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Morphological Characterization of the Hemocytes of the Clam,Ruditapes decussatus(Mollusca: Bivalvia)
Abstract
Hemocytes play an important role in internal defence in molluscs. The morphology of hemolymph cells was studied for the first time in Ruditapes decussatus. Two main types of hemocytes (hyalinocytes and granulocytes) exist in R. decussatus. Three types of granulocytes were identified by light microscopy, in accordance with the presence of basophilic or acidophilic granules or a mixture of both in the cytoplasm. The existence of hyalinocytes and granulocytes was confirmed by electron microscopy. Some monoclonal antibodies (MABs) raised against hemocytes of Crassostrea gigas showed cross-reactivity with the total population of hemocytes of R. decussatus; however, none of the MABs raised against hemocytes of Mytilus edulis showed cross-reactivity. The MABs assayed did not allow us to distinguish hemocyte subpopulations.
... Granulocytes are the large cells with high complexity and hylinocytes are the small cells with low SSC and FSC values. Similar results were also found in oysters (Hegaret et al. 2003), clams (Lopéz et al. 1997;Cima et al. 2000), mussels (Cajaraville et al. 1995), and scallops (Xing and Zhan 2005;Zhang et al. 2006). The differentiation between granulocytes and hyalinocytes could be easily achieved using light microscopy and flow cytometry. ...
... Based on such observation, the hypothesis that phagocytic activity of hyalinocytes is higher than in granulocytes when cells were exposed to CuSO 4 could be advanced. The capacity of R. decussatus hyalinocytes to internalize yeast cells, bacteria, and protozoan parasites was already described (Lopéz et al. 1997;Cima et al. 2000), and Lopéz et al. (1997) showed that the phagocytic index of hyalinocytes was related to the nature of the foreign material. ...
... Based on such observation, the hypothesis that phagocytic activity of hyalinocytes is higher than in granulocytes when cells were exposed to CuSO 4 could be advanced. The capacity of R. decussatus hyalinocytes to internalize yeast cells, bacteria, and protozoan parasites was already described (Lopéz et al. 1997;Cima et al. 2000), and Lopéz et al. (1997) showed that the phagocytic index of hyalinocytes was related to the nature of the foreign material. ...
In ecotoxicology, in vitro testing on cell cultures represents an ideal alternative to in vivo strategies for emerging contaminants. These tests have limited use particularly with marine invertebrates like the clams Ruditapes decussatus. In the present study, a primary culture of R. decussatus haemocytes was realized for the first time in order to determine the effect of metals (copper, zinc, and cobalt) on haemocyte parameters like viability and phagocytosis. Results showed that (i) among the studied medium, the modified Leibovitz (L-15) is the best for R. decussatus haemocytes primary culture. (ii) The primary culture system used here represents a suitable in vitro model for assessing cytotoxic responses, (iii) a decrease of cell viability and phagocytosis after 24 h exposure to 100 μg mL−1 CoSO4 and an increase of phagocytosis after 24 h exposure to 50 μg mL−1CuSO4.
... A l'issue du processus de dégradation des particules, les produits de dégradation peuvent être accumulés dans des vacuoles glycogéniques ou libérées dans le milieu extracellulaire générant également une décharge d'enzymes lysosomales dans l'hémolymphe (Carballal et al., 1997). ...
... Outre des effets sur les paramètres hémolymphatiques, des modifications de température peuvent entraîner des perturbations de fonctionnalités cellulaires. Une diminution de l'activité de phagocytose in vitro a été observée à une température de 10°C chez Mytilus galloprovincialis (Carballal et al., 1997). Une augmentation de l'efficacité de cette fonction a été mesurée entre 20 et 25°C pour les hémocytes de la palourde Ruditapes philippinarum (Cima et al., 2000 ;Allam et al., 2002b). ...
... Changes in morphometrical parameters remain unknown on suspended cells but it was indicated that Ruditapes decussatus hyalinocytes were smaller than granulocytes after cytocentrifugation while an opposite observation was done when cell were fixed in suspension (López et al., 1997). On the other hand, cytocentrifuged cells were fixed with methanol while formaldehyde was used for cell suspension fixation. ...
L’extension des activités humaines est responsable du rejet de molécules et de perturbations climatiques pouvant affecter la physiologie des organismes aquatiques. La moule zébrée possède des caractéristiques biologiques faisant d’elle une espèce intéressante en surveillance environnementale. Chez cet organisme, les hémocytes constituent une cible privilégiée pour la mise en place d’une approche multi-biomarqueurs. En effet, ces cellules à fonctionnalités multiples sont impliquées dans les grandes fonctions physiologiques de l’espèce et la régulation de l’homéostasie des individus. L’objectif de ce travail est de développer les outils analytiques permettant d’étudier les réponses hémocytaires de la moule zébrée. Les expérimentations menées ont permis de caractériser la structure des populations hémocytaires ainsi que leurs fonctionnalités propres en lien avec le processus de phagocytose. L’utilisation de ces biomarqueurs dans divers contextes indique une forte adaptabilité de l’espèce aux conditions environnementales. Les résultats montrent l’intérêt d’analyser les activités hémocytaires à l’échelle des sous populations comparativement à l’approche globale ne tenant pas compte de la diversité cellulaire. Il a été observé que certains facteurs comme le statut reproducteur ou l’espèce échantillonnée (D. polymorpha vs D. bugensis) constituent des facteurs de confusion importants. Il ressort également un positionnement fort du test de phagocytose en tant que marqueur de sensibilité aux contaminants. Ce travail constitue un ensemble de données voué à être utilisé dans des contextes multiples aussi bien en écotoxicologie qu’en écophysiologie.
... Le Foll et al. [56] highlighted quick morphology changes in adherent cells exhibiting condensed and spread phases within few minutes. Changes in morphometrical parameters remain unknown on suspended cells but it was indicated that Ruditapes decussatus hyalinocytes were smaller than granulocytes after cytocentrifugation while an opposite observation was done when cell were fixed in suspension [77]. On the other hand, cytocentrifuged cells were fixed with methanol while formaldehyde was used for cell suspension fixation. ...
... On the other hand, cytocentrifuged cells were fixed with methanol while formaldehyde was used for cell suspension fixation. Differences in fixation protocol used in this study may indicate that this factor may influence cell size parameters [77]. In addition, some studies based on flow cytometry analysis discriminated cell types into different groups of size [42,67,78]. ...
... However, microscopic observations indicated that hyalinocytes were phagocytic cells (data not shown). Other studies indicated that both granulocytes and hyalinocytes were performing phagocytosis with higher activity for granulocytes in Mytilus galloprovincialis [74], Crassostrea ariakensis [44], Cerastoderma glaucum [73], Perna viridis [64,75], Mercenaria mercenaria [79], Saccostrea glomerata [57], Ruditapes decussatus [77]. Moreover, some authors highlighted that only granulocytes possessed this capacity [80,81] while no granular cells are found in some scallops species [35,82] resulting in a phagocytic activity relying on agranular cells as single effector [61,83]. ...
... These cells play a key role in digestion, metabolite transport, shell repair (Armstrong et al. 1971;Pollero et al. 1985;Cheng 1996;Mount et al. 2004) and immune system (Adema et al. 1991;Gopalakrishnan et al. 2009). They are able of executing several immune functions including phagocytosis (Tripp 1961;Cheng 1981;López et al. 1997a) and cytotoxicity through the production of reactive oxygen intermediates (Pipe 1992). The central role of phagocytosis in immune defense and the sensitivity of this biological function to environmental xenobiotics in several species emphasize the usefulness of this approach (Krzystyniak et al. 1989;Voccia et al. 1994;Fugere et al. 1996). ...
... Subpopulations of granulocytes were also observed. In fact, three types of R. decussatus granulocytes were identified, using light microscopy, in accordance with the presence of basophilic or acidophilic granules or a mixture of both in the cytoplasm (López et al. 1997a). Similarly, Cima et al. (2000) characterized the granulocytes of R. philippinarum into three subpopulations: basophils, acidophils and neutrophils. ...
... Recently, Rioult et al. (2013) proposed a showing thin sections of a granulocyte (Gr) and hyalinocyte (Hy). N nucleus, gr electron-dense particles, mi mitochondria, g Golgi complex, ps pseudopods, v electron-lucid vesicles (95800) (López et al. 1997a) Cytotechnology (2016) 68:1669-16851671 novel approach which helped to propose a functional classification of cell subtypes. This approach was based on fluorescence staining and nuclei-tracking to determine migration velocity of haemocytes in vitro. ...
Among aquatic organisms suitable for biological monitoring, molluscs occupy a prominent place due to their wide geographic distribution, their abundance and accessibility in the field as well as in aquaculture. Molluscs reflect the degree of environmental contamination and are the most useful bioindicator tools. The study of modulation of immune system or immunomodulation in marine molluscs has become one of the privileged ways for evaluating the physiological effects of environmental factors. Physiological responses of molluscs to environmental stresses could be mediated by haemocytes. These cells are continually exposed to the external environment due to the open circulatory system of molluscs and are affected by pollutants. In fact, several studies showed the effects of different environmental contaminants on haemocyte functions (viability, phagocytosis, ROS production) as well as on proteins involved in cytoskeletal structure maintenance using the in vitro approaches. In ecotoxicology, in vitro approach is an alternative to animal testing due to the reduced use of experimental animals, low cost and rapid performance. Although several studies showed the importance of using in vitro cell models to determine the effects of different environmental contaminants on haemocyte parameters in marine molluscs, a few reviews highlight these effects. The main purpose of this paper is to summarize the recent data on the effect of some xenobiotics on haemocyte parameters in some mollusc species and then suggest future research prospects.
... Female clams (Ruditapes philippinarum) have been shown to have significantly higher numbers of granulocytes in the haemolymph than males during the pre-spawning period (Matozzo and Marin, 2010). Since granulocytes are considered to be the major effector cells of the innate immune system in bivalve molluscs (Foley and Cheng, 1975;López et al., 1997;Tripp, 1992), the higher proportion of active haemocytes in female clams is thought to lead to higher endocytotic and haemolymph lysozyme activities and more efficient defence pathways against oxidative stress than those of males (Matozzo and Marin, 2010). In oysters (e.g., Saccostrea glomerata and Pinctada fucata), although sex-related differences have not been observed for total and differential haemocyte counts or phagocytosis capacity, the intracellular oxidative metabolism is reportedly higher in males than females (Dang et al., 2012). ...
... antimicrobial activities, especially in phagocytosis (Foley and Cheng, 1975;López et al., 1997;Tripp, 1992). The higher proportion of granulocytes in females than males in the clam R. philippinarum was suggested to lead to higher phagocytosis capacity and more efficient defence pathways against oxidative stress in females than males (Matozzo and Marin, 2010). ...
Massive mortalities due to pathogens are routinely reported in bivalve cultivation that have significant economic consequences for the global aquaculture industry. However, host-pathogen interactions and infection mechanisms that mediate these interactions are poorly understood. In addition, gender-specific immunological responses have been reported for some species, but the reasons for such differences have not been elucidated. In this study, we used a GC/MS-based metabolomics platform and flow cytometry approach to characterize metabolic and immunological responses in haemolymph of male and female mussels (Perna canaliculus) experimentally infected with Vibrio sp. Sex-based differences in immunological responses were identified, with male mussels displaying higher mortality, oxidative stress and apoptosis after pathogen exposure. However, central metabolic processes appeared to be similar between sexes at 24 h post injection with Vibrio sp. DO1. Significant alterations in relative levels of 37 metabolites were detected between infected and uninfected mussels. These metabolites are involved in major perturbations on the host’s innate immune system. In addition, there were alterations of seven metabolites in profiles of mussels sampled on the second day and mussels that survived six days after exposure. These metabolites include itaconic acid, isoleucine, phenylalanine, creatinine, malonic acid, glutaric acid and hydroxyproline. Among these, itaconic acid has the potential to be an important biomarker for Vibrio sp. DO1 infection. These findings provide new insights on the mechanistic relationship between a bivalve host and a pathogenic bacterium and highlight the need to consider host sex as a biological variable in future immunological studies.
... Hemolymph, the circulatory fluid of invertebrates, is a potential source of information of the physiological status of animals, as it transports nutrients, enzymes, gases, hormones and other molecules related to metabolism. In bivalves, hemolymph flows through different sinuses, and its extraction through the abductor muscle sinus have been reported, with no effect on the specimens' survival López et al. 1997;Gustafson et al. 2005). Therefore, besides providing a wide range of information on the individual physiological status, hemolymph analysis offers the possibility to sample the same individuals during long periods of time without adverse effects. ...
... Hemolymph extraction was always performed on tagged animals. 0.5-2 mL were withdrawn from the posterior abductor muscle with a 1 mL syringe through a hole drilled to the shell according to López et al. (1997). Samples were centrifuged (10 min, 2000g, 4°C), and the supernatants were stored at -80°C for further analysis. ...
The concentration of the sex steroids 17β-estradiol, testosterone and progesterone was determined by specific enzyme-linked immunosorbent assays in the hemolymph of the mussel Mytilus
galloprovincialis and related to its sexual maturation. A non-lethal hemolymph sampling procedure was used in mussels matured in controlled conditions of a hatchery. To our knowledge, this is the first study on sexual steroid hormones of a bivalve species using the same specimens in consecutive samplings. Histological data showed that nearly 90 % of the mussels ripened during the experiment. Steroid analyses revealed that 17β-estradiol and testosterone were closely related to the onset of gamete development, while progesterone would have an important role at the end of gonadal maturity. This method can be used to assess sexual maturation in bivalves when conditioning in hatcheries, without killing them.
... Ces 2 principaux types d'hémocytes existeraient chez tous les bivalves (Cheng, 1981;Hine, 1999). Ainsi, plusieurs études ont révélé la présence de granulocytes et de hyalinocytes chez la mye Mya arenaria (Huffman & Tripp, 1982), chez les moules Mytilus edulis (Pipe, 1990a), M. galloprovincialis (Cajaraville et al., 1995;Carballal et al., 1997b), chez les palourdes Ruditapes decussatus (López et al., 1997), Ruditapes philippinarum (Cima et al., 2000) et Mercenaria mercenaria (Tripp, 1992), chez les coques Cerastoderma glaucum (Matozzo et al., 2007), Chamelea gallina (Pampanin et al., 2002), Cerastoderma edule et le couteau Ensis siliqua (Wootton et al., 2003) ainsi que chez les coquilles Argopecten irradians (Zhang et al., 2006a), Patinopecten yessoensis (Nakamura et al., 1985), Pecten maximus (Le Gall et al., 1991), Chlamys farreri (Xing et al., 2002). 7 Chez l'huître plate Ostrea edulis, grâce aux techniques d'immunohistochimie, des granulocytes largement présents dans les tissus conjonctifs des différents organes (glande digestive, le manteau, les branchies et les gonades) ont pu être observés . ...
... De la même manière, les granulocytes sont les cellules les plus actives dans le processus de phagocytose, lors d'injection de particules de zymosan chez la palourde européenne Ruditapes decussatus, ou encore chez la coquille St Jacques Argopecten irradians, lors de stimulations par des souches bactériennes Escherichia coli (López et al., 1997;Zhang et al., 2006b). ...
The chemical communication systems constitute an essential element in the establishment of intra- or inter-species relationships in marine environment, weaving a dense network of relations between individuals, in ecosystem. The invertebrates lacking of an immune system and usually sessile produce this type of bioactive metabolites playing a crucial role in the answer to the environmental pressures like the predation and the defence against potentially pathogens organisms. The aim of this work was to identify antimicrobial peptides in commercially bivalve and gastropod marine molluscs. Thus, the search for antimicrobial molecules from peptidic nature was undertaken in acid extracts of bivalve molluscs Cerastoderma edule, Ruditapes philippinarum, Ostrea edulis, and gastropods Crepidula fornicata, Buccinum undatum and Littorina littorea and from Crassostrea gigas hemolymph. The extracts were pre-purified by Solid Phase Extraction C18 (SPE) and elution was ensured by three successive steps of 10%, 40% and 80% of ACN-0.1% TFA. The antibacterial activities were assayed by determination of the CMI against a panel of target bacteria including Gram+ and Gram- bacteria. In parallel, antiviral activities were assayed in vitro against Herpes simplex virus type 1 and Vero cells by cell viability. The species C. edule, L. littorea and C. gigas proved to be the most effective and non cytotoxic species. A partial characterization of the activity detected in these species allowed determining the protenaceous nature of the active molecules. The purification of antimicrobial peptides realised on the C. gigas hemolymph led us to the identification of a peptide which structure lets foresee a bacterial origin. The hypothese of an association between C. gigas and bacteria led us from non axenic oysters culture to search for antagonist bacteria in C. gigas hemolymph and has conduced to isolate 2 Vibrio spp. and 3 Pseudoalteromonas spp. The Pseudoalteromonas spp. hCg 5 strain, allowed to partially characterized an active compound. Whole of these results suggests that the bacteria associated with the immune system could play an essential function of defence in bivalves.
... At least two kinds of hemocytes have been identified in bivalves and gastropods (hyalinocytes and granulocytes) according to the presence or absence of granules, which are characterized by staining affinity (Chu, 2000;López et al., 1997;Salimi et al., 2009). Furthermore, there is an agreement about the function of hemocytes to repair tissue damage, nutrient transport and digestion, and internal defense against non-self material (Cheng, 1975;Chu, 2000). ...
... As in their molluskan relatives (López et al., 1997) the cellular defense by cephalopod hemocytes involves phagocytosis as well as production of oxygen and nitrogen radicals (Ford, 1992;Malham et al., 1997;Rodríguez-Domínguez et al., 2006). Phagocytosis of microbial agents and non-self materials is an important defense reaction (Cheng, 1975;Chu, 2000). ...
Cephalopod mollusks are an important marine resource for fisheries, and have received marked attention for studies on organismal biology; they are also good candidates for aquaculture. Wild and reared cephalopods are affected by a wide variety of pathogens, mainly bacteria, protozoa and metazoan parasites. Cephalopods do not have acquired immunity and immunological memory; therefore vaccination cannot be used to protect them against infectious diseases. Their defense mechanisms rely only on their innate immunity. In this review, we will summarize and update knowledge on the most common pathogens, the diseases they cause, and on symbionts. In addition, we provide a general overview of the cephalopod immune system, response to pathogens with a short discussion on the gene expression involved in the immune response by these animals.
... Ruditapes decussatus (López et al. 1997), Ruditapes philippinarum (Cima et al., 2000) et ...
... Chez la moule, Mytilus galloprovincialis, la palourde européenne Ruditapes decussatus, ou encore chez la coquille St Jacques Argopecten irradians, seuls les granulocytes sont capables de phagocyter le zymosan 1 et/ou les bactéries (Vibrio tapetis) tandis que les hyalinocytes ne semblent pas posséder la capacité de phagocytose (Carballal et al, 1997bLópez et al., 1997Zhang et al.,2006b). ...
The aim of this work was studying the immune responses at molecular and cellular level of mussels exposed to different types of contaminants (metals and chemicals) by studying the transcriptomic of some genes used as biomarkers like MgBD3 (Antimicrobian peptide), lysozyme, catalase and Superoyxde Dismutase), using RT-PCR. In this work, we tried to understand how aquatic species, bivalves here, "react" in polluted environment polluted by cadmium, Chrysene and TiO2 nanoparticles by studying some of their responses at the molecular level, face to these stresses, these responses are multiple, simultaneous or cascading, and always complex. We have tried throughout this study to understand the resistance mechanisms in these organisms. We are also committed to reproduce the environmental pollution conditions by using during the bivalve laboratory exposure, the same concentrations of those encountered in the natural study sites.
The results obtained in this work show that:
- These contaminants affect the immune parameters that respond differently.
- These parameters are all in a functional relationship with or control the generation of reactive oxygen species.
- There is a threshold concentration responsible for the type variation the biomarkers response (increase or decrease) but must be determined and validated.
- Responses against contaminants influence the generation of reactive oxygen species in the same way.
- The response intensity against heavy metals and PAHs varies between these two.
- Nanoparticles affect the immune response of bivalves.
... Similar increase in hemocytes has been also observed in the shell repairing process in oysters (Ruddell 1971;López et al. 1997), suggesting the involvement of the hemocytes and SMPs in oyster's shell formation and/or repair (Johnstone et al. 2008). Li et al. (2016) also showed evidence that circulating hemocytes can directly participate in the CaCO3 crystal formation in two species of pearl oyster (Pinctada fucata and Crassostrea virginica). ...
