Search

Marine animals in the wild are often difficult to access, so they are studied in captivity. However, the implicit assumption that physiological processes of animals in artificial environments are not different from those in the wild has rarely been tested. Here, we investigate the extent to which an animal is impacted by captivity by comparing global gene expression in wild and captive crown‐of‐thorns starfish (COTS). In a preliminary analysis, we compared transcriptomes of three external tissues obtained from multiple wild COTS with a single captive COTS maintained in aquaria for at least 1 week. On average, an astonishingly large 24% of the coding sequences in the genome were differentially expressed. This led us to conduct a replicated experiment to test more comprehensively the impact of captivity on gene expression. Specifically, a comparison of 13 wild with 8 captive COTS coelomocyte transcriptomes revealed significant differences in the expression of 20% of coding sequences. Coelomocyte transcriptomes in captive COTS remain different from those in wild COTS for more than 30 days and show no indication of reverting back to a wild state (i.e. no evidence of acclimation). Genes upregulated in captivity include those involved in oxidative stress and energy metabolism, whereas genes downregulated are involved in cell signalling. These changes in gene expression indicate that being translocated and maintained in captivity has a marked impact on the physiology and health of these echinoderms. This study suggests that caution should be exercised when extrapolating results from captive aquatic invertebrates to their wild counterparts. [ABSTRACT FROM AUTHOR]

• Pathological changes showed a chlorine substitution-dependent toxicity. • Highly chlorinated PCBs had lower accumulation capacities to earthworms. • Lowly chlorinated PCBs were less toxic to Eisenia fetida. Polychlorinated biphenyls (PCBs) with different chlorine substitution patterns often coexist in e-waste-processing sites. However, the single and combined toxicity of PCBs to soil organisms and the influence of chlorine substitution patterns remain largely unknown. Herein, we evaluated the distinct in vivo toxicity of PCB28 (a trichlorinated PCB), PCB52 (a tetrachlorinated PCB), PCB101 (a pentachlorinated PCB), and their mixture to earthworm Eisenia fetida in soil, and looked into the underlining mechanisms in an in vitro test using coelomocytes. After a 28-days exposure, all PCBs (up to 10 mg/kg) were not fatal to earthworms, but could induce intestinal histopathological changes and microbial community alterations in the drilosphere system, along with a significant weight loss. Notably, pentachlorinated PCBs with a low bioaccumulation ability showed greater inhibitory effects on the growth of earthworm than lowly chlorinated PCBs, suggesting that bioaccumulation was not the main determinant of chlorine substitution-dependent toxicity. Furthermore, in vitro assays showed that the highly chlorinated PCBs induced a high-percentage apoptosis of eleocytes in the coelomocytes and significantly activated antioxidant enzymes, indicating that the distinct cellular vulnerability to lowly/highly chlorinated PCBs was the main contributor to the PCBs toxicity. These findings emphasize the specific advantage of using earthworms in the control of lowly chlorinated PCBs in soil due to their high tolerance and accumulation ability. [Display omitted] [ABSTRACT FROM AUTHOR]

A representative echinoderm, the sea cucumber Apostichopus japonicus has a special regeneration mechanism. The sea cucumber has no specific immune tissues or organs. Coelomocytes of sea cucumbers are involved in their cellular and humoral immunity. In this study, expression profiles of the main immune-related factors in sea cucumber coelomocytes were detected during coelomocyte regeneration after evisceration in A. japonicus. Immune-related factors Cu/Zn superoxide dismutase (Cu/Zn SOD), Catalase (CAT), C-type lysozyme (C-LYZ), Cathepsin D (CTSD), Melanotransferrin (MTF), Toll-like receptor (TLR), C-type lectin (C-LCT), Complement 3 (C3), Myeloid differentiation factor 88 (MYD88), Nuclear factor kappa-B (NF-κB), NF-κB inhibitor (IKB), TNF receptor-associated factor 6 (TRAF6), Peroxiredoxins (PRX), Nitric oxide synthase(NOS), Caspase-2 (CASP-2), Phenoloxidase (PO), and Glutathione Peroxidase(GPX), Heat shock protein 70 (HSP70) were detected by real-time fluorescent quantitative PCR at different time points during regeneration. The main immune-related genes in sea cucumber coelomocytes were significantly differentially expressed after evisceration, and an upregulation was observed for the majority of the considered genes. In summary, the discharge of viscera had a significant effect on expression of immune-related genes of sea cucumber coelomocytes. The expression level of each gene had a certain correlation with the sea cucumber regeneration process. The results provide reference data for the immune response of coelomocytes during regeneration. [ABSTRACT FROM AUTHOR]

