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Sea cucumbers are deposit feeding members of marine benthic communities. The over-exploitation of sea cucumber natural stocks, especially in the Mediterranean basin, is having negative impacts on the marine ecosystem. This concerns had led the Italian government to take legal actions to preserve these important marine resources. The aim of this study was to evaluate the level of genetic diversity and population structures within two sympatric Holothuria sea cucumber species, H. polii and H. tubulosa, across ten Mediterranean areas along Italy. A 323 bp portion of the COI mitochondrial gene was sequenced in a total of 441 holothurians (251 H. polii, 177 H. tubulosa and 13 from other species). Genetic diversity analyses and analysis of molecular variance (AMOVA) suggest that H. polii and H. tubulosa species are distinct, but within and among populations are homogeneous, indicating active gene flows across the Mediterranean areas investigated. H. polii showed a lower genetic diversity than H. tubulosa, probably related to differences in life history traits. Phylogenetic analyses showed a clear differentiation between the two species, even if six specimens morphologically assigned to a species clustered within the other species, indicating a possible occurrence of hybridization events. These data will be useful in implementing conservation actions for these holothurian genetic resources., (© 2024. The Author(s).)

Sea cucumbers are predominant deposit feeders in benthic ecosystems, providing protective benefits to coral reefs by reducing disease prevalence. However, how they receive sufficient nutrition from seabed sediments remains poorly understood. Here, we investigate Holothuria leucospilota, an ecologically significant tropical sea cucumber, to elucidate digestive mechanisms underlying marine deposit-feeding. Genomic analysis reveals intriguing evolutionary adaptation characterized by an expansion of digestive carbohydrase genes and a contraction of digestive protease genes, suggesting specialization in digesting microalgae. Developmentally, two pivotal dietary shifts, namely, from endogenous nutrition to planktonic feeding, and from planktonic feeding to deposit feeding, induce changes in digestive tract enzyme profiles, with adults mainly expressing carbohydrases and lipases. A nuanced symbiotic relationship exists between gut microbiota and the host, namely, specific resident bacteria supply crucial enzymes for food digestion, while other bacteria are digested and provide assimilable nutrients. Our study further identifies Holothuroidea lineage-specific lysozymes that are restrictedly expressed in the intestines to support bacterial digestion. Overall, this work advances our knowledge of the evolutionary innovations in the sea cucumber digestive system which enable them to efficiently utilize nutrients from seabed sediments and promote food recycling within marine ecosystems., (© 2024. The Author(s).)

Apostichopus japonicus is an important marine aquaculture species in China, with high nutritional and economic value. In A. japonicus, there is no obvious sexual dimorphism in external appearance, and sex differentiation primarily relies on the observation of mature gonads after dissection, which leads to difficulties in sex identification. The confusion in sex identification greatly reduces breeding efficiency in the sea cucumber industry. Therefore, developing a reliable sex-specific marker is crucial. In this study, we identified 586 male-specific sequences through whole-genome assembly and sequence alignment, but did not identify any female-specific sequences, inferring an XY-type sex determination system in sea cucumbers. We developed a set of male-specific molecular markers to establish an accurate, stable, and widely adaptable genetic sex identification technique for A. japonicus. The male-specific molecular markers were validated with 100% accuracy in sea cucumber populations from six different geographical regions in China. In conclusion, this study provides further evidence for the XY-type sex determination system in A. japonicus and establishes an effective genetic sex identification method for multi-geographic populations, which benefits future study on reproductive biology and has significant implications in sea cucumber aquaculture industry., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The holothurians, commonly known as sea cucumbers, are marine organisms that possess significant dietary, nutritional, and medicinal value. However, the National Center for Biotechnology Information (NCBI) currently possesses only approximately 70 complete mitochondrial genome datasets of Holothurioidea, which poses limitations on conducting comprehensive research on their genetic resources and evolutionary patterns. In this study, a novel species of sea cucumber belonging to the genus Benthodytes, was discovered in the western Pacific Ocean. The genomic DNA of the novel sea cucumber was extracted, sequenced, assembled and subjected to thorough analysis., Results: The mtDNA of Benthodytes sp. Gxx-2023 (GenBank No. OR992091) exhibits a circular structure spanning 17,386 bp, comprising of 13 protein-coding genes (PCGs), 24 non-coding RNAs (2 rRNA genes and 22 tRNA genes), along with two putative control regions measuring 882 bp and 1153 bp, respectively. It exhibits a high AT% content and negative AT-skew, which distinguishing it from the majority of sea cucumbers in terms of environmental adaptability evolution. The mitochondrial gene homology between Gxx-2023 and other sea cucumbers is significantly low, with less than 91% similarity to Benthodytes marianensis, which exhibits the highest level of homology. Additionally, its homology with other sea cucumbers is below 80%. The mitogenome of this species exhibits a unique pattern in terms of start and stop codons, featuring only two types of start codons (ATG and ATT) and three types of stop codons including the incomplete T. Notably, the abundance of AT in the Second position of the codons surpasses that of the First and Third position. The gene arrangement of PCGs exhibits a relatively conserved pattern, while there exists substantial variability in tRNA. Evolutionary analysis revealed that it formed a distinct cluster with B. marianensis and exhibited relatively distant phylogenetic relationships with other sea cucumbers., Conclusions: These findings contribute to the taxonomic diversity of sea cucumbers in the Elasipodida order, thereby holding significant implications for the conservation of biological genetic resources, evolutionary advancements, and the exploration of novel sea cucumber resources., (© 2024. The Author(s).)

Caspase (CASP) is a protease family that plays a vital role in apoptosis, development, and immune response. Herein, we reported the identification and characterization of two CASPs, AjCASPX1 and AjCASPX2, from the sea cucumber Apostichopus japonicus , an important aquaculture species. AjCASPX1/2 share similar domain organizations with the vertebrate initiator caspases CASP2/9, including the CARD domain and the p20/p10 subunits with conserved functional motifs. However, compared with human CASP2/9, AjCASPX1/2 possess unique structural features in the linker region between p20 and p10. AjCASPX1, but not AjCASPX2, induced marked apoptosis of human cells by activating CASP3/7. The recombinant proteins of AjCASPX2 and the CARD domain of AjCASPX2 were able to bind to a wide range of bacteria, as well as bacterial cell wall components, and inhibit bacterial growth. AjCASPX1, when expressed in Escherichia coli , was able to kill the host bacteria. Under normal conditions, AjCASPX1 and AjCASPX2 expressions were most abundant in sea cucumber muscle and coelomocytes, respectively. After bacterial infection, both AjCASPX1 and AjCASPX2 expressions were significantly upregulated in sea cucumber tissues and cells. Together, these results indicated that AjCASPX1 and AjCASPX2 were initiator caspases with antimicrobial activity and likely functioned in apoptosis and immune defense against pathogen infection.

With the continuous rise of the sea cucumber aquaculture industry in China, the tropical sea cucumber aquaculture industry is also improving. However, research on the gut microorganisms of tropical sea cucumbers in captivity is scarce. In this study, high-throughput sequencing methods were used to analyze the gut microbial composition of Stichopus monotuberculatus and Holothuria scabra in the dry season and wet season of artificial environments. The results showed that 66 phyla were obtained in all samples, of which 59 phyla were obtained in the dry season, and 45 phyla were obtained in the wet season. The Tax4Fun analysis showed that certain gut bacterial communities affect the daily metabolism of two sea cucumber species and are involved in maintaining gut microecological balance in the gut of two sea cucumber species. In addition, compared with differences between species, PCoA and UPGMA clustering analysis showed the gut prokaryotes of the same sea cucumber species varied more in different seasons, indicating that the influence of environment was higher than the feeding choices of sea cucumbers under relatively closed conditions. These results revealed the gut bacterial community composition of S. monotuberculatus and H. scabra and the differences in gut bacterial structure between two sea cucumber species in different seasons were compared, which would provide the foundation for tropical sea cucumber aquaculture in the future.

