Phagocytic activities of hemocytes from the deep-sea symbiotic mussels Bathymodiolus japonicus, B. platifrons, and B. septemdierum (original) (raw)
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Royal Society Open Science
Animals that live in nutrient-poor environments, such as the deep sea, often establish intracellular symbiosis with beneficial bacteria that provide the host with nutrients that are usually inaccessible to them. The deep-sea mussel Bathymodiolus japonicus relies on nutrients from the methane-oxidizing bacteria harboured in epithelial gill cells called bacteriocytes. These symbionts are specific to the host and transmitted horizontally, being acquired from the environment by each generation. Morphological studies in mussels have reported that the host gill cells acquire the symbionts via phagocytosis, a process that facilitates the engulfment and digestion of exogenous microorganisms. However, gill cell phagocytosis has not been well studied, and whether mussels discriminate between the symbionts and other bacteria in the phagocytic process remains unknown. Herein, we aimed to investigate the phagocytic ability of gill cells involved in the acquisition of symbionts by exposing the mu...
Hemocytes released in seawater act as Trojan horses for spreading of bacterial infections in mussels
Scientific Reports, 2020
Global warming has been associated with increased episodes of mass mortality events in invertebrates, most notably in bivalves. Although the spread of pathogens is one of multiple factors that contribute to such mass mortality events, we don't fully understand the pathophysiological consequences of sea warming on invertebrates. In this work, we show that in temperature stress conditions, circulating hemocytes in mussels leave the hemolymph to gain access to the intervalvar fluid before being released in seawater. External hemocytes can survive for several hours in seawater before entering other mussels. When infected by bacteria, externally-infected hemocytes can enter naive mussels and promote bacterial dissemination in the host. These results reveal the existence of a new opportunistic mechanism used by pathogens to disseminate in marine ecosystems. Such mechanisms may explain how thermal anomalies triggered by global warming can favor episodic mass mortality observed in recent years in marine ecosystem. Abbreviations A. ater Aulacomya ater M. desolationis Mytilus edulis desolationis M. marinum Mycobacterium marinum IVF Intervalvar fluid Because of their ability to accumulate xenobiotics in their tissues, their wide distribution, and their ecological importance, bivalve mollusks have long been recognized as good biological indicators for monitoring the effects of pollution and global warming in marine habitats 1. They have also been exploited worldwide for their economic and nutritional values. Given their sensitivity to bacterial pathogens and development of different forms of cancer, including horizontally-transmitted leukemia 2,3 , there has been increasing interest in studying the physiology of their immune system, which has until recently received considerably less attention compared to other Metazoans. All living organisms are constantly threatened by surrounding microorganisms seeking to exploit the same environmental niche. This is particularly true for invertebrates living in marine costal ecosystems where concentrations of bacteria are notoriously high. Survival and spread of marine bacteria, however, have to contend with several antagonistic factors including light exposure, nutrient deprivation, and physical and chemical properties of seawater 4 , notwithstanding viruses, which kill 20-40% of marine bacteria on a daily basis 5. When entering into a host, bacteria further face the innate immune response, a crucial first line of host defense against pathogenic microorganisms. In bivalves, elimination of infectious pathogens by the innate immune system involves physical barriers, production of antimicrobial peptides, and phagocytes, which circulate freely in the hemolymph in direct contact with the animal's tissues where they perform local immune surveillance activities 6. Phagocytic cells are also endowed with the ability to move to other compartments if necessary where they remain functionally competent, allowing them to perform a sentinel role similar to leukocytes in vertebrates 7,8. Several studies have further shown that such migration of hemocytes through the pallial (or intervalvar) epithelium is bi-directional 9. Unfortunately, for pathogens that resist phagocytosis or the action of hydrolytic immune mediators, such bidirectional movement provides an ideal gateway for establishing tissue-specific and/or systemic infection in the host 10. This hypothesis is supported by recent studies showing that infection of hemocytes by some pathogens, such as Perkinsus marinus, can upregulate integrin-mediated cell motility of hemocytes, favoring their
Cell signalling in the immune response of mussel hemocytes
2006
In this work data on immune cell signallling in the circulating hemocytes of the edible bivalve, the mussel Mytilus spp, are summarized. Studies with different bacterial species and strains, heterologous cytokines and natural hormones, as well as with organic environmental chemicals, led to the identification of the role of conserved components of kinase-mediated transduction pathways, including cytosolic kinases (such as MAPKs and PKC) and kinase-activated transcription factors (such as STATs, CREB, NF-kB), in the immune response. From these data a general scenario emerged indicating that close similarities exist in the signalling pathways involved in cell mediated immunity in bivalve and mammalian immunocytes. In particular, the results indicate that both the extent and duration of activation of components of kinase-mediated cascades are crucial in determining the hemocyte response to extracellular stimuli. The identification of the basic mechanisms of immunity and its modulation in mussels can give important information for the possible utilization of these species as an invertebrate model for studies on innate immunity. Moreover, the application of this knowledge to the understanding of the actual adaptive responses of bivalves when exposed to microorganisms in their natural environment can represent significant ecological, economical and public health-related interest.
