Sea cucumber physiological response to abiotic stress: Emergent contaminants and climate change.

The ocean is facing a multitude of abiotic stresses due to factors such as climate change and pollution. Understanding how organisms in the ocean respond to these global changes is vital to better predicting consequences. Sea cucumbers are popular echinoderms with multiple ecological, nutritional, and pharmaceutical benefits. Here, we reviewed the effects of environmental change on an ecologically important echinoderm of the ocean, aiming to understand their response better, which could facilitate healthy culture programs under environmental changes and draw attention to knowledge gaps. After screening articles from the databases, 142 studies were included on the influence of emergent contaminants and climate variation on the early developmental stages and adults of sea cucumbers. We outlined the potential mechanism underlying the physiological response of sea cucumbers to emerging contaminants and climate change. It can be concluded that the physiological response of sea cucumbers to emergent contaminants differs from their response to climate change. Sea cucumbers could accumulate pollutants in their organs but are aestivated when exposed to extreme climate change. Research showed that the physiological response of sea cucumbers to pollutants indicates that these pollutants impair critical physiological processes, particularly during the more susceptible early phases of development compared to adults, and the accumulation of these pollutants in adults is often observed. For climate change, sea cucumbers showed gradual adaptation to the slight variation. However, sea cucumbers undergo aestivation under extreme conditions. Based on this review, critical suggestions for future research are presented, and we call for more efforts focusing on the co-occurrence of different stressors to extend the knowledge regarding the effects of environmental changes on these economically and ecologically important species.

Microplastics in canned, salt-dried, and instant sea cucumbers sold for human consumption.

Determining the amount of microplastics (MPs) in food is key to clarifying their potential toxicity to humans. Here, we collected canned, instant, and salt-dried sea cucumbers Apostichopus japonicus, the most valued sea cucumbers, from Chinese markets to determine their content of MPs. Sea cucumbers contained MPs in the range of 0-4 MP individual-1, an average of 1.44 MP individual-1, and 0.081 MP g-1. Accordingly, consuming 3 g of sea cucumbers could result in an exposure risk of an average of 0.51 MPs, 0.135 MPs, and 0.078 MPs day-1 for canned, instant, and salt-dried sea cucumbers, respectively. MPs were in size range of 12-575 µm, and fibrous shape was dominant. Furthermore, among the five polymers identified, polypropylene showed the highest energy binding with two catalysts engaged in organic chemical oxidation. This study extends the knowledge regarding MPs occurrence in food and provides a theoretical basis for MPs toxicity in humans.

A regulatory network controlling ovarian granulosa cell death.

Follicular atresia triggered by granulosa cell (GC) apoptosis severely reduces female fertility and accelerates reproductive aging. GC apoptosis is a complex process regulated by multiple factors, regulatory axes, and signaling pathways. Here, we report a novel, small regulatory network involved in GC apoptosis and follicular atresia. miR-187, a miRNA down-regulated during follicular atresia in sows, maintains TGFBR2 mRNA stability in sow GCs by directly binding to its 5'-UTR. miR-187 activates the transforming growth factor-ß (TGF-ß) signaling pathway and suppresses GC apoptosis via TGFBR2 activation. NORHA, a pro-apoptotic lncRNA expressed in sow GCs, inhibits TGFBR2-mediated activation of the TGF-ß signaling pathway by sponging miR-187. In contrast, NORFA, a functional lncRNA associated with sow follicular atresia and GC apoptosis, enhances miR-187 and TGFBR2 expression by inhibiting NORHA and activating NFIX. Our findings define a simple regulatory network that controls GC apoptosis and follicular atresia, providing new insights into the mechanisms of GC apoptosis, follicular atresia, and female fertility.

Single and combined effects of microplastics and cadmium on the sea cucumber Apostichopus japonicus.

Microplastic pollution of the ocean has received extensive attention as plastic pollution increases globally, but the potential ecological risks caused by microplastic interactions with trace metals still require further research. In this study, Apostichopus japonicus was used to explore the individual and combined toxicities of cadmium (Cd) and microplastics and their effects on growth, Cd tissue accumulation, digestive enzymes, and gut microbes. The body weight gain and specific growth rate of animals exposed to a combination of high concentrations of Cd and microplastics decreased. The addition of high concentrations of cadmium to the diet led to an increase in cadmium content in the respiratory tree, digestive tract and body wall. Amylase, lipase and trypsin decreased to different degrees in the group treated with high concentrations of Cd/microplastics. Firmicutes were significantly reduced across multiple treatment groups, with the order Lactobacillales being the most significantly affected. Cd is the pollutant causing the greatest negative impact, but the presence of microplastics undoubtedly increases its toxicity.

Association of heavy metals with plastics used in aquaculture.

The association of heavy metals with plastics could represent a source of contamination, presenting acute metal exposure to ecosystems. Here, we aimed to evaluate the level of 20 metals on plastics employed in aquaculture areas and their surrounding water. All the selected metals were detected on six different polymers/types of plastics. Despite the difference in sampling sites, there was no significant difference among metal concentrations on plastics. However, some metals showed a significant difference in the surrounding seawater among sites or were not detected. Additionally, the median concentrations of all heavy metals were higher on plastic than in their surrounding water at all sites. Furthermore, multivariate analysis showed that metals accumulate on plastics in a similar pattern among sites but distinct from the surrounding water. This study contributes to understanding the accumulation of metals on plastics used in aquaculture areas.

Existence of microplastics in the edible part of the sea cucumber Apostichopus japonicus.

Microplastics (MPs; ≤ 5 mm) have become a potential threat to human health due to the widespread detection of MPs in foods consumed by humans. Here, we investigated the potential of MP occurrence in the main edible part of the most valuable species of sea cucumbers (Apostichopus japonicus). Laboratory experiments showed that fluorescent MPs and microfibers (MFs) could transfer into the body wall of the sea cucumber. The evidence revealed that these MPs enter the body wall via the outer surface. Although these MPs decreased after the sea cucumbers were transferred to clean water, traces of MPs (at least one MP particle) were found up to 60 d post-transfer. To validate these laboratory observations, sea cucumber samples were collected from the field. MPs were found in 86% of live and processed sea cucumber samples. The MP abundances in the field samples ranged from 0-15 MPs animal-1 and 0-2 MP g-1. The isolated MPs were mainly MFs, constituting 81% of MPs, followed by fragments, films, and beads. Fourier transform infrared spectroscopy revealed that the polymer composition of the isolated MPs mainly included rayon, followed by polyester and chlorinated polyethylene. The findings of this study demonstrated that the body walls of farmed and processed sea cucumbers contain MPs, thus highlighting the need to control MP pollution during the farming and processing of sea cucumbers.

SMAD4 Inhibits Granulosa Cell Apoptosis via the miR-183-96-182 Cluster and FoxO1 Axis.

The miR-183-96-182 cluster is a polycistronic miRNA cluster necessary for ovarian functions in mammals. However, its transcriptional regulation in the ovary is largely unclear. In this study, we characterized the promoter region of the porcine miR-183-96-182 cluster, and showed that SMAD4 may function as a transcriptional activator of the miR-183-96-182 cluster in GCs through direct binding to SBE motifs in its promoter. SMAD4 may inhibit GC apoptosis via suppression of FoxO1, an effector of GC apoptosis and a direct target of the miR-183-96-182 cluster, by inducing the miR-183-96-182 cluster, and this process may be regulated by the TGF-ß/SMAD signaling pathway. Our findings uncovered the regulatory mechanism of miR-183-96-182 cluster expression in GCs and demonstrated that TGF-ß1/SMAD4/miR-183-96-182 cluster/FoxO1 may be a potential pathway for regulating follicular atresia and female reproduction.

Emerging roles of circRNAs in regulating thermal and hypoxic stresses in Apostichopus japonicus (Echinodermata: Holothuroidea).

