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Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to evaluate phagocytosis, coupled with cytology and Electron Microscopy (TEM), to define animal immunocompetence following infection by P. nobilis Picornavirus (PnPV). The study was performed on 27 animals in July 2021 and May 2022 on two natural population from the Ebro Delta (Catalonia, Spain) and animals maintained in captivity at facilities in Valencia and Murcia Aquarium. Hemolymph was collected in the field and in captivity as a non-destructive sampling method. Based on dimension and internal complexity, flow cytometry identified three haemocyte types, distinguished in granulocytes, hyalinocytes and a third type, biggest in size and with high internal complexity and granularity. Those cells corresponded at ultrastructure to hemocytes with advanced phases of PnPV infection and related to cytopathic effect of the replicating virus displaying numerous Double Membrane Vesicles (DMVs) and cells corpse fusion. The results showed that pen shell in captivity had significantly lower Total Hemocyte Count (THC) compared with natural population of Alfacs Bay (mean number of 7-9 x 10 4 vs 2-5 x 10 5  cells/mL, respectively). FACS (Fluorescence-activated cell sorting) based phagocytosis analysis demonstrate that animals in captivity at IMEDMAR-UCV and Murcia Aquarium, had scarce or absent ability to phagocyte the two stimuli (Staphylococcus aureus and Zymosan A) (10,2 % ± 1,7 of positives) if compared with the natural population in Alfacs Bay (28,5 % ± 5,6 of positive). Ultrastructure images showed that PnPV itself can lead to an alteration of the hemocyte cytoskeleton, impairing the capabilities to perform an active phagocytosis and an efficient phagolysosome fusion., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila , along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

In this work we studied the ability of polystyrene (PS) nanoplastics (NPs) and microplastics (MPs) to transfer benzo(a)pyrene (BaP) to mussel hemocytes and to produce toxic effects in vitro. For this, intracellular fate and toxicity of PS NPs (0.05 μm) and MPs (0.5 and 4.5 μm) alone or with BaP and of BaP alone were assessed. Particles of 0.05 and 0.5 µm largely aggregated in the exposure medium whereas presence of BaP reduced particle aggregation. Cells internalized PS NPs and MPs alone or with BaP and these were found inside and outside lysosomes, depending on their size. PS particles alone or with BaP were cytotoxic to hemocytes only at the highest concentrations tested. The same was true for most sublethal endpoints except for increased phagocytic activity provoked by NPs and 0.5 μm MPs at lower concentrations. Plastic particles appeared to be the main drivers for reduced plasma membrane integrity and increased phagocytic and lysosomal activities whereas BaP appeared to contribute more to reduced cell viability and phagocytosis and increased ROS production and genotoxicity. Overall, PS NPs and MPs can act as carriers of BaP to mussel hemocytes, rising concerns about risks plastics associated to pollutants may pose to aquatic organisms., (© 2021. The Author(s).)

Oncomelania hupensis is the only obligatory intermediate host of Schistosoma japonicum, the pathogen of zoonosis schistosomiasis. Haemocytes play a critical role in the cellular immune defence of O. hupensis against S. japonicum challenge. Here, the morphology and classification of haemocytes of O. hupensis were investigated by Giemsa staining and light microscopy, combining with the scanning and transmission electron microscopy and flow cytometry. Granulocytes and hyalinocytes were confirmed as two main types of haemocytes, account for ~ 10% and ~ 90% of all haemocytes, with size varying in 4.3-10.9 μm and 0.4-30.8 μm, respectively. Subpopulations can be identified further by granule feature, shape, size, and surface and inner structure of cells. The heterogeneity in morphology implied varied developmental process and function of haemocyte subpopulations. After the S. japonicum challenge, haemocytes of O. hupensis respond to S. japonicum invasion immediately. The dynamic change of haemocyte subpopulations indicates that the small hyalinocyte could differentiate into a larger one or granulocyte after S. japonicum challenge, and the granulocytes and larger hyalinocytes play leading roles in early defence reaction, but in different ways. Phagocytosis and apoptosis of haemocytes in O. hupensis were proved to be related to immune defence against S. japonicum, with the combined effect of granulocytes and larger hyalinocytes. However, the main pathway of each subpopulation to take effect in different periods need further investigation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Herbicides have become the most commonly applied agrochemicals in agroecosystems. Thus, basic knowledge of their physiological effects on insects is needed, especially for understanding their impact on beneficial insect species. In this study, we evaluated the effect of a pendimethalin-based herbicide (PND) on the cellular immune response of the carabid beetle Harpalus (Pseudoophonus) rufipes (De Geer 1774) (Coleoptera, Carabidae), acting as biocontrol agent in agroecosystems. Total and differential haemocyte counts and phagocytosis assay, performed by injecting in vivo carboxylate-modified polystyrene latex beads, were measured in beetles exposed to a recommended field dose (4L per ha) of PND to evaluate the exposure effects over the time. The pattern of haemocyte subpopulations and the decrease of the phagocytic index after the exposure to PND suggested a lowering of P. rufipes ability to face an infection performing a cell-mediated response. PND was also found to cause cytotoxic effects on the haemocyte ultrastructure. Ultrastructural alterations such as irregular shape, large vacuolization of the cytoplasm, and condensation of marginated chromatin were recorded from 2d of exposure. The loss of RER, Golgi apparatus, mitochondria integrity and the swelling of the outer nuclear membrane found in some haemocytes suggested an interference of PND with the membrane permeability. Results indicated that the exposure to PND impairs the distribution, morphology and physiological functions of haemocytes causing a decrease of P. rufipes immunocompetence. Moreover, the sensitivity to herbicide exposure makes this species a suitable model and a useful bioindicator for monitoring exposure effects on non-target species. This study provides useful information to protect and preserve biodiversity of insects in agroecosystems., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The terrestrial crustacean Porcellio scaber (Crustacea: Isopoda) is an established invertebrate model in environmental research. Preceding research using isopods did not widely use immune markers. In order to advance their use in research, knowledge of the reference values in control animals as well as variations during infections is of importance. This study presents, for the first time, the morphology, and ultrastructure of the three main haemocyte types of Porcellio scaber as semigranulocytes (SGCs), granulocytes (GCs), and hyalinocytes (HCs), with the latter having two subtypes, using various light and electron microscopy approaches. The modulation of selected immune cellular and humoral parameters of P. scaber in symptomatic phases of Rhabdochlamydia porcellionis and Iridovirus IIV-31 infections is presented. A clear difference in the immune responses of bacterial and viral infections was shown. Remarkable changes in total haemocyte count (THC) values and the proportions of three different haemocyte types were found in animals with a viral infection, which were not as significant in bacterially infected animals. Modified NO levels and SOD activity were more pronounced in cases of bacterial infection. Knowledge of the morphological and ultrastructural features of distinct haemocyte types, understanding the baseline values of immune parameters in control animals without evident symptoms of infection, and the influence that infections can have on these parameters can serve as a basis for the further use of P. scaber immune markers in environmental research., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi-granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule-containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid-Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell-mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase-mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme-agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis., (© 2020 Wiley Periodicals, Inc.)

Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, Crassostrea gigas . However, the autophagy pathway has never been explored in the hemocytes of C. gigas , which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of C. gigas and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level. Abbreviations : MAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NH 4 Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy.

Vibrio coralliilyticus has emerged as a coral pathogen of concern throughout the Indo-Pacific reef. The interest towards understanding its ecology and pathogenic potential has increased since V. coralliilyticus was shown to be strongly virulent also for other species; in particular, it represents a serious threat for bivalve aquaculture, being one of the most important emerging pathogen responsible for oyster larval mortalities worldwide. V. coralliilyticus has a tightly regulated temperature-dependent virulence and it has been related to mass mortalities events of benthic invertebrates also in the temperate northwestern Mediterranean Sea. However, no data are available on the effects of V. coralliilyticus in the mussel Mytilus galloprovincialis, the most abundant aquacultured species in this area. In this work, responses of M. galloprovincialis to challenge with V. coralliilyticus (ATCC BAA-450) were investigated. In vitro, short term responses of mussel hemocytes were evaluated in terms of lysosomal membrane stability, bactericidal activity, lysozyme release, ROS and NO production, and ultrastructural changes, evaluated by TEM. In vivo, hemolymph parameters were measured in mussels challenged with V. coralliilyticus at 24h p.i. Moreover, the effects of V. coralliilyticus on mussel early embryo development (at 48 hpf) were evaluated. The results show that both in vitro and in vivo, mussels were unable to activate immune response towards V. coralliilyticus, and that challenge mainly induced lysosomal stress in the hemocytes. Moreover, V. coralliilyticus showed a strong and concentration-dependent embryotoxicity. Overall, the results indicate that, although M. galloprovincialis is considered a resistant species to vibrio infections, the emerging pathogen V. coralliilyticus can represent a potential threat to mussel aquaculture., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Crustacean hemocytes are known to remove invading pathogens by phagocytosis. In this study, we investigated how the semigranular cells (SGCs) and granular cells (GCs) of crayfish Cherax quadricarinatus participated in this process. By injecting the animals with excessive amounts of fluorescent microspheres (FMs), we showed that only a small portion of the circulating hemocytes were phagocytic cells, and they took up FMs in a size-dependent manner. The 0.2 μm FMs were internalized almost entirely by SGCs, while GCs and SGCs both contributed to the uptake of 2 μm FMs. Further analysis of the hemocytes from the animals injected with a mixture of FMs suggested that there were a subpopulation of SGCs specifically ingesting 0.2 μm FMs. The size-dependent manner was also applied to biological particles. Escherichia coli was internalized by both SGCs and GCs, whereas white spot syndrome virus (WSSV) was mostly ingested by SGCs. However, the bacterial cells were rapidly taken and cleared from the circulation by the hemocytes, while the WSSV virions were gradually internalized and remained in the cells for a relatively longer period of time. These findings provide basic information of the phagocytic hemocytes of crayfish and how they respond to different foreign particles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The use of manufactured nanoparticles (NPs) is spreading rapidly across technology and medicine fields, posing concerns about their consequence on ecosystems and human health. The present study aims to assess the biological responses triggered by iron oxide NPs (IONPs) and iron oxide NPs incorporated into zeolite (IONPZ) in relation to oxidative stress on the land snail Helix aspersa in order to investigate its use as a biomarker for terrestrial environments. Morphology and structure of both NPs were characterized. Snail food was supplemented with a range of concentrations of IONPs and IONPZ and values of the hemocyte lysosomal membranes' destabilization by 50% were estimated by the neutral red retention (NRRT50) assay. Subsequently, snails were fed with NPs concentrations equal to half of the NRRT50 values, 0.05 mg L -1 for IONPs and 1 mg L -1 for IONPZ, for 1, 5, 10 and 20 days. Both effectors induced oxidative stress in snails' hemocytes compared to untreated animals. The latter was detected by NRRT changes, reactive oxygen species (ROS) production, lipid peroxidation estimation, DNA integrity loss, measurement of protein carbonyl content by an enzyme-linked immunoabsorbent assay (ELISA), determination of ubiquitin conjugates and cleaved caspases conjugates levels. The results showed that the simultaneous use of the parameters tested could constitute possible reliable biomarkers for the evaluation of NPs toxicity. However, more research is required in order to enlighten the disposal and toxic impact of iron oxide NPs on the environment to ensure their safe use in the future., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The aim of this study was to evaluate the metabolic condition of Steatoda grossa (Theridiidae) spider, from their hemocytes, after a short-term (four-week) exposure to cadmium and copper in sublethal doses by administering them into the body of the preys. The ultrastructure of the dominant types of hemocytes, such as granulocytes, plasmatocytes and prohemocytes, was evaluated using transmission electron microscope (TEM). Quantitative evaluation of apoptotic and necrotic cells, as well as the ones with depolarized mitochondria in hemolymph, was performed using flow cytometry, while ATP concentration and ADP/ATP ratio in hemocytes were measured by luminescent methods. Cadmium, unlike copper, demonstrated proapoptotic and pronecrotic activity. Low ATP levels and high ADP/ATP ratio in hemocytes indicate a disturbance in the energy metabolism of cells and may account for their qualitative and quantitative degenerative changes. The intensification of death processes in hemocytes after an exposure to cadmium-contaminated food may impair the ability of these cells to fight infectious diseases. Copper at the applied dosage was safe for the spiders without causing visible changes in the hemocyte ultrastructure and in the level of analyzed cell death indices., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5-10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Diplosoma listerianum is a colonial aplousobranch ascidian of the family Didemnidae that is native to the northeast Atlantic and exhibits a cosmopolitan distribution in temperate waters. It lacks a shared colonial circulation crossing the tunic, and the zooids are connected only by the common tunic. In the present study, the haemocytes of this ascidian were analysed via light and electron microscopy. Their phagocytic and enzymatic activities, staining and immunostaining properties, and lectin affinity were examined with various classical methods reconsidered and modified for small marine invertebrates. Eight morphotypes were identified in reference to corresponding cell types described in other ascidians: undifferentiated cells (haemoblasts), storage cells for nitrogenous catabolites (nephrocytes) and immunocytes. The immunocytes are involved in immune responses, acting as (1) phagocytes, rich in hydrolases and involved in the clearance of both foreign particles and effete cells (hyaline amoebocytes and macrophage-like cells); (2) cytotoxic cells, able to degranulate and induce cytotoxicity through the release of the enzyme phenoloxidase after an immune stimulus (granular amoebocytes and morula cells); and (3) basophilic cells with an affinity for ConA and NPA that contain heparin and histamine and that show sensitivity to the compound 48/80, promoting their degranulation (mast cell-like granulocytes). In addition, a particular cell type showing exceptional development of the Golgi apparatus and large vacuoles containing a filamentous material has been recognised (spherule cell), for which a role in tunic repair and fibrogenesis has been hypothesised., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Exposure of the toxin-producing dinoflagellate Alexandrium catenella (A. catenella) was previously demonstrated to cause apoptosis of hemocytes in the oyster species Crassostrea gigas. In this work, a coumarin-labeled saxitoxin appeared to spread throughout the cytoplasm of the hemocytes. PSTs, including saxitoxin, were also shown to be directly responsible for inducing apoptosis in hemocytes, a process dependent on caspase activation and independent of reactive oxygen species (ROS) production. A series of in vitro labelling and microscopy experiments revealed that STX and analogs there of induced nuclear condensation, phosphatidylserine exposure, membrane permeability, and DNA fragmentation of hemocytes. Unlike in vertebrates, gonyautoxin-5 (GTX5), which is present in high concentrations in A. catenella, was found to be more toxic than saxitoxin (STX) to oyster immune cells. Altogether, results show that PSTs produced by toxic dinoflagellates enter the cytoplasm and induce apoptosis of oyster immune cells through a caspase-dependent pathway. Because of the central role of hemocytes in mollusc immune defense, PST-induced death of hemocytes could negatively affect resistance of bivalve molluscs to microbial infection., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

Toxicity of Cd was tested with the hemocytes of the freshwater crab, Sinopotamon henanense, which were exposed to concentrations of 0, 0.725, 1.450, and 2.900mgL -1 Cd for 7, 14 and 21 d. We investigated the effects of Cd on the total antioxidant capacity (TAC), and oxidative damage of biomarkers, such as malondialdehyde (MDA), protein carbonyl derivates (PCO), and DNA-protein crosslink (DPC). Transmission electron microscopy (TEM) was applied to assess ultrastructural changes of hemocytes. The mRNA expression levels of prophenoloxidase (proPO), lysozyme (LSZ), metallothionein (MT), and the activity of phenoloxidase (PO) were also determined. Our results showed that TAC was inhibited by Cd, resulting in an increase of MDA contents, PCO contents, and DPC levels in hemocytes, respectively. Ultrastructural observations revealed that chromatin condensation, nucleus deformation, mitochondrial dilation, rough endoplasmatic reticulum (rER) degranulation and secondary or tertiary lysosomes were observed in hemocytes of crabs exposed to Cd. Meanwhile, the expression levels of proPO were down-regulated, while the activity of PO was up-regulated in hemocytes. The expression levels of LSZ and MT were up-regulated to some extent. Our findings suggest these parameters could be used as biomarkers in the monitoring of heavy metal pollution and quantitative risk assessments of pollutant exposure., (Copyright © 2016. Published by Elsevier Inc.)

Individuals of colonial animals (e.g. zooids) are in continuous turnover. In ascidians colonial or solitary species have evolved by convergence multiple times. Colonial Botryllus and Botrylloides are well-studied genera that exhibit colony-wide developmental mechanisms that regulate synchronous and orchestrated cycles of budding and turnover of zooids. The origins of modular developmental mechanisms that facilitated the evolution of coloniality in this group remain unclear. To reconstruct ancestral states of coloniality we studied Symplegma brakenhielmi, a sister taxon of the botryllids. S. brakenhielmi zooids are embedded in a common tunic and present a similar vascular system as the botrylloides, however development and turnover of zooids occurs asynchronously and in a more independent manner. We generated a table of common stages of budding in Symplegma and Botryllus for comparative studies of asexual development. We tested dependent processes of budding among individuals of the colony by systemic bud or zooid removals. Although our results showed a higher degree of independence in bud development in S. brakenhielmi, we found a subtle colony-wide regulatory mechanism of modular development, i.e. new buds expedited development after the removal of all buds in the colony. Next, we characterized external morphology, ultrastructure, and abundance of circulatory blood cells in the vascular system of S. brakenhielmi. Macrophage-like cells (MLCs) are involved in zooid resorption and turnover. Proportions of MLCs in the blood of S. brakenhielmi corresponded to the peak of occurrence of this cell type during the budding cycle of B. schlosseri. We found several new blood cell types in S. brakenhielmi, including two cell types that resemble circulatory progenitor stem cells of other botryllid colonial ascidians. These cells showed features of undifferentiated cells and expressed mitotic marker Phospho-histone H3. Comparative studies of S. brakenhielmi and B. schlosseri allow us to discuss possible changes in the regulation of modular development (i.e. regulation of life and death in the colony), and a possible contribution of circulatory blood cells in budding processes. We propose that the higher degree of developmental independence in S. brakenhielmi budding is a result of its ancestral solitary mode of development., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The present study was performed to evaluate the effects of CO 2 - or HCl-induced seawater acidification (pH 7.7 or 7.1; control: pH 8.1) on haemocytes of Mytilus edulis, and the changes in the structure and immune function were investigated during a 21-day experiment. The results demonstrated that seawater acidification had little effect on the cellular mortality and granulocyte proportion but damaged the granulocyte ultrastructure. Phagocytosis of haemocytes was also significantly inhibited in a clearly concentration-dependent manner, demonstrating that the immune function was affected. Moreover, ROS production was significantly induced in both CO 2 and HCl treatments, and four antioxidant components, GSH, GST, GR and GPx, had active responses to the acidification stress. Comparatively, CO 2 had more severe destructive effects on haemocytes than HCl at the same pH level, indicating that CO 2 stressed cells in other ways beyond the increasing H + concentration. One possible explanation was that seawater acidification induced ROS overproduction, which damaged the ultrastructure of haemocytes and decreased phagocytosis., Competing Interests: The authors declare no competing financial interests.

