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Bacteria–hemocyte interactions and phagocytosis in marine bivalves
Abstract
Marine bivalves (such as mussels, oysters, and clams) are widespread mollusks in coastal waters at different latitudes; due to their filter-feeding habits, they accumulate large numbers of bacteria from the harvesting waters and may act as passive carriers of human pathogens. To cope with this challenge, bivalves possess both humoral and cellular defense mechanisms with remarkably effective capabilities. The circulating cells, or hemocytes, are primarily responsible for defense against parasites and pathogens; microbial killing results from the combined action of the phagocytic process with humoral defense factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), toxic oxygen intermediates, and various antimicrobial peptides. In this work, current knowledge of the mechanisms underlying the interactions between bacteria and the hemolymph components of marine bivalves is summarized. Bacterial susceptibility to hemolymph killing in different bivalve species may be a consequence of the different ability of bacterial products to attract phagocytes, the presence or absence of specific opsonizing molecules, the hemocyte capability to bind and engulf different bacteria, and the different bacterial sensitivity to intracellular killing. The role of soluble (e.g., agglutinins and opsonins) and surface-bound factors in bacterial phagocytosis by hemocytes of the most common marine bivalve species is described and the possibility that environmental temperatures and other seasonal factors may influence this process is considered. Moreover, the potential strategies used by bacteria to evade phagocytic killing by hemocytes are discussed. From the available data it is clear that several questions need further investigation; the elucidation of the factors influencing phagocytosis in bivalves and the fundamental strategies used by bacteria to escape hemolymph killing are important not only to understand bivalve immune defenses but also to explain the persistence of pathogenic bacteria in bivalve tissues and to predict the consequent impact on human health.
... Phagocytosis is an effective cellular defence against pathogen invaders and foreign material. This process recognizes and eliminates non-self-components, such as invading microorganisms (Canesi et al., 2002;Ottaviani, 2006). In phagocytosis, the phagosome (the vacuole that contains the phagocytosed particles in the cytoplasm) fuses with the lysosome (cell organelle containing hydrolytic enzymes in the membrane) resulting in the intracellular digestion of the ingested microorganisms (Canesi et al., 2002). ...
... This process recognizes and eliminates non-self-components, such as invading microorganisms (Canesi et al., 2002;Ottaviani, 2006). In phagocytosis, the phagosome (the vacuole that contains the phagocytosed particles in the cytoplasm) fuses with the lysosome (cell organelle containing hydrolytic enzymes in the membrane) resulting in the intracellular digestion of the ingested microorganisms (Canesi et al., 2002). Some studies have shown that there are functional differences between bivalve haemocytes to recognize and eliminate pathogens. ...
... finding supports the work of Tame et al. (2015) who also reported that haemocytes are phagocytic cells, which eliminate foreign microorganisms that invade the haemolymph and tissue. According to Canesi et al. (2002) and Pruzzo et al. (2005), bivalves can eliminate pathogens from their tissues through haemocytes, which can bind, phagocytize and kill the bacteria, microbes and other pathogens. ...
Perna canaliculus is endemic to New Zealand and is the top shellfish export product. However, the growth of this industry is being adversely affected by summer mortality events. To assess these health threats, differential haemocyte counts (DHC) were performed on haemolymph smears stained with Giemsa, and in vitro phagocytosis activity assays were applied in P. canaliculus for several months covering three seasons (winter, spring and summer). A new optimized classification scheme was developed for P. canaliculus haemocytes where 55% eosinophilic and 27.2% basophilic granulocytes were identified. Eosinophilic granulocytes were classified as dense, semi and small semi, and these new categories were reported herein for the first time in this species. A new haemocyte type (mixed granulocytes), which contains both acidophilic and basophilic granules, is proposed for P. canaliculus. The phagocytosis percentages were significantly affected by incubation time, indicating an increase in zymosan particle uptake from 18.42 ± 1.7% after 30 min, 32.08 ± 3.1% after 60 min and 44.74 ± 3.5% (maximum) after 120 min of incubation. The lowest phagocytosis was observed in the winter season and the highest phagocytosis in summer. This study findings provide a better understanding of the immune function of P. canaliculus haemocytes and serve as a reference for further investigations on the impacts of seasonal variations on haemocyte activity and phagocytosis.
... Finally, the suitability of label-free nanoplastics for biological studies has been assessed by in vitro exposure experiments with Mytilus galloprovincialis hemolymphatic cells in a time interval up to 6 h. The nanoplastic internalization into these cells, known to be provided with phagocytic activity [35,36], has been assessed by fluorescence microscopy. have a key role in photochemical and photophysical processes in polyethylene terephthalate powder. ...
... Finally, the suitability of label-free nanoplastics for biological studies has been assessed by in vitro exposure experiments with Mytilus galloprovincialis hemolymphatic cells in a time interval up to 6 h. The nanoplastic internalization into these cells, known to be provided with phagocytic activity [35,36], has been assessed by fluorescence microscopy. Scheme 1. Schematic representation of the present study. ...
... Hemolymph, being the internal circulating body fluid of these organisms, is responsible for the pollutant transport and distribution to the whole body. In particular, hemolymphatic cells, being involved in the internal defense system of the organisms, are known to be provided with phagocytic activity [35], whose defensive function accounts for elimination of non-self-components from the body. Nanoparticle endocytosis by mussel hemolymphatic cells has been very recently demonstrated with fluorescent-labeled polystyrene nanoparticles of different sizes, from 50 nm to 1 μm [36]. ...
This work contributes to fill one of the gaps regarding nanoplastic interactions with biological systems by producing polyethylene terephthalate (PET) model nanoplastics, similar to those found in the marine environment, by means of a fast top-down approach based on mechanical fragmentation. Their size distribution and morphology were characterized by laser diffraction and atomic force microscopy (AFM). Their autofluorescence was studied by spectrofluorimetry and fluorescence imaging, being a key property for the evaluation of their interaction with biota. The emission spectra of label-free nanoplastics were comparable with those of PET nanoplastics labeled with Nile red. Finally, the suitability of label-free nanoplastics for biological studies was assessed by in vitro exposure with Mytilus galloprovincialis hemolymphatic cells in a time interval up to 6 h. The nanoplastic internalization into these cells, known to be provided with phagocytic activity, was assessed by fluorescence microscopy. The obtained results underlined that the autofluorescence of the model PET nanoplastics produced in the laboratory was adequate for biological studies having the potential to overcome the disadvantages commonly associated with several fluorescent dyes, such as the tendency to also stain other organic materials different from plastics, to form aggregates due to intermolecular interactions at high concentrations with a consequent decrease in fluorescence intensity, and to dye desorption from nanoparticles. The results of the autofluorescence study provide an innovative approach for plastic risk assessment.
... Bivalves have an open circulatory system populated by hemocytes, the quintessential component of bivalve immunity. Hemocytes function in several additional physiological processes including wound healing, biomineralization, nutrition, and transport (1)(2)(3)(4). Hemocytes in the hemolymph, the blood of bivalves, are well studied and known to serve as the main constituent of innate immunity (3,(5)(6)(7). Hemocytes release a broad range of proteins including immune effectors into the plasma of hemolymph, contributing to humoral immunity of bivalves (1,6,8). ...
... Hemocytes function in several additional physiological processes including wound healing, biomineralization, nutrition, and transport (1)(2)(3)(4). Hemocytes in the hemolymph, the blood of bivalves, are well studied and known to serve as the main constituent of innate immunity (3,(5)(6)(7). Hemocytes release a broad range of proteins including immune effectors into the plasma of hemolymph, contributing to humoral immunity of bivalves (1,6,8). Hemocytes are also found in the extrapallial fluid (EPF), an aqueous microenvironment between the mantle and the shell which is the site of shell formation. ...
... It is already well established that hemocytes in hemolymph are main components of immune response in bivalves (3,6,(61)(62)(63)(64)(65). Both the transcriptome and proteome generated in the current study reflected this, with many immune related genes having higher expression at both the mRNA and protein levels in hemolymph as compared to EPF. ...
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 Ca 2+ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca 2+ 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.
... Hemocytes are an important part of the innate immune response of bivalves. Hemocytes act through phagocytosis (Hanington et al., 2010) and release cytotoxic molecules, such as nitric oxide and hydrogen, to participate in immune response (Canesi et al., 2002). Hemocytes located in areas in close contact with external microorganisms act as sentinels for any impending infection and migrate to any infection site (Allam and Espinosa, 2016). ...
... The autophagy response of hemocytes plays a key role in the process of resisting potential pathogens in bivalves, and it determines the expression of autophagy-related genes and signaling pathways (Moreau et al., 2015;Balbi et al., 2018;Liu et al., 2020). Although hemocytes are known to have an effect on the immune system, the efficiency of defense comes from the activation of hemocytes and the components dissolved in serum, such as lectins, antioxidant enzymes, reactive oxygen species, and lysosomal enzymes (Mitta et al., 2000;Canesi et al., 2002). At the first glance, similar to that of other invertebrates, the structure of the existing immune system of bivalves is relatively simple and contains only the innate immune system, lacking an adaptive immune system (Pipe and Coles, 1995). ...
The polyamine putrescine (Put) is a ubiquitous small cationic amine. It plays an essential role in controlling the innate immune response. However, little is known about its function in mollusks. In this study, the Put content was observed to increase in the serum of pearl oyster Pinctada fucata martensii after 6 and 24 h of lipopolysaccharide (LPS) stimulation. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) increased, and nitric oxide synthase was downregulated in the Put group (i.e., combined treatment with Put and LPS) compared with that in the LPS group (i.e., combined treatment with phosphate-buffered saline and LPS). Furthermore, activities of alkaline phosphatase and acid phosphatase were inhibited after 6 h of LPS stimulation. The expression levels of the nuclear factor kappa B, IκB kinase, Janus kinase, and signal transducer and activator of transcription proteins genes were all significantly suppressed at 12 and 24 h in the Put group. Pseudomonas aeruginosa and Bacillus subtilis grew better after being incubated with the serum from the Put group than that from the LPS group. Additionally, the Put treatment remarkably inhibited the autophagy of hemocytes mediated by the AMP-activated protein kinase-mammalian target of rapamycin-Beclin-1 pathway. This study demonstrated that Put can effectively inhibit the inflammatory response induced by LPS in pearl oysters. These results provide useful information for further exploration of the immunoregulatory functions of polyamines in bivalves and contribute to the development of immunosuppressive agents.
... Hemoglobin of T. granosa contributes to the host antimicrobial defense through the peroxidase activity and certain peptides released from the proteins (Bao et al., 2013;Bao et al., 2016;Wang et al., 2021). Hemocytes in bivalves are capable of encapsulating pathogens and producing cytotoxic molecules, including reactive oxygen species, antimicrobial peptides, and inflammatory cytokines, which are involved in the killing and elimination of pathogens (Canesi et al., 2002;de la Ballina et al., 2022;Song et al., 2010). ...
... Hemoglobin of T. granosa contributes to the host antimicrobial defense through the peroxidase activity and certain peptides released from the proteins (Bao et al., 2013;Bao et al., 2016;Wang et al., 2021). Hemocytes in bivalves are capable of encapsulating pathogens and producing cytotoxic molecules, including reactive oxygen species, antimicrobial peptides, and inflammatory cytokines, which are involved in the killing and elimination of pathogens (Canesi et al., 2002;de la Ballina et al., 2022;Song et al., 2010). ...
... Humoral factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), and various antimicrobial peptides (Canesi et al., 2002) are considered as important indicators to investigate the humoral immunity of invertebrates. Prolonged hypoxia causes significant decrease of antioxidant and other humoral immune responses has been demonstrated in many invertebrates including the bivalves Arctica islandica, P. perna, M. coruscus and Hyriopsis cumingii (Philipp et al., 2012;Hu et al., 2015;Sui et al., 2016;Nogueira et al., 2017;Li et al., 2022b), as well as the crabs M. japonicus, Callinectes sapidus, and Scylla paramamosain (Tanner et al., 2006;Cheng et al., 2020;Wang et al., 2022). ...
Hypoxia and sulfide are inducing potential damage to aquatic organisms. However, the effects of hypoxia and sulfide on their immune systems and molecular mechanisms are not fully understood. In the present study, the clam Tegillarca granosa was exposed to hypoxia alone or in combination with sulfide (0.1, 0.5 mM) to investigate the physiological and transcriptomic responses in haemolymph. The IBR analysis revealed that moderate sulfide stimulated immune responses via increasing the total hemocyte counts, phagocytic activity, antibacterial activity, and antioxidant activity. The transcriptomic analysis revealed many critical signaling pathways (Toll and Imd, FoxO, NLR) and biological processes (antimicrobial/antibacterial peptide, interferon, interleukin, leukoc yt e, lymphocyte, mitophagy) involved in the immunostimulation. Our results would offer insights into the sulfide-tolerant molecular mechanisms in this species and provide a useful tool for assessing the integrated biological impacts of hypoxia and sulfide on shellfish.
... Additionally, it has been demonstrated that changes in salinity, pH and temperature affect immunity in marine bivalves [12]. Since marine invertebrates rely mainly on their innate immune system to confront microorganism infection, phagocytosis is the basal immune mechanism that copes with pathogens [13,14]. During the process of phagocytosis, reactive oxygen species (ROS) production, a process known as respiratory burst (resulting from the interaction of several substrates with the NADPH-oxidase multiprotein complex) is taking place in tissues [7,[15][16][17]. ...
Background: The effects of climate change negatively affect marine bivalves’ health. Lately, the intensity of marine heatwaves poses serious threats to the aquatic environment setting of high-risk bivalve farming. Since temperature increases can jeopardize bivalves’ immunity response, pathogen infection becomes more evident. Reactive oxygen species (ROS) production, increased during the process of phagocytosis, is confronted by the animals’ antioxidant defense system. However, apart from pathogenic infections, antioxidant defense responses are also induced by seawater temperature increases; Methods: To enlighten the antioxidant status of Mytilus galloprovincialis originating from mortality events enhanced by intense heatwaves in Thermaikos Gulf, northern Greece, along with Marteilia refringens infection, we examined the expression of genes related to antioxidant defense (catalase, CuSOD and mt10) along with the lipid peroxidation levels and activity levels of antioxidant enzymes (catalase, SOD and glutathione reductase); Results: Our results exhibited increased levels of all these biomarkers. This increase was intensified in the Marteilia infected individuals; Conclusions: Our findings shed light on the oxidative and antioxidant status of farmed mussels led to mortality in the context of Marteilia infection. The latter is augmented by the synergistic effect of heatwaves causing a significant increase in oxidative damage and subsequent antioxidant defense.
... Phagocytosis is another immune mechanism of bivalves. Hemocytes can efficiently remove pathogens through phagocytosis followed by release of antibacterial compounds, lysosomal degradation and cytotoxic activities including oxidative burst activity (Canesi et al., 2002;Hégaret et al., 2003;Allam and Raftos, 2015). A few in vitro researches have indicated this fact that hemocytes are capable to clear nanoparticles and their aggregates through phagocytosis (Canesi et al., 2010;Sendra et al., 2018;Auguste et al., 2021). ...
... The PI3K-Akt signaling pathway plays a central role in immune regulation by controlling the proliferation, differentiation, and migration of immune cells (Song et al., 2005;Vergadi et al., 2017;Bao et al., 2022a). Previous studies have shown that the PI3K-Akt signaling pathway in mollusks is involved in and regulates immune processes (Canesi et al., 2002;Fukao and Koyasu, 2003;Troutman et al., 2012). For example, the PI3K-Akt signaling pathway can regulate phagocytosis during the immune response elicited by organismal stimulation. ...
Sepia esculenta is a cephalopod widely distributed in the Western Pacific Ocean, and there has been growing research interest due to its high economic and nutritional value. The limited anti-stress capacity of larvae renders challenges for their adaptation to high ambient temperatures. Exposure to high temperatures produces intense stress responses, thereby affecting survival, metabolism, immunity, and other life activities. Notably, the molecular mechanisms by which larval cuttlefish cope with high temperatures are not well understood. As such, in the present study, transcriptome sequencing of S. esculenta larvae was performed and 1,927 differentially expressed genes (DEGs) were identified. DEGs were subjected to functional enrichment analyses using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The top 20 terms of biological processes in GO and 20 high-temperature stress-related pathways in KEGG functional enrichment analysis were identified. A protein-protein interaction network was constructed to investigate the interaction between temperature stress-related genes. A total of 30 key genes with a high degree of participation in KEGG signaling pathways or protein-protein interactions were identified and subsequently validated using quantitative RT-PCR. Through a comprehensive analysis of the protein-protein interaction network and KEGG signaling pathway, the functions of three hub genes (HSP90AA1, PSMD6, and PSMA5), which belong to the heat shock protein family and proteasome, were explored. The present results can facilitate further understanding of the mechanism of high temperature resistance in invertebrates and provide a reference for the S. esculenta industry in the context of global warming.
... These two groups of cells were found in Mya arenaria [2], Mytilus edulis [3], M. galloprovincialis [4], Bathymodiolus japonicus [5], Tridacna derasa, Hippopus hippopus, Corculum cardissa [6], Ruditapes decussatus [7], Mercenaria mercenaria [8], Anodonta cygnaea [9], Meretrix lusoria and Crassostrea gigas [10], Panopea globosa [11], Pinna nobilis [12] and others. However, some authors also had found and described intermediate cell types between granulocytes and agranulocytes [13][14][15]. For example, researchers still can't agree on the existence of granulocytes in scallops, because the cells found in their hemolymph do not have the distinctive features of granulocytes, in contrast to other bivalves. ...
Bivalves, such as Modiolus are used as indicator organisms to monitor the state of the marine environment. Even though hemocytes are known to play a key role in the adaptive and protective mechanisms of bivalves, these cells are poorly studied in horse-mussel Modiolus kurilensis. In this paper, we present classification of horse-mussel hemocytes based on their immune functions, including the production of specific immune-related molecules, as well as their morphological composition after isolation by density gradient centrifugation. An effective fractionation protocol was adapted to separate four hemocyte subpopulations with distinct morphofunctional profiles. First subpopulation consisted of small under-differentiated hemoblasts (2.20 ± 0.85%) with a bromodeoxyuridine positive nucleus, and did not show any immune reactivity. Second was represented by agranulocytes (24.11 ± 2.40%), with evenly filled cytoplasm containing a well-developed protein-synthesizing apparatus, polysomes, smooth endoplasmic reticulum and mitochondria, and positively stained for myeloperoxidase, acidic proteins, glycogen and neutral polysaccharides. Third subpopulation consisted of eosinophilic granulocytes (62.64 ± 9.32%) that contained the largest number of lysosomes, peroxisomes and vesicles with contents of different density, and showed the highest phosphatase, reactive oxygen species (ROS) and phagocytic activities. Lastly, fourth group, basophilic granulocytes (14.21 ± 0.34%), are main producers of lectin-like protein MkC1qDC, recently discovered in M. kurilensis and characterized by pronounced antibacterial and anticancer activity. These cells characterized by intracytoplasmic of the MkC1qDC localization, forming granule-like bodies visualized with specific antibody. Both granulocytes and agranulocytes showed phagocytic activity and ROS production, and these reactions were more pronounced for eosinophilic granulocytes, suggesting that this group is the key element of the cell-mediated immune response of M. kurilensis. Our results support a concept of bivalve's hemocyte specification with distinct phenotypes.
... In most bivalve species, granulocytes generally exhibited higher phagocytosis activity to pathogens or foreign particles than hyalinocytes [36]. Among granulocytes, the eosinophilic granulocytes exhibited a higher phagocytosis activity than the basophilic granulocytes in oyster, clams, mussels and cockles [54][55][56][57][58][59][60][61][62][63][64]. However, only granulocytes exhibited phagocytic activity in oyster C. virginica [65], clam Tridacna crocea [66], cockles Cerastoderma edule [67], mussel M. [31,33,[176][177][178][179][180][181][182]188,189,193,194] IL-17 galloprovincialis [63], mussel Pinna nobilis [68] and the pearl oyster Pinctada fucata [69]. ...
Lophotrochozoa is one of the most species-rich but immunologically poorly explored phyla. Although lack of acquired response in a narrow sense, lophotrochozoans possess various genetic mechanisms that enhance the diversity and specificity of innate immune system. Here, we review the recent advances of comparative immunology studies in lophotrochozoans with focus on immune recognition and effector systems. Haemocytes and coelomocytes are general important yet understudied player. Comparative genomics studies suggest expansion and functional divergence of lophotrochozoan immune reorganization systems is not as “homogeneous and simple” as we thought including the large-scale expansion and molecular divergence of pattern recognition receptors (PRRs) (TLRs, RLRs, lectins, etc.) and signaling adapters (MyD88s etc.), significant domain recombination of immune receptors (RLR, NLRs, lectins, etc.), extensive somatic recombination of fibrinogenrelated proteins (FREPs) in snails. Furthermore, there are repeatedly identified molecular mechanisms that generate immune effector diversity, including high polymorphism of antimicrobial peptides and proteins (AMPs), reactive oxygen and nitrogen species (RONS) and cytokines. Finally, we argue that the next generation omics tools and the recently emerged genome editing technicism will revolutionize our understanding of innate immune system in a comparative immunology perspective.
... Invertebrates are the largest group of animals on earth, accounting approximately 95% of all animal species [11,12]. Unlike vertebrates armed with both innate and antigen-antibody based adaptive immunity, invertebrates such as bivalve mollusks primarily rely on phagocytosis executed by hemocytes to fight against pathogen invasion [13][14][15]. Although relative little is known about the immunomodulatory function of GABA in invertebrate bivalves, one case study conducted in the Pacific oyster, Crassostrea gigas, demonstrated that the lipopolysaccharides (LPS) induced elevation in hemocyte phagocytosis was markedly suppressed by GABA administration, suggesting an inhibitory effect of GABA on the process of phagocytosis [16]. ...
Although accumulating data demonstrated that gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, plays an important regulatory role in immunity of vertebrates, its immunomodulatory function and mechanisms of action remain poorly understood in invertebrates such as bivalve mollusks. In this study, the effect of GABA on phagocytic activity of hemocytes was evaluated in a commercial bivalve species, Tegillarca granosa. Furthermore, the potential regulatory mechanism underpinning was investigated by assessing potential downstream targets. Data obtained demonstrated that in vitro GABA incubation significantly constrained the phagocytic activity of hemocytes. In addition, the GABA-induced suppression of phagocytosis was markedly relieved by blocking of GABAA and GABAB receptors using corresponding antagonists. Hemocytes incubated with lipopolysaccharides (LPS) and GABA had significant higher K+-Cl- cotransporter 2 (KCC2) content compared to the control. In addition, GABA treatment led to an elevation in intracellular Cl-, which was shown to be leveled down to normal by blocking the ionotropic GABAA receptor. Treatment with GABAA receptor antagonist also rescued the suppression of GABAA receptor-associated protein (GABARAP), KCC, TNF receptor associated factor 6 (TRAF6), inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα), and nuclear factor kappa B subunit 1 (NFκB) caused by GABA incubation. Furthermore, incubation of hemocytes with GABA resulted in a decrease in cAMP content, an increase in intracellular Ca2+, and downregulation of cAMP-dependent protein kinase (PKA), calmodulin kinase II (CAMK2), calmodulin (CaM), calcineurin (CaN), TRAF6, IKKα, and NFκB. All these above-mentioned changes were found to be evidently relieved by blocking the metabotropic G-protein-coupled GABAB receptor. Our results suggest GABA may play an inhibitory role on phagocytosis through binding to both GABAA and GABAB receptors, and subsequently regulating corresponding downstream pathways in bivalve invertebrates.
... lectins), lysosomal enzymes (e.g. acid phosphatase, lysozyme), ROS and antimicrobial peptides (AMPs) (Canesi et al., 2002). In the mussel Mytilus galloprovincialis, different haemocyte subpopulations were identified by cytofluorimetric analysis of size, granularity, phagocytic activity, and ROS and nitric oxide (NO) production capacity (García-García et al., 2008). ...
Interaction of engineered nanomaterials (ENMs) with the immune system mainly occurs with cells and molecules of innate immunity, which are present in interface tissues of living organisms. Immuno-nanotoxicological studies aim at understanding if and when such interaction is inconsequential or may cause irreparable damage. Since innate immunity is the first line of immune reactivity towards exogenous agents and is highly conserved throughout evolution, this review focuses on the major effector cells of innate immunity, the phagocytes, and their major sensing receptors, Toll-like receptors (TLRs), for assessing the modes of successful versus pathological interaction between ENMs and host defences. By comparing the phagocyte- and TLR-dependent responses to ENMs in plants, molluscs, annelids, crustaceans, echinoderms and mammals, we aim to highlight common recognition and elimination mechanisms and the general sufficiency of innate immunity for maintaining tissue integrity and homeostasis.
... These constituents are, a basal body that anchors the system to the bacterial membranes, an export apparatus which includes a cytoplasmic sorting platform that selects substrates and provides energy for the secretion process, a needle filament which protrudes toward the outside of the bacterial surface and serves as a passage for translocator proteins and, effectors and a translocation pore formed in the eukaryotic cell membrane that allows the entry of virulence effectors into the host cytoplasm [130]. In addition, pathogenic bacteria employ various strategies to avoid phagocytic killing by hemocytes [131]. Specifically, the Phdd bacterium uses two major strategies of virulence factors, the phospholipase D damselysin (Dly) and the pore-forming toxin phobalysin P (PhlyP) which are encoded within the large conjugative plasmid pPHDD1. ...
Infectious diseases in aquaculture could be associated with high mortalities and morbidity rates, resulting in negative impacts to fish farming industry, consumers, and the environment. Octopods are reared near marine fish farming areas, and this may represent a major risk since fish pathogens may cause pathologies to octopods. Up to date cephalopods immune defense and pathologies, are incompletely understood. Therefore, the aim of this study was to determine the effect of water temperature and challenge route on hemocyte phagocytosis in vitro after experimental challenge of common octopus with Photobacterium damselae subsp. damselae or Vibrio anguillarum O1. Hemolymph was withdrawn at various time-points post-challenge and the number of circulating hemocytes, and phagocytosis ability were determined. No mortalities were recorded irrespective of pathogen, route of challenge and temperature employed. Great variation was observed in the number of circulating hemocytes of both control and challenged specimens in both experiments (1.04 × 10⁵ to 22.33 × 10⁵ hemocytes/ml for the Photobacterium damselae subsp. damselae challenge and 1.35 × 105 to 24.63 × 105 hemocytes/ml for the Vibrio anguillarum O1 and at both studied temperatures). No correlation was found between circulating hemocytes and baseline control specimens body weight. Probably, the number of circulating hemocytes is affected by many extrinsic, and intrinsic factors such as size, age, maturity stage, natural fluctuations and temperature, as indicated in the literature. The hemocyte foreign particles binding ability observed in Photobacterium damselae subsp. damselae experiments, at 21 ± 0.5 °C and 24 ± 0.5 °C, was (mean ± SD) 2.26 ± 2.96 and 11.72 ± 12.36 yeast cells/hemocyte for baseline specimens and 7.84 ± 8.88 and 8.56 ± 9.89 yeast cells/hemocyte for control and challenged specimens, respectively. The corresponding values for Vibrio anguillarum O1 experiments were (mean ± SD) 6.68 ± 9.26 and 7.00 ± 8.11 yeast cells/hemocyte for baseline specimens and 8.82 ± 9.75 and 6.04 ± 7.64 yeast cells/hemocyte for control and challenged specimens, respectively. Hemocytes of the Photobacterium damselae subsp. damselae and Vibrio anguillarum O1 challenged specimens, were more activated at lower temperature. Apparently, temperature is an important factor in hemocyte activation. In addition, our results indicated that time post challenge, route of challenge and pathogen may influence phagocytosis ability.
