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.     

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.     

Hydrolytic enzymes associated with the granular haemocytes of the marine mussel Mytilus edulis

Summary The ultrastructural localization of a range of hydrolytic enzymes has been investigated in the granular haemocytes of the marine musselMytilus edulis. Arylsulphatase activity and immunocytochemical localization of -glucuronidase and elastase were demonstrated within the large granules of the haemocytes. Lysozyme and cathepsin B were both localized within all sizes of granule, however, at high dilutions the primary antibody against lysozyme was also restricted to the large granules. The labelling density for cathepsin B antibody tended to be very low. Antibodies for cathepsin G showed a clear, discrete labelling which was restricted to the granules of haemocytes containing small granules. The fact that antibodies raised against human proteinases recognize invertebrate enzymes suggests that there must be a certain degree of structural similarity between the human proteinases and the enzymes present in the mussel haemocytes indicating either convergence or conservation of the enzyme molecules. The presence of a range of hydrolytic enzymes including proteinases, glycosidases and sulphatases within the large granules shows that these granules are a form of lysosome. The reduction in activity of lysosomal enzymes in haemocytes following adhesion to glass is evidence for release of the enzymes from the granules (degranulation). The possibility of a serine protease being specifically associated with the small granules and its role as a cytolysin are discussed.     

The chemiluminescence response of bivalve haemocytes: utility in screening for immunomodulators and as a biomarker

Resistance to infectious diseases in bivalves depends primarily on the vigour and efficacy of haemocyte-dependent antimicrobial defence mechanisms. Like other phagocytes, haemocytes seem to rely on oxygen-independent (lysosomal hydrolases, lysozyme) and oxygen-dependent (reactive oxygen species) mechanisms to destroy ingested microorganisms. The generation of cytotoxic oxyradicals by haemocytes can be precisely quantified by means of a simple chemiluminescence (CL) assay using luminol or other CL probes. Tributyltin (TBT), and other environmental contaminants, at sublethal levels, will produce dose-dependent suppression of CL activity in haemocytes exposed in short-term, in vitro assays. Presumably, this suppression would find expression as impaired host defence capability. In fact, TBT has been shown to exacerbate progression and lethality of Perkinsus marinus infections in the oyster, Crassostrea virginica. This suggests that CL assays on haemocytes exposed in vitro to single agents or complex mixtures might be useful in screening for aquatic immunomodulators. Statistically significant alterations in CL responses of haemocytes withdrawn from bivalves exposed to xenobiotics in the laboratory or field are more difficult to identify because of high inter-animal variation; however, the use of haemocyte CL as a biomarker of effect merits further investigation.     

Haemocytes of the pink bollworm, Pectinophora gossypiella, during larval development and diapause

Seven types of haemocytes were observed in the last larval instar of the pink bollworm, Pectinophora gossypiella (Saunders): prohaemocytes, plasmatocytes, granular haemocytes, spherule cells, adipohaemocytes, oenocytoids, and podocytes. Total and differential haemocyte counts made from diapausing and non-diapausing larvae showed that during diapause there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. There were no significant differences in the level of haemocytes in the pharate pupae that developed from diapause or non-diapause type larvae, except in the case of adipohaemocytes, which were three times as prevalent in pharate pupae from diapausing larvae. Functional aspects of various types of haemocytes are discussed, and it is suggested that the lower haemocyte level observed during diapause is the result of lower metabolic activity.     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in the crayfish haemocytes

Three types of cells circulate in haemolymph of the crayfish Astacus astacus: agranular haemocytes (HCs I), small-granule haemocytes (HCs II) and large-granule haemocytes (HCs III). Their proliferation, differentiation and function remain poorly understood. By means of light and electron microscopic autoradiography using [3H]-thymidine, we have revealed that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To determine whether the HCs I are proliferating progenitor cells for the granular HCs, we have analyzed autographs of HC population in 1, 2, 7 and 21 days after a single [3H]-thymidine administration. Contrary to the expectation, we have failed to find labeled HCs II and HCs III. These findings raise doubts on the capacity of the HCs I to differentiate into two other types of HCs. By autoradiography using 3H-uridine, it has been detected that intensity of the RNA synthesis was the greatest in HCs I and less by a factor of two and four in HCs II and HCs III, respectively. Additionally, by EM immunocytochemistry, ANP-like immunoreactivity was revealed in the large granules of the HCs III. We assume that availability of ANP in secretory granules extends the possible functions of the crayfish HCs and suggests their participation in regulation of water-salt balance and immune responses.     

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.     

Synthesis of nucleic acids and localization of atrial natriuretic peptide in crayfish haemocytes

Three types of cells circulate in the haemolymph of the crayfish Astacus astacus, i.e., agranular haemocytes (HCs I), small-granule haemocytes (HCs II), and large-granule haemocytes (HCs III). Their proliferation, differentiation, and function remain poorly understood. Using light and electron microscopic autoradiography with [³H]-thymidine, we found that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To verify whether HCs I are proliferating progenitor cells for granular HCs, we have analyzed autographs of the HC population 1, 2, 7, and 21 days after a single [3H]-thymidine administration. Contrary to our expectations, we have failed to find labeled HCs II and HCs III. These findings have raised doubts as to the capacity of HCs I to differentiate into two other types of HCs. With the use of ³H-uridine autoradiography, it was found that RNA synthesis was the most active in HCs I and 2 and 4 times lower in HCs II and HCs III, respectively. ANP-like immunoreactivity was revealed in large granules of the HCs III by electron microscopic immunocytochemistry. We assume that the presence of ANP in secretory granules extends the possible functions of crayfish HCs and suggests their participation in the regulation of the watersalt balance and immune response.     

Ultrastructural study of the neurosecretory granules in the sinus gland of the blue crab,Callinectes sapidus

Summary Seven morphologically different types of neurosecretory granules have been found in the axon terminals of the sinus gland of the blue crab, Callinectes sapidus. They differ from each other in size, shape, staining characteristics, solubility characteristics, core matrix characteristics, axon terminal matrix characteristics, presence or absence of space between the granule membrane and granule core matrix, and frequency of occurrence. Five of the types are segregated in different axon terminals and are believed to represent different hormone-protein complexes. Two of the types, which have lost part or all of their granular contents, are thought to be variants of the other five types. The differences in granular morphology are better revealed by some fixation procedures than others. Palade's acetate-veronal buffered osmium tetroxide, in particular, reveals striking differences. The following observations suggest that different hormone-protein complexes are segregated in different axon terminals and that these complexes may be morphologically distinguished at the level of the electron microscope.Supported by USPHS-NIH Training Grant GM-00669 and Grant GB-7595X from the National Science Foundation.     

Steinernema carpocapsae DD136: metabolites limit the non-self adhesion responses of haemocytes of two lepidopteran larvae, Galleria mellonella (F. Pyralidae) and Malacosoma disstria (F. Lasiocampidae)

Live adult and juvenile entomopathogenic Steinernema carpocapsae DD136 (P. Nematoda) were not subjected to adhesion by haemocytes of lepidopteran insect larvae of Galleria mellonella or Malacosoma disstriain vitro or in vivo. In vitro freeze-killed nematodes exhibited haemocyte attachment, the intensity increasing with time. Accumulation of haemocytes on the dead nematodes was associated with host phenoloxidase activity; live nematodes and their exudates did not activate the enzyme whereas dead nematodes but not their exudate did activate phenoloxidase. Live-nematode exudate inhibited granular cell and some plasmatocyte adhesion to slides, increased granular cell but not plasmatocyte dissociation from preformed haemocyte monolayers and in vivo elevated total haemocyte counts and changed the floating haemocyte types while impairing bacterial removal from the haemolymph. Dead-nematode exudate did not affect these parameters thus immunosuppressant activity by live nematodes may represent the release of inhibitors not associated with their cuticle. The third stage juveniles released the inhibitors.     

Infection with the protozoan parasite Bonamia ostreae modifies in vitro haemocyte activities of flat oyster Ostrea edulis

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy.Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.     

Ageing adipokinetic cells in Locusta migratoria: an ultrastructural morphometric study

Summary A morphometric study was made of the ultrastructure of adipokinetic cells in resting adults of Locusta migratoria at 3, 23, and 43 days after imaginal ecdysis. The nucleus, rough endoplasmic reticulum, and Golgi apparatus enlarge with age, which indicates that the synthesis and packaging of secretory substances increases during ageing. The size of the storage compartment, consisting of secretory and ergastoplasmic granules, does not increase earlier than 23–43 days after imaginal ecdysis. The lysosomal compartment markedly enlarges between 3 and 23 days; later on, the growth of this compartment, especially of autophagosomes, is less prominent. This suggests that lysosomal destruction initially compensates for the production of new secretory granules, assuming that exocytosis of secretory granules by adipokinetic cells is insignificant in resting locusts. Afterwards, lysosomal destruction may no longer be sufficient to prevent over-production of secretory granules, as is suggested by the increase in the number of these granules between 23 and 43 days. This coincides with the appearance of a considerable number of large ergastoplasmic granules, which represent a spatially more efficient form of storage of secretory material than the much smaller secretory granules. The increase with age in the amount of secretory products indicates that the biosynthetic activity of the adipokinetic cells is not (finely) tuned to their releasing activity.     

Localization of xenopsin and xenopsin precursor fragment immunoreactivities in the skin and gastrointestinal tract of Xenopus laevis

Summary Xenopsin (Xp) and xenopsin precursor fragment (XPF) are bioactive peptides derived from a single precursor molecule; both were isolated previously from extracts of Xenopus laevis skin. The present immunohistochemical study was undertaken to determine the specific cellular localization of these two peptides in the skin and also in the gastrointestinal tract of adult Xenopus. We report here that Xp-like and XPF-like immuno-reactivities co-exist in the granular glands of the skin and specific granular cells in the lower esophagus and stomach. However, only Xp-like immunoreactivity, not XPF-like immunoreactivity, was detected in tall, thin cells of the duodenum and in club-shaped cells of the large intestine. The immunochemical co-localization of the two peptides in specific cells of the skin, lower esophagus and stomach suggests that the same gene is expressed in each of these cells, and that the precursor molecule undergoes similar post-translational processing. In contrast, the observation that certain cells of the duodenum and large intestine display only one peptide immunoreactivity suggests an alternative phenomenon, possibly involving selective peptide accumulation or expression of a different gene.     

The structure of the haemocytes of Calpodes ethlius (Lepidoptera)

The haemocytes of Calpodes ethlius are described with the light and electron microscopes. Five fine structurally distinct types are distinguishable. However only three of these, the granular haemocytes, sphaerule cells and oenocytoids can be positively identified using a series of histochemical stains on smears and on thick sections of araldite-embedded material. The classification is based entirely on the structural features even though several suggestions concerning their function can be made from their fine structure. Intermediates having features of more than one cell type suggest developmental relationships.     

The binding of lectins to carbohydrate moieties on haemocytes of the insects,Blaberus craniifer (Dictyoptera) and Extatosoma tiaratum (Phasmida)

Summary The specificity and distribution of carbohydrate moieties, which act as receptors for lectins on the haemocytes of two insect species, Blaberus craniifer and Extatosoma tiaratum, were investigated. Four peroxidase-labelled lectins were utilised as probes: wheat-germ agglutinin, Ricinus communis (120) agglutinin, concanavalin A and Lens culinaris agglutinin, and binding visualised by reaction with DAB/H₂O₂. The lectin-binding studies indicated that Blaberus and Extatosoma plasmatocytes, and Extatosoma spreading granular cells possess surface N-acetyl-D-glucosamine, D-galactose and mannose moieties; Extatosoma cystocytes have D-galactose and mannose; whilst Blaberus granular cells/cystocytes and Extatosoma fine granular cells have mannose determinants.     

The haemocytes of the salp Thalia democratica (Tunicata,Thaliacea): an ultrastructural and histochemical study in the oozoid

In comparative immunology and evolution of the chordate immune system, tunicates hold an important phylogenetic position as sister group of vertebrates. However, knowledge of the tunicate immune system is limited to the class Ascidiacea, in which some species are now considered model organisms. In the class Thaliacea, represented by fragile pelagic species, the few studies on their haemocytes go back to several decades ago and do not consider comparative aspects with ascidian haemocytes. In this study, we identified various haemocyte types and their distribution in the common salp Thalia democratica by comparative observations under light and electron microscopy and by histochemical, histoenzymatic and immunohistochemical techniques. By comparing specialisations with those of ascidian haemocytes, we detected an undifferentiated cell type (lymphocyte‐like cell) and three categories with four cell types, that is, (i) phagocytic line (hyaline amoebocyte and amoebocyte with large vacuoles), (ii) mast cell‐like line (granular cell) and (iii) storage cells (nephrocyte). Both phagocytes and granular cells appear to migrate in the tunic. Phagocytes adhere to the tunic which internally covers the oral siphon, where they probably function as sentinel cells of the pharynx. Results show the variety of haemolymph cells in the salp similar to phlebobranch ascidians.     

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system

The phenoloxidase (PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by means of 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identified by a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatant from hemocyte populations separated on a discontinuous Percoll density gradient. In Styela plicata, the enzyme appeared to be contained in morula cells only. In Ciona intestinalis, PO activity was shown in univacuolar refractile granulocyte and granular hemocyte. In Phallusia mammillata both compartment cell and granular hemocytes were positive. Enzymatic assay following electrophoretic analysis on polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE indicated that hemocyte lysate presented orthodiphenoloxidase (catecholase) activity. The enzymes from the three species differed in molecular size, activating substances and trypsin sensitivity.     

Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea)

A morpho‐functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehyde‐fixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin‐affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti‐CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage‐like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron‐dense content, positive to arylsulphatase activity and binding DBA, UEA‐I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS‐positive vacuoles with arylsulphatase, chloroacetylesterase and β‐glucuronidase activities.     

Immuno-electron microscopy of formation,degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

Summary The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20–36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive.The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules.     

Ultrastructure of the haemocytes of Tetrodontophora bielanensis Waga (Collembola)

Five types of haemocytes: prohaemocytes, plasmatocytes, granular haemocytes, spherule cells and phagocytes, have been distinguished on the basis of ultrastructural studies. Prohaemocytes are ovoid cells with a simple structural organization. Plasmatocytes are larger; their cytoplasm contains well-developed rough endoplasmic reticulum, numerous mitochondria and free ribosomes. Granular haemocytes are the most numerous of the blood cells, characterized by the presence of electron-dense granules. The cytoplasm of spherule cells contains many spherules made up of filamentous material of medium electron density. Rough endoplasmic reticulum, free ribosomes and mitochondria are also found in the cytoplasm. Phagocytes are the largest haemocytes. Their cytoplasm contains an abundance of lysosomes and myelin structures. In addition to haemocytes, cells intermediate between plasmatocytes and granular haemocytes have been observed, which indicates that the granular haemocytes are derived from plasmatocytes.