Steroids play a vital role in animal survival, promoting growth and development when administered appropriate concentration exogenously. However, it remains unclear whether steroids can induce gonadal development and the underlying mechanism. This study assessed sea cucumber weights post-culturing, employing paraffin sections and RNA sequencing (RNA-seq) to explore gonadal changes and gene expression in response to exogenous steroid addition. Testosterone and cholesterol, dissolved in absolute ethanol, were incorporated into sea cucumber diets. After 30 days, testosterone and cholesterol significantly increased sea cucumber weights, with the total weight of experimental groups surpassing the control. The testosterone-fed group exhibited significantly higher eviscerated weight than the control group. In addition, dietary steroids influenced gonad morphology and upregulated genes related to cell proliferation，such as RPL35, PC, eLF-1, MPC2, ADCY10 and CYP2C18. Thees upregulated differentially expressed genes were significantly enriched in the organic system, metabolism, genetic information and environmental information categories. These findings imply that steroids may contribute to the growth and the process of genetic information translation and protein synthesis essential for gonadal development and gametogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Morphologically cryptic and pseudo-cryptic species pose a challenge to taxonomic identification and assessments of species diversity and distributions. Such is the case for the sea cucumber Stichopus horrens, commonly confused with Stichopus monotuberculatus. Here, we used mitochondrial cytochrome oxidase subunit I (COI) and microsatellite markers to examine genetic diversity in Stichopus cf. horrens throughout the Philippine archipelago, to aid species identification and clarify species boundaries. Phylogenetic analysis reveals two recently diverged COI lineages (Clade A and Clade B; c. 1.35-2.54 Mya) corresponding to sequence records for specimens identified as S. monotuberculatus and S. horrens, respectively. Microsatellite markers reveal two significantly differentiated genotype clusters broadly concordant with COI lineages (Cluster 1, Cluster 2). A small proportion of individuals were identified as later-generation hybrids indicating limited contemporary gene flow between genotype clusters, thus confirming species boundaries. Morphological differences in papillae distribution and form are observed for the two species, however tack-like spicules from the dorsal papillae are not a reliable diagnostic character. An additional putative cryptic species was detected within Clade B-Cluster 2 specimens warranting further examination. We propose that these lineages revealed by COI and genotype data be referred to as Stichopus cf. horrens species complex., (© 2024. The Author(s).)

Activator protein-1 subfamily member c-Fos wields significant influence over cellular activities, such as regulation of cell growth and division, cell death, and immune responses under various extracellular situations. In this study, the full-length c-Fos of sea cucumber, Apostichopus japonicus (Ajfos) was successfully cloned and analyzed. The anticipated 306 amino acid sequences of Ajfos displayed a basic-leucine zipper (bZIP) domain, similar to invertebrate counterparts. In addition, the qPCR results suggested Ajfos expressed in all tissues, with the highest level in coelomocytes from polian vesicle (vesicle lumen cells), followed by coelomocytes from coelom (coelomocytes). Moreover, the expression levels of Ajfos in the coelomocytes and vesicle lumen cells of sea cucumber showed significant changes after the Vibrio splendidus challenge, especially reaching a peak at 6 h. Compared with the silencing negative control RNA interference (siNC) group, silencing Ajfos (siAjfos) in vivo decreased the downstream proliferation-related gene expression of vesicle lumen cells after infection with V. splendidus while no significant influence was observed on coelomocytes. Furthermore, the proliferation proportion of vesicle lumen cells in the siAjfos group was significantly reduced under pathogen stimulation conditions. Finally, based on the fluctuation trend of total coelomocyte density (TCD) from coelom and polian vesicle previously discovered, it is evident that Ajfos played a critical role in facilitating the swift proliferation of vesicle lumen cells in response to V. splendidus stimulation. Altogether, this research provided an initial reference of the function of Ajfos in echinoderms, unveiling its participation in host coelomocyte proliferation of sea cucumbers during bacterial challenges., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Sea cucumber Apostichopus japonicus displays the typical circadian rhythms. This present study investigated the molecular regulation of clock genes, as well as monoamines and melatonin, in multiple tissues of A. japonicus, responding to the photoperiod. In order to determine their pivotal role in circadian rhythms, the crucial clock genes, namely AjClock, AjArnt1, AjCry1, and AjTimeless, were identified and a comprehensive analysis of their expressions across various tissues in adult A. japonicus was conducted, revealing the potential existence of central and peripheral oscillators. Results demonstrated that the tissues of polian vesicle and nerve ring exhibited significant clock gene expression associated with the orchestration of circadian regulation, and that environmental light fluctuations exerted influence on the expression of these clock genes. However, a number of genes, such as AjArnt1 and AjCry1, maintained their circadian rhythmicity even under continuous light conditions. Moreover, we further investigated the circadian patterns of melatonin (MT), serotonin (5-HT), and dopamine (DA) secretion in A. japonicus, data that underscored the tissue-specific regulatory differences and the inherent adaptability to dynamic light environments. Collectively, these findings will provide the molecular mechanisms controlling the circadian rhythm in echinoderms and the candidate tissues playing the role of central oscillators in sea cucumbers., Competing Interests: Declaration of Competing Interest All authors disclosed no relevant relationships., (Copyright © 2023. Published by Elsevier Inc.)

Background: Chiridota heheva is a cosmopolitan holothurian well adapted to diverse deep-sea ecosystems, especially chemosynthetic environments. Besides high hydrostatic pressure and limited light, high concentrations of metal ions also represent harsh conditions in hydrothermal environments. Few holothurian species can live in such extreme conditions. Therefore, it is valuable to elucidate the adaptive genetic mechanisms of C. heheva in hydrothermal environments., Findings: Herein, we report a high-quality reference genome assembly of C. heheva from the Kairei vent, which is the first chromosome-level genome of Apodida. The chromosome-level genome size was 1.43 Gb, with a scaffold N50 of 53.24 Mb and BUSCO completeness score of 94.5%. Contig sequences were clustered, ordered, and assembled into 19 natural chromosomes. Comparative genome analysis found that the expanded gene families and positively selected genes of C. heheva were involved in the DNA damage repair process. The expanded gene families and the unique genes contributed to maintaining iron homeostasis in an iron-enriched environment. The positively selected gene RFC2 with 10 positively selected sites played an essential role in DNA repair under extreme environments., Conclusions: This first chromosome-level genome assembly of C. heheva reveals the hydrothermal adaptation of holothurians. As the first chromosome-level genome of order Apodida, this genome will provide the resource for investigating the evolution of class Holothuroidea., (© The Author(s) 2024. Published by Oxford University Press GigaScience.)

The microRNA novel-3 (miRn-3) is a 23-nt small endogenous noncoding RNA of unknown function. To enrich our knowledge of the regulatory function of miRn-3 in the process of wound healing, the sea cucumber Apostichopus japonicus was used as a target model in this study. Gelsolin (AjGSN), a potential target gene of miRn-3, was cloned and characterized, and the interaction between miRn-3 and AjGSN was verified. The function of the miRn-3/AjGSN axis in regulating cutaneous wound healing was explored in the sea cucumber A. japonicus. The results showed that 1) the full-length cDNA of AjGSN was 2935 bp, with a high level of sequence conservation across the echinoderms; 2) miRn-3 could bind to the 3'UTR of AjGSN and negatively regulate the expression of AjGSN; 3) overexpression of miRn-3 and inhibition of the expression of AjGSN suppressed cutaneous wound healing in A. japonicus. In general, all observations of this study suggest that miRn-3 plays an important role in the early process of cutaneous wound healing by negatively targeting AjGSN, and that it may be a potential biomarker in wound healing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The signal recognition particle (SRP) system delivers approximately 30% of the proteome to the endoplasmic reticulum (ER) membrane. SRP receptor alpha (SRα) binds to SRP for targeting nascent secreted proteins to the ER membrane in eukaryotic cells. In this study, the SRα homologous gene was identified in the sea cucumber, Apostichopus japonicus (AjSRα). AjSRα codes for 641 amino acids and has 54.94% identity with its mammalian homologs. Like mammalian SRα, it is expected to contain the SRP-alpha N domain, SRP54_N domain, and SRP54 domain. In addition, AjSRα is ubiquitously expressed in adult tissues and exhibits a sexually dimorphic expression pattern, with significantly higher expression in ovaries compared to testes. As a maternal factor, AjSRα can be continuously detected during embryonic development. Importantly, we first attempted to investigate its function by using lentiviral vectors for delivering SRα gene-specific shRNA, and we revealed that lentiviral vectors do not induce an upregulation of immune-related enzymes in sea cucumbers. However, compared to the dsRNA-based RNA interference (RNAi) method, lentivirus-mediated RNAi caused dynamic changes in gene expression at a later time. This study supplied the technical support for studying the functional mechanism of SRα in sea cucumbers., (© 2023. The Author(s).)

Background: Sea cucumbers exhibit a remarkable ability to regenerate damaged or lost tissues and organs, making them an outstanding model system for investigating processes and mechanisms of regeneration. They can also reproduce asexually by transverse fission, whereby the anterior and posterior bodies can regenerate independently. Despite the recent focus on intestinal regeneration, the molecular mechanisms underlying body wall regeneration in sea cucumbers still remain unclear., Results: In this study, transverse fission was induced in the tropical sea cucumber, Holothuria leucospilota, through constrainment using rubber bands. Histological examination revealed the degradation and loosening of collagen fibers on day-3, followed by increased density but disorganization of the connective tissue on day-7 of regeneration. An Illumina transcriptome analysis was performed on the H. leucospilota at 0-, 3- and 7-days after artificially induced fission. The differential expression genes were classified and enriched by GO terms and KEGG database, respectively. An upregulation of genes associated with extracellular matrix remodeling was observed, while a downregulation of pluripotency factors Myc, Klf2 and Oct1 was detected, although Sox2 showed an upregulation in expression. In addition, this study also identified progressively declining expression of transcription factors in the Wnt, Hippo, TGF-β, and MAPK signaling pathways. Moreover, changes in genes related to development, stress response, apoptosis, and cytoskeleton formation were observed. The localization of the related genes was further confirmed through in situ hybridization., Conclusion: The early regeneration of H. leucospilota body wall is associated with the degradation and subsequent reconstruction of the extracellular matrix. Pluripotency factors participate in the regenerative process. Multiple transcription factors involved in regulating cell proliferation were found to be gradually downregulated, indicating reduced cell proliferation. Moreover, genes related to development, stress response, apoptosis, and cell cytoskeleton formation were also involved in this process. Overall, this study provides new insights into the mechanisms of whole-body regeneration and uncover potential cross-species regenerative-related genes., (© 2023. The Author(s).)

Competing Interests: Declaration of competing interest All the authors have declared that no competing interests exist and approved it for publication.

Sea cucumber is a bioremediator as it can composite organic matter and excrete inorganic matter. Sea cucumber has the potential to serve as a bioindicator in marine habitat as they provide an integrated insight into the status of their environment over long periods. Sea cucumbers are sensitive to the organic concentration in the marine environment and can effectively provide an early warning system for any organic contamination that can negatively impact the ecosystem. The availability of a reference transcriptome for sea cucumber would constitute an essential tool for identifying genes involved in crucial steps of the defence pathway. De novo assembly of RNA-seq data enables researchers to study the transcriptomes without needing a genome sequence. In this study, sea cucumbers fed with Kappaphycus alvarezii powder were treated with 0.20 mg/L copper concentration comprehensive transcriptome data containing 75,149 Unigenes, with a total length of 20,460,032 bp. A total of 8820 genes were predicted from the unigenes, annotated, and functionally categorized into 25 functional groups with approximately 20% cluster in signal transduction mechanism. The reference transcriptome presented and validated in this study is meaningful for identifying a wide range of gene(s) related to the bioindication of sea cucumber in a high copper environment.

The sea cucumber Holothuria leucospilota is an economically and ecologically important tropical species. Following development into juveniles, H. leucospilota undergoes a color change from white to black, involving a pigmentation process for over a period of several months. In this study, a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and Next-Generation sequencing (NGS) were employed to investigate the changes in metabolomic and transcriptomic profiles during pigmentation in H. leucospilota juveniles. The metabolomic analysis identified a total of 341 metabolites, of which 52 were found to be differentially regulated (P < 0.05 and VIP > 1), with 27 being upregulated in white individuals and 25 in black individuals. Additionally, 632 differentially expressed genes (DEGs) were identified, with 380 genes upregulated in white samples and 252 genes upregulated in black samples. Interestingly, the melanin content and tyrosinase transcript levels did not display significant differences between the two groups. Metabolomic data suggested the involvement of the linoleic acid metabolic pathway in pigmentation. Transcriptomic analysis, coupled with realtime PCR validation, revealed a decrease in the transcript levels of digestive enzymes like α-amylase, maltase-glucoamylase, and trehalase after the juveniles changed to black. Furthermore, the mRNA expressions of major yolk proteins showed a decline, indicating a shift in the accumulation of protein nutrient sources. Overall, our findings suggest that during the pigmentation process in H. leucospilota, no significant changes were observed in the classical melanin pathway, while notable alterations were observed in their nutritional status. This study provides valuable insights into the regulatory mechanisms of pigmentation in marine organisms., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The sea cucumber Apostichopus japonicus is an economically important marine species in China, and understanding the mechanisms underlying its gonad development is crucial for successful reproduction and breeding. In this study, we performed transcriptome comparisons and analyses of A. japonicus gonadal and non-gonadal tissues to identify genes and molecular pathways associated with gonadal development. We also supplemented the annotation of the A. japonicus genome. Collectively, results revealed a total of 941 ovary-specific genes and 2499 testis-specific genes through different expression analysis and WGCNA analysis. The most enriched pathways in ovary and testis were "DNA replication" and "purine metabolism", respectively. Additionally, we identified key candidate gene modules that control gonad development and germ cell maturation, with CDT1 and DYNC2LI1 serving as hub genes. Our findings provide important insights into the gonadal development system of A. japonicus and offer valuable references for further research on reproductive biology in this marine invertebrate species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The overexploitation of the western Pacific Ocean has expanded the sea cucumber fishery into new regions to supply the Asian market. In 2013, sea cucumbers were removed from the Croatian marine protected species list, and commercial fishery took place for a short period (2017-2018) in the Eastern Adriatic Sea. However, holothuroid species are difficult to distinguish. Holothuria mammata is a species that has rarely been reported in this region and strongly resembles the common species Holothuria tubulosa . This is the first study to assess the genetic diversity of sea cucumbers in the Adriatic Sea using genetic barcoding of the mitochondrial gene cytochrome c oxidase subunit 1 ( COI ). Specimens for barcoding were collected from the northern and central Adriatic, along with a specimen that had been previously identified as H. sp. cf. mammata based on its morphological characteristics. While genetic analyses showed identified this specimen as H. tubulosa , 30% of the collected specimens were genetically identified as H. mammata . These results call into question the historically accepted sea cucumber assemblage in the Adriatic Sea, which regarded H. mammata as a rare species and generally disregarded its presence in large census studies. Such species distribution data are extremely important in developing and monitoring a sustainable fishery.