Toxic metals, such as mercury, contribute substantially to anthropogenic pollution in many estuarine environments. Animals living in those environments, particularly invertebrate filter feeders like tunicates, can be used as bioindicators. In an attempt to identify cellular markers for revealing pollution, this study examined in vitro the effects of different concentrations of methyl mercury on Styela plicata hemocytes. The harvested hemocytes from S. plicata that were exposed to the metal had a significant mortality, cellular count and morphometric alterations. These findings provided evidence of MeHg immunotoxic effects on S. plicata, resulting in hemocyte death and morphological changes induced by cytoskeleton alterations. Thus, a morphometric cellular parameter, such as spreading ability, was used as a complementary method for differentiation between hemocytes treated with a marine solution (as a negative control) and hemocytes incubated with methylmercury and/or Sicilian seawater samples., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation., Results: Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs., Conclusions: To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

The internal defense system consists of soluble components of hemolymph and circulating cells known as hemocytes. The circulating hemocytes play a central role in innate immunity. This work aimed to study the hemocytes of both susceptible and resistant B. alexandrina snails exposed to S. mansoni infection using light and electron microscopes. Two tested groups were included in the study; 60 susceptible and 60 resistant B. alexandrina snails. Both tested groups were studied as regad the hemocyte count (before and after infection) and the morphological characteristics of both circulating and tissue hemocytes by light and electron microscopes. Before infection, .there was no significant difference between the two groups as regard the hemocyte count, however after infection, there is a significant decrease in the circulating hemocytes of the resistant group. Light microscopy revealed five morphological types of circulating cells of both susceptible and resistant snails. Regarding scannig electron microscopy, hemocytes of susceptible snails appeared rounded with smooth or slightly rough surface. However, that of the resistant snails appeared irregular in shaped with corrugated surface. Furthermore, Light microscopy and the transmission electron microscopy revealed signs of cell activation in the hemocytes of the resistant group. The circulating hemocytes consist of five cell types in both susceptible and resistant B. alexandarina and morphologies of these cells are quite similar, but with more signs of cell activations in the resistant group. More specific studies on the functional activities of the hemocytes and mechanisms that may affect or influence the susceptibility and/or non-susceptibility of molluscs to invade microorganisms is essential and how they can act in the immune response.

The Western Honeybee is a key pollinator for natural as well as agricultural ecosystems. In the last decade massive honeybee colony losses have been observed worldwide, the result of a complex syndrome triggered by multiple stress factors, with the RNA virus Deformed Wing Virus (DWV) and the mite Varroa destructor playing crucial roles. The mite supports replication of DWV to high titers, which exert an immunosuppressive action and correlate with the onset of the disease. The aim of this study was to investigate the effect of 1,3-1,6 β-glucan, a natural innate immune system modulator, on honeybee response to low-titer natural and high-titer experimental DWV infection. As the effects exerted by ß-glucans can be remarkably different, depending on the target organism and the dose administered, two parallel experiments were performed, where 1,3-1,6 ß-glucan at a concentration of 0.5% and 2% respectively, was added to the diet of three cohorts of newly emerged honeybees, which were sampled from a Varroa-free apiary and harboured a low endogenous DWV viral titer. Each cohort was subjected to one of the following experimental treatments: no injection, injection of a high-copy number DWV suspension into the haemocel (experimental DWV infection) or injection of PBS into the haemocoel (physical injury). Control bees fed a ß-glucan-free diet were subjected to the same treatments. Viral load, survival rate, haemocyte populations and phenoloxidase activity of each experimental group were measured and compared. The results indicated that oral administration of 0.5% ß-glucan to naturally infected honeybees was associated with a significantly decrease of the number of infected bees and viral load they carried, and with a significant increase of the survival rate, suggesting that this natural immune modulator molecule might contribute to increase honeybee resistance to viral infection., Competing Interests: The authors have declared that no competing interests exist.

The first part of the study was devoted to test the hypothesis according to which the hemolymph of Lymnaea stagnalis can be collected repeatedly - regardless the time-intervals - at an individual scale without impact on survival nor immunocapacity defined as the hemocyte density and viability. No significant effects on snail survival were observed when repeated hemolymph samplings were performed at frequencies ranging from 96 h up to 24 h. The frequency of hemolymph sampling had no significant effects on hemocyte density but the hemocyte viability was slightly increased for the 24 h frequency group. Hence, we recommend setting the frequency lower than 48 h after two consecutive samplings for further assessment of hemocyte density and viability. Furthermore, a slight "day" effect was observed on snail immunocapacity. These results support the idea that L. stagnalis is a promising gastropod model in environmental immunotoxicology. A time-course analysis of individual hemocytes parameters can be evaluated with a relative confidence in the non-detrimental effect of the sampling. Linear mixed-effect models allow taking the "day" effect into account and so the possible effect of an environmental factor (i.e. xenobiotic exposures) can be analyzed. Statistical inferences indicated that the inter-individual variability for these hemocyte endpoints were on the same order of magnitude than intra-individual variability. The second part of the study was devoted to provide greater insights into the structure/ultrastructure of hemocytes in L. stagnalis. Only one type of hemocyte has been observed. The hemocytes in their free-floating status showed ovoid or spherical shapes. Some hemocytes exerted filopodia and structures shaped like sailboats. Their ultrastructure showed signs of intense cellular activity. Two peculiar organelles were observed. One corresponds to a massive perinuclear structure of dense aspect. The other corresponds to a structure with fibrillary arrangements. These two structures deserve further investigation in order to understand their nature, function and importance in the snails' immunocompetence., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The in vitro model Ostrea edulis hemocyte - Bonamia ostreae is interesting to investigate host-parasite interactions at the cellular level. Indeed, this unicellular parasite infects the flat oyster Ostrea edulis and multiplies within hemocytes, the central effectors of oyster defenses. Apoptosis is a mechanism used by many organisms to eliminate infected cells. In order to study the potential involvement of this mechanism in the oyster response to B. ostreae, in vitro experiments were carried out by exposing hemocytes from the naturally susceptible oyster O. edulis and a resistant oyster species Crassostrea gigas to live and heat-inactivated parasites. Hemocyte apoptotic response was measured using a combination of flow cytometry and microscopy analyses. Whatever the host species was, the parasite was engulfed in hemocytes and induced an increase of apoptotic parameters including intracytoplasmic calcium concentration, mitochondrial membrane potential or phosphatidyl-serine externalization as well as ultrastructural modifications. However, the parasite appears more able to infect flat oyster than cupped oyster hemocytes and the apoptotic response was more important against live than dead parasites in the natural host than in C. gigas. Our results suggest that O. edulis specifically responds to B. ostreae by inducing apoptosis of hemocytes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The fan mussel Pinna nobilis (Linnaeus, 1758) is one of the biggest bivalves worldwide. Currently, no updated information is available in the literature concerning the morpho-functional aspects of haemocytes from this bivalve species. Consequently, in this study, we characterised P. nobilis haemocytes from both a morphological and functional point of view. The mean number of haemocytes was about 5 (×10(5)) cells mL haemolymph(-1), and the cell viability was about 92-100%. Two haemocyte types were distinguished under the light microscope: granulocytes (51.6%), with evident cytoplasmic granules, and hyalinocytes (48.4%), with a few granules. The granules of the granulocytes were mainly lysosomes, as indicated by the in vivo staining with Neutral Red. Haemocytes were further distinguished in basophils (83.75%), acidophils (14.75%) and neutrophils (1.5%). After adhesion to slides and fixation, the cell diameter was approximately 10 μm for granulocytes and 7 μm for hyalinocytes. The granulocytes and hyalinocytes were both positive to the Periodic Acid-Schiff reaction for carbohydrates. Only granulocytes were able to phagocytise yeast cells. The phagocytic index (6%) increased significantly up to twofold after preincubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. In addition, haemocytes produce superoxide anion and acid and alkaline phosphatases. Summarising, this preliminary study indicates that both the granulocytes and hyalinocytes circulate in the haemolymph of P. nobilis and that they are active immunocytes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

TiO2 nanoparticles (TiO2-NP) have been incorporated into a large range of materials for different applications in the last decades and are very likely to appear in wastewater and effluents, eventually reaching the aquatic environment. Therefore, the assessment of the biological impact of TiO2-NP on aquatic ecosystem is of a major concern. The mussels represent a target group for TiO2-NP toxicity, as they are filter feeders and are capable of bioaccumulating toxic compounds. Furthermore, the exotic organism Limnoperna fortunei, golden mussel, is a freshwater bivalve that has been used in biomonitoring environmental conditions. In this work, the TiO2-NP's ability to interact with haemocytes of golden mussel was assessed by transmission electron microscopy. The enzymatic and non-enzymatic antioxidant defenses were evaluated by superoxide dismutase (Sod) and catalase (Cat) activities and protein sulfhydryl content, which were measured after the golden mussel was exposed to TiO2-NP (1, 5, 10 and 50μgmL(-1)). Results demonstrate that TiO2-NP was internalized by cells, causing alterations in haemocytes membrane. Antioxidant activity of Sod and Cat decreased after 2h TiO2-NP exposure. After 4h exposure, the enzymatic antioxidant activity was restored. Notably, the protein sulfhydryl content decreased after 2h to all the TiO2-NP concentrations and no alterations were observed after 4h of TiO2-NP exposure. These results demonstrate the potential of golden mussel as sentinel organism to TiO2-NP exposure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Invertebrates rely on the efficient innate immune mechanisms against invaders, in which the continuous production of hemocytes (hematopoiesis) is indispensable. In the present study, the hematopoietic tissue (HPT) from Chinese mitten crab Eriocheir sinensis was identified and characterized. It was a thin and non-transparent sheet located at the dorsolateral side of the stomach, which was composed of a series of ovoid lobules. Each lobule was surrounded by connective tissue containing a large amount of spherical cells with big nucleus. In HPT, the cells were full of mitochondria and granules, and DNA replication was detected in some cells by EdU labeling technique. Cell proliferation was observed in HPT by transmission electron microscope (TEM). The distribution of two transcription factors, GATA1 and RUNX1, were examined by human GATA1 and RUNX1 antibodies, respectively. Three homologues of RUNX1 were detected in the HPT while no signal of RUNX1 was observed in hemocytes, and GATA1 was detected in both HPT and some hemocytes. The mRNA transcript of a novel hematopoiesis related cytokine EsAst was detected in hepatopancreas and hemocytes, but it was no detectable in HPT. The mRNA expression level of EsAst in hepatopancreas was 1.38-fold higher than that in hemocytes. Total hemocytes counts were related to the mRNA expression level of EsAst post Aeromonas hydrophila challenge. The results suggested that the stem cells in the hematopoietic tissue of Chinese mitten crab E. sinensis were regulated by transcriptional and humoral factors to generate hemocytes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Atlantic jackknife clam, Ensis directus, is currently being researched as a potential species for aquaculture operations in Maine. The goal of this study was to describe the hemocytes of this species for the first time and provide a morphological classification scheme. We viewed hemocytes under light microscopy (using Hemacolor, neutral red, and Pappenheim's stains) as well as transmission electron microscopy (TEM). The 2 main types of hemocytes found were granulocytes and hyalinocytes (agranular cells). The granulocytes were subdivided into large and small granulocytes while the hyalinocytes were subdivided into large and small hyalinocytes. The large hemocytes had both a larger diameter and smaller nucleus to cell diameter ratio than their smaller counterparts. A rare cell type, the vesicular cell, was also observed and it possessed many vesicles but few or no granules. Using TEM, granulocytes were found to contain both electron-lucent and electron-dense granules of various sizes. These numerous granules were the only structures that took up the neutral red stain. Hyalinocytes had few of these granules relative to granulocytes. Large hyalinocytes had both various organelles and large vesicles in their abundant cytoplasm while small hyalinocytes had little room for organelles in their scant cytoplasm. Total hemocyte counts averaged 1.96×10(6) cells mL(-1) while differential hemocyte counts averaged 11% for small hyalinocytes, 12% for large hyalinocytes, 59% for small granulocytes, and 18% for large granulocytes. The results of this study provide a starting point for future studies on E. directus immune function., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, light microscope, scanning and transmission electron microscope, hematoxylin-eosin and fluorescent staining, and mass spectrometry methods were employed to observe the calcium carbonate (CaCO3) crystal formation, hemocyte release and transportation, and hemocyte distribution at the shell regeneration area and to analyse the proteome of hemocytes in the pearl oyster, Pinctada fucata. The results indicated that intracellular CaCO3 crystals were observed in circulating hemocytes in P. fucata, implying that there was a suitable microenvironment for crystal formation in the hemocytes. This conclusion was further supported by the proteome analysis, in which various biomineralization-related proteins were detected. The crystal-bearing hemocytes, mainly granulocytes, may be released to extrapallial fluid (EPF) by the secretory cavities distributed on the outer surface of the mantle centre. These granulocytes in the EPF and between the regenerated shells were abundant and free. In the regenerated prismatic layer, the granulocytes were fused into each column and fragmented with the duration of shell maturation, suggesting the direct involvement of hemocytes in shell regeneration. Overall, this study provided evidence that hemocytes participated in CaCO3 crystal formation, transportation and shell regeneration in the pearl oyster. These results are helpful to further understand the exact mechanism of hemocyte-mediated biomineralization in shelled molluscs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Few studies have examined the cellular immune response of ticks, and further research on the characterization of the hemocytes of ticks is required, particularly on those of Rhipicephalus sanguineus (Latreille) because of the medical and veterinary importance of this tick. The aims of this study were to characterize the morphology and the ultrastructure of the different types of hemocytes of adult R. sanguineus and to determine the population abundance and the ultrastructural changes in the hemocytes of ticks infected with Leishmania infantum. The hemocytes were characterized through light and transmission electron microscopy. Within the variability of circulating cells in the hemolymph of adult R. sanguineus, five cell types were identified, which were the prohemocytes, plasmatocytes, granulocytes, spherulocytes, and adipohemocytes. The prohemocytes were the smallest cells found in the hemolymph. The plasmatocytes had polymorphic morphology with vesicles and cytoplasmic projections. The granulocytes had an elliptical shape with the cytoplasm filled with granules of different sizes and electrodensities. The spherulocytes were characterized by several spherules of uniform shapes and sizes that filled the entire cytoplasm, whereas the adipohemocytes had an irregular shape with multiple lipid inclusions that occupied almost the entire cytoplasmic space. The total counts of the hemocyte population increased in the group that was infected with L. infantum. Among the different cell types, the numbers increased and the ultrastructural changes occurred in the granulocytes and the plasmatocytes in the infected group of ticks., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non-immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott-Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex-independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations., (© 2015 Verboon, Rahe, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The total haemocyte count (THC) and the possible ultrastructural alterations induced in the haemocytes of the fourth larval instars of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), 96 h post-feeding on a semi-synthetic diet, treated with the LC50 of Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) and the LC50 of azadirachtin alone, and the LC25 of SpliMNPV combined with the LC25 of azadirachtin were studied and compared to the control. Single treatment with the virus and azadirachtin or combined treatment significantly decreased the THC compared to the control. There are five types of haemocytes in S. littoralis: prohaemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. The most common symptoms in granulocytes and plasmatocytes, the main affected cell types, due to viral infection were the presence of virogenic stroma, peripheral dispersion of the chromatin and disappearance of the nucleoli. However, the most common symptoms in these two types of haemocytes due to treatment with azadirachtin were the presence of rough endoplasmic reticulum filled with fibrous materials, due to probably apoptosis, in their cisternae and disorganization of mitochondria (looped, vacuolated and swollen). In addition, the cytoplasm of granulocytes was vacuolated with the appearance of autophagic lysosomes, while plasmatocytes showed ruptured cell membrane and folded nuclear envelope. Combined treatment with the NPV and azadirachtin induced the same pathological changes which were recorded from individual treatment with the virus or azadirachtin to the same haemocytes. It can be concluded that the change in the THC and ultrastructure of granulocytes and plasmatocytes may affect the cellular-mediated immune response in S. littoralis. Moreover, it seems likely that mitochondria were the target site of azadirachtin, as they were affected in both granulocytes and plasmatocytes treated with azadirachtin alone or in combination with SpliMNPV., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Eriocheir sinensis (Henri Milne Edwards 1854) is one of the most important aquaculture species in China. In this investigation, we characterised the different types of haemocytes of E. sinensis using light and electron microscopy combined with cytochemical analysis and determined the in vivo phagocytic ability of different haemocyte types by injecting polystyrene beads. The haemocytes of E. sinensis were divided into three types: hyalinocytes, semigranulocytes and granulocytes. The hyalinocytes had no or few cytoplasmic granules; the semigranulocytes contained abundant small granules and a few large refractile cytoplasmic granules; and the granulocytes contained numerous large refractile cytoplasmic granules. The hyalinocytes were demonstrated to be the most abundant circulating haemocytes and the most avid phagocytic haemocytes, accounting for approximately 88.7% of the total phagocytes. The haemocyte-containing granules displayed limited phagocytic ability, with approximately 5.0% of granulocytes and 6.3% of semigranulocytes displaying positive phagocytic ability against the invading polystyrene beads in vivo. After injection with Aeromonas hydrophila, Bacillus subtilis and different concentrations of lipopolysaccharide for 0.25, 0.5, 1, 2, 4, 6 and 8 h, all three types of haemocytes experienced dramatic decline and then rapid recovery to their initial levels. A high concentration of lipopolysaccharide and A. hydrophila were extremely toxic to the crabs, as they induced a more serious loss of haemocytes compared with a low concentration of lipopolysaccharide and B. subtilis. Overall, the results obtained in this study indicate that a small proportion of the haemocytes of E. sinensis contributed to the phagocytic process, and the migration of haemocytes and haemocyte lysis were most likely a prominent pathway for pathogen elimination., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders., (© 2014 Wiley Periodicals, Inc.)

