Types Of Hemocytes Research Articles

Natural variation in differentiated hemocytes is related to parasitoid resistance in Drosophila melanogaster

As a measure of parasitoid resistance, hemocyte load and encapsulation ability were measured in lines collected from natural populations of Drosophila melanogaster in Europe. Results show large geographic variation in resistance against the parasitoid wasp Asobara tabida among the field lines, but there was no clear correlation between resistance and total hemocyte load, neither before nor after parasitization. This was in contrast to the patterns that had been found in a comparison among species of Drosophila, where total hemocyte counts were positively correlated to encapsulation rates. This suggests that the mechanisms underlying between-species variation in parasitoid resistance do not extend to the natural variation that exists within a species. Although hemocyte counts did not correspond to encapsulation ability within D. melanogaster, the ratios of lamellocytes and crystal cells were very similar in lines with successful encapsulation responses. Apart from variation in the hemocytic response of the different hemocyte types, within-species variation was also observed for accurate targeting of the foreign body by the hemocytes. These results are discussed in the context of possible causes of variation in immune functions among natural populations.

Silkworm plasmatocytes are more resistant than other hemocyte morphotypes to Bombyx mori nucleopolyhedrovirus infection

Differences in the viral susceptibility of multiple insect hemocyte morphotypes have not been investigated to date. In this study, a Bombyx mori nucleopolyhedrovirus (BmNPV) derivative possessing a Drosophila hsp70 promoter-driven green fluorescent protein (GFP) gene was used to observe NPV tropism of B. mori larval hemocytes. The experiments clearly revealed that there were fewer GFP-positive plasmatocytes than those observed in other types of hemocytes, such as granulocytes, oenocytoids, and spherulocytes, when infected via the intrahemocoelic or oral route. Our results indicate that silkworm plasmatocytes are more resistant than other hemocyte morphotypes to BmNPV infection.

유세포 분석기와 Neutral Red Retention Assay를 이용한 자연산 참굴(Crassostrea gigas)의 면역력 측정

남해의 광양만과 진해만에 서식하는 자연산 참굴의 건강성 평가를 위해 세포성 면역을 담당하는 혈구의 기능들을 유세포 분석기와 Neutral Red Retention(NRR) assay를 이용하여 신속, 정확하게 측정하였다. 광양만과 진해만의 안쪽과 바깥쪽에 서식하는 자연산 참굴의 혈구를 유세포 분석기를 이용하여 형태학적 특성에 따라 혈구의 종류를 분류하고, 혈구 종류별 수, 사멸률, DNA 손상도, 식세포능을 측정하였다. 또한 NRR assay를 이용하여 혈구의 lysosomal membrane stability를 측정하였다. 참굴의 혈구는 granulocytes, hyalinocytes, blast-like cells의 세 가지 종류로 분류되었다. 조사 지역 간의 혈구의 수, 사멸률, DNA 손상도는 유의적 차이가 없었지만, 식세포율과 lysosomal membrane stability와 같은 면역 관련 기능들은 유의적 차이가 있었다. 진해만은 내만과 외만 지역에 서식하는 참굴의 혈구 면역인자들 간에는 유의적 차이가 관찰되지 않았다. 이에 반해, 광양만에 서식하는 참굴은 내만 지역의 섬진대교가 외만 지역의 평산리 지역에 서식하는 참굴보다 낮은 식세포율과 낮은 lysosomal membrane stability를 보여 면역력이 저하되어 있는 것으로 추정된다. 하지만, 해양환경 변화와 시료의 면역력과의 상관관계를 이해하기 위해서는 조사지역의 환경적 특성이나 오염정도, 그리고 시료 내의 오염물질 축적량 등의 객관적인 분석 결과와의 종합적인 고찰이 필요할 것이다. 유세포 분석기와 NRR assay를 이용한 참굴 혈구 집단의 형태 변화 및 면역능 측정 기술은 시료의 전처리 없이 빠른 시간 내에 세포의 특성을 분석할 수 있는 유용한 분석 tool로 활용될 수 있음을 확인하였다. Hemocyte parameters of the wild Pacific oyster Crassostrea gigas inhabiting intertidal zones in small bays (Gwangyang and Jinhae Bay) on the southern coast of Korea were evaluated using flow cytometry and neutral red retention (NRR) assay. Morphological features, cell count, mortality, DNA damage, phagocytosis, and lysosomal membrane stability of hemocytes were analyzed. Three types of hemocytes were identified in the oyster hemolymph: granulocytes, hyalinocytes, and blast-like cells. Immune related functions of hemocyte including phagocytosis and lysosomal membrane stability were significantly different among the study areas (P<0.05), while cell count, mortality, and DNA damage of hemocytes were not significantly different. In Gwangyang Bay, phagocytosis of granulocytes and lysosomal membrane stability of oyster hemocytes inhabiting inside bay were significantly lower than those of oyster hemocytes in outside bay (P<0.05), indicating that oysters in inside bay of Gwangyang were relatively suppressed the immunological function in hemocytes. Contrary to Gwangyang Bay, immune parameters of oyster hemocytes in Jinhae Bay not showed the difference between sampling sites. In conclusion, flow cytometry and NRR assay using oyster hemocyte has a powerful tool to investigate the cell level in a short time due to no-preprocessing of material.

Looking for putative phenoloxidases of compound ascidians: Haemocyanin-like proteins in Polyandrocarpa misakiensis and Botryllus schlosseri

Phenoloxidases (POs) and haemocyanins constitute a family of copper-containing proteins widely distributed among invertebrates. Both of them are able, under appropriate conditions, to convert polyphenols to quinones and induce cytotoxicity through the production of reactive oxygen species, a fundamental event in many immune responses. In ascidians, PO activity has been described and studied in both solitary and colonial species and the enzyme is involved in inflammatory and cytotoxic reactions against foreign cells or molecules, and in the formation of the cytotoxic foci which characterise the nonfusion reaction of botryllids. Expressed genes for two putative POs (CiPO1 and CiPO2) have been recently identified in C. intestinalis.In the present study, we determined the cDNA sequences of two haemocyanin-like proteins from two colonial ascidians: Botryllus schlosseri from the Mediterranean Sea and Polyandrocarpa misakiensis from Japan. Multiple sequence alignments evidenced the similarity between the above sequences and crustacean proPOs whereas the analysis of the three-dimensional structure reveals high similarity with arthropod haemocyanins which share common precursors with arthropod proPOs. Botryllus HLP grouped in the same cluster with Ciona POs, whereas Polyandrocarpa HLP clustered with arthropod haemocyanins; all of them share the full conservation of the six histidines at the two copper-binding sites as well as of other motifs, also found in arthropod haemocyanin subunits, involved in the regulation of enzyme activity. In situ hybridisation indicated that the genes are transcribed inside morula cells, a characteristic haemocyte type in ascidians where PO activity is located, at the beginning of their differentiation. These results represent a first attempt to identify candidate molecules responsible of the PO activity in compound ascidians.

Effects of Gibberellic Acid on Hemocytes ofGalleria mellonellaL. (Lepidoptera: Pyralidae)

The impacts of different doses of the plant growth regulator gibberellic acid (GA(3)) in diet on the number of total and differential hemocytes, frequency of apoptotic, and necrotic hemocytes, mitotic indices, encapsulation, and melanization responses were investigated using the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. Total hemocyte counts increased in G. mellonella larvae at all treatment doses whereas GA(3) application had no effect on the number of different hemocyte types. The occurrence of apoptosis, necrosis and mitotic indices in GA(3) treated and untreated last instars were detected by acridine orange or ethidium bromide double staining by fluorescence microscopy. While the ratio of necrotic hemocytes increased at all GA(3) treatments, that of late apoptotic cells was only higher at doses >200 ppm when compared with untreated larvae. The percentage of mitotic index also increased at 5,000 ppm. Positively charged DEAE Sephadex A-25 beads were used for analysis of the levels of encapsulation and melanization in GA(3) treated G. mellonella larvae. At four and 24 h posttreatments with Sephadex A-25 bead injection, insects were dissected under a stereomicroscope. Encapsulation rates of larval hemocytes were dependent on the extent of encapsulation and time but not treatment groups. While the extent of melanization of hemocytes showed differences related to time, in general, a decrease was observed at all doses of GA(3) treated larvae at 24 h. We suggest that GA(3) treatment negatively affects hemocyte physiology and cell immune responses inducing cells to die by necrosis and apoptosis in G. mellonella larvae.

Parasitism by Cotesia flavipes alters the haemocyte population and phenoloxidase activity of the sugarcane borer, Diatraea saccharalis

AbstractParasitism of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) larvae by Cotesia flavipes Cameron (Hymenoptera: Braconidae) or injection of C. flavipes polydnavirus causes numerous alterations in host physiology including developmental arrest, abrogation of host immunity, and biochemical changes in proteins, carbohydrates, glycogen, and lipids. This study focused on changes in haemocyte composition occurring in the cellular immune system of parasitised D. saccharalis larvae. We also analysed the effects of parasitisation by C. flavipes on humoral immunity in terms of phenoloxidase (PO) activity in the host. In nonparasitised D. saccharalis larvae, granular cells represented the main haemocyte type (39%) and plasmatocytes were also present at around 35% among the total haemocytes. The percentages of these two major haemocytes decreased significantly in parasitised larvae after 6 days of parasitoid oviposition and the total haemocyte counts exhibited a significant reduction in parasitised larvae 3 and 6 days following parasitisation. The parasitised larvae also showed a significant decrease in humoral immune capacity as evidenced by reduction of PO activity. Moreover, the plasma had more PO activity than the haemocytes and the parasitised larvae showed less PO activity than the control larvae. This research demonstrates that the parasitism of C. flavipes adversely affects the total haemocyte populations and PO activity of D. saccharalis larvae, which would contribute to host immunosuppression.

Influence of low dosage infection on WSSV in latent period and haemocyte of Penaeus monodon

The changes in mortalities of Penaeus monodon(with 1×105 copies/g WSSV),WSSV loads in muscle,THC and the percentage of different types of haemocyte were studied by injection with WSSV solution containing 3×103,6×102,2×102 copies/mL virus and PBS.Real-time PCR,and microscopical examination were used in this experiment.The results showed that the cumulative mortalities were 93.33%±2.89%,56.67%±5.77%,45.00%±5.00% respectively,when the concentration of WSSV solution were 3×103,6×102,2×102 copies/mL.No death was observed in the PBS group.When the concentration of WSSV solution was 3×103 copies/mL,the virus load in the muscle reduced to minimum(3.54×104 copies/g)at 30 min post-injection,and reached its maximum(3.12×108 copies/g)at 48 h post-injection,then decreased again.For the other two experiment groups,the minimal virus load(1.11×105,9.54×104 copies/g)in the muscle was observed at 1 h post-injection and the highest virus load(1.48×107,5.46×106 copies/g)occurred at 48 h post-injection,but a second highest virus load(1.58×105,1.11×105 copies/g)appeared at 6 h post-injection.No significant changes were found in the WSSV load of the control group throughout the experiment.The fluctuation range and the peak time of total haemocyte count(THC)were different between different dosage of WSSV solution.Especially THC reduced to minimum(3.48×106,4.05×106/mL)at 12 h and 48 h post-injection with 3×103 copies/mL solution.The variation of THC and WSSV load were negatively correlated except individual time points.Under all groups,the percentage of semi-granular cells showed upward trend obviously early post infection,but later remained at a high level although fluctuated;the percentage of granular and hyalinocyte cells showed downward trend early post-injection significantly,while increased slightly in the middle period,but the percentage of granular cells at the later stage of post-infection was significantly lower than that of early period,while the levels of hyaline cells between later and early stages of post-injection have no significant difference.

Hemocyte–hemocyte adhesion and nodulation reactions of the greater wax moth, Galleria mellonella are influenced by cholera toxin and its B-subunit

Nodulation, the lepidopteran insect immune response to large numbers of microbes in the blood (hemolymph) consists of the coordination of the blood cell (hemocyte) types the granular cells and plasmatocytes in terms of granular cell–bacteria adhesion and hemocyte–hemocyte adhesion (microaggregation). Hemocyte–microbe adhesion is influenced by the secondary messenger, cAMP, and cAMP-dependent protein kinase A. In the present study, cholera toxin, an AB5 protein known to indirectly stimulate adenylate cyclase, is used to examine the hemocyte responses to glass, bacteria and hemocyte–hemocyte microaggregates. In vitro, this toxin induces a bimodal hemocyte adhesion response that varies with the holotoxin concentration in terms of the individual and aggregated hemocyte adhesion responses: the lower CTX concentration (1.2nM) increases microaggregate adhesion and decreases individual hemocyte binding to glass, as does higher concentrations (6–120nM), however microaggregates induced by lower concentrations do not adhere to glass. Cholera toxin-induced microaggregation is inhibited by RGDS, suggestive of integrin involvement. In vivo, cholera toxin (1.2–120nM) injected into larvae induces also a bimodal hemocytic response: low levels (1.2–6nM) cause reduced hemocyte adhesion, while high levels (12–120nM) increase hemocyte release or mobilization of adhesive hemocyte counts in the hemolymph. Increasing levels of cholera toxin concomitantly injected with the non-pathogenic bacterium, Bacillus subtilis produces a bimodal pattern in bacterial removal from the hemolymph which correlates with nodule frequency in larvae injected with cholera toxin only. The effects of higher concentrations of cholera toxin in vitro (6–120nM) and in vivo (12–120nM) are mediated by the B-subunit, whereas the isolated A-subunit has no effect on hemocyte activity. Cholera toxin and its individual subunits did not detectably alter levels of intracellular cAMP in the hemocytes, suggesting a cAMP-independent mechanism stimulating the nodulation response.

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae): Effects of parasitism on differential hemocyte counts, micro‐ and ultra‐structures of host hemocytes

Abstract Parasitism by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae) by using only its associated venom, can suppress the immunal responses of Pieris rapae (Lepidoptera: Pieridae). However, up to now, current knowledge of the mechanisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy (TEM). Five hemocyte types, prohemocytes (PRs), granulocytes (GRs), plasmatocytes (PLs), oenocytoids (OEs) and coagulocytes (COs), were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.

Insect immune responses to nematode parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis.

Morphological characterization of hemocytes from Biomphalaria glabrata and Biomphalaria straminea

Biomphalaria glabrata and Biomphalaria straminea have been identified as intermediate hosts for Schistosoma mansoni. Several studies have found two cell types in the hemolymph of B. glabrata (hyalinocytes and granulocytes). However, there are no studies describing the hemocytes of B. straminea. With the aim of further describing the hemocyte subsets in B. glabrata and B. straminea, we conducted a detailed study using optical microscopy and transmission electron microscopy. Based on the morphological characteristics of the cells, we identified the same types of hemocytes in two species of molluscs, namely: blast-like cells, granulocytes, type I hyalinocytes, type II hyalinocytes and type III hyalinocytes. Blast-like cells had a spherical profile with a central nucleus filling almost the whole cell. Granulocytes were characterized by presenting variable numbers of granules. Type I hyalinocytes were the most abundant cell type and displayed various cytoplasmic projections. Type II and type III hyalinocytes had never previously been reported. They were few in number and were characterized by having an eccentric nucleus. From these results, it is concluded that there are five types of cells in the hemolymph of B. glabrata and B. straminea. Further studies are now needed to identify the role of these hemocytes in the immune response of these snails.

Immune responses and ultrastructural changes of hemocytes in freshwater crab Sinopotamon henanense exposed to elevated cadmium

Cadmium (Cd) is one of the most toxic heavy metals that can impact immunological parameters in aquatic animals. To investigate the immunotoxicity and ultrastructural changes of hemocytes, specimens of Sinopotamon henanense were exposed to different concentrations of cadmium and the differences in immunologic parameters between Cd exposure groups and control groups were investigated. Total hemocyte count (THC) in Cd-exposure groups were decreased significantly when compared with the control groups, especially in the groups treated with higher Cd concentrations and longer exposure time, while no significant differences were observed in the proportions of the three types of hemocytes. Phenoloxidase (PO) activities were significantly higher in Cd-exposure groups than the control groups. Superoxide dismutase (SOD) activities gradually increased in 7.25 and 14.5 mg L −1 Cd groups, but in other higher Cd groups, they showed first increase and following decrease with the exposure time prolonged. Acid phosphatase (ACP) activities were induced at 48 h, and then decreased, while alkaline phosphatase (AKP) activities increased gradually until 96 h. Electron microscopic results showed that nucleus, mitochondria and rough endoplasm recutulum (rER) of three types of hemocytes were sensitive to acute Cd toxicity. In Cd-exposed groups, chromatin condensation, nucleus deformation and nucleus envelope rupture were noted. Additionally, mitochondrial dilation and rER degranulation were observed in Cd-treated crabs. These results suggested that immune response and organelles of hemocyte of S. henanense were impacted by Cd exposure, and the changes of these immunologic parameters reflect changes in crab immune response capability consequent to Cd exposure.

PGE 2 induces oenocytoid cell lysis via a G protein-coupled receptor in the beet armyworm, Spodoptera exigua

Eicosanoids mediate cellular and humoral immune responses in the beet armyworm, Spodoptera exigua, including activation of prophenoloxidase (PPO). PPO activation begins with release of its inactive zymogen, PPO, from oenocytoids in response to prostaglandins (PGs). Based on the biomedical literature, we hypothesized that PGs exert their actions via specific G protein-coupled receptor(s) in S. exigua. This study reports a G protein-coupled receptor (Se-hcPGGPCR1) gene, which is expressed in the hemocytes of S. exigua. The Se-hcPGGPCR1 consists of 420 amino acids and belongs to rhodopsin-type GPCRs. The high content of hydrophobic amino acid residues within the Se-hcPGGPCR1 protein is explained by prediction of seven-transmembrane domains that are characteristic of these GPCRs. Except for the eggs, Se-hcPGGPCR1 was expressed in all life stages. During the larval stage, it was expressed in hemocytes and gut, but not in fat body nor in epidermis. Real time quantitative RT-PCR showed that bacterial challenge induced more than 20-fold increases in its expression level. Fluorescence in situ hybridization showed that Se-hcPGGPCR1 was expressed in a specific hemocyte type, the oenocytoids. A specific eicosanoid, PGE 2, significantly induced oenocytoid lysis and increased PO activity in the plasma. In contrast, when Se-hcPGGPCR1 expression was suppressed by RNA interference (RNAi), the oenocytoid lysis and the PO activation in response to PGE 2 were not elevated above basal levels. A binding assay using intracellular calcium mobilization showed that the RNAi-treated hemocytes were significantly less responsive to PGE 2 than the control hemocytes. These results support our hypothesis with the specific finding that PGE 2 acts through Se-hcPGGPCR1 to activate PPO by lysing oenocytoids.

Insight on cellular and humoral components of innate immunity in Squilla mantis (Crustacea, Stomatopoda)

For deeper insights into the function of crustacean haemocytes in immune responses, we studied the morphology and enzyme content of circulating cells of the mantis shrimp Squilla mantis from the North Adriatic Sea, together with their ability to phagocytose foreign cells. We also assayed the enzyme content and the agglutinating and haemolytic activities of cell-free haemolymph. Three haemocyte types, i.e., hyalinocytes, semigranulocytes and granulocytes, can be distinguished, according to cell and nuclear morphology and the presence of cytoplasmic granules. All of them share the same patterns of enzyme activities and are recognised by the same lectins. Spreading cells (hyalinocytes and semigranulocytes) can ingest foreign cells; granules of semigranular and granular cells have similar cytochemical properties. Injection of Micrococcus luteus into the heart sinus results in an increase in the frequency of hyaline cells and a decrease in the frequency of granulocytes. After 24 h from the injection, a decrease in the number of phagocytosing hyalinocytes, and a general decrease in the frequency of acid phosphatase-positive cells was reported. Our data match previous results and suggest the existence of a single differentiation pathway for Squilla haemocytes with the three haemocyte morphs as different stages of cell differentiation. Results also indicate that Squilla haemolymph performs immunosurveillance, through rapid changes in haemocyte distribution, increase of antimicrobial and antioxidant enzymes and secretion of lectins stimulating agglutination, phagocytosis and encapsulation.

