Types Of Hemocytes Research Articles

Oxidative burst activity in haemocytes of the freshwater prawn Macrobrachium rosenbergii

Oxidative burst, release of reactive oxygen species/reactive nitrogen species (ROS/RNS) contributed to microorganisms killing, is a vital immune response of crustacean haemocyte. Three morphologic haemocyte types (hyaline cells, HC; semigranular cells, SGC; granular cells, GC) have been defined in crustaceans, and found to play different roles in immune defense. However, oxidative burst activities of different haemocyte subpopulations in crustaceans are currently not documented. In the present study, we investigated the oxidative burst activities of the three haemocyte types in the freshwater prawn Macrobrachium rosenbergii using the common ROS fluorescent probe dichlorofluorescin-diacetate (DCFH-DA). Nitric oxide (NO) donor sodium nitroprusside (SNP) improved the DCF fluorescence in haemocytes, while NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO-synthase inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced the fluorescence, suggesting that DCF fluorescence intensity could also be modified by intracellular NO level and activity of NO-synthase pathway. ROS/RNS was also produced in the untreated haemocytes. GC contained most non-induced ROS/RNS production, while oxidative activity of HC was rather weak. No significant impact of PMA could be observed on ROS/RNS level in all the three cell types. Both zymosan A (ZA) and lipopolysaccharide (LPS) significantly triggered the production of ROS/RNS in SGC and GC, whereas they had no effect on those of HC, suggesting that SGC and GC were the primary cell types involved in pathogens killing by ROS/RNS pathway. Cytochalasin B (Cyt B) inhibited the ZA-induced ROS/RNS production, but could not change the ROS/RNS level stimulated by LPS. For unstimulated haemocytes, ROS/RNS productions decreased 29.6%, 44.1% and 48.6% in SGC, and decreased 44.5%, 28.4% and 57.3% in GC, in the presence of L-NMMA, Fccp and DPI respectively, whereas apocynin could not modulate DCF fluorescence in both SGC and GC, suggesting that mitochondrial oxidative pathway was relatively more dominant in SGC, and NO-synthase (NOS) pathway appeared more active in GC. For LPS-stimulated haemocytes, oxidative activities decreased 22.9%, 42.9%, 29.6% and 60.0% in SGC, and reduced 40.6%, 25.2%, 26.7% and 70.6% in GC with the presence of L-NMMA, apocynin, Fccp and DPI respectively, suggesting that NADPH-oxidase (NOX) pathway in both SGC and GC was activated by LPS, and it became the predominant oxidative pathway in stimulated SGC, while NOS pathway was the relative main source for ROS/RNS production in stimulated GC.

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

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

RNA-seq identifies integrin alpha of kuruma shrimp Marsupenaeus japonicus as a candidate molecular marker for phagocytic hemocytes

Phagocytosis is main cellular immunity, however, it is still unknown or debated upon which types of hemocyte contributes phagocytosis in penaeid shrimps. The hemocyte characterization in kuruma shrimp have been mainly performed based on its morphology by microscopic observation. Therefore, establishment of molecular markers to distinguish phagocytic hemocytes is required. In this study, using magnetic fluorescent beads, we enriched phagocytic hemocytes and conducted RNA-seq analysis between total and enriched phagocytic hemocytes. The data demonstrated functional difference between total and phagocytic hemocytes. In addition, a transcript homologous to integrin-alpha was highly expressed in phagocytic hemocytes, and named Mj-Intgα. Using anti-serum against Mj-Intgα revealed that around 60% of total hemocytes and more than 90% of phagocytic hemocytes showed positive for Mj-Intgα. This study presents Mj-Intgα as a candidate molecular marker for future functional characterization of hemocytes.

Disruptive Impacts of Selected Insecticides on Larval Haemogram parameters of the Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

The present study was conducted to investigate the effect of three insecticides,pyriproxyfen (admeral), neemazal (azadirachtin) and spinetoram (Radiant), against the red palm weevil Rhynchophorus ferrugineus. Two sublethal concentrations of each compound had been estimated in a preliminary test: LC50: 1067.5 ppm, LC75: 2317.5 ppm (pyriproxyfen), LC50: 14600 ppm, LC75: 27100 ppm (neemazal), LC50: 18.37 ppm, LC75: 88.60 ppm (spinetoram). These concentrations were tested against the immune cells (Total hemocyte count, Differential hemocyte count and hemocyte deformations). Depending on the present study, five types of hemocytes were observed as prohemocyte, granulocyte, plasmatocyte, oenocyte and spherulocyte. Total hemocyte count was significantly increased irrespective to the insecticide. All insecticide induced the prohemocyte while the other types were declined. However, the tested insecticide exhibited pathological symptoms on insect haemocytes morphology in the cell membrane, cytoplasm and nucleus. v

IMMUNE PROTECTION OF SALVINIA MOLESTA D.S. MITCHELL IN FRESHWATER CRAB OZIOTELPHUSA SENEX SENEX BACTERIALLY CHALLENGED WITH AEROMONAS HYDROPHILA

Objective: This is aimed to study the immune protection parameters of freshwater weed Salvinia molesta in bacterially challenged freshwater crab Oziotelphusa senex senex.Methods: In this present study, ethanolic extract of freshwater weed S. molesta was tested for its ability to induce immunity in bacterial challenged freshwater crab O. senex senex. Male and female crabs were challenged with Aeromonas hydrophila in relevant concentrations. The treated groups were allowed to withstand for 96 hrs. After relevant incubation time, the hemolymph of the treated crabs was subjected for various hematological, biochemical, and immunological assays.Results: Total hemocyte count increased on infection at 96 hrs, whereas significantly reduced on treatment with S. molesta at 96 hrs. All the three cell types of differential hemocytes showed significant positive changes on treatment. Levels of prophenoloxidase decreased significantly on infection and showed a significant increase in treated groups at 96 hrs of treatment.Conclusion: The present study elucidated the medicinal and pharmaceutical role of S. molesta weed which is been subjected to eradication in the recent days. Thus, the plant source can be utilized as an immunomodulatory agent and a better alternative to treat aquatic diseases.

The Drosophila lymph gland is an ideal model for studying hematopoiesis

Hematopoiesis in Drosophila melanogaster occurs throughout the entire life cycle, from the embryo to adulthood. The healthy lymph gland, as a hematopoietic organ during the larval stage, can give rise to two mature types of hemocytes, plasmatocytes and crystal cells, which persist into the pupal and adult stages. Homeostasis of the lymph gland is tightly controlled by a series of conserved factors and signaling pathways, which also play key roles in mammalian hematopoiesis. Thus, revealing the hematopoietic mechanisms in Drosophila will advance our understanding of hematopoietic stem cells and their niche as well as leukemia in mammals. In addition, the lymph gland employs a battery of strategies to produce lamellocytes, another type of mature hemocyte, to fight against parasitic wasp eggs, making the lymph gland an important immunological organ. In this review, the developmental process of the lymph gland and the regulatory networks of hematopoiesis are summarized. Moreover, we outline the current knowledge and novel insight into homeostasis of the lymph gland.

Transgenic characterization of two silkworm tissue-specific promoters in the haemocyte plasmatocyte cells.

Haemocytes play crucial roles in insect metabolism, metamorphosis, and innate immunity. As a model of lepidopteran insects, the silkworm is a useful model to study the functions of both haematopoiesis and haemocytes. Tissue-specific promoters are excellent tools for genetic manipulation and are widely used in fundamental biological research. Herein, two haemocyte-specific genes, Integrin β2 and Integrin β3, were confirmed. Promoter activities of Integrin β2 and Integrin β3 were evaluated by genetic manipulation. Quantitative real-time PCR and western blotting suggested that both promoters can drive enhanced green fluorescent protein (EGFP) specifically expressed in haemocytes. Further evidence clearly demonstrated that the transgenic silkworm exhibited a high level of EGFP signal in plasmatocytes, but not in other detected haemocyte types. Moreover, EGFP fluorescence signals were observed in the haematopoietic organ of both transgenic strains. Thus, two promoters that enable plasmatocytes to express genes of interest were confirmed in our study. It is expected that the results of this study will facilitate advances in our understanding of insect haematopoiesis and immunity in the silkworm, Bombyx mori.

Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives

For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1–5, 6–10, 11–15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of β-laminarin-treated granulocytes decreased significantly for zymosan and zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of β-glucan receptors for phagocytosis.

ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas

ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraíba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types, including hyalinocytes from the oyster C. gigas. Additionally, our results showed that C. gigas exhibited higher activity of ABCC1-like transporter in all hemocyte types than C. gasar. The present work is the first to characterize ABCB1 and ABCC1-like transporter activity in the immune system cells of sea urchins E. lucunter and E. esculentus and oysters. Our findings encourage the performing studies regarding ABC transporters activity/expression in immune system cells form marine invertebrates under stress conditions and the possible use of ABC transporters as biomarkers.

Various cellular responses of different shrimp haemocyte subpopulations to lipopolysaccharide stimulation

Different haemocyte types have been reported to play diverse roles in immune defense of shrimp. To investigate the roles of the three haemocyte types [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)] of shrimp in immune responses against lipopolysaccharide (LPS), percentage, non-specific esterase activity (EA), reactive oxygen species (ROS) production and nitric oxide (NO) production of the three haemocyte subpopulations were analyzed in LPS-injected Penaeus monodon using flow cytometry. Results showed that percentage of HC increased after 3 h injection, and returned to the original level after 48 h. Proportion of SGC and GC reduced after 6–36 h and 3–12 h respectively, and recovered to the initial level after 48 and 24 h respectively. Loss of SGC and GC might be related to degranulation to release proPO system, and degranulation of GC seemed more sensitive to LPS stimulation. EA of both HC and SGC improved after 3–6 h injection, while EA of GC was induced after 3–24 h. No significant effect of LPS injection could be found in ROS production and NO production of HC. Enhanced ROS levels was observed in SGC and GC after 3–24 h and 3–36 h respectively, and NO production of SGC and GC improved after 3–48 h injection. These results demonstrated that SGC and GC possessed strong capabilities for LPS-induced EA, ROS production and NO production, while HC only displayed EA response to LPS, suggesting that GC and SGC play the main role in immune defense of shrimp against Gram-negative bacteria.

Characterization of novel bangle lectin from Photorhabdus asymbiotica with dual sugar-binding specificity and its effect on host immunity.

Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcβ1–4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed β-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer—the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.

Flow cytometric characterization of hemocytes of the solitary ascidian, Halocynthia roretzi

Internal defense of ascidians relies, at least partially, on cells circulating in body fluids and infiltrating in tissues, referred to as hemocytes, although structure and composition of ascidian hemocytes still remain unclear. In the present study, we investigated hemocyte types and their functions of the solitary ascidian Halocynthia roretzi using flow cytometry. Based on morphology, cellular activities and intracellular parameters from the flow cytometry, we identified eight hemocyte types including, three granulocytes (Gr-1, Gr-2, and Gr-3), 4 hyalinocytes (Hy-1, Hy-1′, Hy-2, and Hy-3) and lymphocyte-like (Ly-like) cells. The granulocyte Gr-1 accounted for 30% of the total circulating hemocytes and exhibited highest density of lysosomes and oxidative activity. Gr-1 was deeply involved in phagocytosis and degradation of foreign material. Hyalinocytes consist of two main populations, Hy-1 and Hy-2, and each accounted for 30% of the circulating hemocyte. Hy-1 displayed lysosomal content, an inducible oxidative activity, and no proteases, while Hy-2 expressed highest density of intracellular proteases, no lysosomes and a low oxidative activity. It was believed that Hy-2 may represent an important link between cellular and humoral immune reactions. Hy-1 did not show phagocytosis activity. Hy-3 and the Ly-like cells presented a similar profile except for their size and complexity, and Hy-3 may represent an intermediate differentiation/maturation step between Ly-like cells and other hemocyte populations. This first characterization of the hemocyte populations of H. roretzi provides a solid basis to investigate further their respective roles and functions in physiological and pathological contexts.

Haemocyte Morphology and Differential Haemocyte Counts of Giant Ladybird Beetle, &lt;i&gt;Anisolemnia dilatata&lt;/i&gt; (F.) (Coleoptera:Coccinellidae):A Unique Predator of Bamboo Woolly Aphids

Changes in haemolymph characteristics such as differential counts of haemocytes have direct bearing on the general performance of insects. The present study was carried out to generate data on the morphology of different haemocytes and their differential counts of giant ladybird predator, Anisolemnia dilatata (F.), unique to woolly aphid pests of bamboo habitat. Five types of haemocytes, viz. prohaemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytes were morphologically characterized in the haemolymph of larvae, pupae, virgin females and males. Among these, plasmatocytes were dominant followed by granulocytes, prohaemocytes, spherulocytes and oenocytes. Granulocytes showed consistency in numbers in all life cycle stages from first instar larva to adults of males and females of the giant ladybird.

Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria.

Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

Ultrastructural and immunocytochemical analysis of circulating hemocytes from Cherax quadricarinatus (von Martens, 1868)

Transmission electron microscopy (TEM) and immunocytochemistry were used to assay the circulating hemocytes characteristics in Cherax quadricarinatus (von Martens, 1868). Three haemocyte types could be identified in circulating hemocytes by TEM, i.e. hyaline cells (HC), semigranular cells (SGC), or granular cells (GC). The circulating hemocytes from C. quadricarinatus could be separated into layers by density gradient centrifugation. The CD34+ cells which are believed to be stem cell-like cells has been discovered in the first layer via immunocytochemical assay, which has not been reported in previous studies. These CD34+ cells may be immature cells derived from a single undifferentiated precursor cell line in specialized hematopoietic tissue (HPT).

The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas

Hemocytes comprise diverse cell types with morphological and functional heterogeneity and play indispensable roles in immunological homeostasis of invertebrates. The morphological classification of different hemocytes in mollusk has been studied since the 1970's, yet the involvement of the different sub-populations in immune functions is far from clear. In the present study, three types of hemocytes were morphologically identified and separated as agranulocytes, semi-granulocytes and granulocytes by flow cytometry and Percoll® density gradient centrifugation. The granulocytes were characterized functionally as the main phagocytic and encapsulating population, while semi-granulocytes and agranulocytes exhibited low or no such capacities, respectively. Meanwhile, the lysosome activity and the productions of ROS and NO were all mainly concentrated in granulocytes under both normal and immune-activated situations. Further, the mRNA transcripts of some immune related genes, including CgTLR, CgClathrin, CgATPeV, CgLysozyme, CgDefensin and CgIL-17, were mainly expressed in granulocytes, lower in semi-granulocytes and agranulocytes. These results collectively suggested that the granulocytes were the main immunocompetent hemocytes in oyster C. gigas, and a differentiation relationship among these three sub-population hemocytes was inferred based on the gradual changes in morphological, functional and molecular features.

Analysis of hemocytes in Lymnaea stagnalis: Characterization and effects of repeated hemolymph collections

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

Optimization of Non-Thermal Plasma Treatment in an In Vivo Model Organism.

Non-thermal plasma is increasingly being recognized for a wide range of medical and biological applications. However, the effect of non-thermal plasma on physiological functions is not well characterized in in vivo model systems. Here we use a genetically amenable, widely used model system, Drosophila melanogaster, to develop an in vivo system, and investigate the role of non-thermal plasma in blood cell differentiation. Although the blood system in Drosophila is primitive, it is an efficient system with three types of hemocytes, functioning during different developmental stages and environmental stimuli. Blood cell differentiation in Drosophila plays an essential role in tissue modeling during embryogenesis, morphogenesis and also in innate immunity. In this study, we optimized distance and frequency for a direct non-thermal plasma application, and standardized doses to treat larvae and adult flies so that there is no effect on the viability, fertility or locomotion of the organism. We discovered that at optimal distance, time and frequency, application of plasma induced blood cell differentiation in the Drosophila larval lymph gland. We articulate that the augmented differentiation could be due to an increase in the levels of reactive oxygen species (ROS) upon non-thermal plasma application. Our studies open avenues to use Drosophila as a model system in plasma medicine to study various genetic disorders and biological processes where non-thermal plasma has a possible therapeutic application.

Endoenzymes and Mitochondrial Membrane Potential Associated with Hemocyte Types inMacrobrachium rosenbergii

ABSTRACT Hemocyte types of the Macrobrachium prawn Macrobrachium rosenbergii were identified using the methods of flow cytometry and light microscopy. The activities of endoenzymes including peroxidase (POD), phenoloxidase (PO), and alkaline phosphatase (ALP) in hemocytes were analyzed by cytochemical and biochemical methods. Mitochondrial membrane potential (MMP) of the hemocyte was probed according to the accumulation of the cyanine dye, 3′, 3′-dihexyloxacarbocyanine iodide, in the mitochondrial matrix. The results showed that there were three types of circulating hemocytes (hyalinocytes, semigranulocytes, and granulocytes) in M. rosenbergii. Enzymes POD, PO, and ALP were more abundant and active in granulocytes and semigranulocytes than in hyalinocytes. The data associated with MMP showed that the fluorescence intensity in the hemocyte was directly correlated to cell complexity. The MMP in granulocytes was significantly greater than that in semigranulocytes, and the latter was significantly greater tha...

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection.

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail.