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Degranulation and Other Changes of Molluscan Granulocytes Associated with Phagocytosis
Abstract
The loss of cytoplasmic granules, which have been shown to be lysosomes, from granulocytes of Mercenaria mercenaria during the in vitro phagocytosis of Bacillus megaterium has been demonstrated semiquantitatively. This process, designated as degranulation, represents the morphological basis of the release of lysosomal enzymes from granulocytes into serum as- sociated with phagocytosis. In addition to degranulation associated with phagocytosis, there is rearrangement of lysosomes, the appearance of large vacuolated areas in the cytoplasm, con- traction of cell margins, and the appearance of atypically large granules in granulocytes of M. mercenaria.
... In phagocytosis a distinct sequence of events can be identified, with particle recognition followed by adherence, uptake, destruction and disposal of the invading body (Feng, 1988). Intracellular degradation occurs by a range of lysosomal enzymes, although extracelluar digestion by elevated lysozyme has been observed in haemolymph samples of Mercenaria mercenaria Foley and Cheng, 1977;Mohandas et al, 1985). Phagocytosis requires the movement of cells -frequently termed amoebocytes for the manner of their locomotion -the spreading and rate of locomotion of which can be quantified as a haemocytic index. ...
... If peroxide production is the more immediate defence mechanism, then it would be expected that lysozyme activity would increase soon afterwards. Lysozyme and related enzymes rely on degranulation of lysosomes found within granulocytes (Foley and Cheng, 1977;Cheng, 1986), and is increased in rapidly phagocytosing cells (Foley and Cheng, 1977). This requires contact with the pathogen which, from the increase in peroxide production, had probably occurred. ...
... If peroxide production is the more immediate defence mechanism, then it would be expected that lysozyme activity would increase soon afterwards. Lysozyme and related enzymes rely on degranulation of lysosomes found within granulocytes (Foley and Cheng, 1977;Cheng, 1986), and is increased in rapidly phagocytosing cells (Foley and Cheng, 1977). This requires contact with the pathogen which, from the increase in peroxide production, had probably occurred. ...
p>The aim of this study was to provide a comprehensive set of quantitative and qualitative baseline responses at physiological, metabolical and immunological levels, in the Pacific oyster Crassostrea gigas (Thunberg), the European flat oyster Ostrea eduli (L.), and the Manila clam Tapes philippinarum (Adams and Reeve). The energetics of these species were compared across a matrix of temperature and salinity conditions. Field trials examined the effect of exposure of three O. edulis populations to infection by the protozoan parasite Bonamia ostreae , and enzyme electrophoresis investigated the genetic basis for any differences. Changes in immunocompetence were monitored from field samples and with controlled Vibrio anguillarum bacterial challenges. Haemolymph and haemocytic responses were recorded.
Filtration rate had the most significant effect on scope for growth (SFG) indices measured in all species. C. gigas showed a much wider range of filtration rates than O. edulis and consequently had much higher SFG. Optimum environmental conditions for C.gigas occurred at 20-25<sup>o</sup>C and 19-25�, compared with 20<sup>o</sup>C and 33� for O. edulis , and 15-20<sup>o</sup>C at 33� in T. philippinarum . Separate winter and summer physiological behaviour was detected in C. gigas and O. edulis , with the change occurring at 15<sup>o</sup>C and 10-12<sup>o</sup>C respectively. Body condition indices were inversely proportional to SFG and were probably related to the reproductive cycle. Temperature was shown to have the most significant influence on energetic factors, with salinity having little effect.
Field trials investigating Bonamia effects in three O. edulis populations found a significant, inverse size relationship with most of the physiological measurements.</p
... Yoshino and Cheng (1976) demonstrated through cytochemistry and electron microscopy that the granules in amebocytes of Mercenaria mercenaria were lysosomes, serving as storage organelles for hydrolytic acids. Foley and Cheng (1977) showed that lysosomes can be stimulated to move to the surface of the amebocyte and expel their contents (Fig. 22), a process that resembles, or is identical to the degranulation process described by both Ruddell (1971) and Brown (1975) for metal-containing amebocyte granules. It can be postulated from these observations that the copper-and zinc-bearing granules in eastern oysters are either lysosomes or closely-related structures. ...
... external contact with certain foreign substances will result in hypersynthesis of intracellular lysosomal enzymes which are released from haemocytes into the serum where digestion of the foreign material, such as bacteria, is initiated. The release of enzymes is effected by what has been termed degranulation (Foley and Cheng, 1977), a process involving the migration of lysosomes to the surface of the cell where the enclosed enzymes are discharged." (Cheng 1981, p. 286) Cheng et al. (1975) demonstrated the extracellular release of lysozyme from lysosomes during phagocytosis by Mercenaria mercenaria amebocytes. ...
The distribution of eastern oysters Crassostrea virginica near terrestrial watersheds has led to a general impression that low or variable salinity is imperative for survival. However, freshwater runoff contains numerous mineral elements from geologic deposits that could play significant roles in oyster physiology. Two metals of terrestrial origin, copper and zinc, are accumulated to extremely high concentrations in eastern oysters, even in the absence of anthropogenic sources. As yet, there has been no defendable demonstration of a physiologic function for such high concentrations. Both copper and zinc, however, are accumulated almost exclusively in the amebocytes and calcareous shell of oysters, a unique distribution that implicates a role in the functions of amebocytes. Amebocytes are migratory, diapedetic cells generally recognized to provide nutriment and defense through phagocytosis, killing, and digestion of invading or ingested microorganisms. There is sufficient evidence in existing literature to suggest that copper and zinc directly contribute to these antimicrobial activities. This review presents historical and recent findings that demonstrate a strong affinity of oyster amebocytes for copper and zinc (even in low ambient concentrations), prolonged retention of the metals despite a potential route of elimination, and strong circumstantial evidence of antimicrobial activity by accumulated copper and zinc. It is proposed that oysters actively concentrate copper and zinc as antimicrobial agents to be used in intracellular and extracellular killing (direct toxicity) as well as extracellular clot formation (precipitation of hemolymph). This potential, combined with evidence of amebocyte involvement in deposition of oyster shell, provides an alternative framework for understanding amebocyte functions, defense activities, and coastal distributions of oyster populations. It also affords some resolution to the apparent contradiction of eastern oysters thriving at seemingly polluted locations.
... Thus, the movement of granulocytes into the hyalinocyte cloud was observed in Mya arenaria in response to exposure to pathogenic microorganisms that caused degranulation and a decrease in the number of lysosomes [21]. The release of granules was accompanied by their displacement to the peripheral region of the cytoplasm [16] that we observed in some neutrophilic and acidophilic granulocytes of C. grayanus. Before exocytosis, granules can merge [27], which also changes the physical properties of granulocyte cytoplasm. ...
Based on light microscopy data, hemocytes of Crenomytilus grayanus were classified into five morphological types common for Bivalvia. In the stage of sexual inertia (late October), the proportions of the cell
types are as follows: (1) hemoblasts (0.2 ± 0.1%), (2) hyalinocytes (1.9 ± 0.3%), and also (3) basophilic
(10.9 ± 1.4%), (4) neutrophilic (13.3 ± 3.0%), and (5) acidophilic (74.1 ± 2.9%) granulocytes. All hemocytes
were divided into four groups on the basis of their size (FSC) and complexity (SSC) by flow cytometry. Correlation analysis has shown that R1 corresponds to hemoblasts, R2 to hyalinocytes, and R4 to granulocytes
and their acidophilic forms. However, these correlations are not observed in the summer season. The hemocyte morphology and quantitative relationships between their structural types confirm Mix’s hematopoietic
model, which postulates histogenetic continuity of hyalinocytes and granulocytes. The arrangement of cells
in the light-scatter dot plots (FSC vs. SSC) indicates their maturity stage; it depends on functional status and
may change with disturbances of the mitotic cycle. The hemocyte population in C. grayanus shows a low rate
of renewal and a dominance of acidophilic granulocytes (up to 99% of all cells in the sexual inertia stage),
which suggests a strategy targeted at long-term maintenance of highly differentiated cells and is consistent
with the long life expectancy of the species.