Some sea cucumbers are economically and ecologically important, but they are threatened by thermal and hypoxic stress in changing oceanographic conditions. We construct circRNAs profiles, reveal circRNAs characters, and illustrate the potential regulatory roles of circRNAs in one commercially important species of sea cucumber, Apostichopus japonicus. Reads are distributed in intergenic (44.14%), exonic (48.26%) and intronic (7.60%) regions of the genome. A total of 1684 circRNAs were identified, and the most common spliced length is 269 nt in the present study. In three treatments (HT [thermal stress], LO [hypoxic stress], and HL [combined thermal and hypoxic stress]), 24, 27 and 27 differentially expressed (DE) circRNAs were identified, respectively. Five novel DE-circRNAs commonly occur in these treatments (novel_circ_0003311, novel_circ_0000229, novel_circ_0003944, novel_circ_0001458 and novel_circ_0000707), and based on them, potential circRNA-miRNA binding pairs were predicted. Sanger sequencing, RNase R treatment experiment and qPCR validation identified the accuracy of the circRNAs. Key circRNAs identified in the present study were covalently closed and were more stable under RNase R treatment than linear RNAs. Based on function analysis, circRNAs could regulate metabolic process, signal transduction, and ion responses in A. japonicus when exposed to thermal and hypoxic stress, and 'regulation of response to stimulus' is a common gene ontology (GO) term that is significantly enriched in each treatment; GO terms for 'DNA' and 'stress' are commonly enriched in heat-related treatments (HT and HL); and GO terms for 'protein' are commonly enriched in hypoxia-related treatments (LO and HL). When environmentally stressed, 'metabolism,' 'transport and catabolism,' 'membrane transport,' and 'signal transduction' were significantly responded in sea cucumber based on KEGG analysis. We provide insights into circRNA functions in stress regulation and lay a foundation for invertebrate circRNA research.

Mechanism underlying the toxicity of the microplastic fibre transfer in the sea cucumber Apostichopus japonicus.

Microscopic plastic particles (0.1 µm-5 mm) are widespread hazardous pollutants, and microfibres (MFs) are their dominant shape in habitats. Previous field and laboratory studies have demonstrated that MFs enter the coelomic fluid of sea cucumbers from the water through the respiratory tree. However, the possible mechanism underlying the toxicity of this process is not well understood. Herein, RNA-Seq was used to examine the responses of the respiratory tree during the MF transfer process in the sea cucumber Apostichopus japonicus. Polyester synthetic MFs were used, and the number of transferred MFs was controlled to the amount reported from the field. The results showed that the MFs altered gene expression as the transfer process increased. The top genes regulated by MF transfer were mainly related to metabolic processes and signal transduction pathways, with upregulated genes following low MF transfer and downregulated genes following high MF transfer. Functional enrichment analysis revealed the pathways in which differentially expressed genes were enriched under different MF transfer scenarios. The transcriptomic findings were further supported by histological observations, which revealed injury and loss of cell components. This study contributes to understanding the effects of MFs in a valuable echinoderm species through transcriptomic and histological examinations.

Effect of chronic exposure to microplastic fibre ingestion in the sea cucumber Apostichopus japonicus.

Microplastics (MPs) in the form of microfibres (MFs) are of great concern because of their size and increasing abundance, which increase their potential to interact with or be ingested by aquatic organisms. Although MFs are the dominant shape of MPs ingested by sea cucumbers in habitats, their effect on sea cucumbers remains unclear. This study examined the effect of dietary exposure to MFs on the growth and physiological status of both juvenile and adult Apostichopus japonicus sea cucumbers. MFs were mixed into the diet of sea cucumbers for 60 d at environmentally relevant concentrations of 0.6 MFs g-1, 1.2 MFs g-1 and 10 MFs g-1. Dietary exposure to MFs, with concentrations at or above those commonly found in the habitats, did not significantly affect the growth and faecal production rate of either juvenile or adult sea cucumbers. However, a disruption in immunity indices (acid phosphatase and alkaline phosphatase activity) and oxidative stress indices (total antioxidant capacity and malondialdehyde content) was observed in juvenile and adult sea cucumbers, indicating that these indices might be useful as potential biomarkers of the exposure to MF ingestion in sea cucumbers. This study provides insights into the toxicity mechanism of MF ingestion in a commercially and ecologically important species.

A deposit-feeder sea cucumber also ingests suspended particles through the mouth.

Although the sea cucumber Apostichopus japonicus has been characterised as a deposit feeder, nutrients sourced from the water column have been recorded in the intestines of this species. However, the mechanisms whereby nutrients in the water enter the intestinal tract of A. japonicus, and whether other suspended particles can be ingested via the mouth of A. japonicus adults, remain unknown. Here, we reveal how A. japonicus ingests suspended particles through the mouth. We used synthetic particles and video recording to confirm the suspension uptake by the sea cucumber. Apostichopus japonicus continued to ingest suspended particles (if present) over time, and the particle ingestion rate was positively correlated with the concentration of suspended particles (Pearson correlation: r=0.808). Additionally, clearance rates of the suspended particles ranged from 0.3 to 0.9 l h-1 The findings of this study thus provide evidence of a previously undescribed particle uptake mechanism in a commercially important species.

Transcriptome analysis of phototransduction-related genes in tentacles of the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus (Selenka)is a typical nocturnal echinoderm, which is believed to be almost completely dependent on light intensity for the regulation of endogenous rhythms. Under conditions of high light intensity, this species shows clear evidence of light avoidance behavior, seeking out shaded areas of reef in which to reside. In this study, we performed RNA-Seq analysis to examine the tentacle transcriptome of A. japonicus specimens that had been subjected to dark and light (5 min and 1 h) conditions. We specifically focused on detecting genes involved in opsin-based light perception, including opsins and members of phototransduction-related pathways. On the basis of comparisons with both vertebrate and invertebrate phototransduction pathways, we determined that components of two of the main metazoan phototransduction pathways were altered in response to illumination. Among the key phototransduction-related genes in tentacles, we identified retinol dehydrogenase, members of the dehydrogenase/reductase family, and myosin III, and also detected a pair of visual pigment-like receptors, peropsin and peropsin-like, the homologous genes of which are believed to have the same function but show opposite expression patterns in response to different light environments. In general, the up-regulation of key genes in sea cucumber exposed to illumination indicated that the tentacles can respond to differences in the light environment at the molecular level.

Microplastic fibers transfer from the water to the internal fluid of the sea cucumber Apostichopus japonicus.

Microplastics (MPs) are small plastic particles less than 5 mm in diameter. MPs in the form of microfibers (MFs) are widely detected in aquatic habitats and are of high environmental concern. Despite many reports on the effects of MFs on marine animals, their effect on sea cucumbers is still unclear. In addition, our previous filed study has shown that MFs may transfer to the coelomic fluid of the sea cucumber Apostichopus japonicus (A. japonicus). Here, we show how MFs transfer to the coelomic fluid of the sea cucumber. We captured the MFs during their transfer from the water to the coelomic fluid through the respiratory tree. A. japonicus ingested in the MFs along with the water during respiration; the MFs got stuck in the respiratory tree or transferred to the coelomic fluid. The transferred MFs increased during 72 h of exposure and persisted for 72 h after the transfer to clean water. Among the immunity indices, lysozyme (LZM) levels increased in response to the transferred MFs, which confirms the defensive role of LZMs against strange substances. Additionally, non-significantly decreased levels of total antioxidant capacity (T-AOC), malondialdehyde (MDA), peroxidase (POD) and phenol oxidase (PPO) were observed at 24 h and 48 h post-exposure, suggesting minimal oxidative imbalance. Furthermore, there were no significant changes in the speed and the total distance moved by A. japonicus post MFs transfer. This study revealed that MFs transfer and accumulate in the coelomic fluid of A. japonicus.

Heavy metals in sediment, microplastic and sea cucumber Apostichopus japonicus from farms in China.

The concentrations of eight heavy metals (As, Cd, Cr, Cu, Mn, Ni, Pb and Zn) were measured in the sediment, the isolated microplastics from the sediment and the body wall of sea cucumbers from farms in China. Accordingly, the heavy metal concentrations in the sediment were below the class I upper limit of Chinese sediment quality guidelines. Among heavy metals, the median concentrations of Cd and As were higher in the body wall than in the corresponding sediment. Additionally, the median concentrations of Cd, Pb, and Zn were higher on the microplastics than in the corresponding sediment. Furthermore, there was no significant correlation among heavy metals in sediment, sea cucumber and microplastics. This study contributes to the understanding of the heavy metal accumulation in the sediment, the microplastics and the body wall of the sea cucumber.

Transcriptome analysis provides insights into the molecular mechanisms responsible for evisceration behavior in the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus (Selenka) is a valuable economic species in Southeast Asia. It has many fascinating behavioral characteristics, such as autolysis, aestivation, regeneration, and evisceration, thus it is a notable species for studies of special behaviors. Evisceration and autotomy are controlled by the neural network and involve a complicated physiological process. The occurrence of evisceration behavior in sea cucumbers is strongly related to their environment, and it negatively impacts their economic value. Evisceration behavior plays a pivotal role in the survival of A. japonicus, and when it is induced by dramatic changes in the coastal ecological environment and the aquaculture setting it can strongly affect the economic performance of this species. Although numerous studies have focused on intestinal regeneration of A. japonicus, less is known about evisceration behavior, especially its underlying molecular mechanisms. Thus, identification of genes that regulate evisceration in the sea cucumber likely will provide a scientific explanation for this significant specific behavior. In this study, Illumina sequencing (RNA-Seq) was performed on A. japonicus specimens in three states: normal (TCQ), eviscerating (TCZ), and 3 h after evisceration (TCH). In total, 129,905 unigenes were generated with an N50 length of 2651 base pairs, and 54,787 unigenes were annotated from seven functional databases (KEGG, KOG, GO, NR, NT, Interpro, and Swiss-Prot). Additionally, 190, 191, and 320 genes were identified as differentially expressed genes (DEGs) in the comparisons of TCQ vs. TCZ, TCZ vs. TCH, and TCQ vs. TCH, respectively. These DEGs mapped to 157, 113, and 190 signaling pathways in the KEGG database, respectively. KEGG analyses also revealed that potential DEGs enriched in the categories of "environmental information processing," "organismal system," "metabolism," and "cellular processes," and they were involved in evisceration behavior in A. japonicus. These DEGs are related to muscle contraction, hormone and neurotransmitter secretion, nerve and muscle damage, energy support, cellular stress, and apoptosis. In conclusion, through our comparative analysis of A. japonicus in different stages, we identified many candidate evisceration-related genes and signaling pathways that likely are involved in evisceration behavior. These results should help further elucidate the mechanisms underlying evisceration behavior in sea cucumbers.

Effects of artificial reefs on the meiofaunal community and benthic environment - A case study in Bohai Sea, China.

Artificial reefs are widely deployed for fishery enhancement and marine conservation. A comprehensive assessment on the effects of artificial reefs could minimize the negative consequence of blindly developing artificial reefs. We examined the meiofaunal community and benthic environment adjacent to and <5 m from artificial reefs in Xiangyun Bay, Bohai Sea, China. We found the highest total meiofaunal abundance beside the artificial reef. Shannon-Wiener and Pielou indexes had no significant difference among different distances from the artificial reefs. The presence of artificial reefs impeded the surrounding flow and provided additional substrate for bivalves and kelps, which could cause finer sediment and organic enrichment around it. Sediment grain size and total organic matter were the most important parameters influencing the meiofauna. We suggest that the shape, material, configuration and location of artificial reefs should be related with a specific goal to avoid mindless proliferation.

Microplastic ingestion by the farmed sea cucumber Apostichopus japonicus in China.

Microplastic ingestion by the farmed sea cucumber is undocumented. Microplastics were isolated from the sea cucumber Apostichopus japonicus that was collected from eight farms along the Bohai Sea and the Yellow Sea in China. To examine microplastic ingestion, the intestines were isolated, digested and then subjected to the floatation test. The microplastic abundance in the sediment ranged from 20 to 1040 particles kg-1 of dry sediment, while the ingested microplastics ranged from 0 to 30 particles intestine-1. After filtering the coelomic fluid, the extracted microplastics from the coelomic fluid ranged from 0 to 19 particles animal-1. Thus, we speculated that microplastics may transfer to the coelomic fluid of sea cucumber. The ingested microplastics did not correlate with the animal body weight but was site dependent, suggesting that sea cucumber may serve as sentinel for microplastic pollution monitoring in the sediment. The microplastics were identified by Fourier transform infrared micro spectroscopy, and the polymer types were mainly cellophane, polyester, and polyethylene terephthalate. This study revealed that, microplastics widely existed in sea cucumber farms, and that sea cucumbers ingest microplastics as suitable with their mouth open. Moreover, the microplastics might transfer to the coelomic fluid of the sea cucumber. Further investigations are needed to assess the chronic effect of the microplastics on the growth and physiological status of the sea cucumber.

