Biomphalaria glabrata amoebocytes: Effect of Schistosoma mansoni infection on in vitro phagocytosis

The rate of phagocytosis by amoebocytes obtained from hemolymph of the pulmonate Biomphalaria glabrata infected with the trematode Schistosoma mansoni for 24 hr and 2, 4, and 6 weeks has been determined using the monolayer assay system. Amoebocyte preparations from snails infected for 4 and 6 weeks showed a gradual decrease in the phagocytic rates compared to those from uninfected controls. Snails harboring the parasite for 4 and 6 weeks also showed a significant increase in the number of amoebocytes in the hemolymph. No significant changes were detected in the rate of phagocytosis or number of amoebocytes in snails infected for 2 weeks or less. Alterations in the morphology and behavior of amoebocytes from infected snails were also noted.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

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.     

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.     

Phagocytic efficiency and cytotoxic responses of Indian freshwater sponge (Eunapius carteri) cells isolated by density gradient centrifugation and flow cytometry: a morphofunctional analysis

The freshwater sponge Eunapius carteri (Porifera: Demospongiae: Spongillidae), a resident of Indian freshwater ecosystems, has pharmaceutical and ecological potential, but there is inadequate information on its cellular spectrum and cell-mediated immune responses. Microscopical analysis revealed the existence of eight distinct cellular variants, i.e. blast-like cells, choanocytes, small amoebocytes, granular cells, pinacocytes, large amoebocytes, archaeocytes and sclerocytes. The cells were isolated by density gradient centrifugation and flow cytometry and used for a morphofunctional analysis. We investigated the phagocytic efficiency of E. carteri cells under the challenge of yeast particles in vitro and spectrophotometrically quantified the generation of cytotoxic molecules (superoxide anions and nitric oxide) in different isolated cellular fractions. The two cell separating technologies did not yield any significant differences in the major findings on morphology, phagocytic response and generation of superoxide anions and nitric oxide. Archaeocytes, granular cells and large amoebocytes were identified as chief phagocytes with a high phagocytic potential as recorded by light microscopy. Archaeocytes were the principal generators of superoxide anions, whereas nitric oxide was recorded in the fractions rich in archaeocytes and large amoebocytes. The present investigation thus provides useful information regarding cellular variation, cytotoxic status and innate phagocytic response of the cells of E. carteri, a common but less studied sponge of India.     

Biomphalaria glabrata amoebocytes: Assay of factors influencing in vitro phagocytosis

A simple and short-term in vitro assay system has been adapted for quantitative evaluation of the rate of phagocytosis of amoebocytes obtained from hemolymph of the pulmonate gastropod Biomphalaria glabrata. The variation in the rate of phagocytosis exhibited by amoebocyte monolayers prepared from individual snails is reduced by utilizing amoebocytes from pooled hemolymph from snails of similar sizes. The study demonstrates that the rate of phagocytosis depends on incubation time, temperature, and pH. Substantial inhibition of phagocytosis is exerted by 2-deoxy-d-glucose and 2-iodoacetamide but not by potassium cyanide, indicating that energy consumed during phagocytosis is generated by the glycolysis pathway.     

Amoebocytes facilitate efficient carbon and nitrogen assimilation in the Cassiopea-Symbiodiniaceae symbiosis

The upside-down jellyfish Cassiopea engages in symbiosis with photosynthetic microalgae that facilitate uptake and recycling of inorganic nutrients. By contrast to most other symbiotic cnidarians, algal endosymbionts in Cassiopea are not restricted to the gastroderm but are found in amoebocyte cells within the mesoglea. While symbiont-bearing amoebocytes are highly abundant, their role in nutrient uptake and cycling in Cassiopea remains unknown. By combining isotopic labelling experiments with correlated scanning electron microscopy, and Nano-scale secondary ion mass spectrometry (NanoSIMS) imaging, we quantified the anabolic assimilation of inorganic carbon and nitrogen at the subcellular level in juvenile Cassiopea medusae bell tissue. Amoebocytes were clustered near the sub-umbrella epidermis and facilitated efficient assimilation of inorganic nutrients. Photosynthetically fixed carbon was efficiently translocated between endosymbionts, amoebocytes and host epidermis at rates similar to or exceeding those observed in corals. The Cassiopea holobionts efficiently assimilated ammonium, while no nitrate assimilation was detected, possibly reflecting adaptation to highly dynamic environmental conditions of their natural habitat. The motile amoebocytes allow Cassiopea medusae to distribute their endosymbiont population to optimize access to light and nutrients, and transport nutrition between tissue areas. Amoebocytes thus play a vital role for the assimilation and translocation of nutrients in Cassiopea, providing an interesting new model for studies of metabolic interactions in photosymbiotic marine organisms.     