... In cluster I, genes associated with phagosome and lysosome were highly expressed in the August samples of resistant oysters, while no obvious waves were detected in susceptible oysters. It has been verified that pathogens bypassing initial defense strategies face phagocytosis by circulating hemocytes (Canesi et al., 2002;Lorgeril et al., 2011). Hydrolytic enzymes, especially lysozymes that contribute to microbial destruction, participate in the elimination of invading bacteria immediately following phagocytosis (Schmitt et al., 2012). ...
Climate change, represented by rising and fluctuating temperature, induces systematic changes in marine organisms and in their bacterial symbionts. However, the role of host-microbiota interactions in the host's response to rising temperature and the underlying mechanisms are incompletely understood in marine organisms. Here, the symbiotic intestinal microbiota and transcriptional responses between diploid and triploid oysters that displayed susceptible and resistant performance under the stress of rising temperature during a summer mortality event were compared to investigate the host-microbiota interactions. The rising and fluctuating temperatures triggered an earlier onset and higher mortality in susceptible oysters (46.7%) than in resistant oysters (17.3%). Correlation analysis between microbial properties and environmental factors showed temperature was strongly correlated with indices of α-diversity and the abundance of top 10 phyla, indicating that temperature significantly shaped the intestinal microbiota of oysters. The microbiota structure of resistant oysters exhibited more rapid changes in composition and diversity compared to susceptible oysters before peak mortality, indicating that resistant oysters possessed a stronger ability to regulate their symbiotic microbiota. Meanwhile, linear discriminant analysis effect size (LefSe) analysis found that the probiotics Verrucomicrobiales and Clostridiales were highly enriched in resistant oysters, and that potential pathogens Betaproteobacteriales and Acidobacteriales were enriched in susceptible oysters. These results implied that the symbiotic microbiota played a significant role in the oysters' adaptation to rising temperature. Accompanying the decrease in unfavorable bacteria before peak mortality, genes related to phagocytosis and lysozymes were upregulated and the xenobiotics elimination pathway was exclusively expressed in resistant oysters, demonstrating the validity of these immunological functions in controlling proliferation of pathogens driven by rising temperature. Compromised immunological functions might lead to proliferation of pathogens in susceptible oysters. This study might uncover a conserved mechanism of adaptation to rising temperature in marine invertebrates from the perspective of interactions between host and symbiotic microbiota.
... At the same time, it can promote inflammatory response by regulating the release of inflammatory factors (88)(89)(90). In mollusks, the PI3K-Akt signaling pathway involved in and regulated many physiological and pathological processes of innate immunity (91,92). For instance, it could regulate phagocytosis to promote innate immune response processes (93). ...
Sepia esculenta is a popular economic cephalopod with high yield, delicious meat, and rich nutrition. With the rapid development of heavy industry and medical industry, a large amount of waste has been released into the ocean recklessly in recent years, inducing a significant increase in the content of heavy metals, especially cadmium (Cd) and copper (Cu), in the ocean. This phenomenon significantly affects the growth and development of S. esculenta, causing a serious blow to its artificial breeding. In this study, transcriptome analysis is used to initially explore immune response mechanisms of Cd and Cu co-exposed juvenile S. esculenta. The results show that 1,088 differentially expressed genes (DEGs) are identified. And DEGs functional enrichment analysis results suggests that co-exposure may promote inflammatory and innate immune responses in juvenile S. esculenta. Fifteen key genes that might regulate the immunity of S. esculenta are identified using protein-protein interaction (PPI) network and KEGG enrichment analyses, of which the three genes with the highest number of interactions or involve in more KEGG pathways are identified as hub genes that might significantly affect the immune response processes. Comprehensive analysis of PPI network and KEGG signaling pathway is used for the first time to explore co-exposed S. esculenta juvenile immune response processes. Our results preliminarily reveal immune response mechanisms of cephalopods exposed to heavy metals and provide a valuable resource for further understanding of mollusk immunity.
... Lysozyme activity was largely used as biomarker of physiological stress in response to organic and inorganic contamination (Stabili and Pagliara, 2009;Tang et al., 2020). Cyclooxygenase (COX) is another key enzyme that plays a crucial role in the inflammatory response by transforming arachidonic acid to prostaglandin (Canesi et al., 2002) in response to organic pollutants (Lin et al., 2022). Thus, it is well known that pollutants such as PAH could have a genotoxic impact in marine organisms (Sarker et al., 2018). ...
This work assessed the impact of polycyclic aromatic hydrocarbons (PAHs) on the polychaeta Marphysa sanguinea in Tunis Lagoon. Highest PAHs concentrations were accumulated at station E with maximum Σ PAH of 6028,87 ng/g DW. Changes in animal physiology were clearly related to bioaccumulated PAH. In fact, high levels of immune biomarkers (cyclooxygenase [COX] and lysozyme activity with maximum of 44631,10 FU/mn/mg protein and 0,017 lysozyme activity/mn/mg protein, respectively) were recorded at stations B and E. Triacylglycerol (TAG), the energy source, was lowest at the most polluted stations (E and B), while phospholipids (PL) were highest at the control station. Statistical analysis revealed a probable effect of both low and high molecular weight PAHs on variations in energy storage lipids (TAG and sterol and wax esters [SE/WE]) and membrane lipids, particularly PL. Our results encourage the use of M. sanguinea to assess pollution levels in coastal ecosystems.
... In bivalve the main cellular mediator of defence system are the haemocyte . However, haemocytes along its immune functions can perform other functions such as wound healing and shell repair (Canesi et al., 2002). Phagocytosis is one of best designated immune functions of haemocytes, which lead to eliminate the microorganisms recognized as non-self. ...
Together with the increase of human population, there is a mathematical increase for food supply. Since 2008, mass mortality events of Pacific oysters (Crassostrea gigas) juveniles caused by the Pacific Oyster Mortality Syndrome (POMS) have threatened the oyster aquaculture industry. Studies on the resistance of C. gigas to POMS has demonstrated a genetic bases and more recently, it was shown to rely on early transcriptomic response to the viral infection. Although data about the involvement of epigenetics in POMS resistance are still scarce, the essential role of the transcriptome, from the basal level to the antiviral response, and the effect of environmental exposure on the resistance of oyster, collectively suggest that epigenetic can play an essential role. Here we propose a framework to study simultaneously the potential role of genetic and epigenetic in the expression of phenotype by using the C.gigas/POMS model at the natural population level. We developed an exome capture approach to obtain genetic (Single Nucleotide Polymorphisms; SNPs) and epigenetic (DNA methylation at CG context; CpGs) information. In the present thesis, the exome capture developed allowed us to capture the genetic and epigenetic variation on more than 65 % of the total exons. We showed that wild oyster populations differentially exposed to the POMS display signatures of selections both in their genome (SNPs) and in epigenome (CpGs). A high number of these SNPs and CpGs were located in genes involved in immune functions. These results confirmed that host population facing pathogen emergence could rely on genetic and epigenetic variation to rapidly adapt to emerging diseases. While our study confirms the essential role played by the DNA sequence it also shows that other mechanisms can interplay with this sequence to encode a resistant phenotype. However, they can also be independent from this DNA sequence and participate to the expressio n of resistance. These results confirm that holistic approaches of the resistance of host population must be envisioned to have access to most of the mechanisms at stake. In addition, it also demonstrates that epigenetic assisted selection would be a way to assist breeding industry without effects on the DNA sequence.
... Another advantage of LB compared to eDNA is that it exploits the phagocytic activity of hemolymphatic circulating hemocytes which engulfed microorganisms (and their genome). Phagocytosis is the most fundamental role for hemocytes in bivalves [82]. Finally, the approach takes advantage of the high filtration capacity of mussels (which pumps an average of 1.5 L/h of seawater) and the bi-diurnal cycles, both of which increases the mixing of different layers of seawater columns, thereby allowing the capture of heterogenous eDNA [83,84]. ...
Liquid biopsy (LB) is a concept that is rapidly gaining ground in the biomedical field. Its concept is largely based on the detection of circulating cell-free DNA (ccfDNA) fragments that are mostly released as small fragments following cell death in various tissues. A small percentage of these fragments are from foreign (nonself) tissues or organisms. In the present work, we applied this concept to mussels, a sentinel species known for its high filtration capacity of seawater. We exploited the capacity of mussels to be used as natural filters to capture environmental DNA fragments of different origins to provide information on the biodiversity of marine coastal ecosystems. Our results showed that hemolymph of mussels contains DNA fragments that varied considerably in size, ranging from 1 to 5 kb. Shotgun sequencing revealed that a significant amount of DNA fragments had a nonself microbial origin. Among these, we found DNA fragments derived from bacteria, archaea, and viruses, including viruses known to infect a variety of hosts that commonly populate coastal marine ecosystems. Taken together, our study shows that the concept of LB applied to mussels provides a rich and yet unexplored source of knowledge regarding the microbial biodiversity of a marine coastal ecosystem.
... Exposure to extremely high or low temperatures, disrupt rates of metabolic and physiological processes, including the immune system. Thus, mechanisms of stress tolerance in land snail offer fundamental insights into the adaptation of the organisms for a wide range of environmental challenges, regulated by physiological adaptation (Canesi, Gallo, Gavioli, & Pruzzo, 2002) of immune response. Less attention has been given to the immunological thermoregulatory abilities of the Giant African Land snail under extreme thermal conditions; therefore, understanding of the defense mechanisms is necessary to the establishment of adaptation limits and possible preparation in the changing climate for better productivity. ...
The study determined immunological indices of Giant African Land snail (Archachatina marginata) improved with fixed dose of vitamin C under acute heat stress (AHS). Prior to the AHS, vitamin C was administered for four weeks to two treatment groups, while other two treatment groups were not. Each treatment was monitored, haemolymph collected at 0, 30 and 60 minutes exposure times. Immunological cytokines: interferon gamma (IFN-γ) and interleukin 2 (IL-2); and total haemocyte counts (THC) were determined. Under AHS, vitamin C elevated (p <0.05) IFN-γ production (606.33 ± 302.86) compared to other groups with or without vitamin C administration (7.20 ± 1.58 vs. 73.20 ± 32.23 vs. 7.80 ± 1.36). IL-2 was not affected (p >0.05) by vitamin C under AHS. Highest (p <0.05) THC values was obtained with vitamin C administration under AHS, but reduced under no AHS. Exposure time affected (p <0.05) IFN-γ production and THC values, but not IL-2 (p >0.05). With fixed dose of vitamin C and exposure time, highest (p <0.05) IFN-γ values were obtained under AHS with vitamin C administration at 30 minutes and at 60 minutes in THC, compared to other groups. The study concluded that fixed dose of vitamin C at 150 mg kg-1 of feed was appropriate under AHS to boost the immune system of the animals.
... Our finding supports the hypothesis of Hine (1999) and the finding of Donaghy et al. (2010) who suggested that one cell type may produce hyalinocytes, the later would then grow into granulocytes. The ability of hemocytes to ingest and then digest foreign material by phagocytosis is important to their role in the cellular immune response of mollusks (Canesi et al., 2002;Hooper et al., 2007). Phagocytosis is the process by which an invading pathogen is killed inside the cell. ...
Organophosphorus pesticides like Chlorpyrifos 48%EC were widely used to control agricultural pests. The present study aimed to evaluate the toxic effects of Chlorpyrifos 48%EC on B. alexandrina snails, the intermediate host of Schistosoma mansoni. After exposure of snails to serial concentrations to determine the LC50, thirty snails for each sublethal concentration (LC10 2.1 and LC25 5.6 mg/l) in each group were exposed for 24 h followed by another 24 h for recovery. After recovery random samples were collected from hemolymph and tissue to measure the impacts on Phagocytic index, histological, biochemical, and molecular parameters. The current results showed a toxic effect of Chlorpyrifos 48%EC on adult B. alexandrina snails after 24 h of exposure at LC50 9.6 mg/l. After exposure to the sub-lethal concentrations of this pesticide, it decreased the total number of hemocytes and the percentage of small cells, while increased the percentage of hyalinocytes. The granulocyte percentage was increased after exposure to LC10, while after LC25, it was decreased compared to the control group. Also, the light microscopical examination showed that some granulocytes have plenty of granules, vacuoles and filopodia. Some hyalinocytes were contained shrinked nuclei, incomplete cell division and forming pseudopodia. Besides, the phagocytic index of hemocytes was significantly increased than control in all treated groups. Also, these sub-lethal concentrations increased MDA and SOD activities, while, tissue NO, GST and TAC contents were significantly decreased after exposure. Levels of Testosterone (T) and Estradiol (E) were increased significantly after exposure compared with control group. The present results showed that the concentration of DNA and RNA was highly decreased after exposure to LC10, 25 than the control group. Therefore, B. alexandrina snails could be used as a bio monitor of the chemical pollution. Besides, this pesticide could reduce the transmission of schistosomiasis as it altered the biological system of these snails.
... Lysosymal enzymes are proteins involved in phagocytosis (26,43). ST vs SS and ST vs AT had downregulated lysosymal enzyme genes. ...
Teneurin C-terminal associated peptide (TCAP) is an ancient bioactive peptide that is highly conserved in metazoans. TCAP administration reduces cellular and behavioral stress in vertebrate and urochordate models. There is little information for invertebrates regarding the existence or function of a TCAP. This study used the Sydney rock oyster (SRO) as a molluscan model to characterize an invertebrate TCAP, from molecular gene analysis to its physiological effects associated with hemocyte phagocytosis. We report a single teneurin gene (and 4 teneurin splice variants), which encodes a precursor with TCAP that shares a vertebrate-like motif, and is similar to that of other molluscan classes (gastropod, cephalopod), arthropods and echinoderms. TCAP was identified in all SRO tissues using western blotting at 1-2 different molecular weights (~22 kDa and ~37kDa), supporting precursor cleavage variation. In SRO hemolymph, TCAP was spatially localized to the cytosol of hemocytes, and with particularly high density immunoreactivity in granules. Based on ‘pull-down’ assays, the SRO TCAP binds to GAPDH, suggesting that TCAP may protect cells from apoptosis under oxidative stress. Compared to sham injection, the intramuscular administration of TCAP (5 pmol) into oysters modulated their immune system by significantly reducing hemocyte phagocytosis under stress conditions (low salinity and high temperature). TCAP administration also significantly reduced hemocyte reactive oxygen species production at ambient conditions and after 48 h stress, compared to sham injection. Transcriptomic hemocyte analysis of stressed oysters administered with TCAP demonstrated significant changes in expression of genes associated with key metabolic, protective and immune functions. In summary, this study established a role for TCAP in oysters through modulation of physiological and molecular functions associated with energy conservation, stress and cellular defense.
... In our study, the highest expression levels of AwMyD88 were detected in hemocytes, which was consistent with the results in Portunus trituberculatus (42) and C. sinensi (28). It is generally believed that hemocytes are one of the key immune tissues in bivalve mollusks and play crucial roles in the recognition and elimination of bacterial pathogens via phagocytic activity and the production of lectins, antimicrobial peptides and lysosomal enzymes (47)(48)(49)(50). Additionally, a relatively high transcript level of AwMyD88s was observed in immune-related gill and hepatopancreas tissues, which are responsible for directly contacting the pathogens from the external environment and acting on the last immune barrier in the body (44,51), respectively. ...
Myeloid differentiation factor 88 (MyD88) is a key adapter molecule in Toll-like receptor signal transduction that triggers downstream immune cascades involved in the host defense response to exogenous pathogens. However, the function of MyD88s in mollusks, especially in freshwater shellfish, remains poorly understood. In this study, a novel freshwater shellfish MyD88 (denoted Aw MyD88) was characterized from Anodonta woodiana . The present Aw MyD88 protein consists of 474 amino acids and contains a conserved a typical death domain (DD) and a conservative Toll/IL-1R (TIR) domain with three typical boxes. Quantitative real-time PCR (qRT-PCR) analysis showed that Aw MyD88 was broadly expressed in all the examined tissues, and the highest expression level was observed in hemocytes of A. woodiana . When challenged with Aeromonas hydrophila and lipopolysaccharide (LPS), the mRNA expression levels of Aw MyD88 were significantly induced in hemocytes of A. woodiana in vivo and in vitro . In addition, in vivo injection experiments revealed that MyD88 signaling pathway genes showed strong responsiveness to A. hydrophila challenge, and their expression levels were significantly upregulated in hemocytes. Knockdown of Aw MyD88 reduced the transcript levels of immune related transcription factors ( Aw NF-κB and Aw AP-1) and effectors ( Aw TNF, Aw LYZ, Aw Defense and Aw AIF) during A. hydrophila infection. Moreover, subcellular localization analysis indicated that Aw MyD88 was mainly localized to the cytoplasm in HEK293T cells. Finally, luciferase reporter assays revealed that Aw MyD88 associates with Aw TLR to activate the NF-κB and AP-1 signaling pathways in HEK293T cells. These results suggested that Aw MyD88 might be involved in the host defense response to bacterial challenge, providing new insight into the immune function of the MyD88 signaling pathway in freshwater shellfish.
... In bivalves, autophagy plays a key role in maintaining cell homeostasis (Carella et al., 2015). This mechanism has been used as an indicator of cell injury in response to different stressors (Moore, 2004;Picot et al., 2019), such as environmental changes (Moore, 2008), and the innate-immune response to pathogens (Canesi et al., 2002;Moreau et al., 2015;Canesi et al., 2016;Balbi et al., 2018) However, nothing is still known on the role of autophagy in ingestion, mobilization and excretion of phycotoxins in these organisms. ...
Domoic acid (DA), the phycotoxin responsible for amnesic shellfish poisoning (ASP), is an excitatory amino acid naturally produced by at least twenty-eight species of the bloom-forming marine diatoms Pseudo-nitzschia spp. Suspension feeders, such as bivalve mollusks, can accumulate and lengthy retain high amounts of DA in their tissues, threatening human health and leading to extensive-prolonged fishery closures, and severe economic losses. This is particularly problematic for the king scallop Pecten maximus, which retains high burdens of DA from months to years compared to other fast-depurator bivalves. Nonetheless, the physiological and cellular processes responsible for this retention are still unknown. In this work, for the first time, a novel immunohistochemical techniques based on the use of an anti-DA antibody was successfully developed and applied for DA-detection in bivalve tissues at a subcellular level. Our results show that in naturally contaminated P. maximus following a Pseudo-nitzschia australis outbreak, DA is visualized mainly within small membrane-bounded vesicles (1 – 2.5 µm) within the digestive gland cells, identified as autophagosomic structures by means of immune-electron microscopy, as well as in the mucus-producing cells, particularly those from gonad ducts and digestive tract. Trapping of DA in autophagososomes may be a key mechanism in the long retention of DA in scallops. These results and the development of DA-immunodetection are essential to provide a better understanding of the fate of DA, and further characterize DA contamination-decontamination kinetics in marine bivalves, as well as the main mechanisms involved in the long retention of this toxin in P. maximus.
... Phagocytosis is the internalization of pathogens within hemocytes, an essential process during which hemocyte membranes engulf microbial agents through endocytosis and contain them within an internal membrane called the phagosome. The cytoskeleton, an intracellular network of microtubules responsible for the cell structure and movement of cellular components, is implicated in the migration of lysosomes toward the phagosome, allowing for the fusion of these two membranes so that the lysosomal contents can initiate pathogen neutralization and digestion (Canesi et al., 2002). Bivalve pathogens, including V. tapetis and P. olseni in the Manila clam, can be intracellular, and some have been shown to develop mechanisms to evade phagocytosis and inhibit the fusion of lysosomes with the phagosome (Carella et al., 2015;Flannagan et al., 2009). ...
The Manila clam is a bivalve species of particular commercial interest, currently representing about a quarter of total bivalve aquaculture worldwide. European production of this species has suffered greatly from the negative impact of infectious diseases, namely due to the protozoan parasite Perkinsus olseni and the Gram-negative bacteria Vibrio tapetis. These pathogens induce chronic infections that have been associated with reduced growth, fecundity, spat production, and overall health, as well as triggering mass mortality in culturedclam populations. The difficulties inherent in treating diseases in commercial bivalve production have led tothe search for alternative solutions to limit the impact of disease by 1) improving our understanding of virulence,resistance, and host-pathogen interactions and 2) developing the tools necessary for hatcheries to initiateselection for traits of commercial interest. This PhD, in joint agreement between the University of Padova andthe University of Brest, and within the framework of the EU Horizon2020 project VIVALDI, seeks to investigatethe potential for selection to increase resistance to disease in the Manila clam, as well as examine the functional mechanisms associated with resistance. In order to lay the basis for understanding the genetic factors of resistance a novel tool for parentage assignment was developed and applied for the first time to several populations of clams from hatchery broodstock. This made it possible to estimate genetic parameters for multiple traits including resistance to disease in large-scale field experiments, revealing the high potential formultiple trait selection in this species for the first time, and raising interesting questions regarding impact of genotype-by environment on selection strategies. Furthermore, the molecular basis for resistance was investigated through a relevant proteomics approach, shedding light on the metabolisms at play in long-term resistant juvenile clams. Overall, the work carried out in these studies highlights the feasibility of future selection methods based on both genetic information and clear molecular markers as a solution forincreasing disease resistance in the Manila clam.
... Haemocytes constitute the cellular component of the haemolymph; they move through the circulatory system and migrate to other locations, such as the connective tissue and epithelia (19,20). Among the important functions they perform in bivalves, haemocytes are best known for their primary role in phagocytosis, encapsulation and production of cytotoxic molecules, such as reactive oxygen species, antimicrobial peptides (AMPs) and secretion of inflammatory cytokines involved in pathogen killing and elimination (9,19,(21)(22)(23)(24)(25)(26)(27). In addition to their role in host defence, bivalve haemocytes perform various important physiological functions, including nutrient digestion, transportation and distribution, wound healing, detoxification processes, shell mineralisation and excretion (25,28,29). ...
Bivalve molluscs stand out for their ecological success and their key role in the functioning of aquatic ecosystems, while also constituting a very valuable commercial resource. Both ecological success and production of bivalves depend on their effective immune defence function, in which haemocytes play a central role acting as both the undertaker of the cellular immunity and supplier of the humoral immunity. Bivalves have different types of haemocytes, which perform different functions. Hence, identification of cell subpopulations and their functional characterisation in immune responses is essential to fully understand the immune system in bivalves. Nowadays, there is not a unified nomenclature that applies to all bivalves. Characterisation of bivalve haemocyte subpopulations is often combined with 1) other multiple parameter assays to determine differences between cell types in immune-related physiological activities, such as phagocytosis, oxidative stress and apoptosis; and 2) immune response to different stressors such as pathogens, temperature, acidification and pollution. This review summarises the major and most recent findings in classification and functional characterisation of the main haemocyte types of bivalve molluscs.
... Aquatic invertebrates have been affected by alterations of abiotic parameters including salinity (Nunes et al., 2021;Pérez-Velasco et al., 2021), temperature (Matoo et al. 2021;Wu et al., 2021), pollutants (Baussant et al., 2009;Tresnakova et al., 2020;Günal et al., 2021) and by biotic factors such as bacteria (Canesi et al., 2002;Ciacci et al. 2009), invasive species (Berber et al., 2018) in the aquatic environment. Therefore, their immune system may be also affected by these factors. ...
The overuse of pesticides has been increasing since the 20th century. Depending on this use, non-target organisms are also affected apart from target organisms. Cyfluthrin is a synthetic pyrethroid pesticide used in agriculture, domestic and veterinary medicine against insects. It may also affect non-target aquatic organisms as a result of mixing with aquatic ecosystems. This study was aimed to investigate the effect of cyfluthrin on freshwater mussels, one of the aquatic invertebrate species, with hemocyte parameters. Total hemolymph counts, hemolymph cell morphology, and differential hemocyte counts were performed from hemolymph taken from mussels exposed to different cyfluthrin doses exposures for 24 and 48 hours. Compared to the control group, the total hemocyte counts of the experimental groups were found to increase in 24h and decrease in 48h significantly (p<0.05). In the examination of hemocyte morphologies, granular, semi granular, and hyalinocyte cells were observed. Similar values of differential hemocyte counts were found both 24 and 48h exposure times. As a result, in aquatic toxicology studies, besides total hemocyte count, analysis of hemocyte morphologies and differential hemocyte counts are found to be good biomarkers.
... Ces agglutinines permettent d'améliorer la prise en charge de bactéries par les hémocytes (Hardy et al. 1977). Cette activité opsonique pourrait également jouer sur l'activité de phagocytose des hémocytes (Canesi et al. 2002). ...
Les importantes mortalités observées sur le naissain de l’huître creuse, Crassostrea gigas, ont fortement affecté l’économie aquacole de plusieurs pays dans le monde. Les causes de ces mortalités sont complexes, mais un agent viral appartenant à la famille des herpèsvirus, appelé ostreid herpesvirus 1 (OsHV-1), a été identifié comme étant le principal facteur responsable de ces mortalités. Les moyens disponibles pour lutter contre le virus OsHV-1 restent limités et une meilleure connaissance des interactions entre l’huître creuse et le virus est nécessaire. Récemment, les résultats de plusieurs études et la caractérisation du génome de C. gigas ont démontré l’existence potentielle chez cette espèce de plusieurs voies antivirales connues chez les mammifères. La voie de l’autophagie est impliquée dans de nombreux processus cellulaires dont la défense immunitaire. Cette voie serait fonctionnelle dans le manteau de C. gigas et impliquée dans la réponse de l’huître creuse à différentes pathologies incluant les infections virales. Dans le cadre de la thèse, un travail a été réalisé afin d’approfondir les connaissances sur le mécanisme de l’autophagie chez C. gigas et sur sa régulation au cours d’une infection par le virus OsHV-1. Ces travaux de thèse ont permis de mettre en évidence une forte conservation de la voie de l’autophagie au niveau moléculaire. Pour la première fois chez C. gigas, il a été observé des structures autophagiques chez les hémocytes. Ce résultat a permis de développer de nouvelles approches afin de détecter et suivre la régulation de l’autophagie chez l’huître creuse. Un suivi de l’autophagie au cours d’une infection par le virus OsHV-1 a montré une réplication virale suivie d’une modulation de l’autophagie dans le manteau et dans l’hémolymphe. Enfin, il a été montré une régulation différentielle de l’autophagie au niveau transcriptiomique dans le manteau et dans l’hémolymphe.
... The release of lysosomal hydrolytic enzymes is an important part of the extracellular killing of bacteria through the hydrolysis of components of bacterial cell walls (Canesi et al. 2002, Monari et al. 2007. Lysozyme is an bacteriolytic agents acting against several species of Gram-positive and Gramnegative bacteria; it is synthesized in bivalve haemocytes and subsequently secreted into haemolymph during phagocytosis (Cheng et al. 1975). ...
... The release of lysosomal hydrolytic enzymes is an important part of the extracellular killing of bacteria through the hydrolysis of components of bacterial cell walls (Canesi et al. 2002, Monari et al. 2007. Lysozyme is an bacteriolytic agents acting against several species of Gram-positive and Gramnegative bacteria; it is synthesized in bivalve haemocytes and subsequently secreted into haemolymph during phagocytosis (Cheng et al. 1975). ...
... The release of lysosomal hydrolytic enzymes is an important part of the extracellular killing of bacteria through the hydrolysis of components of bacterial cell walls (Canesi et al. 2002, Monari et al. 2007. Lysozyme is an bacteriolytic agents acting against several species of Gram-positive and Gramnegative bacteria; it is synthesized in bivalve haemocytes and subsequently secreted into haemolymph during phagocytosis (Cheng et al. 1975). ...
The aim of this study was to investigate the immunotoxic effects of acute temperature challenge in the carpet shell clam, Ruditapes decussatus. Haemolymph samples were incubated at 20 and 30 °C for 24 h. Total haemocyte count (THC), percentage of dead cells (PDC), phenoloxidase (PO), lysozyme (LYZ), alkaline phosphatase (ALP), esterase (EST), lectin (LCT), and antibacterial (AB) activities were chosen as biomarkers of temperature stress. Our results revealed that exposure to high temperatures increases the AB, and the LCT activities while it decreases the THC, the PDC, and the ALP activity. Meanwhile, no obvious negative effect of acute temperature stress was detected on PO, LYZ and EST activities.
... [99][100][101] Haemocyte proliferation accompanied by an increase in antioxidant activity has been related to PAMP recognition in marine bivalves. [102][103][104][105] Endocytosis process is a mechanism to internalise and, in some cases, include the elimination of non-self-molecules. 106,107 These results suggest that ViP and ViA at decimal (1D) and centesimal (7C) dilution have the potential to modulate mechanisms related to immune response in marine organisms (fish, crustaceans and molluscs) and may explain why HBDC increase organism survival. ...
Highly diluted bioactive compounds (HDBC) and highly diluted immunomodulatory compounds (HDIC) are treatments that cover a wide variety of concentrations, starting from molar to yoctomolar concentrations or even lower where the presence of nanoparticles has been detected. The prophylactic use of these compounds has improved nutrition, growth, overall condition, defence mechanisms regulation and increased marine organism survival during challenge against pathogens. The HDBC used in marine organisms are formulated from Vibrio spp. lysates, sodium metasilicates, phosphoric acid and highly diluted drugs for humans as Silicea terra®, Phosphoricum acidum®, Passival® and VidatoX®, as well as homotoxic Heel® complexes. Because HDBC are innocuous, inexpensive and easy to use by immersion or ingestion, they have been proposed as a potential sustainable alternative to improve marine organism production under hatchery conditions and reduce the use of chemotherapy, including antibiotics and disinfectants. Nonetheless, articles documenting their mode of action (biological process and metabolic pathways activated) are scarce. Some studies have proposed that certain HDBC activate the sentinel system of marine organisms, such as non‐self‐recognition and endocytosis mechanisms without completely activating the immune defence mechanisms. This contribution brings a new insight on increasing productivity and cost effectiveness during hatchery operations and at the same time strengthening the immune system response and survival of marine organisms against infections. Therefore, this review discusses and analyses published results of HDBC in species of commercial importance worldwide and also emerging species of interest for aquaculture reared in Mexico.
Microplastics (MPs) are widely distributed in marine environments, and they are easily attached by various microorganisms, including pathogenic bacteria. When bivalves mistakenly eat MPs, pathogenic bacteria attached to MPs enter their bodies through the Trojan horse effect, causing adverse effects. In this study, the mussel Mytilus galloprovincialis was exposed to aged polymethylmethacrylate MPs (PMMA-MPs, 20 μm) and Vibrio parahaemolyticus attached to PMMA-MPs to explore the effect of synergistic exposure by measuring lysosomal membrane stability, ROS content, phagocytosis, apoptosis in hemocytes, antioxidative enzyme activities and apoptosis-related gene expression in gills and digestive glands. The results showed that MP exposure alone did not cause significant oxidative stress in mussels, but after long-term coexposure to MPs and V. parahaemolyticus, the activities of antioxidant enzymes were significantly inhibited in the gills of mussels. Both single MP exposure and coexposure will affect hemocyte function. Coexposure can induce hemocytes to produce higher ROS, improve phagocytosis, significantly reduce the stability of the lysosome membrane, and induce the expression of apoptosis-related genes, causing apoptosis of hemocytes compared with single MP exposure. Our results demonstrate that MPs attached to pathogenic bacteria have stronger toxic effects on mussels, which also suggests that MPs with pathogenic bacteria might have an influence on the immune system and cause disease in mollusks. Thus, MPs may mediate the transmission of pathogens in marine environments, posing a threat to marine animals and human health. This study provides a scientific basis for the ecological risk assessment of MP pollution in marine environments.
As a well-known marine metal element, Cd can significantly affect bivalve mollusk life processes such as growth and development. However, the effects of Cd on the molecular mechanisms of the economically important cephalopod species Sepia esculenta remain unclear. In this study, S. esculenta larval immunity exposed to Cd is explored based on RNA-Seq. The analyses of GO, KEGG, and protein-protein interaction (PPI) network of 1,471 differentially expressed genes (DEGs) reveal that multiple immune processes are affected by exposure such as inflammatory reaction and cell adhesion. Comprehensive analyses of KEGG signaling pathways and the PPI network are first used to explore Cd-exposed S. esculenta larval immunity, revealing the presence of 16 immune-related key and hub genes involved in exposure response. Results of gene and pathway functional analyses increase our understanding of Cd-exposed S. esculenta larval immunity and improve our overall understanding of mollusk immune functions.
The protein inhibitor of activated STAT (PIAS) family proteins act as the important negative regulators in janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which can be also involved in regulating the expression of interferon-stimulated genes (ISGs). In the present study, a PIAS homologue (designated as CgPIAS) was identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgPIAS cDNA was of 1887 bp encoding a peptide of 628 amino acid residues. The CgPIAS protein contains a conserved scaffold attachment factor A/B/acinus/PIAS (SAP) domain, a Pro-Ile-Asn-Ile-Thr (PINIT) motif, a RING-finger-like zinc-binding domain (RLD) and two SUMO-interaction Motifs (SIMs). The deduced amino acid sequence of CgPIAS shared 74.58–81.36% similarity with other PIAS family members in the RLD domain. The mRNA transcripts of CgPIAS were detected in all the tested tissues with highest level in haemocytes (32.98-fold of mantles, p < 0.001). After poly (I:C) and recombinant Interferon-like protein (rCgIFNLP) stimulation, the mRNA expression of CgPIAS in haemocytes significantly up-regulated to the highest level at 48 h (7.38-fold, p < 0.001) and at 24 h (13.08-fold, p < 0.01), respectively. Moreover, the nuclear translocation of CgPIAS was observed in haemocytes after poly (I:C) stimulation. Biolayer Interferometry (BLI) assay revealed that the recombinant protein CgPIAS-RLD could interact with the recombinant protein CgSTAT in vitro with the KD value of 3.88×10⁻⁸ M. In the CgPIAS-RNAi oysters, the green signals of CgSTAT protein in nucleus of haemocytes increased compared with that in NC-RNAi group, and the mRNA expression of myxovirus resistance (CgMx1), oligoadenylate synthase-like proteins (CgOASL), CgViperin and IFN-induced protein 44-like (CgIFI44L-1) in haemocytes significantly increased at 12 h after poly (I:C) stimulation, which were 2.39-fold (p < 0.05), 2.18-fold (p < 0.001), 1.74-fold (p < 0.05), and 2.89-fold (p < 0.01) of that in control group, respectively. The above results indicated that CgPIAS negatively regulated the ISG expression by inhibiting STAT activation in oyster C. gigas.
In this study, seven transcripts representing a novel antimicrobial peptide (AMP) family with structural features similar to those of arthropod defensins were identified from Mytilus coruscus. These novel defensins from the Mytilus AMP family were named myticofensins. To explore the possible immune-related functions of these myticofensins, we examined their expression profiles in different tissues and larval stages, as well as in three immune-related tissues under the threat of different microbes. Our data revealed that the seven myticofensins had relatively high expression levels in immune-related tissues. Most myticofensins were undetectable, or had low expression levels, in different larval mussel stages. Additionally, in vivo microbial challenges significantly increased the expression levels of myticofensins in M. coruscus hemocytes, gills, and digestive glands, showing different immune response patterns under challenges from different microbes. Our data indicates that different myticofensins may have different immune functions in different tissues. Furthermore, peptide sequences corresponding to the beta-hairpin, alpha-helix, and N-terminal loop of myticofensin were synthesized and the antimicrobial activities of these peptide fragments were tested. Our data confirms the diversity of defensins in Mytilus and reports the complex regulation of these defensins in the mussel immune response to different microbes in immune-related tissues. The immune system of Mytilus has been studied for years as they are a species with strong environmental adaptations. Our data can be regarded as a step forward in the study of the adaptation of Mytilus spp. to an evolving microbial world.
Research to assess the impacts of mariculture on the microbiota of the surrounding environment is still inadequate. Here, we examined the effects of Mytilus coruscus farming on the diversity of bacterial community in surrounding seawater using field investigations and indoor simulations, focusing on the variation of members of aerobic anoxygenic photoheterotrophic (AAP) bacteria. In the field, Mytilus farming shaped bacterial community and significantly increased their diversity, including biomass, OTUs, Shannon, relative abundance, number of enriched species, as compared with the non-farming area. Higher abundance of AAP related genera was observed in the Mytilus farming seawater. Under the controlled condition, the presence of M. coruscus significantly shaped the bacterial community composition and caused species composition to become similar after 10 days. Furthermore, the presence of M. coruscus consistently strengthened local diversity in seawater bacterial community, with linkages to the recruitment of AAP members as well. In addition, the tissue-related composition of M. coruscus significantly differed from those in seawater. Our findings highlight a ecological importance of Mytilus farming, as process that shape surrounding water-cultured bacterial community and offer experimental evidence for the accumulation of AAP-related genera in aquaculture systems.
Sepia esculenta is a common economic cephalopod that has received extensive attention due to the tender meat, rich protein content and certain medicinal value thereof. Over the past decade, multiple industries have discharged waste into the ocean in large quantities, thereby significantly increasing the concentration of heavy metals in the ocean. Copper (Cu) is a common heavy metal in the ocean. The increase of Cu content will affect numerous biological processes such as immunity and metabolism of marine organisms. High concentrations of Cu may inhibit S. esculenta growth, development, swimming, and other processes, which would significantly affect its culture. In this research, transcriptome analysis is used to initially explore Cu-exposed S. esculenta larval immune response mechanisms. And compared to control group with normally growing larvae, 2056 differentially expressed genes (DEGs) are identified in experimental group with Cu-exposed larvae. The results of DEGs functional enrichment analyses including GO and KEGG indicate that Cu exposure might promote inflammatory and innate immune responses in cuttlefish larvae. Then, 10 key genes that might regulate larval immunity are identified using a comprehensive analysis that combines protein-protein interaction (PPI) network and KEGG functional enrichment analyses, of which three genes with the highest number of protein interactions or involve in more KEGG signaling pathways are identified as hub genes that might significantly affect larval immune response processes. Comprehensive analysis of PPI network and KEGG signaling pathway are used for the first time to explore Cu-exposed S. esculenta larval immune response mechanisms. Our results preliminarily reveal immune response mechanisms of cephalopods exposed to heavy metals and provide valuable resources for further understanding mollusk immunity.
Disruptions to reproductive health in wildlife species inhabiting polluted environments is often found to occur alongside compromised immunity. However, research on impacts of aquatic pollution on freshwater mollusc immune responses is limited despite their importance as vectors of disease (Schistosomiasis) in humans, cattle and wild mammals. We developed an in vitro ‘tool-kit’ of well-characterized quantitative immune tests using Biomphalaria glabrata hemocytes. We exposed hemocytes to environmentally-relevant concentrations of common aquatic pollutants (17β-estradiol, Bisphenol-A and p,p’-DDE) and measured key innate immune responses including motility, phagocytosis and encapsulation. Additionally, we tested an extract of a typical domestic tertiary treated effluent as representative of a ‘real-world’ mixture of chemicals. Encapsulation responses were stimulated by p,p’-DDE at low doses but were suppressed at higher doses. Concentrations of BPA (above 200 ng/L) and p,p’-DDE (above 500 ng/L) significantly inhibited phagocytosis compared to controls, whilst hemocyte motility was reduced by all test chemicals and the effluent extract in a dose-dependent manner. All responses occurred at chemical concentrations considered to be below the cytotoxic thresholds of hemocytes. This is the first time a suite of in vitro tests has been developed specifically in B. glabrata with the purpose of investigating the impacts of chemical pollutants and an effluent extract on immunity. Our findings indicate that common aquatic pollutants alter innate immune responses in B. glabrata, suggesting that pollutants may be a critical, yet overlooked, factor impacting disease by modulating the dynamics of parasite transmission between molluscs and humans.
Understanding the impacts of environmental pollutants on immune systems is indispensable in ecological and health risk assessments due to the significance of normal immunological functions in all living organisms. Bivalves as sentinel organisms with vital ecological importance are widely distributed in aquatic environments and their innate immune systems are the sensitive targets of environmental pollutants. As the central component of innate immunity, bivalve hemocytes are endowed with specialized endolysosomal systems for particle internalization and metal detoxification. These intrinsic biological features make them a unique cellular model for metal- and nano-immunotoxicology research. In this review, we firstly provided a general overview of bivalve's innate immunity and the classification and immune functions of hemocytes. We then summarized the recent progress on the interactions of metals and nanoparticles with bivalve hemocytes, with emphasis on the involvement of hemocytes in metal regulation and detoxification, the interactions of hemocytes and nanoparticles at eco/bio-nano interface and hemocyte-mediated immune responses to the exposure of metals and nanoparticles. Finally, we proposed the key knowledge gaps and future research priorities in deciphering the fundamental biological processes of the interactions of environmental pollutants with the innate immune system of bivalves as well as in developing bivalve hemocytes into a promising cellular model for nano-immuno-safety assessment.
