Morphology and ultrastructure of digestive system in pre-zoea and zoea I larvae of red king crab, Paralithodes camtschaticus (Tilesius, 1815).

The digestive system structure in pre-zoea and zoea I larvae of the red king crab Paralithodes camtschaticus has been examined. During this development period, the digestive system consists of an esophagus, a stomach, a midgut (where the hepatopancreas ducts open), and a hindgut. The esophagus begins from the oral slit on the animal's ventral side and extends vertically up to the junction with the cardiac stomach. The latter is followed by the pyloric stomach. At the stages under study, crabs have a cardiac-pyloric valve and a pyloric filter in the stomach already developed. The midgut begins with an expansion in the cephalothorax, enters the pleon, grows narrower there, and extends to somite 3 of pleon. The hepatopancreas is represented by a symmetrical paired gland which occupies almost the entire cephalothorax space and opens with its ducts at the junction of the pyloric stomach with the midgut. The hepatopancreas is divided into the anterior and posterior lobes. At the pre-zoea stage, the anterior lobes are large and filled with yolk. At the zoea I stage, the anterior lobes are smaller relative to the entire hepatopancreas, and the posterior lobes increase and form tubular outgrowths. It has been shown that during the transition from pre-zoea to zoea I, the number of mitochondria in enterocytes increases and a peritrophic membrane forms in the midgut. These changes are probably associated with the transition to independent living and feeding.

Extracellular Matrix of Echinoderms.

This review considers available data on the composition of the extracellular matrix (ECM) in echinoderms. The connective tissue in these animals has a rather complex organization. It includes a wide range of structural ECM proteins, as well as various proteases and their inhibitors. Members of almost all major groups of collagens, various glycoproteins, and proteoglycans have been found in echinoderms. There are enzymes for the synthesis of structural proteins and their modification by polysaccharides. However, the ECM of echinoderms substantially differs from that of vertebrates by the lack of elastin, fibronectins, tenascins, and some other glycoproteins and proteoglycans. Echinoderms have a wide variety of proteinases, with serine, cysteine, aspartic, and metal peptidases identified among them. Their active centers have a typical structure and can break down various ECM molecules. Echinoderms are also distinguished by a wide range of proteinase inhibitors. The complex ECM structure and the variety of intermolecular interactions evidently explain the complexity of the mechanisms responsible for variations in the mechanical properties of connective tissue in echinoderms. These mechanisms probably depend not only on the number of cross-links between the molecules, but also on the composition of ECM and the properties of its proteins.

Muscle Regeneration in Holothurians without the Upregulation of Muscle Genes.

The holothurian Eupentacta fraudatrix is capable of fully restoring its muscles after transverse dissection. Although the regeneration of these structures is well studied at the cellular level, the molecular basis of the process remains poorly understood. To identify genes that may be involved in the regulation of muscle regeneration, the transcriptome of the longitudinal muscle band of E. fraudatrix has been sequenced at different time periods post-injury. An analysis of the map of biological processes and pathways has shown that most genes associated with myogenesis decrease their expression during the regeneration. The only exception is the genes united by the GO term "heart valve development". This may indicate the antiquity of mechanisms of mesodermal structure transformation, which was co-opted into various morphogeneses in deuterostomes. Two groups of genes that play a key role in the regeneration have been analyzed: transcription factors and matrix metalloproteinases. A total of six transcription factor genes (Ef-HOX5, Ef-ZEB2, Ef-RARB, Ef-RUNX1, Ef-SOX17, and Ef-ZNF318) and seven matrix metalloproteinase genes (Ef-MMP11, Ef-MMP13, Ef-MMP13-1, Ef-MMP16-2, Ef-MMP16-3, Ef-MMP24, and Ef-MMP24-1) showing differential expression during myogenesis have been revealed. The identified genes are assumed to be involved in the muscle regeneration in holothurians.

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Echinoderms: Structure and Possible Functions.

