Epidermal ultrastructure of Anoplodium stichopi (Plathelminthes,Dalyellioida), a flatworm endosymbiotic in Stichopus tremulus (Holothurioida)

Summary The epidermal ultrastructure of Anoplodium stichopi Bock 1925 (Platyhelminthes, Dalyellioida, Umagillidae) was studied using transmission and scanning electron microscopy. The species lives in the perivisceral coelom of the aspidochirote holothurian Stichopus tremulus Gunnerus 1767. Two types of cells were observed in the epidermis of A. stichopi: ciliated cuboidal epithelial cells and nonciliated pear-shaped cells. The surface of the ciliated epidermal cells is folded into anastomosing ridges. Numerous coated vesicles are subjacent to the surface folds and mitochondria are abundant just below them. Observations indicate that A. stichopi takes up nutrients pinocytically from the coelomic fluid of the host. The ciliation of A. stichopi is sparse.     

海参微卫星DNA的多态性

微卫星DNA(microsatellite DNA)广泛存在于真核生物的基因组中。由于其具有突变频率快、多态性丰富、呈共显性遗传、通用性等特点,已成为近年来被广泛应用的分子遗传标记。本研究对10个海参微卫星DNA进行了克隆与测序。结果表明:90%的微卫星DNA序列存在长度多态性,这为进一步研究海参的分子标记辅助育种奠定了基础。     

Ultrastructure of the Statocysts in the Apodous Sea Cucumber Leptosynapta inhaerens (Holothuroidea, Echinodermata)

The burrowing sea cucumber Leptosynapta inhaerens possesses five pairs of statocysts, one pair on either side of each radial nerve cord where it arises from the circumoral nerve ring. The nerve cords exhibit only ectoneural components at the level of the statocysts. A sinus-like epineural canal lies superjacent to each cord. This canal is lined by a robust monociliated neuroepithelium which lacks any special support cells. Beneath the neuroepithelium, the somata of the ectoneural neurons form a perikaryal layer whereas the axons are located within the proximal parts of the cords. Glial cells have not been found. Each statocyst is a hollow sense organ. Its central cavity is lined by a monolayer of monociliated parietal cells. Axons of these parietal cells extend towards the statocyst nerve which connects each statocyst with the ectoneural pathways of the cord. A single lithocyte floats within each central statocyst cavity. This unciliated cell contains a voluminous vacuole with the statolith and several smaller vacuoles. It is concluded that statocysts do not belong to the basic organization of the Holothuroidea but have been evolved within this group. The statement, that the statocysts of apodous sea cucumbers and that of the enigmatic Xenoturbella bocki are homologous organs, is rejected.     

Sea cucumbers of the genus Stichopus Brandt, 1835 (Holothuroidea,Stichopodidae) in Straits of Malacca with description of a new species

Five sea cucumber species including one new species of the genus Stichopus are reported from the shallow coral reefs of Straits of Malacca. The new species Stichopus fusiformiossa has unusual fusiform spicules in the tentacles, which are not found in the other species of the genus. Pseudo-tables and large perforated plates are newly recorded for Stichopus hermanni Semper, 1868 and Stichopus vastus Sluiter, 1887, respectively.     

海参i型溶菌酶基因及其编码产物的结构特点

通过RT-PCR 和 RACE PCR技术,从海参(Stichopus japonicus)体壁中克隆得到一种溶菌酶基因(GenBank：EF036468).生物信息软件分析表明,其中全长cDNA为 713 bp,5′非编码区（UTR）246 bp,3′UTR 29 bp,开放阅读框438 bp,编码145个氨基酸,包括溶菌酶成熟肽124个氨基酸和信号肽21个氨基酸.对海参溶菌酶与多种无脊椎动物的c、g和i型溶菌酶进行分析比较,发现它与i型溶菌酶有较高的同源性,并具有i型溶菌酶高度保守的2个活性位点,即Glu34和Ser50.活性位点附近具有i型溶菌酶的一段特有的氨基酸保守序列MDVGSLSCG(P/Y)(Y/F)QIK,所以推断克隆的海参溶菌酶为i型.另外,通过搜索蛋白保守结构域数据库,发现海参溶菌酶与医用水蛭失稳酶相似性最高,并且这2个酶的三级结构模型也极其相似.因此推测,海参i型溶菌酶具有双功能特性,既能作用于细菌细胞壁的糖苷键使细胞裂解,又具有失稳酶的一些生化功能,能够水解纤维蛋白,这些特点在海参自溶过程中发挥重要的作用.     

