Immunomodulatory effects of temperature and pH of water in an Indian freshwater sponge

Eunapius carteri, a freshwater sponge of India, inhabits the ponds and lakes and experiences variations of temperature and pH of water throughout the year. Sponges bear evolutionary and ecological importance with limited information on their immunological attribute and adaptational resilience in a changing environment. This paper reports temperature and pH specific responses of immune related parameters in sponge maintained in the experimental conditions of laboratory. Innate immunological parameters like phagocytosis and generation of cytotoxic molecules like superoxide anion, nitric oxide and phenoloxidase activity were estimated in E. carteri at different environmentally realistic water temperatures (10, 20, 30 and 40 °C) and pH (6.4, 7.4 and 8.4). Phagocytosis and cytotoxicity are established as important immune parameters of invertebrates. Calalase, an antioxidant enzyme and phosphatases are involved in pathogen destruction and are considered as components of innate immunity. Activities of catalase, acid and alkaline phosphatases were estimated in E. carteri at different thermal regimes and pH. Modulation of phagocytic and cytotoxic responses and the activities of catalase and phosphatases at different water temperatures and pH indicated temperature and pH specific immunological status of E. carteri. Present investigation deals with the effects of selected hydrological parameters on the fundamental immune related parameters in sponge indicating its adaptational plasticity. Immunological resilience of this species in the face of variation of water temperature and pH is thought to be a special adaptive feature of sponge, a reported “living fossil”.     

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.     

Purification and in vitro cultivation of archaeocytes (stem cells) of the marine sponge <Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> (Demospongiae)

Marine sponges (Porifera) are the best source of marine bioactive metabolites for drug discovery and development, although the sustainable production of most sponge-derived metabolites remains a difficult task. In vitro cultivation of sponge cells in bioreactors has been proposed as a promising technology. However, no continuous cell line has as yet been developed. Archaeocytes are considered to be toti/multipotent stem cells in sponges and, when purified, may allow the development of continuous sponge cell lines. As a prerequisite, we have developed a novel four-step protocol for the purification of archaeocytes from a marine sponge, Hymeniacidon perleve: (1) differential centrifugation to separate large sponge cells including archaeocytes; (2) selective agglomeration in low-Ca²⁺/Mg²⁺ artificial seawater in which living archaeocytes form small loose aggregates with some pinacocytes and collencytes; (3) differential adherence to remove anchorage-dependent pinacocytes, collencytes and other mesohyl cells; (4) Ficoll-Vrografin density gradient centrifugation to purify archaeocytes. The final purity of archaeocytes is greater than 80%. The proliferation potential of the archaeocytes has been demonstrated by high levels of BrdU incorporation, PCNA expression and telomerase activity. In 4-day primary cultures, the purified archaeocytes show a 2.5-fold increase in total cell number. This study opens an important avenue towards developing sponge cell cultures for the commercial exploitation of sponge-derived drugs. The authors are grateful for the financial support of the Chinese Academy of Sciences under the “100 Talent Project”, the “Innovation Fund” from the Dalian Institute of Chemical Physics, the “Hi-Tech Research and Development Program of China” (2001AA620404), and the European Commission (project: Silicon Biotechnology).     

Phagocytic activities of the gorgonian coral Swiftia exserta

The cellular response component of body defense in gorgonians and other cnidarians is thought to be carried out by cells with phagocytic capabilities. To test for the phagocytic character of cells, the introduction of foreign particles was employed and observed in both living cells and histological preparations of the gorgonian coral Swiftia exserta. Observations of untreated tissues revealed normal cells and tissue morphologies. A microscopic observation of living cells following the introduction of particles in a cut revealed that only a mixed population of colorless cells phagocytized the particles. Also particles or clumps of particles were seen on the surface of the colorless cells. Subsequent histological observations allowed identity of colorless cells to be inferred as granular amoebocytes, ectodermal cells, and gastrodermal cells. Cells stained for localization of peroxidase (indicative of phagocytic activity) demonstrated the presence of peroxidase-positive cells. Histological preparations revealed that major phagocytosis of particles was associated with tissue trauma. When particles were introduced by means of a cut or inserted thread, phagocytic activity was detected within 2 h. However, it was confined to the granular amoebocytes in the immediate site of trauma. After 24 h, extensive phagocytosis spread throughout a relatively large area surrounding the wound. At that later time, phagocytic cell types included granular amoebocytes, epidermal cells, sclerocytes, mesogleal cells, and gastrodermal cells of the solenia. Observations suggest that trauma induces phagocytosis in cells not normally phagocytic in S. exserta. No localization of phagocytic cells and no mitotic cells were observed at either 2 or 24 h after particle introduction.     

