Insight on cellular and humoral components of innate immunity in Squilla mantis (Crustacea, Stomatopoda)

For deeper insights into the function of crustacean haemocytes in immune responses, we studied the morphology and enzyme content of circulating cells of the mantis shrimp Squilla mantis from the North Adriatic Sea, together with their ability to phagocytose foreign cells. We also assayed the enzyme content and the agglutinating and haemolytic activities of cell-free haemolymph. Three haemocyte types, i.e., hyalinocytes, semigranulocytes and granulocytes, can be distinguished, according to cell and nuclear morphology and the presence of cytoplasmic granules. All of them share the same patterns of enzyme activities and are recognised by the same lectins. Spreading cells (hyalinocytes and semigranulocytes) can ingest foreign cells; granules of semigranular and granular cells have similar cytochemical properties. Injection of Micrococcus luteus into the heart sinus results in an increase in the frequency of hyaline cells and a decrease in the frequency of granulocytes. After 24 h from the injection, a decrease in the number of phagocytosing hyalinocytes, and a general decrease in the frequency of acid phosphatase-positive cells was reported. Our data match previous results and suggest the existence of a single differentiation pathway for Squilla haemocytes with the three haemocyte morphs as different stages of cell differentiation. Results also indicate that Squilla haemolymph performs immunosurveillance, through rapid changes in haemocyte distribution, increase of antimicrobial and antioxidant enzymes and secretion of lectins stimulating agglutination, phagocytosis and encapsulation.     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda)

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3–5 µm in diameter and 30–50 fL in volume, the second was of about 6–12 µm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20±1.7 µm and 272.30±143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94±1.43 µm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38±1.76 µm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88±1.6 µm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25–30 µm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa’s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim’s panoptical staining and Ehrlich’s triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.Key words: Carcinus aestuarii, crab, haemocytes, light microscopy, cytochemical assays, morphological characterisation.     

Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation

Tapes philippinarum is a bivalve mollusc of the Pacific Ocean, successfully imported for human consumption into the northern Adriatic Sea (Europe). For better knowledge of its considerable adaptive ability in comparison with similar autochthonous species, a morpho-functional characterisation of its haemocytes was carried out with the establishment of short-term cell cultures (60 min at 25 degrees C). Various methods of cytochemical staining identified four cell types in the haemolymph: granulocytes (48.05% +/- 1.43), hyalinocytes (32.18% +/- 0.99), haemoblasts (18.97% +/- 0.63) and serous cells (0.8% +/- 0.19). The granulocytes, possessing cytoplasmic granules with differing dye affinity, included basophils, neutrophils and acidophils. Such granules stained vitally with Neutral Red, and correspond to lysosomes. Hydrolytic and oxidative enzymes were mainly detectable after stimulation in the presence of yeast cells. Both granulocytes and hyalinocytes were positive for alkaline phosphatase, non-specific esterase, peroxidase, and cytochrome C oxidase, whereas only granulocytes were positive for beta-glucuronidase, acid esterase, and arylsulphatase. Both cell types were competent phagocytes towards yeast and plasma had an opsonising effect. Moreover, the respiratory burst accompanied phagocytosis with superoxide anion production, recognisable through cytoplasmic deposits of formazan after treatment with nitro blue tetrazolium. Haemoblasts were small undifferentiated cells which, due to their morphology and positivity to the anti-CD34 antibody, show the typical features of stem cells. Serous cells, probably arising from Keber's gland and belonging to another differentiation pathway, contained non-sulphate acid mucopolysaccharides and play an important role in early defence mechanisms, taking part in the formation of clots.     

Effects of high temperatures on functional responses of haemocytes in the clam Chamelea gallina

The effects of high temperatures on the clam, Chamelea gallina, generally recognised as a low tolerant bivalve species, were studied by evaluating some functional responses of the haemocytes. The animals were kept for 7days at 20, 25 and 30 degrees C and total haemocyte count (THC), phagocytosis, lysozyme activity (in both haemocyte lysate and cell-free haemolymph), activity and expression of the antioxidant enzyme superoxide dismutase (SOD) (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of exposure to high temperatures. The survival-in-air test was also performed. During the experiment, the clams showed differing burrowing behaviour: the animals kept at 20 and 25 degrees C burrowed completely, whereas at 30 degrees C the clams progressively emerged from the sediment and then remained on the surface. The highest temperature significantly increased THC, whereas it decreased the phagocytic activity of haemocytes. The haemocyte size frequency distribution in clams kept at 30 degrees C showed that the cell population of about 8-10microm was markedly reduced compared to clams kept at 20 and 25 degrees C. In clams maintained at 25 degrees C, lysozyme activity was significantly increased in haemocyte lysate, whereas it was markedly decreased in cell-free haemolymph. Total SOD activity significantly decreased in haemocytes from clams held at 30 degrees C whereas it increased in cell-free haemolymph from clams held at 25 degrees C and 30 degrees C. A significant decrease in haemocyte Mn-SOD and Cu/Zn-SOD activities was found with increasing temperature. In cell-free haemolymph, the highest Mn-SOD activity was recorded at 30 degrees C, whereas the Cu/Zn-SOD activity showed no significant changes in clams maintained at different temperatures. SOD isoform expression exhibited different patterns in haemocyte lysate and cell-free haemolymph. The resistance to air exposure of clams kept at 30 degrees C was shown to decrease significantly, LT(50) values fell from 6days in clams kept at 20 degrees C and 25 degrees C to 4days in those kept at 30 degrees C.     

Morphological and cytoenzymatic characterization of haemocytes of the venus clam Chamelea gallina

A morphological and enzymatic characterization of Chamelea gallina haemocytes was carried out as a prerequisite for further studies on venus clam immunobiology. Two main types of circulating haemocytes were identified (1) hyalinocytes (79.2%), agranular cells with a central nucleus surrounded by a little cytoplasm, and (2) granulocytes (16.5%), smaller granular cells with smaller nuclei. Small cells with a strongly basophilic nucleus and a thin layer of peripheral cytoplasm, probably undifferentiated blast cells (4.3%), were also observed. Both granulocytes and hyalinocytes can assume a spreading or round morphology. The enzymatic activities of haemocytes were also investigated. Some of the granulocytes and hyalinocytes were positive for hydrolytic enzymes, suggesting a role for these cells in phagocytosis; no oxidative enzymes were detected in C. gallina haemocytes. Granulocytes and hyalinocytes can easily adhere to the substratum and exhibit a low phagocytosis activity towards foreign particles (6.3%), whereas the fraction of cells containing ingested material significantly increased after pre-incubation of test particles with cell-free haemolymph, which suggests the presence of opsonin(s) in the haemolymph.     

Haemocyte morphology and function in the Akoya Pearl Oyster, Pinctada imbricata

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62 ± 2.81%), followed by hyalinocytes (36 ± 2.35%), and serous cells (2 ± 0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8 ± 3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 μm in diameter, with small granules (0.05-0.5 μm in diameter), large (5.00-9.00 μm in diameter, with large granules (0.50-2.50 μm in diameter) and combination (5.00-9.00 μm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.     

Monoclonal antibodies specific to haemocytes of black tiger prawn Penaeus monodon