A continuous cell line, SYSU-OfHe-C, from larval hemocytes of corn borer, Ostrinia furnacalis was established. With increasing passages, the cells grew increasingly faster, and approximately 45% of the cells were in division at passage 55. The culture was mainly composed of two types of cells, granulocytes and plasmatocytes, which showed different division and proliferation behaviors, but possessed similar phagocytic ability. Its spreading ability was significantly weaker than that of hemocytes from naïve larva; however, it could be promoted by larval plasma. Furthermore, its encapsulation ability was also promoted by larval plasma to form multilayer capsules on Sephadex A-25 beads. Finally, the expression of several immune-related genes was verified after provocation by microbes or Sephadex beads. These results indicated that the cell line possessed immune ability depending on the presence of plasma of naïve larvae and are beneficial to studies of insect cellular systems., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Spiroplasma is a novel pathogen of commercially exploited crustaceans. To more full clarify its pathogenic mechanism at the molecular level in vitro, a spiroplasma-infected hemocytes model was developed as described here, using a modified method of hemocytes culture from the Chinese mitten crab Eriocheir sinensis. It has been standardized by employing L-15 growth medium supplemented with 15% fetal bovine serum along with 0.15% glucose, 0.75% NaCl, antibiotics (100 U ml(-1) penicillin, 100 U ml(-1) streptomycin) and a suitable pH of 7.20-7.40, incubated at 28°C without the requirement for 5% carbon dioxide. Cytopathic effects of Spiroplasma eriocheiris in the cultures, including cell debris and cellular exudates, were observed as early as 36 h post-inoculation. The green fluorescent dye Alex-488 was used as an immunofluorescence marker of S. eriocheiris to study its adhesion to, and infection of, the host cell. At about 24-h post-inoculation, a large number of spiroplasmas were observed infecting the host hemocytes cells, which became slug-like and widely distributed in the culture. Intracellular inclusion bodies containing spiroplasmas, and a large number of free spiroplasmas released from the ruptured cells, were observed using transmission electron microscopy. All the results show that S. eriocheiris can invade E. sinensis hemocytes in vitro, and this can provide a significant step forward toward further study of the relationship between the novel pathogen spiroplasma and its hosts in laboratory experimental studies., (Copyright © 2014. Published by Elsevier B.V.)

