Fungal infection causes changes in the number,morphology and spreading ability of Galleria mellonella haemocytes

The most effective and important strategy in the insect immune response is based on cellular reactions incorporating haemocytes. The present study uses Galleria mellonella (Lepidoptera: Pyralidae) as a host to study the pathogenesis caused by the entomopthoralean fungus Conidiobolus coronatus (Entomophthorales). Five types of haemocytes with different morphologies and behaviour are observed in the haemolymph of G. mellonella: granulocytes (GRs), plasmatocytes (PLs), spherulocytes (SPs), oenocytes (OEs) and prohaemocytes (PRs). During in vitro cultivation, three morphological subtypes of PLs are distinguished: flattened PLs, sun‐like PLs and oval PLs. In fresh smears of haemolymph observed under phase‐contrast microscopy, only flattened PLs are identified. No morphological changes are observed between fresh smears and in vitro cultures for GR, OE, SP and PR. Haemocytes cultured in vitro form a cellular network composed of PLs and GRs. Changes in the numbers, morphology and behaviour of haemocytes induced by fungal infection are compared with those observed in normally‐developing untreated larvae. Infection results in a significant drop in the number of haemocyte types. Fresh smears of haemocytes from mycosed larvae reveal malformed OEs, vacuolized PLs and GRs, as well as PLs with apoptotic blebs. Haemocytes from mycosed larvae incubated in vitro look similar, with degranulated GRs and vacuolized PLs forming microaggregations, as well as deformed OEs; only the SPs remain unharmed. Fungal infection impairs the ability of haemocytes to attach and spread on the culture dish. The actin cytoskeleton of haemocytes from mycosed larvae appear disorganized.     

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the musselMytilus galloprovincialis

Summary Cell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesiles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cystoskeleton inMytilus galloprovincialis haemocytes are discussed.     

In vitro interactions between several species of harmful algae and haemocytes of bivalve molluscs

Harmful algal blooms (HABs) can have both lethal and sublethal impacts on shellfish. To understand the possible roles of haemocytes in bivalve immune responses to HABs and how the algae are affected by these cells (haemocytes), in vitro tests between cultured harmful algal species and haemocytes of the northern quahog (= hard clam) Mercenaria mercenaria, the soft-shell clam Mya arenaria, the eastern and Pacific oysters Crassostrea virginica and Crassostrea gigas and the Manila clam Ruditapes philippinarum were carried out. Within their respective ranges of distribution, these shellfish species can experience blooms of several HAB species, including Prorocentrum minimum, Heterosigma akashiwo, Alexandrium fundyense, Alexandrium minutum and Karenia spp.; thus, these algal species were chosen for testing. Possible differences in haemocyte variables attributable to harmful algae and also effects of haemolymph and haemocytes on the algae themselves were measured. Using microscopic and flow cytometric observations, changes were measured in haemocytes, including cell morphology, mortality, phagocytosis, adhesion and reactive oxygen species (ROS) production, as well as changes in the physiology and the characteristics of the algal cells, including mortality, size, internal complexity and chlorophyll fluorescence. These experiments suggest different effects of the several species of harmful algae upon bivalve haemocytes. Some harmful algae act as immunostimulants, whereas others are immunosuppressive. P. minimum appears to activate haemocytes, but the other harmful algal species tested seem to cause a suppression of immune functions, generally consisting of decreases in phagocytosis, production of ROS and cell adhesion and besides cause an increase in the percentage of dead haemocytes, which could be attributable to the action of chemical toxins. Microalgal cells exposed to shellfish haemolymph generally showed evidence of algal degradation, e.g. loss of chlorophyll fluorescence and modification of cell shape. Thus, in vitro tests allow a better understanding of the role of the haemocytes and the haemolymph in the defence mechanisms protecting molluscan shellfish from harmful algal cells and could also be further developed to estimate the effects of HABs on bivalve molluscs in vivo.     

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

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

Altered actin polymerization of Plutella xylostella (L.) in response to ovarian calyx components of an endoparasitoid Cotesia plutellae (Kurdjumov)

Abstract Cotesia plutellae (Kurdjumov) (Hymenoptera: Braconidae), a solitary braconid endoparasitoid wasp, parasitizes the diamondback moth Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) by suppressing the host defense response, thereby resulting in successful parasitization. During parasitization, ovarian calyx fluid is also delivered into the haemocoel of the host along with the wasp egg. The effect of calyx fluid constituents on haemocyte‐spreading behaviour of P. xylostella is analysed by measuring F‐actin development in the haemocytes. For this purpose, the calyx fluid of C. plutellae is separated into ovarian protein and C. plutellae bracovirus (CpBV). The ovarian protein consists of a wide range of molecular weight proteins, which are apparently different from those of CpBV. When nonparasitized P. xylostella haemocytes are incubated with either ovarian protein or CpBV for 1 or 2 h, haemocytes lose their responsiveness to a cytokine, plasmatocyte‐spreading peptide, in a dose‐dependent manner for each calyx component and fail to exhibit haemocyte‐spreading behaviour. Some CpBV genes are expressed within 1 h of parasitization. The inhibition of haemocyte‐spreading could be explained by measuring F‐actin contents, in which parasitization by C. plutellae inhibits F‐actin development in the haemocytes of P. xylostella. Either ovarian protein or CpBV could inhibit F‐actin development in the nonparasitized haemocytes. In addition, co‐incubation of ovarian protein and CpBV results in significant additive inhibition of both haemocyte‐spreading and F‐actin development in the haemocytes in response to cytokine. These results suggest that both components of C. plutellae calyx fluid function in a synergistic manner, leading to immunosuppression during the early stage of parasitization.     

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

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

Regulation of nitric oxide production in snail (Lymnaea stagnalis) defence cells: a role for PKC and ERK signalling pathways

Background information. Nitric oxide (NO) is an important molecule in innate immune responses. In molluscs NO is produced by mobile defence cells called haemocytes; however, the molecular mechanisms that regulate NO production in these cells is poorly understood. The present study focused on the role of cell signalling pathways in NO production by primary haemocytes from the snail Lymnaea stagnalis. Results. When haemocytes were challenged with PMA (10 μM) or the β‐1,3‐glucan laminarin (10 mg/ml), an 8‐fold and 4‐fold increase in NO production were observed after 60 min respectively. Moreover, the NOS (NO synthase) inhibitors L‐NAME (N^G‐nitro‐L‐arginine methyl ester) and L‐NMMA (N^G‐monomethyl‐L‐arginine) were found to block laminarin‐ and PMA‐induced NO synthesis. Treatment of haemocytes with PMA or laminarin also increased the phosphorylation (activation) status of PKC (protein kinase C). When haemocytes were preincubated with PKC inhibitors (calphostin C or GF109203X) or inhibitors of the ERK (extracellular‐signal‐regulated kinase) pathway (PD98059 or U0126) prior to challenge, significant reductions in PKC and ERK phosphorylation and NO production were observed following exposure to laminarin or PMA. The greatest effect on NO production was seen with GF109203X and U0126, with PMA‐induced NO production inhibited by 94% and 87% and laminarin‐induced NO production by 50% and 91% respectively. Conclusions. These data suggest that ERK and PKC comprise part of the signalling machinery that regulates NOS activation and subsequent production of NO in molluscan haemocytes. To our knowledge, this is the first report that shows a role for these signalling proteins in the generation of NO in invertebrate defence cells.     

Detection of serpins involved in cellular immune response of Rhipicephalus microplus challenged with fungi

The understanding of tick physiology and immune system is important to improve the effective control of this ectoparasite. Invertebrates' innate immune response is activated when the organism is challenged with pathogens. The present study describes the changes of serine proteinase inhibitors (serpins) and in the number of circulating haemocytes involved in cellular immune defence of Rhipicephalus microplus engorged females challenged with the entomopathogenic fungi Metarhizium anisopliae or Beauveria bassiana, or with the non-entomopathogenic fungus Fusarium oxysporum. The cell-free haemolymph was separated from haemocytes by centrifugation and cells were re-suspended in phosphate buffer pH 7.2. The proteins of haemocytes were analysed by SDS-PAGE and the segments of the 1D gel were submitted to protein digestion with trypsin. The peptides were analysed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS). The analysis by mass spectrometry allowed the identification of several proteins through the search in the database built based on public banks of Ixodidae and Argasidae. In haemocytes, many proteins were identified highlighting serpins. The results showed that the entomopathogenic fungi M. anisopliae or B. bassiana reduced the amount of serpins, while F. oxysporum increased. The present study reports, for the first time, the variation of serpins in haemocytes of R. microplus engorged females infected by fungi.     

Drosophila Rab14 mediates phagocytosis in the immune response to Staphylococcus aureus

Drosophila haemocytes are essential for the animal to resist Staphylococcus aureus infections. Phagocytosis is a central component of the haemocyte‐mediated immune response. It involves regulated interaction between the phagocytic and the endocytic compartments. RabGTPases are pivotal for the membrane trafficking and fusion events, and thus are often targets of intracellular pathogens that subvert phagocytosis. An in vivo screen identified Rab2 and Rab14 as candidates for proteins regulating phagosome maturation. Since Rab14 is often targeted by intracellular pathogens, an understanding of its function during phagocytosis and the overall immune response can give insight into the pathogenesis of intracellular microbes. We generated a Drosophila Rab14 mutant and characterized the resulting immune defects in animals and specifically in haemocytes in response to an S. aureus infection. Haemocyte based immunofluorescence studies indicate that Rab14 is recruited to the phagosome and like Rab7, a well‐characterized regulator of the phagocytic pathway, is essential for progression of phagosome maturation. Rab14 mutant haemocytes show impaired recruitment of Rab7 and of a lysosomal marker onto S. aureus phagosomes. The defect in phagocytosis is associated with higher bacterial load and increased susceptibility to S. aureus in the animal.     

The use of the neutral red retention assay to examine the effects of temperature and salinity on haemocytes of the European flat oyster Ostrea edulis (L)

The neutral red retention assay was adapted to assess the effects of a matrix of temperature and salinity combinations on haemocytes of the European flat oyster Ostrea edulis (L.). Oysters were collected from a commercially fished site in the western Solent and were acclimated to different temperature and salinity combinations in the laboratory. The results indicate that the haemocytes are most stable at high salinity and intermediate temperature (28‰/15°C). Furthermore it is evident from the data that at low salinities the adaptive capacity of the cellular processes is reduced to such an extent that the haemocytes are unable to respond to favourable temperature regimes. The use of neutral red as a probe to monitor cellular stability is discussed.     

Resistance of Drosophila suzukii to the larval parasitoids Leptopilina heterotoma and Asobara japonica is related to haemocyte load

Unlike other Drosophila species, the invasive Drosophila suzukii Matsumura (Diptera: Drosophilidae) shows a remarkable pest status. Among the physiological traits that may explain the high level of resistance to parasitoids of Drosophila larvae, the haemocyte load is shown repeatedly to play an important role. To determine whether haemocyte load can explain immunity resistance of D. suzukii to parasitoids, the haemocytes of parasitized and healthy larvae are quantified in two Japanese and three French populations of D. suzukii. Parasitization tests are conducted with two larval parasitoids: the paleartic Leptopilina heterotoma Thomson (Hymenoptera: Figitidae) and the Asian Asobara japonica Belokobylskij (Hymenoptera: Braconidae). Based on morphological and functional criteria, D. suzukii has classes of haemocytes similar to those described in Drosophila melanogaster. However, healthy larvae of the five populations tested possess particularly large numbers of haemocytes compared with D. melanogaster. Haemocyte load is also higher in larvae from the French populations than in the Japanese strains. The ability of D. suzukii larvae to encapsulate eggs of L. heterotoma is associated with a particularly high load of circulating haemocytes. However, it is notable that A. japonica induces a strong depression of the haemocyte population in this resistant host associated with an inability to encapsulate parasitoid eggs. The results show that the cellular immune system plays a major role in the failure of larval parasitoids to develop in most instances in larvae of D. suzukii, possibly contributing to the success of this species as an invader.     

Particle recognition by haemocytes from the colonial ascidianBotrylloides leachii: Evidence that theB. leachii HA-2 agglutinin is opsonic

Summary Haemocytes from the ascidianBotrylloides leachii were observed in vivo to phagocytose sheep erythrocytes. The possibility that a sheep erythrocyte agglutinin (the HA-2 agglutinin) previously purified fromB. leachii haemolymph functions as a recognition molecule for the phagocytosis of these erythrocytes was investigated. Untreated sheep erythrocytes were found to adhere toB. leachii haemocytes in vitro. Adherence appeared to be mediated by the HA-2 agglutinin, as evidenced by the inhibition of adhesion by lactose (which is a specific inhibitor of the HA-2 agglutinin) and by an anti-HA-2 IgG preparation. Immunofluorescence studies indicated that HA-2 molecules secreted by the haemocytes bound to unsensitised erythrocytes, causing them to adhere to haemocytes. No HA-2 agglutinin could be detected on the surface of the haemocytes in the absence of erythrocytes but receptors for the agglutinin were detected. The results suggest that the HA-2 agglutinin can function as a recognition molecule for sheep erythrocytes and other particles bearing the appropriate carbohydrate moieties on their surfaces. At least one of two other lectins purified from haemolymph (HA-1 and LBP-3) was detected by immunofluorescence on the surface of haemocytes. The function(2) of these latter molecules, neither of which binds to sheep erythrocytes, is not known.     

Larval excretory-secretory products from the parasite <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> modulate HSP70 protein expression in defence cells of its snail host, <Emphasis Type="Italic">Biomphalaria glabrata</Emphasis>

Synthesis of heat shock proteins (HSPs) following cellular stress is a response shared by many organisms. Amongst the HSP family, the ∼70 kDa HSPs are the most evolutionarily conserved with intracellular chaperone and extracellular immunoregulatory functions. This study focused on the effects of larval excretory-secretory products (ESPs) from the parasite Schistosoma mansoni on HSP70 protein expression levels in haemocytes (defence cells) from its snail intermediate host Biomphalaria glabrata. S. mansoni larval stage ESPs are known to interfere with haemocyte physiology and behaviour. Haemocytes from two different B. glabrata strains, one which is susceptible to S. mansoni infection and one which is resistant, both showed reduced HSP70 protein levels following 1 h challenge with S. mansoni ESPs when compared to unchallenged controls; however, the reduction observed in the resistant strain was less marked. The decline in intracellular HSP70 protein persisted for at least 5 h in resistant snail haemocytes only. Furthermore, in schistosome-susceptible snails infected by S. mansoni for 35 days, haemocytes possessed approximately 70% less HSP70. The proteasome inhibitor, MG132, partially restored HSP70 protein levels in ESP-challenged haemocytes, demonstrating that the decrease in HSP70 was in part due to intracellular degradation. The extracellular signal-regulated kinase (ERK) signalling pathway appears to regulate HSP70 protein expression in these cells, as the mitogen-activated protein-ERK kinase 1/2 (MEK1/2) inhibitor, U0126, significantly reduced HSP70 protein levels. Disruption of intracellular HSP70 protein expression in B. glabrata haemocytes by S. mansoni ESPs may be a strategy employed by the parasite to manipulate the immune response of the intermediate snail host.     

Ultrastructural,cytochemical, and immunocytochemical characterization of haemocytes of the hard tick Ixodes ricinus (Acari; Chelicerata)

Haemocytes of the hard tick Ixodes ricinus were characterized on the basis of their ultrastructure, their ability to ingest foreign material, and to produce or store molecules of the immune defence. Distinction was made between types of haemocytes according to the absence or presence of granular inclusions, shape and size of the lysosomal compartment or the rough endoplasmic reticulum, and ultrastructural and functional similarity to the corresponding haemocytes of insects. Three types of haemocytes were found in adult ticks: plasmatocytes and type-I and type-II granular haemocytes, respectively. The precipitated reaction product of acid phosphatase activity revealed the shape of the lysosomal compartment. The additional injection of particulate materials into the haemocoel further revealed the endocytic activity of the haemocytes. The lysozyme-like immunoreactivity of the haemocytes suggests bactericidal potential. Detection of immunoreactivity in haemocytes to a 25 kDa antigenic protein involved in cuticle formation further suggests their involvement in wound healing and encapsulation.     

Ontogeny of protein concentration,haemocyte concentration and antimicrobial activity against Escherichia coli in haemolymph of the invasive harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae)

The harlequin ladybird is considered to be one of the most successful invasive insect species. Among other traits, its invasive success is considered to be caused by a powerful immune system. In the present study, we investigate the ontogenetic profile of protein concentration, concentration of circulating haemocytes and constitutive antimicrobial activity against Escherichia coli in Harmonia axyridis haemolymph during late larval development and early adult life. Protein concentration increases during the first 32 days of adult life from 45 to 100 mg per mL of haemolymph and reaches intermediate values during larval stages. The concentration of circulating haemocytes is very low (5000 haemocytes per μL of haemolymph) in late larval stages and increases strongly during first 8 days of adult life to values of approximately 30 000 haemocytes per μL of haemolymph. The killing efficiency of haemolymph against E. coli is lowest in larval stages, rapidly increases in the prepupal stage and then steadily grows during the whole period of adult life. There are no significant effects of sex on any of the investigated physiological or immune parameters. In general, the patterns observed for H. axyridis contrast with many results that are reported for other insects (e.g. bees, fruit flies, crickets or mosquitoes). One possible explanation is the contrasting life history of H. axyridis, with a fast preimaginal development and a long adult lifespan being linked to a long reproductive period. Substantial variation in physiological and immune parameters during ontogeny also has important methodological implications because individuals of exactly the same stage/age have to be employed for comparative studies.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

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

Haemocyte involvement in the repair of the insect central nervous system after selective glial disruption

Summary Injection of physiologically inert particles (fluorescent microspheres) has a profound effect on neural repair of central nervous connectives of the cockroach Periplaneta americana following selective glial disruption. The injected particles, which do not gain direct access to the central nervous tissues, are taken up by a relatively small proportion (< 10%) of the haemocytes. This interference with haemocyte function virtually abolishes the appearance of the granule-containing cells (which are prominently involved in normal glial repair) and produces abnormal reorganization of the superficial glial elements. These results are interpreted as evidence that the granule-containing cells are derived from haemocytes which are critically involved in glial repair.     

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

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

Immunodetection of haemocyte subpopulations by N-acetylmuramic acid antibody in Planorbarius corneus (L.) (Gastropoda, Pulmonata)

Summary The cell subpopulations in the haemolymph ofPlanorbarius corneus were distinguished by means of flow cytometry. An antibody againstn-acetylmuramic acid was prepared and used as a cellular marker to recognize the cell types forming the subpopulations. The spreading haemocytes showed a positive reaction for anti-n-acetylmuramic acid; round haemocytes gave a negative reaction.