucumber intestinal flora as an indicator to study the impact of environmental microorganisms on the deterioration process of sea cucumbers.
This study aims to investigate the involvement of gut microbiota composition in the deterioration of sea cucumbers during preservation. The changes of gut microbial diversity during the routine storage of sea cucumbers were identified by 16S rRNA gene sequencing. Moreover, mixed storage of fresh sea cucumbers and the metamorphic sea cucumbers was performed to reveal the effects of gut microbiota composition change on the fresh individuals' deterioration during storage.

Materials
Sexually mature adult sea cucumbers (A. japonicus, 100-150 g; 12-16 cm total body length) were collected by divers in the Changhai Sea Area, the Yellow Sea of China. Sea cucumbers were packed without seawater in the absence of light and transported to the lab within two hours. BCA protein concentration determination kits purchased from China Beijing Solarbio Technology Co., Ltd. Kits for bacterial genomic DNA extraction were purchased from China Tiangen Biochemical Technology (Beijing) Co., Ltd. Ammonium chloride, salicylic acid, potassium sodium tartrate, copper sulfate, sodium hydroxide, sodium hypochlorite, and sodium nitroso ferricyanide were all analytical grade and obtained from China Tianjin Komiou Chemical Reagent Co., Ltd.

Live sea cucumber storage and the seawater determination
The live sea cucumbers were immediately put into a tank filled with fresh seawater after arrival at the lab. The 30 sea cucumbers were evenly distributed in a tank with 20 L of seawater and stored in a cold room at a temperature range of 8-12°C. The top of the tank was left open and protected from light to simulate the real transportation circumstances. The fresh seawater was not replaced and stored in a static state for up to 7 days. Three sea cucumbers were grouped randomly and sampled every day for testing according to storage time, such as Day 0 (storage 0 d), Day 1 (storage 1 d), Day 2 (storage 2 d), Day 3 (storage 3 d), Day 4 (storage 4 d), Day 5 (storage 5 d), Day 6 (storage 6 d), and Day 7 (storage 7 d). Set 6 parallels per group. During storage, photographs were taken every day to record the deterioration process of sea cucumbers. Seawater and intestinal content of sea cucumbers was sampled every day. A pH meter (PHS-3, Shanghai Lei Magnetic Instrument Factory, China) was used to determine the pH of each seawater sample. A dissolved oxygen meter (HQ430d, HACH Company, USA) was used to determine the dissolved oxygen content of each sample. The BCA protein content detection kit was used to determine the watersoluble protein content of each sample.

Total ammonia nitrogen measurement in seawater
The ammonia nitrogen level is related to the acceleration of biological degradation in metabolism ). The total ammonia nitrogen content reflects the deterioration shift of sea cucumber. The photometric salicylic acid flow injection method was used to determine the ammonia nitrogen content in each group's seawater sample (Zeng et al. 2014). The standard ammonium solution with an ammonium nitrogen (NH 4 Cl) concentration of 1 μg/mL and sodium nitroso ferricyanide solution of 0.01 g/mL were prepared. The 100 mL salicylic acid solution (0.5 g/mL) and 160 mL NaOH solution (2 mol/L) were mixed. Potassium sodium tartrate of 50 g was dissolved in 500 mL of water and mixed with the above solution to 1 L to obtain a color developing solution. Serial dilution of ammonia nitrogen was used to establish a standard curve. The storage water sample was adjusted to pH above 10.5 with 2 mol/L NaOH and filtered to obtain a clear liquid. Then, 1 mL of color developing solution, 2 drops of sodium nitroferricyanide solution, and NaClO solution was added to 1 mL of the filtered water sample. The mixture was diluted to constant volume of 10 mL with distilled water. After mixing uniformly, the reaction mixture was left in the dark at 25°C for 1 h. The absorbance was measured at a wavelength of 697 nm. The total ammonia nitrogen content of the water sample was calculated by the standard curve.

DNA extraction from sea cucumber intestinal content
The microbial DNA in the sea cucumber intestinal content was extracted using a bacterial genomic DNA extraction kit (China Tiangen Biochemical Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions.

16S rRNA gene sequencing and data analysis
A high-throughput sequencing and bioinformatics analysis of the bacterial 16S RNA gene regions V3-V4 in the gut microbial genome of sea cucumbers was performed to investigate the gut bacterial community composition during the deterioration of sea cucumbers. Amplification and sequencing of the V3-V4 region of the bacterial 16S DNA gene was performed by Beijing Nuohe Zhiyuan Technology Co., Ltd. (Beijing, China). Sea cucumber intestinal samples DNA (1 ng/μL) was used as template, and barcoded fusion primers 515F and 806 R were used as primers for PCR amplification. The primer sequences of 515F and 806 R were: 5'-GTGCCAGCMGCCGCGGTAA-3' and 5'-GGACTACHVGGGTWTCTAAT-3', respectively (Caporaso et al. 2012;Cole et al. 2014). The conditions of PCR amplification cycle were: 94°C predenaturation hold for 1 min, 94°C denaturation for 20 s, annealing at 53°C for 25 s; and lastly extension at 68°C for 45 s. After 30 cycles, an extension was performed at 68°C for 10 min. Library construction was performed using the TruSeq® DNA PCR-Free Sample Preparation Kit. The constructed 16S rDNA library was quantified using Qubit and Q-PCR. Once the library was established, the NovaSeq6000 system was used for second-generation sequencing.
Raw Tags data were obtained by splicing the reads of each sample using FLASH (V1.2.7, http://ccb. jhu.edu/software/FLASH/) (Magoč and Salzberg 2011). Raw data was filtered using the Tags quality control process of Qiime (V1.9.1, http://qiime.org/scripts/split_libraries_fastq.html) (Caporaso et al. 2010) for the Tags quality control process to obtain high quality Tags data. Cluster analysis was performed on the entire sequences of the sample using Uparse software (v7.0.1001, http://www.drive5. com/uparse/) (Haas et al. 2011). The sequences were clustered into OTUs with 97% identity. The highest frequency sequences in OTUs were screened as representative sequences. Species annotation analysis was performed using the Mothur method with the SSUrRNA database (Wang et al. 2007) of SILVA132 (http://www.arb-silva.de/) (Edgar 2013). Chao1, Shannon, Simpson, ACE indices were calculated using Qiime software (version 1.9.1). R software was used to perform between-group difference analysis of alpha diversity index. UPGMA sample clustering trees were constructed according to the Unifrac distances calculated by Qiime software (version 1.9.1). Principal component analysis (PCA) was performed using the ade4 and ggplot2 packages of R software. Principal coordinate analysis (PCoA) was performed using the WGCNA, stats, and ggplot2 packages of R software (version 2.15.3). LEfSe analysis was performed using LEfSe software with a default setting of 4 for the LDA Score screening.

Mixed storage of the deteriorated and healthy sea cucumbers
The mixed storage of the deteriorated and healthy sea cucumbers was carried out to investigate the microbial flora role during the sea cucumber deterioration procedure. Six fresh sea cucumbers were previously stored for 6 days to obtain the deteriorated sea cucumbers. The six deteriorated sea cucumbers were mixed equally with six fresh sea cucumbers to store for 6 days (mixing group). Another twelve fresh sea cucumbers were stored for 6 days for comparison and named as control group (unmixed group). The environmental temperature during storage ranged from 8 to 12°C, and the density was 0.67 L/animal. The seawater pH value, dissolved oxygen content, soluble protein content, total ammonia nitrogen content, and the morphologic change were measured.

Data processing and statistical analysis
The discovered data obtained in the experiment was graphed by Origin 8.5 software (OriginLab Corporation, Northampton, MA, USA) and statistically processed with SPSS 20.0 (SPSS Inc., Chicago, IL, USA). The significant difference analysis of the data adopts the method of one-way analysis of variance followed by Duncan's test. The signs are expressed as X ± S, p < .05 as significant, and p < .01 as highly significant.

The deterioration pattern of live sea cucumber during storage
Photographs were taken daily to document the structural modifications of the sea cucumber epidermis under natural storage environment. The results showed that the skin of sea cucumbers is severely damaged after 7 days of storage ( Figure 1a). Fresh sea cucumbers had complete skin, deep color, and hard spines at day 0. The sea cucumber body wall began to develop white spots on day 4, and the epidermis dissolved on day 7. Within 7 days of natural storage, the body wall of sea cucumber was generally flabby, the spines became softer, and the skin produced more mucus. This demonstrates that the live sea cucumbers deteriorate after 7 days of natural storage. Figure 1b-e illustrates the changes in the stored seawater. As shown in Figure 1b, the pH of seawater decreased and remained at low level from day 1 to day 5. It increased at day 6 and pH reached 7.56 ± 0.25 at day 7. Total ammonia nitrogen in seawater increased from day 1 to day 7 ( Figure 1c). The concentration of ammonia nitrogen reached 6.19

Alpha diversity of microbiota and OTU clusters in intestinal contents
The composition and species distribution of intestinal microbial in live sea cucumber was examined using 16S rRNA sequencing. A total of 90,301 tags were obtained by metabarcoding sequencing, and 85,535 valid data were retained after quality filtering. Samples from sea cucumber gut were analyzed at a 97% identity level by assigning OTUs.
At the phylum level, the dominant flora in the epidermolysis process of sea cucumber were mainly Proteobacteria, unidentified bacteria, Bacteroidetes, and Firmicutes ( Figure 2). With prolonged storage, the abundance of Bacteroidetes in the sea cucumber intestinal flora increased from 4.17% (day 0) to 12.68% (day 1), then reduced to 1.70% (day 5); the abundance of Actinobacteria in the sea cucumber intestinal flora decreased from 1.74% (day 0) to 0.11% (day 3); the abundance of Tenericutes decreased from 3.19% (day 0) to 1.79% (day 3); and the abundance of Firmicutes decreased from 13.37% (day 0) to 7.36% (day 1) (Figure 2). The composition of sea cucumber intestinal microbes at the phylum level indicated that Actinobacteria, Firmicutes and Tenericutes are related to the deterioration of sea cucumbers.
The intestinal flora diversity of sea cucumbers changed significantly during sea cucumber deterioration. The Chao1, Simpson, ACE, and Shannon indices of α-diversity show a trend of initial decrease and then increase with prolonged storage (Figure 3). Clustering was performed from two levels of species and samples, and the top 35 abundance genera in the intestinal flora of sea cucumbers were selected to create the heat map (Figure 4a). The species information of these genera is shown in Table S1. The accumulation of bacteria was analyzed at the genus level based on the heat map. At the genus level, the dominant flora in sea cucumber changed from Roseovarius, Weissella, Aerococcus, Lutoreibacte, Planococcus, Fusobacterium, Ruegeris, Lutibacter at day 0 to Bacteroides, Bizionia, Pseudofulvibacter, Shewanella, Colwellia, Romboutsia, Unidentified_Enterobacteriaceae during deterioration at Day 7. Moreover, compared to the fresh sea cucumbers (Day 0), the individuals that have undergone deterioration (Day 7) contained higher levels of corruption related pathogens, such as Shewanella and Arcobacter. Yet, the abundance of some beneficial bacteria, such as Lactobacillus and Weissella, decreased in the deteriorated sea cucumber (Day 7). The unique and standard OTUs of sea cucumbers at different periods was analyzed. The number of OTUs shared by all samples was 296; the number of unique OTUs on day 7 was as high as 2609, while the number of distinctive OTUs on day 0 was 124 ( Figure 4b). This suggests that the occurrence of sea cucumbers deteriorating produces a distinctive intestinal microbial composition.

Correlation analyses of sea cucumber intestinal microbial composition and deterioration
The hierarchical clustering of the β-diversity distance matrix of intestinal microbial composition during the sea cucumber deterioration was analyzed to explore the composition quality of the species. The similarity of microbial communities among each group was determined by the branch tree ( Figure 5). The hierarchical clustering results showed the samples cluster of Day 1 and Day 3 and the samples cluster of Day 4 and Day 6. The samples from Day 0 and Day 7 were not clustered together ( Figure 5). In addition, we performed principal component analysis and principal coordinate analysis based on the unweighted unifrac arithmetic to compare the diversity of each intestinal flora sample. The purpose of PCA and PCoA analysis was to explore similarities in species composition among taxa. PCA analysis showed that the first and second principal coordinates account for 44.08% and 8.39% (Figure 6a). PCoA analysis showed that the first, second, and third principal coordinates account for 18.33%, 10.77%, and  6.76% (Figure 6b). The PCA analysis showed a significant overlap between these groups (Figure 6a). However, the 3D PCoA analysis showed that the Day 7 group is significantly separated from the other groups (Figure 6b). These results suggest that the composition of the gut flora of sea cucumbers is similar during metamorphosis, but deteriorated sea cucumbers possess a specific gut microbiota.  The LEfSe analysis of intestinal microbial composition during the deterioration of sea cucumber is presented in Figure 7a,b. The Day 4 group had no specific species compared to other groups. At Day 0, we identified 10 specific species, among which Firmicutes and Bacilli have the most influence ( Figure 7a). However, the Day 7 group displayed another 5 specific species, among which Clostridiales had the most influence (Figure 7a). These results suggest that sea cucumbers have different biomarkers at different times of storage.
The fresh sea cucumber group (Day 0) was significantly different from the deteriorated sea cucumber group (Day 7) at the levels of phylum and order (Figure 7b). Firmicutes were the most differentially abundant bacterial phylum in the intestinal flora of the Day 0 group; whereas, Actinobacteria dominated the intestinal flora of the Day 7 group at the phylum level. At the order level, the bacterial orders with the highest differential abundance in the intestinal flora of the Day 0 group were Lactobacillus, Rhodobacterales and Bacillales. The bacterial orders with the highest differential abundance in the intestinal flora of the Day 7 group was Clostridiales. This demonstrates  that the gut microbial community of sea cucumbers undergoes significant differences in composition and distribution during metamorphosis.

The effect of mixed storage on healthy sea cucumbers
The deterioration of the preservative sea cucumbers is associated with the intestinal microbiota composition change. To reveal whether the intestinal microbiota composition change is involved in the deterioration process of storage sea cucumbers, the fresh and the deteriorated sea cucumbers were stored together for 6 days. Sea cucumbers in the unmixed and mixed groups exhibited epidermal dissolution at day 5 and day 2, respectively. The mixed group had a faster deterioration speed compared to the unmixed group. The body wall of sea cucumbers in the mixed group became soft and thinner at day 3. On the contrary, the body wall of sea cucumbers in the unmixed group became soft and thin at day 6. The seawater pH of the mixed sea cucumber was 5.64 ± 0.02, significantly lower than the unmixed sea cucumber of 5.81 ± 0.04 (Figure 8b). The dissolved oxygen of the mixed group was close to 0.06 ± 0.00 and significantly lower than that in the unmixed group of 0.30 ± 0.08 after storage for 6 days (Figure 8c). The total ammonia-nitrogen of the mixed group was 467.98 ± 29.77 mg/ L, significantly higher than the unmixed group of 352.12 ± 13.09 mg/L (Figure 8d). However, the water-soluble protein was not significantly different between the two groups ( Figure 8e).

Discussion
In the actual transportation environment, the storage temperature of sea cucumbers is about 8-12°C. Therefore, this study chose this temperature range as the storage temperature to simulate the sea cucumber transportation and preservation environment. The deterioration of sea cucumbers is related to many factors during storage. This study focused on the microorganism's effects on sea cucumber deterioration during storage. The microbes in the water environment change rapidly (Fei et al. 2014). Therefore, the intestinal microbiota was determined to be indicative to the microbial changes in the water environment.
The deterioration pattern of live sea cucumber during 7 days of storage was observed. The release of ammonia nitrogen and free protein are the hallmarks of live sea cucumber deterioration. The ammonia nitrogen level increase is related to the acceleration of biological degradation in metabolism . The significant increase of ammonia nitrogen indicates that the sea cucumber metabolism starts to deteriorate. High levels of total ammonia nitrogen in the storage environment can feedback to damage the respiratory system of aquatic animals, leading to mortality (Wang et al. 2021). When the sea cucumber commences to autolysis, the tissue protein would decompose and release the free protein outside the body (Xing et al. 2021). Thus, the free protein increase in seawater indicates that the sea cucumbers have severely deteriorated. Our results showed that the live sea cucumbers increased the release of ammonia nitrogen from day 4 under the seawater preservation at 8-12°C. The dissolved oxygen was nearly exhausted at day 5, which led to tissue degradation and free protein release at day 6. The seawater pH rose significantly at day 6, demonstrating the deterioration of sea cucumbers. The sea cucumber storage state diagram shows that the deterioration from day 1 to day 3 is not obvious, and sea cucumbers were observed to rot and produce mucus from day 4 to day 7 ( Figure 1). Therefore, day 1 to day 3 was considered as the early phase of deterioration, and day 4 to day 7 was considered as the late phase of deterioration.
Alpha diversity is mainly related to richness and homogeneity in the distribution of individuals in the community (Li et al. 2022). Shannon index and Simpson index results showed that the community diversity of sea cucumbers intestinal flora dropped within 3 days but improved from day 4 to day 7. The decrease in community diversity in the early phase indicated that the intestinal flora gradually changed but remained steady. The increase in community diversity from day 4 to day 7 (the late phase) demonstrated that the flora composition began to be replaced dramatically compared to the first 3 days. Both Chao1 and ACE indexes showed a decline from day 0 to day 4 but increased significantly on day 7, suggesting the sea cucumber intestinal flora richness tends to decrease within 4 days of storage. The intestinal flora diversity and richness decreases due to the lack of food, which leads to the shrinkage of flora during storage. Although the reduction of diversity and richness does not influence the condition of sea cucumber, the increase of diversity and richness at late phase in our study indicated the blooming of new bacteria species. The blooming of new bacteria species replaced the original flora composition and harmed the sea cucumber health. These results suggest that the sea cucumbers remain healthy for the first 3 days but deteriorate severely at day 7. These results are consistent with the seawater substance analysis, showing that the sea cucumbers maintain good condition at the early phase (0 to 3 days) and start to deteriorate at the late phase (4 to 7 days). The number of OTUs shared by all samples was 296, while the number of distinctive OTUs from day 0 to day 4 were 124, 81, 58, 38, and 29, respectively. The distinctive OTUs kept a relatively low level within the 4 days of storage but increased on day 5 and day 7. The distinctive OTUs at day 7 reached 2609, which was 8.8-fold of the shared OTUs. Moreover, species abundance cluster results showed that the dominant flora at the genus level in sea cucumber changes from day 0 to day 7. The hierarchical clustering of the β-diversity distance matrix results confirmed that the samples of Day 0 and Day 7 are not clustered together. Also, the 3D PCoA analysis showed that the Day 7 group is significantly separated from the other groups, indicating that the Day 7 group has no similarities in species composition among taxa. Gut microbiota is associated with the health of a wide range of animals . Research has shown that the gut microbiota of sea cucumbers is specific when their body states are different (Song et al. 2019). The deteriorative sea cucumbers (day 7) have a completely different approach to dominant intestinal flora of healthy individuals.
Shewanella and Arcobacter are considered to be pathogenic bacteria that cause the deterioration of sea cucumbers during cultivation (Mizutani et al. 2019;Wang et al. 2018;Zhao et al. 2019). The abundance of Shewanella and Arcobacter increased significantly on day 7 compared to day 0 ( Figure 4a). Hence, these two bacteria may play a role in the deterioration process of stored sea cucumbers.
Previous research showed that LEfSe assessment can identify potential biomarkers from the gut microbiota (Kong et al. 2019). The Psychrobacter-cibarius, Ralstonia-pickettii, and Clostridiales were identified as specific species in the late deterioration period (Day 4 -Day 7). Psychrobacter species are commonly found in seafood and can cause acid formation from carbohydrates. Broekaert et al. (2013) suggested that there is a potential risk of Psychrobacter-cibarius to promote spoilage of brown shrimp. The Psychrobacter-cibarius was the species with the most significant difference in abundance on day 5. The abundance of Psychrobacter-cibarius increased obviously on day 5 ( Figure S1). The markedness of Psychrobacter-cibarius at late phase reinforces the sea cucumber deterioration, and it is an indicative species during the deteriorative process. Fowler et al. (2021) suggested that Ralstonia pickettii may be a potential pathogen affecting the hybrid striped bass. Clostridiales are microbes that are very prone to food spoilage (Green et al. 2020). The abundance of Ralstonia-pickettii and Clostridiales increased significantly on day 6 and day 7 ( Figure S1). This change could be a sign of deterioration of sea cucumber. Therefore, it is reasonable to consider Psychrobacter-cibarius, Ralstonia-pickettii, and Clostridiales as biomarkers indicating sea cucumber deterioration.
In addition, Vibrio splendidus is a pathogen that has an immense influence on the health of sea cucumbers (Liu et al. 2020). Vibrio is also considered to be the main pathogen causing the deterioration of sea cucumbers in aquaculture. (Kim et al. 2017;Yamazaki et al. 2016;Zeng et al. 2020). Our study found that Vibrio increased sharply on Day 2, but its abundance decreased on Day 5 (Figure 4a). Although the LEfSe analysis indicated Vibrio is the most influential in the early phase of deterioration (Day 2), the sea cucumber did not begin to deteriorate at this stage ( Figure 1a). Therefore, Vibrio did not cause the deterioration of sea cucumber directly in the storage and transportation. This may be because Vibrio is gradually reduced by competition from other flora at the late phase of deterioration. As a result, other bacteria could cause the deterioration of sea cucumber during transportation.
The hypothesis of deteriorated sea cucumbers will accelerate the deterioration of healthy sea cucumbers is verified by the mixed storage study. The sea cucumbers in the mixed group developed skin peeling on the second day, which is much earlier than sea cucumbers in the unmixed group. This indicates that the deterioration of the sea cucumber occurred earlier after being mixed with the metamorphic sea cucumber. Previous studies have shown that excessive acidity in the environment can promote acidosis of sea cucumbers (Collard et al. 2014), and low oxygen content is not conducive to sea cucumber survival (Zamora and Jeffs 2015). This study found that mixed storage of fresh and unfresh sea cucumbers had lower dissolved oxygen content and pH value than the unmixed storage group, and mixed storage released more ammonia nitrogen than the unmixed storage group. This study reveals that the mixed storage of healthy and deteriorated sea cucumbers accelerates the deterioration of healthy sea cucumbers. Rigos and Katharios (2010) suggested that individuals infected with pathogenic microorganism may also affect the health of other individuals when aquatic animals are in the same environment. Therefore, the reason for the deterioration of sea cucumbers may be the migration of microorganisms from degraded sea cucumbers to normal sea cucumbers through the water environment.
In summary, sea cucumbers began to deteriorate on day 4 and completely deteriorated on day 7 in normal storage environment. The deterioration of sea cucumbers promoted the abundance of pathogenic bacteria (Shewanella and Arcobacter). The s-Psychrobacter-cibarius, s-Ralstonia-picketti, and o-clostridiales can be used as biomarkers to diagnose deteriorative proceeding of sea cucumbers. The mixed storage of fresh and deteriorated sea cucumbers accelerates the deterioration of sea cucumbers. This study provides information that can be used to regulate the storage environment and extend the transportation time of live sea cucumbers.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by China's National Key R&D Program (2018YFD0901001) and the National Natural Science Foundation (No. 31771998).