Interactive effects of chronic ocean acidification and warming on the growth, survival, and physiological responses of adults of the temperate sea urchin Strongylocentrotusintermedius.

To investigate the interactive effects of chronic ocean acidification and warming (OAW) on the growth, survival, and physiological responses of sea urchins, adults of the temperate sea urchin Strongylocentrotus intermedius were incubated separately/jointly in acidic (ΔpHNBS = -0.5 units) and thermal (ΔT = +3.0 °C) seawater for 120 days under lab-controlled conditions based on the projected ocean pH and temperature for 2100 put forward by the Intergovernmental Panel on Climate Change (IPCC). Survival rate (SR), average food consumption rate (FCR), gut index (GuI), specific growth rate (SGR), digestive capability, energy production, and antioxidant capability were subsequently determined. The results showed that 1) the SR, FCR, GuI and SGR decreased sharply under OAW conditions. Significant interactive effects of OAW on SR and SGR were observed at 120 days post-incubation (dpi), and on FCR this occurred at 90 dpi. 2) OAW altered the activities of both digestive and antioxidant enzymes. There were significant interaction effects of OAW on the activities of amylase, trehalase, and superoxide dismutase. 3) The relative gene expression levels and activities of key enzymes involved in glycometabolism pathways (i.e., glycolysis and the tricarboxylic acid cycle) were significantly affected by OAW, resulting in an alteration of the total ATP content in the sea urchins. Interaction effects of OAW were observed in both relative gene expression and the activity of enzymes involved in glycolysis (hexokinase), the transformation of glycolysis end-products (lactate dehydrogenase), the tricarboxylic acid cycle (citrate synthetase), and ATP production (Na+/K+-ATPase). The data from this study will enrich our knowledge concerning the combined effects of global climate change on the survival, growth, and physiological responses of echinoderms.

miRn-3 inhibits cutaneous wound healing by targeting gelsolin in the sea cucumber Apostichopus japonicus.

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.

Living in a hypoxic world: A review of the impacts of hypoxia on aquaculture.

Hypoxia is a harmful result of anthropogenic climate change. With the expansion of global low-oxygen zones (LOZs), many organisms have faced unprecedented challenges affecting their survival and reproduction. Extensive research has indicated that oxygen limitation has drastic effects on aquatic animals, including on their development, morphology, behavior, reproduction, and physiological metabolism. In this review, the global distribution and formation of LOZs were analyzed, and the impacts of hypoxia on aquatic animals and the molecular responses of aquatic animals to hypoxia were then summarized. The commonalities and specificities of the response to hypoxia in aquatic animals in different LOZs were discussed lastly. In general, this review will deepen the knowledge of the impacts of hypoxia on aquaculture and provide more information and research directions for the development of fishery resource protection strategies.

Non-coding RNAs targeting NF-κB pathways in aquatic animals: A review.

Nuclear factor-kappa B (NF-κB) pathways have a close relationship with many diseases, especially in terms of the regulation of inflammation and the immune response. Non-coding RNAs (ncRNAs) are a heterogeneous subset of endogenous RNAs that directly affect cellular function in the absence of proteins or peptide products; these include microRNAs (miRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), etc. Studies on the roles of ncRNAs in targeting the NF-κB pathways in aquatic animals are scarce. A few research studies have confirmed detailed regulatory mechanisms among ncRNAs and the NF-κB pathways in aquatic animals. This comprehensive review is presented concerning ncRNAs targeting the NF-κB pathway in aquatic animals and provides new insights into NF-κB pathways regulatory mechanisms of aquatic animals. The review discusses new possibilities for developing non-coding-RNA-based antiviral applications in fisheries.

MiR-7 Regulates Pathogen-Induced Immune Response via PAK1 in the Sea Cucumber Apostichopus japonicus.

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.

Isolation and characterization of four pathogenic strains of Vibrio spp. from farmed sea urchins, Strongylocentrotus intermedius, in China.

The sea urchin Strongylocentrotus intermedius, famous for its gonadal quality, is one of the most important farmed species in the sea area of northern China. Since 2020, outbreaks of black peristomial membrane disease (commonly called black mouth disease) have frequently occurred in spring and winter in cultured S. intermedius. In this study, we isolated the predominant bacteria from different tissues of diseased sea urchins from a North China farm in the spring of 2021. Four pathogenic strains (named SIBMPM01, SIBMPM02, SIBMPM03 and SIBMCF01) were obtained and characterized by Gram staining, morphological observation, artificial infection tests, and metabolic characteristics. Our results showed that: 1) all obtained strains belonged to the genus Vibrio and had morphological differences. Phylogenetic analysis indicated that the four obtained strains might be novel Vibrio species. 2) Laboratory-based artificial infection tests showed that sea urchins infected with either SIBMPM01, SIBMPM02, SIBMPM03 or SIBMCF01 exhibited pathological symptoms of a black peristomial membrane in a dosage-dependent and temperature-dependent manner. The virulence of SIBMCF01 was greater than those of the others. 3) Metabolic characterization data showed that SIBMPM01, SIBMPM02, SIBMPM03 and SIBMCF01 shared similar metabolic characteristics. 4) Antimicrobial susceptibility tests demonstrated that the four obtained strains were all sensitive to ampicillin, doxycycline, norfloxacin, ofloxacin, furazolidone and chloramphenicol. SIBMPM01 was specifically sensitive to neomycin, and SIBMCF01 was specifically sensitive to carboxybenzyl penicillin.

Responses of sea urchins (Strongylocentrotus intermedius) with different sexes to CO2-induced seawater acidification: Histology, physiology, and metabolomics.

Responses of different sexes of farmed Strongylocentrotus intermedius to chronic CO2-induced seawater acidification were investigated in 120-day lab-based experiments. Four experimental groups were set up as one control group and three seawater acidification groups. The results showed that 1) Specific growth rate and the numbers of mature gamete cells declined in a pH-dependent way in both sexes of adult S. intermedius. 2) There were differences in SDMs identified in females and males reared in acidified seawater reflecting sex-specific response variation in adult S. intermedius. 3) The number of altered metabolic pathways exhibited a linear increasing trend as seawater pH declined in both sexes of adult S. intermedius. Meanwhile seawater acidification might affect metabolic processes via changing the relative expression and activity of key enzymes controlling the corresponding metabolic pathways of adult S. intermedius.

Identification of a novel RhoA gene in the sea cucumber Apostichopus japonicus and its immune regulatory function via interacting with miR-2012-5p.

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.

Integrated miRNA-mRNA analysis provides potential biomarkers for selective breeding in bay scallop (Argopecten irradians).

Growth traits were compared between selected Argopecten irradians (BA) and non-selected A. irradians (NA; as a control). The results indicated that 1) the BA line exhibited greater average body weight and adductor muscle wet weight increase compared with the NA line at the same age of 10 months. 2) Comparative and integrated microRNA (miRNA) and mRNA transcriptome analyses identified 3373 differentially expressed genes (DEGs), 33 differentially expressed miRNAs (DEMs), and 39 "DEM-DEG" pairs in the BA line compared with the control. DEGs, DEMs, and "DEM-DEG" pairs involved in insulin signaling, immune related pathways, and actin cytoskeleton regulation were identified as candidates correlated with growth improvement in the BA line. A total of 259 positively selected genes were also identified. Collectively, our observations in this study will enrich the molecular information for A. irradians and provide potential biomarkers for future selective breeding and new seed creation in scallops.

Distant hybrids of Heliocidaris crassispina (♀) and Strongylocentrotus intermedius (♂): identification and mtDNA heteroplasmy analysis.

BACKGROUND: Distant hybridization between the sea urchin Heliocidaris crassispina (♀) and the sea urchin Strongylocentrotus intermedius (♂) was successfully performed under laboratory conditions. A new variety of hybrid sea urchin (HS hybrid) was obtained. However, the early-development success rates for the HS hybrids were significantly lower than those of purebred H. crassispina or S. intermedius offspring. In addition, it was difficult to distinguish the HS-hybrid adults from the pure H. crassispina adults, which might lead to confusion in subsequent breeding attempts. In this study, we attempted to develop a method to quickly and effectively identify HS hybrids, and to preliminarily investigate the molecular mechanisms underlying the poor early-development success rates in the HS hybrids. RESULTS: The hybrid sea urchins (HS hybrids) were identified both morphologically and molecularly. There were no significant differences in the test height to test diameter ratios between the HS hybrids and the parents. The number and arrangement of ambulacral pore pairs in the HS hybrids differed from those of the parental lines, which might serve as a useful morphological character for the identification of the HS hybrids. A primer pair that identified the HS hybrids was screened by comparing the mitochondrial genomes of the parental lines. Moreover, paternal leakage induced mitochondrial DNA heteroplasmy in the HS hybrids, which might explain the low rates of early development success in these hybrids. CONCLUSIONS: The distant-hybrid sea urchins were accurately identified using comparative morphological and molecular genetic methods. The first evidence of mtDNA heteroplasmy after the distant hybridization of an echinoderm was also provided.

Comparative metabolome analysis provides new insights into increased larval mortality under seawater acidification in the sea urchin Strongylocentrotus intermedius.

Mortality and metabolic responses of four-armed larvae of Strongylocentrotus intermedius under CO2-induced seawater acidification were investigated. Gametes of S. intermedius were fertilized and developed to the four-armed larval stage in either current natural seawater pH levels (as Control; pH = 7.99 ± 0.01) or laboratory-controlled acidified conditions (OA1: ΔpH = -0.3 units; OA2: ΔpH = -0.4 units; OA3: ΔpH = -0.5 units) according to the predictions of the Intergovernmental Panel on Climate Change (IPCC). The degrees of spicule exposure and asymmetry and mortality of four-armed larvae of S. intermedius were observed; each had a significant linearly increasing trend as the seawater pH level decreased. Comparative metabolome analysis identified a total of 87 significantly differentially expressed metabolites (SDMs, UP: 57, DOWN: 30) in OA-treated groups compared with the control group. Twenty-three SDMs, including carnitine, lysophosphatidylcholine (LPC) 18:3, lysophosphatidyl ethanolamine (LPE) 16:1, glutathione (GSH) and L-ascorbate, exhibited a linear increasing trend with decreasing seawater pH. Nine SDMs exhibited a linear decreasing trend as the seawater pH declined, including hypoxanthine, guanine and thymidine. Among all SDMs, we further mined 48 potential metabolite biomarkers responding to seawater acidification in four-armed larvae of S. intermedius. These potential metabolite biomarkers were mainly enriched in five pathways: glycerophospholipid metabolism, glutathione metabolism, purine metabolism, pyrimidine metabolism and the tricarboxylic acid cycle (TCA cycle). Our results will enrich our knowledge of the molecular mechanisms employed by sea urchins in response to CO2-induced seawater acidification.

Identification of molecular markers for superior quantitative traits in a novel sea cucumber strain by comparative microRNA-mRNA expression profiling.

To investigate the adaptability of Apostichopus japonicus (A. japonicus) strain "Anyuan No. 1" in the South China Sea, field monitoring and microRNA-mRNA integrated analyses were conducted between "Anyuan No. 1" and a regular A. japonicus population from Wendeng (Shandong Province, as a control) in the Xiapu farming area in Fujian Province, China. The results showed that "Anyuan No. 1" exhibited greater body weight increase and a higher number of papillae compared to the control during two and a half months of field monitoring. Comparative microRNA (miRNA) and mRNA transcriptome analyses identified 12 differentially expressed miRNAs (DEMs) and 165 differentially expressed genes (DEGs) in "Anyuan No. 1" compared to the control. Long-chain specific acyl-CoA dehydrogenase (ACADL), transmembrane protein 251 (TMEM251), dehydrogenase/reductase SDR family protein 7-like (Dhrs7), insulin-like growth factor-binding protein 7 (IGFBP-7), CDK5 regulatory subunit-associated protein 1 (CDK5RAP1), visual pigment-like receptor peropsin, 39S ribosomal protein, miR-10, miR-153, miR-7, and miR-3529 were identified as gene and miRNA candidates correlated with superior economic traits in "Anyuan No. 1". Collectively, "Anyuan No. 1" is suitable for large-scale cultivation extension due to its better adaptability to the South China Sea area. Furthermore, we identified "miR10-ACADL" as a potential module for further molecular marker-assisted selective breeding of A. japonicus.

CO2-driven ocean acidification repressed the growth of adult sea urchin Strongylocentrotus intermedius by impairing intestine function.