Molluscs are the largest and most well-known marine phylum of invertebrates.
Nudibranchs are an interesting group of gastropod molluscs because the loss of the shell in this group allows the development of a wide variety of defense mechanisms. In the study of this group, the external anatomy is important to determine taxonomic relationships, but in most cases, the species show a very similar morphology which makes them very difficult to identify. Thus, the use of molecular techniques and the study of their internal anatomy are necessary for their correct identification. The internal anatomy of these animals is normally studied by means of invasive techniques as dissection or classical histology, which are complex processes resulting in the sample being destroyed. In many cases the samples are very small, are just one or very limited in number, and the use of
these techniques poses a problem.
Micro-computed X-ray tomography (Micro-CT or μCT) is a non-invasive technique which
allows to get information in 2D and 3D reconstruction of the external and internal anatomy of the animal without destroying the specimen (Golding & Jones, 2007; Candás et al., 2016; Parapar et al., 2017). This characteristic makes it a good alternative to the classical methods when studying specimens which are to be kept intact.
In this contribution, the internal anatomy of gastropod Doris ocelligera (Bergh, 1881)
(Nudibranchia: Dorididae) was studied with micro-CT techniques focusing on the
digestive and reproductive system and using 2D and 3D reconstructions. The images of the sections obtained are of high quality. This technique obtains excellent results, not only
regarding 2D but also 3D images. These results show that micro-CT is a tool of great
potential for the study of the anatomy of Nudibranchs.
Benzo[a]pyrene (B[a]P), a typical PAHs widely existing in the marine environment, has been extensively studied for its immunotoxicity due to its persistence and high toxicity. Nevertheless, the immunotoxicity mechanism remain incompletely understood. In this study, isolated hemocytes of Chlamys farreri were exposed at three concentrations of B[a]P (5, 10 and 15 μg/mL), and the effects of B[a]P on detoxification metabolism, signal transduction, humoral immune factors, exocytosis and phagocytosis relevant proteins and immune function at 0, 6, 12, 24 h were studied. Results illustrated the AhR, ARNT and CYP1A1 were significantly induced by B[a]P at 12 h. Additionally, the content of B[a]P metabolite BPDE increased in a dose-dependent manner with pollutants. Under B[a]P stimulation, the expressions of PTK (Src, Fyn) and PLC-Ca²⁺-PKC pathway gene increased significantly, while the transcription level of AC-cAMP-PKA pathway gene decreased remarkably. Additionally, the expressions of nuclear transcription factors (CREB, NF-κB), complement system genes and C-type lectin genes up-regulated obviously. The gene expressions of phagocytosis and exocytosis related proteins were also notably affected. 5 μg/mL B[a]P could promote phagocytosis in a transitory time, but with the increase of exposure time and concentration of B[a]P, the phagocytosis, antibacterial and bacteriolytic activities gradually decreased. These results indicated that similar to vertebrates, BPDE, the metabolite of B[a]P, mediated downstream signal transduction via PTK in bivalves. The declined of the immune defense ability of hemocytes might be closely related to the inhibition of AC-cAMP-PKA pathway and the imbalance of intracellular Ca²⁺ pathway. In addition, the results manifested that complement and lectin systems play a significant role in regulating immune response. In this study, the direct relationship between detoxification metabolism and immune signal transduction in bivalves under B[a]P stress was demonstrated for the first time, which provided important information for the potential molecular mechanism of B[a]P-induced immune system disorder in bivalves.
Bacterial infections and the rise of antibiotic-resistance pose significant threats to public health, which calls for a profound change in diagnosis and treatment. Considering this, we develop two multi-functional light-up metabolic probes, namely TPEPy-Ala and TPAPy-Kdo, based on aggregation-induced emission (AIE) luminogens with photosensitizing properties. These AIE light-up probes can not only metabolically label both Gram-positive and Gram-negative bacteria with a high signal-to-noise contrast but also visualize the bacteria internalized in cells. Furthermore, these photosensitizing probes could in situ generate reactive oxygen species (ROS) upon light irradiation to effectively kill the intracellular bacteria. More importantly, both TPEPy-Ala and TPAPy-Kdo only exert minimal side effects on the host macrophages. This work presents a one-step light-up metabolic approach to discriminate and kill bacteria simultaneously with high specificity by utilizing their respective metabolic pathways.
Bivalves are increasingly being included as display animals in homes and public aquaria, and as invertebrate alternatives for biomedical research studies. This chapter is an introduction to their basic anatomy and physiology, their maintenance requirements, and standard aquaculture techniques. It provides detailed descriptions of economically relevant bivalve pathogens as examples of the types of problems that may affect bivalve health in both closed recirculating and open systems. Hemolymph cell and biochemical composition play a key role in bivalve health assessments. Bivalves provide a lengthy list of ecosystem services. The culture of bivalves for human consumption is a global enterprise. Oysters, clams, scallops, and other species are actively cultured for local, regional, and global food markets. Although the specific growth requirements of individual species vary, three universal basic requirements drive the effective selection of rearing locations: water quality, physical and chemical conditions that define the rearing site, and adequate diet.
Antimicrobial peptides (AMPs) play a fundamental role in mussels' innate immunity, preventing the invasion of potential pathogens. Previous research has shown that AMPs are abundant in Mytilus species. A mussel with important economic value and limited distribution in the East China Sea, M. coruscus, also contains abundant AMPs, including the mytichitin and myticusin identified previously in this species. However, the molecular diversity and expression pattern of M. coruscus AMPs remain largely unknown. Based on the dataset of the M. coruscus hemocyte transcriptome, we identified twelve AMPs representing the main AMP families present in M. coruscus, including three novel AMPs (two arthropod-like and one crustin-like). The variations in the expression of the genes encoding these AMPs have been analyzed in different tissues, different larval development stages, and different M. coruscus individuals, as well as in hemocytes and gonads under the threat of different microbes. The results suggest that in adult M. coruscus, the gonads and hemocytes are the sources of AMPs for mussels collected in winter and summer, respectively. The expression of AMPs is developmentally regulated, and most AMPs are undetectable in larvae until after larval settlement and metamorphosis. In vivo microbial challenges significantly increased the expression of AMPs in M. coruscus hemocytes, and different AMP response patterns under challenges from different microbes were observed, showing a rapid, strong response to gram-positive strains, a weak response to gram-negative strains, and a long-term response to fungus. While the gonads showed a different response pattern than the hemocytes, with stronger changes in expression relative to the control and stronger fluctuation in the expression level of some AMPs. Finally, LC-MS/MS was used for peptidomic analysis of the AMPs in M. coruscus, and the results confirmed the presence of several AMP families in the mussel serum
A review of the literature on shellfish depuration and relaying revealed wide diversity in microbial uptake and elimination among shellfish species and for different microorganisms. Information on relaying of five commercial shellfish species and on controlled purification (depuration) of 11 species indicates that such processes are effective in reducing the levels of bioconcentrated bacteria and viruses from shellfish. The degree of bacterial and viral bioconcentration varies with shellfish species; however, the primary sites of bioconcentration are the hepatopancreas and digestive diverticula. Low levels of enteric viruses and coliphage may be sequestered in shellfish hemolymph and tissues, thus protecting them from elimination through depurative processes. Vibrio spp. appear to proliferate when closely associated with intestinal cells of shellfish. Shellfish relaying techniques offer effective microbial depletion provided water quality is acceptable and shellfish remain physiologically active. The current body of literature on controlled purification demonstrates a broad spectrum of conditions under which shellfish are depurated. Optimal times, temperatures and salinities for effective depuration vary among shellfish species. Proper design and operation of depuration plants is crucial to insure process integrity. Recirculating and flowthrough purification systems are effective in reducing the levels of pathogenic and indicator microorganisms from shellfish, but the extent to which they reduce viruses from shellfish is uncertain. Studies are needed to validate the effectiveness of depuration processes in eliminating pathogenic viruses and to address the adequacy of indicator bacteria as measures of enteric virus contamination.
Hemolymph characteristics were examined in experimentally cultured blue mussels Mytilus galloprovincialis Lmk. from 2 locations in the Ria de Vigo (NW Spain). The highest protein, lysozyme and agglutinin values were found in July. The increase of agranular hemocytes and the decrease of granular blood cells in April was associated with parasitism by Mytilicola intestinalis. No linkage was found between the number of circulating hemocytes and hemolymph component (lysozyme, agglutinins and proteins) concentrations with parasitism. It is suggested that the number of circulating hemocytes, lysozyme and proteins levels and agglutination titer were related to the reproductive cycle and varied according to the location of mussels.
Mytilus hemolymph was found to contain an agglutinin which could be inhibited by mucin. The agglutinin was isolated by affinity chromatography using neuraminidase-treated mucin/Sepharose.In vitro phagocytosis experiments revealed that only about 5% of washed hemocytes phagocytosed yeast cells suspended in a Tris-buffered NaCl-solution, whereas yeast suspended in hemolymph was normally ingested by more than 50% of the hemocytes. This relatively high phagocytic activity was shown to depend on the presence of two serum factors: When purified agglutinin was added to saline-suspended yeast, phagocytosis rates returned to normal, demonstrating opsonizing properties of the purified agglutinin. — On the other hand, addition of Ca++-ions to saline caused an increase of the phagocytic activity of hemocytes. This was interpreted to indicate the activation of divalent cation-dependent recognition molecules at the hemocyte surface. The function of these postulated recognition factors was demonstrated by phagocytosis inhibition tests. Their location at the hemocyte membrane became evident by binding of specific antiagglutinin IgG purified by help of an agglutinin/Sepharose column from an antiserum raised againstMytilus serum proteins. Consequently, humoral as well as cell bound agglutinin molecules are involved in the attachment of yeast cells toMytilus hemocytes which subsequently internalize foreign cells.
Lectins have been implicated as recognition molecules in the invertebrate immune response, yet their capacity to recognize (agglutinate and/or opsonize) potentially invasive microorganisms is largely unknown. In this study, sera from six species of marine molluscs (oyster, clam, octopus, squid, cuttlefish, and sea hare) were found to agglutinate 64 of 94 bacterial isolates (15 genera, 36 species) and seven types of vertebrate erythrocytes. Oyster, clam, and octopus sera agglutinated the greatest number of bacterial isolates and oyster serum exhibited the highest intensity and titer of agglutination. Agglutination was isolate dependent, implying high lectin specificity. Titers were highly variable for wild populations of oysters and sea hares and relatively constant for F4-generation cuttlefish reared in aquaculture systems. Simultaneous agglutination of specific bacterial isolates indicated conservation of lectin specificity between oyster and clam sera (bivalves) and between squid and cuttlefish sera (cephalopods).
Cell-free hemolymph (serum) of the eastern oyster, Crassostrea virginica, agglutinated Vibrio cholerae, including all O1 serovars and biovars. Seventy-nine other strains of bacteria, including 14 genera and 26 species, were not agglutinated. The A, B, and C factors of O1 antigen were not involved in agglutination. Bacterial agglutinating (BA) activity was demonstrated for oysters inhabiting different environments of the U.S. Atlantic and Gulf coasts. Oyster serum BA titers showed high individual variation. The serum component(s) involved in BA was inhibited by 80 degrees C heat, pronase, EDTA, mucin, and fetuin treatments. N-Acetylneuraminic acid (10 mg/ml) weakly inhibited BA activity. Ligands of V. cholerae were sensitive to neuraminidase and resistant to 80 degrees C and pronase. High salinities (24 and 30%) enhanced BA. Cross-adsorption tests with V. cholerae and human O+ erythrocytes indicated that BA and hemagglutinating activities may involve different serum components. These results imply that the ecology of V. cholerae in C. virginica is influenced by agglutinating activity of oyster serum.
Nonopsonic phagocytosis mediated by phagocyte receptors that recognize corresponding adhesins on microbial surfaces has attracted increasing interest as a potential host defense mechanism against extracellular pathogens and as a means of survival in the host for intracellular pathogens. Three types of nonopsonic phagocytosis involving carbohydrate-protein interactions (also termed lectinophagocytosis), protein-protein interactions, and hydrophobic interactions are discussed. A prominent receptor on phagocytic cells involved in recognizing pathogens belongs to the CD11/CD18 integrins. It mediates both opsonophagocytosis and nonopsonic phagocytosis and exhibits multiple specificity for different microbial adhesins. In other cases, similar specificity toward a microbial ligand (e.g. the Klebsiella pneumoniae capsule) is shared by dual molecules, one of which (e.g. the mannose-binding protein in serum) mediates opsonophagocytosis and the other (e.g. the macrophage mannose receptor) mediates nonopsonic phagocytosis of the microorganisms. In addition, we discuss how nonopsonic phagocytosis can trigger the phagocytes to release inflammatory agents and cause tissue injury. Further studies of the molecular mechanisms of nonopsonic phagocytosis, in particular those underlying the up-regulation of the phagocytic receptors by various agents, should lead to the development of new approaches for the prevention of infectious diseases.
In summary, the encounter between the microorganism and the phagocytic cell is a central feature of infection and pathogenicity. Phagocytes are designed to ingest, kill and digest invaders, and the course of the infection depends on the success with which this is carried out.
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.
Summary The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs. In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria. To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M. galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells). We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M. galloprovincialis haemocytes. Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M. galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M. galloprovincialis .