One of the distinguishing morphological features of conchiferan mollusks is the presence of a calcified exterior shell. Despite being conchiferans, most extant cephalopods have lost, internalized, or degraded their shells, with the exception of the basally divergent nautiloids. We compiled the shell matrix protein (SMP) data from several Nautilus pompilius studies and compared them with publicly available conchiferan SMP data, including those of Sepia pharaonis and Spirula spirula, two decapodiform cephalopods with internalized and partially internalized calcified shells, respectively. The recompilation of N. pompilius data revealed the presence of 85 distinct SMPs. Reciprocal homology searches suggested that N. pompilius shares 27 proteins known for their significant roles in shell formation and biomineralization, such as Pif/Pif-like, with other conchiferans. This is in agreement with our previous results, which suggested that the main functional domains of the SMPs in N. pompilius were also found in the conchiferans, suggesting that the domains were present in the ancestral conchiferans, with some recruited ancestrally, and some taxon-specifically. Meanwhile, 16 N. pompilius proteins are shared among the conchiferans, with only six proteins present in the cephalopods. indicating that some proteins were lost or co-opted in the course of their evolution. Three proteins shared among the conchiferans (CD109 antigen, Chitinase, and Tyrosinase) and one shared among the cephalopods (SOUL Domain-containing protein) apparently have dual functions of immunity and shell biomineralization. Intriguingly, four proteins are shared only between the two decapodiforms, indicating that they were co-opted in the decapodiform lineage with internalized shells.
... At the end of the experiment, 100 μL of haemolymph were taken by carefully inserting a 27G needle with an insulin syringe in the abductor muscle of each clam (n = 8 clams from each treatment). Haemolymph was immediately mixed with 325 μL of an Anti-aggregant solution (AAS)fromLopez et al. (1997) (AAS: 20.80 g/L glucose, 8.00 g/L sodium citrate, 3.36 g/L EDTA, 22.50 g/L sodium chloride in distilled water) ...
Extreme events like Marine Heatwaves (MHWs) are becoming more intense, severe, and frequent, threatening benthic communities, specifically bivalves. However, the consequences of non-lethal MHWs on animals are still poorly understood. Here, we exposed the Manila clam Ruditapes philippinarum to non-lethal MHW for 30 days and provided an integrative view of its effects. Our result indicated that albeit non-lethal, MHW reduced clam's energy reserves (by reducing their hepato-somatic index), triggered antioxidant defenses (particularly in males), impaired reproduction (via the production of smaller oocytes in females), triggered dysbiosis in the digestive gland microbiota and altered animals' behaviour (by impacting their burying capacity) and filtration rate. Such effects were seen also at RNA-seq (i.e. many down-regulated genes belonged to reproduction) and metabolome level. Interestingly, negative effects were more pronounced in males than in females. Our results show that MHWs influence animal physiology at multiple levels, likely impacting its fitness and its ecosystem services.
... After the corresponding exposure treatment, the total counts, phagocytic activity, and cell type composition of haemocytes were analysed on the basis of previously reported methods (López et al., 1997;Nakayama et al., 1997;Sun et al., 2020Sun et al., , 2021Tang et al., 2020). Briefly, haemolymph extracted individually (n = 6 for each parameter analysed) from the pericardial cavity of blood clams was centrifuged at 4 ℃ to collect haemocytes. ...
The ubiquitous presence of pentachlorophenol (PCP) in ocean environments threatens marine organisms. However, its effects on immunity of marine invertebrates at environmentally realistic levels are still largely unknown. In this study, the immunotoxicity of PCP to a representative bivalve species was evaluated. In addition, its impacts on metabolism, energy supply, detoxification, and oxidative stress status were also analysed by physiological examination as well as comparative transcriptomic and metabolomic analyses to reveal potential mechanisms underpinning. Results illustrated that the immunity of blood clams was evidently hampered upon PCP exposure. Additionally, significant alterations in energy metabolism were detected in PCP-exposed clams. Meanwhile, the expressions of key detoxification genes and the in vivo contents (or activity) of key detoxification enzymes were markedly altered. Exposure to PCP also triggered significant elevations in intracellular ROS and MDA whereas evident suppression of haemocyte viability. The abovementioned findings were further supported by transcriptomic and metabolomic analyses. Our results suggest that PCP may hamper the immunity of the blood clam by (i) constraining the cellular energy supply through disrupting metabolism; and (ii) damaging haemocytes through inducing oxidative stress. Considering the high similarity of immunity among species, many marine invertebrates may be threatened by PCP, which deserves more attention.
... These include eosinophilic granular, agranular, lymphoid, large granular, small granular and morula-like haemocytes. According to the staining affinities of their cytoplasmic granules, granulocytes may be further sub-classified into eosinophilic, basophilic and neutrophilic haemocytes, depending on the bivalve species (Hine, 1999;López et al., 1997a;McCormick-Ray & Howard, 1991). Indeed, Hine (1999) described granulocytes as having many cytoplasmic granules and being the most abundant cell type. ...
Perna canaliculus is endemic to New Zealand and is the top shellfish export product. However, the growth of this industry is being adversely affected by summer mortality events. To assess these health threats, differential haemocyte counts (DHC) were performed on haemolymph smears stained with Giemsa, and in vitro phagocytosis activity assays were applied in P. canaliculus for several months covering three seasons (winter, spring and summer). A new optimized classification scheme was developed for P. canaliculus haemocytes where 55% eosinophilic and 27.2% basophilic granulocytes were identified. Eosinophilic granulocytes were classified as dense, semi and small semi, and these new categories were reported herein for the first time in this species. A new haemocyte type (mixed granulocytes), which contains both acidophilic and basophilic granules, is proposed for P. canaliculus. The phagocytosis percentages were significantly affected by incubation time, indicating an increase in zymosan particle uptake from 18.42 ± 1.7% after 30 min, 32.08 ± 3.1% after 60 min and 44.74 ± 3.5% (maximum) after 120 min of incubation. The lowest phagocytosis was observed in the winter season and the highest phagocytosis in summer. This study findings provide a better understanding of the immune function of P. canaliculus haemocytes and serve as a reference for further investigations on the impacts of seasonal variations on haemocyte activity and phagocytosis.
... Even for the same species, different haemocyte classification has been proposed, as shown in Table 1 (species in bold). This diversity may be in part due to some true differences, but also resulting from the use of different classification criteria or experimental procedures, endogenous and exogenous factors, like age, pollution or the high inter-individual variability (71,77,79,112,196,197). Additionally, different nomenclatures adopted by various researchers due to lack of biological markers for specific cell lineages or maturation stages contribute to the problem (198). ...
Bivalve molluscs stand out for their ecological success and their key role in the functioning of aquatic ecosystems, while also constituting a very valuable commercial resource. Both ecological success and production of bivalves depend on their effective immune defence function, in which haemocytes play a central role acting as both the undertaker of the cellular immunity and supplier of the humoral immunity. Bivalves have different types of haemocytes, which perform different functions. Hence, identification of cell subpopulations and their functional characterisation in immune responses is essential to fully understand the immune system in bivalves. Nowadays, there is not a unified nomenclature that applies to all bivalves. Characterisation of bivalve haemocyte subpopulations is often combined with 1) other multiple parameter assays to determine differences between cell types in immune-related physiological activities, such as phagocytosis, oxidative stress and apoptosis; and 2) immune response to different stressors such as pathogens, temperature, acidification and pollution. This review summarises the major and most recent findings in classification and functional characterisation of the main haemocyte types of bivalve molluscs.
... Os hemócitos de bivalves consistem em uma população heterogênea de células que apresentam diferentes morfotipos (ANISINOVA, 2013), sendo a identificação dependente das diferentes técnicas de obtenção e identificação dos hemócitos utilizadas pelos autores (LÓPEZ et al., 1997). ...
... Os hemócitos de bivalves consistem em uma população heterogênea de células que apresentam diferentes morfotipos (ANISINOVA, 2013), sendo a identificação dependente das diferentes técnicas de obtenção e identificação dos hemócitos utilizadas pelos autores (LÓPEZ et al., 1997). ...
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... To characterize each hemocyte subpopulation, cells were separated by centrifugation in a discontinuous Percoll gradient according to the protocol used for molluskan hemocytes (López et al., 1997). 0.5 ml of hemocytes suspensions was layered over a 1.5 ml of discontinuous gradient and centrifuged at 400 g for 10 min in centrifuge Elmi CM-80 (Russia). ...
Hemolymph cellular composition, morphology and functional properties of the Pacific oyster (Crassostrea gigas) hemocytes were studied. Three hemocyte types (agranulocytes, hyalinocytes and granulocytes) were described in hemolymph. The morphology of each type was characterized by light microscopy and flow cytometry. Agranular cells (agranulocytes and hyalinocytes) were the dominant type of cells in hemolymph; their number was 86.7±2.7% of total cell count. Under hypoxia the number of agranulocytes increased (37.4% for control group versus 95.3% for hypoxic probes), whereas granulocyte and hyalinocyte number decreased up to 3.9% and 0.7% in hypoxic specimens respectively. The spontaneous ROS production decreased in each hemocyte type after exposure to hypoxia. Low dissolved oxygen did not influence hemocyte proliferation and mortality level.
... Conventionally, the presence or absence of cytoplasmic granules as an intuitive criterion has inspired the classification of hemocytes into granulocytes and hyalinocytes, as mentioned above. These two cell types have been reported in many species, including Mytilus edulis (20), Tapes philippinarum (21), Biomphalaria glabrata (22), Ruditapes decussatus (23), and Crassostrea gigas (24). Of the two, hyalinocytes are cells that are smaller and harbor few or no cytoplasmic granules. ...
Hemocytes play unequivocally central roles in host immune defense of bivalve mollusks, though the exact mechanisms underlying their functional differentiation are only partially understood. To this end, granulocytes and hyalinocytes were sorted via flow cytometry from hemocytes of the Pacific oyster Crassostrea gigas, and consequently quantitative transcriptomic analysis revealed a striking array of differentially expressed genes (DEGs), which were globally upregulated in granulocytes, dedicating to functional differentiation among oyster hemocytes. Our network of DEGs illustrated actively engaged signaling pathways, with Cdc42/Cdc42l being a core regulator of pathway network, which was validated by a dramatically reduced capacity for hemocyte phagocytosis in the presence of Cdc42 inhibitors. Additionally, a number of transcription factors were identified among DEGs, including ELK, HELT, and Fos, which were predominantly expressed in granulocytes. The AP-1 transcription factor Fos was confirmed to facilitate functional differentiation of hemocytes in an assay on binding to target genes by the AP-1 binding site, consistent with downstream phagocytosis and ROS production. Importantly, Cdc42/Cdc42l were also regulated by the expression of Fos, providing a possible regulatory mechanism-guided hemocyte functional differentiation. Findings in this study have bridged a knowledge gap on the mechanistic underpinnings of functional differentiation of hemocytes in a marine invertebrate C. gigas, which promise to facilitate research on the evolution of immune defense and functional differentiation of phagocyte in higher-order and more recent phyla.
... veneriformis by morphological and biochemical examinations. Bivalve haemocytes are classified into two major cell types, granulocyte and agranulocyte based on existence of cytoplasmic granules (Cheng, 1981;López et al., 1997b;Barracco et al., 1999;Hunakoshi 2000;Park et al., 2000 andPampanin et al., 2002;Dyrynda et al., 2003;Chou et al., 2005). In the present study, two types of haemocytes, granulocytes and agranulocytes, occurring in M. veneriformis were distinguishable by the presence or absence of cytoplasmic granules both with a light microscope and electron microscope. ...
... Granulocytes and hyalinocytes were competent phagocytes towards yeast as experimental target and were able to produce hydrolytic and oxidative enzymes (mostly after stimulation with yeast) and superoxide anions. Interestingly, the presence of both granulocytes and hyalinocytes has been confirmed in the haemolymph of the congenus species Ruditapes decussatus [2,4,5]. ...
Ultrastructural investigation confirmed the presence of four cell types (granulocytes, hyalinocytes, serous cells, and haemoblasts) in the haemolymph of the Manila clam, Ruditapes philippinarum. Granulocytes were characterised by numerous electron-dense granules, whereas hyalinocytes had a considerable number of small clear vesicles. Serous cells exhibited large vacuoles, which filled the cytoplasm, and haemoblasts (the undifferentiated cells) were small roundish cells characterised by a high nucleus/cytoplasm ratio. The presence of circulating haemoblasts was observed at various phases of mitosis. Updated data concerning the proliferation and differentiation of circulating haemocytes were obtained after both in vitro and in vivo bacterial challenge. The results demonstrated that cell proliferation occurred within 15 h of exposure, and most haemocyte types responded to the stimuli. The number of granulocytes significantly decreased after massive phagocytosis and ultrastructural observations confirmed that they were active phagocytic cells against both Gram-positive and Gram-negative bacteria, which were rapidly engulfed into large phagosomes. Granulocyte lysis may represent a protection response against bacterial proliferation inside phagosomes. The number of serous cells significantly increased, suggesting a previously unreported pivotal immune role during bacterial infection. A panel of lectins was used as probes to further characterise haemocytes and their relationships. Only hyalinocytes were not positive for the lectins assayed, whereas all lectins labelled serous cells, suggesting that these cells have a variety of specific carbohydrates, which are shared with certain haemoblasts. The hypothesis of the existence of a prospective haemoblast for serous cell origin is discussed.
... The endoplasm was the part where the cytoplasmic granules were present, whereas the ectoplasm showed the hyaline area where granulocytes showed the ability to produce pseudopods (Figure 4). Multinucleate structures could be showed in the monolayer, which could be the result of the fusion of granulocytes ( Lopez et al, 1997). Basophilic granulocytes appeared to be blue colored while acidophilic ones showed to be dark pink. ...
Nanoparticles toxicity (1-100nm) to living organisms so far reported to affect different organismal levels, from whole organism, cellular to molecular levels. An integrative approach was followed in this thesis to investigate the role of nanoparticle size, exposure time and uptake routes in determining the immunotoxicity of silver nanoparticles to Mytilus galloprovincialis.
In so doing, in vivo exposures for 3, 6 and 12 hr to a single sublethal dose (100 µg/L) of two size of silver nanoparticles (AgNP: < 50 nm and < 100 nm) prior to and after inhibition of clathrin- and caveolae-mediated endocytosis uptake routes, were designed. Cytotoxicity assay and the hemocytic parameters used as markers to assess the sub-cellular responses. Histopathological analyses of inflammation in the gills and digestive gland were done at tissues level. Redox-based proteomic investigations conducted to assess the proteome status after exposure to AgNPs.
The results showed a significant cytotoxic effect on immune cells of mussels exposed to both sizes of AgNP for different timeframes (p < 0.01); the greater effect was with the smaller size. However, hemocytes seemed to be more sensitive to the larger size of AgNP after clathrin-mediated endocytosis was blocked (p < 0.01); this was not so with inhibition of caveolae-mediated endocytosis. The percentages of different hemocytes sub-populations showed to be varied after exposure to AgNP; these variations seemed to be decreased as exposure length progressed and noted to be delayed after blockade of either clathrin- or caveolae-mediated endocytosis (p < 0.01). Histological analyses showed that AgNP impacted on inflammation morphology and intensity dependently to exposure time, nanoparticle size (Highest response recorded with Ag-NPs <100nm alone) and uptake pathways (with Ag-NPs <50nm after blockade of uptake routes).
Overall, it was concluded that AgNP toxicity was size- as well as exposure time-dependent. It was proven that endocytotic routes were deeply involved in determining redox-based changes and immune response face to NP toxicity. Interestingly, NP uptake pathways found to be more impactful than the size. Lastly, a protective role of clathrin-mediated endocytosis route against NP toxicity was reported.
... A sampling pool per time was done from the hemolymph of five scallops. The samples were incubated at 37°C for 4 h López et al., 1997;Luna-González et al., 2002. Sixty five microlliters of the sample were added to each well (including the control well) followed by incubation at 35°C for 4 h. ...
Susceptibility and immune response of Argopecten ventricosus challenged with Vibrio parahaemolyticus were determined. Mortality and median lethal concentration of V. parahaemolyticus were assessed in scallop seeds and adults challenged by immersion. Histopathological analysis was done in adults challenged by immersion. The immune response was analyzed in adults by injecting heat-inactivated bacteria in the adductor muscle. Hemolymph samples were taken at 0, 8, 24, 48, and 72 h. Time zero corresponded to samples of scallop hemolymph before challenging with vibrio. In bioassays, scallops were fed with microalgae concentrate Shellfish Diet 1800®. Seeds and adults of A. ventricosus were susceptible to V. parahaemolyticus showing a LC50 of 623 CFU mL− 1 and 62,853 CFU mL− 1, respectively. V. parahaemolyticus toxins caused lesions in digestive glands of adults analyzed. Significant variations were observed in hemocyte number, phenoloxidase activity, and lysosomal hydrolytic enzymes, mainly at 8 and 24 h, post infection. Results confirm that seeds and adults of A. ventricosus are susceptible to V. parahaemolyticus infection, but seeds are more susceptible than adults. In adults, the immune response of A. ventricosus was modulated by V. parahaemolyticus.
... In these studies, hyalinocytes are characterized as cells with fewer granules in their cytoplasm and a higher nucleus to cell ratio, when compared with the granulocytes, which have abundant granules. In other studies, density gradient centrifugation Friebel and Renwrantz 1995;Bachère et al. 1988), flow cytometry (Ashton-Alcox and Ford 1998; Hégaret et al. 2003a;Hégaret et al. 2003b;Xue et al. 2001;Allam et al. 2002) and functional assays have been also used to separate and identify haemocyte populations, from which more haemocyte types have been proposed (Pipe et al. 1997;Nakayama et al. 1997;Carballal et al. 1997;Lopez et al. 1997). However, morphological characteristics are difficult to analyse since they often vary with taxa, life stage, with the different haemocytes sampled and culture techniques (Ittoop et al. 2001;Carballal et al. 1997). ...
: Haemocytes play a major role in molluscs immunity. Functional studies are, however, impaired by limited available
experimental tools to identify and sort distinct haemocyte populations. Therefore, using nonlethal methods, we aimed at
evaluating whether lectin staining combined with flow cytometry could be used to distinguish circulating haemocyte populations
from two freshwater bivalves of the family Unionidae, the duck mussel (Anodonta anatina (L., 1758)) and the swan mussel
(Anodonta cygnea (L., 1758)). Based on classical classification, haemocytes were distinguished as granulocytes and hyalinocytes and
cytological features were visualized using transmission microscopy and staining techniques. Size, granularity, viability, and
surface staining using lectins as specific probes were analysed by flow cytometry and fluorescence microscopy. The microscopic
proportions of granulocytes and hyalinocytes significantly differed, being of 70% and 30% for A. cygnea and of 85% and 15% for
A. anatina, respectively. Two haemocyte populations were sorted by flow cytometry based on size and granularity and confirmed
as granulocytes and hyalinocytes. Interestingly, two different granulocyte populations could be further discriminated in
A. cygnea according to their binding affinity to wheat-germ agglutinin (WGA), whereas granulocytes of A. anatina all stained
similarly. Our results show that WGA labelling combined with flow cytometry can be used to better discriminate Anodonta
haemocyte populations and obtain purified populations for functional studies.
... Based on their morphological and cytochemical features, mollusk hemocytes are mainly categorized into 2 types: granulocytes, with numerous granules in the cytoplasm, and hyalinocytes, with no or few granules (Hine 1999, Cima et al. 2000, Aladaileh et al. 2007, Donaghy et al. 2009, Astuya et al. 2015. Moreover, subpopulations of hyalinocytes and granulocytes are also observed in several mollusk species, such as the clam Ruditapes philippinarum (López et al. 1997, Cima et al. 2000, Lambert et al. 2003 and abalone Haliotis tuberculata (Hine 1999, Travers et al. 2008. In addition to the common granulocytes and hyalinocytes, a small quantity of large hyalinocytes possessing a phagocytic function are deemed to be macrophages in the Zhikong scallop Chlamys farreri (Sun & Li 2001). ...
Primary cultured cells can be a useful tool in the studies on physiology, virology and toxicology. Hemocytes play an important role in animal rapid response to pathogen invasion. In this study, an appropriate medium for primary culture of the hemocytes of bivalve Chlamys farreri was developed by supplementing 5% fetal bovine serum and 1% C. farreri serum to Leibovitz L-15 medium. These primary-cultured hemocytes were maintained for more than 40 days in vitro, and were classified into three types: (1) granulocytes containing numerous granules in the cytoplasm, (2) hyalinocytes with none or fewer granules, (3) less macrophage-like cells. Furthermore, the primary-cultured hemocytes were observed to be sensitive to bacterial and viral challenges. These hemocytes could phagocytose bacteria Vibrio anguillarum, and presented cytopathic effects to the extracellular products (ECPs) of V. anguillarum; the mRNA level of QM, which plays an important role in immune response, also significantly increased 12 h after infection. When these hemocytes were challenged with Ostreid herpesvirus-1 (OsHV-1), virus particles and empty capsids in the cells infected for 48 h were observed by transmission electron microscope, and the QM mRNA level increased significantly at 12 h and 24 h following OsHV-1 challenge. This primary culture system is available for C. farreri hemocytes which can be used in the future to study on host–pathogen interactions.
