Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process.

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing revealed six types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and predicted the differentiation pathways involved in their maturation. We also predicted cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

Differences in proteomic profile between two haemocyte types, granulocytes and hyalinocytes, of the flat oyster Ostrea edulis.

Haemocytes play a dominant role in shellfish immunity, being considered the main defence effector cells in molluscs. These cells are known to be responsible for many functions, including chemotaxis, cellular recognition, attachment, aggregation, shell repair and nutrient transport and digestion. There are two basic cell types of bivalve haemocytes morphologically distinguishable, hyalinocytes and granulocytes; however, functional differences and specific abilities are poorly understood: granulocytes are believed to be more efficient in killing microorganisms, while hyalinocytes are thought to be more specialised in clotting and wound healing. A proteomic approach was implemented to find qualitative differences in the protein profile between granulocytes and hyalinocytes of the European flat oyster, Ostrea edulis, as a way to evaluate functional differences. Oyster haemolymph cells were differentially separated by Percoll® density gradient centrifugation. Granulocyte and hyalinocyte proteins were separated by 2D-PAGE and their protein profiles were analysed and compared with PD Quest software; the protein spots exclusive for each haemocyte type were excised from gels and analysed by MALDI-TOF/TOF with a combination of mass spectrometry (MS) and MS/MS for sequencing and protein identification. A total of 34 proteins were identified, 20 unique to granulocytes and 14 to hyalinocytes. The results suggested differences between the haemocyte types in signal transduction, apoptosis, oxidation reduction processes, cytoskeleton, phagocytosis and pathogen recognition. These results contribute to identify differential roles of each haemocyte type and to better understand the oyster immunity mechanisms, which should help to fight oyster diseases.

Hemocyte cell types of the Cortes Geoduck, Panopea globosa (Dall 1898), from the Gulf of California, Mexico.

The geoduck Panopea globosa is an endemic and economic valuable species from the Mexican Northwest coast whose biology has been little studied. No information exists about their hemocytes to date, which is highly important to assess the welfare of wild and cultured organisms. In this study, hemocytes of adult P. globosa were characterized at the morphological, ultrastructural and functional level. The mean number of hemocytes in the hemolymph of P. globosa was 6 × 105 ± 2 × 105 cells mL-1. The cells were identified as granulocytes (Gr) and hyalinocytes (H). The former accounted for 28% of adhered cells in the hemolymph, measured 6-18 µm, showed numerous basophilic granules in the cytoplasm, with round and eccentric nuclei, and a nucleus:cytoplasm ratio of 0.44 ± 0.01. Hyalinocytes were the most abundant cells in the hemolymph of P. globosa (72% adhered cells) and were subdivided, according to their size, in small (Hs) 4-12 µm and large (HL) 6-18 µm. Hyalinocytes were eosinophilic round or ovoid cells with a central or eccentric nucleus, few or no granules in the cytoplasm and similar nucleus:cytoplasm ratio (Hs: 0.63 and HL: 061). Lysosomes and lipids were observed in Gr, while carbohydrates were the most abundant energy substrate in H. Both hemocytic cell types, mainly Gr, were capable to ingest particles and yield superoxide (P > 0.05). The present study shows for the first time the cell types, abundance and immune activities of hemocytes present in the hemolymph of P. globosa. This information provides a useful baseline to carry out further research on the cellular immune response of the clam to potential pathogens or changes in environmental factors.

Morphological and functional characterization of circulating hemocytes using microscopy techniques.

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi-granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule-containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid-Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell-mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase-mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme-agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.

Flow cytometric characterization of hemocytes of the flat oyster, Ostrea chilensis.

The flat oyster, Ostrea chilensis, native to New Zealand (NZ) and Chile is considered an important ecological, cultural and fisheries resource. Currently, commercial landings of this species in NZ are restricted due to low population numbers caused by ongoing mortalities resulting from the presence of the haplosporidian parasite, Bonamia exitiosa. More recently, the arrival of B. ostreae in NZ led to major mortalities in farmed stocks. To understand how diseases caused by Bonamia spp. affect this oyster species, a more complete understanding of its biology, physiology and immune system is needed. The present study characterized, for the first time, hemocytes of adult O. chilensis, from the Foveaux Strait, NZ, using flow cytometry (FCM) and histology. Based on the internal complexity of the hemocytes, two main circulating hemocyte populations were identified: granulocytes and hyalinocytes (accounting for ~30% and ~70% of the total circulating hemocyte population, respectively). These were further divided into two sub-populations of each cell type using FCM. A third sub-population of granulocytes was identified using histology. Using FCM, functional and metabolic characteristics were investigated for the two main hemocyte types. Granulocytes showed higher phagocytic capabilities, lysosomal content, neutral lipid content and reactive oxygen species production compared to hyalinocytes, indicating their important role in cellular immune defence in this species. Methods of hemocyte sampling and storage were also investigated and flow cytometric protocols were detailed and verified to allow effective future investigations into the health status of this important species.

Classification and characterization of hemocytes from two Asian horseshoe crab species Tachypleus tridentatus and Carcinoscorpius rotundicauda.

In present study, transmission electron microscopy and flow cytometry were utilized to investigate the classification, characterization and immune functions of hemocytes from horseshoe crab, Tachypleus tridentatus and Carcinoscorpius rotundicauda. Three types of hemocytes were distinguished respectively: the granular cell, the semi-granular cell and the hyaline cell by transmission electron microscopy, while three hemocyte subpopulations (Gate 1 cell, Gate 2 cell, Gate 3 cell) were classified by flow cytometry. Hyaline cell was the major cell type with the highest nuclear-cytoplasmic ratio and granular cell and semi-granular cell showed lower ratios. Immune parameters of hemocytes in horseshoe crabs were investigated by flow cytometry. Different hemocyte subpopulations respond for diverse functions. Lysosomal contents and hemocyte mortality in Gate 3 cell subpopulation were higher than that in other subpopulations, while reactive oxygen species, phagocytosis and non-specific esterase, in Gate 1 cell subpopulation, were higher than those in other subpopulations. The hemocyte types between the two species had no significant differences in staining or morphology.

Morphological and functional characterization of hemocytes from two deep-sea vesicomyid clams Phreagena okutanii and Abyssogena phaseoliformis.

Deep-sea vesicomyid clams harboring intracellular symbiotic sulfur-oxidizing bacteria are often dominant in chemosynthetic animal communities. Although they are known to have erythrocytes, little is known about other hemocytes. To investigate the types and roles of various hemocytes in vesicomyid clams, we performed morphological, histochemical and functional characterization of the hemocytes in two species, Phreagena okutanii, collected from 873 to 978 m depth, and Abyssogena phaseoliformis, from 5199 to 5355 m. Both were found to have three types of hemocytes: erythrocytes (ERCs), eosinophilic granulocytes (EGs), and basophilic granulocytes (BGs). The ERCs contain hemoglobin in the cytoplasm, with basophilic vacuoles containing acid polysaccharide, neutral lipids, and peroxidase. The EGs were found to contain acid polysaccharides and eosinophilic granules containing lysosomal enzymes, acid and alkaline phosphatases, chloroacetate esterase, and peroxidase. Although BGs had some basophilic granules with alkaline phosphatase, they lacked acid phosphatase and acid polysaccharides. The EGs and BGs were shown to have phagocytic ability, while the ERCs exhibited no phagocytosis. The EGs showed higher phagocytic activity as well as a higher phagosome-lysosome fusion rate than BGs. The hemocytes of the two vesicomyid species differed in the intracellular structures. In A. phaseoliformis, ERCs additionally contained neutral polysaccharides in vacuoles and had vesicles with acinus-like acidic mucus in the cytoplasm, neither of which were observed in P. okutanii. The eosinophilic granules in the EGs had heteromorphically-elongated shapes containing homogeneously electron-dense material in P. okutanii, but were more spherical and composed of fibrous structures in A. phaseoliformis. The difference in hemocytes between the two clams seems to be reflective of phylogenetically differentiated lineages adapting to differing conditions in their respective deep-sea environments, such as dissolved oxygen, hydrogen sulfide concentration, and hydrostatic pressure. In the view of phylogeny of veneroida clams including two vesicomyids, their hemocytes appear to be categorizable into three basic types, with the first containing ERCs and agranulocytes, the second including EGs, and the third comprised of BGs, small eosinophilic granulocytes, and other granulocytes. The present data showed no phagocytic activity of ERCs and a lack of agranulocytes in both vesicomyid species, and when combined with previous reports that other veneroid clams show low or no phagocytic activity, this suggests that ERCs have become evolutionarily differentiated from agranulocytes in the ancestral vesicomyid clam.

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.

Oxidative burst activity in haemocytes of the freshwater prawn Macrobrachium rosenbergii.