This study focused on an isolation and characterization of the circulating hemocytes in mud crab, Scylla olivacea. Isolation of specific cell types of hemocytes from crab hemolymph was accomplished by using 60% Percoll density gradient centrifugation. Four separated bands of the hemocytes were successfully obtained. Characterization of these isolated hemocytes by light microscope using trypan blue-rose bengal staining, rose bengal-hematoxilin staining, and phase contrast revealed four distinct types of hemocyte cells. Using their specific morphology and granularity, they were identified as hyaline cell (HC), small granular cell (SGC), large granular cell (LGC) and mixed granular cell (MGC). Transmission electron microscopy (TEM) revealed more details on specific cell size, size of cytoplasmic granule, and nuclear to cytoplasmic ratio, and confirmed the classification. Relative abundance of these cells types in the hemolymph of an adult crab were 15.50±8.22% for HC, 55.50±7.15% for SGC, 13.50±5.28% for LGC, and 15.50±3.50% for MGC. Proteomic analysis of protein expression for each specific cell types by two-dimensional electrophoresis identified two highly abundant proteins, prophenoloxidase (ProPO) and peroxinectin in LGC. Determination of phenoloxidase (PO) activity in each isolated cell types using in vitro and in situ chemical assays confirmed the presence of PO activity only in LGC. Based on an increased PO activity of crab hemolymph during the course of White Spot Syndrome Virus (WSSV) infection, these results suggest that prophenoloxidase pathway was employed for host defense mechanism against WSSV and it may link to the role of large granular hemocyte., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Some snails of Biomphalaria alexandrina can resist the infection of Schistosoma mansoni so this study aimed to clearly this mechanism by using light and electron microscopy (EM) and determine the role of Nitric oxide in this mechanism. B. alexandrina snails used in this study were exposed individually to S. mansoni infection according to their response they were classified into susceptible group (shed cercariae) and resistant group (failed to shed cercariae). Snails not exposed to infection were included in this study as control group. Nitric oxide (NO) level was assayed directly in the soluble fraction of B. alexandrina haemolymph supernatants collected from each group of B. alexandrina snails were subjected to NO assay by the Greiss reaction. The level of NO in haemolymph of infected snails was significantly increased (p < 0.001) than both control and non infected snails groups, however, in non infected snails group had significantly (p < 0.05) compared to control group. This study when correlated the changes recognized by EM with NO level the pro apoptotic effect of high level of NO on the haemocytes. Characterization and identification of cell shape of haemocytes in both haemolymph and tissue were examined by light and electron microscopy. Examination of B. alexandrina snail's haemocytes revealed three types of different cells classified according to their shape and granular contents. These cells are granulocytes, amoebocytes and hyalineocytes. Electron microscope study also revealed the important role of granulocytes and amoebocytes as defense mechanism against snail infection. NO is considered an important anti parasite molecule; intra-molluscan stages of parasites switch off host NO defense response.

The influence of infection with an entomopathogenic strain of Pseudomonas aeruginosa on Galleria mellonella hemocytes was investigated. Extensive bacteriaemia developed 18 h after infection. This was correlated with significant changes in morphology, viability and the spreading ability of immunocompetent hemocytes, namely granulocytes and plasmatocytes. Since bacteriaemia developed, membrane blebbing, cytoplasm vacuolization, cell and organelle swelling, and chromatin condensation were observed among others. These features are typical for apoptotic and autophagal cell death. A gradually increasing level of procaspase and its activation as well as lack of DNA degradation were also detected. Propidium iodide and acridine orange staining indicated that hemocytes become dead ultimately. Infection of G. mellonella larvae with P. aeruginosa also caused significant changes in the arrangement of the actin cytoskeleton in the hemocytes, which might be correlated with their restricted spreading ability., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The hemocytic count and defense reaction within 4 families of higher Diptera: Tabanidae, Syrphidae, Muscidae and Sarcophagidae, whose larvae inhabit bacterially aggressive environment, were investigated. The least hemocytes types (3) were revealed in Tabanidae and Syrphidae larvae--prohemocytes, plasmatocytes and prophenoloxydase-containing unstable hyaline cells (oenocytoids). In Sarcophaga crassipalpis and Musca domestica stable hyaline cells and thrombocytoids or podocytoid-like cells can be added to this set. At the time of pupariation in Sarcophaga, new generation of prohemocytes is segregated into the hemolymph, which form small round or spindle-shaped hyaline cells. So, the number of plasmatocyte types in Sarcophaga increase to six. Typical to Calliphoridae juvenile plasmatocytes in the members of investigated families are absent. Among the one hemocyte type morphology also can vary, especially in unstable prophenoloxydase hyaline cells. In Drosophila there are crystal cells containing in the cytoplasm paracrystalloidal inclusions. In Calliphoridae there are big hyaline cells with homogenous cytoplasm producing circumferential bubbles. Both in Sarcophaga and Tabanidae they contain in their cytoplasm big globules. However in Sarcophaga they rapidly disintegrate, while in Tabanidae are maintained unchanged during hours. In Muscidae and Syrphidae prophenoloxydase extrusion occurs very early and these cells obtain pycnotic nuclei and very liquid cytoplasm with strings of granules. Thrombocytoids in Musca larvae are represented by big flattened anucleated irregular cytoplasm and "naked" nuclei and cytoplasmic fragments often with fan-like projections. Plasmatocytes in all species studied are the cells with pronounced phylopodies. In larvae they contain cytoplasmic catabolic inclusions and in pupa--ragments of apoptotic tissues. Clearance of hemolymph from alien particles in Sarcophagidae and Muscidae occur by thrombocytoides, while in Tabanidae by plasmatocyte nodulation. A differing case is Syrphidae whe-e charcoal injection produce depletion of hemolymph both from particles and all types of hemocytes. So the specimen of different higher Diptera families can use different schemes of cellular defense reaction.

In the hemolymph of surgical maggots Lucilia sericata seven types of hemocytes were revealed. These are prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation. In contrast to Calliphora hyaline cells, this type of hemocytes in cropemptying larvae of Lucilia is elongated or vermiform in shape. Hyaline cells may be transformed to both prothrombocytoids and unstable prophenoloxydase-producing cells. Appearance and differentiation of each hemocyte type is rigidly linked with a definite stage of development. In cellular defense the main role play juvenile plasmatocytes, plasmatocytes II and III and trombocytoides. Juvenile plasmatocytes are the most active ones. After charcoal particles injection they were instantly surrounded by the thick envelope of adhered alien particles and form uniform morules aggregations or conglomerates together with thrombocytoidal agglutinates. Plasmatocytes II and III during the early stages of differentiation may be involved in adhesion and phagocytosis of alien particles and during the last stages in the engulfing of apoptose desintegrated tissues. Thus the cellular defense reaction is assisted by 4 hemocyte types--prophenoloxydase-unstable hyaline cells, thrombocytoids, juvenile plasmatocytes and plasmatocytes I-IV.

To date nothing is known about the subacute phase of neurodegeneration following injury in invertebrates. Among few clues available are the results published by our group reporting hemocytes and activated glial cells at chronic and acute phases of the lesion. In vertebrates, glial activation and recruitment of immunological cells are crucial events during neurodegeneration. Here, we aimed to study the subacute stage of neurodegeneration in the crab Ucides cordatus, investigating the cellular/molecular strategy employed 48 hours following ablation of the protocerebral tract (PCT). We also explored the expression of nitric oxide (NO) and histamine in the PCT during this phase of neurodegeneration. Three immune cellular features which seem to characterize the subacute phase of neurodegeneration were revealed by: 1) the recruitment of granulocytes and secondarily of hyalinocytes to the lesion site (inducible NO synthase- and histamine-positive cells); 2) the attraction of a larger number of cells than observed in the acute phase; 3) the presence of activated glial cells as shown by the round shaped nuclei and increased expression of glial fibrillary acidic protein. We suggest that molecules released from granulocytes in the acute phase attract the hyalinocytes thus moving the degeneration process to the subacute phase. The importance of our study resides in the characterization of cellular and biochemical strategies peculiar to the subacute stage of the neurodegeneration in invertebrates. Such events are worth studying in crustaceans because in invertebrates this issue may be addressed with less interference from complex strategies resulting from the acquired immune system.

We explored the pathogenic mechanism of Vibrio alginolyticus in the stone crab Charybdis japonica by studying the hemolymph of C. japonica artificially infected by V. alginolyticus. To this end, Wright-Geimsa staining and electron microscopy were used, and phenoloxidase (PO) activity and the immune protection rate of C. japonica injected with immune polysaccharide during infection were analyzed. The results indicated that the total hemocyte and hyaline hemocyte (HH) counts in diseased crabs were significantly lower than those in healthy crabs (P < 0.05), whereas the large granule hemocytes (LGHs) were significantly higher in diseased crabs than in healthy crabs (P < 0.05). The cellular sizes of HHs and LGHs showed an increasing trend after V. alginolyticus infection, while the nuclear/cytoplasmic ratio (NP) of these cells showed a sharp decline after V. alginolyticus infection (P < 0.05). Micro-pathological analysis of hemocytes revealed fewer hemocytes in the hemolymph of diseased crabs and the presence of disintegrated cells. Ultrastructural and micro-pathological analyses showed damage in all types of hemocytes. The mitochondria were damaged and incomplete in structure, parts of the nuclear membrane were anamorphic and parts of the nuclei had shrunk, hematocyte nuclei exhibited heterochromatinization, hemocyte granules were increased in the polysaccharide-treated group infected with V. alginolyticus, and the numbers of mitochondria and rough endoplasmic reticulum were also increased. PO activity in the two Vibrio-infected groups peaked at 6 h and 24 h after infection, respectively, and PO activity increased in the hemolymph of infected crabs but sharply decreased with prolonged infection. Finally, the PO activities in the two Vibrio-infected groups were significantly lower than controls at 120 h post-infection (P < 0.05). Interestingly, PO activity was higher in polysaccharide-treated crabs than non-polysaccharide-challenged infected crabs, resulting in an immunoprotective rate of 69.64% at 7 days post-infection. This phenomenon suggests that polysaccharides could enhance the organism's antibacterial defenses by improving immune-related enzyme activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This paper is the first descriptive review of hemolymph cell types in the circulation of the tarantula spider Lasiodora sp. These animals are more long-lived than other arthropods, and may live for approximately twenty years. Such remarkable longevity may result from a highly successful immune system, which in turn is directly correlated with hemocyte function. Since the literature on the genus Lasiodora sp. is limited, the main goal of the present study was to identify the different cell types by optical and transmission microscope. Six hemocyte types were characterized and called prohemocyte, granulocyte type I, granulocyte type II, spherulocyte, oenocytoid and plasmatocyte. Prohemocytes presented a large nucleus, elongated granulocytes type I showed the nucleus with the same cell format, elliptical granulocytes type II showed the central nucleus of identical shape, spherulocytes exhibited the nucleus filling almost the whole cell, oval oenocytoids showed eccentric nucleus and less dense cytoplasm, and irregular plasmatocytes showed a nucleus and no granules in cytoplasm. These polymorphic granulocytes presented a round, elongated, elliptical, oval or irregular profile with large and varied numbers of granules, except for plasmatocytes, that were agranular. Different densities and different concentrations of these granules were found at the periphery of the cell. The possible reasons and implications of differences and similarities between arthropods hemocytes are discussed. It can be concluded that there are six cell types in Lasiodora sp. This study is of the first step in the elucidation of the role these cells play in the circulatory and immune system in spiders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches. Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This study examined the effects of an acoustic stimulus on the haemolymph and agonistic behaviour of the red swamp crayfish, Procambarus clarkii. The experiment was conducted in a tank equipped with a video recording system using six groups (three control and three test groups) of five adult crayfish (30 specimens in total). After 1 h of habituation, the behaviour of the crayfish was monitored for 2 h. During the second hour, the animals in the test groups were exposed to a linear sweep (frequency range 0.1-25 kHz; peak amplitude 148 dB(rms) re. 1 μPa at 12 kHz) acoustic stimulus for 30 min. Exposure to the noise produced significant variations in haemato-immunological parameters as well as a reduction in agonistic behaviour.

A comparative analyses of hemocytes of molluscs, Pila globosa (Gastropoda: Prosobranchia), Bellamya bengalensis (Gastropoda: Prosobranchia) and Lamellidens marginalis (Bivalvia: Eulamellibranchiata) were carried out for morphotype and subpopulation identification, analyses of phagocytosis and generation of cytotoxic agents. Flow cytometry and microscopic analyses of hemocytes revealed the existence of agranulocytes (blast like cells, round hyalinocytes and spindle hyalinocytes), semigranulocytes (semigranular asterocytes and round semigranulocytes) and granulocytes (round granulocytes, spindle granulocytes and granular asterocytes) as three morphotypes. In P. globosa, granulocytes and semigranulocytes and in B. bengalensis granulocytes and agranulocytes are the chief phagocytes and major producers of superoxide anion and nitric oxide. In L. marginalis, granulocytes were identified as principal phagocytes with prominent activity of superoxide anion and nitric oxide. Highest activity of phenoloxidase was recorded in the agranulocytes of P. globosa with moderate activities among other morphotypes of all three species. Differential result may be due to species specific response, non-identical habitat preference and related adaptation of the species to their different ecological niches., (Copyright © 2012 Elsevier Ltd. All rights reserved.)