The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps.

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.

Polystyrene nanoplastics and microplastics can act as Trojan horse carriers of benzo(a)pyrene to mussel hemocytes in vitro.

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.

Morphology and activities of cell populations of haemocytes in Oncomelania hupensis following Schistosoma japonicum infection.

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.

Pendimethalin-based herbicide impairs cellular immune response and haemocyte morphology in a beneficial ground beetle.

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.

Modulations of immune parameters caused by bacterial and viral infections in the terrestrial crustacean Porcellio scaber: Implications for potential markers in environmental research.

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.

Morphological and functional characterization of circulating hemocytes using microscopy techniques.

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

A study of autophagy in hemocytes of the Pacific oyster, Crassostrea gigas.

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; NH4Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy.

Responses of Mytilus galloprovincialis to challenge with the emerging marine pathogen Vibrio coralliilyticus.

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.

Different roles of crayfish hemocytes in the uptake of foreign particles.

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.

Insights into the toxicity of iron oxides nanoparticles in land snails.

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.

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses.

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

Effects of food contaminated with cadmium and copper on hemocytes of Steatoda grossa (Araneae: Theridiidae).

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.

The haemocytes of the colonial aplousobranch ascidian Diplosoma listerianum: Structural, cytochemical and functional analyses.

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.

The paralytic shellfish toxin, saxitoxin, enters the cytoplasm and induces apoptosis of oyster immune cells through a caspase-dependent pathway.

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.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior.

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.

Relevant principal factors affecting the reproducibility of insect primary culture.

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.

Seawater acidification induced immune function changes of haemocytes in Mytilus edulis: a comparative study of CO2 and HCl enrichment.

The present study was performed to evaluate the effects of CO2- 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 CO2 and HCl treatments, and four antioxidant components, GSH, GST, GR and GPx, had active responses to the acidification stress. Comparatively, CO2 had more severe destructive effects on haemocytes than HCl at the same pH level, indicating that CO2 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.

Vascular budding in Symplegma brakenhielmi and the evolution of coloniality in styelid ascidians.

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.

Oxidative damage, ultrastructural alterations and gene expressions of hemocytes in the freshwater crab Sinopotamon henanense exposed to cadmium.

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.

The ascidian Styela plicata hemocytes as a potential biomarker of marine pollution: In vitro effects of seawater and organic mercury.

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.