Comparative Phospho- and Acetyl Proteomics Analysis of Posttranslational Modifications Regulating Intestine Regeneration in Sea Cucumbers.

Sea cucumbers exposed to stressful circumstances eviscerate most internal organs, and then regenerate them rapidly under favorable environments. Reversible protein phosphorylation and acetylation are major modifications regulating protein function. Herein, for the first time, we perform quantitative phospho- and acetyl proteomics analyses of intestine regeneration in a sea cucumber species Apostichopus japonicus. We identified 1,862 phosphorylation sites in 1,169 proteins, and 712 acetylation sites in 470 proteins. Of the 147 and 251 proteins differentially modified by phosphorylation and acetylation, respectively, most were related to cytoskeleton biogenesis, protein synthesis and modification, signal recognition and transduction, energy production and conversion, or substance transport and metabolism. Phosphorylation appears to play a more important role in signal recognition and transduction than acetylation, while acetylation is of greater importance in posttranslational modification, protein turnover, chaperones; energy production and conversion; amino acid and lipid transport and metabolism. These results expanded our understanding of the regulatory mechanisms of posttranslational modifications in intestine regeneration of sea cucumbers after evisceration.

Impact of hypoxia stress on the physiological responses of sea cucumber Apostichopus japonicus: respiration, digestion, immunity and oxidative damage.

Hypoxia is one of the most frequently occurring stressors confronted by industrial cultures of sea cucumber and can cause large economic losses and resource degradation. However, its responsive mechanisms are still lacking. In this paper, the physiological responses of Apostichopus japonicus to oxygen deficiency was illustrated, including induced oxidative response and immune defense and changed digestive enzymes activities. Significantly increased activities of alpha-amylase (AMS), acid phosphatase (ACP), lactate dehydrogenase, catalase, peroxidase, succinate dehydrogenase and higher content of malondialdehyde, and decreased activities of lipase and trypsin (TRY) were observed after hypoxia exposure (dissolved oxygen [DO] 2 mg/L). Expressions of key genes showed that AMS, peptidase, ACP, alkaline phosphatase, lysozyme, heat shock protein 70 and glutathione peroxidase were increased and TRY was decreased under hypoxia. With the decline of the DO level, the decreased tendency of oxygen consumption rates was different in varied weight groups. Moreover, respiratory trees were observed degraded under long-term hypoxia stress, thus leading a negative effect of respiration. These results could help to develop a better understanding of the responsive mechanism of sea cucumber under hypoxia stress and provide a theoretical basis for the prevention of hypoxia risk.

Effect of water temperature on diel feeding, locomotion behaviour and digestive physiology in the sea cucumber Apostichopus japonicus.

This study used controlled laboratory conditions to directly assess the role of water temperature in controlling diel feeding and locomotion behaviours, and digestive physiology, in the sea cucumber Apostichopus japonicus The results revealed that both the proportion of feeding individuals and ingestion rate were highest at 16°C. Regardless of water temperature, sea cucumbers appeared to be nocturnal and their peak feeding activity occurred at 00:00 h to 04:00 h. Tentacle insertion rate was not significantly correlated with water temperature (<24°C). In all temperature treatments except 24°C, the proportion of moving sea cucumbers was also observed to be higher at night than during the day. The water temperature above thermal threshold (24°C) for aestivation may alter the diel locomotion rhythm. The highest lipase and amylase activities were both observed at 20°C. The highest activities of lipase and amylase at all temperature treatments were observed at 22:00 h to 02:00 h, which was slightly earlier than the feeding peak. In conclusion, even in total darkness, A. japonicus showed more active feeding and moving activities, and higher digestive enzyme activities, at night than during the day. These results demonstrated that diel feeding and locomotion behaviours, at least in the short term, were not controlled by light or low water temperature (<24°C) but by an endogenous rhythm, and A. japonicus had the ability to optimize the digestive function for the coming feeding peak. These findings should provide valuable information for the development of the aquaculture of this species.