Hemocyte Subpopulations Research Articles

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5–10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs.

Characterization of hemocytes from different body fluids of the eastern oyster Crassostrea virginica

Bivalve hemocytes are involved in a variety of physiological and immunological functions. Circulating hemocytes in the hemolymph represent the main component of the internal self-defense system while hemocytes present in the extrapallial space (between the mantle and the shell) are actively involved in biomineralization and shell formation. This study focused on the characterization of hemocytes from different body fluids of the eastern oyster Crassostrea virginica. Hemocytes present in the hemolymph were compared to those contained in the extrapallial fluid. Hemocytes associated with the mucus layer covering pallial organs (mantle, gills, body wall) were also investigated because of their potential role as sentinel cells. Hemocytes were characterized using flow cytometry in conjunction with fluorescent epitope markers (clusters of differentiation, lectins) as well as functional assays (i.e. phagocytosis and reactive oxygen species -ROS). Compared with the hemolymph, there was a significantly greater percentage of granulocytes and agranulocytes among extrapallial and pallial hemocytes, respectively. Accounting for the different percentages of hemocyte sub-populations, significant differences in surface carbohydrate and clusters of differentiation signatures were also revealed between the different fluids. Most informative epitope markers included concanavalin A, peanut agglutinin, soybean agglutinin, CD11b and CD14. Functional assays revealed significant differences in phagocytic activity and ROS production between hemocytes from the extrapallial fluid and hemolymph; however, less robust differences were observed between hemolymph cells and hemocytes associated with the pallial mucus. Findings from this study suggest that there are markedly different hemocyte populations in the three body fluids. The role of peripheral cells, particularly those associated with the pallial mucus, requires further investigations.

Comparative proteomic analysis between two haemocyte subpopulations in shrimp Fenneropenaeus chinensis

In our previous work, granulocytes and hyalinocytes were successfully separated by immunomagnetic bead (IMB) method using monoclonal antibodies (mAbs) against granulocytes of shrimp (Fenneropenaeus chinensis). In order to elucidate the proteomic differentiation between granulocytes and hyalinocytes, in this paper, the differentially expressed proteins were analyzed between non-fixed/un-permeabilized (NFP) haemocytes and fixed/permeabilized (FP) haemocytes using two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS). Then the FP haemocytes were separated into two haemocyte subpopulations using IMB method, and the comparative proteome between granulocytes and hyalinocytes was investigated. The results showed that 10 differentially expressed protein spots were detected and identified as 4 proteins in the NFP haemocytes. Twenty one differentially expressed proteins were successfully identified between granulocytes and hyalinocytes, which include 4 unique expressed proteins in granulocytes, 4 significantly highly expressed proteins in granulocytes, and 13 significantly high expressed proteins in hyalinocytes. According to Gene Ontology annotation, the identified proteins between granulocytes and hyalinocytes were classified into six categories, including binding proteins, proteins involved in catalytic activity, enzyme regulator activity, structural molecule activity, translation regulator activity, and ungrouped proteins. Furthermore, quantitative PCR confirmed that the trend of transcription levels of three selected genes were consistent with the proteomic data from 2-DE. The results may lead to better understanding of the functions of haemocyte subpopulations.

Various cellular responses of different shrimp haemocyte subpopulations to lipopolysaccharide stimulation

Different haemocyte types have been reported to play diverse roles in immune defense of shrimp. To investigate the roles of the three haemocyte types [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)] of shrimp in immune responses against lipopolysaccharide (LPS), percentage, non-specific esterase activity (EA), reactive oxygen species (ROS) production and nitric oxide (NO) production of the three haemocyte subpopulations were analyzed in LPS-injected Penaeus monodon using flow cytometry. Results showed that percentage of HC increased after 3 h injection, and returned to the original level after 48 h. Proportion of SGC and GC reduced after 6–36 h and 3–12 h respectively, and recovered to the initial level after 48 and 24 h respectively. Loss of SGC and GC might be related to degranulation to release proPO system, and degranulation of GC seemed more sensitive to LPS stimulation. EA of both HC and SGC improved after 3–6 h injection, while EA of GC was induced after 3–24 h. No significant effect of LPS injection could be found in ROS production and NO production of HC. Enhanced ROS levels was observed in SGC and GC after 3–24 h and 3–36 h respectively, and NO production of SGC and GC improved after 3–48 h injection. These results demonstrated that SGC and GC possessed strong capabilities for LPS-induced EA, ROS production and NO production, while HC only displayed EA response to LPS, suggesting that GC and SGC play the main role in immune defense of shrimp against Gram-negative bacteria.

Two hemocyte sub-populations of kuruma shrimp Marsupenaeus japonicus

Hemocytes in the circulating hemolymph play important roles for immune responses in shrimp. Previous studies on immune responses by hemocytes in penaeid shrimp were based on gene expression analyses of the entire population of hemocytes and thus may have missed different immune responses of different hemocyte sub-populations. In this study, we separated hemocytes into two sub-populations by Percoll gradient centrifugation, morphological characteristics of each population were then analyzed by May-Giemsa staining, flow cytometry, and FACSCalibur. Results showed hemocytes were divided into an upper layer basophilic, and lower layer of eosinophilic hemocytes. Basophilic hemocytes were larger in size compared to eosinophilic hemocytes, which were more granulated than the basophilic hemocytes. Transcriptome analysis was then conducted through RNA-seq analysis by Miseq, which revealed 16 differentially-expressed transcripts between the two sub-populations. In the upper-layer, the highly expressed transcripts that were homologous to immune-related genes that suggest hemocytes from this layer may play as the regulator of immune system and control the action of other cells to eliminate pathogen. On the other hand, transcripts that were highly expressed in the lower-layer were homologous to the antimicrobial peptide (AMP) crustin, which supports that hemocytes on this layer have granules as crustins are normally secreted from hemocyte granules. The high expression of crustin in the lower-layer also provides insight on the mechanism of the anti-microbial function, where hemocytes produce and store AMPs in its granules. These differentially expressed genes are potential hemocyte molecular markers, and among them we identified one of the highly expressed genes in the hemocytes from the upper-layer (c11736_g1) to be a promising candidate molecular marker predicted to be a surface molecule, which is a common characteristic for molecular markers.

Differential sensitivity to cadmium of immunomarkers measured in hemocyte subpopulations of zebra mussel Dreissena polymorpha

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals.The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10-6 M to 10-3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC50 of 3.71±0.53×10-4M for phagocytic activity and 2.79±0.19×10-4M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies.

Separation of haemocyte subpopulations in shrimp Fenneropenaeus chinensis by immunomagnetic bead using monoclonal antibody against granulocytes

In our previous work, two monoclonal antibodies (Mabs) against granulocytes of shrimp (Fenneropenaeus chinensis) had been produced, in this paper, haemocyte subpopulations were analyzed by flow cytometry (FCM) using the Mabs. Then immunomagnetic bead (IMB) method was applied for separation hyalinocytes and granulocytes using the Mabs. The separated hyalinocytes and granulocytes were analyzed by FCM, indirect immunofluorescence assay, Giemsa staining and transmission electron microscopy, respectively. The results showed the proportion of hyalinocytes in haemolymph of F. chinensis was 15.14 ± 1.22%, and that of granulocytes was 75.43 ± 2.31%. After two times separation by IMB, the purity rate of hyalinocytes and granulocytes was 96.27 ± 1.06% and 98.13 ± 0.86%, respectively. The hyalinocytes possessed 0.60–0.85 in nucleus/cytoplasm (N/C) ratio and had few granule in cytoplasm, whereas the separated granulocytes with N/C ratio of 0.12–0.36 and high electronic density of double membrane granules. The results reported the separation of haemocyte subpopulations using Mabs in shrimp for the first time, and the hyalinocytes and granulocytes isolated by IMB could be used for their differential protein analysis.

Functional features of hemocyte subpopulations of the invasive mollusk species Dreissena polymorpha.

Dreissena polymorpha is a mussel species that invaded many lotic and lentic inland waters in Western Europe and North America. Its positive or negative interactions with biotic and abiotic components of ecosystems are numerous, making this bivalve the subject of numerous studies in ecology, ecophysiology and ecotoxicology. In these contexts, the functional characterization of the zebra mussel hemocytes is of particular interest, as hemocytes are central cells involved in vital functions (immunity, growth, reproduction) of molluscan physiology. Dreissena polymorpha circulating hemocytes populations were characterized by a combination of structural and functional analysis. Assessments were performed during two contrasted physiological periods for mussels (gametogenesis and spawning). Three hemocyte types were identified as hyalinocytes and blast-like cells for agranular hemocytes and one granulocyte population. Flow cytometry analysis of hemocytes functionalities indicated that blast-like cells had low oxidative and mitochondrial activities and low lysosomal content. Hyalinocytes and granulocytes are fully equipped to perform innate immune response. Hyalinocytes exhibit higher oxidative activity than granulocytes. Such observation is not common since numerous studies show that granulocytes are usually cells that have the highest cellular activities. This result demonstrates the significant functional variability of hemocyte subpopulations. Moreover, our findings reveal that spawning period of Dreissena polymorpha was associated with an increase of hyalinocyte percentage in relation to low levels of biological activities in hemocytes. This reduction in hemocyte activity would reflect the important physiological changes associated with the spawning period of this invasive species known for its high reproductive potential.

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei (Boone), and the outcome for virus and cell.

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)-killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV-killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV-killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV-killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60min post-inoculation) and that afterwards the capsid started to become disintegrated (from 360min post-inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV-killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular-sensing molecules.

Monoclonal antibodies to hemocytes of the deep-sea symbiotic mussel, <i>Bathymodiolus japonicus</i>

The deep-sea mytilid mussel, Bathymodiolus japonicus, harbors methane-oxidizing bacteria in the gill epithelial cells that are transmitted horizontally or environmentally to the next generation of the mussel. It remains to be elucidated how the symbiotic bacteria are maintained under the immune defense system of the host mussels. As hemocytes generally play a major role in the immune system, their characterization is important in understanding the immune defense system of the symbiotic mussel. In hemocytes of B. japonicus, two types of granulocytes and one type of agranulocyte have been reported. To develop biomarkers to identify these hemocyte subpopulations, we generated a monoclonal antibody (mAb) library against mussel hemocytes. We obtained 16 hybridoma clones producing mAbs against the hemocytes. These were divided into six categories based on reactivity to the hemocyte subpopulations. Ten out of 16 mAbs reacted to all of the hemocytes. Four of the remaining six mAbs respectively and exclusively reacted to granulocytes, agranulocytes, a subset of granulocytes, and a subset of agranulocytes. Two of them recognized both granulocytes and agranulocytes but only their subsets. These suggested that granulocytes and agranulocytes are respectively composed of hetetogeneous subsets. The present mAb library may be useful not only for the classification of the hemocytes, but also for investigating their functions, differentiation and localizations in the body. The information obtained from the mAbs will be a baseline for understanding the relationship between the immunological defense system and stable maintenance of symbiotic bacteria in the deep-sea symbiotic bivalves.

Differences in uptake and killing of pathogenic and non-pathogenic bacteria by haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone).

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non-pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi-granulocytes) have the main function in phagocytosis of both pathogenic and non-pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non-virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post-inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non-virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.