... These histomorphological alterations are mainly attributable to the immune system activation process, in fact haemocytes, due to their phagocytic activity, are the most important cells involved in immune mechanism of the bivalves Pruzzo et al. 2005). This evidence is also confirmed by TEM analysis showing that haemocytes were granulocytes (López et al. 1997). Moreover, ultrastructural observation has highlighted important morphological alterations typical of an advanced apoptotic conditions. ...
Despite the wide use of nanoscale materials in several fields, some aspects of the nanoparticle behavior have to be still investigated. In this work, we faced the aspect of environmental effects of increasing concentrations of TiO2NPs using the Mytilus galloprovincialis as animal model and carrying out a multidisciplinary approach to better explain the results. Bioaccumulation suggested that the gills and digestive gland are the most sensitive organs to TiO2NP exposure. Histological observations have evidenced an altered tissue organization and a consistent infiltration of haemocytes, as a consequence of the immune system activation, even though an increase in lipid peroxidation is uncertain and DNA damage became relevant only at high exposure dose (10 mg/L) or for longer exposure time (96 h). However, the over expression of SOD1 mRNA, strengthen the concept that the toxicity of TiO2NPs could occur indirectly by ROS production. TEM analysis showed the presence of multilamellar bodies, RER fragmentation and cytoplasmic vacuolization within relevant presence of dense granules, residual bodies and lipid inclusions. These findings support the evidence of an initial inflammatory response by the cells of the target organs leading to apoptosis. In conclusion, we can state that, certainly the exposure to TiO2NPs has affected our animal model from cellular to molecular levels. Interestingly, the same responses are caused by lower TiO2NP concentration and longer exposure time as well as higher doses and shorter exposure. We do not know if some of the conditions detected are reversible, then further studies are required to clarify this aspect.
... Morphological characterisation of the circulating haemocytes of Ruditapes decussatus and phagocytic ability had been studied. Two cell types were found, hyalinocytes and granulocytes (López et al., 1997). Granulocytes have a greater phagocytic ability than hyalinocytes (López et al., in press). ...
... Morphological characterisation of the circulating haemocytes of Ruditapes decussatus and phagocytic ability had been studied. Two cell types were found, hyalinocytes and granulocytes (López et al., 1997). Granulocytes have a greater phagocytic ability than hyalinocytes (López et al., in press). ...
... Morphological characterisation of the circulating haemocytes of Ruditapes decussatus and phagocytic ability had been studied. Two cell types were found, hyalinocytes and granulocytes (López et al., 1997). Granulocytes have a greater phagocytic ability than hyalinocytes (López et al., in press). ...
... There is an ongoing contention regarding hemocyte classification in bivalve mollusks, and widely accepted nomenclature standards are lacking [8,26,43]. In previous studies, two basic types of hemocytes, namely, hyalinocytes and granulocytes, were defined in the Mollusca phylum based on morphological features [44,45]. ...
The presence of microglial cells as resident macrophage population in the Central Nervous System (CNS) is well documented from the study of repairing of lesions in CNS that varies widely throughout the animal kingdom. The existence of neuroglia cells similar to vertebrate microglia and small mobile phagocytes and hemocytes were documented from ganglia of some invertebrate animal models like leech (H. medicinalis), insects (P. americana and D. melanogaster) and mollusca (M. edulis). Neuronal replacement and migration of immunocompetent cells (macrophage, microglia, ependymal cells etc.) after surgical lesions in CNS of non-mammals (fishes, reptiles and aves) are much restricted to specific neurogenic niches associated to the neural regeneration and migration of cells in invertebrates. Microglial presence is largely restricted in the optic tract of fish and amphibian ganglionic cells because they have a surprising capacity to regenerate their neurons after lesions. Hence the CNS of both invertebrates and vertebrates contain microglia like mononuclear phagocytes, ensheathing glia and reticular glia, which indicate about the evolutionary conserved innate immune response to maintain CNS development and health. But the presence and gradual changes in the structure and function of microglia and neuron-microglia relationship in the CNS along the phylogeny need to be focused thoroughly.
Molluscan hemolymph is a unique kind of body fluid, which in many respects is analogous to human blood, although there are several crucial differences. Here, for the first time, we critically analyze the prospects for applications of this fluid in modern biomaterials science. Particular attention is paid to the biochemistry and chemistry of molluscan hemolymph, as well as to hemocytes and hemocyanins as key functional players within this unique biological fluid. We focus on hemocytes as multifunctional hemolytic cells involved in immune response, and especially in the biomineralization process. The next part of the review contains a discussion of molluscan shell formation and regeneration from different points of view. Finally, we consider the challenges, solutions, and future directions in the application of molluscan hemolymph for bioinspired material chemistry and biomedicine.
The razor clam Sinonovacula constricta is widely distributed in the intertidal zones and estuarine waters along the coast of western Pacific Ocean and is extensively cultured. Even though haemocytes are known to play an important role in the immune mechanisms of bivalves, these cells are poorly studied in S. constricta. We researched the morphology and immunological activities of haemocytes in S. constricta using light and electron microscopy and flow cytometry. Three major subpopulations of haemocytes were identified in the haemolymph: granulocytes, semigranulocytes, and hyalinocytes. These subpopulations were divided using flow cytometry, but not satisfactorily. Therefore, the flow cytometry findings were combined with the light and electron microscopy findings, as well as Percoll density-gradient centrifugation findings, to classify and distinguish between the cell types more effectively. The combined findings showed that granulocytes was larger cells, while semigranulocytes was smaller and more abundant. Further, granulocytes had numerous granules in the cytoplasm, semigranulocytes contained fewer and smaller granules, and hyalinocytes was smaller and less abundant with no or a few granules. Both granulocytes and semigranulocytes had greater phagocytotic activity and a higher lysosomal content than hyalinocytes. The results declared that granulocytes and semigranulocytes were the main haemocytes involved in the cellular defence mechanism in S. constricta.
Recent advances in high-throughput technologies for omics analyses and bioinformatics for data interpretation have led to the application of omics approaches across all fields of life sciences. There has also been an expansion of omics research in immune studies of marine invertebrates, such as bivalves to gain insights into pathogenic infections and disease progression. Many of these omics studies have been conducted on haemocytes and haemolymph, which are the most important components of the bivalve immune system. Characterization of transcriptomes, proteomes and metabolomes of bivalve haemocytes in response to pathogenic infections and other environmental stressors have revealed valuable information regarding the mechanisms that drive the innate immune system in response to stress challenges, as well as insights regarding complex host-pathogen-environment interactions across bivalve species. For instance, detailed analysis of haemocyte transcriptomes has resulted in the discovery of a number of coding and non-coding transcripts involved in immune and stress responses. In addition, comprehensive examination of the proteome and metabolome of bivalve haemocytes following stress exposure has helped identify changes in the physiological status of the organism, including specific molecular pathways involved in these processes. Furthermore, the differently expressed molecules that have been identified through these omics studies can be used as candidate biomarkers with applications in breeding selection programs, disease diagnosis and environmental monitoring. However, despite these significant biotechnological advances, the application of omics tools for bivalve haemocyte research is currently hindered by several challenges and bottlenecks. In this contribution, we aim to review the major advances, current perspectives and future directions of three main omics (transcriptomics, proteomics and metabolomics) with regards to their application in bivalve haemocyte and aquaculture research.
Marine invertebrates, like bivalves depend exclusively on their innate immunity system to deal with pathogen challenges. In Atlantic mussels it has been shown that hemocytes present in haemolymph, synthesize a variety of antimicrobial peptides, which are released to haemolymph. In endemic mussels of the Chilean coasts, the innate immune system has not been characterized, therefore, the antimicrobial activity of haemolymph components of C. chorus was evaluated in this work.
Using the classic acid and solid phase extractions to isolate antimicrobial peptides from haemolymph, we detected a powerful antibacterial activity in fractions which present very low and medium hydrophobicity These results indicate that in C. chorus would contain antimicrobial peptides with different features than those described in other mussels.
Using May-Grünwald-Giemsa staining haemocytes population was studied and as well as in the other mythilids, basophylic, granular eosinophylic and hialine, were observed. In addition, hemocytes in animals challenged whith E. coli significant morphology change in haemocytes, including degranulation and appearence of a brown pigment, corresponding to melanin were observed. This brown melanin is synthesized by an ancient defense mechanism called the prophenoloxidase reaction. On the other hand, the most abundant protein of haemolymph of C. chorus has a molecular weight of 75 kDa, contrasting with what was described for other mussels, in which a 37 kDa protein is the predominant. This protein was isolated by Ni-IMAC chromatography, which indicates that it may belong to the Histidine Rich Glycoprotein family (HRG), described in other mussels. The apparent molecular weight for this protein determined by gel filtration chromatography under native conditions was more than 1 MDa. Immunodetection of this protein with anti-key hole limpet and C. concholepas haemocyanin antibodies by Western blot, suggests that this protein may have haeomcyanin features. Interestingly, this putative HRG- haemocyanin from C. chorus exhibits antibacterial activity, suggesting that it may be a component of the innate immunity system.
Because it is widely recognized that the mantle tissue controls nacre precipitation, intensive studies have been conducted to unravel the regulatory mechanism underlying nacre formation. In this chapter, we will focus on the cellular regulation of shell formation in the pearl oyster Pinctada fucata. First, the morphology and proliferation rate in different parts of the mantle tissue have been investigated, and a proliferation hot spot located in the center of the thinnest mantle region was found. However, mantle tissues mainly composed of the mantle edge were used for primary cell culture, due to their high yield of migrated mantle cells. The primary mantle cell culture is used to study how the mantle cells regulate calcium carbonate precipitation. It was found that the physiological functions of the mantle cells were maintained in vitro. High expression of many shell matrix proteins, including ACCBP, Pif80, and nacrein, and high activities of carbonic anhydrase and alkaline phosphatase were detected. Numerous crystals were found inside the cultured cells by polar light microscopy and scanning electron microscopy, and FTIR and XRD analysis demonstrated that these particles were amorphous calcium carbonate (ACC). What’s more, the cultured mantle cells promoted and regulated calcium carbonate precipitation in the culture dishes. These results showed that mantle cells may directly participate in shell formation. In addition, we have studied the function of hemocytes in shell formation. Hemocytes were identified from P. fucata and found to be present in the extrapallial space (EPS). Many components involved in immunity and calcification were identified by proteomics analysis. Poststimulation of lipopolysaccharide and shell damage, most of the tested immune genes and calcification, was upregulated. Moreover, polar light microscopy, scanning electron microscopy, and energy spectrum showed that some hemocytes carried crystals of calcium carbonate, indicating they participated in both immunity and biomineralization. Our studies elucidated the vital roles of mantle cells and hemocytes in regulating shell growth and nacre formation, which would shed light on the improvement of the cultured pearl quality.
Clam Ruditapes philippinarum is one of the most important commercial aquaculture species in China. The haemocytes play vital roles in internal defense of the calm. In this investigation, classification and immune functions of R. philippinarum haemocytes were identified. The haemocyte density was (8.28 ± 1.42) × 106/mL and two major haemocyte types basophilic hyalinocytes and eosinophilic granulocytes were recognized based on the presence or absence of granules and staining affinities of their cytoplasm. Granulocytes were the most common cell type (73.08 ± 3.23%). The hyalinocytes and granulocytes could be divided into eight subtypes respectively according to N/C ratio as well as the nucleus shape and number by light microscope. Fourteen types of granules were identified and the multivesicular body and R-body were first found in bivalve, moreover, transmission electron microscope observation was consistent with the results from light microscope. Also eight different external forms of haemocytes could be identified by scanning electron microscope. Both granulocytes and hyalinocytes showed the abilities of phagocytosis and reactive oxygen species (ROS) production which were higher in granulocytes than that in hyalinocytes. The phagocytic rate of the total haemocytes and the granulocytes was about 45.06% and 40.74% respectively. The ROS production of the total haemocytes and granulocytes was 58.7% and 51.19% respectively. Although the hyalinocytes showed less ability in phagocytosis and ROS production, they played important roles in agglutination. This investigation provided a fundamental knowledge for future study of the immune function of haemocytes in clam R. philippinarum.
Hemocytes are the first line of defence of the innate immune system of molluscs. For the first time hemocytes of Crassostrea hongkongensis were morphologically and functionally characterized, identifying circulating cell types and studying their involvement in immune responses. In the present study, two main populations, hyalinocytes and granulocytes, were characterized based on the presence or absence of cytoplasmic granules, using light and electron microscopy (TEM), and flow cytometry analyses. Granulocytes are 7-13 μm in diameter and present evident cytoplasmic granules, and hyalinocytes, 6-15 μm in diameter, with a few or no granules. The mean number of circulating hemocytes in the hemolymph was 2.52 × 106 cells/mL. Flow cytometry indicated that both granulocytes and hyalinocytes showed cell phagocytosis and reactive oxygen species (ROS) production. However, phagocytosis and spontaneous production of reactive oxygen species (ROS) in granulocytes are much more active compared with hyalinocytes, which demonstrated that the granulocytes are the main hemocytes involved in the immune response of Hong Kong oyster. Moreover, the cell-free hemolymph showed antibacterial activity against Vibrio alginolyticus. Our results provide the basic information of hemocytes population of Hong Kong oyster for further investigations associated with innate immunity.
Non‐destructive, sub‐lethal, and sensitive health monitoring tools are needed for the family Unionidae (freshwater mussels). Recent developments to standardize hemocyte characterization have paved the way for hematologic assessment of wild and captive freshwater mussels. In this study, preliminary baseline hematological reference ranges were established for wild Mapleleaf Mussels, (Quadrula quadrula, n = 14) and Threeridge Mussels, (Amblema plicata, n = 20) collected from the Muskingum River in Devola, Ohio, USA. Mussels were collected from the wild and hemolymph was sampled from each mussel in the field upon capture (baseline). They were then transported live to a propagation facility. Subsequent hemolymph samples were collected at 2 weeks, 4 weeks, and quarterly thereafter for 11 months following translocation. Hemocyte counts, hemocyte morphology, and hemolymph chemistry [Na⁺, Cl‐, Mg²⁺, P3‐, K⁺, Ca²⁺, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP)] were measured from each sample on each sampling occasion. Hemocyte counts were consistently greater in Q. quadrula than A. plicata following transfer to captivity. Baseline hemocyte morphology and hemolymph chemistry varied between species. This study provides a foundation of reference ranges for hemocyte characterization for Q. quadrula, and A. plicata and a preliminary understanding of how hemocyte character might be expected to change when wild mussels are translocated into captivity, which may be a useful technique for monitoring health in freshwater mussels.
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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.
The Unionidae family, or commonly referred as freshwater pearly mussels or naiades, are one of the most impaired groups of the Animalia kingdom. Although it includes hundreds of different species all over the world, it is an unknown group for many, but for the minority that spend their lives studying these organisms, they are quite amazing and surrounded by many open questions. They are extremely interesting from a biological perspective; first, as important providers of aquatic ecosystem services; secondly, they require a host for larval development and dispersal; third, they are powerful ecotoxicological bioindicators; and were already used until exhaustion for commercial purposes (pearl and button industry).
Freshwater mussel’s immunology is a field with great potential, since little is known. However, the lack of suitable culture media and cell markers greatly difficult the achievement of more functional knowledge, concerning the immune elements and understanding how the immune system operate. The aim of this thesis is to contribute with new strategies to evaluate the fitness of freshwater mussels, by demonstrating the important relationship between immunity and ecology for these organisms. Their capacity to adapt is closely link to the well-functioning of their immune system. The more we know about their immune system, better tools can be provided to preserve them.
The work presented here has a multidisciplinary approach integrating different scientific areas like immunology, cell biology, toxicology and microbiology. In this perspective, this work explores immune elements, like the blood cells (haemocytes), fluids (plasma) and micropearls, to stablish the best conditions to study them and explore their potential as complementary biomarkers when analysing the health status of the bivalves. From these studies stands out the potential source of new antimicrobial compounds, present in the plasma from these species, probably similar to the ones already identified in marine species.
Haemocytes are the cells responsible for the cellular immune response and were key elements in the different studies conducted. Their concentration, ratios, cell types and viability, vary greatly among organisms and under experimental conditions. Thus, they firstly were well characterized to become helpful elements in posterior studies. Besides the morphological features, the functional capacity to have affinity with specific lectins like WGA, found only for one cell type (granulocytes) in the two species studied here (Anodonta cygnea and Anodonta anatina) was a new characteristic found, important to distinguish between haemocytes.
Micropearls or calcium carbonate concretions, are a peculiarity to highlight when studying these animals, can easily be extracted and analysed by multiple techniques, involved in the biomineralization process and can also function as bioindicators of the habitat conditions, since many toxic compounds can be trapped inside, even their origin may be closely linked to the haemocytes.
Unionids are an important faunistic group. Their ecological relevance should be by itself important to promote their preservation. Nonetheless these animals may also be a source of new antimicrobial compounds, adding further interest to their protection. The antibacterial capacity of the fluids and haemocytes of several unionid species is shown here. Even if the elements involved are still unidentified, their action is proved with the inhibition of bacterial growth and biofilm formation, using human opportunistic gram negative bacteria: Escherichia coli and Pseudomonas aeruginosa and gram positive bacteria: Staphylococcus aureus.
Ecotoxicological assays give important information surrounding the threats that these animals are constantly being exposed to, and the implications in terms of behaviour, fitness and immune response. Although the results obtained in the combined ecotoxicologic and immunologic study were not very consolidated, however this multifactor approach is more realistic, these organisms tend to have a large plasticity among them, giving rise to inconclusive results, the effects noticed were not always in the same direction, but reflect imbalance instead of homeostasis.
The combination of immunological and ecological knowledge will thus greatly benefit on the measures to implement to deal with factors threatening the naiades.
Various diagnostic techniques have been developed in ecotoxicology to assess the negative effects of pollutants on organisms and populations. Among invertebrate species, mollusks are widely used as sentinel organisms worldwide. In this invertebrate group, many biomarkers have been examined to understand the negative effects of pollutants. In particular, at the immune level, end points, such as hemocyte count, viability and locomotion, phagocytic activity, and the production of hydrolytic enzymes and cytotoxic molecules, have been used. This chapter intends to provide examples of immunomarkers that have been used to assess the immunotoxicity of emerging contaminants in mollusks and to present new diagnostic approaches from recent immunotoxicogenomic investigations for biomarker discovery, albeit still in its infancy. Immunotoxicogenomic studies identified several specific gene targets responding to exposure to complex municipal effluents, selected pharmaceuticals, and nanotechnology. Finally, some attention is given to the use of immunocompetence end points to predict impacts at the higher levels of biological organization up to bivalve population status.
Hemocytes are the effective immunocytes in bivalves, which have been reported to be derived from stem-like cells in gill epithelium of oyster. In the present work, a conserved haematopoietic transcription factor Tal-1/Scl (Stem Cell Leukemia) was identified in Pacific oyster (Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. Cg-SCL was highly distributed in the hemocytes as well as gill and mantle. The hemocyte specific genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. During the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage (10 hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage (15 hpf) before resuming in umbo larvae (120 hpf). Whole-mount immunofluorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were significantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. These results demonstrated that Cg-SCL could be used as haematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae.
To help understand the cellular defense mechanism of Meretrix lusoria hemocytes withdrawn from the posterior adductor muscle were classified on the basis of their morphology, cytochemistry and phagocytic characteristics under a light microscope. Two cell types, granulocytes and hyalinocytes, were found in the hemoctyes. Granulocytes, 13.1 ± 2.6 μm ∼ 16.2 ± 3.3 μm in size, contain some large or small eosinophlic granules in their cytoplasm. Granuloctyes were subclassified into small eosinophilic granulocytes and large eosinophilic granulocytes based on the size of the granules. Granulocytes were positive to PAS, acid phosphatase and phenoloxidase but negative to Sudan black B. Granulocytes showed positive phagocytic activity to zymosan particles. Hyalinocytes, that is, agranulocytes that did not contain granules in the basophilic small cytoplasm, were 11.3 ± 2.8 μm in diameter and had an oval or irregular nucleus at the center of the cell. The ratio of nucleus to the cytoplasm was very high. Hyalinocytes were weakly positive to PAS but negative to Sudan black B and acid phosphatase. Phenoloxidase acivity was not detected in hyalinocytes. Hyalinocytes phagocytized zymosan particles but the activity was very low compared with that of granulocytes.
known as garden snail, is a species of land snail, a pulmonate gastropod. This snail is very common and widespread in Mediterranean region and western Europe. This species is characterized by a spherical in shape shell with a short spire and a rough surface. The shell color is pale brown, or yellow with a number of broken dark bands, which give the shell it, is blotchy appearance. This study investigates the effect of emperature on phagocytosis activity in garden snails H. aspersa. Low and high temperatures depressed phagocytosis activity and decrease the number of yeast cells phagocyted by H.aspersa hemocytes. The phagocytosis activity at 25C° is (50.8±4.5), 35 C° (19.2 ± 3.9), 40 C° (3.6±0.89), and 15 C° (4.4±1.1).Two types of hemocytes cell GLC (Granulocyte like Cell), and HLC (Hyalinocyte like Cell) are diagnosed
Many species of bivalve mollusks (phylum Mollusca, class Bivalvia) are important in fisheries and aquaculture, whilst others are critical to ecosystem structure and function. These crucial roles mean that considerable attention has been paid to the immune responses of bivalves such as oysters, clams and mussels against infectious diseases that can threaten the viability of entire populations. As with many invertebrates, bivalves have a comprehensive repertoire of immune cells, genes and proteins. Hemocytes represent the backbone of the bivalve immune system. However, it is clear that mucosal tissues at the interface with the environment also play a critical role in host defense. Bivalve immune cells express a range of pattern recognition receptors and are highly responsive to the recognition of microbe-associated molecular patterns. Their responses to infection include chemotaxis, phagolysosomal activity, encapsulation, complex intracellular signaling and transcriptional activity, apoptosis, and the induction of anti-viral states. Bivalves also express a range of inducible extracellular recognition and effector proteins, such as lectins, peptidoglycan-recognition proteins, thioester bearing proteins, lipopolysaccharide and β1,3-glucan-binding proteins, fibrinogen-related proteins (FREPs) and antimicrobial proteins. The identification of FREPs and other highly diversified gene families in bivalves leaves open the possibility that some of their responses to infection may involve a high degree of pathogen specificity and immune priming. The current review article provides a comprehensive, but not exhaustive, description of these factors and how they are regulated by infectious agents. It concludes that one of the remaining challenges is to use new "omics" technologies to understand how this diverse array of factors is integrated and controlled during infection.
Copyright © 2015. Published by Elsevier Inc.
In order to characterize in more detail hemocytes from Mytilus edulis and establish links between histological and cytological featues, monoclonal antibodies (MABs) were prepared using hybridoma technology. Five MABs were identified and their reactivity patterns were analyzed with both immunoperoxidase and ultrastructural immunogold labelling. Four MAB classes were identified by their immunostaining patterns. Optical and ultrastructural examination revealed that Class I MAB reacted specifically with basophilic granulocytes (granulocytes containing small granules). Class II and III MABs differed in the intensity of their indirect immunofluorescence and degree of immunoperoxidase labelling, but all recognized both basophilic and eosinophilic granulocytes. Epitopes recognized by Class IV MABs were not always present in basophilic granulocytes, whereas they were always expressed in the eosinophilic granulocytes (granulocytes containing large granules) and could constitute differentiation antigens which progressively form during the maturation process. Molecular weights of the proteins recognized by these MABs were determined by Western blotting. It is concluded that immunostaining can identify at least three hemocyte types in M. edulis.
The protozoan Bonamia ostreae (Ascetospora), a paasite of the flat oyster Ostrea edulis, was purified by differential and isopycnic centrifugations. Mice of the strain Balb/c were immunized with purified parasites and hybridomas were prepared by fusion of immunized mouse splenocytes with the mouse myeloma cell line P3-X63-Ag8-653. From the fusion, 12 clones were isolated and saved. The resulting antibodies were characterized with a solid phase radioimmunoassay (RIA). Antibodies from 7 clones reacted only with B. ostreae and not with normal host tissue. Although some of the antibiodies identified. One of these antibodies (20B2-1B12) has an apparent association constant of ca 3 X 10(8) M-1 and bound to a number of different sites on the parasite. These monoclonal antibodies should prove to be of great value as diagnostic and research tools.
Three monoclonal antibodies, designated RM-1, TRPM-1, and TRPM-2, were raised against rat peritoneal macrophages. By the immunoperoxidase method, antigens recognized by these antibodies were distributed throughout most tissue and free macrophages examined, including those of splenic red pulp, lymphatic sinus, connective tissue, and peritoneal cavity, as well as Kupffer cells of liver and alveolar macrophages. The numbers of positive cells were different for each antibody. RM-1 and TRPM-1 were also reactive with interdigitating cells (IDCs) in the thymus-dependent area and with Langerhans cells in the skin, whereas TRPM-2 failed to demonstrate IDCs in thymic medulla and Langerhans cells. The reactions of each antibody were observed by immunoelectron microscopy in the different ultrastructural compartments of the cells. RM-1 recognized a cell surface antigen; reaction products for TRPM-1 were found on a part of the cell membrane and in the cytoplasmic vacuoles; and those of TRPM-2 were present along the nuclear envelope and intracytoplasmic vacuoles. These antibodies seem to be useful not only for the detection of macrophages in tissue sections but also for investigation of macrophage heterogeneity in different tissues.
A new apicomplexan species, Perkinsus atlanticus, is described from gill filaments of the clam Ruditapes decussatus (Bivalvia) from Portugal, where it causes great mortality. The zoospores differ from those of other species of Perkinsus in size and shape, dimensions, insertion of the 2 flagella, and in the identity of the host. On the other hand, the life cycle stages showed some ultrastructural differences compared with Perkinsus marinus, the only species previously studied in detail. When the clams were parasitized heavily, ultrastructurally similar life cycle stages were found in foot and mantle tissues.
Examination of Mercenaria mercenaria hemocyte preparations on glass cover slips showed that adherent, macrophage-like cells tended to form multinucleated gi ant cells morphologically similar to classical Langhans cells and foreign body in flammatory giant cells, as seen in vertebrates. The frequency ofgiant cell formation varied from clam to clam, possibly reflecting each animal's physiological state, as influenced by pollutant exposure, other environmental stressors and disease. Treat ment of the hemocytes with concanavalin A promoted hemocyte aggregation and increased the rate ofgiant cell formation. Attempts to increase polykaryon formation by in vivo or in vitro phagocytic stimulation ofthe hemocytes were generally unsuc cessful.
Automated techniques for counting molluscan hemocytes and measuring certain of their characteristics are becoming more common, but there has not been a concerted attempt to compare results with more traditional microscopy. We compared electrical resistance (Coulter counter) and light-scatter (flow-cytometry) measurements of eastern oyster, Crassostrea virginica, hemocytes with those from phase-contrast light microscopy. Our findings showed that the clarity of results from the two automated measurements was inversely related to the complexity, in type and size, of the cell population being analyzed. Microscopy produced the most detailed information, but flow-cytometric measurements, even when relying on light scatter alone, clearly surpassed the discriminatory ability of the Coulter counter. Microscopic measurements documented considerable size overlap among morphologically different types of hemocytes. Because it measures size only, the Coulter counter is relatively insensitive to these differences. The flow cytometer consistently described three subpopulations: two granular and one agranular. Because of the wide range and overlap in hemocyte size and intracellular contents (the two criteria measured by light scatter) within individuals, however, we found only a loose correlation between subpopulation counts from microscopy and from flow cytometry. By combining light scatter with the flow cytometer's ability to detect specific fluorescent dyes and its capacity to physically sort cells according to defined parameters, however, we expect to greatly increase its usefulness for studying molluscan hemocytes.
The hematological parameters of Mercenaria mercenaria of similar size from two geographical areas as well as the morphology and behavior of their leucocytes were studied. On the basis of qualitative and quantitative characteristics, three types of leucocytes, designated as granulocytes, fibrocytes, and hyalinocytes, can be distinguished in both living and stained preparations.A correlation matrix computed between all parameters considered has revealed an insignificant correlation between the dimensions of whole animal and the differential count, packed cell volume, and total cell count as well as an insignificant correlation between the hematological parameters themselves. The only exception is that there is a positive correlation between the packed cell volume and the total cell count.
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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.
Circulating leucocytes of the mussel, Mytilus edulis, were studied by electron microscopy. Based on morphological criteria, the leucocytes were classified as agranular and granular leucocytes, dependent upon the presence or absence of specific granules in their cytoplasm. Furthermore, the existing literature is being critically revised, and it is suggested that agranular and granular leucocytes might belong to the same cell line.
The hemolymph cells of Mercenaria mercenaria were studied with the transmission electron microscope. Three morphological types of cells, granulocytes, hyalinocytes, and fibrocytes, are distinguishable and their fine structural characteristics are described. However, as a result of analyzing the fine structural features of the so-called fibrocytes of M. mercenaria, i.e., the inclusion of large aggregates of glycogen granules in their cytoplasm and the occurrence of primary phagosomes enclosing partially degraded exogenous material and digestive lamellae, it is suggested that fibrocytes are actually granulocytes which are at the terminal phase of their physiologic cycle relative to the degradation of phagocytized nonself materials. The cytoplasmic granules of M. mercenaria granulocytes are structurally different from those of Crassostrea virginica in that they are delimited by a unit membrane, rather than by a complex wall, and include a homogenously electron-dense material. Lipidlike droplets are reported from both granulocytes and hyalinocytes of M. mercenaria for the first time.
Glutaraldehyde-fixed hemocytes of Crassostreu t,irginiccr were subjected to differential centrifugation
on a 5, 10, 15, and 25% discontinuous sucrose gradient. Five subpopulations of cells were
separated by this technique. Subpopulation 1 coincides with the small granulocytes, subpopulation
2 is comprised of hyalinocytes, subpopulation 3 of medium-sized granulocytes, subpopulation
4 of large granulocytes, and subpopulation 5 of a mixture of very large granulocytes
and aggregates of small cells. By using several plant and animal lectins, it was ascertained
that cells of subpopulations 1, 3, and 4 were agglutinated with Con A and extracts of the
albumin glands of Helix pomutirr and Cepcrea nemoralis while those of subpopulation 2
were agglutinated by the same three lectins as well as wheat germ agglutinin. By applying
the Con A-peroxidase cytochemical technique, it was determined that approximately 20%
of the granulocytes of subpopulations 1 and 3 do not possess Con A-binding sites and only 18%
of the large cells comprising subpopulation 5 possess such sites. These results suggest that the
subpopulations of C. virginica granulocytes distinguishable by their dimensions and densities may
be further subdivided by differences in specific surface binding sites.
