Hemocyte Phagocytosis Research Articles

Hemocyte-Mediated Phagocytosis in Crustaceans.

Phagocytosis is an ancient, highly conserved process in all multicellular organisms, through which the host can protect itself against invading microorganisms and environmental particles, as well as remove self-apoptotic cells/cell debris to maintain tissue homeostasis. In crustacean, phagocytosis by hemocyte has also been well-recognized as a crucial defense mechanism for the host against infectious agents such as bacteria and viruses. In this review, we summarized the current knowledge of hemocyte-mediated phagocytosis, in particular focusing on the related receptors for recognition and internalization of pathogens as well as the downstream signal pathways and intracellular regulators involved in the process of hemocyte phagocytosis. We attempted to gain a deeper understanding of the phagocytic mechanism of different hemocytes and their contribution to the host defense immunity in crustaceans.

Trpml controls actomyosin contractility and couples migration to phagocytosis in fly macrophages.

Phagocytes use their actomyosin cytoskeleton to migrate as well as to probe their environment by phagocytosis or macropinocytosis. Although migration and extracellular material uptake have been shown to be coupled in some immune cells, the mechanisms involved in such coupling are largely unknown. By combining time-lapse imaging with genetics, we here identify the lysosomal Ca2+ channel Trpml as an essential player in the coupling of cell locomotion and phagocytosis in hemocytes, the Drosophila macrophage-like immune cells. Trpml is needed for both hemocyte migration and phagocytic processing at distinct subcellular localizations: Trpml regulates hemocyte migration by controlling actomyosin contractility at the cell rear, whereas its role in phagocytic processing lies near the phagocytic cup in a myosin-independent fashion. We further highlight that Vamp7 also regulates phagocytic processing and locomotion but uses pathways distinct from those of Trpml. Our results suggest that multiple mechanisms may have emerged during evolution to couple phagocytic processing to cell migration and facilitate space exploration by immune cells.

An Ancient BCR-like Signaling Promotes ICP Production and Hemocyte Phagocytosis in Oyster.

SummaryBCR/TCR-based adaptive immune systems arise in the jawed vertebrates, and B cell receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. The existence of BCR-like molecule and the activation mechanism of the downstream response are still not clear in invertebrates. In this study, an ancient BCR-like molecule (designated as CgIgR) with an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail was identified from the Pacific oyster Crassostrea gigas to investigate its involvement in immune response. CgIgR could bind different bacteria through five extracellular Ig domains and formed dimers. The activated CgIgR recruited CgSyk to promote CgERK phosphorylation. The CgIgR-mediated signaling promoted the production of immunoglobulin domain-containing proteins (CgICP-2 and CgLRRIG-1) through inducing CgH3K4me2. The produced CgICPs eventually facilitated hemocytes to phagocytize and eliminate V. splendidus. This study proposed that there was an ancient BCR-like molecule and BCR-like signaling in molluscs.

A novel C-type lectin with a YPD motif from Portunus trituberculatus (PtCLec1) mediating pathogen recognition and opsonization

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins that function as pattern recognition receptors (PRRs) in innate immune system. In this study, a new C-type lectin was identified from the swimming crab Portunus trituberculatus (PtCLec1). The full-length cDNA of PtCLec1 was 873 bp encoding 176 amino acids. The predicted PtCLec1 protein contained a signal peptide and a single carbohydrate-recognition domain with a special YPD motif. The PtCLec1 transcripts were mainly detected in hepatopancreas and its relative expression levels were significantly up-regulated after the challenges of Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtCLec1 (rPtCLec1) could bind all the tested pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan (GLU), and microorganisms, including V. alginolyticus, V. parahaemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, M. luteus and P. pastoris. It also exhibited strong activity to agglutinate bacteria and yeast in a Ca2+-dependent manner, and such agglutinating activity could be inhibited by d-galactose and LPS. Moreover, rPtCLec1 revealed antimicrobial activity against the tested Gram-negative (V. alginolyticus, V. parahaemolyticus and P. aeruginosa) and Gram-positive bacteria (S. aureus and M. luteus), and promoted the clearance of V. alginolyticus in vivo and hemocyte phagocytosis in vitro. Knockdown of PtCLec1 could down-regulate the expression of phagocytosis-related genes, but enhance the expression levels of prophenoloxidase (proPO) system-related genes, mannose-binding lectin (MBL), antimicrobial peptides (AMPs), MyD88 and Relish. All these results indicate that PtCLec1 might act as a PRR in immune recognition and an opsonin in pathogen elimination.

Pea Aphid Rearing, Bacterial Infection and Hemocyte Phagocytosis Assay.

Insects rely on the simple but effective innate immune system to combat infection. Cellular and humoral responses are interconnected and synergistic in insects' innate immune system. Phagocytosis is one major cellular response. It is difficult to collect clean hemolymph from the small insect like pea aphid. Here, we provide a practicable method for small insects hemocyte phagocytosis assay by taking pea aphid as an example. Furthermore, we provide the protocols for pea aphid rearing and bacterial infection, which offer referential method for related research.

Clic2α Chloride Channel Orchestrates Immunomodulation of Hemocyte Phagocytosis and Bactericidal Activity in <i>Crassostrea Gigas</i>

Chloride ion plays critical roles in modulating immunological interactions. Herein, we demonstrated that the anion channel CLIC2α mediates Cl- flux to regulate hemocytes functions in the Pacific oyster (Crassostrea gigas). Specifically, during infection by Vibrio parahemolyticus, chloride influx was activated following onset of phagocytosis. The phosphorylation of Akt was stimulated by Cl- ions entering host cells, further contributing to signal transduction regulating internalization of bacteria through the PI3K/Akt signaling pathway. Concomitantly, Cl- entered phagosomes, promoted the acidification and maturation of phagosomes, and contributed to production of HOCl to eradicate engulfed bacteria. Finally, genomic screening reveals CLIC2α as a major Cl- channel gene responsible for regulating Cl- influx in oysters. Knockdown of CLIC2α predictably impeded phagosome acidification and restricted bacterial killing in oysters. In conclusion, our work has established CLIC2α as a prominent regulator of Cl- influx and thus Cl- function in C. gigas in bacterial infection contexts.

Opsonic character of the plasma proteins in phagocytosis-dependent host response to bacterial infection in a marine invertebrate, Crassostrea gigas

Phagocytosis is an evolutionarily conserved immune response, whose efficiency is fundamentally coupled with opsonization of extracellular microbes. How marine mollusks cells recognize and selectively capture pathogens during phagocytosis to clear them is not completely understood. In this study, we observed that plasma is extremely effective for oyster hemocyte phagocytosis, so we investigated candidate proteins among plasma proteins with binding affinity for Vibrio parahaemolyticus in Pacific oyster (Crassostrea gigas) by subjecting them to mass spectroscopy analysis for protein identification and characterization, and address the complex regulatory network to engulf invaders. There were 620 identified proteins potentially associated with bacteria binding and phagocytosis which could be quantified. Our results showed that C1q and lectins identified in Pacific oyster plasma held binding ability to bacteria, clearly suggesting their potent to be opsonins. The dominant expressed plasma protein p1-CgC1q (Complement component 1q)-like protein was identified and its opsonic role was confirmed in this study. The cell surface receptor Cgintegrin interacts directly with p1-CgC1q to mediate phagocytosis. We further confirmed that the interaction between C1q and integrin not rely on the typical recognition site RGD but on the RGE. Evidence exist revealed that p1-CgC1q could coat bacteria via the endotoxin LPS (lipopolysaccharide) and subsequently bind the receptor integrin to significantly enhance hemocytic phagocytosis and bacteria clearance. This study has thus furnished clear evidence for the importance of plasma proteins in mollusk, shedding light on the humoral immunity and an underappreciated strategy in marine host-pathogen interactions.

Comparison of viability and phagocytic responses of hemocytes withdrawn from the bivalves Mytilus edulis and Dreissena polymorpha, and exposed to human parasitic protozoa

Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.

Polystyrene microplastics increase uptake, elimination and cytotoxicity of decabromodiphenyl ether (BDE-209) in the marine scallop Chlamys farreri

Microplastics are a growing problem in marine environments due to their ubiquitous occurrence and affinity for chemical pollutants. However, the influence of microplastics on the uptake, depuration and toxicity of decabromodiphenyl ether (BDE-209) in marine organisms is unclear. We exposed the marine scallop Chlamys farreri to polystyrene microplastics (PS; 125 μg/L) combined with BDE-209 (10 and 100 μg/L) to determine their toxicokinetics, cellular toxicity and histopathological effects. The results showed that PS acted as both a carrier and a scavenger for the bioaccumulation of BDE-209. Importantly, the carrier role of PS was greater than scavenger one. PS increased the negative effect of BDE-209 (100 μg/L) on hemocyte phagocytosis, and ultrastructural changes in gills and digestive gland of scallops due to their carrier role for the bioaccumulation of BDE-209. However, PS did not increase the DNA damage of BDE-209 on the hemocytes. These findings are evidence of microplastics transferring adsorbed pollutants to marine organisms, and increasing their toxicity.

Metallothionein, hemocyte status and superoxide dismutase/aspartate aminotransferase activity are sensitive biomarkers of cadmium stress in Onchidium reevesii

Metal pollution in the environment is a serious threat to the biological sustainability of coastal ecosystems. However, our current understanding of the biological effects of metals in these ecosystems is limited. Herein, we investigated the responses of the sea slug Onchidium reevesii to persistent sublethal Cd environmental stress. Dynamic expression was analyzed using various biomarkers. The full-length cDNA of O. reevesii metallothionein (MT) was cloned and consists of 1639 nucleotides encoding a 65 amino acid polypeptide. Phylogenetic analysis showed that Or-MT has conserved Cys residues typical of MTs, including a typical Cys–X–Cys motif, implying that it can function the same as the MT of other shellfish. Expression of Or-MT in response to Cd varied in different tissues, and was highest in gastropod tissues. Thus, regiotemporal expression of MT may be useful for assessing pollution in coastal areas. Cellular immunity (in the hemolymph) and enzyme activity (in the hepatopancreas) were investigated along with hemocyte viability, hemocyte phagocytosis, and superoxide dismutase (SOD) and aspartate aminotransferase (AST) activities. Hemocyte viability was elevated under continuous Cd exposure but hemocyte phagocytosis was decreased. SOD and AST activities in the hepatopancreas fluctuated considerably, and SOD activity was more sensitive. SOD activity was lowest at 4 h and highest at 12 h, while AST activity peaked at 2 h and was lowest at 48 h. Thus, changes in enzyme activity may reveal adaptation to stress. Furthermore, the response patterns of certain enzymes, cellular immunity, and MT expression in O. reevesii could serve as biomarkers of Cd pollution in aquatic environments.

SpBark Suppresses Bacterial Infection by Mediating Hemocyte Phagocytosis in an Invertebrate Model, Scylla paramamosain.

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, SpBark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. SpBark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that SpBark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca2+ by incubating microorganisms with SpBark CTLD instead of SRCRDs. These results suggested that SpBark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with SpBark CTLD promoted bacterial clearance in vivo. This finding indicated that SpBark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by SpBark was further determined using SpBark-silenced crabs injected with V. parahemolyticus. SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by SpBark was closely related to SpBark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by SpBark was as follows: SpBark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.

Effects of dopamine on immune signaling pathway factors, phagocytosis and exocytosis in hemocytes of Litopenaeus vannamei

Dopamine (DA) is an important neuroendocrine factor, which can act as neurotransmitter and neurohormone. In this study, we explored the immune defense mechanism in Litopenaeus vannamei with injection of dopamine at 10−7 and 10−6 mol shrimp−1, respectively. The genes expressions of dopamine receptor (DAR), G proteins (Gs, Gi, Gq), phagocytosis and exocytosis-related proteins, as well as intracellular signaling pathway factors, and immune defense parameters were measured. Results showed that mRNA expression levels of dopamine receptor D4 (D4), Gi, nuclear transcription factors and exocytosis-related proteins decreased significantly and reached the minimum at 3 h, while the genes expressions of Gs, Gq and phagocytosis-related proteins reached the highest and lowest levels at 3 h and 6 h, respectively. The second messenger synthetases increased significantly in treatment groups within 3 h. Simultaneously, the second messengers and protein kinases shared a similar trend, which were significantly elevated and reached the peak value at 3 h. Ultimately lead to the total hemocyte count (THC), proPO activity and phagocytic activity decreased significantly, reaching minimum values at 3 h, 3 h and 6 h, respectively. While PO activity showed obvious peak changes, which maximum value reached at 3 h. These results suggested that DA receptor could couple with G protein after DA injection and might regulate immunity through cAMP-PKA, DAG-PKC or CaM pathway.

Hemolin, an immunoglobulin-like peptide, opsonizes nonself targets for phagocytosis and encapsulation in Spodoptera exigua, a lepidopteran insect

Abstract Hemolin is an immunoglobulin-like peptide that plays crucial role in microbial pathogen surveillance. However, its specific molecular actions in immune responses remain largely unclear. This study focused on the modulation of hemocyte phagocytosis of hemolin via its opsonin activity. A full length open reading frame (ORF) of hemolin (SeHem) was identified in the beet armyworm, Spodoptera exigua. Its ORF encoded 418 amino acids and shared 37.3–93.3% sequence similarities with other lepidopteran hemolins. SeHem was expressed in all developmental stages of S. exigua. In larval stage, SeHem was mostly expressed in hemocytes. Immune challenge significantly increased SeHem expression in hemocytes, fat body, midgut, and epidermis. RNA interference (RNAi) was performed by injecting SeHem-specific double-stranded RNA (dsRNA) into hemocoel, resulting in 60–80% reduction of SeHem transcript levels at 24–48 h after injection. Under RNAi condition, larvae showed significant loss of phagocytotic activities. In addition, hemocytes collected from control larvae exhibited significant reduction in phagocytosis when they were overlaid with plasma obtained from RNAi-treated larvae. RNAi treatment also suppressed encapsulation against injected beads. Furthermore, RNAi-treated larvae became highly susceptible to entomopathogenic bacteria. These results suggest that hemolin can act as opsonin in cellular immune responses to protect S. exigua against infections by entomopathogens.

Molecular characteristics of AMPK and its role in regulating the phagocytosis of oyster hemocytes

Phagocytosis is one of the fundamental cellular immune defense parameter that helps in the elimination of the invading pathogens in both vertebrates and invertebrates, which require plenty of energy for functioning. In the present study, we identified the critical energy regulator AMP-activated protein kinase (AMPK) in Crassostrea hongkongensis which is composed of three subunits, named ChAMPK-α, ChAMPK-β, and ChAMPK-γ, and then analyzed the function of AMPK in regulating hemocyte phagocytosis. All the three ChAMPK subunits mRNA were detected to be expressed at various embryological stages, and also constitutively expressed in multiple tissues with high expression in gill and mantle. The phylogenetic tree showed that the three subunits of AMPK were correspondingly clustered with its orthologue branches. Furthermore Western Blot analysis revealed that the AMPK pharmacological inhibitors Compound C could effectively down-regulate the Thr172 phosphorylation level of AMPK-α, and the hemocyte phagocytosis was inhibited by Compound C (CC), which indicate its existence in the oyster. Our results showed that treatment of AMPK inhibitors significantly attenuated the capacity of hemocytes phagocytosis. Moreover, Compound C could also change the organization of actin cytoskeleton in the oyster hemocytes, demonstrating the crucial role of AMPK signaling in control of phagocytosis.

Effects of crustacean hyperglycemic hormone (CHH) on regulation of hemocyte intracellular signaling pathways and phagocytosis in white shrimp Litopenaeus vannamei

Shrimps like other arthropods rely on innate immune system, and may have some form of adaptive immunity in defending against pathogens. Phagocytosis is one of the oldest cellular processes, serving as a development process, a feeding mechanism and especially as a key defense reaction in innate immunity of all multicellular organisms. It is confirmed that crustacean hyperglycemic hormone (CHH) is one of the most important neuropeptides produced by Neuro-endocrine Immune (NEI) regulatory network, which undertakes important roles in various biological processes, especially in immune function and stress response. In this study, the recombinant Litopenaeus vannamei CHH (rLvCHH) was obtained from a bacterial expression system and the intracellular signaling pathways involved in the mechanism of phagocytosis after rLvCHH injection was investigated. The results showed that the contents of adenylyl cyclase (AC), phospholipase C (PLC) and calmodulin (CaM) in hemocytes were increased significantly after rLvCHH injection. Furthermore, the mRNA expression levels of NF-kB family members (relish and dorsal) and phagocytosis-related proteins in hemocytes were basically overexpressed after rLvCHH stimulation, while the expression level of NF-kB repressing factor (NKRF) gene was down-regulated significantly. Eventually, the total hemocyte count and phagocytic activity of hemocyte were dramatically enhanced within 3 h. Collectively, these results indicate that shrimps L. vannamei could carry out a simple but ‘smart’ NEI regulation through the action of neuroendocrine factors, which could couple with their receptors and trigger the downstream signaling pathways during the phagocytic responses of hemocytes.

Effect of Ulva reticulata Extract in Increasing Hemocyte Count and Phagocytosis Activity in Tiger Shrimp (Penaeus monodon)

Effect of Ulva reticulata Extract in Increasing Hemocyte Count and Phagocytosis Activity in Tiger Shrimp (Penaeus monodon)

Persistent symbiont colonization leads to a maturation of hemocyte response in the Euprymna scolopes/Vibrio fischeri symbiosis.

The binary association between the squid, Euprymna scolopes, and its symbiont, Vibrio fischeri, serves as a model system to study interactions between beneficial bacteria and the innate immune system. Previous research demonstrated that binding of the squid's immune cells, hemocytes, to V. fischeri is altered if the symbiont is removed from the light organ, suggesting that host colonization alters hemocyte recognition of V. fischeri. To investigate the influence of symbiosis on immune maturation during development, we characterized hemocyte binding and phagocytosis of V. fischeri and nonsymbiotic Vibrio harveyi from symbiotic (sym) and aposymbiotic (apo) juveniles, and wild‐caught and laboratory‐raised sym and apo adults. Our results demonstrate that while light organ colonization by V. fischeri did not alter juvenile hemocyte response, these cells bound a similar number of V. fischeri and V. harveyi yet phagocytosed only V. harveyi. Our results also indicate that long‐term colonization altered the adult hemocyte response to V. fischeri but not V. harveyi. All hemocytes from adult squid, regardless of apo or sym state, both bound and phagocytosed a similar number of V. harveyi while hemocytes from both wild‐caught and sym‐raised adults bound significantly fewer V. fischeri, although more V. fischeri were phagocytosed by hemocytes from wild‐caught animals. In contrast, hemocytes from apo‐raised squid bound similar numbers of both V. fischeri and V. harveyi, although more V. harveyi cells were engulfed, suggesting that blood cells from apo‐raised adults behaved similarly to juvenile hosts. Taken together, these data suggest that persistent colonization by the light organ symbiont is required for hemocytes to differentially bind and phagocytose V. fischeri. The cellular immune system of E. scolopes likely possesses multiple mechanisms at different developmental stages to promote a specific and life‐long interaction with the symbiont.

A C1qDC (CgC1qDC-6) with a collagen-like domain mediates hemocyte phagocytosis and migration in oysters

Most of the bivalve C1q domain containing proteins (C1qDCs) are either only composed of the globular head domain, or contain an N-terminal coiled-coil domain, presumed to cover a role in oligomerization. On the other hand, collagen regions, widespread in vertebrate C1qDCs, are very uncommon in bivalves. In the present study, a C1qDC with a collagen-like domain (designated CgC1qDC-6) was identified from the Pacific oyster Crassostrea gigas and its possible involvement in immune responses was also characterized. The coding sequence of CgC1qDC-6 was of 756 bp, encoding a peptide of 251 amino acids with an N-terminal signal peptide, a central collagen-like domain, and a C-terminal ghC1q domain. CgC1qDC-6 was clustered with the C1qDCs from several mollusks in the phylogenetic tree. CgC1qDC-6 was detected at both mRNA and protein levels in all tested tissues including hepatopancreas, gonad, gill, mantle, adductor muscle, and hemocytes. The recombinant CgC1qDC-6 protein (rCgC1qDC-6) exhibited binding activity to various pathogen-associated molecular patterns (PAMPs) including LPS, PGN, mannose and Poly I:C, and microorganisms including Gram-negative bacteria (Escherichia coli and Vibrio splendidus), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus), and fungus (Pichia pastoris). The phagocytic rates of oyster hemocytes towards V. splendidus pre-incubation with rCgC1qDC-6 were significantly enhanced (p < 0.05). In the chemotaxis assay, rCgC1qDC-6 could mediate the migration of oyster hemocytes in a dose-dependent manner, which exhibited a positive chemotactic effect at low concentration (<10 nM). These results collectively indicated that CgC1qDC-6 could serve as a pattern recognition receptor and mediate the hemocyte phagocytosis and migration to eliminate the invading pathogens.

Hemolymph C1qDC promotes the phagocytosis of oyster Crassostrea gigas hemocytes by interacting with the membrane receptor β-integrin

Phagocytosis constitutes a conserved cellular process for multicellular animals to ingest or engulf other cells or particles, which is facilitated by the use of opsonins to bind foreign particles and interact with cell surface receptors. The invertebrate secreted C1q domain-containing proteins (C1qDCs) have been reported to exhibit opsonic activity, while the detailed mechanisms of opsonization still remain unclear. In the present study, a C1qDC (designated as CgC1qDC-5) with opsonic activity was identified from the hemolymph of oyster Crassostrea gigas. CgC1qDC-5 exhibited the ability to bind pathogen-associated molecular patterns (PAMPs) of lipopolysaccharides (LPS) and Lipid A. It could also bind and agglutinate Gram-negative bacteria Escherichia coli, Vibrio splendidus and Vibrio anguillarum, whereas the agglutinating activity could be inhibited by LPS. In addition, CgC1qDC-5 could enhance the phagocytosis of hemocytes toward E. coli, V. splendidus, and V. anguillarum. GST pull-down and surface plasmon resonance assays in vitro revealed that CgC1qDC-5 could interact with β-integrin (CgIntegrin). In vivo, CgC1qDC-5 was observed to bind hemocytes and co-localized with CgIntegrin on the cell membrane of hemocytes. Antibody-mediated blockage of CgIntegrin hindered the CgC1qDC-5-enhanced hemocytic phagocytosis. CgIntegrin also exhibited the ability to bind the Gram-negative bacteria E. coli, V. splendidus, V. anguillarum and Vibrio parahaemolyticus, and PAMP of LPS, but not Lipid A. A phagocytosis assay demonstrated that CgIntegrin could directly mediate phagocytosis toward bacteria as a phagocytic receptor. These results collectively suggested that CgC1qDC-5 could serve as an opsonin to recognize and bind bacteria, and subsequently interact with CgIntegrin on the hemocyte surface to enhance the CgIntegrin-mediated phagocytosis in oyster.

Flow cytometric characterization of hemocytes of the abalone Haliotis diversicolor (Reeve, 1846) and effects of air exposure stresses on hemocyte parameters

The variously colored abalone Haliotis diversicolor is widely distributed in the northwest Pacific region and commonly occurs in shallow subtidal regions of Jeju Island, off the south coast of Korea. In this study, we first characterized the morphology and immune-related activities of H. diversicolor hemocytes using flow cytometry and light microscopy. Flow cytometry revealed three types of hemocytes in the hemolymph: granulocytes, hyalinocytes, and blast-like cells. Hyalinocytes were the largest and most abundant, while granulocytes were intermediate-sized and contained many granules in the cytoplasm. Flow cytometry indicated that granulocytes were mainly involved in phagocytosis and oxidative activities, although hyalinocytes also exhibited a certain level of immune-related activities. Blast-like cells were the smallest and exhibited a lack of phagocytosis and oxidative capacities. To understand the adaptive capacity to air exposure, abalones were exposed to air or humidity over 30 h. All abalones perished within 24 h after dry-air exposure, while 20% of abalones had survived under humid conditions by the end of the experiment. After 6 h of air exposure and 12 h of humidity, total hemocyte counts and phagocytosis capacity declined dramatically, and the level of hemocyte DNA damage increased significantly (P < .05). This study suggests that sufficient oxygen supply during transportation could enhance the survival of H. diversicolor, thereby improving its market value.