Morphological and ultrastructural characterization of the coelomocytes in Apostichopus japonicus

The coelomocytes suspended in the coelomic fluid and occurring in the coelomic epithelial layer of the sea cucumber Apostichopus japonicus (Selenka) (Holothuroidea: Aspidochirota: Stichopodidae) function as mediators of the immune system, trephocytic cells and nutrient transport cells. Types of coelomocytes are characterized based on their morphological and ultrastructural features. Flow cytometry plus light and electron microscopic analyses were conducted in order to characterize the coelomocytes of A. japonicus. Six types of coelomocytes were identified: lymphocytes, morula cells, amoebocytes, crystal cells, fusiform cells and vibratile cells. Within these major categories, several distinctive cell types occurred that might represent developmental stages. The mean +/- SD coelomocyte concentration in the individuals (body length: 10 to 15 cm; weight: 100 to 150 g) was (3.79 +/- 0.65) X 10(6) cells ml(-1). The coelomic fluid contained mainly hyalinocytes (76.69%) and granulocytes (23.31 %).

CLONING AND EXPRESSION ANALYSIS OF ALLOGRAFT INFLAMMATORY FACTOR TYPE 1 IN COELOMOCYTES OF ANTARCTIC SEA URCHIN (STERECHINUS NEUMAYERI)

We have cloned and characterized for the first time an allograft inflammatory factor 1 (Sn-AIF-1) from the Antarctic sea urchin. We report the cloning of Sn-AIF-1 cDNA and the characterization of its expression in coelomocytes after a bacterial challenge. The cDNA Sn-AIF-1 has a size of 608 bp and encodes a polypeptide of 151 aa. The deduced amino acid sequence has a putative size of 17.430 Da, an isoelectric point of 4.92, and shows 2 elongation factor handlike motifs that normally bind calcium ions. BLAST analysis revealed close matches with other known AIF-1. The deduced amino acid sequence of Sn-AIF-1 showed high homology with AIF-1 in vertebrates such as fish, mice, and humans; and in the case of invertebrates, the major degree of identity (55%) was with a predicted sequence of the purple sea urchin AIF-1, and 52% corresponded to a sponge. Expression of Sn-AIF-1 mRNA was analyzed by qPCR. Sn-AIF-1 mRNA expression was measured from coelomocytes after a bacterial challenge using RT-PCR and revealed that the gene was upregulated after 24 h. Sn-AIF-1 could participate in the inflammatory response, particularly in the activation of coelomocytes and their survival.

Two Components of Actin-based Retrograde Flow in Sea Urchin Coelomocytes

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase–specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a “push–pull” mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin

Coelomocytes, the heterogeneous population of sea urchin putative immune cells, were found to express a complex set of transcripts featuring scavenger receptor cysteine-rich (SRCR) repeats. SRCR domains define a metazoan superfamily of proteins, many of which are implicated in development and regulation of the immune system of vertebrates. Coelomocytes transcribe multiple SRCR genes from among a multigene family encoding an estimated number of 1,200 SRCR domains in specific patterns particular to each individual. Transcription levels for given SRCR genes may range from pronounced to undetectable, yet all tested animals harbor the genomic loci encoding these genes. Analysis of several SRCR genes revealed multiple loci corresponding to each type. In the case of one SRCR type, a cluster of at least three genes was detected within a 133-kb bacterial artificial chromosome insert, and conserved as well as unique regions were identified in sequences of three genomic clones derived from a single animal. Array hybridizations with repeated samples of coelomocyte messages revealed substantial alterations in levels of expression of many SRCR genes, with fluctuations of up to 10-fold in 1 week and up to 30-fold over a period of 3 months. This report is the first demonstration of genomic and transcriptional complexity in molecules expressed by invertebrate coelomocytes. The mechanisms controlling SRCR gene expression and the functional significance of this dynamic system await elucidation.

一种发光蚯蚓——磷微蠕蚓(Microscolex phosphoreus)体腔细胞的超微结构

下载PDF阅读器应用光学显微镜、扫描电镜和透射电镜对磷微蠕蚓(Microscolex phosphoreus)的体腔细胞进行观察,基于各种细胞的形态特征、胞质内细胞器和颗粒组成、伪足形状及行为特征等将其归纳为三类:黄色细胞、阿米巴样细胞、粒细胞.从外现土看,黄色细胞由几个个体较大的黄色细胞体组成,其细胞质内缺少细胞器;阿米巴样细胞呈明显球形或椭圆形的花朵状,经常可见几个至十几个细胞形成聚合体,胞质内细胞器丰富;粒细胞胞质的颗粒是其主要特征,其星状伪足常处于细胞一端,细胞质内细胞器含量比较丰富.本文将磷微蠕蚓的三类细胞与其它种蚯蚓中相关体腔细胞的形态和超微结构进行了异同比较.

Fridericia liangi sp nov (Enchytraeidae, Oligochaeta) from Changbaishan Mountain, China

We describe one new enchytraeid species, Fridericia liangi sp. nov., from Mt. Changbaishan, Jilin Province, northeastern China. It was collected from soils at the foot of Changbaishan Mountain and is distinguished from all known congeners by the following combination of characters: 1) no lateral chaetae, only ventral chaetae throughout, 2) a maximum of four chaetae in ventral preclitellar bundles, 3) one chaeta in ventral postclitellar bundles, 4) dorsal pores from VII on, 5) esophageal appendages unbranched, 6) coelomocytes without refractile vesicles, 7) clitellum girdle shaped, well developed, 8) no subneural glands, and 9) spermathecae simple.

Fridericia dianchiensis sp nov (Enchytraeidae, oligochaeta) from Yunnan Province, China

Fridericia dianchiensis, a new enchytraeid species collected from Yunnan Province, is described here. It is characterized by a combination of the following characters: 1) lateral bundles containing maximum 3 chaetae; 2) esophageal appendages with 3-4 simple, terminal branches; 3) dorsal vessel originating in XX-XXIII; 4) sub-neural glands absent; 5) seminal vesicle large, occupying two segments; 6)clitellum girdle-shaped or gland cells absent between bursal slits and pre-middle ventrally; 7) coelomocytes without refractile vesicles, 8) spermatheca without diverticula and both ampullae broadly united; and 9) long spermathecal ectal duct without gland at the orifice.

The diverse Grania fauna (Clitellata : Enchytraeidae) of the Esperance area, Western Australia, with descriptions of two new species

Seven species of the marine enchytraeid genus Grania Southern, 1913 are described from sediments sampled during the 2003 International Workshop on the Marine Flora and Fauna of Esperance Bay and the Recherche Archipelago, on the southern coast of Western Australia. Two species are new to science, the euryhaline Tasmanian G. dolichura Rota and Erseus, 2000 represents a new record for the state, and the remaining four species were known from other parts of Western Australia. Grania quaerens sp. n. is recognized by having a high chaetal index (= 5 short chaetal foot), small coelomocytes, penial apparati with long whip-like terminal stylets, conspicuous spermathecae with ectally bulbous ducts, and ectally granulated ampullae housing sperm rings in their ental region. Grania sperantia sp. n. is readily distinguishable by the complete lack of lateral chaetae, a multiple-banded pattern of the clitellum, extremely long sperm funnels, and the intrasegmental location of the spermathecal pores. The latter new species and four others in the collection (G. bykane Coates, 1990, G. crassiducta Coates, 1990, G. dolichura, and G. ersei Coates, 1990) are remarkable in possessing the head organ, a sensory structure unique to Grania that was not noted previously in Western Australian species. When considering the whole genus, the geographic pattern of the head organ appears southern-centred: of the 17 species of Grania reported to possess it, as many as 13 inhabit the southern latitudes. The seventh species of the Esperance collection, G. vacivasa Coates and Stacey, 1993, is notable for the kind of items found in its gut and the unusual appearance of its pygidium.

Bryodrilus fuscistriatus, a new enchytraeid species (Oligochaeta : Enchytraeidae) from northeastern China

Bryodrilus fuscistriatus, a new enchytraeid species from Mt. Changbaishan, Jilin Province, north-eastern China, is described. It is characterized by brown epidermal glands, 7 pairs of preclitellar nephridia, poorly-developed clitellar glands, spermatheca with 2 sessile globular diverticula, and a long sperm funnel with a very broad collar. It is similar to the Alaskan B. tunicatus Dozsa-Farkas & Christensen, 2002 in possessing two diverticula in the spermathecal ampulla and the origin of the dorsal vessel, and the Chinese B. longifistulatus and B. macrotheca Xie et al., 2000c in body size, long sperm funnel and undeveloped clitellar glands, but it differs from B. tunicatus by the presence of brown-striped epidermal gland cells in III-V, a poorly-elevated clitellum, the absence of copulatory glands in XIII-XIV, the regular outline of coelomocytes, and 7 pairs of preclitellar nephridia; from B. longifistulatus and B. macrotheca by the shape of spermatheca, the color of epidermal gland cells, the position of the first pair nephridia, and the origin of dorsal vessel.

Four new terrestrial species of Marionina (Clitellata, Enchytraeidae) from China and re-examination of M. hoVbaueri Moller

Enchytraeid surveys were made in China, mainly along the Changjiang (Yangtze) River Basin, during the period 1991-1999. Among the findings, four terrestrial species of Marionina are new to science and well illustrate the taxonomic complexity of the genus as currently defined. Marionina sinica sp. n. is characterized by a specific chaetal distribution, the marionine pattern of the dorsal blood vessel, and elongate, fusiform, spermathecal ectal ducts. Marionina sacculata sp. n. is distinguished by the possession of a pair of pouch-like oesophageal appendages in IV, the lack of lateral chaetae in VII-XI, a marionine pattern of the dorsal blood vessel, and short spermathecal ectal ducts gradually expanding into spherical ampullae. Both M. sinica and M. sacculata have minute bodies (2-3 mm long in vivo) and lack spermathecal accessory glands. The former species shows its closest aYnities with the European M. brendae Rota, 1995, whereas the latter is closest to the German M. hoVbaueri Moller, 1971, for which an amended diagnosis is provided. Marionina seminuda sp. n. has only ventral chaetal bundles, distributed from III onwards and bisetose. It is similar to the Holarctic M. subterranea (Knollner, 1935) in lacking entirely the lateral chaetae and in having the brain incised posteriorly, the dorsal vessel bifurcating behind the pharynx, and coelomocytes containing opaque granules, but diVers from it in having the longest chaetae in preclitellar segments and gland cells distributed all over the spermathecal ectal ducts. Marionina righiana sp. n. is diagnosed by the location of the head pore on the prostomium, the absence of lateral chaetae from VIII ( VII or IX) onwards, the possession of free spermathecae extending backwards through one to four segments, the brain deeply incised posteriorly, the lumbricilline pattern of the dorsal blood vessel, and the opacity of coelomocytes in vivo. Prior to this study, members of the genus so atypical as M. righiana with respect to the position of the head pore and the structure of the spermathecae were known only from South American soils. Until the taxonomy of Marionina has been more thoroughly assessed and revised, the assignment of the four species to this large assemblage should be regarded as tentative.

Fridericia nanningensis, a new terrestrial enchytraeid species (Oligochaeta) from southwestern China

Fridericia nanningensis, a new species from wetland soil of Nanhu Park, Nanning city, the capital of Guangxi Zhuang Autonomous Region in southwest China, is described. It is characterized by 2-4 chaetae per bundle, poorly-developed clitellar glands, slender, unbranched peptonephridia, and spermathecae with 2 ampullar diverticula, a deep constriction in the middle of the ampulla and one large ectal gland. It is closely related to the European species, F. alata Nielsen & Christensen, 1959 and the East European species, F. tubulosa Dozsa-Farkas, 1972 by the shape of peptonephridia and the undeveloped clitellar glands. It differs from F. alata by its shorter body length and fewer chaetae per bundle, its type of coelomocytes (type "c"), its deep constriction in the middle of the spermathecae ampulla and a larger ectal gland, and it differs from F. tubulosa by its pale epidermal glands, its more anterior origin of the dorsal vessel, a deep constriction in the middle of the spermathecae ampulla, shorter ectal duct, and only one ectal gland at the spermathecal orifice.

Immune condition of Apostichopus japonicus during aestivation

Aestivation is an indispensable state in the life history of sea cucumbers, Apostichopus japonicus. The immune characteristics of the coelomic fluid of A. japonicus, were investigated during aestivation. Samples were collected between July and November 2006 from a coastal pond located off the Yellow Sea in Jiaonan, Shandong Province, China. The total coelomocytes counts (TCC), total superoxide dismutase (T-SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (IZM) in the coelomic fluid were measured. The activities of catecholamines, [adrenaline (AD), noradrenaline (NA), and dopamine (DOP)] were estimated. TCC decreased from July to September, indicating weakness of the cellular immune activity at that time. Activities of SOD, CAT, MPO, and LZM changed significantly from July to October. Catecholamines AD and NA in coelomic fluid were significantly higher on August 21 and November 27. There was no significant variation in DOP during the sampling period. Thus, immune characters in coelomic fluid of A. japonicus changed significantly during aestivation. Water temperature was significantly and negatively correlated with TCC, and salinity was significantly and positively correlated with AD and NA. The mechanism of aestivation in A. japonicus is complex and might not be attributed only to environmental changes, such as temperature and salinity, as shown in previous studies. (C) 2008 Elsevier B.V. All rights reserved.

Immunofluorescence detection and localization of B[a]P and TCDD in earthworm tissues

An immunohistochemical method using antibodies against polycyclic aromatic hydrocarbons (PAHs) and dioxins was developed on frozen tissue sections of the earthworm Eisenia andrei exposed to environmentally relevant concentrations of benzo[a]pyrene (B[a]P) (0.1, 10, 50 ppm) and 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) (0.01, 0.1, 2 ppb) in spiked standard soils. The concentrations of B[a]P and TCDD in E. andrei exposed to the same conditions were also measured using analytical chemical procedures. The results demonstrated that tissues of worms exposed to even minimal amount of B[a]P and TCDD reacted positively and specifically to anti-PAHs and -dioxins antibody. Immunofluorescence revealed a much more intense staining for the gut compared to the body wall; moreover, positively immunoreactive amoeboid coelomocytes were also observed, i.e. cells in which we have previously demonstrated the occurrence of genotoxic damage. The double immunolabelling with antibodies against B[a]P/TCDD and the lysosomal enzyme cathepsin D demonstrated the lysosomal accumulation of the organic xenobiotic compounds, in particular in the cells of the chloragogenous tissue as well as in coelomocytes, involved into detoxification and protection of animals against toxic chemicals. The method described is timesaving, not expensive and easily applicable.

Expression pattern of arenicins - the antimicrobial peptides of polychaete Arenicola marina

Immune responses of invertebrate animals are mediated through innate mechanisms, among which production of antimicrobial peptides play an important role. Although evolutionary Polychaetes represent an interesting group closely related to a putative common ancestor of other coelomates, their immune mechanisms still remain scarcely investigated. Previously our group has identified arenicins - new antimicrobial peptides of the lugworm Arenicola marina, since then these peptides were thoroughly characterized in terms of their structure and inhibitory potential. In the present study we addressed the question of the physiological functions of arenicins in the lugworm body. Using molecular and immunocytochemical methods we demonstrated that arencins are expressed in the wide range of the lugworm tissues - coelomocytes, body wall, extravasal tissue and the gut. The expression of arenicins is constitutive and does not depend on stimulation of various infectious stimuli. Most intensively arenicins are produced by mature coelomocytes where they function as killing agents inside the phagolysosome. In the gut and the body wall epithelia arenicins are released from producing cells via secretion as they are found both inside the epithelial cells and in the contents of the cuticle. Collectively our study showed that arenicins are found in different body compartments responsible for providing a first line of defence against infections, which implies their important role as key components of both epithelial and systemic branches of host defence.

Innate immune response in the sea urchin Echinometra lucunter (Echinodermata)

Echinometra lucunter, (Pinda) is a sea urchin encountered in the Brazilian coast and exposed to high and low temperatures related to low and high tides. Despite their great distribution and importance, few studies have been done on the biological function of their coelomocytes. Thus, Echinometra lucunter perivisceral coelomocytes were characterized under optical and transmission electron microscopy. Phagocytic amoebocytes in the perivisceral coelom were labelled by injecting ferritin, and ferritin labelled phagocytic amoebocytes were found in the peristomial connective tissue after injecting India ink into the tissue, indicating the amoebocytes ability to respond to an inflammatory stimulus. Results showed that the phagocytic amoebocytes were the main inflammatory cells found in the innate immune response of E lucunter. While other works have recorded these phenomena in sea urchins found in moderate and constant temperature, this study reports on these same phenomena in a tropical sea urchin under great variation of temperature, thus providing new data to inflammatory studies in invertebrate pathology. (C) 2007 Elsevier Inc. All rights reserved.