Influence of reproductive condition of the Manila clam Ruditapes philippinarum on hemocyte parameters during early post-spawning period

In marine bivalve aquaculture, mortality is a major problem for sustainability of the industry. Mortalities usually result from complex interactions in which the physiological status is suspected to play a major role. In many bivalves, spawning appears as a critical period. Spawning is an energy demanding process potentially resulting in immunodepression and higher susceptibility to pathogenic infection. In an effort to understand the actual impacts of spawning on the cellular defense system (i.e., hemocytes) of the Manila clam Ruditapes philippinarum, we collected clams on a one-time sampling during early post-spawning period. We then applied, for the first time, a histology–flow cytometry combined approach to these animals. Hemocyte parameters, including concentration, sub-populations, mortality, phagocytosis capacity and reactive oxygen species production were analyzed along with gender, condition index as well as the tissue protein and carbohydrate levels. Among the 200 clams analyzed, 60.5% were spent, 24% spawning, 12% resting and 3.5% ripe and ready for spawning. No gender differences could be observed among all parameters and no correlation was determined between the protozoan parasite Perkinsus olseni infection intensity and the immune parameters. CI and tissue protein level of spawning and post-spawning (spent and resting) clams were significantly lower than those of pre-spawning (ripe) clams. The concentration of hemocytes tended to decline from ripe to spent clams, partly because of a concomitant increase of cell mortality and, most of all, a decrease in the quantity of circulating granulocytes. The decline in circulating granulocytes also resulted in a reduced phagocytosis capacity (% of cells able to phagocytise). At the animal scale, these observations support a depression of the global immune defense of spawning and post-spawning clams. However, at the cellular scale, remaining circulating cells did not display signs of stress nor altered metabolism. Indeed, phagocytosis, as the number of beads ingested per cell, was normal and no oxidative stress could be detected. Further studies will be necessary to determine if these hemocytes already reached their limit of stress tolerance, and to potentially help develop immunoprotective food additives for the aquaculture industry.

Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV, but in different ways

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish.

Adult somatic progenitor cells and hematopoiesis in oysters.

Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills.

The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes

In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK571) blockers, we show that MXR activity is only carried out by MRP-type transporters in M. edulis hemocytes. In addition, cell-type-gated flow cytometry and calculation of the MXR activity factor indicate that ABCC-efflux activity is higher and more inducible in eosinophilic granulocytes than in other hemocyte subtypes. We conclude that, in the hemocytes of M. edulis, MXR phenotype is mediated by an ABCC/MRP-type transporter activity principally supported by eosinophilic granulocytes. A role for ABC transporters in hemocyte migration is discussed.

Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the Mitten Crab, Eriocheir sinensis

Hemocytes are the main immune defense cells in crustacean, and its in vitro culture can be a useful tool for the study of host and pathogen interaction. In the present study, the primary hemocyte culture of Chinese mitten crab (Eriocheir sinensis), including mixed and single hemocyte, was set up for the first time. In this study, different pH (6.4, 6.8, 7.2, 7.6, and 8.0), temperature (26, 28, and 30°C), and osmolarity (500, 700, 900, 1,100, and 1,300 mOsm kg(-1)) values were tested. Moreover, the effects of two types of medium (1× L-15 and 3× L-15) with the same osmolarity on hemocyte culture were evaluated. After incubation at different culture conditions, the morphological changes (degranulation, lysis, shrinkage, and detachment) and survival rate of hemocytes were taken into account in order to evaluate the culture condition effect. Our results showed that the total hemocyte counts of Chinese mitten crab were about 2.5 × 10(7) cells ml(-1), and three subpopulations of hemocytes were distinguished as granulocytes (43.46 ± 4.98%), semigranulocytes (31.04 ± 1.95%), and hyalinocytes (25.50 ± 4.89%). The optimal culture condition for primary hemocytes of Chinese mitten crab was 3× L-15 medium, 1,100 mOsm kg(-1), pH 6.8 at 28°C. Hemocytes at optimal culture condition could retain a better morphology and higher survival rate: hemocytes retained a survival rate >60% after 5 d and >40% after 7 d. Furthermore, the hemocyte subpopulations were isolated by Percoll step gradient centrifugation and cultured in optimized hemocyte culture conditions. The results showed that hyalinocytes and semigranulocytes could maintain a survival rate of >50% after 15 d, while granulocytes only retained a survival rate of 26% after 5 d.

Separation of Penaeus vannamei haemocyte subpopulations by iodixanol density gradient centrifugation

Methodologies for separation of immune cell subpopulations are essential tools in immunology studies. Up to date, only one methodology for separating crustacean haemocyte subpopulations using Percoll density gradient centrifugation has been described.In the present work, a new methodology to separate Penaeus vannamei haemocyte subpopulations was developed, using a two-step iodixanol density gradient centrifugation. P. vannamei haemolymph was collected with anticoagulant and centrifuged through a first gradient (densities from 1.063 to 1.109g/ml) for 10min at 2000g. Three bands were formed: two bands with lower density close together, and a third band with higher density. The first two were collected together whilst the third band was collected separately. The volume fraction in-between these bands contained dispersed cells and was also collected. The suspension containing the mixture of the first two bands was centrifuged through a second gradient (densities from 1.047 to1.087g/ml) for 15min at 2000g. Two bands were formed and collected individually. All the cell suspensions were used for in vitro culture (cell survival evaluation) and for evaluation of cell morphology by flow cytometry and light microscopy. Each of the three bands contained a major cell type with distinct morphology and behaviour. The dispersed cell fraction contained a mixture of two different cell types, which were distinct from the cell types in the bands. By order of appearance from the top of the gradient, the cell types were named: subpopulations (Sub) 1 (band 1), Sub 2 (band 2), Sub 3+4 (dispersed cells) and Sub 5 (band 3). The purity level (percentage of the major cell type) of Sub 1, 2 and 5 was 95.0±1.0%, 97.7±1.2% and 99.4±0.8%, respectively. Cells of Sub 2 showed the best survival time in vitro (up to 96h) followed by cells from Sub 1, Sub 3+4 and Sub 5. Phagocytic activity was detected in Sub 1 and 4.This methodology allowed the separation and characterization of five morphologically distinct and physiologically active P. vannamei haemocyte subpopulations, from which three were isolated with a very high degree of purity. Therefore, we consider this methodology a valuable alternative for the traditional crustacean haemocyte separation procedure in Percoll.

New insights from the oyster Crassostrea rhizophorae on bivalve circulating hemocytes.

Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge.

Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates.

Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval Schistosoma mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins, as well as the ability of hemocytes to acquire shared glycans by the selective binding of parasite-released LTP. Unraveling the functional significance of these naturally expressed and acquired shared glycans on specific hemocyte populations represents an important challenge for future investigations.

Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum

The detailed sterol (free sterol proportions and compositions) and phospholipid (PL) compositions (relative proportions of PL classes and subclasses and their respective fatty acid (FA) compositions) of hemocyte membranes were investigated in two bivalve mollusks: the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum. Hemocyte membrane lipids of both species revealed similar general composition: i) their free sterol/PL ratio was above 0.4 and ii) their PL were predominated by the diacyl + alkyl forms of glycerophosphatidylcholine (PC), the plasmalogen form of glycerophosphatidylethanolamine (PE) and ceramide aminoethylphosphonate (CAEP). Free sterols were predominated by cholesterol in both species. Plasmalogen forms of PE and glycerophosphatidylserine (PS) represented 82–83% and 46–55% of total PE and PS, respectively. When compared to their respective diacyl + alkyl forms, plasmalogen forms of PE and PS were specifically enriched in non-methylene-interrupted (NMI) FA and 20:1n-11, suggesting a functional significance of these PL molecular species in bivalve hemocytes. Lysoglycerophosphatidylcholine (LysoPC) levels were found to be fairly high in hemocytes, accounting for about 8% of the PL. Some species-specific features were also found. LysoPC and glycerophosphatidylinositol (PI) FA compositions differed between Ruditapes philippinarum and Crassostrea gigas. CAEP proportion was higher in R. philippinarum than in C. gigas (14.5% and 27.9% of the PL, respectively). Hemolymph cell monolayer observations and flow-cytometric analyses revealed species-specific hemocyte morphology and sub-populations which could account for some of the observed species-specific membrane lipid compositions.