The course of infection in oysters initiated by intracardial injection of Bacillus thuringiensis (spores and vegetative cells), Mycobacterium smegmatis, and bacterium A-3 (a Pseudomonas-like organism) was monitored periodically by plate counts of whole oyster homogenate and of heart blood samples. All three species of bacteria used in the present study, when injected into the oysters, were eventually rendered nonviable by the host. However, the susceptibility of these microorganisms to host defensive action was quite different and presumably reflected their potentials as prospective oyster pathogens. Prior to the 5th day, M. smegmatis apparently suffer little or no loss in the oyster. The bacteria, however, were rapidly destroyed by the host thereafter. Oysters injected with bacterium A-3 ran a relapsing course of infection which was influenced by the ambient temperatures of sea water in which the oysters were maintained. Apparently oysters were not affected by the toxin produced by B. thuringiensis.Histological studies of tissues of oysters injected with B. thuringiensis and M. smegmatis showed that most of the vegetative bacteria were quickly ingested by the leucocytes in the circulatory system, and were eventually rendered nonviable by intracellular digestion. Migration of microorganism-laden leucocytes through the intestinal epithelium was seen occasionally. Occlusion of visceral mass blood vessels by leucocytes began 4 hours post-injection of B. thuringiensis and remained for at least 2 days before it was resolved. Injection of M. smegmatis did not elicit such a host response.
Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.
A cytotoxic protein complex of 320 kDA was isolated from dialyzed plasma of the edible mussel, Mytilus galloprovincialis. Constituted by the assembly of several different proteins, the complex exhibits selective killing against eukaryotic cells, including erythrocytes, mouse tumor cells and protozoan parasites. High variability, which was not correlated with protein concentration, suggested that the immune response of naive mussels was in various stages of activation. Stimulation assays by different treatments in vivo resulted in significant increases in the activity of the plasma suggesting that cytotoxic complexes are involved in immune defense. Lytic activity appears to involve binding of cytotoxic complexes onto target cell membranes and the formation of transmembrane pores. This research provides more evidence that the innate immune system of invertebrates involves large cytotoxic proteins with a broad range of recognitive specificities in addition to small antibacterial, antifungal peptides.
The cytoplasmic and cell wall components of the Gram-positive bacterium Bacillus megaterium and the cytoplasmic and cell envelope components of the Gram-negative bacterium Escherichia coli were assayed for chemotactic activity for the hemocytes of Crassostrea virginica. The cellular components were separated by differential centrifugation and gel filtration was used to determine the approximate molecular weights of the chemoattractant molecules. Active fractions were assayed for glycoproteins and lipoproteins. As a result, it is known that hemocytes are chemotactically attracted to proteins of approximately 10,000 daltons which are associated with the cell wall of B. megaterium and the cell envelope of E. coli.
Forty-seven carbohydrates failed to inhibit the cell aggregation and substrate adhesion behaviors of Mytilus californianus hemocytes. Treatment of hemocytes with sodium periodate and with YV-acetyl glucosaminidase interfered with these behaviors. Trypsin slightly reduced adhesion, and had no effect on aggregation. Hyaluronidase and neuraminidase failed to inhibit either behavior, and slightly enhanced them. An inhibitory effect of caffeine was not altered by hyaluronidase of N-acetyl-glucosaminidase, but neuraminidase partially reversed this inhibition. Inhibition by the calcium channel blocker verapamil was reversed by hyaluronidase. Cell-cell and cell-substrate adhesion behaviors probably involve complex carbohydrates in molluscan hemocytes.
The biological roles of lysozymes in bivalves are believed to be involved with the mechanisms of host defense and digestion. Experiments were carried out to examine hemolymph lysozyme activity and protein in oysters over a 1-year period to determine their relationship with seasonal environmental variables (temperature, salinity, and food availability) and whether they correlate with parasitism by Perkinsus marinus. Hemolymph lysozyme and protein exhibited seasonal fluctuations. Hemolymph lysozyme varied greatly between individual oysters, and it was higher in winter months than in summer months. No linkage was found between hemolymph lysozyme and protein concentration and infection of oysters by P. marinus. It is argued that the observed seasonal changes in hemolymph lysozyme and protein may relate to the oyster reproductive cycle.
Results are summarized which prove the highly developed discriminative
ability of the clearance mechanisms of invertebrates. Not
only hemocytes but also various organs are probably involved in the
elimination of foreign substances from the circulation of mollusks
and crustaceans. The binding of non-self materials is based on
the presence of carbohydrate-specific recognition molecules. Lectins
- or agglutinins - are known to occur in the serum of a
variety of invertebrates, and have also been detected at the membrane
of hemocytes. Since lectin-like molecules are situated at
the surface of viruses, bacteria, amoebae as well as at the surface
of some vertebrate leukocytes and liver cells involved in the
blood clearance, the carbohydrate specific mechanism may represent
an evolutionary ancient binding principle.
Monolayers of blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were incubated in vitro in snail Ringer as well as in snail serum. Sheep erythrocytes and yeast cells were added and allowed to react with the amoebocytes. The results showed that one or more serum factors promote recognition and subsequent phagocytosis of foreign particles by amoebocytes. Moreover, amoebocytes may possess plasmamembrane receptors for foreign particles.
As lectins are believed to mediate non-self-recognition in molluscs, carbohydrate-binding proteins (CBP) from the circulating plasma of the gastropod Biomphalaria glabrata were harvested by affinity chromatography using six different monosaccharides as ligands. Pools of plasma were derived from B. glabrata of either the M line strain, which is susceptible to infection with the PR1 strain of the digenetic trematode Schistosoma mansoni, or from the 13-16-R1 strain, which is resistant to infection. For each strain, plasma was obtained from control snails and from snails exposed to infection 1 or 8 days previously with S. mansoni or the related digenean, Echinostoma paraensei, which is able to develop in either host strain. For control snails, only minor interstrain differences were noted. In M line snails exposed 8 days previously to either parasite, marked changes in CBP populations were observed. Only E. paraensei infections produced comparable alterations in 13-16-R1 snails. The most conspicuous changes noted were the increased production of 80-120 kDa CBP in both strains, 150-210 kDa in 13-16-R1 snails, and 190-210 kDa in M line snails. The results demonstrate 1) interstrain differences in CBP, particularly following exposure to trematodes; 2) that infection provokes increased production and diversity of CBP that bind with greater affinity to the columns; and 3) that snails of the same strain respond differently to the two parasites used.
Hemocytes from adult and juvenile specimens of a brackish-water clam, Corbicula japonica, were attracted chemotactically to live cells of Vibrio parahaemolyticus and Escherichia coli strains in a balanced salt solution, which was enhanced significantly in the presence of respective C. japonica plasma. Chemotactic attractions of adult's and juvenile's hemocytes were seen also in artificial seawater at a similar level to those in the balanced salt solution. Chemotactic attractions of juvenile's hemocytes to these strains were lower in level than those of adult's hemocytes. C. japonica plasma seems to facilitate for C. japonica hemocytes to recognize these organisms.
Hemolymph from the Pacific oyster (Crassostrea gigas) contains lectins that agglutinate horse (Gigalin E) and human (Gigalin H) erythrocytes. The gigalins also agglutinate bacteria, including Vibrio anguillarum, and were adsorbed from oyster hemolymph at different temperatures by living, heat-killed, and freeze-dried V. anguillarum cells. Baseline activities of the two gigalins were established by measuring their activities in oyster hemolymph over a period of 4 years. A normal distribution of Gigalin H activity (mean titer 139) was found, whereas the distribution of Gigalin E activity in the same samples was skew (mean titer 512). No covariance was observed between the two agglutinin activities. Increased lectin activity above this baseline was found in oysters exposed for varying time intervals to V. anguillarum at different seasons and temperatures over a period of 2 years. Such exposure resulted in an increase in activity (titer) of four- to nine-fold for Gigalin E and three- to seven-fold for Gigalin H when compared with controls, and in augmentation in the hemolymph of a protein with the same electrophoretic mobility as affinity-purified oyster lectins (gigalins). Challenge with either living or heat-killed bacteria resulted in a significant increase of Gigalin E activity, whereas results for Gigalin H were variable. Oysters challenged with bacteria were observed to filter normally with open shells during the experiments. Also, no increase was found in hemolymph calcium that could indicate anoxia following bacterial challenge (0.49 +/- 0.004 mg mL-1) compared to unexposed oysters (0.50 +/- 0.001 mg mL-1). Increase in the concentration of free amino acids in oyster hemolymph was observed following exposure to bacteria (15.05 mM) and anaerobiosis (13.51 mM) compared to controls (9.06 mM), and changes (in mol %) of individual amino acids differed considerably between hemolymph from animals challenged with bacteria and animals kept anaerobic. The augmented lectin activity in oyster hemolymph, following in vivo exposure to increased bacteria in the seawater, suggests their involvement in enhancing bacterial clearance and defense in the oyster.
Lectins in the serum of the clam Mercenaria mercenaria agglutinate some red blood cells, bacteria, and yeast. The interaction of these substances with particles is affected by sugars, ions, temperature, and alteration of particle surfaces. Lectins are not needed for phagocytosis of foreign particles in vitro. In M. mercenaria these recognition molecules do not enhance defense mechanisms.
Large granular hemocytes of Mercenaria mercenaria avidly phagocytose a variety of biological particles (red blood cells of six species, yeast, and gram-positive and gram-negative bacteria) as well as polystyrene spheres. Clam hemolymph is not necessary for phagocytosis but may have some opsonic effect in certain circumstances (e.g., low temperature and low particle density). Formaldehyde treatment of red blood cells enhances susceptibility to phagocytosis. Phagocytosis by Mercenaria hemocytes in vitro appears to be a nonspecific process.
Hemocytes of Crassostrea virginica were video recorded and tracked to determine their locomotive rates and to assign these rates to Wright-stained morphological variants. From 24 oysters examined in January, February, March, and May, 1571 hemocytes were video recorded, identified, and their rate of locomotion (ROL) measured. Granulocytes (three types) and agranulocytes (one lymphoid and three nonlymphoid types) were recognized. Focusing on 15 oysters in March and May, 20,318 hemocytes were counted from duplicate slides to verify the classification and to show that predominant hemocytes vary greatly between samples and among individual oysters, yet population differences can be detected. Measured rates of locomotion indicate that the granulocyte subpopulation moved significantly faster (3.3 microns/min) than the agranulocyte subpopulation (0.7 microns/min) because most (81%) agranulocytes were not mobile. Of the mobile hemocytes, granulocytes were also significantly faster (4.8 microns/min vs. 3.5 microns/min, P less than 0.0001), and basophilic granulocytes (BASOs) were the most active and abundant cell type. Examination of monthly percentages of cells and ROL indicates, however, that granulocyte dominance and ROL are not invariable. Granulocyte percentages of more than 60% in January, February, and March decreased to 32% in May, and BASO dominance was reduced to 15%. Further, percentages of mobile granulocytes decreased from greater than 65% in January, February, and March to 50% in May. ROL for all cells decreased from greater than 2.3 microns/min in these months to 1.0 microns/min in May. The fewer mobile hemocytes tracked in May had significantly (P less than .05) lower average ROL (4.0 microns/min) than those in January and March (4.7 microns/min each). Agranulocytes increased in May due to an increase in nonlymphoid cells.
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.
Opsonizing and agglutinating activities of plasma from the freshwater clam, Corbicula fluminea, were found to be inhibited by the sugars, 2-deoxy-D-glucose (deoxy-Glu) and N-acetyl-D-galactosamine (GalNAc). The plasma opsonin/agglutinin was subsequently isolated by a two-step separation procedure. Aldehyde-fixed rabbit erythrocytes (RRBC) were used as a solid-phase plasma opsonin affinity absorbant, and deoxyGlu and GalNAc were used in the eluting buffer to desorb several RRBC-binding plasma proteins. The second step involved the further separation of sugar-eluted proteins by Sephacryl S-200 gel filtration. A plasma protein with an apparent molecular weight of 40 kd on SDS-PAGE under nonreducing conditions was found to possess both agglutinating and opsonizing activities. It was further shown to be composed of two identical 20 kd subunits associated through disulfide linkage(s). Although this protein shares some structural similarity with other bivalve opsonins, differences in native molecular size or subunit structure, agglutinating properties and/or sugar binding specificity support the current hypothesis that naturally occurring plasma opsonins of molluscs represent a heterogeneous group of proteins unified primarily through their lectin-like characteristic of binding specific carbohydrate determinants.
To elucidate a mutual correlation between the hemolymph lectin and hemocytes of the pearl oyster, Pinctada fucata martensii, we searched for common epitopes and ligands. Neither the hemocyte plasma membrane nor cytoplasm was immunoreactive to anti-hemolymph lectin antibody. The hemolymph lectin strongly bound to D-galactose and N-acetyl-D-galactosamine, and the plasma membrane of both granulocytes and agranulocytes had affinity only for D-mannose and N-acetyl-D-glucosamine-binding plant lectins. In the gonad, the hemolymph lectin selectively adsorbed injected horse red blood cells (HRBC), and its hemagglutinating activity probably prevented them from dispersing. Pinctada sp. may possess system of recognition of non-self by the hemolymph lectin.
The juvenile-bivalve pathogen Vibrio alginolyticus NCMB 1339 was toxic, in vitro, to hemocytes from adult Mytilus edulis. Toxicity was mediated by both washed bacterial cells and culture supernates. Washed cells of an environmental isolate of V. alginolyticus, Strain PS-1, were 2.5 times less toxic to Mytilus hemocytes, but this strain did produce a lethal extracellular factor(s) in broth culture, albeit at lower levels than V. alginolyticus NCMB 1339. Hemolymph fractions from Mytilus exerted a reciprocal toxic effect on the bacteria. Hemocytes were responsible for most of this bacteriocidal activity and toxic oxygen intermediates were involved in the phagocytic defense mechanisms of these cells.