A representative echinoderm, the sea cucumber Apostichopus japonicus has a special regeneration mechanism. The sea cucumber has no specific immune tissues or organs. Coelomocytes of sea cucumbers are involved in their cellular and humoral immunity. In this study, expression profiles of the main immune-related factors in sea cucumber coelomocytes were detected during coelomocyte regeneration after evisceration in A. japonicus. Immune-related factors Cu/Zn superoxide dismutase (Cu/Zn SOD), Catalase (CAT), C-type lysozyme (C-LYZ), Cathepsin D (CTSD), Melanotransferrin (MTF), Toll-like receptor (TLR), C-type lectin (C-LCT), Complement 3 (C3), Myeloid differentiation factor 88 (MYD88), Nuclear factor kappa-B (NF-κB), NF-κB inhibitor (IKB), TNF receptor-associated factor 6 (TRAF6), Peroxiredoxins (PRX), Nitric oxide synthase(NOS), Caspase-2 (CASP-2), Phenoloxidase (PO), and Glutathione Peroxidase(GPX), Heat shock protein 70 (HSP70) were detected by real-time fluorescent quantitative PCR at different time points during regeneration. The main immune-related genes in sea cucumber coelomocytes were significantly differentially expressed after evisceration, and an upregulation was observed for the majority of the considered genes. In summary, the discharge of viscera had a significant effect on expression of immune-related genes of sea cucumber coelomocytes. The expression level of each gene had a certain correlation with the sea cucumber regeneration process. The results provide reference data for the immune response of coelomocytes during regeneration. [ABSTRACT FROM AUTHOR]

To characterize the immune functions of the coelomic fluid of echiuran worms (Urechis unicinctus), their cellular composition and biochemical index were initially analyzed. Meanwhile, the phagocytic activity of coelomocytes and the impact of pathogenic infection immuno-related enzymes were explored. Lastly, coelomocyte density and humoral immune factor levels were detected under pathogenic conditions. The results revealed three major coelomocyte types in the coelomic fluid, comprising granular cells, hyaline cells and thrombocytes. Granulosa cells exhibited ability to adsorb and phagocytose with bacteria, whereas hyaline cells and thrombocytes manifested no adsorption and phagocytosis abilities. Additionally, numerous immune factors (such as albumin, alanine aminotransferase, and alkaline phosphatase) were detected in the coelomic fluid. Following pathogenic stimulation, the coelomocyte density and level of humoral immune factors (SOD, LSZ, ACP, and AKP) were changed. Collectively, the results suggested that humoral immune factors and coelomocytes were sensitive to environmental factors. Taken together, our study indicated that coelomocytes, in conjunction with immune factors in the coelomic fluid, formed an immune barrier in U. unicinctus. • The prime coelomocytes types were granular cells, hyaline cells, and thrombocytes. • Granulosa cells exhibited adsorption and phagocytosis abilities with bacteria. • The humoral immune factors levels were altered after pathogen stimulation. • The coelomocytes and immune factors support the defense system of U. unicinctus. [ABSTRACT FROM AUTHOR]

The immune system of the sea urchin species Paracentrotus lividus is highly complex and, as yet, poorly understood. P. lividus coelomocytes mediate immune response through phagocytosis and encapsulation of non-self particles, in addition to the production of antimicrobial molecules. Despite this understanding, details of exactly how these processes occur and the mechanisms which drive them are still in need of clarification. In this study, we show how the bacterial lipopolysaccharides (LPS) is able to induce a stress response which increases the levels of the heat shock proteins HSP70 and HSP90 only a few hours after treatment. This study also shows that LPS treatment increases the expression of the β-thymosin-derivated protein paracentrin, the precursor of antimicrobial peptides. • Immune response in Paracentrotus lividus after stimulation with LPS. • Echinoderm immune response and HSP90 and HSP70 expression. • β-Thymosin modulation following LPS treatment. [ABSTRACT FROM AUTHOR]

Abstract Sea cucumber Apostichopus japonicus rely on the efficient innate immune mechanisms against invaders, in which the consumption and regeneration of coelomocytes take place at the same time. In the present study, histological features of putative hematopoietic tissues (HPTs) including the rete mirabile, the respiratory tree, the polian vesicle and the coelomic epithelium were characterized. The distribution of transcription factor GATA1 in coelomocytes and putative HPTs was examined by immunohistochemistry. In addition, cell proliferation using EdU labeling and coelomocyte distribution in different tissues using monoclonal antibody labeling were analyzed to further confirm the HPTs. The results showed that two homologs of GATA1 were detected with molecular weight of 43 and 90 kDa in coelomocytes, rete mirabile, respiratory tree and polian vesicle, whereas no signals were detected in the coelomic epithelium. A few cells were detected to be EdU-positive for coelomocytes, which accounted for approximately 9.5%. In the rete mirabile and the respiratory tree, the EdU signals were strong in cells of the tube wall. In the polian vesicle, numerous EdU-positive cells were detected in the cyst wall. In the coelomic epithelium, little EdU signaling was detected. Immunohistochemistry analysis by mAb 3F6 against A. japonicus coelomocytes showed that positive signals were observed in the tube wall of the rete mirabile, respiratory tree, cyst wall of the polian vesicle and in the coelomocyte antrum of coelomic epithelium. These results suggest that the rete mirabile, respiratory tree and polian vesicle are the HPTs of A. japonicus. Highlights • Rete mirabile, respiratory tree, and polian vesicle were suggested as HPTs. • Two homologs of transcription factor GATA1 were detected in the HPTs. • EdU signals were strong in coelomocytes and HPTs. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are Ca 2+ dependent carbohydrate-binding proteins that share structural homology in their carbohydrate-recognition domains (CRDs). In the present study, a novel CTL was identified from sea cucumber Apostichopus japonicus (named as Aj CTL-2). The deduced amino acid sequence of Aj CTL-2 was homologous to CTLs from other animals with the identities ranging from 33% to 40%. It contained a canonical signal peptide at the N-terminus, a low density lipoprotein receptor class A (LDLa), a C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB), and a CRD with two motifs Glu-Pro-Asn (EPN) and Trp-Asn-Asp (WND) in Ca 2+ binding site 2. The mRNA transcripts of Aj CTL-2 were extensively expressed in all the tested tissues including respiratory tree, muscle, gut, coelomocyte, tube-foot, body wall and gonad, and the highest expression level of Aj CTL-2 in coelomocyte was about 4.2-fold ( p  < 0.05) of that in body wall. The mRNA expression level of Aj CTL-2 in coelomocyte increased significantly after Vibrio splendidus stimulation, and dramatically peaked at 12 h, which was 206.4-fold ( p  < 0.05) of that in control group. Aj CTL-2 protein was mainly detected in cytoplasm of coelomocyte by immunofluorescence. The recombinant Aj CTL-2 (r Aj CTL-2) displayed binding activity to d -galactose independent of Ca 2+ , while the binding activity to other tested pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS), peptidoglycan (PGN), and mannose (Man) could not be detected. Surface plasmon resonance (SPR) analysis further revealed the high binding specificity and moderate binding affinity of r Aj CTL-2 to d -galactose (KD = 4.093 × 10 −6  M). After r Aj CTL-2 was blocked by its polyclonal antibody, the binding activity to d -galactose could not be detected by using a blocking ELISA (B-ELISA). Moreover, r Aj CTL-2 could bind various microorganisms including V. splendidus , V. anguillarum , Staphylococcus aureus , Bifidobacterium breve and Yarrowia lipolytica with the strongest binding activity to B. breve . These results collectively suggested that Aj CTL-2 was a member of CTL superfamily (CTLs) with preferential binding of d -galactose and participated in the immune response of sea cucumber. [ABSTRACT FROM AUTHOR]

Sea cucumber, Apostichopus japonicus , is one of the most important holothurian species cultured in China. Severe evisceration induced by various natural and artificial factors commonly occurs during transport and culture of A. japonicus. Evisceration causes higher mortality and lower yield. Along with the visceral regeneration process, sea cucumbers also regenerate coelomocytes in order to recover immune function. In this study, evisceration of A. japonicus was induced by intracoelomic injection of 0.35 M KCl. Regeneration of coelomocytes was investigated by time course cell counting as well as detection of DNA replication by the EdU labeling technique. Coelomic fluid volume was restored to the pre-evisceration level within 2 h after evisceration. Total coelomocyte count (TCC) reached a peak at 6 h post-evisceration, followed decreased and then increased with a slight fluctuation, restored to the pre-evisceration level at 35 d post-evisceration. The change in different subtypes of coelomocytes was consistent with that of total coelomocytes. However, there were some variations in the regeneration of coelomocyte subtypes. At the end of the study, only the counts of amoebocytes and morula cells recovered to the pre-evisceration level. DNA replication assay showed EdU-positive cells accounted for 9.5% before evisceration and 4.7% at 6 h post-evisceration. However, the percentage of EdU-positive cells significantly increased, reaching 18.6% at 3 d after evisceration, then declined. Therefore, we analyzed the observed increase in coelomocytes at 6 h post-evisceration, which may be due to coelomocyte migration from the water-vascular system into the coelom rather than de novo cell proliferation. [ABSTRACT FROM AUTHOR]