Differential involvement of mussel hemocyte sub-populations in the clearance of bacteria
Fish & Shellfish Immunology, 2008
Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12 h post-injection for Micrococcus lysodeikticus, 24 h for Vibrio splendidus and more than 48 h for V ibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with À66% 9 h post-injection of living bacteria and À56% 3 h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9 h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (þ90% at 12 h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.
Developmental & Comparative Immunology, 2008
Despite the lack of an adaptive immune system, invertebrates are able to survive among potentially pathogenic viruses, bacteria, and fungi [1, 2], and respond to infection by activating various defence mechanisms [2, 3]. This suggests a key role for their apparently limited innate immune system, in protecting these invertebrates from infections. Among the defence mechanisms described in invertebrates are phagocytosis, activation of the phenoloxydase system, encapsulation, the respiratory burst, nitric oxide production, and the production and release of various microbicidal molecules [3]. It is however unclear which of the different cells integrating the invertebrate innate immune system, usually named hemocytes, are responsible for these cell responses. Different types of hemocytes have been described in various invertebrate species, but an accurate universal classification of invertebrate hemocytes is still lacking. A consensus hemocyte classification is not available yet, due in part to intrinsic differences in the immune systems of even closely related invertebrate species [4-6], and also to differences in the experimental methods used to study them [7-11]. Depending on the species, and the experimental methodology, hemocytes have been classified in as few as two, or up to eleven categories [7, 11, 12]. In molluscs, hemocyte classification has largely depended on cell granularity, determined either by microscopy or by flow cytometry [6, 13-16]. However, as in most invertebrate phyla, no definitive mollusc hemocyte classification exists. Extensive studies have shown that blood cells appear to have conserved roles in vertebrate organisms ranging from fish to mammals [17-19]. In contrast, few attempts
Helgoländer Meeresuntersuchungen, 1984
Hemocytes represent one of the most important defense mechanisms against foreign material in Mollusca. The morphology, hematological parameters and behaviour of hemolymph cells were studied in the southern quahog Mercenaria campechiensis, the eastern oyster Crassostrea virginica, and the blood ark Anadara ovalis challenged with the bacteria Vibrio vulnificus and V. anguillarum. Two general classes of hemocytes (granular and agranular) exist in C. virginica and M. campechiensis. In contrast, A. ovalis possesses 3 general classes (granular, agranular and erythrocytes). Three types of granules were identified by light microscopy. When hemolymph cells were studied by transmission electron microscopy, the cytoplasm of hemolymph cells was noted to contain many organelles, including electron dense granules. Both agranular and granular hemolymph cells were capable of colchicine-sensitive pseudopodial movement and spreading. The results indicate that marine bivalves possess hemolymph blood cells which may play a role in the internal defense paralleling mammalian phagocytes. The morphology of these cells, as determined by light, scanning and transmission electron microscopy, showed some similarity to mammalianmononuclear phagocytes. The sub-cellular events of molluscan hemocyte phagocytosis of V. vulnificus and V~ anguillarum were studied by both scanning and transmission electron microscopy. The role of these cells and the factors which govern their behavior are of economic and public health importance.
Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus
… and Physiology-Part A: …, 2009
The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca 2+ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and wholemount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components.
Hemolymph cell types of the mussel Mytilus galloprovincialis
Diseases of Aquatic Organisms, 1997
Two types of circulating hemocytes were identified in Mytilus galloprovincialis hemolymph: hyalinocytes and granulocytes. The hyalinocytes are agranular cells and show a central nucleus surrounded by relatively small cytoplasm. The granulocytes are larger than hyalinocytes and have smaller nuclei. Three classes of granulocytes (acidophilic granulocytes, basophilic granulocytes and granulocytes containing both types of granules) were distinguished based on staining properties of their cytoplasmic granules. At the ultrastructural level, the hyahnocytes show characteristics of undifferentiated cells. The granulocytes are more differentiated and have membrane-limited cytoplasmic granules. Some granulocytes contain only small granules; others have numerous large granules and some of them contain a mix of granule sizes. Density grad~ent centnfugation allowed the separation of acidophilic granulocytes from the other types. Immunocharacterization demonstrated that the granulocytes are an antigenically heterogeneous population.