... 2. Death of a certain type of hemocytes [23]; 3. Functional changeovers of one type of hemocytes into another (granulation vs. degranu lation) [36,37]; ...
... Differences between populations of hemocytes have primarily focused on morphological diversity and classification into the subpopulations granulocytes and agranulocytes (10)(11)(12). These classifications are based on size and presence or absence of granules. ...
Circulating hemocytes in the hemolymph represent the backbone of innate immunity in bivalves. Hemocytes are also found in the extrapallial fluid (EPF), the space delimited between the shell and the mantle, which is the site of shell biomineralization. This study investigated the transcriptome, proteome, and function of EPF and hemolymph in the hard clam Mercenaria mercenaria. Total and differential hemocyte counts were similar between EPF and hemolymph. Overexpressed genes in the EPF were found to have domains previously identified as being part of the “biomineralization toolkit” and involved in bivalve shell formation. Biomineralization related genes included chitin-metabolism genes, carbonic anhydrase, perlucin, and insoluble shell matrix protein genes. Overexpressed genes in the EPF encoded proteins present at higher abundances in the EPF proteome, specifically those related to shell formation such as carbonic anhydrase and insoluble shell matrix proteins. Genes coding for bicarbonate and ion transporters were also overexpressed, suggesting that EPF hemocytes are involved in regulating the availability of ions critical for biomineralization. Functional assays also showed that Ca²⁺ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca²⁺ during biomineralization. Overexpressed genes and proteins also contained domains such as C1q that have dual functions in biomineralization and immune response. The percent of phagocytic granulocytes was not significantly different between EPF and hemolymph. Together, these findings suggest that hemocytes in EPF play a central role in both biomineralization and immunity.
... For example, homologues of human LYZ were first reported in abalone (Haliotis discus hannai) (Ding et al., 2011) and LYG in the Zhikong scallop (Chlamys farreri) where it was proposed to be a multifunctional lysozyme (Van Herreweghe and Michiels, 2012;Zhao et al., 2007b). The invertebrate specific LYI was first isolated and characterized in the Japanese clam (Ruditapes philippinarum) and shown to be important for defence against bacteria but was also suggested to have digestive functions (Foley and Cheng, 1977;Ito et al., 1999;Pipe, 1990). Overall, our phylogenetic analysis suggested that the molluscs and vertebrate Ctype and G-type lysozymes shared a common origin. ...
Lysozymes are an ancient group of antimicrobial enzymes of the innate immune system. Here we provide a comparative analysis of the evolution and function of lysozymes during early development in fish, the most speciose vertebrate group. In fishes, lineage and species-specific evolution of both C-type (chicken or conventional) and G-type (goose type) genes occurred. Phylogenetic analysis revealed that the teleost lysozyme G-type members group with the tetrapod homologues but the teleost C-type form three different clusters with the tetrapods. Most of the teleost C-type cluster with tetrapod Lyz but there are some that group with the mammalian Lyzl1/2 and LALBA. This suggests that early in gnathostome evolution these genes already existed and that lyzl1/2 and lalba genes are present in fish and tetrapods. Gene synteny analysis to confirm sequence orthologies failed to identify conserved genome regions between teleosts and other vertebrates lysozyme gene regions suggesting that in the ancestral bony fish genome lyz, lyzl1/2, lalba and lyg precursor genes were transposed to different chromosome regions. The homologue of the mammalian lactalbumin (LALBA) gene was identified for the first time in teleosts and was expressed in skin and during egg and larval development. Lysozyme activity was detected in teleost eggs and varied between species and in the gilthead sea bream lyg and lalba transcript abundance differed in eggs and larvae from different brood stock suggesting differences exist in maternal innate immune protection.
... Morphological diversity of hemocytes that include cells of different sizes and internal complexity (commonly defined as granulocytes, agranulocytes, and/or hyalinocytes) is a well-known trait of bivalves, including oysters ( Allam et al., 2002;Foley and Cheng, 1977;Hegaret et al., 2003;Kennedy et al., 1996). However, the functional differentiation of hemocytes remains elusive, owing in part to difficulties of separating different subpopulations of live hemocytes for functional analysis ( Goedken and De Guise, 2004;Terahara et al., 2006;Wang et al., 2017a). ...
Molluscan exoskeleton (shell) plays multiple important roles including structural support, protection from predators and stressors, and physiological homeostasis. Shell formation is a tightly regulated biological process that allows mollusks to build their shells even in environments unfavorable for mineral precipitation. Outer mantle edge epithelial cells (OME) and hemocytes were implicated in this process; however, the exact functions of these cell types in biomineralization are not clear. The Pacific oysters Crassostrea gigas were used to study differences in the expression profiles of selected biomineralization-related genes in hemocytes and mantle cells, and the functional characteristics of hemocytes such as adhesion, motility and phagocytosis. The specialized role of OME in shell formation was supported by high expression levels of the extracellular matrix (ECM) related and cell-cell interaction genes. Density gradient separation of hemocytes revealed four distinct phenotypes based on the cell morphology, gene expression patterns, motility and adhesion characteristics. These hemocyte fractions can be categorized into two functional groups, i.e. biomineralization and immune response cells. Gene expression profiles of the putative biomineralizing hemocytes indicate that in addition to their proposed role in the mineral transport, hemocytes also contribute to the formation of the ECM, thus challenging the current paradigm of the mantle as the sole source of the ECM for shell formation. Our findings corroborate the specialized roles of hemocytes and the OME in biomineralization and emphasize complexity of the biological controls over the shell formation in bivalves.
... This requires that hemocytes be able to recognize, bind, ingest and dispose of exogenous or nonself particles (Fisher 1988a). In addition, hemocytes are believed to release lysosomal enzymes into the hemolymph to destroy bacteria or alter foreign particles so they can be recognized as nonself (Foley and Cheng 1977). It has been hypothesized that lectins (nonimmunoglobulin proteins) in invertebrate serum can enhance the phagocytic response by marking, or "recognizing" nonself material (Olafsen 1988). ...
... Some non phagocytizing granulocytes showed gigantic granules of high electron density which were probably the result of R granules' fusion. It is likely that these merging granules were participating in a kind of 'compound exocytosis' [42] resulting in granulocyte degranulation, as it has been shown in bivalves (e.g., [43,[44][45][46][47]) and has been related to the release of lysozyme and other hydrolytic enzymes that may kill bacteria, either in the circulation or in tissue. Release of lyzozyme immunoreactive molecules was increased in serum of an heterobranch gastropod after bacterial challenge [48]. ...
Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes.
... Nevertheless the loss of granules from granulocytes of Ruditapes decussatus during the in vitro phagocytosis assays could also be due to the release of lysosomal enzymes from granulocytes into the serum (degranulation). A degranulation process associated with phagocytosis was reported by Foley et al. (1977) in Mercenana mercenaria and Hinsch & Hunte (1990) in Crassostrea virginica. ...
Phagocytosis of foreign materials by haemocytes is an important aspect of the internal defence of bivalve molluscs. Two main haemocyte types can be distinguished in the haemolymph of the clam Ruditapes decussatus: granulocytes and hyalinocytes. The ability of clam haemocytes to phagocytose zymosan particles, Vibrio P1 cells and trophozoites of the protistan parasite Perkinsus atlanticus was demonstrated by means of in vitro assays. However, clam haemocytes did not phagocytose P. atlanticus zoospores in the assays. Granulocytes showed the highest phagocytic capacity in each assay. Phagocytic capacity of haemocytes was not significantly affected by clam age. An ultrastructural study of phagocytosis showed the following sequence of events: engulfment of particles by pseudopods, formation of a phagocytic vacuole, fusion of lysosomes with the phagocytic vacuole, and digestion of the particles giving rise to residual bodies that might be discharged.
... Another explanation for V. vulnificus persistence inside these phagocytic cells is the inability of some hemocytes to kill V. vulnificus. While granular hemocytes are quite lethal to V. vulnificus, agranular hemocytes are not, possibly due to the lack of the lysosomal enzymes responsible for bacterial degradation828384 . In the summer, the percentage of agranular hemocytes in oysters is greater than granular hemocytes , possibly contributing to the increase of V. vulnificus seen within oysters in the summer months [82, 85]. ...