Echinoderms are one of the most ancient groups of invertebrates. The study of their genomes has made it possible to conclude that these animals have a wide variety of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The phylogenetic analysis shows that the MMPs and TIMPs underwent repeated duplication and active divergence after the separation of Ambulacraria (Echinodermata+Hemichordata) from the Chordata. In this regard the homology of the proteinases and their inhibitors between these groups of animals cannot be established. However, the MMPs of echinoderms and vertebrates have a similar domain structure. Echinoderm proteinases can be structurally divided into three groups-archetypal MMPs, matrilysins, and furin-activatable MMPs. Gelatinases homologous to those of vertebrates were not found in genomes of studied species and are probably absent in echinoderms. The MMPs of echinoderms possess lytic activity toward collagen type I and gelatin and play an important role in the mechanisms of development, asexual reproduction and regeneration. Echinoderms have a large number of genes encoding TIMPs and TIMP-like proteins. TIMPs of these animals, with a few exceptions, have a structure typical for this class of proteins. They contain an NTR domain and 10-12 conservatively located cysteine residues. Repeated duplication and divergence of TIMP genes of echinoderms was probably associated with an increase in the functional importance of the proteins encoded by them in the physiology of the animals.

Expression of Piwi, MMP, TIMP, and Sox during Gut Regeneration in Holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirotida).

Mesodermal cells of holothurian Eupentacta fraudatrix can transdifferentiate into enterocytes during the regeneration of the digestive system. In this study, we investigated the expression of several genes involved in gut regeneration in E. fraudatrix. Moreover, the localization of progenitor cells of coelomocytes, juvenile cells, and their participation in the formation of the luminal epithelium of the digestive tube were studied. It was shown that Piwi-positive cells were not involved in the formation of the luminal epithelium of the digestive tube. Ef-72 kDa type IV collagenase and Ef-MMP16 had an individual expression profile and possibly different functions. The Ef-tensilin3 gene exhibited the highest expression and indicates its potential role in regeneration. Ef-Sox9/10 and Ef-Sox17 in E. fraudatrix may participate in the mechanism of transdifferentiation of coelomic epithelial cells. Their transcripts mark the cells that plunge into the connective tissue of the gut anlage and give rise to enterocytes. Ef-Sox9/10 probably controls the switching of mesodermal cells to the enterocyte phenotype, while Ef-Sox17 may be involved in the regulation of the initial stages of transdifferentiation.

Molecular Aspects of Regeneration Mechanisms in Holothurians.

Holothurians, or sea cucumbers, belong to the phylum Echinodermata. They show good regenerative abilities. The present review provides an analysis of available data on the molecular aspects of regeneration mechanisms in holothurians. The genes and signaling pathways activated during the asexual reproduction and the formation of the anterior and posterior parts of the body, as well as the molecular mechanisms that provide regeneration of the nervous and digestive systems, are considered here. Damage causes a strong stress response, the signs of which are recorded even at late regeneration stages. In holothurian tissues, the concentrations of reactive oxygen species and antioxidant enzymes increase. Furthermore, the cellular and humoral components of the immune system are activated. Extracellular matrix remodeling and Wnt signaling play a major role in the regeneration in holothurians. All available morphological and molecular data show that the dedifferentiation of specialized cells in the remnant of the organ and the epithelial morphogenesis constitute the basis of regeneration in holothurians. However, depending on the type of damage, the mechanisms of regeneration may differ significantly in the spatial organization of regeneration process, the involvement of different cell types, and the depth of reprogramming of their genome (dedifferentiation or transdifferentiation).

The Eupentacta fraudatrix transcriptome provides insights into regulation of cell transdifferentiation.

The holothurian Eupentacta fraudatrix is a unique organism for studying regeneration mechanisms. Moreover, E. fraudatrix can quickly restore parts of its body and entire organ systems, yet at the moment, there is no data on the participation of stem cells in the process. To the contrary, it has been repeatedly confirmed that this process is only due to the transformation of terminally differentiated cells. In this study, we examine changes in gene expression during gut regeneration of the holothurian E. fraudatrix. Transcriptomes of intestinal anlage of the three stages of regeneration, as well as the normal gut, were sequenced with an Illumina sequencer (San Diego, CA, USA). We identified 14,617 sea urchin protein homologs, of which 308 were transcription factors. After analysing the dynamics of gene expression during regeneration and the map of biological processes in which they participate, we identified 11 factors: Ef-EGR1, Ef-ELF, Ef-GATA3, Ef-ID2, Ef-KLF1/2/4, Ef-MSC, Ef-PCGF2, Ef-PRDM9, Ef-SNAI2, Ef-TBX20, and Ef-TCF24. With the exception of TCF24, they are all involved in the regeneration, development, epithelial-mesenchymal transition, and immune response in other animals. We suggest that these transcription factors may also be involved in the transdifferentiation of coelomic epithelial cells into enterocytes in holothurians.

Different Macrophage Type Triggering as Target of the Action of Biologically Active Substances from Marine Invertebrates.

Macrophages play a fundamental role in the immune system. Depending on the microenvironment stimuli, macrophages can acquire distinct phenotypes characterized with different sets of the markers of their functional activities. Polarization of macrophages towards M1 type (classical activation) is involved in inflammation and the related progression of diseases, while, in contrast, alternatively activated M2 macrophages are associated with the anti-inflammatory mechanisms. Reprogramming macrophages to switch their phenotypes could provide a new therapeutic strategy, and targeting the M1/M2 macrophage balance is a promising current trend in pharmacology. Marine invertebrates are a vast source of the variety of structurally diverse compounds with potent pharmacological activities. For years, a large number of studies concerning the immunomodulatory properties of the marine substances have been run with using some intracellular markers of immune stimulation or suppression irrespective of the possible application of marine compounds in reprogramming of macrophage activation, and only few reports clearly demonstrated the macrophage-polarizing activities of some marine compounds during the last decade. In this review, the data on the immunomodulating effects of the extracts and pure compounds of a variety of chemical structure from species of different classes of marine invertebrates are described with focus on their potential in shifting M1/M2 macrophage balance towards M1 or M2 phenotype.

Reference assembly and gene expression analysis of Apostichopus japonicus larval development.

The transcriptome of the holothurian Apostichopus japonicus was sequenced at four developmental stages-blastula, gastrula, auricularia, pentactula-on an Illumina sequencer. Based on our RNA-seq data and the paired-end reads from 16 libraries obtained by other researchers earlier, we have achieved the currently most complete transcriptome assembly for A. japonicus with the best basic statistical parameters. An analysis of the obtained transcriptome has revealed 174 differentially expressed transcription factors, as well as stage-specific transcription factors that are most promising for further study. In addition, a total of 1,174,999 high-quality single nucleotide polymorphisms have been identified, including 58,932 indels. A GO enrichment analysis of contigs containing polymorphic loci shows the predominance of GO terms associated with immune response. The data obtained by us provide an additional basis for a deeper study of the mechanisms of the planktotrophic-type development in holothurians and can be used in commercial sea cucumber breeding programs.

Cladolosides C4, D1, D2, M, M1, M2, N and Q, new triterpene glycosides with diverse carbohydrate chains from sea cucumber Cladolabes schmeltzii. An uncommon 20,21,22,23,24,25,26,27-okta-nor-lanostane aglycone. The synergism of inhibitory action of non-toxic dose of the glycosides and radioactive irradiation on colony formation of HT-29 cancer cells.

Eight new triterpene olygoglycosides, cladolosides C4 (1), D1 (2), D2 (3), M (4), M1 (5), M2 (6), N (7) and Q (8), were isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Sclerodactylidae, Dendrochirotida). Structures of these glycosides were elucidated by 2D NMR spectroscopy and HR ESI mass spectrometry. A novel hexasaccharide carbohydrate chain having xylose residues as the first, second and third sugars was found in the glycoside 7. Cladoloside C4 (1) contains an uncommon 20,21,22,23,24,25,26,27-octa-norlanostane aglycone. Cladolosides D1 (2), D2 (3) and Q (8) were new representatives of the hexaosides with a non-methylated terminal sugar unit in the "upper" half-chain. Cytotoxic activities of the isolated compounds against ascite form of mouse Ehrlich carcinoma cells, mouse erythrocytes and human colorectal adenocarcinoma HT-29 cells were examined and their structure-activity relationships were analyzed. In addition, the majority of tested compounds, except for cladoloside D2 (3), inhibited the colony formation and growth of HT-29 cells at non-cytotoxic concentrations. The highest inhibitory activity was demonstrated by cladoloside M1 (5). Moreover, synergism of effects of radioactive irradiation and non-toxic dose of compounds 1-8 decreasing the number of colonies of HT-29 cells was observed. Cladoloside N (7) was the most active and increased the inhibitory effect from radiation by 75%. The biosynthetic transformations of the aglycones are discussed.