Sea urchin is one of marine animals with high economic and great scientific research values. Axial organ is a glandular organ that has been presumed as coelomocytes origin site. In this paper, two monoclonal antibodies (3G10 and 6B3) against coelomocytes of sea urchin Strongylocentrotus intermedius were developed by hybridoma technique. The mAbs were characterized by indirect immunofluorescence assay test (IIFAT), flow cytometry (FCM) and western blot assay. Results showed that mAb 3G10 recognized a protein of a molecular weight of 17 kDa in the spherule cells, while mAb 6B3 reacted with a protein of a molecular weight of 35 kDa in the phagocytes. Furthermore, specificity analysis revealed that the two mAbs could react with the coelomocytes of sea urchin S. nudus and Hemicentrotus pulcherrimus , but not with those of other common echinoderms including sea cucumber Apostichopus japonicus and starfish Asterias rollestoni. To determine whether the coelomocytes exist in the axial organ of sea urchin, the IIFAT assays were carried out based on the two mAbs. Result showed that positive fluorescence signals were distributed in the organ. It was revealed that the axial organ was rich in coelomocytes, which suggests that the organ may play as a producing source or reservoir in the ontogenesis of coelomocytes of sea urchin. [ABSTRACT FROM AUTHOR]

Proper development of a multicellular organism relies on well-coordinated regulation of cell fate specification, cell proliferation and cell differentiation. The C. elegans postembryonic mesoderm provides a useful system for uncovering factors involved in these processes and for further dissecting their regulatory relationships. The single Spalt-like zinc finger containing protein SEM-4/SALL is known to be involved in specifying the proliferative sex myoblast (SM) fate. We have found that SEM-4/SALL is sufficient to promote the SM fate and that it does so in a cell autonomous manner. We further showed that SEM-4/SALL acts through the SoxC transcription factor SEM-2 to promote the SM fate. SEM-2 is known to promote the SM fate by inhibiting the expression of two BWM-specifying transcription factors. In light of recent findings in mammals showing that Sall4, one of the mammalian homologs of SEM-4, contributes to pluripotency regulation by inhibiting differentiation, our work suggests that the function of SEM-4/SALL proteins in regulating pluripotency versus differentiation appears to be evolutionarily conserved. [ABSTRACT FROM AUTHOR]

Lysosomes, the major membrane-bound degradative organelles, have a multitude of functions in eukaryotic cells. Lysosomes are the terminal compartments in the endocytic pathway, though they display highly dynamic behaviors, fusing with each other and with late endosomes in the endocytic pathway, and with the plasma membrane during regulated exocytosis and for wound repair. After fusing with late endosomes, lysosomes are reformed from the resulting hybrid organelles through a process that involves budding of a nascent lysosome, extension of the nascent lysosome from the hybrid organelle, while remaining connected by a membrane bridge, and scission of the membrane bridge to release the newly formed lysosome. The newly formed lysosomes undergo cycles of homotypic fusion and fission reactions to form mature lysosomes. In this study, we used a forward genetic screen in Caenorhabditis elegans to identify six regulators of lysosome biology. We show that these proteins function in different steps of lysosome biology, regulating lysosome formation, lysosome fusion, and lysosome degradation. [ABSTRACT FROM AUTHOR]

Echinoderms have many types of coelomocytes, which have been known to form aggregates immediately after they are removed from the coelom. To assess the roles that each type of coelomocyte plays in aggregate formation, cellular components of coelomocyte aggregates of the Japanese sea cucumber, Apostichopus japonicus , were investigated. The coelomocytes were tentatively classified into 12 types based on May-Grunwald/Giemsa staining. After the coelom was incubated for 30 min or 6 h, the aggregates were disaggregated completely with 200 mM EDTA. Differential counts of the dissociated cells indicated that the largest component of the aggregates was amoebocytes (67.8%) and the second-largest component of the aggregates incubated 30 min was a type of basophilic granulocyte. In the 6h-incubated aggregates, the fraction of amoebocytes decreased to 59.0%, while that of lymphoid cells significantly increased, which suggests that lymphoid cells participate in late-stage aggregation. After 24-h incubation, only a portion of the aggregated cells could be disaggregated with EDTA. After 48 h, most of the cells could not be detached from the aggregates. Microscopy of frozen sections of the aggregates after 6-h incubation revealed that amoebocytes constructed a mesh-like structure to which other types of cells adhered. After 48 h, the borders of the cells and the intracellular granules were not recognizable. In time-lapse microscopy, the aggregates were observed to move on a glass slide, which suggests that aggregates can “crawl” on the intraluminal surface of the coelom toward, for example, injured regions in the body of the sea cucumber. [ABSTRACT FROM AUTHOR]

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains ( Escherichia coli and Bacillus subtilis ) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil. [ABSTRACT FROM AUTHOR]

Three proteins with PO-like activities in the coelomocytes of sea urchin Strongylocentrotus nudus were identified using electrophoretic method and named as SnPO1, SnPO2 and SnPO3 according to their molecular mass from high to low. The SnPOs were characterized for substrate specificity and the effects of temperature, pH, divalent metal ions and inhibitors on PO activities. They showed oxidative activities to L-3,4-dihydroxyphenylalanine. ( l -DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine, which illustrated the three proteins had laccase-like PO activities. The optimum temperature for the activities of SnPO1, SnPO2 and SnPO3 was 75 °C, 70 °C, 40 °C, and the optimum pH was 7.0, 9.0, 8.0, respectively. The SnPOs were notably activated after being incubated in boiled water for 60 min, suggesting that the three proteins are thermophilic. The activity of SnPO1 was greatly enhanced by Cu 2+ , Mn 2+ and Fe 2+ and inhibited by Pb 2+ , Cd 2+ , EDTA, DETC, sodium sulfite and ascorbic acid, but SnPO2 and SnPO3 were not obviously affected by Pb 2+ and Cd 2+ , suggesting the three proteins are copper-containing, and the catalytic properties of SnPO1 might be different from those of SnPO2 and SnPO3. Taken together, SnPO1, SnPO2 and SnPO3 might play different roles in the immune and physiological processes of S. nudus . [ABSTRACT FROM AUTHOR]

The essential mechanism within immune systems is the recognition of pathogens and parasites by the immune system cells, which attach to their targets and destroy them. Glycans are fundamental macromolecular components of all cells, and are important in the vertebrate immunity. But, glycans have been investigated rarely in coelomocytes of echinoids. Hence, the aim of this study is to determine the monosaccharides which form glycan chains on the sea urchin immune system cells, coelomocytes, via analytical and lectin histochemistry methods. The study material is the coelomocytes obtained from adult sea urchin Paracentrotus lividus . In order to analyze the monosaccharides with the Capillary Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (CapLC-ESI-MS/MS) system, the samples underwent hydrolysation, reacetylation and derivatization steps. In order to determine the monosaccharides with the lectin histochemistry, the cells were incubated with fluorescein isothiocyanate (FITC) conjugated PNA, HPA, WGA-suc, WGA, and PSL lectins and then photographed with the fluorescence microscope. As a result of the CapLC-ESI-MS/MS analysis; mannose, ribose, N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, arabinose, xylose and fucose monosaccharides were detected. A peak area calculation analysis revealed the most prevalent saccharides as glucose, galactose and fucose, respectively. Lectin histochemistry came out with higher intensity emission signals obtained from the FITC-conjugated lectin WGA, which is specific to N-acetylglucosamine and sialic acid in comparison to the emission obtained from the sialic acid unspecific WGA-suc lectin. This finding indicates the existence of sialic acid within coelomocytes. Fluorescent emissions from other lectins were detected at lower levels. Determination of the monosaccharides which form glycan chains of the sea urchin coelomocytes and elucidating their similarities among other invertebrate and vertebrate systems is vital in terms of understanding the uncovered complex features of the immune systems of higher vertebrates. [ABSTRACT FROM AUTHOR]

Abstract: The nematode Caenorhabditis elegans continues to serve as a useful model of life extension caused by dietary restriction. Using this model, downstream effectors of dietary restriction-induced longevity have been elucidated, including neuropeptides and cell-surface receptors. Although it remains possible that different forms of dietary restriction may utilize both specific and overlapping mechanisms to promote longevity, the nematode model has revealed roles for autophagy, metabolic energy-sensing and the hypoxic response. The nematode has also been used to identify specific tissues required for life extension via DR, including coelomocytes, intestine, and neurons. [Copyright &y& Elsevier]

Abstract: Anthropogenic CO2 emissions are acidifying the world''s oceans. A growing body of evidence demonstrates that ocean acidification can impact survival, growth, development and physiology of marine invertebrates. However, little is known on the impact of elevated pCO2 on immune-response. Here we investigate the impact of short-term (5–7days) exposure to elevated pCO2 (1275μatm compared to 350μatm in the control) on extracellular pH (pHe) and cellular immune response in two polar echinoderm species, the green sea urchin Strongylocentrotus droebachiensis and the seastar Leptasterias polaris. Both species experienced extracellular acidosis following short term exposure to elevated pCO2. While this acidosis remained uncompensated within 7days for L. polaris, pHe was fully compensated after 5days for S. droebachiensis. For both species, coelomic fluid acidosis was associated with an increase in total coelomocyte number and a reduction in vibratile cells in S. droebachiensis. A relationship between pHe and phagocyte numbers was observed in S. droebachiensis suggesting a direct link between pHe and cellular immune-response. Further studies would require the coordinated effort of ecologists and immunologists to understand the role of elevated pCO2 on the host–pathogen interactions that are involved in the stability of ecosystems. [Copyright &y& Elsevier]