Strongylocentrotus intermedius cultured in the northern Yellow Sea in China was utilized to evaluate the effects of chronic CO2-driven ocean acidification (OA) on adult sea urchins. Based on the projection of the Intergovernmental Panel on Climate Change (IPCC), present natural seawater conditions (pHNBS = 8.10 ± 0.03) and three laboratory-controlled OA conditions (OA1, ΔpHNBS = - 0.3 units; OA2, ΔpHNBS = - 0.4 units; OA3, ΔpHNBS = - 0.5 units) were employed. After 60-day incubation, our results showed that (1) OA significantly repressed the growth of adult S. intermedius; (2) food consumption tended to be decreased with pH decline; (3) intestinal morphology was changed, and activities of intestinal cellulase and lipase were decreased under acidified conditions; (4) expression levels of two immune-related genes (SiTNF14 and SiTGF-ß) were altered; (5) rate-limiting enzyme activities of the glycolytic pathway and tricarboxylic acid cycle (TAC) were changed in all OA treatments compared to those of controls.

MicroRNAs involved in innate immunity regulation in the sea cucumber: A review.

The sea cucumber is one of the most economically significant echinoderms. The immunity against exogenous stimulation of sea cucumber is of great academic and economic importance. MicroRNAs (miRNAs) are a class of short endogenous non-coding RNAs (ncRNAs) that are considered as vital regulators of both innate and adaptive immune responses in most eukaryotes. In sea cucumbers, some miRNAs (such as miR-133, miR-137, and miR-2008, among others) that participate in the regulation of innate immunity have been recently identified and characterized. This review focuses on those known miRNAs and their corresponding target genes that participate in the regulation of the complement system, Toll-like receptor (TLR) pathway, reactive oxygen species (ROS) production and apoptosis pathways in sea cucumbers. Moreover, we cover immune-related miRNA investigations in sea cucumbers that provide insights into developing more miRNA-based biomarkers and therapeutic strategies for sea cucumber diseases.

The Impact of Chronic Heat Stress on the Growth, Survival, Feeding, and Differential Gene Expression in the Sea Urchin Strongylocentrotus intermedius.

To explore the impact of chronic heat stress on commercial echinoderms, the present study assessed the effects of chronic high temperature on the growth, survival, feeding, and differential gene expression in the sea urchin Strongylocentrotus intermedius cultured in northern Yellow Sea in China. One suitable seawater condition (20°C) and one laboratory-controlled high temperature condition (25°C) were set up. After 28 days incubation, our results showed that: (1) The specific growth, survival, and ingestion rates of S. intermedius reared under high temperature (25°C) decreased compared to those reared under optimal temperature (20°C) conditions; (2) comparative transcriptome analysis identified 2,125 differentially expressed genes (DEGs) in S. intermedius reared under high temperature (25°C) compared to those subjected to optimal temperature condition (20°C), which included 1,015 upregulated and 1,100 downregulated genes. The accuracy of the transcriptome profiles was verified by quantitative real-time PCR (qRT-PCR). Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses revealed that these DEGs mainly enriched the functional categories of ribosome, protein processing in endoplasmic reticulum, and prion diseases. A total of 732 temperature-induced expressed genes, such as ATP5, heat shock protein 70, and heat shock protein 90, were identified as candidates that were closely correlated with heat resistance in S. intermedius. Differentially expressed transcription factors (TFs), such as AP-1, Fos, CREB, and ZNF, were also identified as potential regulators that regulate the molecular network that was associated with responses to heat stress in sea urchins. Observations in the present study provide additional information that improves our understanding of the molecular mechanism of temperate echinoid species in response to heat stress, as well as theoretical basis for the molecular-assisted breeding of heat-resistant sea urchins.

Comparative transcriptome analysis identifies genes associated with papilla development in the sea cucumber Apostichopus japonicus.

To explore the molecular mechanisms underlying the initial differentiation and formation of papillae in sea cucumbers, the transcriptomes of Apostichopus japonicus pentactulae (without papillae) were compared to those of A. japonicus juveniles (with papillae). From the RNA of the three pentactula libraries and the three juvenile libraries, we obtained 41-46 million raw reads, yielding 39-45 million clean reads. From these, we generated 599,673 transcripts and identified 230,604 unigenes. Across all six transcriptomes, we identified 246,207 single nucleotide polymorphisms (SNPs) and 80,455 single sequence repeats (SSRs). There were more transition SNPs (60.74%) than transversion SNPs (39.26%). The mononucleotide repeat was the most abundant SSR motif. We identified 7965 differentially expressed unigenes (DEGs) in the juveniles and the pentactulae; 2421 DEGs were expressed only in the juveniles, and 1238 were expressed only in the pentactulae. Of all DEGs, 5215 were significantly upregulated and 2750 were significantly downregulated in the juveniles as compared to the pentactulae. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that the DEGs were primarily enriched in ribosome synthesis, cell proliferation, tight junction formation, collagen fibrillogenesis, and neurogenesis.