Ultrastructural study of differentiation processes during aggregation of purified sponge archaeocytes

Archaeocytes from the spongeEphydatia fluviatilis were dissociated and then isolated on Ficoll density gradients. Their aggregation and reconstitution processes were studied by transmission electron microscopy to determine their capabilities for differentiation.Archaeocyte aggregates follow a well defined sequence of differentiation to generate the characteristic structures of a sponge. Pinacoderm is the first structure to be regenerated and appears progressively at the surface of the 12 h aggregates. Pinacocytes which have differentiated in archaeocyte aggregates are identical to native ones except that the nucleolus remains in most cells. The choanocytes appear only after 24 h by a two step process. First, small cells (choanoblasts) are formed from archaeocytes by mitosis. These cells then transform into fully differentiated choanocytes possessing collars and flagella. The early choanocyte chambers are small, irregular and randomly dispersed in the aggregates. Finally, collencytes and sclerocytes begin to appear just before the aggregates spread on the substrate.The differentiation of a suspension of pure archaeocytes is a unique model system to study sponge cell differentiation and has allowed us to demonstrate that archaeocytes isolated from developed sponges maintain the capacity to differentiate even though this capacity is not usually expressed.     

Evaluation of phagocytic activity and nitric oxide generation by molluscan haemocytes as biomarkers of inorganic arsenic exposure

The natural habitats of the freshwater bivalve Lamellidens marginalis face the risk of contamination by the toxic metalloid arsenic. Haemocyte-mediated non-self phagocytosis and generation of nitric oxide (NO) as reactive nitrogen intermediate were examined to establish the reliability of the parameters as biomarkers of sodium arsenite-induced stress on the animal at sublethal concentrations. The studies suggest imposition of a remarkable immune compromise/immune suppression on the animal by the natural contaminant. The animal expressed partial recovery in its phagocytic potential and NO generation over a period of 30 days. Quantitation of phagocytic efficiency and intrahaemocyte NO generation indicates the possibility of the parameters be accepted as cellular biomarkers to estimate and characterize the vulnerability of the freshwater organisms to sodium arsenite-induced stress.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

The blood cells of the sea squirt, Ciona intestinalis: Morphology,differential counts,and in vitro phagocytic activity

The sea squirt, Ciona intestinalis, contains several types of blood cells: stem cells, hyaline, granular, and refractile amoebocytes, signet ring cells, morula cells, small and large compartment cells, and orange cells. Of these cell types, only the hyaline and granular amoebocytes are capable of phagocytosing formalized sheep erythrocytes in vitro. After the addition of erythrocytes to blood cell monolayers, the attachment and ingestion of these particles occurs rapidly. The interrelationships of the various blood cell types are discussed.     

Allograft rejection, autograft fusion and inflammatory responses to injury in Callyspongia diffusa (Porifera; Demospongia)

Sponges exhibit a variety of swift, cellular defence responses to protect self integrity. The sponge Callyspongia diffusa has been used to characterize the cytological changes that occur during allograft rejection, autograft fusion, and inflammation. Allogeneic contact results in fusion of the two exopinacoderms followed by an infiltration of mesohyl cells into the graft zone. As mesohyl cells accumulate, they form tissue bridges that span the graft interface. After a few days, the tissue bridges and the nearby cellular infiltrate become necrotic and slough off, which separates the allogeneic tissues. Autograft fusion begins similarly but cellular infiltration does not follow exopinacoderm fusion. Contacted exopinacocytes are redistributed, the endopinacoderms and choanosomes come into contact, and the grafted sponge tissues merge. Tissue damage exposes internal regions of the sponge to the external environment. In many areas of injury, exposed choanosome is sealed by infiltrating mesohyl cells. In other areas, exposed endopinacoderm appears to serve as new exopinacoderm. Cellular debris is removed by phagocytic archaeocytes and new exopinacoderm is regenerated over the damaged choanosome. No scars remain once the inflammatory infiltrate has dispersed. In general, mesohyl cells are involved in defence responses without an observed enrichment of any specific cell type. However, archaeocytes from rejecting sponges appear to line both sides of the allogeneic interface.     

Agglutinin-mediated phagocytosis-associated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyte mediated phagocytosis is one of the vital components of innate defence mechanisms in crustaceans and this phagocytic process is aided by serum agglutinins. However, literature on agglutinin mediated opsono-phagocytosis is unclear in the case of Macrobrachium rosenbergii hemocytes. Further, very few studies in the case of superoxide anion generation and none with regard to nitric oxide generation during phagocytosis exist among crustaceans. We investigated the occurrence of agglutinins in the serum and the role of serum agglutinins in mediating phagocytosis by the hemocytes. We show that the prawn serum possesses agglutinins that function as opsonins during phagocytosis of HB RBC by the hemocytes. Hemagglutination-inhibition assays revealed the specificity of serum agglutinins for N-acetylated hexoses, namely GalNAc, GlcNAc and ManNAc, with a higher affinity for ManNAc. In addition, ManNAc was able to inhibit the phagocytic response (by about 60%) of the hemocytes against serum pretreated HB RBC, wherein the serum was previously treated with ManNAc. We next investigated the ability of the hemocytes to generate superoxide anion and nitric oxide during HB RBC phagocytosis and results show generation of both these free radicals. In addition, there was an enhancement in generation (75% increase) of these free radicals during agglutinin mediated opsonophagocytosis, when compared to buffer treated targets and interestingly this enhanced generation was inhibited by ManNAc (27% for superoxide anion and 36% for nitric oxide), an inhibitory sugar for phagocytosis. Inhibition of phagocytosis induced superoxide anion generation by DPI (53%), sodium azide (56%) and tropolone (61%), reveals the possible involvement of NADPH-oxidases, peroxidases and probably phenoloxidases, respectively, in the generation of superoxide anion. Similarly, decrease in nitric oxide generation in the presence of l-NIO (47%) during phagocytosis lends support to the role of nitric oxide generation during cellular immune processes. These findings thus suggest a role for superoxide anion and nitric oxide in the innate defense mechanism, namely phagocytosis, in Macrobrachium rosenbergii.     

Cellular location of (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol, a potent antimicrobial metabolite produced by the Caribbean sponge Haliclona vansoesti

The Caribbean sponge Haliclona vansoesti has been found to contain large amounts of a new sphingosine derivative, (2R, 3R, 7Z)-2-aminotetradec-7-ene-1, 3-diol (compound 1). To determine the localization of this compound within the organism, cell distribution and quantitative determination of the aminodiol content of cell fractions obtained by differential centrifugation have been performed. Results show that choanocytes and archaeocytes are the major sponge cell types and that H. vansoesti harbour small photosynthetic symbionts (cyanobacteria) and few heterotrophic bacteria. Reverse-phase HPLC analyses of the cell fractions reveal that the aminodiol 1 is not associated with the prokaryotic endobionts but with the sponge cells, in particular the archaeocytes. This is clearly established by the positive significant correlation existing between the numbers of archaeocytes and the amounts of aminodiol 1. The mean aminodiol concentration is estimated to be 2 microg/10(5) archaeocytes. The aminodiol 1 is also found in substantial amounts in primary cell cultures, so that cell culture can be envisaged as an option for its production. Sponge cell suspensions display potent antibacterial and antiyeast activities, in correlation with their aminodiol content, indicating that this compound is at least in part responsible for these activities in the sponge. The release of the aminodiol I into the external medium suggests that this substance may be involved in the defence mechanisms of the sponge.     

Correlative Fine Structural,Behavioral, and Histochemical Analysis of Ascidian Blood Cells

Abstract The blood cells of a solitary ascidian, Halocynthia roretzi, were examined by electron microscopy (EM) with reference to their appearance by light microscopy (LM). In addition, their movement and stainability by vital dyes was observed by phase-contrast microscopy, and their stainability by Giemsa was also examined. Nine cell types were recognized: vacuolated cells, hyaline amoebocytes, small amoebocytes, granular amoebocytes, macrogranular cells, globular cells, lymphocyte-like cells, large basophilic cells and large granular cells. Vacuolated cells were found to possess various numbers of vacuoles containing strongly electron-dense materials and could be divided into at least three subgroups. Granular amoebocytes contained microfilaments and many granules of uniform size. Hyaline amoebocytes and small amoebocytes seemed to be specialized as phagocytes. Macrogranular cells and globular cells were not well characterized. In the blood of adult individuals, hemoblasts were rarely found, although lymphocyte-like cells were present. Each of two large cells, large basophilic cells and large granular cells, possessed novel granules or vacuoles, whose functions remain to be elucidated. The possible functions and relationships of these cells among various ascidian species are discussed.     