Cladolosides O, P, P1-P3 and R, triterpene glycosides with two novel types of carbohydrate chains from the sea cucumberCladolabes schmeltzii. Inhibition of cancer cells colony formation and its synergy with radioactive irradiation.

The sea cucumber Cladolabes schmeltzii (Sclerodactylidae, Dendrochirotida) contains diverse glycosides, including several dozen individual compounds. Six new triterpene oligoglycosides, cladolosides O (1), P (2), P1 (3), P2 (4), P3 (5) and R (6), were isolated from this sea cucumber. Their structures were elucidated by 2D NMR spectroscopy and HR ESI mass spectrometry. Cladoloside O (1) has a pentasaccharide carbohydrate chain. Cladolosides of the group P and cladoloside R (6) include novel hexasaccharide carbohydrate chains with different positions of non-methylated terminal sugar units. All the isolated compounds demonstrate strong cytotoxic activities against cells of mouse Ehrlich carcinoma cells (ascite form) and mouse erythrocytes. The cytotoxicity of these compounds against human colorectal adenocarcinoma HT-29 cells was somewhat lower. The compounds 1-6 also inhibit the colony formation and growth of HT-29 cells at non-cytotoxic concentrations. The highest inhibition effect was demonstrated by cladoloside P1 (3). Moreover, synergism of effects of radioactive irradiation and non-toxic dose of compounds 1-6 on colony formation of HT-29 cells was observed. Cladolosides P2 (4) and P3 (5) were the most active and increased the inhibitory effect of radiation by more than 70%. The metabolic network demonstrating the biosynthetic pathways to carbohydrate chains of the glycosides of C. schmeltzii, based on a comparison of their structures, was constructed.

Molecular mechanisms of fission in echinoderms: Transcriptome analysis.

Echinoderms are capable of asexual reproduction by fission. An individual divides into parts due to changes in the strength of connective tissue of the body wall. The structure of connective tissue and the mechanisms of variations in its strength in echinoderms remain poorly studied. An analysis of transcriptomes of individuals during the process of fission provides a new opportunity to understand the mechanisms of connective tissue mutability. In the holothurian Cladolabes schmeltzii, we have found a rather complex organization of connective tissue. Transcripts of genes encoding a wide range of structural proteins of extracellular matrix, as well as various proteases and their inhibitors, have been discovered. All these molecules may constitute a part of the mechanism of connective tissue mutability. According to our data, the extracellular matrix of echinoderms is substantially distinguished from that of vertebrates by the lack of elastin, fibronectins, and tenascins. In case of fission, a large number of genes of transcription factors and components of different signaling pathways are expressed. Products of these genes are probably involved in regulation of asexual reproduction, connective tissue mutability, and preparation of tissues for subsequent regeneration. It has been shown that holothurian tensilins are a special group of tissue inhibitors of metalloproteinases, which has formed within the class Holothuroidea and is absent from other echinoderms. Our data can serve a basis for the further study of the mechanisms of extracellular matrix mutability, as well as the mechanisms responsible for asexual reproduction in echinoderms.

Metabolite Profiling of Triterpene Glycosides of the Far Eastern Sea Cucumber Eupentacta fraudatrix and Their Distribution in Various Body Components Using LC-ESI QTOF-MS.

The Far Eastern sea cucumber Eupentacta fraudatrix is an inhabitant of shallow waters of the south part of the Sea of Japan. This animal is an interesting and rich source of triterpene glycosides with unique chemical structures and various biological activities. The objective of this study was to investigate composition and distribution in various body components of triterpene glycosides of the sea cucumber E. fraudatrix. We applied LC-ESI MS (liquid chromatography-electrospray mass spectrometry) of whole body extract and extracts of various body components for metabolic profiling and structure elucidation of triterpene glycosides from the E. fraudatrix. Totally, 54 compounds, including 26 sulfated, 18 non-sulfated and 10 disulfated glycosides were detected and described. Triterpene glycosides from the body walls, gonads, aquapharyngeal bulbs, guts and respiratory trees were extracted separately and the distributions of the detected compounds in various body components were analyzed. Series of new glycosides with unusual structural features were described in E. fraudatrix, which allow clarifying the biosynthesis of these compounds. Comparison of the triterpene glycosides contents from the five different body components revealed that the profiles of triterpene glycosides were qualitatively similar, and only some quantitative variabilities for minor compounds were observed.