Oxidative burst, release of reactive oxygen species/reactive nitrogen species (ROS/RNS) contributed to microorganisms killing, is a vital immune response of crustacean haemocyte. Three morphologic haemocyte types (hyaline cells, HC; semigranular cells, SGC; granular cells, GC) have been defined in crustaceans, and found to play different roles in immune defense. However, oxidative burst activities of different haemocyte subpopulations in crustaceans are currently not documented. In the present study, we investigated the oxidative burst activities of the three haemocyte types in the freshwater prawn Macrobrachium rosenbergii using the common ROS fluorescent probe dichlorofluorescin-diacetate (DCFH-DA). Nitric oxide (NO) donor sodium nitroprusside (SNP) improved the DCF fluorescence in haemocytes, while NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO-synthase inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced the fluorescence, suggesting that DCF fluorescence intensity could also be modified by intracellular NO level and activity of NO-synthase pathway. ROS/RNS was also produced in the untreated haemocytes. GC contained most non-induced ROS/RNS production, while oxidative activity of HC was rather weak. No significant impact of PMA could be observed on ROS/RNS level in all the three cell types. Both zymosan A (ZA) and lipopolysaccharide (LPS) significantly triggered the production of ROS/RNS in SGC and GC, whereas they had no effect on those of HC, suggesting that SGC and GC were the primary cell types involved in pathogens killing by ROS/RNS pathway. Cytochalasin B (Cyt B) inhibited the ZA-induced ROS/RNS production, but could not change the ROS/RNS level stimulated by LPS. For unstimulated haemocytes, ROS/RNS productions decreased 29.6%, 44.1% and 48.6% in SGC, and decreased 44.5%, 28.4% and 57.3% in GC, in the presence of L-NMMA, Fccp and DPI respectively, whereas apocynin could not modulate DCF fluorescence in both SGC and GC, suggesting that mitochondrial oxidative pathway was relatively more dominant in SGC, and NO-synthase (NOS) pathway appeared more active in GC. For LPS-stimulated haemocytes, oxidative activities decreased 22.9%, 42.9%, 29.6% and 60.0% in SGC, and reduced 40.6%, 25.2%, 26.7% and 70.6% in GC with the presence of L-NMMA, apocynin, Fccp and DPI respectively, suggesting that NADPH-oxidase (NOX) pathway in both SGC and GC was activated by LPS, and it became the predominant oxidative pathway in stimulated SGC, while NOS pathway was the relative main source for ROS/RNS production in stimulated GC.

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.

Production a monoclonal antibody specific to granulocytes of swimming crab (Portunus trituberculatus) and its cross reactivity with other crustaceans.

In this study, a monoclonal antibody (mAb) 3F4 specific to granulocytes of swimming crab, Portunus trituberculatus, was obtained by immunizing mice with whole haemocytes. mAb 3F4 showed strong immunofluorescent reaction with granulocytes, but no reaction with hyalinocytes. The positive cell percentage of granulocytes was 86.3% detected by Flow cytometry (FCM). A special antigen with molecular weight of about 26kDa was further recognized by mAb 3F4 in haemocytes of P. trituberculatus. mAb 3F4 also showed strong cross-reactivity with haemocytes of Eriocheir sinensis and Petalomera japonica, but no reaction with other crustaceans tested. In E. sinensis, the positive cell percentage was 73.4% for granulocytes and 59.8% for hyalinocytes; while in P. japonica, the positive cell percentage was 81.2% for granulocytes and 7.1% for hyalinocytes. There was also a special antigen with molecular weight of about 31kDa identified by mAb 3F4 in haemocytes of E.sinensis, but no corresponding protein band in P. japonica haemocytes. These results demonstrated that mAb 3F4 can be used as a marker for granulocytes of crabs.

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.

Wright-Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum.

Hemocytes are the first line of defense in the invertebrate immune system. Understanding their roles in cellular immunity is important for developing more efficient mycoinsecticides. However, the exact classification of hemocytes has been inconsistent and the various types of phagocytes in Locusta migratoria are poorly defined. Herein, the Wright-Giemsa staining method and microscopy were employed to characterize the hemocytes of L. migratoria following infection by Metarhizium acridum. Hemocytes were classified into four types, including granulocytes, plasmatocytes, prohemocytes, and oenocytoids, based on size, morphology, and dye-staining properties. Each type of hemocyte was classified into several subtypes according to different ultrastructural features. At least four subtypes of granulocytes or plasmatocytes, including small-nucleus plasmatocytes, basophil vacuolated plasmatocytes, homogeneous plasmatocytes, and eosinophilic granulocytes, carried out phagocytosis. The percentage of total phagocytes increased two days after infection by M. acridum, then gradually declined during the next two days, and then increased sharply again at the fifth day. Our data suggested that plasmatocytes and granulocytes may be the major phagocytes that protect against invasion by a fungal pathogen in L. migratoria. Total hemocytes in locusts significantly increased in the initial days after infection and decreased in the late period of infection compared to controls. In the hemocoel, hyphal bodies were recognized, enwrapped, and digested by the phagocytes. Then, the broken hyphal pieces were packaged as vesicles to be secreted from the cell. Moreover, locusts might have a sensitive and efficient cellular immune system that can regulate phagocyte differentiation and proliferation before fungi colonize the host hemolymph.

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet.

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae's investment into the different types of hemocytes.

Morphology and classification of hemocytes in Pinctada fucata and their responses to ocean acidification and warming.

Hemocytes play important roles in the innate immune response and biomineralization of bivalve mollusks. However, the hemocytes in pearl oysters are poorly understood. In the present study, we investigated the morphology and classification of hemocytes in the pearl oyster, Pinctada fucata. Three types of hemocytes were successfully obtained by light microscopy, electron microscopy and flow cytometry methods: small hyalinocytes, large hyalinocytes and granulocytes. The small hyalinocytes are the major hemocyte population. Morphological analyses indicated that these hemocytes have species-specific characterizations. In addition, we assessed the potential effects of ocean acidification (OA) and ocean warming (OW) on the immune parameters and calcium homeostasis of the hemocytes. OA and OW (31 °C) altered pH value of hemolymph, increased the total hemocyte count, total protein content, and percentage of large hyalinocytes and granulocytes, while it decreased the neutral red uptake ability, suggesting active stress responses of P. fucata to these stressors. Exposure to OW (25 °C) resulted in no significant differences, indicating an excellent immune defense to heat stress at this level. The outflow of calcium from hemocytes to hemolymph was also determined, implying the potential impact of OA and OW on hemocyte-mediated biomineralization. This study, therefore, provides insight into the classification and characterization of hemocyte in the pearl oyster, P. fucata, and also reveals the immune responses of hemocytes to OA and OW, which are helpful for a comprehensive understanding of the effects of global climate change on pearl oysters.

A first insight into haemocytes of the smooth venus clam Callista chione.

The smooth venus clam Callista chione is a commercially exploited bivalve species that lives on the sandy bottom of the Italian coast of the Northern Adriatic Sea. Currently, no information is available in the literature about the haemocytes of this bivalve species. In this study, we performed a morpho-functional characterisation of the haemocytes of C. chione. In freshly collected haemocytes, the total haemocyte count (THC) (measured by a Coulter Counter) varied markedly among individuals, and the mean number of haemocytes was 1.2 (×10(6)) cells mL haemolymph(-1). The mean values for the haemocyte diameter and volume were 4.2 µm and 77.8 fL, respectively. In some cases, higher THC values were related to a smaller haemocyte size, but no correlation was detected between the THC and haemocyte diameter or between THC and cell volume. Conversely, a positive correlation was observed between cell diameter and volume. Two haemocyte types were distinguished by light microscopy: granulocytes (76%), with evident cytoplasmic granules, and hyalinocytes (24%), with a few or no granules. After adhesion to slides and fixation, the cell diameter was approximately 10 µm for granulocytes and 7 µm for hyalinocytes. The granules of the granulocytes were stained in vivo with Neutral Red, indicating that they were lysosomes. The granulocytes and hyalinocytes were further distinguished as basophils and acidophils. Both the granulocytes and the hyalinocytes were able to phagocytise yeast cells. Of 2643 cells that were counted, 2007 (76%) showed phagocytic activity. The granulocytes and hyalinocytes were both positive for some hydrolytic enzymes, whereas they were not positive for peroxidase or phenoloxidase. The two types of haemocytes also produced superoxide anion. Overall, this preliminary study indicates that both the granulocytes and hyalinocytes of C. chione are immune effector cells.

Classification and phagocytosis of circulating haemocytes in Chinese mitten crab (Eriocheir sinensis) and the effect of extrinsic stimulation on circulating haemocytes in vivo.

Eriocheir sinensis (Henri Milne Edwards 1854) is one of the most important aquaculture species in China. In this investigation, we characterised the different types of haemocytes of E. sinensis using light and electron microscopy combined with cytochemical analysis and determined the in vivo phagocytic ability of different haemocyte types by injecting polystyrene beads. The haemocytes of E. sinensis were divided into three types: hyalinocytes, semigranulocytes and granulocytes. The hyalinocytes had no or few cytoplasmic granules; the semigranulocytes contained abundant small granules and a few large refractile cytoplasmic granules; and the granulocytes contained numerous large refractile cytoplasmic granules. The hyalinocytes were demonstrated to be the most abundant circulating haemocytes and the most avid phagocytic haemocytes, accounting for approximately 88.7% of the total phagocytes. The haemocyte-containing granules displayed limited phagocytic ability, with approximately 5.0% of granulocytes and 6.3% of semigranulocytes displaying positive phagocytic ability against the invading polystyrene beads in vivo. After injection with Aeromonas hydrophila, Bacillus subtilis and different concentrations of lipopolysaccharide for 0.25, 0.5, 1, 2, 4, 6 and 8 h, all three types of haemocytes experienced dramatic decline and then rapid recovery to their initial levels. A high concentration of lipopolysaccharide and A. hydrophila were extremely toxic to the crabs, as they induced a more serious loss of haemocytes compared with a low concentration of lipopolysaccharide and B. subtilis. Overall, the results obtained in this study indicate that a small proportion of the haemocytes of E. sinensis contributed to the phagocytic process, and the migration of haemocytes and haemocyte lysis were most likely a prominent pathway for pathogen elimination.

Morphofunctional study of hemocytes from lions-paw scallop Nodipecten subnodosus.

Hemocytes play an important role in internal defense in mollusk bivalves; they are generally divided into granular and hyaline types, where the granules possess molecules to combat foreign particles. We investigated the morphology, staining reactions, and immune-related activities of hemocytes from the pectinid lions-paw scallop Nodipecten subnodosus. We showed that, in contrast to the conditions in most bivalves and similar to other pectinids, no granular cells were observed; only very few semi-granular cells were present and the large majority of cells were hyalinocytes and blast-like cells. The cytoplasm in many hyalinocytes showed vesicles of various sizes, but none with the staining characteristics of typical granules. When hemolymph was removed from these scallops, the plasma did not clot and the hemocytes rapidly adhered to one another in suspension. When living hemocytes were placed on a glass surface, they clustered into groups of various sizes; within minutes, pseudopods extended from the cells and outward migration began. Many cells spread to form extensive networks of flat cells, and other undifferentiated cells did not spread and kept a rounded morphology. Some hemocytes were immunologically active because they phagocytize Escherichia coli bacteria. Hemocytes expressed the carbohydrates residues N-acetyl-D-glucosamine, α-D-glucose, α-D-mannose, and α-L-fucose moieties, and cytochemical assays showed activity of acid phosphatase, specific esterase, and non-specific esterase, enzymes commonly associated with lysosomes and immunologic activity. These observations serve as a useful tool for further investigation of morphofunctional studies and physiological status of scallops.

Ultrastructural characterization of the hemocytes of Lasiodora sp. (Koch, 1850) (Araneae: Theraphosidae).

This paper is the first descriptive review of hemolymph cell types in the circulation of the tarantula spider Lasiodora sp. These animals are more long-lived than other arthropods, and may live for approximately twenty years. Such remarkable longevity may result from a highly successful immune system, which in turn is directly correlated with hemocyte function. Since the literature on the genus Lasiodora sp. is limited, the main goal of the present study was to identify the different cell types by optical and transmission microscope. Six hemocyte types were characterized and called prohemocyte, granulocyte type I, granulocyte type II, spherulocyte, oenocytoid and plasmatocyte. Prohemocytes presented a large nucleus, elongated granulocytes type I showed the nucleus with the same cell format, elliptical granulocytes type II showed the central nucleus of identical shape, spherulocytes exhibited the nucleus filling almost the whole cell, oval oenocytoids showed eccentric nucleus and less dense cytoplasm, and irregular plasmatocytes showed a nucleus and no granules in cytoplasm. These polymorphic granulocytes presented a round, elongated, elliptical, oval or irregular profile with large and varied numbers of granules, except for plasmatocytes, that were agranular. Different densities and different concentrations of these granules were found at the periphery of the cell. The possible reasons and implications of differences and similarities between arthropods hemocytes are discussed. It can be concluded that there are six cell types in Lasiodora sp. This study is of the first step in the elucidation of the role these cells play in the circulatory and immune system in spiders.

[Study on morphological and functional characterization of hemocytes in snails].

Snails are the intermediate host of Schistosoma and they play an important role in the transmission of schistosomiasis. The snails in different areas present various susceptibilities, which are related to the different hemocytes in the snails. There is not a common standard in the classification of hemocytes, and generally the hemocytes are divided into two categories: a granulocyte and a hyalinocyte. The granulocyte plays an important role in immunization, while hyalinocyte shows a less effect. Besides, soluble factors may also play a role in immunization.