The human bacterial pathogen, Vibrio vulnificus, is found in brackish waters and is concentrated by filter-feeding molluscan shellfish, especially oysters, which inhabit those waters. Ingestion of raw or undercooked oysters containing virulent strains of V. vulnificus can result in rapid septicemia and death in 50 % of victims. This review summarizes the current knowledge of the environmental interactions between these two organisms, including the effects of salinity and temperature on colonization, uptake, and depuration rates of various phenotypes and genotypes of the bacterium, and host-microbe immunological interactions.
... The biological function of lysozyme in marine mollusks is important in their self-defense against bacterial infection [23,24]. On the other hand, the digestive function of lysozyme in marine mollusks was also suggested. ...
Lysozymes were purified from three invertebrates: a marine bivalve, a marine conch, and an earthworm. The purified lysozymes all showed a similar molecular weight of 13 kDa on SDS/PAGE. Their N-terminal sequences up to the 33rd residue determined here were apparently homologous among them; in addition, they had a homology with a partial sequence of a starfish lysozyme which had been reported before. The complete sequence of the bivalve lysozyme was determined by peptide mapping and subsequent sequence analysis. This was composed of 123 amino acids including as many as 14 cysteine residues and did not show a clear homology with the known types of lysozymes. However, the homology search of this protein on the protein or nucleic acid database revealed two homologous proteins. One of them was a gene product, CELF22 A3.6 of C. elegans, which was a functionally unknown protein. The other was an isopeptidase of a medicinal leech, named destabilase. Thus, a new type of lysozyme found in at least four species across the three classes of the invertebrates demonstrates a novel class of protein/lysozyme family in invertebrates. The bivalve lysozyme, first characterized here, showed extremely high protein stability and hen lysozyme-like enzymatic features.
... The decrease in melanin concentration in M. faveolata and the lack of variation of melanin among treatments for S. intersepta and P. astreoides may be due to the release of melanin via degranulation (Xian et al., 2009;Palmer et al., 2011b). The degranulation of redoxactive melanin (Nappi and Ottaviani, 2000) from melanin-containing cells to aid during phagocytosis and barrier formation (Foley and C. V. Palmer and othersCheng, 1977;Xian et al., 2009) would therefore lead to a reduced concentration within the host tissue. In addition, melanin degranulation may be triggered to mitigate thermal stress (Palmer et al., 2010) by forming a photo-protective layer. ...
Disease epizootics are increasing with climatic shifts, yet within each system only a subset of species are identified as the most vulnerable. Understanding ecological immunology patterns as well as environmental influences on immune defenses will provide insight into the persistence of a functional system through adverse conditions. Amongst the most threatened ecosystems are coral reefs, with coral disease epizootics and thermal stress jeopardizing their survival. Immune defenses were investigated within three Caribbean corals, Montastraea faveolata, Stephanocoenia intersepta and Porites astreoides, which represent a range of disease and bleaching susceptibilities. Levels of several immune parameters were measured in response to elevated water temperature and the presence of a commercial pathogen-associated molecular pattern (PAMP) - lipopolysaccharide (LPS) - as an elicitor of the innate immune response. Immune parameters included prophenoloxidase (PPO) activity, melanin concentration, bactericidal activity, the antioxidants peroxidase and catalase, and fluorescent protein (FP) concentration. LPS induced an immune response in all three corals, although each species responded differently to the experimental treatments. For example, M. faveolata, a disease-susceptible species, experienced significant decreases in bactericidal activity and melanin concentration after exposure to LPS and elevated temperature alone. Porites astreoides, a disease-resistant species, showed increased levels of enzymatic antioxidants upon exposure to LPS independently and increased PPO activity in response to the combination of LPS and elevated water temperature. This study demonstrates the ability of reef-building corals to induce immune responses in the presence of PAMPs, indicating activation of PAMP receptors and the transduction of appropriate signals leading to immune effector responses. Furthermore, these data address the emerging field of ecological immunology by highlighting interspecific differences in immunity and immunocompetences among Caribbean corals, which are reflected in their life-history characteristics, disease susceptibilities and bleaching-induced mortality.
... Invertebrate granular cells, which are sometimes referred to as granulocytes, are extensively documented in literature on immunity [30,49,50,51,52], where they are reported to contain many active products involved in immunity, including phenoloxidase, melanin, peroxidase and lysozyme [21,52,53]. Degranulation of granular cells during wound healing [37,52,54,55] releases antimicrobial and cytotoxic material, including melanin, which can kill foreign organisms. This mechanism is also documented for mammalian mast cells and granulocytes [45] and for crystal cells of insects [33,56]. ...
Sessile animals, like corals, frequently suffer physical injury from a variety of sources, thus wound-healing mechanisms that restore tissue integrity and prevent infection are vitally important for defence. Despite the ecological importance of reef-building corals, little is known about the cells and processes involved in wound healing in this group or in phylogenetically basal metazoans in general.
A histological investigation into wound healing of the scleractinian coral Porites cylindrica at 0 h, 6 h, 24 h and 48 h after injury revealed differences in cellular components between injured and healthy tissues. Cell counts of the obligate endosymbiont, Symbiodinium, and melanin volume fraction analysis revealed rapid declines in both Symbiodinium abundance and tissue cross-sectional area occupied by melanin-containing granular cells after injury. Four phases of wound healing were identified, which are similar to phases described for both vertebrates and invertebrates.
The four phases included (i) plug formation via the degranulation of melanin-containing granular cells; (ii) immune cell infiltration (inflammation); (iii) granular tissue formation (proliferation); and (iv) maturation. This study provides detailed documentation of the processes involved in scleractinian wound healing for the first time and further elucidates the roles of previously-described immune cells, such as fibroblasts. These results demonstrate the conservation of wound healing processes from anthozoans to humans.
... Some of the enzymes in our samples may have come from non-epithelial sources. For example, lysozyme and lipase occur in molluscan hemocytes (Cheng and Yoshino, 1976;Foley and Cheng, 1977) and N-acetyl-β-glucosaminidase, acid phosphatase, β-glucuronidase and esterases have been reported as indications of hemocyte function in adult gastropods and bivalves Luna-Gonzalez et al., 2004). In addition, we cannot distinguish between enzymes to be released into the gut lumen to aide in extracellular digestion and those retained within cells for possible intracellular digestion. ...
The esophagus and intestine form the longest regions of the digestive tract in the giant keyhole limpet and are lined by epithelial cells sharing a common morphology and releasing materials into the gut lumen by apocrine secretion. The purpose of this study was to determine if these morphologically similar regions release similar digestive enzymes and compare their contributions to digestive enzymes released from other regions of the gut. Principal component analysis of enzymes detected by the API ZYM system for 19 enzymes plus EnzChek assays for protease, α-amylase, lipase, cellulase, and lysozyme identify four distinct regions of the gut: 1) crystalline style and style sac, 2) digestive gland, 3) salivary glands, and 4) esophagus and intestine. Heterogeneity in enzymatic activity was observed in regions of the gut with similar cell morphology (middle and posterior esophagus and intestine) as well as regions with different cell morphology (salivary glands, digestive gland and crystalline style). Enzyme activity in each of these regions is compared to other gastropods, in particular the abalone. Although much of the length of the digestive tract is lined by a morphologically similar epithelium, different regions of the alimentary tract produce a different suite of enzymes which may contribute to the digestive process. These data will help enhance our limited understanding of the digestive physiology of Megathura crenulata and lead to improvement of its culture for clinical research.