Salinity is important abiotic factor influencing sea cucumber aquaculture. This study aimed to identify and functional study of a novel transient receptor potential cation channel subfamily A member 1 (TRPA1) involved in salinity stress through interaction with miR-2013 in the sea cucumber. The full-length cDNA sequence was 1369 bp in length and encoded 138 amino acids. The TRPA1 homolog protein was a hydrophilic protein without a signal peptide and was predicted to a spatial structure of seven helices and eight random coils and two major ANK functional domains. Bioinformatic analysis and luciferase reporter assays confirmed TRPA1 as a target gene of miR-2013. Quantitative PCR revealed that miR-2013 was induced upregulation after salinity stress, while TRPA1 showed upregulated expression with maximum expression at 24 h. The expression of miR-2013 and TRPA1 was negatively regulated. Transfection experiments were conducted to validate the role of miR-2013 and TRPA1 in salinity response. The results showed that miR-2013 was upregulated and TRPA1 was downregulated after transfection with miR-2013 mimics, while miR-2013 was downregulated and TRPA1 was upregulated after transfection with miR-2013 inhibitor. Transfection with si-TRPA1 homolog resulted in upregulation of miR-2013 and downregulation of TRPA1 homolog. These findings suggest that miR-2013 can regulate the expression of TRPA1 under salt stress, and highlight the importance of miR-2013 and TRPA1 in salt stress response. miR-2013 mimics improved the survival rate, while miR-2013 inhibitor and si-TRPA1 reduced it. These findings suggest that miR-2013 and TRPA1 play important roles in sea cucumbers adaptation to salinity changes., (© 2023. The Author(s), under exclusive licence to Cell Stress Society International.)

The tropical sea cucumber Holothuria scabra is naturally found in the Indo-West Pacific. However, due to their commercial value, natural H. scabra populations have declined significantly in recent years, resulting in its status as an endangered species. Surveys of H. scabra resource pose a challenge due to its specific characteristics, such as sand-burrowing behavior. To overcome this problem, our study established a convenient and feasible method for assessing H. scabra resources using environmental DNA (eDNA) monitoring technology. First, H. scabra-specific TaqMan primers and probe were designed based on its cox1 gene, followed by the development of an eDNA monitoring method for H. scabra in two separate sea areas (Xuwen and Daya Bay). The method was subsequently employed to investigate the distribution of H. scabra and assess the effects of aquaculture stock enhancement through juvenile releasing in the Weizhou Island sea area. The H. scabra eDNA monitoring approach was found to be more appropriate and credible than traditional methods, and a positive impact of stocking on H. scabra populations was observed. In summary, this is the first report to quantify eDNA concentration in a Holothuroidea species, and it provides a convenient and accurate method for surveying H. scabra resources. This study introduces novel concepts for eDNA-based detection of endangered marine benthic animals and monitoring their population distribution and abundance., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A Gram-staining-negative, oxidase-positive, strictly aerobic rod-shaped bacterium, designated strain PT1T, was isolated from the laboratory-reared larvae of the sea cucumber Apostichopus japonicus. A phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT1T was closely related to Neptuniibacter marinus ATR 1.1T (= CECT 8938T = DSM 100783T) and Neptuniibacter caesariensis MED92T (= CECT 7075T = CCUG 52065T) showing 98.2% and 98.1% sequence similarity, respectively. However, the average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values among these three strains were 72.0%-74.8% and 18.3%-19.5% among related Neptuniibacter species, which were below 95% and 70%, respectively, confirming the novel status of PT1T. The average amino acid identity (AAI) values of PT1T showing 74-77% among those strains indicated PT1T is a new species in the genus Neptuniibacter. Based on the genome-based taxonomic approach, Neptuniibacter victor sp. nov. is proposed for PT1T. The type strain is PT1T (JCM 35563T = LMG 32868T)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kudo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Sea cucumbers are one of the most common large animals in the deep-sea benthic communities, and those of the genus Pannychia are particularly abundant in the bathyal North Pacific Ocean. In Japanese waters, three species of Pannychia have been reported, mainly from the northern and eastern parts of the country. Here, we describe two new species from the western part of Japan. These two new species were placed in Pannychia by the presence of dorsal papillae on the ventrolateral radii and the presence of large wheel ossicles with rim teeth. They are distinguished from all other nominal species of Pannychia on the basis of the morphological characteristics of their body wheel ossicles. Pannychia nagasakimaruae sp. nov. has a unique ossicle composition in its dorsal and ventral body walls: four forms of wheel ossicles. Pannychia rinkaimaruae sp. nov. differs from other congeners in its small wheel ossicles in the dorsal and ventral body walls, which have conical rim teeth. We barcoded partial COI gene sequences from holotypes and paratypes of two new Japanese species. Our molecular phylogenetic analysis showed that these two new morphologically recognized Japanese species form distinct clades separated from other Pacific Pannychia species, so that the two new Japanese species were also supported by genetic results. In addition, we describe and visualize the poorly calcified calcareous rings of the two new species using X-ray μCT system, a useful method for observing such structures, which are important structures in holothurian anatomy.

The sea cucumber Apostichopus japonicus has important nutritional and medicinal value. Unfortunately, we know little of the source of active chemicals in this animal, but the plentiful pigments of these animals are thought to function in intriguing ways for translation into clinical and food chemistry usage. Here, we found key cell groups with the gene activity predicted for the color morphology of sea cucumber body using single-cell RNA-seq. We refer to these cell populations as melanocytes and quinocytes, which are responsible for the synthesis of melanin and quinone pigments, respectively. We integrated analysis of pigment biochemistry with the transcript profiles to illuminate the molecular mechanisms regulating distinct pigment formation in echinoderms. In concert with the correlated pigment analysis from each color morph, this study expands our understanding of medically important pigment production, as well as the genetic mechanisms for color morphs, and provides deep datasets for exploring advancements in the fields of bioactives and nutraceuticals.

Sea cucumber is a morphologically diverse and ecologically important clade of echinoderms. The sea cucumber Apostichopus japonicus is the most economically valuable species of sea cucumber. The initial assembly of the A. japonicus genome was released in 2017. However, this genome assembly is fragmented and lacks relative position information of genes on chromosomes. In this study, we produced a high-quality chromosome-level genome of A. japonicus using Pacbio HiFi long-reads and Hi-C sequencing data. The assembled A. japonicus genome spanned 671.60 Mb with a contig N50 size of 17.20 Mb and scaffold N50 size of 29.65 Mb. A total of 99.9% of the assembly was anchored to 23 chromosomes. In total, 19,828 genes were annotated, and 97.2% of BUSCO genes were fully represented. This high-quality genome of A. japonicus will not only aid in the development of sustainable aquaculture practices, but also lay a foundation for a deeper understanding of their genetic makeup, evolutionary history, and ecological adaptation., (© 2023. The Author(s).)

The genus Thalassotalea is ubiquitous in marine environments, and up to 20 species have been described so far. A Gram-staining-negative, aerobic bacterium, designated strain PTE2T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PTE2T was closely related to Thalassotalea sediminis N211T (= KCTC 42588T = MCCC 1H00116T) with 97.9% sequence similarity. ANI and in silico DDH values against Thalassotalea species were 68.5-77.0% and 19.7-24.6%, respectively, indicating the novelty of PTE2T. Based on genome-based taxonomic approaches, strain PTE2T (= JCM 34608T = KCTC 82592T) is proposed as a new species, Thalassotalea hakodatensis sp. nov., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Yamano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study aimed to assess the genetic differentiation and relationship among five sea cucumber species from the Red Sea in Egypt, namely Holothuria atra, H. impatiens, H. leucospilota, Actinopyga crassa and A. mauritiana, using Inter Simple Sequence Repeated (ISSR) and Start Codon Targeted (SCoT) markers. A collection of 100 specimens, with 20 individuals per species, was gathered for the analysis. With ten ISSR primers, 135 amplified bands were detected, including 11 distinct species-specific bands, indicating high-level polymorphism among species. Using ten SCoT primers, 151 amplicons were generated, including 30 species-specific bands, with 52% polymorphic bands indicating high-level polymorphism among species. The degree of genetic similarity (GS) among the different genotypes of species was calculated based on ISSR bands analysis, which ranged from 93% between H. atra and H. impatiens to 86% between H. atra and A. crassa. The highest genetic similarity was observed between H. atra and H. impatiens (90%), while the lowest was identified between A. crassa and A. mauritiana (75%) using SCoT bands. Notably, the ISSR and SCoT-based DNA analysis revealed similar genetic relationships between H. atra and H. impatiens compared to other sea cucumber species studied. This study provides new insights into the genetic diversity and relationship among sea cucumber species in the Red Sea, which could have implications for their conservation and management.

A growing body of evidence suggests that spatial population structure can develop in marine species despite large population sizes and high gene flow. Characterizing population structure is important for the effective management of exploited species, as it can be used to identify appropriate scales of management in fishery and aquaculture contexts. The California sea cucumber, Apostichopus californicus, is one such exploited species whose management could benefit from further characterization of population structure. Using restriction site-associated DNA (RAD) sequencing, we developed 2075 single nucleotide polymorphisms (SNPs) to quantify genetic structure over a broad section of the species' range along the North American west coast and within the Salish Sea, a region supporting the Washington State A. californicus fishery and developing aquaculture production of the species. We found evidence for population structure (global fixation index (FST) = 0.0068) with limited dispersal driving two patterns of differentiation: isolation-by-distance and a latitudinal gradient of differentiation. Notably, we found detectable population differences among collection sites within the Salish Sea (pairwise FST = 0.001-0.006). Using FST outlier detection and gene-environment association, we identified 10.2% of total SNPs as putatively adaptive. Environmental variables (e.g., temperature, salinity) from the sea surface were more correlated with genetic variation than those same variables measured near the benthos, suggesting that selection on pelagic larvae may drive adaptive differentiation to a greater degree than selection on adults. Our results were consistent with previous estimates of and patterns in population structure for this species in other extents of the range. Additionally, we found that patterns of neutral and adaptive differentiation co-varied, suggesting that adaptive barriers may limit dispersal. Our study provides guidance to decision-makers regarding the designation of management units for A. californicus and adds to the growing body of literature identifying genetic population differentiation in marine species despite large, nominally connected populations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lowell et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Holothuria (Halodeima) atra Jaeger, 1833 is a tropical sea cucumber usually harvested for the "bêche-de-mer" trade market. It has been reported to reproduce both sexually, through gamete spawning, or asexually, through fission. To date, no study has ever investigated clonality, nor genetic connectivity, among its populations, using microsatellite markers., Methods and Results: We isolated the first 21 microsatellite loci specific for H. atra, which were then used to investigate clonal diversity, genetic structure and diversity among 44 H. atra individuals sampled in Reunion Island (southwestern Indian Ocean), over two seasons. All 21 loci were polymorphic, with number of alleles per locus ranging from 2 to 10. No repetitive multi-locus genotype (MLG) and few clonal lineages (MLL) were found. Observed heterozygosities per locus and season ranged from 0.000 to 0.909, while expected heterozygosities ranged from 0.290 to 0.882. Four loci were at Hardy-Weinberg equilibrium for both seasons, all others presenting a deficit of heterozygotes in one or both seasons. Meanwhile, no genetic differentiation was detected between seasons, according to assignment tests and global F ST ., Conclusions: These results suggest low asexual propagation in this population. These loci represent useful tools to better understand reproductive strategies and population connectivity of H. atra, and thus provide relevant knowledge for efficient management., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Circular RNAs (circRNAs) are a kind of extensive and diverse covalently closed circular endogenous RNA, which exert crucial functions in immune regulation in mammals. However, the functions and mechanisms of circRNAs in invertebrates are largely unclarified. In our previous work, 261 differentially expressed circRNAs including circRNA432 (circ432) were identified from skin ulcer syndrome (SUS) diseased sea cucumber Apostichopus japonicus by RNA-seq. To better address the functional role of sea cucumber circRNAs, circ432 was first found to be significantly induced by Vibrio splendidus challenge and LPS exposure in this study. Knock-down circ432 could depress the V. splendidus-induced coelomocytes phagocytosis. Moreover, circ432 is validated to serve as the sponge of miR-2008, a differential expressed miRNA in SUS-diseased sea cucumbers, by Argonaute 2-RNA immunoprecipitation (AGO2-RIP) assay, luciferase reporter assay and RNA fluorescence in situ hybridization (FISH) in vitro. Engulfment and cell motility protein 1 (AjELMO1) is further demonstrated to be the target of miR-2008, and silencing AjELMO1 inhibits the V. splendidus-induced coelomocytes phagocytosis, and this phenomenon could be further suppressed by supplementing with miR-2008 mimics, suggesting that circ432 might regulate coelomocytes phagocytosis via miR-2008-AjELMO1 axis. We further confirm that the depressed coelomocytes' phagocytosis by circ432 silencing is consistent with the decreased abundance of AjELMO1, and could be recovered by miR-2008 inhibitors transfection. All our results provide the evidence that circ432 is involved in regulating pathogen-induced coelomocyte phagocytosis via sponge miR-2008 and promotes the abundance of AjELMO1. These findings will enrich the regulatory mechanism of phagocytosis in echinoderm and provide theoretical data for SUS disease prevention and control in sea cucumbers., (© 2023. The Author(s).)

Ever-increasing consumer demand for sea cucumbers mainly leads to huge damage to wild sea cucumber resources, including Stichopus monotuberculatus , which in turn exerts negative impacts on marine environments due to the lack of ecological functions performed by sea cucumbers. Aquaculture of sea cucumbers is an effective way to meet consumer demand and restore their resources. Unsynchronous growth is a prominent problem in the aquaculture of sea cucumbers which has concealed unelucidated molecular mechanisms until now. In this study, we carried out an integrative analysis of transcriptomics and metabolomics on fast-growing (SMF) and slow-growing (SMS) groups of S. monotuberculatus cultured in the same environmental conditions. The results revealed that a total of 2054 significantly differentially expressed genes (DEGs) were identified, which are mainly involved in fat digestion and absorption, histidine metabolism, arachidonic acid metabolism, and glutathione metabolism. 368 differential metabolites (DMs) were screened out between the SMF group and the SMS group; these metabolites are mainly involved in glycerophospholipid metabolism, purine metabolism, biosynthesis of unsaturated fatty acids, pyrimidine metabolism, arachidonic acid metabolism, and other metabolic pathways. The integrative analysis of transcriptomics and metabolomics of S. monotuberculatus suggested that the SMF group had a higher capacity for lipid metabolism and protein synthesis, and had a more frequent occurrence of apoptosis events, which are likely to be related to coping with environmental stresses. The results of this study provide potential values for the aquaculture of sea cucumbers which may promote their resource enhancement.

The lysin motif (LysM)-containing protein is one of widespread pattern-recognition receptors in prokaryotes and eukaryotes. Numerous LysM-containing gene sequences are present in gene databases; however, few have been well characterized, especially in echinoderms. In this study, the full-length cDNA of a novel LysM-containing gene was obtained from the sea cucumber Apostichopus japonicus, named AjLysM-1, using polymerase chain reaction (PCR) combined with rapid amplification of cDNA ends. We prepared and expressed recombinant AjLysM-1 protein (rAjLysM-1) and determined its pathogen-recognition ability by enzyme-linked immunosorbent and immunofluorescence assays. We also analyzed the tissue expression pattern and response to immune challenges of AjLysM-1 using quantitative real-time reverse transcription-PCR and in situ hybridization. The AjLysM-1 protein was predicted to be an intracellular non-secreted LysM-containing protein, highly homologous to the same protein in other marine echinoderms. AjLysM-1 transcripts were highest expressed in coelomocytes and were strikingly induced by challenge with representative bacterial and fungal polysaccharides. rAjLysM-1 showed weak binding to mannan, Pseudoalteromonas nigrifaciens, and Shewanella baltica, implying that AjLysM-1 might provide inadequate defense against Gram-negative bacteria and fungi. Notably, rAjLysM-1 also interacted with tyrosine protein kinase and filamin-B, indicating that it could be involved in focal adhesion in A. japonicus. These findings improve our understanding of the functions of LysM-containing proteins in marine echinoderms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pacifastin family proteins play a crucial role in regulating innate immune responses such as phagocytosis in invertebrates. However, the function of the Ajpacifastin-like counterpart in the sea cucumber Apostichopus japonicus remains elusive. In this study, the pacifastin gene of A. japonicus was cloned, characterized and named Ajpacifastin-like. The open reading frame of Ajpacifastin-like is 1497 bp in length and encodes a polypeptide containing 498 amino acid residues. Structural analysis revealed that the protein encoded by Ajpacifastin-like contains two pacifastin light chain domains (amino acids 287-322 and amino acids 376-407). Real-time reverse transcriptase PCR showed that Ajpacifastin-like mRNA is ubiquitously expressed in all tissues examined, with the highest expression in muscle. Ajpacifastin-like mRNA expression was significantly upregulated to 3.27-fold after challenge with Vibrio splendidus for 24 h. To explore the function of the Ajpacifastin-like protein in the immune response of A. japonicus, dsRNA interference with Ajpacifastin-like expression and with the expression of its postulated target gene was performed. Flow cytometry analysis showed that the rate of phagocytosis by coelomocytes increased to 1.21-fold in individuals treated with specific Ajpacifastin-like siRNA. However, rate of phagocytosis by coelomocytes decreased to 86% in individuals treated with Ajphenoloxidase siRNA. These results show that the Ajpacifastin-like gene is ubiquitously expressed in almost all tissues and that Ajpacifastin-like protein acts as an immunomodulatory factor via phenoloxidase to mediate phagocytosis by coelomocytes in pathogen-challenged A. japonicus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The sea cucumber Holothuria (Metriatyla) scabra, known as sandfish, is a high-value tropical echinoderm central to the global bêche-de-mer (BDM) trade. This species has been heavily exploited across its natural range, with overharvesting and ineffective fishery management leaving stocks in the Pacific region heavily depleted. In Fiji, sandfish stocks have not recovered since a 1988 harvest ban, with surveys reporting declining populations and recruitment failure. Therefore, to inform fishery management policy for the wild sandfish resource and to guide hatchery-based restocking efforts, a high-resolution genomic audit of Fijian populations was carried out. A total of 6,896 selectively-neutral and 186 putatively-adaptive genome-wide SNPs (DArTseq) together with an independent oceanographic particle dispersal model were used to investigate genetic structure, diversity, signatures of selection, relatedness and connectivity in six wild populations. Three genetically distinct populations were identified with shallow but significant differentiation (average Fst = 0.034, p≤0.05), comprising (1) Lakeba island (Lau archipelago), (2) Macuata (Vanua Levu), and (3) individuals from Yasawa, Ra, Serua island and Kadavu comprising the final unit. Small reductions in allelic diversity were observed in marginal populations in eastern Fiji (overall mean A = 1.956 vs. Lau, A = 1.912 and Macuata, A = 1.939). Signatures of putative local adaptation were also discovered in individuals from Lakeba island, suggesting that they be managed as a discrete unit. An isolation-by-distance model of genetic structure for Fijian sandfish is apparent, with population fragmentation occurring towards the east. Hatchery-based production of juveniles is promising for stock replenishment, however great care is required during broodstock source population selection and juvenile releases into source areas only. The successful use of genomic data here has the potential to be applied to other sea cucumber species in Fiji, and other regions involved in the global BDM trade. While preliminary insights into the genetic structure and connectivity of sandfish in Fiji have been obtained, further local, regional and distribution-wide investigations are required to better inform conservation efforts, wild stock management and hatchery-based restocking interventions for this valuable invertebrate., Competing Interests: The authors have declared that no competing interests exist.

Apostichopus japonicus achieves intestinal regeneration in a short period after evisceration, and multiple genes are involved in this process. The transcriptome of A. japonicus was screened for regeneration-associated protein (Aj-Orpin), a gene that is specifically upregulated during intestinal regeneration. The expression and function of Aj-Orpin were identified and investigated in this study. The 5' and 3' RACE polymerase chain reaction (PCR) was used to clone the full-length cDNA of Aj-Orpin. The open reading frame codes for a 164 amino-acid protein with an EF-hand&#95;7 domain and overlapping signal peptides and transmembrane regions. Moreover, Aj-Orpin mRNA and protein expression during intestinal regeneration was investigated using real-time quantitative PCR and Western blot. The expression pattern of Aj-Orpin in the regenerating intestine was investigated using immunohistochemistry. The results showed that Aj-Orpin is an exocrine protein with two EF-hand-like calcium-binding domains. Expression levels were higher in the regenerating intestine than in the normal intestine, but protein expression changes lagged behind mRNA expression changes. Aj-Orpin was found to play a role in the formation of blastema and lumen. It was primarily expressed in the serosal layer and submucosa, suggesting that it might be involved in proliferation. These observations lay the foundation for understanding the role of Orpin-like in echinoderm intestinal regeneration.

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The functional characteristics of neuropeptides in marine invertebrates have attracted significant attention recently although functional studies of luqin-type neuropeptides are still very limited, especially in deuterostomes. The sea cucumber, Apostichopus japonicus, is a representative species of deuterostomian Holothurian invertebrates. The species has high nutritional and medicinal value in China. In this study, we report the first comprehensive histological, biochemical and pharmacological characterization of luqin-type neuropeptide signaling in the sea cucumber A. japonicus. The A. japonicus luqin-like neuropeptide precursor (AjLQP) contains a single typical deuterostomian luqin-like neuropeptide AjLQ with an xFxRWamide motif. AjLQ was identified as the ligand for a luqin-type neuropeptide receptor AjLQR, that was previously predicted to be a tachykinin-type receptor, and triggers a rapid intracellular mobilization of Ca 2+ , followed by receptor internalization and a transient increase in ERK1/2 phosphorylation. In situ hybridization, immunohistochemistry and qRT-PCR analysis revealed extensive expression of AjLQP and AjLQ in A. japonicus tissues, especially in locomotion-related organs. In vitro pharmacological tests revealed that AjLQ caused 12.69% ± 1.99% (p < 0.01) relaxation of longitudinal muscle preparations at 10 -7 M concentration. Furthermore, we observed significantly increased expression of AjLQP (about 17.63 fold, p < 0.01) in intestine of deeply aestivating sea cucumbers, which suggests that AjLQ might be involved in feeding inhibition during aestivation. The present study provides a first insight into the experimental characterization of luqin-type neuropeptide signaling in a sea cucumber. The results will broaden our understanding of the potential function of neuropeptides during important biological processes in marine invertebrates and provide theoretical support for optimizing sea cucumber aquaculture technology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m 6 A modification has been found in circRNA, and m 6 A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m 6 A levels are largely unknown. Here, we firstly investigated the genome-wide map of m 6 A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m 6 A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m 6 A peak and about 55% of m 6 A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m 6 A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m 6 A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m 6 A circRNAs showed a positive correlation between the circRNAs expression and m 6 A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m 6 A modification was constructed based on conjoint analysis. Our results indicated that several selected m 6 A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m 6 A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m 6 A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m 6 A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

N 6 -methyladenosine (m 6 A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA (mRNA), plays important roles in regulation of gene expression for fundamental biological processes and diverse physiological functions, including combating with pathogen infection. Here, we were first profile transcriptome-wide m 6 A sequencing in four stages of skin ulceration syndrome-diseased Apostichopus japonicus following Vibrio splendidus infection, including Control (healthy), Early (small ulcer), Later (extensive ulcer), and Resistant (no ulcer) groups. Our results revealed that three experimental groups were all extensively methylated by m 6 A and the proportion of the m 6 A modified genes were also significantly increased to 28.90% (Early), 27.97% (Later), and 29.98% (Resistant) when compared with Control group (15.15%), indicating m 6 A modification could be induced by V. splendidus infection. Intriguingly, we discovered a positive correlation between the m 6 A methylation level and mRNA abundance, indicating a positive regulatory role of m 6 A in sea cucumber gene expression during V. splendidus infection. Moreover, genes with specific and differentially expressed m 6 A methylation in Later group were both enriched in cell adhesion, while Early and Resistant groups were both mainly involved in DNA conformation change and chromosome organization when compared with Control, suggesting the higher-methylated m 6 A might serve as "conformational marker" and associated to the initiation of related anti-disease genes transcription in order to improve disease resistance of sea cucumber. Subsequently, we selected the pivotal genes enriched in cell adhesion pathway and found that the IggFc-binding protein (FcGBP) and Fibrocystin-L both had higher levels of m 6 A methylation and higher level of mRNA expressions in Later group. Conversely, Fibrinogen C domain-containing protein 1 (F1BCD1) gene presented as an antibacterial role in sea cucumber and showed higher mRNA expression and higher m 6 A methylation in Resistant group and lower mRNA level in Later group. The levels of m 6 A methylation and mRNA abundance of FcGBP and F1BCD1 genes indicates disease occurrence or disease resistant were also verified by MeRIP-qPCR. Overall, our study presents the first comprehensive characterize of dynamic m 6 A methylation modification in the different stages of disease in sea cucumber. These data provide an invaluable resource for future studies of function and biological significance of m 6 A in mRNA in marine invertebrates., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

MicroRNA-7 (miR-7) is a highly conserved short non-coding RNA involved in various bioprocesses via the regulation of multiple target genes. To enrich our knowledge of the functions of miR-7 in innate immune regulation in echinoderms, we first investigated the targeting relationship between miR-7 and PAK1 in the sea cucumber Apostichopus japonicus and then explored the functions of miR-7, the PAK1 gene, and the miR-7/ PAK1 axis in the pathogen-induced immune response of A. japonicus . Our results showed that miR-7 can bind to the 3'UTR of PAK1 and negatively regulate the expression of PAK1 in A. japonicus . Overexpression and inhibition of miR-7 and inhibition of the expression of PAK1 can alter phagocytosis, cellular agglutination, and lysozyme contents in A. japonicus . Both miR-7 and the PAK1 gene are involved in immune defense against Vibrio splendidus infection; the miR-7/ AjPAK1 axis showed immune regulatory function at 48 to 72 h post-infection (hpi) after V. splendidus infection in A. japonicus. In summary, the results of this study established that miR-7 regulates the pathogen-induced immune response by targeting PAK1 in A. japonicus ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhao, Ren, Li, Liu, Zou, Yan, Zhan and Chang.)

Heavy-metal pollution has increasingly jeopardized the habitats of marine organisms including the sea cucumber, a seafloor scavenger vital to seawater bio-decontamination, ocean de-acidification and coral-reef protection. Normal physiology including immune functions of sea cucumbers is toxicologically modulated by marine metal pollutants such as cadmium (Cd). The processes underpinning Cd's toxic effects on immune systems in the sea cucumber, Holothuria leucospilota , are still poorly understood. To this end, we cloned and characterized a full-length caspase-9 ( Hl-CASP9 ) cDNA in the sea cucumber, Holothuria leucospilota . Hl-CASP9 mRNA levels evolved dynamically during embryonic development. Coelomocytes, a type of phagocytic immune effectors central to H. leucospilota immunity, were found to express Hl-CASP9 mRNA most abundantly. Hl-CASP9 protein structurally resembles caspases-2 and -9 in both invertebrate and vertebrate species, comprising a CARD domain and a CASc domain. Remarkably, Hl-CASP9 was transcriptionally sensitive to abiotic oxidative stress inducers including hydrogen peroxide (H 2 O 2 ), nitric oxide ( • NO) and cadmium (Cd), but insensitive to immunostimulants including lipopolysaccharide (LPS), and poly(I:C). Overexpression of Hl-CASP9 augmented mitochondria-dependent apoptosis in HEK293T cells, while knock-down of Hl-CASP9 blunted Cd-induced coelomocyte apoptosis in vivo . Overall, we illustrate that an evolutionarily ancient caspase-9-dependent pathway exists to sensitize coelomocytes to premature cell death precipitated by heavy metal pollutants, with important implications for negative modulation of organismal immune response in marine invertebrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Chen, Wu, Li, Zhang, Jiang, Luo, Hu, Wong and Ren.)

High-throughput single nucleotide polymorphism (SNP) genotyping assays are powerful tools for genetic studies and genomic breeding applications for many species. Though large numbers of SNPs have been identified in sea cucumber (Apostichopus japonicus), but, as yet, no high-throughput genotyping platform is available for this species. In this study, we designed and developed a high-throughput 24 K SNP genotyping array named HaishenSNP24K for A. japonicus, based on the multi-objective-local optimization (MOLO) algorithm and HD-Marker genotyping method. The SNP array exhibited a relatively high genotyping call rate (> 96%), genotyping accuracy (>95%) and exhibited highly polymorphic in sea cucumber populations. In addition, we also assessed its application in genomic selection (GS). Deep neural networks (DNN) that can capture the complicated interactions of genes have been proposed as a promising tool in GS for SNP-based genomic prediction of complex traits in animal breeding. To overcome the problem of over-fitting when using the HaishenSNP24K array as high-dimensional DNN input, we developed minmax concave penalty (MCP) regularization for sparse deep neural networks (DNN-MCP) that finds an optimal sparse structure of a DNN by minimizing the square error subject to the non-convex penalty MCP on the parameters (weights and biases). Compared to two linear models, namely RR-GBLUP and Bayes B, and the nonlinear model DNN, DNN-MCP has greatly improved the genomic prediction ability for three quantitative traits (e.g., wet weight, dry weight and survival time) in the sea cucumber population. To the best of our knowledge, this is the first work to develop a high-throughput SNP array for A. japonicus and a new model DNN-MCP for genomic prediction of complex traits in GS. The present results provide evidence that supports the HaishenSNP24K array with DNN-MCP will be valuable for genetic studies and molecular breeding in A. japonicus., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

DNAJ proteins function as co-chaperones of HSP70 and play key roles in cell physiology to promote protein folding and degradation, especially under environmental stress. Based on our previous study on HSP70, a systematic study of DNAJ was performed in sea cucumber Apostichopus japonicus using the transcriptomic and genomic data, identifying 43 AjDNAJ genes, including six AjDNAJA genes, eight AjDNAJB genes, and 29 AjDNAJC genes. Slight expansion and conserved genomic structure were observed using the phylogenetic and syntenic analysis. Differential period-specific and tissue-specific expression patterns of AjDNAJs were observed between adult and juvenile individuals during aestivation. Strong tissue-specific expression correlations between AjDNAJ and AjHSP70 genes were found, indicating that the involvements of AjHSP70IVAs in the aestivation of sea cucumbers were regulated by AjDNAJs. Several key genes with significant expression correlations, such as AjDNAJB4L and AjHSP70IVAs, were suggested to function together under heat stress. Together, these findings provide early insight into the involvement of AjDNAJs in the aestivation and their roles as co-chaperones of AjHSP70s., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

During rearing in hatcheries and transportation to restocking sites, sea cucumbers are often exposed to air for several hours, which may depress their non-specific immunity and lead to mass mortality. We performed transcriptome analysis of Apostichopus japonicus coelomocytes after air exposure to identify stress-related genes and pathways. After exposure to air for 1 h, individuals were re-submerged in aerated seawater and coelomocytes were collected at 0, 1, 4, and 16 h (B, H1, H4, and H16, respectively). We identified 6148 differentially expressed genes, of which 3216 were upregulated and 2932 were downregulated. Many genes involved in the immune response, antioxidant defense, and apoptosis were highly induced in response to air exposure. Enrichment analysis of Gene Ontology terms showed that the most abundant terms in the biological process category were oxidation-reduction process, protein folding and phosphorylation, and receptor-mediated endocytosis for the comparison of H1 vs. B, H4 vs. H1, and H16 vs. H4, respectively. Kyoto Eecyclopedia of Genes and Genomes enrichment analysis showed that six pathways related to the metabolism of proteins, fats, and carbohydrates were shared among the three comparisons. These results indicated that sea cucumbers regulate the expression of genes related to the antioxidant system and energy metabolism to resist the negative effects of air exposure stress. These findings may be applied to optimize juvenile sea cucumber production, and facilitate molecular marker-assisted selective breeding of an anoxia-resistant strain., (Copyright © 2022. Published by Elsevier Inc.)

Background: Sex determination mechanisms are complicated and diverse across taxonomic categories. Sea cucumber Apostichopus japonicus is a benthic echinoderm, which is the closest group of invertebrates to chordate, and important economic and ecologically aquaculture species in China. A. japonicus is dioecious, and no phenotypic differences between males and females can be detected before sexual maturation. Identification of sex determination locus will broaden knowledge about sex-determination mechanism in echinoderms, which allows for the identification of sex-linked markers and increases the efficiency of sea cucumber breeding industry., Results: Here, we integrated assembly of a novel chromosome-level genome and resequencing of female and male populations to investigate the sex determination mechanisms of A. japonicus. We built a chromosome-level genome assembly AJH1.0 using Hi-C technology. The assembly AJH1.0 consists of 23 chromosomes ranging from 22.4 to 60.4 Mb. To identify the sex-determination locus of A. japonicus, we conducted genome-wide association study (GWAS) and analyses of distribution characteristics of sex-specific SNPs and fixation index F ST . The GWAS analysis showed that multiple sex-associated loci were located on several chromosomes, including chromosome 4 (24.8%), followed by chromosome 9 (10.7%), chromosome 17 (10.4%), and chromosome 18 (14.1%). Furthermore, analyzing the homozygous and heterozygous genotypes of plenty of sex-specific SNPs in females and males confirmed that A. japonicus might have a XX/XY sex determination system. As a physical region of 10 Mb on chromosome 4 included the highest number of sex-specific SNPs and higher F ST values, this region was considered as the candidate sex determination region (SDR) in A. japonicus., Conclusions: In the present study, we integrated genome-wide association study and analyses of sex-specific variations to investigate sex determination mechanisms. This will bring novel insights into gene regulation during primitive gonadogenesis and differentiation and identification of master sex determination gene in sea cucumber. In the sea cucumber industry, investigation of molecular mechanisms of sex determination will be helpful for artificial fertilization and precise breeding., (© 2022. The Author(s).)

Heat stress poses an increasing threat for the marine invertebrate Apostichopus japonicus. Histone lysine acetylation is a central chromatin modification for epigenetic regulation of gene expression during stress response. In this study, a genome-wide characterization for acetylated lysine 9 on histone H3 (H3K9ac) binding regions in normal temperature (18 °C) and heat-stress conditions (26 °C) via ChIP-seq were carried out. The results that revealed H3K9ac was an extensive epigenetic modulation in A. japonicus. The GO terms "regulation of transcription, DNA-templated" and "transcription coactivator activity" were significantly enriched in both groups. Particularly, various transcriptional factors (TFs) families showed notable modification of H3K9ac. Differentially acetylated regions (DARs) with H3K9ac modification under heat stress were identified with 24 hyperacetylated and 23 hypoacetylated peaks, respectively. We further examined the transcriptional expression for 13 genes with dysregulated H3K9ac level in the promoter regions by qRT-PCR. Combined H3K9ac ChIP-seq characteristics with the transcriptional expression, 5 up-up genes (ZCCHC3, RPA70, MTRR, β-Gal and PHTF2) and 2 down-down genes (PRPF39 and BSL78&#95;10147) were identified. Surprisingly, the increasing mRNA expression of NECAP1 under heat stress was negatively related to the decreasing H3K9ac level in its promoter region. Our research is the first genome-wide characterization for the epigenetic modification H3K9ac in A. japonicus, and will help to advance the understanding of the roles of H3K9ac in transcriptional regulation under heat-stress condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The mitochondrial genome is widely used to study the molecular evolution of and perform phylogenetic analyses on animals. In this study, the complete mitochondrial genome (mitogenome) of Stichopus naso was sequenced. The mitogenome was 16,239 bp in length and contained 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and 2 ribosomal RNA genes (rRNAs). The genome composition showed positive AT-skew (0.023) and negative GC-skew (-0.158). The order of the mitochondrial genes was consistent with those from the Stichopus and Isostichopus species, whereas it was different from those of other species of Aspidochirotida. The phylogenetic analysis, based on the nucleotide sequences of 13 PCGs through the methods of Bayesian inference (BI) and maximum likelihood (ML), indicated that S. naso has close relationships with S. horrens and S. monotuberculatus , and belongs to a member of Stichopodidae. Our study provides a reference mitogenome for further molecular evolution studies and phylogenetic research on sea cucumbers.

RhoA (Ras homolog A) protein is a representative member of the Rho GTPase family and is involved in various cellular processes. The function of RhoA in sea cucumbers is unclear. In this study, we hypothesized that RhoA may regulate the innate immune response of Apostichopus japonicus. Our data showed that 1) the complete sequence of RhoA from A. japonicus (named AjRhoA) was 968 bp, with a high level sequence conservation across the echinoderms and other phyla; 2) tissue expression analysis showed that AjRhoA transcripts and protein exhibited higher abundance in coelomocytes, whereas the relative expression of miR-2012-5p was lower in coelomocytes; 3) interactive binding sites and a negative regulatory targeting relationship between AjRhoA and miR-2012-5p were confirmed through a dual-luciferase reporter assay and functional validation in vivo; 4) the relative expression levels of AjRhoA transcripts and protein were upregulated in coelomocytes 4- and 72-hour post infection (hpi) with Vibrio splendidus, whereas miR-2012-5p was expressed in the opposite pattern; 5) both AjRhoA silencing and miR-2012-5p overexpression suppressed the phagocytic capacity of A. japonicus compared with the control at 4 and 72 hpi. Our observations suggest that AjRhoA can regulate the pathogen-induced immune response of A. japonicus through the "AjRhoA-miR-2012-5p" module during the early infection, while miR-2012-5p plays a direct immunomodulatory role as the infection progresses., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mutable collagenous tissues (MCTs) from echinoderms (e.g., sea stars, sea urchins) possess the remarkable ability to change their mechanical properties rapidly and reversibly thanks to the release of effector molecules regulating the number of cross-links between collagen fibrils. Among these effector molecules, tensilin has been identified as a stiffening factor in sea cucumber MCTs. Since its discovery and description twenty years ago, tensilin orthologs have been identified in a few sea cucumber species but no novel information about its molecular mode of action has been reported. In this study, using a combination of in silico analyses, we identified the tensilin present in the dermis of Holothuria forskali, Hf-(D)Tensilin. Anti-peptide antibodies showed that this protein is localised in the secretory granules of type 2 juxtaligamental-like cells, a MCT specific cell type. We then used the bacterium E. coli to produce recombinantly Hf-(D)Tensilin and confirmed its stiffening effect on pieces of the dermis and its aggregation effect on collagen fibrils extracted from the sea cucumber dermis. To investigate how tensilin can cross-bridge collagen fibrils, truncated recombinant tensilins were also produced and used in combination with various compounds. Results suggest that two types of interactions contribute to the aggregation effect of tensilin on the fibrils: (1) the N-terminal NTR TIMP like domain of the protein interacts strongly with sulfated GAGs attached to the surface of the collagen fibrils, and (2) the C-terminal part of the protein is involved in its dimerisation/oligomerisation through ionic but possibly also cation-π and hydrophobic interactions., Competing Interests: Declaration of competing interests Authors declare that they have no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)