Abstract: One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA), Western blotting and fluorescenceactivated cell sorter (FACS), mAb 3F6 showed specific for spherulocytes of A. japonicus. The mAb 3F6 recognized an antigen of molecular weight 136kDa in Western blotting. Isotype analysis revealed mAb 3F6 as IgG type. The flow cytometry assay confirmed the microscopy observations and showed coelomocytes positive to mAb 3F6. The antigencity of haemocytes or coelomocytes of Hemicentrotus pulcherrimus, Scapharca subcrenata, Asterina pectinifera, Asterias rollestoni, Ruditapes philipinarum, Patinopecten yessoensis and Mytilus edulis was compared and the result showed that none of them was positive with mAb 3F6. Most of cells free in polian vesicle were positive with mAb 3F6. The positive cells are in spherical shape, 5–7μm in diameter, smaller than coelomic spherulocytes of A. japonicus. The result of immunofluorescent staining with cells in hemal vessel showed that there were strong positive signals on cytoplasm of some spherical cells with diameter of 7–8μm. Some other cells with higher nucelo-plasmic ratio, about 5–6μm in diameter showed weak positive signals on membrane. Immunohistochemistry assay revealed that positive signals were mostly observed in the lumen structure of rete mirabile and haemocoel of the respiratory tree. In addition, the outer epidermis of body wall and tentacle also showed positive. [Copyright &y& Elsevier]

Abstract: A full length cDNA sequence expressed in coelomocytes shows significant sequence match to vertebrate Tie1 and Tie2/TEK. Vertebrate Tie2/TEK is the receptor for the angiopoietins and plays an important role in angiogenesis and hematopoiesis, whereas Tie1 regulates the activity of Tie2. The deduced sequence of the SpTie1/2 protein has a similar order and organization of domains to the homologous vertebrate proteins including a highly conserved receptor tyrosine kinase domain in the cytoplasmic tail. The N terminus of the ectodomain has one immunoglobulin (Ig)-Tie2_1 domain, followed by an Ig domain, four epidermal growth factor domains, a second Ig domain, and three fibronectin type III domains. The SpTie1/2 gene is expressed in coelomocytes and the axial organ, whereas other organs do not show significant expression. The timing of embryonic expression corresponds with the differentiation of blastocoelar cells, the embryonic and larval immune cells. Searches of the sea urchin genome show several gene models encoding putative ligands and signaling proteins that might interact with SpTie1/2. We speculate that SpTie1/2 may be involved in the proliferation of sea urchin immune cells in both adults and embryos. [Copyright &y& Elsevier]

Abstract: The Sp185/333 system of genes, messages and proteins are expressed in the coelomocytes of the purple sea urchin, Strongylocentrotus purpuratus, and is an extraordinary example of diversification of a putative innate immune response system in an invertebrate. Reviewed here, is the current understanding of this complex system as illustrated by sequence comparisons of the genes, messages and deduced proteins with descriptions of diversity, including preliminary results on genomic organization and descriptions of 185/333 in other echinoids. Sp185/333 gene expression in adults and embryos occurs in response to immune challenge and includes changes in the frequencies of Sp185/333-positive coelomocytes in the adults. The diversity of the Sp185/333 protein repertoire in coelomocytes is far greater than the sequence diversity encoded in the genes, which may be the result of rapid gene recombination, RNA editing and/or low-fidelity transcription, plus post-translational modifications. This review concludes with preliminary results and speculations on protein function. [Copyright &y& Elsevier]

Abstract: Ultrastructural characteristics of coelomocytes of the sipunculid Phascolosoma esculenta were studied by transmission electron microscopy. There are several cell types in the coelomic fluid, including three kinds of granulocytes, vesicular cells, germ cells, amoebocytes, phagocytes, and erythrocytes; there are also a new cell complex which is composed of podocytes and granular cells. And several other cell types (erythrocyte and different kinds of granulocytes) gathering together was discovered in the coelomic fluid of P. esculenta. Functional interpretations were provided for these cells using morphological evidence. The coelomocytes from different sipunculid genera and Annelida were compared. The structural diversity of coelomocytes provides both taxonomic characteristics for discriminative identification and phylogenetic markers in Phascolosoma and other sipunculid taxa. [Copyright &y& Elsevier]

Regeneration is a biological phenomenon that occurs in a wide range of animals, and is considered to involve different types of cells including those that are considered to be stem cells. Among the echinoderms, which is a phylum with many regenerating members, crinoids (feather stars and sea lilies) are known to possess high potential of regeneration and are able to regenerate most of their organs. In particular, arm regeneration has been studied using the feather star. During regeneration, coelomocytes and amoebocytes originating from the coelomic canal and the brachial nerve, respectively, migrate to the distal wound area and are involved in the regenerative process. A blastema is formed at the regenerating tip and is derived from migratory amoebocytes. On the other hand, migratory coelomocytes contribute to regenerate the coelomic system. Cells proliferate at the blastema, coelomic canals and brachial nerve. Since the migrating cells differentiate into new structures of the arm, they are considered presumably undifferentiated multipotent stem cells. To deepen our understanding of stem cells in general, we may benefit from an approach from a comparative point of view. Further molecular analyses would increase our knowledge of stem cells in crinoids and allow comparative studies to be possible. [ABSTRACT FROM AUTHOR]

Abstract: The subdivision of mesodermal cells into muscle and non-muscle cells is crucial to animal development. In the C. elegans postembryonic mesoderm, this subdivision is a result of an asymmetric cell division that leads to the formation of striated body wall muscles and non-muscle coelomocytes. Here we report that the Six homeodomain protein CEH-34 and its cofactor Eyes Absent, EYA-1, function synergistically to promote the non-muscle fate in cells also competent to form muscles. We further show that the asymmetric expression of ceh-34 and eya-1 is regulated by a combination of 1) mesodermal intrinsic factors MAB-5, HLH-1 and FOZI-1, 2) differential POP-1 (TCF/LEF) transcriptional activity along the anterior–posterior axis, and 3) coelomocyte competence factor(s). These factors are conserved in both vertebrates and invertebrates, suggesting a conserved paradigm for mesoderm development in metazoans. [Copyright &y& Elsevier]

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration. [ABSTRACT FROM AUTHOR]

Abstract: Manganese (Mn) is a naturally abundant metal in marine sediments where it mainly occurs as MnO2. During hypoxic conditions it is converted into a bioavailable state, Mn2+, and can reach levels that previously have shown effects on immune competent cells of the crustacean, Nephrops norvegicus. Here we investigated if Mn also affects circulating coelomocytes and their renewal in the common sea star, Asterias rubens, when exposed to concentrations of Mn that can be found in nature. When the sea stars were exposed to Mn it accumulated in the coelomic fluid and the number of circulating coelomocytes, in contrast to what was recorded in Nephrops, increased significantly. By using the substitute nucleotide, 5-bromo-2′-deoxyuridine, BrdU, for tracing cell division and by recording mitotic index by nuclei staining, we found that Mn induced proliferation of cells from a putative haematopoietic tissue, the coelomic epithelium. In addition, the haematopoietic tissue and coelomocytes showed stress response in terms of changes in HSP70 levels and protein carbonyls, as judged by immunohistochemistry and Western blotting. Measurement of dehydrogenase activity, using MTS/PMS, revealed that Mn showed cytotoxic properties. We also found that the phagocytotic capacity of coelomocytes was significantly inhibited by Mn. It was concluded that the exposure of A. rubens to Mn induced renewal of coelomocytes and impaired their immune response. [Copyright &y& Elsevier]

Summary: Previous studies evidenced that cystatin B-like gene is specifically expressed and induced in large circulating cœlomic cells following bacterial challenge in the leech Theromyzon tessulatum. In order to understand the role of that cysteine proteinase inhibitor during immune response, we investigated the existence of members of cathepsin family. We cloned a cathepsin L-like gene and studied its tissue distribution. Immunohistochemical studies using anti-cathepsin L and anti-cystatin B antibodies and ultrastructural results demonstrated the presence of three distinct cœlomic cell populations: (1) the chloragocytes, which were initially defined as large cœlomocytes, (2) the granular amœbocytes and (3) small cœlomic cells. Among those cells, while chloragocytes contain cystatin B and cathepsin L, granular amœbocytes contain only cathepsin L and the third cell population contains neither cathepsin nor inhibitor. Finally, results evidenced that cathepsin L immunopositive granular amœbocytes are chemoattracted to the site of injury and phagocyte bacteria. [Copyright &y& Elsevier]

Red spherule coelomocytes are immune cells in the sea urchin Lytechinus variegatus that have been characterized as motile O2 transport cells. Video microscopy of living red spherule coelomocytes reveals a constitutive, dynamic array of cellular morphologies and movements. Cells continuously send out and retract membrane blebs all over the cell surface as part of their normal cellular physiology. Disruption of microtubules by perfusion with either nocodazole or taxol had no effect on bleb formation or motility. Perfusion with cytochalasin B abated bleb formation and revealed cells that exhibited multiple small spheres attached by short membrane extensions. Attenuation of blebbing and intracellular organelle motility were restored by washing out with cytochalasin B. Treatment with phalloidin also abated bleb formation and revealed a smooth, spherical cellular morphology. The effects of phalloidin were completely reversible after washout. Red spherule coelomocytes treated with blebbistatin rounded up with an irreversible retraction of blebs into surface blebs that were greatly reduced in size, number and motility. Normal cell surface bleb formation and intracellular organelle motility were not restored after washout of the drug. These results indicate that the acto-myosin contractile mechanism contributes to the dynamics of constitutive cell surface membrane blebbing in invertebrate immune cells. [ABSTRACT FROM AUTHOR]

Sulfide-tolerant marine invertebrates employ a variety of mechanisms to detoxify sulfide once it has entered their bodies, but their integumentary, respiratory epithelium and circulatory cells may still be exposed to toxic sulfide concentrations. To investigate whether sulfide exposure is toxic to mitochondria of a sulfide-tolerant invertebrate, we used the fluorescent dyes JC-1 and TMRM to determine the effect of sulfide exposure on mitochondrial depolarization in erythrocytes from the annelid Glycera dibranchiata. In erythrocytes exposed to 0.11–1.9 mmol l-1 sulfide for 1 h, the dyes showed fluorescence changes consistent with sulfide-induced mitochondrial depolarization. At the highest sulfide concentration, the extent of depolarization was equivalent to that caused by the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Even when induced by as little as 0.3 mmol l-1 sulfide, the depolarization was not reversible over a subsequent 5 h recovery period. The mechanism of toxicity was likely not via inhibition of cytochrome c oxidase (COX), since other COX inhibitors and other mitochondrial electron transport chain inhibitors did not produce similar effects. Furthermore, pharmacological inhibition of the mitochondrial permeability transition pore failed to prevent sulfide-induced depolarization. Finally, increased oxidation of the free radical indicators H2DCFDA and MitoSOX™ in erythrocytes exposed to sulfide suggests that sulfide oxidation increased oxidative stress and superoxide production, respectively. Together, these results indicate that sulfide exposure causes mitochondrial depolarization in cells of a sulfide-tolerant annelid, and that this effect, which differs from the actions of other COX inhibitors, may be via increased free radical damage. [ABSTRACT FROM AUTHOR]

Earthworm leukocytes (coelomocytes) are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinins (e.show $132#g., lectins), lysosomal enzymes (e.show $132#g., acid phosphatase, lysozyme), and various cytotoxic and antimicrobial molecules. There is also evidence of weak adaptive immune responses against foreign transplants. This study focused on aspects of the innate immune response. First, anti-human acid phosphatase (anti-AcP) polyclonal antibody characterized different acid hydrolase patterns in coelomocytes. Second, flow cytometry identified a strongly immunoreactive coelomocyte population. Third, ultrastructural and cytochemical analyses revealed acid phosphatase in discrete granules (lysosomes) of effector hyaline and granular coelomocytes but not in mature chloragocytes. Coelomocytes were exposed to bacteria to assess how phagocytosis influences: (a) the production of acid phosphatase using Western blot, and (b) release of acid phosphatase using ELISA from cell-free coelomic fluid. Fourth, after phagocytosis, acid phosphatase levels differed between controls and experimentals. Fifth, we found a 39-kDa molecule that reacted intensely with anti-AcP. Our results suggest that effector earthworm coelomocytes may not eliminate pathogens only by phagocytosis but also by extracellular lysis. [ABSTRACT FROM AUTHOR]

Titanium dioxide nanoparticles (TiO2 NPs) are manufactured worldwide. Once they arrive in the soil environment, they can endanger living organisms. Hence, monitoring and assessing the effects of these nanoparticles is required. We focus on the Eisenia andrei earthworm immune cells exposed to sublethal concentrations of TiO2 NPs (1, 10, and 100 µg/mL) for 2, 6, and 24 h. TiO2 NPs at all concentrations did not affect cell viability. Further, TiO2 NPs did not cause changes in reactive oxygen species (ROS) production, malondialdehyde (MDA) production, and phagocytic activity. Similarly, they did not elicit DNA damage. Overall, we did not detect any toxic effects of TiO2 NPs at the cellular level. At the gene expression level, slight changes were detected. Metallothionein, fetidin/lysenin, lumbricin and MEK kinase I were upregulated in coelomocytes after exposure to 10 µg/mL TiO2 NPs for 6 h. Antioxidant enzyme expression was similar in exposed and control cells. TiO2 NPs were detected on coelomocyte membranes. However, our results do not show any strong effects of these nanoparticles on coelomocytes at both the cellular and molecular levels. [ABSTRACT FROM AUTHOR]