Percentage Of Hemocytes Research Articles

Histone lysine-specific demethylase 1 regulates the proliferation of hemocytes in the oyster Crassostrea gigas.

Lysine-specific demethylase 1 (LSD1) is an essential epigenetic regulator of hematopoietic differentiation, which can specifically mono-methylate H3K4 (H3K4me1) and di-methylate H3K4 (H3K4me2) as a transcriptional corepressor. Previous reports have been suggested that it participated in hematopoiesis and embryonic development process. Here, a conserved LSD1 (CgLSD1) with a SWIRM domain and an amino oxidase (AO) domain was identified from the Pacific oyster Crassostrea gigas. We conducted a comprehensive analysis by various means to verify the function of CgLSD1 in hematopoietic process, including quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, immunofluorescence assay, RNA interference (RNAi) and flow cytometry. The qRT-PCR analysis revealed that the transcripts of CgLSD1 were widely expressed in oyster tissues with the highest level in the mantle. And the transcripts of CgLSD1 were ubiquitously expressed during larval development with the highest expression level at the early D-veliger larvae stage. In hemocytes after Vibrio splendidus stimulation, the transcripts of CgLSD1 were significantly downregulated at 3, 6, 24, and 48 h with the lowest level at 3 h compared to that in the Seawater group (SW group). Immunocytochemical analysis showed that CgLSD1 was mainly distributed in the nucleus of hemocytes. After the CgLSD1 was knocked down by RNAi, the H3K4me1 and H3K4me2 methylation level significantly increased in hemocyte protein. Besides, the percentage of hemocytes with EdU-positive signals in the total circulating hemocytes significantly increased after V. splendidus stimulation. After RNAi of CgLSD1, the expression of potential granulocyte markers CgSOX11 and CgAATase as well as oyster cytokine-like factor CgAstakine were increased significantly in mRNA level, while the transcripts of potential agranulocyte marker CgCD9 was decreased significantly after V. splendidus stimulation. The above results demonstrated that CgLSD1 was a conserved member of lysine demethylate enzymes that regulate hemocyte proliferation during the hematopoietic process.

ROS function as an inducer of autophagy to promote granulocyte proliferation in Pacific oyster Crassostrea gigas

Hematopoiesis is the biological process to generate new blood cells in the living body and reactive oxygen species (ROS) contribute significantly to the regulation of haematopoietic cell homeostasis. In the present study, the involvement of ROS in the proliferation of haemocytes was examined in Pacific oyster Crassostrea gigas. The ROS content in haemocytes increased significantly after lipopolysaccharide (LPS) treatment, but decreased after the treatment with antioxidant N-Acetyl-L-cysteine (NAC, a scavenger of ROS). The percentage of 5-ethynyl-2′-deoxyuridine labeled (EdU+) granulocytes in total haemocytes significantly increased at 12 h (4.12-fold, p < 0.001) and 24 h (2.36-fold, p < 0.001) after LPS treatment, while decreased at 12 h (0.26-fold, p < 0.001) and 24 h (0.61-fold, p < 0.05) after NAC treatment, respectively. Meanwhile, the percentage of haemocytes with autophagosome positive signals significantly increased at 12 h (1.17-fold, p < 0.01) and 24 h (1.19-fold, p < 0.05) after LPS treatment, but significantly reduced at 12 h (0.41-fold, p < 0.001) and 24 h (0.28-fold, p < 0.001) after the NAC treatment, respectively. After ammonium chloride (NH4Cl) treatment, the percentage of haemocytes with autophagosome and EdU+ granulocytes significantly increased at 12 h, which was 1.27-fold (p < 0.01) and 1.70-fold (p < 0.01) of control group, respectively. These results collectively suggested that ROS produced after LPS treatment could act as an inducer for autophagy and involved in regulating the proliferation of some granulocytes in C. gigas.

Immunotoxicity in ascidians: Antifouling compounds alternative to organotins—IV. The case of zinc pyrithione

New biocides such as the organometallic compound zinc pyrithione (ZnP) have been massively introduced by many countries in formulations of antifouling paints following the ban on tributyltin (TBT). The effects of sublethal concentrations (LC50=82.5μM, i.e., 26.2mg/l) on cultured haemocytes of the ascidian Botryllus schlosseri have been investigated and compared with TBT. The percentage of haemocytes with amoeboid morphology and containing phagocytised yeast cells were significantly (p<0.05) reduced after exposure to 0.1 (31.7μg/l) and 0.5μM (158μg/l), respectively. An antagonistic interaction in inducing cytoskeletal alterations was observed when ZnP and TBT were co-present in the exposure medium. ZnP affected only the actin component. As caused by TBT, ZnP induced apoptosis and inhibited both oxidative phosphorylation and lysosomal activities. In contrast to the case of TBT, a decrement in Ca2+-ATPase activity and a decrease in cytosolic Ca2+ were detected after incubation at the highest concentration (1μM, i.e., 317.7μg/l) used. In comparison with other antifouling compounds, ZnP shows as much toxicity as TBT to cultured haemocytes at extremely low concentrations interfering with fundamental cell activities.

Fate of GFP-expressing Escherichia coli in the midgut and response to ingestion in a tick, Ornithodoros moubata (Acari: Argasidae)

Ticks are well-known vectors of various pathogens but migration of the pathogens in the tick midgut is not fully understood. In the present study, the fate of microbes in the midgut of Ornithodoros moubata was observed using green fluorescent protein (GFP)-expressing Escherichia coli. Fluctuations in the percentage of hemocytes in the hemolymph (Hc) and expression of an antimicrobial peptide, defensin, in the midgut was also investigated. Most E. coli gradually disappeared in the midgut after ingestion fluctuations in Hc coincided with the changes. Expression of defensin was also confirmed and slightly up-regulated after E. coli ingestion. Moreover, it was demonstrated that E. coli can not pass through the tick midgut epithelium after ingestion by the hemolymph cultures. It is known that various pathogens and host immunoglobulins ingested with a blood meal can enter into the hemocoel, which suggests the presence of unique and complex passage mechanisms for each molecule and organism. The results obtained here help to clarify that digestion enzymes is an important function of the tick midgut to protect against invading molecules and organisms.

A brief review of the immunobiology of Eledone cirrhosa

Blood can be sampled repeatedly from the lesser octopus Eledone cirrhosa (Lamarck) and the haemocytes cultured for up to 72 h. Sampling results in an increase in the number of circulating haemocytes per mland in the percentage of haemocytes containing cytoplasmic granules, and a change in the staining pattern of the haemocytes. It also causes a decrease in the quantity of copper in the haemolymph. The haemocytes phagocytose bacteria (Vibrio anguillarum) in vitro in the absence of haemolymph, but there is enhanced phagocytosis in the presence of haemolymph. Haemocytes migrate towards low concentrations of various blood preparations, to lipopolysaccharide (LPS) and to preparations that had contained live bacteria. They also have an apparent bacteriostatic effect on the growth of live bacteria. Haemocytes produce intracellular reactive oxygen species after incubation with dead bacteria in particular, but also live bacteria and LPS. E. cirrhosa haemolymph agglutinates the bacteria V. anguillarum, V. parahaemolyticus and Aeromonas salmonicida and exerts a bacteriostatic effect on them. The haemolymph, haemocytes and certain tissues from E. cirrhosa exhibit lysozyme and antiprotease activity. Injection of live V. anguillarum causes an increase in lysozyme activity in the branchial heart (after 48 h) and a decrease in the number of circulating haemocytes (after 24 h). Antiprotease activity increases in the haemocytes (4 h) after injection of bacteria but decreases in the haemolymph. Live bacteria cause an increase in the number of circulating haemocytes, but are cleared from the circulation in about 4 h by both the haemocytes and tissues (branchial heart, branchial heart appendage and white body), where they are degraded. Ultrastructural changes were observed in the branchial heart cells and the haemocyte nucleus after injection of bacteria. Following injection, colloidal graphite is aggregated in blood vessels only.

Effects of repeated sampling on the haemocytes and haemolymph of Eledone cirrhosa (Lam.)

Sampling blood has an effect on both the number of circulating haemocytes and the concentration of copper present in the haemolymph of Eledone cirrhosa. Haemocytes were sampled over 4 h and over 7, 10 and 24 days. The number of haemocytes per millilitre significantly increases ( P<0.05) within 2 h of the 4-h sample period and within 24 h of the 7-, 10- and 24-day sampling periods. Haemocyte counts, however, significantly ( P<0.05) decreased within 4 h of the 4-h sample and within 3 days of the 7- and 10-day sampling periods. Over the 24-day sampling periods, haemocyte counts per millilitre decreased significantly ( P<0.05) by day 5 but had significantly ( P<0.05) increased again by day 17. The percentage of haemocytes with Giemsa-positive cytoplasmic granules significantly increased ( P<0.05) over the 4-h sampling period and over 24 h for both the 10- and 24-day sampling period. Methods for acid phosphatase, peroxidase, protein and carbohydrate gave variable staining results over 10 and 24 days. Increased staining was observed on the second and third sampling times for both protein and acid phosphatase over the 10-day sampling period whereas increased positive results were observed for all stains over the 24-day sampling period. The concentration of copper in the haemolymph decreases within 4 h of the 4-h sampling period and continues to decrease over an 11-day sampling period. Protein values decreased over the 4-h sampling period but showed no significant change between the first and last samples over 7 days.

Hemocyte Alterations during Melanotic Encapsulation of Brugia malayi in the Mosquito Armigeres subalbatus

The involvement of hemocytes in melanotic encapsulation reactions against Brugia malayi was assessed in Armigeres subalbatus. Hemocyte populations, epitope changes, phenol oxidase (PO) activity, and the presence of an 84-kDa polypeptide were investigated in mosquitoes exposed to a B. malayi-infective bloodmeal (= immune-activated), in mosquitoes given a noninfective bloodmeal (= controls), in nonbloodfed mosquitoes (= naive), or in some combination of these. Total hemocyte populations in immune-activated mosquitoes significantly decreased at 24 hr postbloodmeal (PB) as compared with controls. At 48 and 72 hr PB, hemocyte population levels in immune-activated mosquitoes increased to control levels. Epitope changes, as indicated by wheat germ agglutinin (WGA) binding, also were observed. There was a significant increase in the percentage of hemocytes binding WGA in immune-activated mosquitoes at 24 hr PB as compared with controls. Furthermore, the activity of hemocyte PO, an enzyme involved in the melanotic encapsulation pathway, was significantly elevated at 12 hr PB in immune-activated mosquitoes as compared with controls. Analysis for the presence of an 84-kDa polypeptide in A. subalbatus indicates that a 2.0-kb message in total RNA hybridized to D6.12, an Aedes aegypti cDNA encoding an 84-kDa polypeptide that is associated with melanotic encapsulation responses. The hybridization of D6.12 to RNA was not greater in immune-activated as compared to control A. subalbatus, as has been observed in A. aegypti. Results indicate that these hemocyte changes correspond in time with the melanotic encapsulation reactions of A. subalbatus against filarial worms.

Hemocyte surface changes in the migratory grasshopper, Melanoplus sanguinipes in response to wounding and infection with Beauveria bassiana

AbstractSurface changes of hemocytes from the migratory grasshopper, Melanoplus sanguinipes (Fab.), following wounding and/or infection with Beauveria bassiana (Bals.) Vuillemin were assessed using fluorescein labelled wheat germ agglutinin (WGA) and concanavalin A (Con A). Binding was observed on outer wall of hemocytes when either Con A or WGA conjugates were tested. After infection there was an increase in the percentage of hemocytes which bound to WGA and which remained so for 24 h. However, after wounding this increase in binding subsided after only 1 h. Furthermore, it was found that the increase in WGA‐binding occurred within the first 15 min of wounding or infection. In addition, ConA binding in males only increased 3‐fold in fungus‐injected insects within 1 h of treatment and remained high for 24 h. These results indicated the presence of mannose (and/or glucose) residues (Con A specificity) and N‐acetyl‐D‐glucosamine residues (wheat germ agglutinin; WGA specificity) on outer wall surfaces of the hemocytes. The implications of these results in the immune response of M. sanguinipes to infection with B. bassiana are discussed.

The Effect of haemolymph extraction on distribution of lysosomal enzymes in Lymnaea stagnalis haemocytes: A cytochemical study

An enzyme cytochemical, light microscopy study was undertaken to evaluate the effect of haemolymph extraction on distribution of acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase in haemocytes of Lymnaea stagnalis. The study revealed that discrete acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase-staining subpopulations of haemocytes do exist in L. stagnalis, and that there are differences in distribution. A high percentage, about 96%, of haemocytes showed positive reaction for peroxidase, the percentage of haemocytes showing activity for alpha-naphthyl acetate esterase, acid phosphatase, and alkaline phosphatase, was about 54, 42, and 34, respectively. Haemolymph extraction by foot retraction, significantly affected enzyme distribution. Whereas distribution of acid phosphatase increased significantly from day 1 to 5 after extraction of haemolymph, that of all other enzymes showed significant decrease from day 1 to 7 depending upon the enzyme. The distribution of all enzymes stabilized by day 8 after extraction of haemolymph. Probable reasons for the observed fluctuations in the distribution of enzymes subsequent to haemolymph extraction are discussed.

Phagocytic Activity of Hemocytes of M-Line Biomphalaria glabrata Snails: Effect of Exposure to the Trematode Echinostoma paraensei

The phagocytic activity of hemocytes from 6-8-mm M-line Biomphalaria glabrata snails was studied in an in vitro assay using glutaraldehyde-fixed sheep erythrocytes (SRBC) as target cells. For individual snails, the percentage of hemocytes ingesting SRBC during a 1-hr interval, termed the phagocytic activity index (PAI), was determined. Hemocytes from snails infected for 1 day with Echinostoma paraensei had a slightly elevated PAI, but at both 8 and 30 days postexposure (DPE), hemocytes from infected snails had a significantly lower PAI than controls. Hemocytes taken from snails at 8 DPE also had a low PAI using rabbit erythrocytes and yeast as target cells. The low PAI at 8 DPE is attributed to the presence of large numbers of poorly spreading hemocytes with low phagocytic activity. Hemocytes from snails with 30-day infections were well spread but nonetheless had a low PAI. The presence of plasma from 8-day infected snails did not alter the PAI of hemocytes from control snails, nor was the PAI of hemocytes from infected snails changed by plasma from control snails. SRBC preincubated for 60 min in plasma from various groups of M-line snails did not elicit an increase in PAI when presented to hemocytes from control snails; in some cases, as with plasma from 6-8-mm control snails, such preincubation significantly reduced the PAI below levels obtained using SRBC preincubated in culture medium. As compared to hemocytes from snails with normally developing, 8-day-old intraventricular sporocysts (IS), hemocytes from snails exposed to infection but subsequently lacking IS had a significantly higher PAI.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenicity of lipopolysaccharide mutants of Pseudomonas aeruginosa for larvae of Galleria mellonella: Hemocyte interaction with the bacteria

There was no correlation between the virulence of smooth and rough Pseudomonas aeruginosa strains for Galleria mellonella and the rate of clearance of these strains from the hemolymph. The total hemocyte counts varied with the bacterial strain and generally did not correlate with the bacterial clearance profiles. All test strains at their LD 50 values induced secondary bacteremias in the insect. The number of bacteria adhering to the hemocytes and the level of responding hemocytes were a function of the bacterial strain and hemocyte type. Evidence is presented for functional heterogeneity of the granulocytes. Carbohydrates were shown to influence the bacterial number per hemocyte type and the percentage of hemocytes with bacteria according to the bacterial strain and hemocyte type. Bacteria treated with trypsin showed reduced adhesion to the plasmatocytes but not to the granulocytes. A virulence mechanism is proposed.