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.     

Pathology and host responses induced by Schistosomatium douthitti in the freshwater snail Lymnaea catascopium.

Most Schitosomatium douthitti miracidia penetrated the esophageal wall of Lymnaea catascopium without provoking amoebocyte encapsulation responses or extensive pathological changes. Amoebocytes frequently attached to developing mother and daughter sporocysts, but did not encapsulate or destroy them. Pressure resulting from extensive growth of mother sporocysts ruptured the transverse membrane of some snails. After releasing daughter sporocysts, mother sporocysts in some snails were destroyed by amoebocytes. Many migrating cercariae became trapped in the tissues of L. catascopium, particularly in the posterior portion of the foot, and were encapsulated and destroyed. Large increases in numbers of amoebocytes in the anterior portion of the lung roof of infected snails were noted, even before cercarial production had been initiated. Atrophy of the digestive gland occurred in infected snails.     

The occurrence of inflammatory granulomas in the ctenidial marsupium of Corbicula fluminea (mollusca: Bivalvia): A consequence of larval incubation

A number of tumor-like cysts in the ctenidia of the freshwater bivalve Corbicula fluminea are described. The cysts result from the encapsulation of incubated larvae within the female inner demibranchs which are modified to form a marsupium. Corbicula fluminea is a protandric consecutive hermaphrodite, and larval incubation commences with sex reversal and the release of the first eggs. For some reason a few of the larvae are not released, and their retention and subsequent death results in the mobilization of the innate cellular defensive mechanism of the parent. This process involves, initially, an invasion of epithelioid cells and amoebocytes (granulocytes), resulting in hyperplasia. This is followed by structural changes to the epithelium bordering the interlamellar spaces (within which the larvae are incubated) to form ultimately the cyst wall. The epithelium is surrounded externally by layers of fibrocytes which eventually form a thick capsule. The autolyzed larval tissues are themselves invaded by fibrocytes, epithelioid cells, and amoebocytes and, in one specimen, had formed a three-layered capsule within the surrounding capsule. The amoebocytes probably reabsorb the larval cellular debris. This unusual example of a molluscan cellular defensive mechanism may assist in the diagnosis and separation of hyperplastic injury responses from neoplasmic conditions in invertebrates.     

Cellular responses in sea fan corals: granular amoebocytes react to pathogen and climate stressors

Background

Climate warming is causing environmental change making both marine and terrestrial organisms, and even humans, more susceptible to emerging diseases. Coral reefs are among the most impacted ecosystems by climate stress, and immunity of corals, the most ancient of metazoans, is poorly known. Although coral mortality due to infectious diseases and temperature-related stress is on the rise, the immune effector mechanisms that contribute to the resistance of corals to such events remain elusive. In the Caribbean sea fan corals (Anthozoa, Alcyonacea: Gorgoniidae), the cell-based immune defenses are granular acidophilic amoebocytes, which are known to be involved in wound repair and histocompatibility.

Methodology/Principal Findings

We demonstrate for the first time in corals that these cells are involved in the organismal response to pathogenic and temperature stress. In sea fans with both naturally occurring infections and experimental inoculations with the fungal pathogen Aspergillus sydowii, an inflammatory response, characterized by a massive increase of amoebocytes, was evident near infections. Melanosomes were detected in amoebocytes adjacent to protective melanin bands in infected sea fans; neither was present in uninfected fans. In naturally infected sea fans a concurrent increase in prophenoloxidase activity was detected in infected tissues with dense amoebocytes. Sea fans sampled in the field during the 2005 Caribbean Bleaching Event (a once-in-hundred-year climate event) responded to heat stress with a systemic increase in amoebocytes and amoebocyte densities were also increased by elevated temperature stress in lab experiments.