Structural and functional characterization of pseudopodocyte, a shaggy immune cell produced by two Drosophila species of the obscura group

We recently reported that most of the Drosophila species of the obscura group were unable to mount cellular capsules and no lamellocyte was ever found in the hemolymph of any of the tested species. Only three species were able to encapsulate, despite lacking lamellocytes. Their encapsulation ability was always associated with the presence of an unpreviously described kind of capsule-forming immunocytes designated as “atypical hemocytes”. Here, we describe the ultrastructural and functional characteristics of this type of hemocyte. We show that these cells share many ultrastructural and morphological features with Drosophila melanogaster plasmatocytes, although they are involved in the formation of the external layers of the cellular capsule, a functional property exhibited by lamellocytes in D. melanogaster. Due to the high number of pseudopodes in these cells, we suggest to name them “pseudopodocytes”. After structural and functional characterization of these atypical hemocytes, their ambiguous status between plasmatocytes and lamellocytes is discussed.

Localization of antimicrobial peptides in the tunic of Ciona intestinalis (Ascidiacea, Tunicata) and their involvement in local inflammatory-like reactions

Tunicates comprising a wide variety of different species synthesize antimicrobial peptides as important effector molecules of the innate immune system. Recently, two putative gene families coding for antimicrobial peptides were identified in the expressed sequence tag database of the tunicate Ciona intestinalis. Two synthetic peptides representing the cationic core region of one member of each of the families displayed potent antibacterial and antifungal activities. Moreover, the natural peptides were demonstrated to be synthesized and stored in distinct hemocyte types. Here, we investigated the presence of these natural peptides, namely Ci-MAM-A and Ci-PAP-A, in the tunic of C. intestinalis considering that the ascidian tunic is a body surface barrier exposed to constant microbial assault. Furthermore, as the tunic may represent a major route of entry for pathogen invasion after its damage we monitored the location of these peptides upon a local inflammatory-like reaction induced by injection of foreign cells. Using immunocytochemistry and electron microscopy both peptides were localized to the tunic and were massively present in granulocytes of inflamed tissue. Conclusively, antimicrobial peptides may constitute a chemical barrier within the tunic of urochordates.

Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the Drosophila lymph gland

The Drosophila melanogaster hematopoietic organ, called lymph gland, proliferates and differentiates throughout the larval period. The lymph gland of the late larva is comprised of a large primary lobe and several smaller secondary lobes. Differentiation into two types of hemocytes, plasmatocytes and crystal cells, is confined to the outer layer (cortical zone) of the primary lobe; the center of the primary lobe (medullary zone), as well as the secondary lobes, contain only proliferating prohemocytes. A small cluster of cells located at the posterior tip of the primary lobe serves as a signaling center (PSC) that inhibits precocious differentiation of the medullary zone. The larval lymph gland is stabilized by layers of extracellular matrix (basement membranes) that surround individual hemocytes, groups of hemocytes, as well as the lymph gland as a whole. In this paper, we investigated the events shaping the lymph gland in the early pupa. The lymph gland dissociates and hemocytes disperse during the first 12h after puparium formation (APF), leaving behind empty husks of basement membrane. Prior to lymph gland dissociation, cells of the medullary zone differentiate, expressing the early differentiation marker Peroxidasin (Pxn), as well as, in part, the late differentiation marker P1. Cells of the PSC spread throughout the pupal lymph gland prior to their dispersal. Cells of the secondary lobes undergo a rapid phase of proliferation that lasts until 8h APF, followed by expression of Pxn and dispersal. These hemocytes do not express P1, indicating that they disperse prior to full maturation.

Losing the battle against fungal infection: Suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2×103, 2×106 or 2×108 conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.

The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia.

BackgroundMost of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes.Methodology/Principal FindingsIn A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×103 Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson's test = 12 822.98, df = 2, p<0.001).Conclusions/SignificanceThe characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.