Wnt and frizzled expression during regeneration of internal organs in the holothurian Eupentacta fraudatrix.

Several genes of the Wnt and Frizzled families in the holothurian Eupentacta fraudatrix are characterized, and the complete coding sequences of wntA, wnt4, wnt6, wnt16, frizzled1/2/7, frizzled4, and frizzled5/8 are obtained. The dynamics of expression of these genes during regeneration of internal organs after evisceration are studied. Evisceration and the associated damages supposedly induce the expression of wnt16 on third day after evisceration. Genes wntA, wnt4, wnt6, and frizzled1/2/7 up-regulate during the period of active morphogenesis (5-7 days after evisceration) and might participate in regulation of tissue and organ formation. The signaling induced via Frizzled5/8 is could be necessary for formation of the anterior (ectodermal) part of the digestive system and development of the calcareous ring on 10th day after evisceration. Our data suggest that the Wnt signaling pathway plays a significant role in the regulation of regeneration of internal organs in holothurians.

Regeneration of the digestive system in the crinoid Himerometra robustipinna occurs by transdifferentiation of neurosecretory-like cells.

The structure and regeneration of the digestive system in the crinoid Himerometra robustipinna (Carpenter, 1881) were studied. The gut comprises a spiral tube forming radial lateral processes, which gives it a five-lobed shape. The digestive tube consists of three segments: esophagus, intestine, and rectum. The epithelia of these segments have different cell compositions. Regeneration of the gut after autotomy of the visceral mass progresses very rapidly. Within 6 h after autotomy, an aggregation consisting of amoebocytes, coelomic epithelial cells and juxtaligamental cells (neurosecretory neurons) forms on the inner surface of the skeletal calyx. At 12 h post-autotomy, transdifferentiation of the juxtaligamental cells starts. At 24 h post-autotomy these cells undergo a mesenchymal-epithelial-like transition, resulting in the formation of the luminal epithelium of the gut. Specialization of the intestinal epithelial cells begins on day 2 post-autotomy. At this stage animals acquire the mouth and anal opening. On day 4 post-autotomy the height of both the enterocytes and the visceral mass gradually increases. Proliferation does not play any noticeable role in gut regeneration. The immersion of animals in a 10-7 M solution of colchicine neither stopped formation of the lost structures nor caused accumulation of mitoses in tissues. Weakly EdU-labeled nuclei were observed in the gut only on day 2 post-autotomy and were not detected at later regeneration stages. Single mitotically dividing cells were recorded during the same period. It is concluded that juxtaligamental cells play a major role in gut regeneration in H. robustipinna. The main mechanisms of morphogenesis are cell migration and transdifferentiation.

Cladolosides I1, I2, J1, K1, K2 and L1, monosulfated triterpene glycosides with new carbohydrate chains from the sea cucumber Cladolabes schmeltzii.

Six new monosulfated triterpene glycosides, cladolosides I1 (1), I2 (2), J1 (3), K1 (4), K2 (5) and L1 (6) were isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida). Structures of these glycosides were elucidated by 2D NMR spectroscopy and mass spectrometry. Four new types of carbohydrate chains have been found in 1-6. Cladolosides of the groups I and J are characterized by pentasaccharide carbohydrate chains sulfated at a terminal 3-O-methylglucose residue and branched by the position 4 of the first xylose residue, but differing from each other in the lengths of the main and side carbohydrate chains. Cladolosides of the groups K and L contain hexasaccharide chains with different positions of a sulfated terminal 3-O-methylglucose residue (as the fourth or the sixth monosaccharide residue). Sulfated hexasaccharide carbohydrate chains were found in the sea cucumbers glycosides for the first time. A pentasaccharide carbohydrate chain of cladoloside J1 (3) having a disaccharide moiety of glucose and a sulfated 3-O-methylglucose linked to the first xylose residue in a linear trisaccharide fragment is also unusual. All substances studied demonstrated strong or moderate hemolytic and cytotoxic effects.

Colochiroside E, an Unusual Non-holostane Triterpene Sulfated Trioside from the Sea Cucumber Colochirus robustus and Evidence of the Impossibility of a 7(8)-Double Bond Migration in Lanostane Derivatives having an 18(16)-Lactone.

The unusual non-holostane triterpene glycoside, colochiroside E (1) was isolated from the sea cucumber Colochirus robustus (Cucumariidae, Dendrochirotida). The structure of 1 was established by analysis of 1D, 2D NMR and HRESI MS data. Colochiroside E (1) belongs to a rare group of glycosylated 9ß-H-lanosta-18(16)-lactones and has an unprecedented sulfated trisaccharide carbohydrate chain consisting of two glucose and one xylose units. In contrast with (9ß-H)-7(8)-unsaturated holostane glycosides, the 7(8)-double bond in the having (9ß-H)-configuration aglycone of colochiroside E is not capable of migration into the 8(9)- and then into the 9(11)-position on treatment with HCl. The formation of a chlorine derivative of 1 was observed under these conditions.

Colochirosides A1, A2, A3, and D, Four Novel Sulfated Triterpene Glycosides from the Sea Cucumber Colochirus robustus (Cucumariidae, Dendrochirotida).

Four new triterpene glycosides, colochirosides A1 (1), A2 (2), A3 (3) and D (4), have been isolated from the sea cucumber Colochirus robustus (Cucumariidae, Dendrochirotida). Structures of the glycosides have been elucidated by 2D NMR spectroscopy and mass-spectrometry. Colochiroside D (4) has a new type of carbohydrate chain having the only sulfate group attached to C-6 of the third (glucose) monosaccharide residue. Cytotoxic activities of glycosides 1-4 against the ascite form of mouse Ehrlich carcinoma cells and hemolytic activity against mouse erythrocytes have been studied. Hemolytic activity of the glycosides was higher than cytotoxic. Glycosides 1, 3 and 4 demonstrated strong effects, whereas compound 2 showed only moderate activity.

Colochirosides B1, B2, B3 and C, Novel Sulfated Triterpene Glycosides from the Sea Cucumber Colochirus robustus (Cucumariidae, Dendrochirotida).

Four new triterpene glycosides, colochirosides B1 (1), B2 (2), B3 (3) and C (4) have been isolated from the sea cucumber Colochirus robustus (Cucumariidae, Dendrochirotida). Six known earlier glycosides from representatives of two families of the order Dendrochirotida have also been found in C. robustus. Structures of the glycosides have been elucidated by 2D NMR spectroscopy and mass spectrometry. All the glycosides belong to the holostane series and contain tetrasaccharide linear carbohydrate chains with one or two sulfate groups. Cytotoxic activities of glycosides 1-4 against the ascite form of mouse Ehrlich carcinoma cells and hemolytic activities against mouse erythrocytes have been studied. Hemolytic activity of the glycosides was higher than cytotoxic. Glycosides 3 and 4 demonstrated strong effects, whereas compounds 1 and 2 containing the hydroxy-group in the side chains showed moderate hemolytic activity and were not cytotoxic.

Structures and biological activities of cladolosides C3, E1, E2, F1, F2, G, H1 and H2, eight triterpene glycosides from the sea cucumber Cladolabes schmeltzii with one known and four new carbohydrate chains.

Eight new nonsulfated triterpene glycosides, cladolosides C3(1), E1(2), E2(3), F1(4), F2(5), G(6), H1(7) and H2(8) have been isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida) collected in the Vietnamese shallow waters. The structures of the glycosides were elucidated by 2D NMR spectroscopy and mass-spectrometry. Glycosides 2, 3, 4, and 5 have pentasaccharide branched carbohydrate moieties and differ from each other by monosaccharide compositions and aglycone structures. At that, glycosides 2 and 3 contain three xylose, one 3-O-methyl-glucose and one quinovose residues, while glycosides 4 and 5 have two quinovose, two xylose and one 3-O-methyl-glucose residues. Compounds 1 and 6-8 are hexaosides differing from each other by aglycone structures and by the fifth monosaccharide residue, which proved to be glucose in cladoloside C3(1), xylose in cladoloside G(6) and quinovose in cladolosides H1(7) and H2(8). The presence of quinovose residue in the fifth position, as in 4, 5, 7 and 8 has never been earlier found in carbohydrate chains of triterpene glycosides from sea cucumbers. The carbohydrate chains with xylose in the fifth position of pentaosides and hexaosides are also very unusual for holothurious glycosides. All the substances demonstrate strong or moderate cytotoxic and hemolytic effects with hexaosides being more active than the corresponding pentaosides. Peculiarities of the biosynthesis and biochemical evolution of glycosides of this type are discussed.