Conclusions/Significance

The observed amoebocyte responses indicate that sea fan corals use cellular defenses to combat fungal infection and temperature stress. The ability to mount an inflammatory response may be a contributing factor that allowed the survival of even infected sea fan corals during a stressful climate event.     

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

Summary Blood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per l haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of ³H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.     

The distribution of the Wnt5 protein in the tissues of the holothurian Eupentacta fraudatrix (Djakonov et Baranova, 1958) (Holothuroidea: Dendrochirotida) in the norm and during regeneration

The Wnt5 protein localization in holothurian Eupentacta fraudatrix tissues was examined in the norm and during regeneration. In healthy E. fraudatrix, Wnt5 was found in solitary cells of the hypodermis and in the radial nerve cords. During regeneration, the number of Wnt5 positive cells increased. They were observed in the connective tissue of the body wall and the pharyngeal bulb, in nervous system tissues, the coelomic epithelium, and amoebocytes. The Wnt5 protein may participate in regulating the regeneration in the holothurian E. fraudatrix; it probably modulates cell migration, extracellular matrix reorganization, and neurogenesis.     

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.     

Antioxidant Enzymatic Activity of Coelomocytes of the Far East Sea Cucumber Eupentacta fraudatrix

The basal activity of superoxide dismutase (SOD), glutathione reductase (GR), and catalase in a pool of coelomocytes as well as in the fraction of amoebocytes and the mixed fraction of amoebocytes and morula-like cells of the sea cucumber Eupentacta fraudatrix is studied. For SOD and catalase, pH optima are in the range of values of pH optimum for tissues of mammals, the pH optimum for GR is shifted to a more acidic region in comparison with the latter enzymes. Temperature optima for all studied enzymes are higher than the usual temperature values of the sea cucumber habitation. A pronounced temperature dependence of all three enzymes is revealed. In coelomocytes, the activities of SOD and catalase, but not of GR, are lower than in the fraction of amoebocytes, but higher than in the mixed fraction of amoebocytes and morula-like cells. The rate of production of active forms of oxygen (AFO) is three times higher in amoebocytes than in the fraction relatively enriched in morula-like cells. Apparently, the main part of the SOD and catalase activities, as well as AFO production in coelomocytes is located in amoebocytes, which confirms the existence of cytophagic function in the latter cells as well as argues in favor of functional differentiation between individual types of coelomocytes.     

Amoebocytes in the Lining of the Body Cavity and Mesenteries of Priapulus caudatus (Priapulida)

Nuclei associated with membranes lining the body cavity in Priapulus caudatus are shown by electron microscopy to be the nuclei of amoebocytes. The membranes themselves are not cellular and do not constitute a peritoneum. Mesenteries, present in the body cavity, also are not cellular; amoebocytes occasionally are found within them. These findings weaken the idea that priapulids are coelomates, although unequivocal proof that priapulids are pseudocoelomates would require a fine structural study of the early developmental stages.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Resistance to schistosome infection in Biomphalaria glabrata induced by gamma radiation

Two strains of Biomphalaria glabrata were studied with respect to the effects of ionizing radiation on their susceptibility to Schistosoma mansoni infection. Gamma radiation at levels of 3.5 and 5 krad did not induce susceptibility in the resistant S-3 strain, but was found to initiate resistance in the susceptible PR-1 strain. In an attempt to understand the induced resistance in irradiated snails, histopathologic examinations and analyses of snail hemolymph were performed. Results indicated that miracidia invading irradiated snails were quickly surrounded and encapsulated by amoebocytes. Similarly, alterations in the hemolymph of irradiated snails suggested that radiation induced aging. It is suggested that radiation-altered snails may be of value in studying the defense mechanisms of these organisms.