Isolation and Characterization of Twenty Microsatellite Loci in Japanese Sea Cucumber (Stichopus japonicus)

Twenty microsatellite markers were first developed from the Japanese sea cucumber Stichopus japonicus using an enrichment protocol. Of the 20 microsatellite loci, 19 loci were polymorphic in the population examined. At these polymorphic loci, the number of alleles per locus varied from 2 to 15, and the observed heterozygosities ranged from 0.03 to 0.97, which is considerably higher than those previously found for allozymes. The high variability of the microsatellite markers identified in this study will make them excellent tools for genetic analyses of S. japonicus.     

Microsatellite Analysis of Japanese Sea Cucumber, Stichopus (Apostichopus) japonicus, Supports Reproductive Isolation in Color Variants

The genetic relationship among the three color variants (Red, Green, and Black) of the Japanese sea cucumber, S. japonicus, was investigated using 11 microsatellite markers. Genetic differentiation testing among the three sympatric color types showed the strong heterogeneity of Red (p < 0.001), while no significant difference was observed between Green and Black (p = 0.301 to 0.961). UPGMA trees constructed from 10 sample lots from 5 localities showed two distinct clusters, one from the Red types and the other from the Green and Black types. In addition, the sympatric Green and Black formed one subcluster with strong bootstrap support at each locality. These results indicate the separate species status of Red and the other color types, and also support the population identity of sympatric Green and Black.     

Purification and Characterization of a Cathepsin L-Like Enzyme from the Body Wall of the Sea Cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS–PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine

7-amido-4-methylcoumarin with K _m (69.92 μM) and k _cat (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 °C. It showed thermal stability below 40 °C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

Identification of Hox Gene Sequences in the Sea Cucumber Holothuria glaberrima Selenka (Holothuroidea: Echinodermata)

The Echinodermata is a unique animal group forming an early branch in the deuterostomes phylogenetic tree. In echinoids and asteroids a single Hox cluster with nine cognates of the vertebrate Hox paralogous groups has been reported, but no data are available from other echinoderm classes. We report here nine Hox-type sequences from the sea cucumber Holothuria glaberrima, a member of the class Holothuroidea. Partial homeodomain sequences were amplified by polymerase chain reaction from genomic DNA and from a regenerating gastrointestinal tract complementary DNA library. Sequence analyses suggest that the holothuroid cluster has at least three genes of the anterior, one of the medial, and five of the posterior groups. This is the first evidence of five posterior sequences in echinoderms. Received July 19, 1999; accepted October 18, 1999.     

受精前后烟草卵细胞内玉米素和三类酸性植物激素分布的免疫金电镜观察

用胶体免疫金电镜技术观察了受精前后烟草（Nicotianatabacumvarmacrophylla）卵细胞内玉米素（t－Z）、GA7与GA4、（＋）ABA与IAA分布的变化。受精前卵细胞内有大量t－Z,主要位于细胞核、内质网与线粒体上;与卵细胞相邻的助细胞合点端及中央细胞珠孔端亦有较多t-Z。受精后,合子与宿存助细胞t－z显著减少,正加厚的合子细胞壁中有t－Z分布。未受精卵细胞内GA7与GA4及（＋）ABA减少,IAA更少。合子内GA7与GA4及（＋）ABA略有增加,IAA仍然很少。初步讨论了上述植物激素与受精作用的关系。     

Asexual reproduction, evisceration, and regeneration in holothurians (Holothuroidea) from Nha Trang Bay of the South China Sea

Peculiarities of asexual reproduction, evisceration, and regeneration were studied in 13 holothurian species of the orders Dendrochirotida and Aspidochirotida from Nha Trang Bay of the South China Sea. Asexual reproduction by fission has been described for the first time for Cladolabes schmeltzii. It has been shown for the first time that transected C. schmeltzii and Colochirus robustus successfully regenerate not only their anterior, but also their posterior structures. It was found that Pseudocolochirus violaceus was able to regenerate the anterior part of the body and to eject viscera through the anus. The modes of evisceration and regeneration of the gut have been elucidated for ten species of aspidochirotids. It has been shown that the intestine of Holothuria scabra forms from two anlagen rather than from a single one, as previously believed.     

Eelgrass Detritus as a Food Source for the Sea Cucumber Apostichopus japonicus Selenka (Echinidermata: Holothuroidea) in Coastal Waters of North China: An Experimental Study in Flow-Through Systems

Eelgrass ecosystems have a wide variety of ecological functions in which living tissues and detritus may be a food source for many marine animals. In this study, we conducted a laboratory simulating experiment to understand the trophic relationship between the eelgrass Zostera marina L and the sea cucumber Apostichopus japonicus. A mixture of decaying eelgrass debris and seafloor surface muddy sediments was used as food to feed A. japonicus, and then specific growth rates (SGR) and fecal production rates (FPR) were measured. According to the proportion of eelgrass debris, we designed five treatment diets, i.e., ES0, ES10, ES20, ES40, and ES100, with eelgrass debris accounting for 0%, 10%, 20%, 40%, and 100% in dry weight, respectively. Results showed that diet composition had a great influence on the growth of A. japonicus. Sea cucumbers could use decaying eelgrass debris as their food source; and when the organic content of a mixture of eelgrass debris and sediment was 19.6% (ES40), a relatively high SGR (1.54%·d⁻¹) and FPR (1.31 g·ind.⁻¹ d⁻¹) of A. japonicus were obtained. It is suggested that eelgrass beds can not only provide habitat for the sea cucumber A. japonicus but can also provide an indirect food source for the deposit feeder. This means that the restoration and reconstruction of eelgrass beds, especially in coastal waters of China, would be a potential and effective measure for sea-cucumber fisheries, in respect to both resource restoration and aquaculture of this valuable species.     

pH-Dependent Conformational Transitions in Conalbumin (Ovotransferrin), a Metalloproteinase from Hen Egg White

Acid unfolding pathway of conalbumin (CA), a monomeric glycoprotein from hen egg white, has been investigated using far- and near-UV CD spectroscopy, intrinsic fluorescence emission, extrinsic fluorescence probe 1-anilino-8-napthalene sulfonate (ANS) and dynamic light scattering (DLS). We observe pH-dependent changes in secondary and tertiary structure of CA. It has native-like α-helical secondary structure at pH 4.0 but loss structure at pH 3.0. The CA existed exclusively as a pre-molten globule state and molten globule state in solution at pH 4.0 and pH 3.0, respectively. The effect of pH on the conformation and thermostability of CA points toward its heat resistance at neutral pH. DLS results show that MG state existed as compact form in aqueous solutions with hydrodynamic radii of 4.7 nm. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states.     

Characterization of the Adhesive from Cuvierian Tubules of the Sea Cucumber <Emphasis Type="Italic">Holothuria forskali</Emphasis> (Echinodermata,Holothuroidea)

Abstract Sea cucumbers possess a peculiar specialized defense system: the so-called Cuvierian tubules. The system is mobilized when the animal is mechanically stimulated, resulting in the discharge of a few white filaments, the tubules. Their great adhesivity, combined with their high tensile strength, allows Cuvierian tubules to entangle and immobilize potential predators. The cellular origin and composition of the Cuvierian tubule adhesive were investigated in the species Holothuria forskali by studying prints left on the substratum after mechanical detachment of the tubule. Polyclonal antibodies raised against tubule print material were used to locate the origin of tubule print constituents in the tubules. Extensive immunoreactivity was detected in the secretory granules of mesothelial granular cells, suggesting that their secretions make up the bulk of the adhesive material. Tubule print material consists of 60% proteins and 40% carbohydrates, a composition that is unique among the adhesive secretions of marine invertebrates. Although it is highly insoluble, a small fraction of this material can be extracted using denaturing buffers. Electrophoretic analysis of the extracts revealed that it contains about 10 proteins with apparent molecular masses ranging from 17 to 220 kDa and with closely related amino acid compositions, rich in acidic and in small side-chain amino acids. The adhesive from the Cuvierian tubules of H. forskali shares these characteristics with many marine bioadhesives and structural biomaterials.     

Electron Microscopy of the Centrifuged Sea Urchin Egg, with a Note on the Structure of the Ground Cytoplasm

Centrifuged, unfertilized eggs of the sea urchin, Arbacia punctulata, have been studied with the electron microscope. Subcellular particles were stratified by centrifuging living cells, known to be normally fertilizable, for five minutes at 3,000 g. The layered subcellular particles, including cortical granules, 16 mµ RNP particles, pigment, yolk, mitochondria, and oil droplets, possess characteristic ultrastructural features by which they may be identified in situ. The clear zone contains 16 mµ particles, most of them freely dispersed, scattered mitochondria, and a few composite structures made up of annulate lamellae in parallel layers or in association with dense, spherical aggregates of the RNP particles. Free 16 mµ particles are found, in addition, throughout the cell, in the interstices between the stratified larger particles. They show a tendency to form ramifying aggregates resulting from certain types of injury to the cell. A few vesicular structures, found mainly in the clear zone, have attached RNP particles, and appear to be related to the ER of tissue cells. Other vesicles, bounded by smooth membranes, are found throughout the cell. These are extremely variable in size, number, and distribution; their total number appears to depend upon conditions of fixation. It is suggested that limited formation of such structures is a normal property of the ground cytoplasm in this cell, but that fixed cells with very large numbers of smooth surfaced vesicles have produced the latter as a response to chemical injury. A model of the ground cytoplasm is proposed whose aim is to reconcile the rheological behavior of the living cell with the ultrastructural features observed.     

Spermatozoon Ultrastructure of Two Holothurian Species of the Genus Cucumaria (Dendrochirotida,Holothuroidea) from the Sea of Japan

The ultrastructure of spermatozoa of Cucumaria japonica and a congeneric morphologically similar deep-sea species was studied. The spermatozoa of both C. japonica and C. conicospermium are similar to those of other holothurians: the acrosome is composed of an acrosomal granule and periacrosomal material; the centrioles lie at an acute angle to one another; and the proximal centriole is connected to the nuclear envelope by a flagellar rootlet. The spermatozoa of C. japonica differ from those of C. conicospermium in the shape of the head and the dimensions and position of the acrosome. In C. japonica, the acrosome is completely embedded in the nuclear fossa and measures 0.7 m. In C. conicospermium, only one-third of the acrosome is embedded in the nuclear fossa; this acrosome measures 1.3 m. A correlation between the structure of the sperm acrosome and that of the egg envelope is discussed.     

An Electron Microscopic Study of Spermatogenesis in Marenzelleria viridis (Verrill, 1873) (Polychaeta; Spionidae)

Abstract Spermatogenesis in Marenzelleria viridis was studied by ultrastructural investigation. The testes are formed on the greatly ramified nephridial blood vessel and are enveloped by a thin layer of peritoneal cells. The spermatogonia vary in shape, are about 10 μm in diameter and are not linked by intercellular bridges. Pairs or tetrads of spermatocytes connected by intercellular bridges float freely in the coelomic cavity. A complex acrosome is produced by a Golgi complex. The acrosome consists of four to five different structures, forms cisternae and, in the mature spermatozoon, lies deep in an invagination of the nucleus. Two centrioles are also situated in a deep centriolar fossa, the proximal centriole being perpendicular to the distal one. The mature spermatozoon is an ect-aquasperm measuring about 5 μm in length and 2.5 μm in width. The midpiece consists of five spherical mitochondria arranged around the axoneme behind the nucleus. The axoneme is connected to the plasma membrane by a satellite complex. The microtubules of the flagellum are arranged in a typical 9 × 2 + 2 configuration. The spermatogenesis and the sperm morphology of M. viridis were compared with those of other members of the family Spionidae. Copyright © 1996 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

Light and Electron Microscopic Observations on the Intraerythrocytic Development of Babesiosoma stableri (Apicomplexa,Dactylosomatidae) in Frogs from Algonquin Park,Ontario1

ABSTRACT. The intraerythrocytic development and ultrastructure of Babesiosoma stableri Schmittner & McGhee, 1961 are described from Rana catesbeiana and Rana septentrionalis from Algonquin Park, Ontario. Morphometric and chronological observations on B. stableri in an experimentally infected Rana pipiens support the hypothesis that two successive types of merogonic cycles occur within the erythrocytes of infected frogs; the first cycle gives rise to the second and the second cycle produces merozoites destined to become gamonts. Merozoites, meronts, and gamonts are described by light and electron microscopy. Merozoites are typically coccidian and have a trilaminate pellicle with micropores, approximately 40 sub-pellicular microtubules, an apical and posterior polar ring, a conoid with two accessory rings and a pair of intra-conoidal microtubules, three rhoptries and numerous micronemes, and a nucleus with a nucleolus and a paranuclear Golgi body. The gamonts are larger than merogonic stages and are isogamous. They have approximately 55 sub-pellicular microtubules and large stores of amylopectin. These observations indicate that the genus Babesiosoma should be transferred from the Family Haemohormidiidae (Piroplasmida, Piroplasmia) to the Family Dactylosomatidae (Eucoccidiida, Coccidia).     

The Light and Electron Microscopic Cytology of Janacekia adipophila N. Sp. (Microspora, Tuzetiidae), a Microsporidian Parasite of Ptychoptera paludosa (Diptera, Ptychopteridae)

ABSTRACT. The microsporidium Janacekia adipophila n. sp., a parasite of Ptychoptera paludosa larvae in Sweden, is described based on light microscopic and ultrastructural characteristics. Merogonial stages and sporonts are diplokaryotic. Merozoites are formed by rosette-like division. Sporonts develop into sporogonial plasmodia with isolated nuclei. These plasmodia give rise to 8–16 sporoblasts by rosette-like budding. A sporophorous vesicle is initiated by the sporogonial plasmodium. Sporoblasts and spores are enclosed in individual sporophorous vesicles. Granular inclusions of the vesicles, visible using light microscopy, discriminate sporogonial stages from stages of the merogony. The monokaryotic, fresh spores are oval with blunt ends, measuring 4.2-6.3 × 9.1-11.2 μm. Macrospores are formed in small numbers. The spore wall has three subdivisions and the exospore is electron-dense. The polaroplast has two parts: closely arranged lamellae anteriorly, wider sac-like compartments posteriorly. The isofilar polar filament, 191–264 nm wide, has 12-13 coils, which are arranged in one layer in the posterior half of the spore. The electron-dense inclusions of the sporophorous vesicle are modified during sporogony, and vesicles with mature spores are traversed by 21–27 nm wide tubules, which connect the exospore with the envelope of the vesicle. The walls of the tubules, the envelope of the vesicles, and the surface layer of the exospore are all identical double-layered structures. The microsporidium is compared to microsporidia of Ptychopteridae and Tipulidae and to related microsporidia of the family Tuzetiidae.     

Electron Microscopic,Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5–6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.