Monoclonal antibodies specific to haemocytes of Penaeus monodon were generated from a mouse immunized with a mixture of SDS-treated and formalin-fixed haemocytes. Hybridoma clones were selected by immunohistochemistry against fixed haemocytes, heart, lymphoid organ, and haemopoietic tissue, and Western blot against haemocyte extract and haemolymph. Sixteen monoclonal antibodies specific to haemocytes were obtained and could be divided into six groups according to their binding capacities to various haemocyte proteins in Western blot analyses, 102, 43, approximately 20, 61, 175 and approximately 230 kDa, and their differences in recognition of haemocyte sub-populations. The first group of antibodies strongly recognized a small subset of semi-granulocytes (SG) and hyalinocytes (H) but occasionally stained lightly a very small population of granulocytes (G). The antibodies also bound to a group of cells in haemopoietic tissue as well as cells located at the inner layers of the tubules in the lymphoid organ but not in the spheroid. The second group of antibodies strongly bound to a large sub-population of G and SG with coarse granules but did not bind to most of the H. This group of antibodies also cross-reacted with cells in the outer layer of the tubules in the lymphoid organ. The third group of antibodies recognized all G and only a small portion of SG. The fourth, fifth and sixth groups bound to sub-populations of G, SG and H in similar proportions. None of the antibodies showed any cross-reactivity to other components in haemolymph. The common antigens recognized by the first and the second groups of antibodies in the haemopoietic tissue and the lymphoid organ may reflect relationships among these organs in the development of the sub-populations of G and SG. Haemopoietic tissue may be the site for haemocyte production and the lymphoid organ may be the site for further differentiation of at least two different lines of haemocytes.     

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte subpopulations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.     

Toxicological impact of butralin,glyphosate-isopropylammonium and pendimethalin herbicides on physiological parameters of Biomphalaria alexandrina snails

ABSTRACT

Biomphalaria alexandrina snails have been used as bioindicators for freshwater qaulity and the effects of some herbicides such as butralin, glyphosate-isopropylammonium and pendimethalin). In the present study the effect of these three herbicides on snail biochemistry was examined. The results indicated that the herbicides increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the haemolymph of B. alexandrina snails and significantly decreased total protein and albumin content. Light microscopical examinations of haemocytes monolayers of B. alexandrina snails showed three different cell types (small cells, granulocytes and hyalinocytes). All three herbicides caused abnormalities in cell shapes. Flow cytometric analysis of haemocytes from B. alexandrina demonstrated that circulating haemocyte populations could be divided into two main subtypes differing in their granularity (granulocytes or hyalinocytes) and size (large and small cells). In addition, the flow cytometric analysis showed that the total number of dead haemocytes in the haemolymph was significantly increased in treated groups compared to the control group. Phagocytosis in groups treated with the herbicides was highly significantly increased compared to the control indicating a very strong response of the treated snails. The results of the alkaline comet assay of DNA damage demonstrated that these herbicides have a genotoxic effect.     

Cytometric,morphologic and enzymatic characterisation of haemocytes in Anodonta cygnea

The haemocytes in bivalve mussels are involved in many processes such as lesion repair, shell repair, elimination of small particles and toxic substances. In Anodonta cygnea there are two categories of haemolymph cells, the granulocytes and hyalinocytes. Two groups of cells were identified by flow cytometry and morphological studies: one with larger size and granularity representing 75%, and another group of cells (25%) which were approximately half the size. The cytochemical reactions showed peroxidase activity in the larger cells and a weak prophenoloxidase activity in the smaller cells. These characteristics suggest that the most common haemocytes are granulocytes and hyalinocytes are less common. Enzymatic studies showed clear activities of few enzymes in different compartments of the mantle. Both haemocytes presented significant variations for alpha-manosidase and beta-glucurosidase activities depending on the acid or alkaline pH. Almost all were sensitive to the pH changes, mainly the beta-galactosidase in the haemolymph plasma. On the contrary, the same enzymatic analysis in the extrapallial elements showed more stabilised activities. The simulation of acidic and alkaline condition with the observation of significant morphological and enzymatic activity changes, allow us to speculate some functional role, mainly in the haemolymph elements. The granulocytes may be speculated to have intense involvement in the digestion of small residues with the formation of calcareous stores while the hyalinocytes are more responsible for the elimination of soluble cytotoxic compounds.     

Electron microscope study of haemolymph cells of Metrioptera roeselii (Orthoptera,Tettigoniidae)

On the basis of patterns of haemocyte ultrastructure and functions at preimago and imago stages of Metrioptera roeselii, secretory cells of granulocyte type were recognized in the haemolymph. The development of granulocytes was traced starting from their formation up to cell death and destruction. The haemocytes develop as "dark" and "light" cells, differing in their functional activity, although their ultrastructure is similar. In the cytoplasm of granulocytes, granules of both mitochondrial and nuclear origin were detected. Simultaneously two processes occur in the cells--the accumulation and discharge of granules.     

Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation: II. Haemocyte functions: aggregation, viability, phagocytosis, and respiratory burst

The capability of an oyster to respond to environmental stresses, such as periodically high summer temperatures, as well as disease or parasite infections, depends, in large measure, upon the viability and functional capability of haemocytes. Eastern oysters (Crassostrea virginica) were subjected to a sudden increase in temperature from 20 to 28 °C for 1 week, and several haemocyte functions were determined before and after the temperature elevation using the flow cytometer. Previously, we described the characterization of different haemocyte types using new and modified flow cytometric methods. In this report, we provide detailed protocols for flow cytometric methods to: (1) determine haemocyte aggregation using paired samples with or without an antiaggregant solution; (2) assess haemocyte viability using propidium iodide (PI); (3) quantify haemocyte phagocytosis with fluorescent microbeads; and (4) measure the respiratory burst response of individual haemocytes using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and zymosan to activate the release of reactive oxygen species (ROS).The temperature increase caused no significant change in haemocyte aggregation, although there was a trend of increasing aggregation in granulocytes and small granulocytes, but a slight decrease in hyalinocyte aggregation. Phagocytosis of all haemocyte types decreased after the temperature increase. Significantly higher percentages of dead haemocytes in all haemocyte types (attributable to a large increase in mortality of hyalinocytes, the most numerous cells) were found after the temperature increase, suggesting generally less capable immune function. Numbers of dead small granulocytes and granulocytes tended to decrease, but this was not statistically significant. Effects of temperature elevation upon respiratory burst were not statistically significant; however, a trend of increased ROS production after temperature elevation was consistent for all haemocyte types. Granulocytes, hyalinocytes, and small granulocytes showed increased production of ROS in the presence of zymosan; granulocytes showed the highest induced fluorescence.     

First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum

In the present study, we report on haemocyte distribution, determined by a Coulter Counter, in the clam Tapes philippinarum. In addition, cytoskeleton components of haemocytes were examined using specific probes for F-actin and alpha-tubulin. The mean number of circulating haemocytes was 5 (x10(6))cells/ml haemolymph. Two main haemocyte populations were found in the haemolymph: small cells, 2-3microm in diameter and 10-100fl in volume; and large cells, 6-10microm in diameter and 150-400fl in volume. Analysis of the haemocyte cytoskeleton revealed bundles of actin filaments oriented according to the cell major axis, and microtubules radiating from the microtubule-organizing centre in proximity of the nucleus. Interestingly, mitotic spindles were also found radiating from the microtubule-organizing centres, located at the spindle poles (centrosomes) of undifferentiated cells. On the basis of both our previous findings regarding circulating stem cells (Cima, F., Matozzo, V., Marin, M.G., Ballarin, L., 2000. Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation. Fish Shellfish Immunol 10, 677-693) and new information from the present study, we suggest that haemoblasts are able to divide in the haemolymph of T. philippinarum. To our knowledge, this is the first report of mitotic spindles in circulating haemocytes from a bivalve species.     

Structural and functional characterisation of the blood cells of the bivalve mollusc,Scrobicularia plana

Light and electron microscopical studies were carried out in order to characterise the blood cells of the bivalve mollusc, Scrobicularia plana. Three types of haemocytes were recognised: eosinophilic granular haemocytes, basophilic granular haemocytes and basophilic agranular haemocytes. The eosinophilic granulocytes were vesicular and contained large granules whereas the basophilic granulocytes were found to contain small granules and glycogen 'lakes'. The basophilic agranular haemocytes were significantly smaller than the granular haemocytes and had a high nucleus to cytoplasm ratio. Functional characterisation of the blood cells identified activity for the lysosomal enzymes: acid phosphatase, beta-glucuronidase, non-specific esterase and arylsulphatase. There was also a weak staining reaction for phenoloxidase and peroxidase activities. Phagocytosis of Gram-positive bacteria was demonstrated by the haemocytes and antibacterial activity was shown by cell-free haemolymph. Assays to determine release of reactive oxygen species from the haemocytes did not detect any reactive oxygen generation.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

Enzyme characterisation of the circulating haemocytes of the carpet shell clam,Ruditapes decussatus(Mollusca:bivalvia)

The occurrence of a number of lysosomal enzymes (Proteases, glycosidases, phosphatases, and esterases) inRuditapes decussatushaemocytes was demonstrated by cytochemical and colorimetric techniques. The levels of 18 enzymes tested monthly varied through the study period (18 months), although they did not conform to a seasonal pattern of variation. No important effect of clam age on enzyme activity levels of haemocytes was detected. In those cytochemical assays in which distinction between granulocytes and hyalinocytes was possible, lysosomal enzymes were only found in granulocytes. Phosphatase was detected inside cytoplasmic granules of granulocytes, suggesting the granules to be lysosomes. NADPH oxidase was not detected in clam haemocytes, which is consistent with the absence of oxidative metabolism coupled with phagocytosis in haemocytes of this clam species. Levels of lysozyme detected inside haemocytes were higher than in serum.     

内脏团插核术刺激对三角帆蚌血细胞的影响

为了探讨三角帆蚌(Hyriopsis cumingii Lea)血细胞的类型及内脏团插核手术刺激对血细胞形态结构和数量的影响,研究利用相差显微镜、光学显微镜、透射电子显微镜和流式细胞仪对三角帆蚌血细胞进行了形态学研究。流式细胞术光散射图谱显示血细胞被分两类,一类为颗粒度高的大细胞,另外一类为颗粒度低的小细胞;相差显微镜观察显示,血细胞可分为胞体暗、折光性差和胞体明亮、折光性强的两类;Giemsa和H.E染色显示细胞分为胞质染色不均一、胞内颗粒明显和胞质染色均一、胞内颗粒不明显的两类;透射电镜超薄切片观察显示,颗粒明显的细胞胞质内线粒体、高尔基体等细胞器较丰富,颗粒不明显的细胞胞质内细胞器较少;负染结果表明血细胞主要分为表面不光滑、突起明显和细胞表面光滑、突起较不明显的两类。综合上述实验结果可见,三角帆蚌血细胞分为颗粒明显的细胞和颗粒不明显的透明细胞两大类。内脏团插核术刺激后,血细胞的形态和比例均发生显著变化。血细胞形态更多样,伪足状突起更明显,细胞内囊泡状物质增多,血细胞密度显著增高(PP<0.01)。研究表明,作为三角帆蚌免疫系统重要组成部分的血细胞,在插核手术后,其类型、形态结构和数量均产生明显变化,这是机体对外界刺激产生的免疫防御反应,其中颗粒细胞担负着主要的免疫功能。       

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.     

贝类血细胞硫代黄素T荧光染色方法

硫代黄素T( thioflavin T,TFT)是一种用于组织学的苯并噻唑荧光染料,因其对淀粉样蛋白有高亲和性而主要被用于淀粉样病变的荧光显微检测.本研究分别以软体动物门双壳纲的栉孔扇贝(Chlamys farreri)和中国蛤蜊(Mactra chinensis)、腹足纲的拟紫口玉螺(Natica janthosto...