A bacterial pathogen is a highly adapted microorganism which has the capacity to cause disease. The mechanisms used by pathogenic bacteria to cause infection and disease usually include an interactive group of virulence determinants, sometimes coregulated, which are suited for the interaction of a particular microorganism with a specific host. Because pathogens must overcome similar host barriers, common themes in microbial pathogenesis have evolved. However, these mechanisms are diverse between species and not necessarily conserved; instead, convergent evolution has developed several different mechanisms to overcome host barriers. The success of a bacterial pathogen can be measured by the degree with which it replicates after entering the host and reaching its specific niche. Successful microbial infection reflects persistence within a host and avoidance or neutralization of the specific and nonspecific defense mechanisms of the host. The degree of success of a pathogen is dependent upon the status of the host. As pathogens pass through a host, they are exposed to new environments. Highly adapted pathogenic organisms have developed biochemical sensors exquisitely designed to measure and respond to such environmental stimuli and accordingly to regulate a cascade of virulence determinants essential for life within the host. The pathogenic state is the product of dynamic selective pressures on microbial populations.
The interactions between bacteria and mammalian cells are important events both for harmless and necessary colonization of mucosal membranes, and in the pathogenesis of infections. Colonization, invasion and tissue damage depend both on bacterial virulence factors and defense mechanisms of the challenged host. This encounter involves not only specific ligand-receptor interactions determining the tropism of the infection, but also non-specific, physicochemical factors.
Hemolymph of Mytilus edulis is known to contain cytotoxic hemocytes and a serum agglutinin. This study describes the occurrence of strong hemolytic properties as well as agglutinating activity of a hemocyte extract against different types of erythrocytes. The hemolysin purified by preparative disc-electrophoresis and subjected to PAGE reveals one band which is also visible in the electropherogram of the extract or serum, respectively. Likewise, the serum agglutinin corresponds to another distinct fraction of extract and serum. Both, hemolytic and agglutinating molecules are detectable in the supernatant of a short-term hemocyte culture indicating that these substances are actively secreted by Mytilus blood cells.
Phagocytosis of human erythrocytes (rbc) by hemocytes of the mussel Mytilus edulis was found to be influenced by four heterologous lectins. The effects were examined in the absence of Ca++ ions under three experimental conditions: when the lectins were bound to 1) both hemocytes and rbc, 2) only hemocytes, but not to rbc, and 3) only rbc, but not to hemocytes. The lectins used included: albumen gland agglutinin from Helix pomatia (HPA), wheat germ agglutinin (WGA), Ricinus-120 (Ric-120) and Concanavalin-A (Con A). HPA, WGA and Ric-120, for which both hemocytes and A-rbc possess receptors, strongly enhanced uptake of A-rbc. This lectin-mediated phagocytosis was abolished by addition of specific sugars either to lectin-pretreated rbc (HPA, WGA) or to a pretreated hemocyte monolayer (Ric-120); this indicated the stimulation of phagocytosis by the binding of lectin to carbohydrate determinants at the surface of hemocytes and target cells. On the other hand, HPA which binds to hemocytes, but not to O-rbc, did not influence phagocytosis of these rbc; and Con A which binds to A-rbc, but not to hemocytes, also failed to stimulate phagocytosis. These findings reveal the importance of carbohydrate determinants on the surface of hemocytes as well as on target cells in recognition and in lectin-mediated phagocytosis of foreign cells by Mytilus hemocytes.
Lectin receptors for WGA, Ricinus 60, Ricinus 120, and for the agglutinin from the albumin gland of Helix pomatia were detected on the surface of Mytilus hemocytes by an agglutination assay. When hemocyte monolayers on slides were incubated with these lectins, strong rosette formation was obtained after addition of human erythrocytes. The lowest lectin concentration which caused formation of rosettes was determined to be 5 micrograms/ml of WGA or of Helix agglutinin, and 20 micrograms/ml of Ricinus 60 or 40 micrograms/ml of Ricinus 120, respectively. In contrast to these results, no binding of indicator cells to Mytilus hemocytes occurred with various other lectins, including Con A.
Lysozyme activity has been demonstrated in both the supernatant and pellet fractions of whole hemolymph of the American oyster, Crassostrea virginica, subjected to centrifugation at 4000 and 10,000 × g. In each case the enzyme activity is greater in the supernatant than in the pellet.The lytic activity of the molluscan lysozyme on Micrococcus lysodeikticus, like that of egg-white lysozyme, is salt dependent, is relatively heat stable, and is very sensitive to changes in ionic concentration. The optimal pH of the molluscan enzyme, however, ranges from 5.0 to 5.5, depending on the buffer employed.When tested against a number of bacteria, the oyster lysozyme has been found to be active against not only M. lysodeikticus but also Bacillus subtilis, B. megaterium, Escherichia coli, Gaffkya tetragena, Salmonella pullorum, and Shigella sonnei, although it is less active against the last four mentioned. It is not active against Staphylococcus aureus.It is postulated that the lysozyme in the serum of C. virginica has its origin in cytoplasmic phagosomes of granulocytes and is released when these organelles become ruptured.
Using antisera produced against a serum lectin we have shown by employing immunocytofluorescence that hemocytes from the oyster, Crassostrea virginica, possess a lectin which is situated on the external surface of the cell membrane. The antisera block the binding of hemocyte microsomes to protease-treated vertebrate erythrocytes, thus confirming that the hemocyte membrane lectin is serologically related to the serum lectin. The major serum lectin has an apparent mass of 34,000. Flow cytometry has revealed that the distribution of the surface lectin on hemocytes represents a heterogeneous expression on a population basis, but no discrete cell subpopulations can be identified.
Vibrio cholerae 01, the causative agent of cholera, is known to persist in estuarine environments as endogenous microflora. The recent introduction of V. cholerae 01 into estuaries of the North and South American continents has stimulated the need to determine the effect of controlled purification on reducing this pathogen in edible molluscan shellfish. Experiments defined parameters for the uptake and retention of V. cholerae 01 in tissues of Crassostrea virginica, and these parameters were compared with those for Escherichia coli and Salmonella tallahassee, bacteria which are usually eliminated from moderately contaminated shellfish within 48 h. Oysters accumulated greater concentrations of V. cholerae 01 than E. coli and S. tallahassee. When V. cholerae 01 was exposed to controlled purification at 15, 19 and 25 degrees C over 48 h, it persisted in oysters at markedly higher levels than E. coli and S. tallahassee. The concentration of a V. cholerae 01-specific agglutinin did not positively correlate with the uptake or retention of V. cholerae 01. These data show that state and federally approved controlled purification techniques are not effective at reducing V. cholerae 01 in oysters.
Hemocytes of the hard clam Mercenaria mercenaria migrate toward secreted bacterial products in vitro by chemotaxis (i.e., by detection of an increasing chemical gradient of attractant). The attractants produced by Escherichia coli are peptides or small proteins. Clam hemocytes also migrate toward formyl-methionyl-leucyl-phenylalanine (fMLF), a mammalian neutrophil chemoattractant produced by bacteria, but not toward the related compound formyl-methionyl-valine. Migration of hemocytes to fMLF was blocked with the neutrophil fMLF receptor antagonist, t-Boc-MLF, suggesting that the hemocytes possess this receptor and that the response is receptor-mediated. However, fMLF is not the major bacterial chemoattractant for clam hemocytes, as t-Boc-MLF did not block migration of these cells to secreted bacterial chemoattractants.
Luminol-dependent chemiluminescence (LDCL) and nitroblue tetrazolium (NBT) reduction assays have been used to measure reactive oxygen intermediate (ROI) production by oyster (Crassostrea virginica) hemocytes, as well as ROI modulation caused by disease or exposure to environmental toxicants. However, ROI responses measured by these tests apparently vary considerably among other bivalve species. In all species tested, unstimulated hemocytes produced small quantities of ROIs. In C. virginica and Geukensia demissa phagocytosis or treatment with phorbol myristate acetate triggered significantly augmented, but kinetically dissimilar, ROI responses; however, no induction was recorded in two clam species (Mya arenaria and Mercenaria mercenaria). This was supported by both LDCL and NBT assays, measuring activity of the myeloperoxidase/hydrogen peroxide system and production of intracellular superoxide anion, respectively. The failure of the clams to respond to standard ROI-eliciting procedures is possibly indicative of interspecies differences in hemocyte-mediated antimicrobial defense mechanisms.
Boyden chamber assays were performed to test the stimulatory effect of different bacterial products on the migratory activity of Mytilus haemocytes. The results indicate that these blood cells exhibit chemotactic as well as chemokinetic reactions. Lipopolysaccharides (LPS) from both Serratia marcescens and Escherichia coli stimulated the migration of cells through the membrane of the Boyden chamber when LPS was present in the lower compartment only. In contrast, addition of LPS to the lower as well as to the upper chamber did not increase the rate of migrating cells. Thus, LPS seemed to act as a chemotaxis-stimulating substance. Further analysis indicated that complete LPS molecules are required for cell stimulation because this did not occur when either the lipid or polysaccharide moieties of LPS were tested alone. Unlike LPS, the formylated tripeptide N-FMLP stimulated random cell migration. The peptide, which is released by bacteria, induced a higher haemocyte motility when present in both wells of the Boyden chamber than in tests where it was added to the lower compartment only. This chemokinetic response was not stimulated by the tetrapeptide N-FMLPLys. These findings demonstrate that bacterial products may elicit chemotactic and/or chemokinetic reactions in haemocytes from an invertebrate, and that the type of reaction that occurs is dependent upon the nature of the molecules presented.
Hemocytes of two marine molluscs, Nerita albicilla (gastropod) and Mytilus edulis (bivalve), were stimulated in vitro with zymosan and live cells of Vibrio parahaemolyticus and Escherichia coli as determined by luminol-dependent chemiluminescence (CL). The CL response was enhanced in the presence of the respective molluscan plasma. Hemocytes of an estuarine gastropod, Clithon retropictus, showed low CL response to zymosan and V. parahaemolyticus, which was slightly enhanced in the presence of C. retropictus plasma. Hemocytes of an estuarine bivalve, Corbicula japonica, showed no CL response. CL response of hemocytes might be a useful tool to analyze defense mechanisms of estaurine molluscs.
Lectins, sugar-binding proteins or glycoproteins of non-immune origin that agglutinate cells or precipitate glycoconjugates, are found in species of all taxa from viruses and bacteria to vertebrates. Their presence in the body-fluids of invertebrates is well documented, but their biological role(s) are not yet firmly established. Invertebrates phylogenetically below the Agnatha lack immunoglobulins, T-cell receptors or lymphoid cells, and thus have no protection due to clonal selection and immune memory as we have observed from studies with vertebrates. However, these primitive invertebrates have survived with success in environments where they are intimately exposed to bacteria, and yet we have relatively scarce information about their humoral defense reactions.
Movements of tissue hemocytes in the Eastern oyster Crassostrea virginica were monitored and quantified by image analysis of sections following inoculation with agar cores containing Escherichia coli or cell-free medium on which the bacteria had previously grown. Hemocytes respond to the presence of live bacteria by accumulating in widely dispersed areas of tissue surrounding the gut and digestive diverticula. The response is rapid and evident within 40 min, is maximal at 1 hr, and declines by 3 hr after inoculation. Sterile implanted agar cores do not produce a response. Bacteria killed with ozone elicit a response when inoculated together with the medium on which they had grown while bacteria killed by heat or formalin do not. Killed bacteria suspended in saline fail to stimulate hemocyte chemokinesis. Cell-free medium applied externally produces a response equal to that measured with live bacteria inoculated internally. Extraction of bacteria-free medium with hexane does not significantly reduce hemocyte chemokinesis. Digestion of bacteria-free medium with pronase completely eliminates chemokinesis. Molecular filtrates of bacteria-free medium induce maximal chemokinetic response at molecular weight as low as 1 kDa. These data show that the oyster hemocyte activators produced by E. coli are most likely low-molecular-weight polypeptides which diffuse from the site of inoculation and can pass through the intact external surface epithelium to induce a chemokinetic response.
The main results published on the production of reactive oxygen intermediates by hemocytes and digestive glands of marine bivalves such as mussels, oysters or clams have been reviewed and discussed. Mussel and oyster hemocytes respond to appropriate stimuli with a burst of respiratory activity and the generation of reactive oxygen intermediates in a manner resembling the respiratory burst of mammalian phagocytes. However, interspecies differences in hemocytes-mediated antimicrobial defense mechanisms occur since clam hemocytes do not show any increase of reactive oxygen intermediate production upon similar stimulations. Hepatopancreatic gland of bivalves, as mammalian and fish liver produce reactive oxygen species during the one-electron reduction of xenobiotics, and mechanistic differences appear between bivalves and mammals. Thus, it appears that, in spite of some interspecies differences, the generation of cytotoxic reactive oxygen species is a general protective mechanism of most, if not all, animal species.
The phylum Mollusca is second only to the Arthropoda in both number and diversity of living species. Representatives include not only the readily recognized gastropods (snails, slugs and limpets) and bivalves (e.g., oysters, mussels and clams) but also the ‘brainy’ cephalopods including octopus and squid, and more primitive representatives such as chitons. These animals occur in a huge variety of terrestrial, freshwater and marine habitats. Interest in their pathogens and parasites arises primarily from the role of gastropods in the transmission of trematodes of medical and veterinary importance. Perhaps the most important of these are the human infecting schistosomes, estimated to parasitize 200 million people world wide. In addition, molluscs such as oysters, clams, mussels as well as gastropod “escargots” are increasingly being raised for human consumption, in both the developed and developing world. There are thus clear medical and economic needs to obtain a full understanding of molluscan pathogens, the capabilities of the molluscan immune systems, and how these interact to determine the outcome of a host-parasite encounter.
Lymphoid organs in Crustacea and Insecta were first discovered by L. Cuénot between 1890 and 1910. For many years, however, little research was done into the immune processes of invertebrates (which represent 95% of animal species), as it was assumed that with a short life-span and a high rate of reproduction, a complex and highly efficient immune system was not required.
Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics.