Nitric oxide production in blowfly hemolymph after yeast inoculation

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses that include both cellular and humoral components. Cellular responses are mediated by hemocytes and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In this work, we determined NO production in Chrysomya megacephala hemolymph and hemocytes after yeast inoculation. Assays were performed with non-infected controls (NIL), saline-injected larvae (SIL) or larvae injected with Saccharomyces cerevisiae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24 or 48h post-injection. NO levels in SIL were comparable to those measured in NIL until 12h, which might be considered the basal production, increasing at 24 and 48h post-injection, probably in response to the increased larval fragility after cuticle rupture. YIL exhibited significantly higher levels of NO than were found in other groups, peaking at 24h. l-NAME and EDTA caused a significant reduction of NO production in YIL at this time, suggesting the activity of a Ca(2+)-dependent NOS. Plasmatocytes and granular cells phagocytosed the yeasts. Plasmatocytes initiated the nodule formation and granular cells were the only hemocyte type to produce NO. These results permit us to conclude that yeasts induced augmented NO production in C. megacephala hemolymph and granular cells are the hemocyte type involved with the generation of this molecule.     

Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.     

Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti

Hemocytes are an essential component of the mosquito immune system but current knowledge of the types of hemocytes mosquitoes produce, their relative abundance, and their functions is limited. Addressing these issues requires improved methods for collecting and maintaining mosquito hemocytes in vitro, and comparative data that address whether important vector species produce similar or different hemocyte types. Toward this end, we conducted a comparative study with Anopheles gambiae and Aedes aegypti. Collection method greatly affected the number of hemocytes and contaminants obtained from adult females of each species. Using a collection method called high injection/recovery, we concluded that hemolymph from An. gambiae and Ae. aegypti adult females contains three hemocyte types (granulocytes, oenocytoids and prohemocytes) that were distinguished from one another by a combination of morphological and functional markers. Significantly more hemocytes were recovered from An. gambiae females than Ae. aegypti. However, granulocytes were the most abundant cell type in both species while oenocytoids and prohemocytes comprised less than 10% of the total hemocyte population. The same hemocyte types were collected from larvae, pupae and adult males albeit the absolute number and proportion of each hemocyte type differed from adult females. The number of hemocytes recovered from sugar fed females declined with age but blood feeding transiently increased hemocyte abundance. Two antibodies tested as potential hemocyte markers (anti-PP06 and anti-Dox-A2) also exhibited alterations in staining patterns following immune challenge with the bacterium Escherichia coli.     

Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects, the cell-mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H. illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell-mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte-like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell-mediated immune response by eicosanoids was investigated.     

Lipopolysaccharide evokes microaggregation reactions in hemocytes isolated from tobacco hornworms, Manduca sexta

Insect cellular immune reactions to bacterial infection include nodule formation. Eicosanoids mediate several cellular actions in the nodulation process, including formation of hemocyte microaggregates, an early step. In previous work, we reported that isolated hemocytes produce and secrete eicosanoids that influence hemocyte behavior in response to bacterial challenge. We also reported that microaggregate formation in response to challenge was mediated by prostaglandins (PGs), but not by products of the lipoxygenase (LOX) pathways. In this paper we describe experiments designed to test the idea that exposing isolated hemocytes to lipopolysaccharide (LPS) evokes formation of hemocyte microaggregates and this cellular action is mediated by PGs. Results show that isolated hemocyte preparations challenged with LPS formed more hemocyte microaggregates than unchallenged preparations (6.9x10(3) microaggregates/ml hemolymph vs. 2.5x10(3) microaggregates/ml hemolymph). LPS challenge stimulated formation of hemocyte microaggregates in a dose dependent manner. Experimental groups pretreated with cyclooxygenase inhibitors produced fewer hemocyte microaggregates in response to LPS challenge than untreated control groups. The formation of hemocyte microaggregates was not influenced by LOX inhibitors. Furthermore, the influence of dexamethasone was reversed by supplementing the experimental groups with the eicosanoid precursor fatty acid molecule, arachidonic acid and PGH(2). Palmitic acid, which is not substrate for eicosanoid biosynthesis, did not reverse the effects of dexamethasone on the formation of microaggregates. The LOX product 5(S)hydroperoxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid also did not reverse the effects of dexamethasone. These results are consistent with similar investigations performed with bacterial suspensions. We infer that isolated hemocyte preparations recognize and react to LPS by forming microaggregates and this reaction is mediated by PGs, but not products of the LOX pathway.     

The insect cellular immune response

The innate immune system of insects is divided into humoral defenses that include the production of soluble effector molecules and cellular defenses like phagocytosis and encapsulation that are mediated by hemocytes. This review summarizes current understand- ing of the cellular immune response. Insects produce several terminally differentiated types of hemocytes that are distinguished by morphology, molecular and antigenic markers, and function. The differentiated hemocytes that circulate in larval or nymphal stage insects arise from two sources： progenitor cells produced during embryogenesis and mesodermally derived hematopoietic organs. Regulation of hematopoiesis and hemocyte differentiation also involves several different signaling pathways. Phagocytosis and encapsulation require that hemocytes first recognize a given target as foreign followed by activation of downstream signaling and effector responses. A number of humoral and cellular receptors have been identified that recognize different microbes and multicellular parasites. In turn, activation of these receptors stimulates a number of signaling pathways that regulate different hemocyte functions. Recent studies also identify hemocytes as important sources Of a number of humoral effector molecules required for killing different foreign invaders.     

Inhibition of fungal growth in thermoregulating locusts,Locusta migratoria,infected by the fungus Metarhizium anisopliae var acridum

The locust, Locusta migratoria, has the capacity to develop a behavioural fever which reduces fungal infection by Metarhizium anisopliae var acridum. We investigated hemocyte and blastospore kinetics in infected insects under conditions that did or did not allow thermoregulation. Hemocyte concentrations were severely reduced in inoculated insects that did not thermoregulate but remained similar to those of controls in inoculated insects that were allowed to thermoregulate. Reductions in hemocyte counts were accompanied by an increase in the concentration of blastospores. In non-thermoregulating insects, circulating blastospores were first observed two days post-inoculation and had heavily colonized the hemolymph by day 5; in contrast, no blastospores were recovered from hemolymph of inoculated-thermoregulating insects. We used fluorescein isothiocyanate (FITC)-labelled silica beads to examine in vivo phagocytosis in thermoregulating and non-thermoregulating locusts. In the absence of fungus, a greater proportion of beads were engulfed by hemocytes in thermoregulating than in non-thermoregulating locusts early (4 and 24h) after bead injection, but the proportions were similar thereafter. In infected locusts, phagocytosis in non-thermoregulating insects was progressively impaired; such impairment, however, was not observed in challenged, thermoregulating insects. Our results suggest that thermoregulation helped keep fungal growth in check, apparently through the maintenance of hemocyte population levels and the direct inhibition of blastospore propagation by elevated temperatures.     

Ultrastructure of the melanization response of Aedes trivittatus against inoculated Dirofilaria immitis microfilariae

Scanning and transmission electron microscopy revealed that intrathoracically-inoculated microfilariae (mff) of Dirofilaria immitis elicited a rapid and effective immune response in the hemocoel of Aedes trivittatus mosquitoes. Hemocyte lysis and melanization of inoculated mff began immediately following exposure to the hemolymph environment. Initial melanin accumulation occurred at any site along the surface of mff and rapidly increased in thickness. Hemocyte encapsulation generally described for insects did not occur, but hemocytes might be necessary for activation of the melanization response. Although intact hemocytes were never abundant, those that were present seemed to show an active secretion of membrane-bound vacuoles directed toward mff. Activated hemocytes were in close association, but never in direct contact with the parasite, and were most commonly seen in various stages of lysis. Numerous cell remnants were noted throughout the developing melanin capsule. Parasites were completely melanized by 24 hr postinoculation (PI). By about 3 days PI, a membrane began to form around deposited melanin and hemocyte remnants. This developed into a double membrane-like structure of 25-30 nm thickness and resulted in the enclosure and isolation of the mff, melanin deposits, and cellular remnants from hemolymph components. It is suggested that this membrane functions as a boundary to isolate the melanized parasite and prevents additional hemocyte involvement.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.     

Regulation of melanization by glutathione in the moth Pseudoplusia includens

The phenoloxidase (PO) cascade regulates the melanization of hemolymph, which serves as a conserved humoral immune response in insects and other arthropods. The reductant glutathione (GSH) has long been used to inhibit melanization of hemolymph from insects but whether GSH levels in hemolymph are sufficient to play a physiological role in regulating melanization is unknown. Here, we characterized the abundance and effects of GSH on the melanization of plasma from larval stage Pseudoplusia includens (Lepidoptera: Noctuidae). GSH concentration in newly collected plasma from day two fifth instars ranged from 50 to 115 μM, while the titer of tyrosine, a substrate for the PO cascade, was 141 μM. GSH titers rapidly declined in plasma after collection from larvae, but no melanin formation occurred until GSH levels fell below 20 μM. Added GSH dose-dependently blocked melanization while PO substrates overrode GSH inhibition. Experiments conducted in the absence of oxygen and presence of PO cascade inhibitors further suggested that depletion of GSH from plasma was primarily due to formation of reactive intermediates produced by activated PO. Additional studies identified hemocytes as a potential source of plasma GSH. Hemocyte lysates recycled oxidized glutathione (GSSG) into GSH using NADPH, while intact hemocytes released GSH into the medium. These results suggest that in addition to protease cascade-releated mechanisms that regulate phenoloxidase, GSH exerts another level of control on melanization of insect hemolymph.     

Adult honeybees (Apis mellifera L.) abandon hemocytic, but not phenoloxidase-based immunity

Hemocytes and the (prophenol-) phenoloxidase system constitute the immediate innate immune system in insects. These components of insect immunity are present at any post-embryonic life stage without previous infection. Differences between individuals and species in these immune parameters can reflect differences in infection risk, life expectancy, and biological function. In honeybees which show an age-related division of labor within the worker caste, previous studies demonstrated that foragers show a strongly reduced number of hemoctyes compared to the younger nurse bees. This loss of immune competence has been regarded advantageous with respect to an already high mortality rate due to foraging and to redistribution of energy costs at the colony level. Based on the idea that abandoning hemocytes in all adults would be a reasonably direct regulatory mechanism, we posed the hypothesis that abandoning hemocytic immunity is not restricted to worker honeybees. We tested our hypotheses by performing a comprehensive analysis of hemocyte number and phenoloxidase (PO)-activity levels in immunologically naive workers, queens, and drones. We found that in all three adult phenotypes hemocyte number is dramatically reduced in early adult life. In contrast, we found that the dynamics of PO-activity levels have sex and caste-specific characteristics. In workers, PO activity reached a plateau within the first week of adult life, and in queens enzyme levels continuously increased with age and reached levels twice as high as those found in workers. PO-activity levels slightly declined with age in drones. These data support our hypothesis, from which we infer that the previously reported reduction of hemocyte in foragers is not worker specific but represents a general phenomenon occurring in all honeybee adult phenotypes.     

Cellular encapsulation in the eastern subterranean termite, Reticulitermes flavipes (Isoptera), against infection by the entomopathogenic fungus Metarhizium anisopliae

Reticulitermes flavipes workers were topically inoculated with ≈10,000 conidia of the entomopathogenic fungus Metarhizium anisopliae. After being kept in groups of 20 individuals for 1-9 d, histopathological examination showed that termites had an individual immune reaction. The nodule formation at the point of entrance of the fungal hyphae was identified as a cellular encapsulation and the different steps in the nodule formation are described. The relative number of hemocytes per termite increased 24 h after fungal exposure and remained high in the hemolymph for at least 3 d before decreasing back to pre-exposure levels. The role of an individual immune cellular reaction in social insects is discussed.     

The strepsipteran endoparasite Xenos vesparum alters the immunocompetence of its host, the paper wasp Polistes dominulus

It is unexplained how strepsipteran insects manipulate the physiology of their hosts in order to undergo endoparasitic development without being entrapped by the innate immune defences of the host. Here we present pioneering work that aimed to explore for the first time several components of the cellular and humoral immune response among immature stages of the paper wasp Polistes dominulus, in both unparasitized insects and after infection by the strepsipteran endoparasite Xenos vesparum. We carried out hemocyte counts, phagocytosis assays in vitro and antibacterial response in vivo. On the whole, hemocyte load does not seem to be drastically affected by parasitization: a non-significant increase in hemocyte numbers was observed in parasitized wasps as respect to control, while the two dominant hemocyte types were present with similar proportions in both groups. On the other hand, phagocytosis was significantly reduced in hemocytes from parasitized wasps while the antibacterial response seemed to be less effective in control. These somewhat unexpected results are discussed, along with the implications of a multiple approach in immune response studies.     

Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea

During oviposition, the endoparasitic wasp Pimpla hypochondriaca injects its pupal hosts with venom. This complex fluid has toxic properties and recently several venom components were characterized. In addition, it was suggested that venom might be involved in host immune suppression. For this to be the case, venom would have to adversely affect hemocytes and this aspect was further addressed in the current study utilizing the larval stage of the tomato moth Lacanobia oleracea as a model system. Using sublethal venom injections we investigated the effects of venom on encapsulation and hemocyte concentration. Additionally, the effects of venom on hemocyte morphology, viability, and phagocytic capability were determined in vitro. Injection of 16 microg of venom protein into sixth instar larvae was sufficient to reduce the ability of hemocytes to encapsulate Sephadex A25 beads by more than 50% in four of five insects examined. Hemocyte concentration in sixth instar larvae 32 h after injection with 16 microg of venom was reduced by 56% compared to that in controls. Damaged hemocytes and cell debris were also observed in hemolymph from venom-treated insects, suggesting that P. hypochondriaca venom has cytotoxic properties. In vitro incubation of washed hemocytes for 20 h with 500 ng/microl venom resulted in disintegration of a high proportion of hemocytes, leaving only parts of the plasma membrane and nucleus intact. Treatment with low concentrations of venom (1.6 ng/microl) resulted in an absence of spread plasmatocytes, which were abundant on control monolayers. High-resolution microscopy of hemocyte cultures exposed to 320 ng/microl venom for 3.5 h on glass slides indicated that venom induced a variety of effects on cellular morphology, including blebbing of the plasma membrane, degranulation, and the formation of cytoplasmic vacuoles. Incubation of hemocytes with 320, 64, or 3.2 ng/microl venom for 3.5 h reduced cell viability to 70, 90, and 92%, respectively, confirming that venom is cytotoxic to hemocytes. Treatment with 320 ng/microl venom reduced the capacity of hemocytes to phagocytose Escherichia coli by 85%. Together, these results demonstrate that at sublethal doses venom has a potent anti-hemocyte action and can impair hemocyte-mediated immune responses.     

Immune defense mechanisms of Culex quinquefasciatus (Diptera: Culicidae) against Candida albicans infection

Mosquitoes have an efficient defense system against infection. The cellular immune defense mechanism initiated by the mosquito Culex quinquefasciatus infected with the fungus Candida albicans was investigated in this study. Differences in the hemocyte counts in hemolymph perfused from uninoculated, saline-inoculated, and C. albicans-infected mosquitoes were compared using a light microscope. Phagocytosis was also investigated using electron microscopy. Four types of hemocytes were identified in control mosquitoes: prohemocytes (9.8%), plasmatocytes (38.8%), granular cells (44.2%), and oenocytoids (7.3%). Between 3 and 18 h postinoculation the total hemocyte count was significantly higher in infected, compared to uninfected, mosquitoes. Differential hemocyte counts from infected mosquitoes at 3, 6, and 18 h after inoculation showed that the relative proportion of plasmatocytes (48.6, 50.7, 45%) was higher and, concomitantly, the proportion of granular cells was lower (38, 36.8, 35%, respectively). Yeast cells were phagocytosed and limited growth was observed within the plasmatocytes. Melanized nodules were found attached to different insect tissues at 24 to 72 h following infection. These results suggest that phagocytosis, followed by nodule formation, was capable of clearing the hemolymph of yeast cells.     

The long‐term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor

The subject of this article is a search for the long‐term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro‐apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p‐NH₂)¹]‐ and [Phe(p‐OMe)¹]‐alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria‐challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide‐untreated bacteria‐challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide‐induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half‐life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N‐terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.     

Biological effects of a 765-kV, 60-Hz transmission line on honey bees (Apis mellifera L.): hemolymph as a possible stress indicator

Number of circulating hemocytes and hemolymph protein patterns of adult worker honey bees were analyzed as possible indicators of stress resulting from colony placement under a 765-kV transmission line. Although exposure to 55, 80, and 95 microA total induced hive current (THC) produced colony behavioral disturbance, there were no consistent effects on mean hemocyte counts at 55- or 95-microA THC. Age-dependent declines in circulating hemocyte number were similar in all exposure groups. There were no consistent differences in tube-gel electropherograms. No consistent differences were found in two-density slab-gel electropherograms based on ultrasensitive silver stain. The 67 positively charged and four negatively charged protein fractions from overwintering bees are two- to threefold more than currently reported in the literature.     

Characterisation of Mytilus edulis hemocyte subpopulations by single cell time-lapse motility imaging

In bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 μm min^?1. Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte–hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed.