Morphology of the Respiratory Trees in the Holothurians Apostichopus japonicus and Cucumaria japonica

The morphology of the respiratory trees in the holothurians Apostichopus japonicus and Cucumaria japonica was studied using histochemical and electron microscopic techniques. The epithelium of the respiratory tree cavity in A. japonicus consists of columnar cells about 17–20 m high. In C. japonica, this epithelium is composed of two cell types: bulbous cells embedded in the connective tissue layer and secretory cells; cells are 1–12 m high. A characteristic feature of cells of the respiratory tree cavity epithelium in these species is the presence of numerous phagosomes and coated vesicles in the apical cytoplasm. The cell surface has many microvilli and a single cilium. Cells of the coelomic epithelium contain vacuoles in the apical part and myofibrils in the basal region; the thickness of this epithelium in A. japonicus and C. japonica is 9–16 and 10–30 m, respectively. Based on the different structure of the respiratory trees and the absence of hemocytes in A. japonicus, it is suggested that the holothurian species studied have different routes of oxygen transport from the environment to the internal organs.     

Mitotic Activity in Tissues of the Regenerating Respiratory Tree of the Holothurian Apostichopus japonicus (Holothuroidea,Aspidochirota)

Mitotic activity in the regenerating respiratory tree was studied in the holothurian Apostichopus japonicus after evisceration. Significant proliferation was recorded in the regenerating tissues. The highest number of mitoses was revealed in the inner epithelium, the mitotic index (MI) of which reached 3.44 ± 0.01% on the 10th day of regeneration, which was comparable to MI values of blastema of a regenerating amphibian limb. In the coelomic epithelium the number of dividing cells varied from 0.49% in the upper part (20th day) up to 2.87% in the base (10th day). The proliferative activity gradually decreased in the further course of regeneration. In spite of high MI values, the distribution of mitoses in tissues was even at all stages of regeneration and blastema did not form.     

Structure of Genital Papillae in the Far Eastern Holothurians of the Genus Cucumaria (Dendrochirotida: Cucumariidae)

The genital papilla structure of female and male specimens is studied in four species of large holothurians of the genus Cucumaria from the Far Eastern seas of Russia: Cucumaria japonica (Semper), C. okhotensis Levin et Stepanov, C. anivaensis Levin, C. djakonovi Baranova. The papillary forms in these species are extremely various and cannot be considered as characteristic for the species.     

Cucumaria anivaensis (Holothuroidea: Dendrochirotida)—A New Species of Holothurians from the Coastal Waters of Sakhalin

Cucumaria anivaensis sp. nov. is described from Aniva Bay (Sakhalin). This species is characterized by having large lengthened plates in the body skin with large apertures in the medial region and small apertures at both edges, very long plates with an outgrowth on one side in the tentacles, and an expanded basis of the radial plates of the pharyngeal ring. Some plates of the oral disk have strong enlargement in the medial region.     

Ultrastructural Study of Relationships between Germinal Bodies and Mitochondria in Apostichopus japonicus (Echinodermata: Holothuroidea) and Pleuronectes asper (Teleostei: Pleuronectidae)

The relationships between germinal bodies and mitochondria were studied in the holothurian Apostichopus japonicus and the flounder Pleuronectes asper using TEM. In the gonial cells of both species the mitochondria are arranged around germinal bodies and are in contact with the latter. A gradual disappearance of the outer membrane is found in the mitochondria that interact with the germinal substance. Later on, dispersion of the globules of the mitochondrial matrix containing mitochondrial cristae occurs. It is supposed that the substance of the mitochondrial matrix takes part in the development and functioning of the germinal plasm in both invertebrates and vertebrates.     

Participation of the coelomic epithelium of the body wall in muscle regeneration in <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Holothuroidea: Aspidochirota)

The cellular sources of regeneration of longitudinal muscles were studied in the holothurian Apostichopus japonicus. An autoradiographic method tracing the distribution of cells labeled with tritiated thymidine (³HT) revealed that the majority of ³HT-cells, which were initially localized in the coelomic epithelium of muscles and the body wall at the beginning of active morphogenesis, were then found in the structure of new muscular bundles during subsequent terms of restoration. Thus, the coelomic epithelium of the body wall participated in the regeneration of muscle tissue concurrently with the coelomic epithelium of muscle, contributing to the recruitment of a pool of myogenic cells.     

Comparative analysis of genes expressed in regenerating intestine and non-eviscerated intestine of Apostichopus japonicus Selenka (Aspidochirotida: Stichopodidae) and cloning of ependymin gene

The regeneration of the intestine of sea cucumber (Apostichopus japonicus) was studied by describing historically the changes that occurred during intestine regeneration on the fifth day after chemically-induced evisceration. An expressed sequence tag (EST) analysis was undertaken to identify major genes, which might be involved in intestine regeneration of A. japonicus. Two cDNA libraries were constructed with directional cloning method, one for regenerating intestine collected on the third, fourth and fifth day after evisceration (post-evisceration, PE), and the other for the non-eviscerated (NE). A total of 730 ESTs were generated by sequencing cDNA clones from the two libraries (372 from PE and 358 from NE). The results showed that the number of genes that were involved in primary metabolism of PE library was less than that of NE library, while the number of genes involved in cell defense/immunity, cell division, cell signal transduction/communication of PE library was more than that of NE library. The results also revealed that the expression of the genes which might be involved in regeneration was enhanced to some extent after evisceration. Only about 11.54% of the sequenced clones were shared by two libraries, which provided some clues for the existence of differential gene expression between PE and NE intestines. A gene named epenAj was also characterized in this study.     

仿刺参多糖抗肿瘤及对荷瘤小鼠免疫调节作用

目的 观察仿刺参多糖(AJPS)抗肿瘤及免疫调节作用.方法 采用MTT法检测AJPS对人肝癌HepG-2细胞抑制率;以Hca-F肝癌小鼠为模型,采用MTT法、放免法测定荷瘤小鼠细胞免疫指标.结果 AJPS抑制HepG-2细胞生长,抑制小鼠移植瘤生长;增强脾淋巴细胞和巨噬细胞活性,促进TNF-α和IL-2的产生.结论 AJPS具有对HepG-2细胞的直接杀伤作用;AJPS对荷瘤小鼠有免疫调节活性,在肿瘤的免疫治疗中发挥作用.     

夏眠对刺参(Apostichopus japonicus (Selenka))能量收支的影响

夏眠是刺参最重要的生理特征;水温升高是其夏眠的主要诱发因子,而夏眠的临界温度与刺参体重密切相关。为揭示刺参夏眠对其能量利用对策的影响,测定了2种体重规格(134.0±13.5)g和(73.6±2.2)g刺参在10、15、20、25℃和30℃5个温度梯度下的能量收支。结果表明,温度和体重及其交互作用对刺参能量的摄入均有显著影响;而温度是影响其摄食能分配的主要因素。研究发现,刺参在非夏眠期、夏眠临界期和完全夏眠期的能量利用对策有所不同:在非夏眠期,刺参摄食能支出的最大组分是粪便能,占摄食能的比例超过50%,其次为呼吸耗能,占19.8%～39.4%,而生长能和排泄能占的比例较小,分别为5.7%～10.7%和2.9%～3.7%;在夏眠临界温度下,呼吸和排泄耗能占摄食能的比例均显著增大(分别为88.3%和13.6%),而生长能所占比例降为负值(-55.3%),刺参表现为负生长;而在夏眠期,刺参的摄食能和排粪能为零,为维持其基本生理活动,不得不动用以往贮存于体内的能量,消耗于呼吸和排泄等生理过程,供维持生命之用。总之,从能量生物学的角度看,夏眠的主要生态学意义在于刺参长时间处于相对高温环境,进而导致摄食受阻条件下的一种能量节约方式。     

Post-autotomy regeneration of respiratory trees in the holothurian Apostichopus japonicus (Holothuroidea, Aspidochirotida)

Specialised respiratory organs, viz. the respiratory trees attached to the dorsal part of the cloaca, are present in most holothurians. These organs evolved within the class Holothuroidea and are absent in other echinoderms. Some holothurian species can regenerate their respiratory trees but others lack this ability. Respiratory trees therefore provide a model for investigating the origin and evolution of repair mechanisms in animals. We conducted a detailed morphological study of the regeneration of respiratory trees after their evisceration in the holothurian Apostichopus japonicus. Regeneration of the respiratory trees occurred rapidly and, on the 15th day after evisceration, their length reached 15–20 mm. Repair involved cells of the coelomic and luminal epithelia of the cloaca. Peritoneocytes and myoepithelial cells behaved differently during regeneration: the peritoneocytes kept their intercellular junctions and migrated as a united layer, whereas groups of myoepithelial cells disaggregated and migrated as individual cells. Although myoepithelial cells did not divide during regeneration, the peritoneocytes proliferated actively. The contractile system of the respiratory trees was assumed to develop during regeneration by the migration of myoepithelial cells from the coelomic epithelium of the cloaca. The luminal epithelium of the respiratory trees formed as a result of dedifferentiation, migration and transformation of cells of the cloaca lining. The mode of regeneration of holothurian respiratory trees is discussed. This work was funded by a grant from the Russian Foundation for Basic Research (project no. 08–04–00284) to I.Y.D. and by a grant from the Far Eastern Branch of the Russian Academy of Sciences and the Russian Foundation for Basic Research (project no. 09–04–98547) to T.T.G.     

The Syndrome of Sea Cucumber (Apostichopus japonicus) Infected by Virus and Bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added,healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.     

The syndrome of sea cucumber (Apostichopus japonicus) infected by virus and bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75–200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%–100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%–80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal. Fundation items: Supported by commonweal program of State Oceanic Administration of China (200705007), Science and Technology Project of Liaoning Province (2004203001) and Science and Technology Project of Dlian (2004 BINS030).     

The Syndrome of Sea Cucumber （Apostichopusjaponicus） Infected by Virus and Bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.     

Structure of the Digestive Tube in the Holothurian Eupentacta fraudatrix (Holothuroidea: Dendrochirota)

In the holothurian Eupentacta fraudatrix,the gut wall exhibits trilaminar organization. It consists of an inner digestive epithelium, a middle layer of connective tissue, and an outer mesothelium (coelomic epithelium). The pharynx, esophagus, and stomach are lined with a cuticular epithelium composed of T-shaped cells. The lining epithelium of the intestine and cloaca lacks a cuticle and consists of columnar vesicular enterocytes. Mucocytes are also encountered in the digestive epithelium. The connective tissue layer is composed of a ground substance, which houses collagen fibers, amoebocytes, morula cells, and fibroblasts. The gut mesothelium is a pseudostratified epithelium, which is dominated by peritoneal and myoepithelial cells and also includes the perikarya and processes of the neurons of the hyponeural plexus and vacuolated cells.     

Development and characterization of 70 novel microsatellite markers for the sea cucumber (Apostichopus japonicus)

The sea cucumber (Apostichopus japonicus) is an important item in Asian cuisine. It is currently produced through aquaculture, especially in China, after being overexploited in the wild in the 1990s. We isolated 70 novel polymorphic microsatellite loci using an enrichment-colony hybridization protocol. All loci were characterized in 48 individuals from a natural population in Rongcheng (Shandong, China) using genomic DNA isolated from muscle tissue. The number of alleles ranged from 2 to 17 (mean 7.0), and the observed and expected heterozygosities varied from 0.0010 to 1.0000 and from 0.2125 to 0.9477, respectively. Thirty-one of the 70 loci exhibited departure from Hardy-Weinberg equilibrium. These microsatellite markers should be useful resources for population genetic studies and for molecular marker-assisted breeding of A. japonicus.     

Reproductive cycle of the sea cucumber Holothuria (Platyperona) sanctori (Holothuroidea: Echinodermata) in the southwestern Mediterranean Sea: interpopulation variability

This study is a first contribution on the reproductive biology of Holothuria (Platyperona) sanctori from the Algerian coastline in the southwestern Mediterranean. Sampling was conducted at two sites in central Algeria, one (Ain Taggourait, w. Tipasa) where there is little anthropogenic influence and another (Tamentefoust, w. Alger) where there are two major sources of pollution from Oued el Harrach and Oued el Hamiz effluents which contain particulate organic matter. Significant differences in sea cucumber reproduction were observed between the two sites. This could be due to the difference in organic matter noted in the sediment of the two study sites. However, temporal similarities of the gonad index (GI) and the sexual maturity stages suggest that the reproductive cycle of H. (P.) sanctori is annual with the main spawning event taking place from June to October. Spawning is of high intensity in the Bay of Bou Ismail (Ain Taggourait) and spread out over time in the Bay of Algiers (Tamentefoust), reflecting an environment sufficiently rich in food to insure reproduction. We also show that gonad maturation of males and females was synchronized. Temperature is the most likely factor influencing the reproductive cycle and spawning. At the study sites, H. (P.) sanctori had a winter sexual resting phase, followed by maturation during spring, before spawning in summer. However, there was a slight lag to the start of spawning at the two study sites.