A novel p38 MAPK gene in the sea cucumber Apostichopus japonicus (Ajp38) is associated with the immune response to pathogenic challenge.

The p38 mitogen-activated protein kinase (MAPK), an important component of the MAPK signal cascade, is activated by extracellular stimuli, such as environmental stress and pathogenic infection. To clarify the function of p38 MAPKs in echinoderms, we used transcriptome database mining and rapid amplification of cDNA ends (RACE) to identify a novel p38 MAPK gene in the sea cucumber Apostichopus japonicus (here designated Ajp38). The full-length cDNA of Ajp38 was 2231 bp, including an open reading frame encoding 356 amino acid residues. Our sequence analysis indicated that the predicted Ajp38 protein contained the dual phosphorylation site Thr-Gly-Tyr (TGY) and was similar to the p38 homolog in sea urchins. Quantitative real-time PCR analysis showed that Ajp38 was ubiquitously expressed in all examined tissues of healthy adult A. japonicus, with the highest level of expression identified in the coelomocytes. Ajp38 mRNA expression was significantly upregulated in the coelomocytes 4, 12, and 72 h post in vivo infection with Vibrio splendidus. Our results provide more information about the characteristics and immune functions of the p38 homolog in sea cucumbers.

Identification and characterization of microRNAs from the tube foot in the sea urchin Strongylocentrotus intermedius.

MicroRNAs (miRNAs) play critical roles in regulating many bio-processes of eukaryotes. The sea urchin Strongylocentrotus intermedius (an important fishery resource) is of great economic importance in Japan, North Korea, Russia, and China. In the current study, miRNAs of tube foot in S. intermedius were firstly identified and characterized. Data in this study can provide more genomic information for the further understanding of the complex regulation network in sea urchins and present a new way for monitoring the health status of cultured sea urchins.

Sea cucumber genome provides insights into saponin biosynthesis and aestivation regulation.

Echinoderms exhibit several fascinating evolutionary innovations that are rarely seen in the animal kingdom, but how these animals attained such features is not well understood. Here we report the sequencing and analysis of the genome and extensive transcriptomes of the sea cucumber Apostichopus japonicus, a species from a special echinoderm group with extraordinary potential for saponin synthesis, aestivation and organ regeneration. The sea cucumber does not possess a reorganized Hox cluster as previously assumed for all echinoderms, and the spatial expression of Hox7 and Hox11/13b potentially guides the embryo-to-larva axial transformation. Contrary to the typical production of lanosterol in animal cholesterol synthesis, the oxidosqualene cyclase of sea cucumber produces parkeol for saponin synthesis and has "plant-like" motifs suggestive of convergent evolution. The transcriptional factors Klf2 and Egr1 are identified as key regulators of aestivation, probably exerting their effects through a clock gene-controlled process. Intestinal hypometabolism during aestivation is driven by the DNA hypermethylation of various metabolic gene pathways, whereas the transcriptional network of intestine regeneration involves diverse signaling pathways, including Wnt, Hippo and FGF. Decoding the sea cucumber genome provides a new avenue for an in-depth understanding of the extraordinary features of sea cucumbers and other echinoderms.

Integrative mRNA-miRNA interaction analysis associate with immune response of sea cucumber Apostichopus japonicus based on transcriptome database.

MicroRNAs (miRNAs) constitute a family of endogenous non-coding small RNAs that have been demonstrated to be the key effectors in mediating host-pathogen interactions. Additionally, high-throughput sequencing provides unexampled opportunities to identify the pathogenic mechanism underlying miRNAs. In the present study, the target genes of immune-related miRNAs (miR-31, miR-2008, miR-92a, miR-210 and miR-7) and specific miRNAs (miR-2004) in Echinodermata were predicted in silico and validated. Gene ontology (GO) analysis of the target genes of these six miRNAs were conducted to further understand the regulatory function in the host immunity of Apostichopus japonicus (A. japonicus). Among the putative target genes of the six miRNAs, various immune-related targets were annotated, such as Nephl, SEC14Ll, p105, GL2, LYS, FNIAL, mTOR, LITAF, SLC44, TLR3, Apaf-1, and CNTN4. This work will provide valuable genetic resources to understand the interaction of multiple mRNA-miRNAs and the regulation mechanism in the anti-bacterial process in the sea cucumber.