A comparative analyses of morphological variations,phagocytosis and generation of cytotoxic agents in flow cytometrically isolated hemocytes of Indian molluscs

A comparative analyses of hemocytes of molluscs, Pila globosa (Gastropoda: Prosobranchia), Bellamya bengalensis (Gastropoda: Prosobranchia) and Lamellidens marginalis (Bivalvia: Eulamellibranchiata) were carried out for morphotype and subpopulation identification, analyses of phagocytosis and generation of cytotoxic agents. Flow cytometry and microscopic analyses of hemocytes revealed the existence of agranulocytes (blast like cells, round hyalinocytes and spindle hyalinocytes), semigranulocytes (semigranular asterocytes and round semigranulocytes) and granulocytes (round granulocytes, spindle granulocytes and granular asterocytes) as three morphotypes. In P. globosa, granulocytes and semigranulocytes and in B. bengalensis granulocytes and agranulocytes are the chief phagocytes and major producers of superoxide anion and nitric oxide. In L. marginalis, granulocytes were identified as principal phagocytes with prominent activity of superoxide anion and nitric oxide. Highest activity of phenoloxidase was recorded in the agranulocytes of P. globosa with moderate activities among other morphotypes of all three species. Differential result may be due to species specific response, non-identical habitat preference and related adaptation of the species to their different ecological niches.     

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis.

The phagocytic activity of Mytilus galloprovincialis hemocytes is thought to be associated with NADPH-oxidase activity of the plasma membrane, thus producing superoxide anions. Few studies, however, have been devoted to nitric oxide release by these haemocytes. We investigated NO generation in M. galloprovincialis in order to understand its role in the defensive mechanisms of these organisms. The presence of NO-synthase-like enzymatic activity in protein homogenates from M. galloprovincialis hemocytes was revealed by the conversion of radiolabelled L-arginine to L-citrulline. We observed partial inhibition of the luminol-dependent chemiluminescence of stimulated M. galloprovincialis hemocytes by both NO-synthase inhibitors and superoxide dismutase, indicating that peroxynitrite (which results from the reaction between nitric oxide and superoxide anions) partially mediated this chemiluminescence. Furthermore, we confirmed the production of nitric oxide by M. galloprovincialis by highlighting the nitric oxide-synthase-dependence of the nitrate and nitrite production of stimulated hemocytes.     

Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera,Carabidae)

The present study focuses on the ability of Pterostichus melas italicus Dejean to mount cellular and humoral immune responses against invading pathogens. Ultrastructural analyses revealed the presence of five morphologically distinct types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. Differential hemocyte counts showed that plasmatocytes and granulocytes were the most abundant circulating cell types and plasmatocytes exhibited phagocytic activity following the latex bead immune challenge. Macrophage-like cells were recruited after the immune challenge to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules formed to immobilize the latex beads. Total hemocyte counts showed a significant reduction of hemocytes after latex bead treatment. Phenoloxidase (PO) assays revealed an increase of total PO in hemolymph after immune system activation with lipopolysaccharide (LPS). Moreover, the LPS-stimulated hemocytes showed increased protein expression of inducible nitric oxide synthase, indicating that the cytotoxic action of nitric oxide was engaged in this antimicrobial collaborative response. These results provide a knowledge base for further studies on the sensitivity of the P. melas italicus immune system to the environmental perturbation in order to evaluate the effect of chemicals on non-target species in agroecosystems.     

The stem cell concept in sponges (Porifera): Metazoan traits

Sponges are considered the oldest living animal group and provide important insights into the earliest evolutionary processes in the Metazoa. This paper reviews the evidence that sponge stem cells have essential roles in cellular specialization, embryogenesis and Bauplan formation. Data indicate that sponge archaeocytes not only represent germ cells but also totipotent stem cells. Marker genes have been identified which are expressed in totipotent stem cells and gemmule cells. Furthermore, genes are described for the three main cell lineages in sponge, which share a common origin from archaeocytes and result in the differentiation of skeletal, epithelial, and contractile cells.     

Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea)

A morpho‐functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehyde‐fixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin‐affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti‐CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage‐like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron‐dense content, positive to arylsulphatase activity and binding DBA, UEA‐I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS‐positive vacuoles with arylsulphatase, chloroacetylesterase and β‐glucuronidase activities.     

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

Echinometra lucunter, (Pindá) 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.     

In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes upon incubation with PMA or laminarin or during yeast phagocytosis

The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs. In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria. To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M. galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells). We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M. galloprovincialis haemocytes. Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M. galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M. galloprovincialis.