The hemocytes of the hard clam M. mercenaria were of three types: an agranulocyte, a small, and a large granulocyte. The agranulocyte, with only a thin periphery of cytoplasm surrounding the nucleus, had no visible cytoplasmic granules in living preparations but did exhibit a few centers of nonspecific esterase activity. This cell type represented 2% of the hemocyte population. The small granulocyte possessed four distinct granule types and comprised 61% of the total cell population. Large granulocytes accounted fro 37% of all hemocytes. While they contained the same four granule types identified in the small granulocyte, only one-third the total number were present. The nucleus of all three hemocyte types appeared morphologically similar. The four types of granules observed were a blunt, dot-like, a refractile and a filamentous granule. Blunt granules were identified as mitochondria, based on their ability to reduce Janus Green B to diethyl safranin, the presence of NADH dehydrogenase activity and boundary staining with Sudan black B. Dot-like granules were identified as lysosomes on the basis of neutral red staining, localization of acid phosphatase and nonspecific esterase activity and staining with Sudan black B. Refractile granules were demonstrated to be membrane-bound, lipid-filled structures that reacted positively with Sudan black B and Oil red O, respectively; these granules act as lipid storage centers. Nuclear similarity of the three cell types suggest that these cells might represent different stages of maturity, rather than three distinct cell lines. This was also indicated by the similar yet graded cytochemical reactions and the varying degree of motility and phagocytic activity demonstrated by hemocyte types.
The present study describes the immunomagnetic isolation of human natural killer (NK) and lymphokine activated killer (LAK) cells. Antibodies against CD56 and sheep anti-mouse IgG-coated magnetic monodisperse particles (Dynabeads M-450) were used for the positive isolation of CD56+ cells from unstimulated mononuclear cells (PBMC). A highly enriched population of CD56+ cells (less than or equal to 3% contaminating cells) was obtained with this method. The cellular yield of CD56+ cells was high (5.3% of the unseparated PBMC). The CD56+ cells remained unactivated after separation and preserved their functional characteristics, as measured by cytotoxic activity against the NK sensitive K562 cells. Incubating the CD56+ cells with IL-2 resulted in high LAK activity, as measured by cytotoxic activity against Daudi cells. Large numbers of functionally active CD56+ cells were obtained from IL-2 stimulated lymphocytes using anti-CD56 coated Dynabeads 450. A further enrichment of effector cells with LAK activity was accomplished by depleting the CD56+ cells for T-cells by anti-CD3 coated Dynabeads M450. The immunomagnetic isolation technique described was easy to perform, did not require expensive equipment and yielded NK and LAK cells of satisfactory purity.
We have developed a standardized procedure for the isolation of monocytes from peripheral blood by negative selection using magnetic polymer particles coated with monoclonal antibodies against T and B lymphocytes. The average purity of the monocyte suspension was 85%, and monocyte recovery was 72% from Ficoll-Hypaque gradient separated mononuclear cells and 32% from whole blood. In a lucigenin enhanced chemiluminescence assay there was no significant difference between cells separated immunomagnetically and those separated on a gradient. Nor did electron microscopy show any significant difference in morphology between such monocytes. Negative selection using magnetic polymer particles is an efficient method for the separation of monocytes with intact morphology and function as measured by chemiluminescence.
Guinea-pig bone marrow megakaryocytes were isolated using an antibody to platelet glycoprotein Ib and a second antibody conjugated to magnetic beads. The procedure yielded an average of 644,800 megakaryocytes from two guinea-pigs with an average viability of 83%. All of the platelet glycoprotein Ib positive cells also expressed the platelet glycoprotein IIb-IIIa complex. The size and ploidy of megakaryocytes isolated by this technique were analysed in the presence of 10 ng/ml of interleukin-6 (IL-6). Without IL-6 megakaryocyte size increased significantly after 24 h, but an even larger increase in size occurred in the presence of IL-6. The modal ploidy class was 16N with an average of 19% 2N, 2.6% 4N, 16.4% 8N, 50.8% 16N and 11.1% 32N cells as determined by flow cytometry. Measurements made by microspectrophotometry were in close agreement. After 24 h incubation there was a significant rise in the percentage of 2N and 32N cells. The ploidy distribution after 24 h with IL-6 was the same as the control. Megakaryocytes cultured in the absence of serum on collagen gels did not form pseudopods and fragment, as occurs with serum (Leven et al, 1987). Addition of IL-6 to the serum-free cultures caused megakaryocytes to form extensive proplatelet extensions. We conclude that large numbers of pure guinea-pig bone marrow megakaryocytes can be isolated by immunomagnetic bead selection, including low ploidy immature megakaryocytes. Spontaneous maturation occurred as evidenced by the increase in megakaryocyte size and ploidy. IL-6 altered megakaryocyte size and morphology but not ploidy, indicating that these different characteristics of megakaryocytes may be regulated separately.
The ultrastructural localization of a range of hydrolytic enzymes has been investigated in the granular haemocytes of the marine mussel Mytilus edulis. Arylsulphatase activity and immunocytochemical localization of beta-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 number and types of hemocytes in four size groups of the clam species Sunetta scripta and Villorita cyprinoides var. cochinensis, and leukocytosis in the 38- to 40-mm-size-group clams of both species subsequent to challenge with Vibrio alginolyticus at a concentration of 1 x 10(8) cells/0.02 ml of sterile 2% saline was investigated. In S. scripta, the mean total hemocyte count in the 42- to 44-mm size group was significantly lower than that of the three other size groups but there was no significant variation in total cell counts in the four size groups of V. cyprinoides var. cochinensis. Only two types of hemocytes, granulocytes and agranulocytes, occur and the percentage of agranulocytes was roughly half of that of granulocytes. The data on the effects of sham injection and Vibrio injection suggest that there is significant leukocytosis early in both clam species as a result of sham injection; the bacterial challenge produces significant leukocytosis in V. cyprinoides var. cochinensis both early (6 and 12 hr) and later (48,96 and 120 hr), but only at 48 hr in S. scripta; and in both clam species there is significant increase in total cell counts in Vibrio-injected ones than in untampered controls at various time intervals.
A protocol is described to separate several subpopulations of hemocytes in a unique medium which avoids cell aggregation and retains cell-viability. Isopycnic centrifugation in Percoll followed by counterflow centrifugal elutriation provides large quantities of separated granulocyte and hyalinocyte subpopulations.
Digestive gland tissues from the gastropods Helisoma duryi normale (strain HI-3), Helisoma trivolvis, Tarebia granifera, and Melania newcombi were transplanted into the cephalopedal sinuses of H. duryi normale (strain HI-2), and their fates were traced by examining histological sections fixed at 24, 48, 96, 192, and 384 hr post-transplantation. It was apparent that the recipients were capable of differentiating between allografts and xenografts since host reactions directed at the latter were more rapid and generally more severe. The rapid dissociation of the acinar cells of transplanted H. trivolvis and T. granifera suggested that some yet undetermined factor of host origin may be responsible for the lysis of these xenogenic tissues within 24 hr. Cellular capsules surrounding the dissociated cells of H. trivolvis and T. granifera transplants were in the form of hypertrophic fibroblasts and epithelioid cells. Evidences indicated that the latter differentiated from the former which, in turn, had differentiated from wandering leukocytes. Both H. trivolvis and T. granifera cells were encapsulated by the 48th hour. Eventually, the cellular remnants of these were removed by leukocytic phagocytosis. In the case of M. newcombi xenografts, the primary type of host reaction was in the form of myofibrous encapsulation, which was initiated by the 24th hour. Multinucleate macrophages were also associated with these xenografts at the same time. The transplants, however, were not dissociated during the span of the experiment. In the case of H. duryi normale allografts, epithelioid cell encapsulation occurred by the 192nd hour but the acini were still not dissociated by the 394th hour.
The morphology and known functions of leucocytes have been tabulated and compared for most invertebrate phyla. The leucocytes of the Manila clam are similar in form to molluscan leucocytes described by other investigators. Two general types were seen. One was a hyaline leucocyte ("leucocyte") and the other a granular leucocyte ("granulocyte"). Cells in these categories showed extreme variations in size and staining characteristics. A decrease in nuclear size often was correlated with an increase in nuclear heterochromasia and eccentricity and cytoplasmic granularity. The average cell count for all circulating leucocytes was 1650 ± 180 per mm3.
Five monoclonal antibodies (mABs) against surface antigens on circulating, glass-adherent hemocytes of the snail, Biomphalaria glabrata, were produced by somatic cell hybridization methods. Two mABs (IID2.6-Bg and IID4.8-Bg) are pan-hemocytic, reacting uniformly with epitopes shared by all adherent hemocytes. Determinants recognized by these mABs also are present in soluble form and appear to be associated with a hemoglobin-depleted ultracentrifuged fraction of snail hemolymph. Hybridoma-derived mABs IIC6.8-Bg and VB10.3-Bg recognize hemocyte surface epitopes expressed by only 50-60% of the adherent cell population. These mABs also are reactive with soluble hemolymph antigens but apparently recognize determinants which are different from the IID2.6-Bg and IID4.8-Bg reactive sites. Another antigenically distinct hemocyte subpopulation is recognized by mAB IID7.1-Bg. Epitopes that are reactive with this mAB differ from the previously described determinants by their asymmetrical distribution on the surface of positive cells and the absence of soluble antigenic components in hemolymph. Furthermore, unlike the other mABs, the prevalence of hemocytes staining with IID7.1-Bg antibodies differed between two strains of B. glabrata. Results of this study clearly demonstrate that circulating B. glabrata hemocytes, consisting of a single, predominant population of adherent cells, is composed of several distinct antigenic subpopulations based on the specific binding of anti-hemocyte mAB probes. Our successful application of hybridoma techniques to the study of molluscan hemocyte surface antigens underscores further the great potential usefulness of this method in analysing the molecular basis of hemocyte reactivity.
Normal neutrophil function requires the cooperation and coordination of many cellular activities within the neutrophil and cells of the vascular system. The free flowing neutrophil begins the process by "rolling" along the vessel wall. This first step in migration is mediated by selectins on the surface of neutrophils, platelets, and endothelial cells. Subsequent firm adhesion requires the collaboration of neutrophil integrins and membrane-expressed cellular adhesion molecules. The activated neutrophil also changes shape from a sphere to an elongated motile cell by the assembly of actin filaments. At the inflammatory focus the neutrophil may encounter material to be ingested and killed. The ingestion process is mediated by the same receptors that stimulate firm adherence to the vessel wall. The ingestion process stimulates the formation of active NADPH oxidase, which is critical for the formation of superoxide and subsequent components of the killing mechanism. The process of normal neutrophil function is complex and multifaceted, but luckily, defects in this system are extremely rare. Patients with persistent and recurrent infections should be carefully evaluated for underlying disease before primary neutrophil dysfunction is pursued.
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