Oysters are an excellent biomonitor of coastal pollution and the hyper-accumulator of toxic metals such as copper and zinc (Zn). One unique feature of molluscs is their hemocytes which are mainly involved in immune defenses. Different subpopulations of hemocytes have been identified, but their functions in metal transport and detoxification are not clear. In this study, we examined the immune responses of different subpopulations of oyster Crassostrea hongkongensis hemocytes under different periods of Zn exposure by using flow cytometer and confocal microscopy. In vitro exposure to Zn resulted in acute immune responses by increasing the reactive oxygen species (ROS) production and phagocytosis and decreased number of granulocytes and mitochondrial membrane potential (MMP) within 3 h. Granulocyte mortality and lysosomal pH increased whereas glutathione (GSH) decreased within 1 h of in vitro exposure, indicating the immune stimulation of granulocytes. Within the first 7 days of in vivo exposure, immunocompetence of granulocytes was inhibited with increasing granulocyte mortality but decreasing ROS production and phagocytosis. However, with a further extension of Zn exposure to 14 days, both phagocytosis and lysosomal content increased with an increasing number of granulocytes, indicating the increase of hemocyte-mediated immunity. Our study demonstrated that granulocytes played important roles in oyster immune defenses while other subpopulations may also participate in immune functions. The degranulation and granulation due to transition between semigranulocytes and granulocytes after Zn exposure were important in metal detoxification. The study contributed to our understanding of the immune phenomena and the adaptive capability of oysters in metal contaminated environments.
Ampullariids are freshwater gastropods bearing a gill and a lung, thus showing different degrees of amphibiousness. In particular, Pomacea canaliculata (Caenogastropoda, Ampullariidae) is an obligate air-breather that relies mainly or solely on the lung for dwelling in poorly oxygenated water, for avoiding predators, while burying in the mud during aestivation, and for oviposition above water level. In this paper, we studied the morphological peculiarities of the gill in this species. We found (1) the gill and lung vasculature and innervation are
intimately related, allowing alternation between water and air respiration; (2) the gill epithelium has features typical of a transporting rather than a respiratory epithelium; and (3) the gill has resident granulocytes within intraepithelial spaces that may serve a role for immune defence. Thus, the role in oxygen uptake may be less significant than the roles in ionic/osmotic regulation and immunity. Also, our results provide a morphological background to understand the dependence on aerial respiration of Pomacea canaliculata. Finally, we consider these findings from a functional perspective in the light of the evolution of amphibiousness in the Ampullariidae, and discuss that master regulators may explain the phenotypic convergence of gill
structures amongst this molluscan species and those in other phyla.
Cellular inflammatory response to nonself materials is a universal phenomenon in the Animal Kingdom. To refresh our memories, inflammation, as generally defined, is the reaction of tissues to insult. The process is characterized by local heat, swelling, redness, and pain. These characteristics have been defined as a result of observations on vertebrates, particularly mammals. As our knowledge of inflammation as revealed by studying invertebrates, especially molluscs, has progressed, it is becoming increasingly evident that these characteristics need not all be present, especially conspicuous swelling and redness. Also, I would like to advance the idea that nonspecific inflammatory response does not occur in molluscs or any other group of animals. In other words, there is some degree of specificity at the molecular level in all instances of cellular inflammation. The evidences for this statement are presented later. At this point, I wish to review in a conceptual manner the various phenomena which collectively comprise cellular response to insult in molluscs. Specifically, as depicted in Fig. 1, I intend to point out that cellular infiltration and subsequent events in molluscs, as in other groups of animals, involves (1) chemotactic attraction of reaction cells to the insulting agent, (2) surface recognition of self from nonself on the part of reaction cells and attachment of the nonself insulting agent to such cells, (3) endocytosis or encapsulation, and (4) intracelluar events leading to degradation and elimination of the insulting agent. As will become apparent at a later point, the reason for reviewing these phases of cellular reaction to challenge is that in terms of modern cell biology, there appears to be specific recognition sites involved at each phase, hence my thesis that nonspecific inflammatory response per se does not occur.
The researcher must exercise care when using terminology that has been developed for use in mammalian pathology when describing what appear to be similar lesions and outcomes within invertebrate species. This theme has been discussed by several authors (53, 54) and is critical when making comparisons between mammalian and invertebrate species. As stated above there is no justification for the use of mammalian hematopoietic terms in describing invertebrate blood cells. This problem can be illustrated by the incorrect usage by some authors of the term lymphocyte in invertebrate literature. There is no evidence that the invertebrate blood cells are homologous to either the mammalian lymphocyte or any of the other formed blood elements. With this theme in mind several pathologic terms that can be used for describing lesions in both vertebrates and invertebrates are defined below. A tumor is any swelling and should not be equated with the word neoplasm. Neoplasia literally means new growth and is the pathologic process resulting in a neoplasm.
There are a number of characteristics which should be considered in a definitive classification of hemocytes [40], but the initial criterion is most often morphology. Although no general agreement on the number of different cell types in oyster hemolymph has been reached, most investigators divide the hemocytes into at least two major classifications: the granular and the agranular [7,40]. There are apparent differences in the roles that granular and agranular cells play in any organism, and those roles are not necessarily the same for each species. The granular hemocytes are generally larger than the agranular cells and contain walled vesicles (granules) in the cytoplasm. Using light microscopy, investigators have also been able to distinguish acidophilic and basophilic granulocytes [7], stem cells [2], slightly granular cells [40], and differences based on nuclear size [28]. But even with ultrastructural investigation, these studies of oyster hemocytes have not led to a generally accepted scheme of nomenclature and classification. Rather, they have emphasized differences between oyster species and raised conflicting evidence within species. For example, there are major disagreements between invertigations examining the ultrastructure of Crassostrea virginica agranular hemocytes [27,34,48]. Also, the easily distinguishable C. virginia granulocytes was first beleived to be two cell types [30] due to its altered morphology upon degranulation [7].
Endocytosis is a widespread cellular function in which vesicles and vacuoles formed by the plasma membrane regulate the uptake of molecules in a cell’s environment. Most eucaryotic cells have this function; however, among mammalian cells, in which it has been mostly thoroughly studied, it is most prominent in leucocytes, ma-crophages, capillary endothelial cells, thyroid epithelial cells, yolk sac cells, and oocytes (Silverstein et al., 1977). Endocytic activity is usually divided into two categories. Phagocytosis, or “eating”, is used to describe the uptake of large particles. This uptake occurs by close apposition of a segment of the plasma membrane to the particle’s surface, while excluding most, if not all, of the surrounding fluid (Silverstein et al., 1977). Particle size has been described as having the range of 0.01 μm to 10 μm and may include microorganisms (from viruses to bacteria and fungi), as well as inert particles (Cohn, 1972). The term pinocytosis, or “drinking”, is used to describe vesicular uptake of everything else. This may include small particles (such as lipoproteins, ferritin, colloids, and immune complexes), soluble macromolecules (e.g., enzymes, hormones, antibodies, yolk proteins, and toxins), and low molecular weight solutes. It is assumed that uptake of extracellular fluid is always included in pinocytosis (Silverstein et al., 1977). Since little information exists on in vitro pinocytosis in molluscan hemocytes, aside from the work of Feng (1965), this discussion will focus on that part of the endocytic process termed phagocytosis for which more information is available.
The mosquito is an ideal laboratory model for the study of innate cellular defense mechanisms among the Metozoa. Although there may be nonspecific protective humoral factors present in the hemolymph of mosquitoes, there is presumably no specific humoral or antibody system as part of their arsenal of internal defense. In addition, both man and mosquitoes are susceptible to infection by parasites of the genus Plasmodium. In both mammals and mosquitoes, the malaria sporozoite must migrate through the circulation to reach a target organ (liver or salivary gland), and is therefore potentially susceptible to the cellular defense mechanisms of both hosts. This fortunate circumstance affords an opportunity to study the response of a purely cellular internal defense mechanism to the same stage of the same parasite which successfully evades the defense mechanisms of both mammals and mosquitoes.
: Contributed herein is an ultrastructural description of Haplosporidium nelsoni (MSX), a highly pathogenic parasite of the American oyster, Crassostrea virginica. In addition, the results of an electron microscope study of host cells, particularly hemocytes, which interact with H. nelsoni , are being reported. A hypothesis is being advanced as to the pathogenicity of the MSX disease once it penetrates the basal lamina and reaches the systemic stage.
Perkinsus marinus can exist in huge numbers in the hemolymph of its host, Crassostrea virginica. It is readily recognized and phagocytized by the hemocytes; however, these putative immunocytes seem unable to destroy this parasite, since the infection is rarely controlled. Some of the defense responses known to be available to the oyster are discussed in relation to P. marinus infection. The picture that emerges is far from clear, and it seems likely that new approaches to studying oyster defense physiology will be required before the mechanisms underlying susceptibility/resistance to dermo disease will be defined. The situation is further complicated by the fact that ambient environmental conditions exert profound effects on both the parasite and the activity of the oyster's defense system. Perhaps the parasite plays a direct role in suppressing the host's defenses by not triggering or by scavenging the production of reactive oxygen species, by regulating immune responses by excretory/secretory factors, or by releasing proteolytic enzymes. The dynamic interplay of other factors of host and/or parasite origin may also influence the progression of dermo disease.
Foreign elements, molecules, or organisms naturally or experimentally introduced into naive molluscs are, although with exceptions, phagocytosed (see reviews by Cheng, 1975, 1981). Of the two categories of hemocytes common to all bivalves, hyalinocytes and granulocytes (Cheng, 1981), the latter in Crassostrea virginica and Mercenaria mercenaria are found to be the most active from the standpoint of phagocytosis (Foley and Cheng, 1975). There are three ways, involving semi-permanent filopodia, classical endocytosis and afunnel-like pseudopodia, by which bacteria are engulfed by hemocytes. The uptake mechanism has been studied at the electron microscope level in Crassostrea gigas (Ruddell, 1971; Feng et al., 1977), C. virginica (Cheng and Cali, 1974; Cheng, 1975), and Mytilus coruscus (Feng et al., 1977). Furthermore, as a rule, such phagocytosed materials, if digestible, are degraded by intracellularly, and some are removed when cells containing such materials migrate to the exterior through epithelial borders (Cheng, 1977a). Since granulocytes are more phagocytic than hyalinocytes, and one of the major differences between these two types of cells is the occurrence of large numbers of cytoplasmic granules in granulocytes, further studies to resolve the nature of these granules revealed that those in C. virginica are electron-lucid vesicles each of which possesses a complex wall (Feng et al., 1971; Cheng and Cali, 1974; Cheng et al., 1974; Cheng, 1975). However, the granules of M. mercenaria granulocytes, which are membrane-bound vesicles containing a homogeneously electron-dense substance (Cheng, 1975; Cheng and Foley, 1975), have subsequently been demonstrated to be true lysosomes (Yoshino and Cheng, 1976). Furthermore, it has been established that when challenged with foreign materials, there is hypersynthesis of certain lysosomal hydrolases within the hemocytes and their subsequent release into serum (Cheng et al., 1975; Cheng and Butler, 1979; Cheng and Mohandas, 1985; Mohandas and Cheng, 1985). These lysosomal hydrolases, which are associated with intracellular degradation (Cheng and Cali, 1974; Cheng et al., 1974), have also been demonstrated to have antimicrobial properties (McDade and Tripp, 1967; Cheng, 1978).
Although extensive studies have been performed on human erythrocytes, there is a shortage of information on marsupial erythrocytes. Studies on haematology and biochemistry are useful in the ecomanagement of these animals especially those in wildlife parks and zoos. The present review summarises our findings from ∼30 species of marsupials. As marsupials show great diversity in physical and behavioural characteristics, it is not surprising that examination of their red blood cells reveals variation in the biochemical features. Many variations in red cell biochemistry appear to be species specific and, although interesting, are too numerous to list here and have been discussed in the relevant sections. Red blood cells of several species of marsupial differ from those in placental mammals by having higher haemoglobin concentration and haematocrit, lower ATP and higher 2,3-diphosphoglycerate concentrations. These features are consistent across the majority of marsupial species, but the relevance of these variations from red cell metabolism in the placental mammals is unknown.
In this study, the subpopulations of coelomocytes of sea cucumber Apostichopus japonicus were identified with differential-interference microscope and flow cytometry (FCM). The immunostimulatory potential of crude lentinan (cLNT) from shiitake mushrooms (Lentinus edodes) on A. japonicus was then investigated both in vitro and in vivo. In vitro, the coelomocytes of A. japonicus were incubated with different concentrations of cLNT (0, 2.5, 7.5, 22.5 mg/L) for 1 h, and immuno assays were performed. Flow cytometric analysis revealed that phagocytic activity and viability of coelomocytes increased significantly with cLNT treatment. Biochemical analysis showed a significant increase in peroxidase and alkaline phosphatase activity in coelomocytes. In vivo, the immunostimulatory effect of cLNT on coelomocytes was investigated with FCM by feeding A. japonicus with cLNT-supplemented or control diet for 24 days. The phagocytic activity of coelomocytes in cLNT treatment group was significantly increased at 4, 8 and 12 days of feeding. The viability of coelomocytes was increased after 8 days of feeding. At 4, 8, 12 and 18 days of feeding, the number of small-sized and low granularity coelomocytes was reduced, while the number of large-sized and high granularity coelomocytes was increased compared to control diet. Collectively, our in vitro and in vivo studies suggest that cLNT derived from L. edodes clearly reveals immunostimulating effects on A. japonicus by increasing the viability and phagocytic activity of coelomocytes, and these findings suggest that dietary supplementation of cLNT might further improve the commercial production of sea cucumbers.
1.1. Acid phosphatase is naturally present in the haemocytes and haemolymph of Patella vulgata.2.2. Haemocyte number and acid phosphatase level in the haemocytes and haemolymph rose after challenge with bacteria.3.3. Haemocytes cultured in vitro with bacteria did not secrete acid phosphatase into the culture medium, but changes in cellular acid phosphatase levels did occur.
This study was carried out to ascertain whether any significant fluctuations in the levels of activity of acid phosphatase, alkaline phosphatase, lysozyme, β-glucuronidase, glutamic-pyruvic transaminase, and glutamic-oxalacetic transaminase from head-foot tissues of the terrestrial gastropod Theba pisana from Israel could be correlated with the winter mass mortalities. It has been determined that there is an elevation in alkaline phosphatase activity during the colder seasons (spring, autumn, and winter) but this cannot be associated with mass mortality. Also, there is a steady rise in glutamic-pyruvic transaminase activity in the spring, terminating in winter prior to death. This is interpreted to reflect a conpensatory metabolic shift prior to death. It is now known that the head-foot tissues in T. pisana can contribute certain lysosomal enzymes to the serum via the open circulatory system.
Anti-Bacillus megaterium activity was measured in unfractionated plasma withdrawn from three common US East Coast bivalve molluscs: an oyster Crassostrea virginica and the mussels Geukensia demissa and Mytilus edulis. The activities of the plasma samples from these bivalves were also measured against a C. virginica pathogen Perkinsus marinus. Strong anti-B. megaterium activity was measured in plasma from C. virginica and M. edulis, but was not detected in G. demissa. Bactericidal activity was found in hemocyte extracts from all bivalves in this study, suggesting a cellular origin of cytotoxic humoral factors. Peptides (
Many bivalve species are cultured widely around the world due to their commercial importance. As a result of the increases in areas and production associated with intensive culture, mass mortalities of bivalves caused by a wide variety of agents have become a serious problem. Various infectious diseases represented by protozoan and bacterial diseases have been threatening shellfish culture industries, and hence, many investigations have been made on the defense mechanisms, as well as diseases, in bivalves. The molluscan defense mechanism is comprised of the only innate immunity, which is also dependent on cellular components such as hemocytes possessing phagocytic and bactericidal activities and on humoral defense molecules such as lectin and lysozyme. In this review, we summarize cellular defense mechanisms in bivalve molluscs focusing on hemocytes: 1) morphology and classification, 2) functional profiles (phagocytosis, encapsulation, wound repair), 3) reactive oxygen species, 4) effects of natural and anthropogenic environmental factors, and 5) stress and signaling pathways.
The internal defence system of gastropod molluscs is able to discriminate between self and non‐self. The recognition is carried out principally by both the cellular and humoral components of the haemolymph. Together with freely circulating haemocytes, other defence cells are found scattered throughout the tissue or localized in organs. The present review refers primarily to Planorbarius corneus, since the defence mechanisms presented by this animal are typical of those of the other gastropods studied. P. corneus presents two circulating haemocytes: the spreading (SH) and the round haemocytes (RH); in other gastropods only one cell type is described, and this can be considered as a spreading haemocyte. The haemocytes derive both from a haematopoietic organ and from mature circulating haemocytes. The SH show phagocytic properties, adhere to glass, produce agglutinins, bind Con A and contain muramic acid. The RH have non phagocytic properties, do not adhere to glass, form rosettes with sheep red blood cells, are stimulated to proliferate by PHA and present numerous typical markers of vertebrate T lymphocytes. RH are also able to lyse Cr pre‐labeled K562 target cells in a classical, short‐term, natural cytotoxicity test, and this function is modulated by human recombinant interleukin‐2. Furthermore, SH and RH play a role in the recognition of foreign tissue, the SH are able to encapsulate and phagocytize foreign material, and the RH, with their NK (natural killer)‐like activity, may act like the vertebrate cytotoxic T lymphocytes or NK cells. Thus, it is possible to conclude that RH have characteristics reminiscent of vertebrate T lymphocytes, while SH belong to the category of macrophages. With regards to the humoral component, different factors, such as lysosomal enzymes, lysins and agglutinins or lectins, have been described. In P. corneus, a natural glycoprotein agglutinin has been isolated, whose carbohydrate component contains muramic rather than sialic acid. Moreover, an induced bacterial agglutinin has been purified, although this induction is relatively rare in gastropods. Lysozyme‐like molecules have also been detected and they act like alarm molecules in inflammatory reactions. Taken together, the humoral and cellular investigations, the bacterial clearance studies and the specific responses observed in transplantation experiments are all in favour of the presence of a memory‐type response of short duration. Finally, interrelations appear to exist between the immune and neuroendocrine systems. ACTH and ß‐endorphin‐immunoreactive molecules have been detected in serum and SH, and these molecules appear to play a physiological role in the process of phagocytosis and in stress response.
Anti-Bacillus megaterium activity was measured in unfractionated plasma withdrawn from three common US East Coast bivalve molluscs: an oyster Crassostrea virginica and the mussels Geukensia demissa and Mytilus edulis. The activities of the plasma samples from these bivalves were also measured against a C. virginica pathogen Perkinsus marinus. Strong anti-B. megaterium activity was measured in plasma from C. virginica and M. edulis, but was not detected in G. demissa. Bactericidal activity was found in hemocyte extracts from all bivalves in this study, suggesting a cellular origin of cytotoxic humoral factors. Peptides (< 10 kDa) were separated from the plasma samples by ultrafiltration; weak antibacterial peptide activity was quantified in C. virginica peptides, but not in peptides from the mussels. In the case of P. marinus, plasma from M. edulis or G. demissa was strongly cidal as compared to plasma from C. virginica. This difference in activity probably reflects the low pathogenicity of this oyster parasite for the mussel species tested. In summary, the bactericidal activity of plasma proteins from these bivalves showed considerable interspecies variation and did not necessarily correlate directly with antiprotistan activity. When present, antibacterial and antiprotistan activities seemed to be associated with plasma proteins rather than < 10-kDa plasma peptides, with the possible exception of C. virginica anti-B. megaterium activity and the occasionally expressed anti-P. marinus activity of M. edulis peptides. The precise identity of the plasma protein(s) responsible for the antimicrobial activities measured have yet to be determined, but it is likely that agents other than, or in addition to, lysozyme play significant roles in the process.
The two in vivo bleeding techniques currently in use in our laboratory to diagnose a hematopoietic neoplasm in Mya arenaria are: (1) phase-contrast microscopy with fresh unstained hemocytes, and (2) bright-field microscopy with Giemsa-stained hemocytes. All in vivo diagnoses were checked by histopathological studies on tissues of the same mollusc. For both methods the correct diagnosis (true + or true −) was made in 94 out of 100 clams examined. A gradation of tissue involvement was observed in the diseased clams and the accuracy of the in vivo diagnosis is related to the disease severity. There is a positive correlation between the degree of tissue involvement and the number of circulating neoplastic cells. For this reason the more extensive the neoplasm the better is the ability to diagnose the neoplasm by the in vivo bleeding techniques. Depending on the percentage of neoplastic cells present in the hemolymph, the neoplasm was graded from level 1 to 5, with 5 being the most severe. In general, at level 1, the accuracy of a single in vivo diagnosis varied from 66 to 71% and at level 2, the accuracy of diagnosis varied from 76 to 93%, while at all other levels the accuracy was 100%. The percentage of diseased clams detected by the in vivo bleeding technique was 89–91% and the percentage of nondiseased clams detected was 95%. These values can be further improved by combining the two tests and/or through multiple bleedings. Between the two types of in vivo tests, the Giemsa-stained hemocytes provided better precision of diagnosis than the fresh unstained cells, although the differences were slight.
Particulate components of the coelomic fluid of the marine worm Sipunculus nudus were studied by light microscopy and their phagocytic and enzymatic activities examined. Coelomocytes include haemerythrocytes (about 90%), granulocytes and hyalinocytes (about 9%). In addition, multicellular structures, i.e., urn cell complexes are found with low frequency (about 1%). Haemerythrocytes are nucleated cells, containing the respiratory pigment haemerythrin, which show neither phagocytic nor enzymatic activities. Granulocytes and hyalinocytes (about 7% and 2% of total coelomocytes, respectively) may be distinguished into two different subpopulations, small and large cells, which may appear as round or amoeboid. Small cells appear positive to the anti‐CD34 antibody for stem cells. The present study demonstrated that both circulating granulocytes and hyalinocytes represented main phagocytes, since they were able to recognise and engulf yeast cells in vitro and contain inducible hydrolytic and oxidative enzymes. In addition, both phagocytes produced superoxide anion, and the neutral red retention suggested that granulocyte granules were lysosomes. The phagocytic ability of urn cell complexes appears controversial: their ciliated basal cells can trap numerous yeast cells and show positivity for some hydrolases, but evidence of engulfment has never been observed. Lysozyme‐like activity was found in coelomocyte lysate and to a lesser extent in cell‐free coelomic fluid.
It has been 100 years since the publication of the landmark work of Kellog. Although much has been l e a r n e d about the anatomy and histology of Mercenaria mercenaria since that seminal work, we have a long way to travel on that tortuous pathway known as research. A case in point: although we now know much about the cytology, fine understand the life cycle very well and can spawn animals upon demand throughout the year independent of latitude; yet we have no knowledge concerning the fine structure and functions of follicle and nutritive cells of gonadal acini. In the past 15 years, however, substantial strides have been made in renal cytology, fine structure and general function largely due to the work of M.R Morse and her students. It is expected that investigators will direct concentrated studies of this type to all organ systems and cell types. A Mercenaria genome project should not be in the too distant future. A little more than 50 years ago, Thurlow Nelson stated that the eastern oyster, Crassostrea virginica, was the best known marine animal in the world; I expect that with continued and expanded research on thiscomplex, hearty and commercially impo mercenaria may well become the marine animal about which we have the most scientific information.
This chapter discusses the pests, parasites, diseases, and defense mechanisms of the hard clam, Mercenaria mercenaria. Clams, mussels, scallops, and oysters are harvested commercially for many centuries and have suffered from disease-caused mortalities throughout that time. The study of bivalve parasites, diseases, and defense mechanisms is relatively recent, however, driven largely by epizootic mortalities of oysters in the United States and Europe in the past half century. Investigations show that each molluskan group becomes infected by a similar array of organisms from viruses to copepods, although relatively few cause disease. The distinction between infection and disease is important. Infection refers to the establishment of a foreign organism in host tissues. Disease indicates damage to a body part, organ, or system, which may or may not be caused by an infectious agent, such that the affected organism no longer functions normally. In fact, an infection does not necessarily lead to disease. Many infectious agents are parasites that may cause localized tissue damage, but relatively little overall harm to their hosts.
The ultrastructure of hemocytes of the freshwater snail Viviparus ater was studied. A single cell type characterized by the presence of thin pseudopods, polymorphic nucleus, and α-glycogen granules was found. Moreover, cytoplasmic granules with different morphologies were observed, suggesting different stages of granule maturation. These granules show variable amounts of acid phosphatase reaction products. Due to its morphological and functional characteristics, this cell type belongs to the spreading hemocytes described in other gastropod molluscs, confirming light microscopy observations.
Large numbers of the soft-shelled clam Mya arenaria from New Bedford Harbor, Massachusetts, exhibit a blood cell disease referred to as hematopoietic neoplasia (HN). Diseased hemocytes differ morphologically from normal hemocytes by their high nucleocytoplasmic ratio. The phagocytic activity, one of the internal defense mechanisms, was compared in the two populations. Surface receptors, essential for recognition during phagocytosis, were demonstrated for both populations using Con A staining. Adherence and uptake were measured after in vitro incubation with yeast. Examination of hemocytes with scanning electron microscopy showed that yeast cells adhered only to the surface of normal hemocytes, and not to diseased hemocytes. Quantitative analysis of adherence and uptake of yeast by hemocytes showed that diseased hemocytes were unable to adhere and ingest yeast. This is thought to be caused by differences in cytoskeletal organization. Finally, the activity of four lysosomal enzymes was used to evaluate intracellular degradation in normal and diseased hemocytes. Histochemical analyses showed diseased hemocytes to have higher than normal acid phosphatase, nonspecific esterases and β-glucuronidase activity. These results indicate that enzyme activity cannot be directly correlated to intracellular degradation. However, it is possible that stress on the lysosomal system may activate certain enzymes.
Plasma from naı̈ve Penaeus vannamei juveniles lacked antibacterial factors and significantly enhanced the growth of Escherichia coli strain XL1-Blue MRF′ compared with sea water complex (SWC) nutrient medium. In contrast, plasma from prawns previously injected with both live, non pathogenic Vibrio harveyi strain DPEX and formalin-killed cells of V. harveyi strains BP04, DPEX, E. coli and sterile shrimp salt solution (SSS), inhibited bacterial growth. Antibacterial activity appeared within 6 h and was detectable until 7 days after “vaccine” administration. Lysozyme activity was not detected in plasma from vaccinated prawns. Both in vitro and in vivo bacterial killing were facilitated by vaccine administration. Activation of the prophenoloxidase (proPO) system was independent of bacterial virulence.
1.1. Planorbarius corneus were injected at three different times with living Staphylococcus aureus, to study the rates of bacterial clearance from circulation.2.2. After the second (14 days) and third (73 days) bacterial injections, clearance rates were faster and clearance patterns markedly different than after the first one.3.3. The clearance correlated with an increase in haemocyte acid phosphatase and serum LDH activities, while acid and alkaline phosphatase were decreased and α-amylase remained constant.4.4. Variations in free amino acid concentrations were found 2 hr after primary stimulation.5.5. Relevance of these data to the primary and secondary immune responses is discussed.
A scanning electron microscope study of Mercenaria mercenaria granulocytes at 1 and 2 hr postchallenge with a 0.02-ml Millipore-filtered, sterile sea water suspension of heat-killed Bacillus megaterium at a concentration of 4 × 106 bacteria/ml revealed that (1) granulocytes challenged with bacteria revealed more and larger lysosomes protruding from their surfaces than those of the control groups; (2) release of intact lysosomes from granulocytes into serum occurred concurrently with phagocytosis; (3) enzymes released from discharged lysosomes acted on bacterial cell walls causing their partial degradation, thereby enhancing endocytosis; and (4) degranulation is a normal process but is greatly enhanced when stimulated by phagocytosis of bacteria. This study also demonstrated that lysosomes budded off from the plasma membrane of granulocytes into serum, although the actual biochemical mechanism(s) that causes the detachment of lysosomes from the plasma membrane and the subsequent lysis of the double-membraned lysosomes to release the hydrolases remains unresolved.
A monoclonal antibody (MAb 6H7) specific to granulocytes of scallop Chlamys farreri was produced by immunising mice with separated granulocytes as an antigen. Characterised using a flow cytometric immunofluorescence assay, MAb 6H7 reacted to granulocytes by 87.1% of total positive haemocytes. At the ultrastructural level, MAb 6H7 demonstrated epitope in cytoplasmic granules of granulocytes. Western blotting analysis indicated that a peptide of 155 kDa was recognised by MAb 6H7. It was therefore used to investigate granulocyte variation in C. farreri after acute viral necrobiotic virus (AVNV) infection using an enzyme-linked immunosorbent assay. The result illustrated that granulocytes varied greatly by AVNV infection, and their amount significantly increased on day 1 post-injection, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8.
L'étude de la variabilité génétique de la compatibilité entre diverses populations de #Schistosoma haematobium$, #S. bovis$ et #S. curassoni$ et les bulins d'Afrique de l'Ouest (Niger, Côte d'Ivoire, Togo et Sénégal) a été effectuée par la détermination du taux d'infestation des mollusques. Les recherches montrent que des facteurs tels que la structure génétique de l'infrapopulation de schistosomes adultes, le temps de conservation des oeufs, l'âge et la taille du mollusque, la dose miracidiale et l'entretien des parasites sur des hôtes définitifs expérimentaux influencent la réussite de l'infestation miracidiale. L'étude de la compatibilité mollusque-schistosome apparaît essentielle pour l'évaluation des potentialités de transmission de différents mollusques hôtes intermédiaires vis-à-vis des 3 espèces de schistosomes étudiées, pour l'estimation des risques potentiels d'extension des bilharzioses ainsi que pour l'appréciation de la biodiversité des populations de mollusques et de schistosomes. De plus, le marqueur se révèle un outil de choix pour évaluer l'efficacité d'une prophylaxie vaccinale sur le potentiel de transmission de #S. haematobium$ d'un hôte primate au mollusque, après l'immunisation du vertébré avec une protéine recombinante (protéine 5M 28GST). (Résumé d'auteur)
The ultrastructural localization of a range of hydrolytic enzymes has been investigated in the granular haemocytes of the marine mussel Mytilus edulis. Arylsulphatase activity and immunocytochemical localization of beta-glucuronidase and elastase were demonstrated within the large granules of the haemocytes. Lysozyme and cathepsin B were both localized within all sizes of granule, however, at high dilutions the primary antibody against lysozyme was also restricted to the large granules. The labelling density for cathepsin B antibody tended to be very low. Antibodies for cathepsin G showed a clear, discrete labelling which was restricted to the granules of haemocytes containing small granules. The fact that antibodies raised against human proteinases recognize invertebrate enzymes suggests that there must be a certain degree of structural similarity between the human proteinases and the enzymes present in the mussel haemocytes indicating either convergence or conservation of the enzyme molecules. The presence of a range of hydrolytic enzymes including proteinases, glycosidases and sulphatases within the large granules shows that these granules are a form of lysosome. The reduction in activity of lysosomal enzymes in haemocytes following adhesion to glass is evidence for release of the enzymes from the granules (degranulation). The possibility of a serine protease being specifically associated with the small granules and its role as a cytolysin are discussed.
Specimens of Biomphalaria glabrata challenged with 2 μl of heat-killed Bacillus megaterium at a concentration of 9 × 109 bacteria/ml showed a significant elevation of acid phosphatase activity level at 2 hr post-challenge but there were no significant fluctuations of enzyme activity at any of the other four time intervals post-challenge. Substantiating the earlier hypothesis of Cheng [Amer. Zool., 23, 129–144 (1983b); Mar. Tech. Soc. J., 17, 18–25 (1983c)], it is postulated that if the dosage of the challenging agent is high, most of the recognition sites on the cell, nuclear, and/or lysosomal membranes become saturated and hence are unavailable for the recognition of subsequent challenge. Also, there is a burst of enzyme hypersynthesis and subsequent release which is reflected by a significant elevation of serum enzyme activity level which, in the present case, occurred at 2 hr post-challenge.
Pseudomonas aeruginosa, which can induce elevated immunity in Helix, is agglutinated by the snail plasma. Helix clears this microbe from its circulation in typical fashion; clearance kinetics may be slightly more rapid in immunized snails. Such snails may also be more efficient at killing P. aeruginosa in the digestive gland.
The question of utilization of zooxanthellae as a holozoic food source by their tridacnid clam hosts was explored utilizing techniques of electron microscopy and electron microscopical histochemistry. It is apparent from the results that older or senescent zooxanthellae are selectively culled from the algal population of the mantle edge by amoebocytes and are intracellularly digested via amoebocyte lysosomes both in the circulatory system and the interdiverticular spaces of the digestive gland. This process cannot be considered "farming," as figured by earlier work, but rather the slow systematic removal and utilization of degenerate zooxanthellae from the algal population of the clam's mantle edge.Electron photomicrographs of the microvillous surface of the hypertrophied siphons of the Tridacna revealed extensive pinocytosis of fluid and particulate material from seawater bathing the clam. It is suggested a priori that this endocytosed material contributes to the nutrition of the clam.
The hematological parameters of Mercenaria mercenaria of similar size from two geographical areas as well as the morphology and behavior of their leucocytes were studied. On the basis of qualitative and quantitative characteristics, three types of leucocytes, designated as granulocytes, fibrocytes, and hyalinocytes, can be distinguished in both living and stained preparations.A correlation matrix computed between all parameters considered has revealed an insignificant correlation between the dimensions of whole animal and the differential count, packed cell volume, and total cell count as well as an insignificant correlation between the hematological parameters themselves. The only exception is that there is a positive correlation between the packed cell volume and the total cell count.
The hemolymph cells of Mercenaria mercenaria were studied with the transmission electron microscope. Three morphological types of cells, granulocytes, hyalinocytes, and fibrocytes, are distinguishable and their fine structural characteristics are described. However, as a result of analyzing the fine structural features of the so-called fibrocytes of M. mercenaria, i.e., the inclusion of large aggregates of glycogen granules in their cytoplasm and the occurrence of primary phagosomes enclosing partially degraded exogenous material and digestive lamellae, it is suggested that fibrocytes are actually granulocytes which are at the terminal phase of their physiologic cycle relative to the degradation of phagocytized nonself materials. The cytoplasmic granules of M. mercenaria granulocytes are structurally different from those of Crassostrea virginica in that they are delimited by a unit membrane, rather than by a complex wall, and include a homogenously electron-dense material. Lipidlike droplets are reported from both granulocytes and hyalinocytes of M. mercenaria for the first time.
Levels of lysozyme activity have been determined in the serum and cells of untreated Biomphalaria glabrata and in snails that had been challenged with heat-killed Bacillus megaterium and water at 1, 2, and 4 hr postinjection. Lysozyme activities have also been ascertained in sham-injected snails at 1, 2, and 4 hr postchallenge. Our results indicate significant alterations in the serum lysozyme activity levels at 2 and 4 hr postchallenge with bacteria and at 1 hr postinjection of water. Also, there is a significant increase in cell lysozyme activity at 1 hr postchallenge with B. megaterium. It is concluded that lysozyme is released from phagocytes into serum as a result of challenge with B. megaterium. Although the exact role of the released enzyme is uncertain, it is hypothesized that it may serve as a humoral defense molecule.
Specimens of Crassostrea virginica were injected with 14C-labelled Bacillus megaterium, and glycogen was extracted from their hemolymph cells, sera, and body tissues at 6, 16, 24, and 72 hr postinjection.14C was first detected in glycogen extracted from the hemolymph cells and body tissues at 16 hr postinjection and at 24 postinjection in the case of serum glycogen.The data presented support earlier observations that the degradation of phagocytosed bacteria in molluscan hemolymph cells leads to the synthesis of glycogen from sugar of bactirial origin and its eventual release from phagocytes.
The levels of lipase activity in both the cellular and serum constituents of the hemolymph of Biomphalaria glabrata that had been challenged in vitro to beat-killed and sonicated Bacillus megaterium as well as samples challenged with live B. megaterium were ascertained. There were no significant alterations in the levels of enzyme activity in both cells and serum of the samples that had been challenged with sonicated bacteria; however, there was a signficant elevation in the enzyme activity associated with both the cells and serum of hemolymph that had been challenged with live bacteria. It has been concluded that live B. megaterium can stimulate hypersynthesis of lipase, a lysosomal enzyme, in phagocytes of B. glabrata and that this enzyme subsequently is released into serum. Consequently, the hydrolysis of lipid constituents of bacteria could theoretically occur in serum as well as within phagocytes.
The levels of aminopeptidase activity in the serum and hemolymph cells of the American oyster, Crassostrea virginica, have been determined to be 2.89 ± 2.25 and 0.62 ± 0.39 Sigma units/ml, respectively. Low levels of aminopeptidase activity have been demonstrated cytochemically within cytoplasmic granules, i.e., secondary phagosomes, of circulating hemolymph cells. Exposure of whole hemolymph to heat-killed Bacillus megaterium or to sterile sea water results in a significant increase in cellular aminopeptidase activity, with the level of activity being the highest in cells that had been exposed to bacteria. The level of aminopeptidase activity in serum is unaltered in similarly challenged whole hemolymph. It is concluded that aminopeptidase synthesized in cells during phagocytosis or as a result of stimulation by exposure to sea water is not released into the serum but is retained intracellularly. Our studies suggest that it is the intracellular and not the serum aminopeptidases whith are of primary importance in the degradation of B. megaterium.
Activity of the lysosomal enzyme, lysozyme, has been quantitatively determined in the serum and cells of the hemolymph of Mercenaria mercenaria which had been exposed to known quantities of Bacillus megaterium and also in the serum and cells of hemolymph which had not been exposed to bacteria. The results indicate that the level of enzyme activity is greater in serum of hemolymph that had been exposed to B. megaterium and concurrently, there is an equivalent decrease in the level of activity in the cells. This evidence indicates that the amount of lysozyme released from cells into serum is enhanced during phagocytosis of the bacteria.It has also been demonstrated that the release of lysozyme from cells occurs during the process of phagocytosis and is not a delayed phenomenon.Enzyme release by secondary phagosomes is reflected morphologically by what is commonly referred to as degranulation. This process does not involve the rupture of the plasma membrane of the hemolymph cells since biochemical studies have revealed that there is no release of the cytoplasmic enzyme, lactate dehydrogenase.
Fresh hemolymph cells of the pelecypods Crassostrea virginica and Mercenaria mercenaria were exposed to known concentrations of Bacillus megaterium, Escherichia coli, and Staphylococcus aureus in vitro and it was ascertained that all four types of cells of C. virginica and all three types of M. mercenaria became associated with the bacteria. Association is defined as either the first, i.e., contact and adherence, or second, i.e., engulfment, phase of phagocytosis. However, when the surfaces of each type of cell, as well as the percentages of each type in whole hemolymph, from both species of molluscs are taken into consideration, it is concluded that the granulocytes are the most important from the standpoint of phagocytosis.When hemocytes of M. mercenaria were exposed to Bacillus megaterium at 4°, 22°, and 37°C, it was found that the association indices were higher at the latter two temperatures. It is postulated, because of the results of Feng and Feng (1974), that nonself materials adhere with less frequency at 4°C and hence are not phagocytosed at this lower temperature.
Quantitative determinations of lipase activity in the sera and hemolymph cell homogenates of Mya arenaria that had been challenged with heat-killed Bacillus megaterium and those of control clams have revealed that there is an increase in both fractions of the hemolymph during phagocytosis.The occurrence of lipase in serum and cells suggests that suitable substrates could be degraded by hydrolysis extracellularly as well as within phagocytes.
Acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase) activity within electron-opaque, membrane-bound vesicles of Mercenaria mercenaria granulocytes has been localized by employing cytochemistry at the light and electron microscope levels. These vesicles can now be considered lysosomes. They presumably function, at least in part, as storage organelles for acid hydrolases, and are therefore analogous to the granules in mammalian polymorphonuclear and monocytic leucocytes. Lysosomes containing acid phosphatase are probably the sources of this enzyme found in cellular and serum fractions of the hemolymph of M. mercenaria, although the mechanism for enzyme release remains uncertain.