Activity Of Hemocytes Research Articles

Identification and cloning of the second type transglutaminase from Litopenaeus vannamei, and its transcription following pathogen infection and in relation to the haemolymph coagulation

Complementary (c)DNA encoding transglutaminaseII (TGII) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134), tiger shrimp, Penaeus monodon (AAV49005; AAO33455), kuruma shrimp, Marsupenaeus japonicus (BAD36808) and Pacifastacus leniusculus (AAK69205) TG. The 2405-bp cDNA contained an open reading frame (ORF) of 2292 bp, a 31-bp 5′-untranslated region (UTR), and an 82-bp 3′-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (764 aa) was 85.9 kDa with an estimated pI of 5.32. The L. vannamei TGII (abbreviated LvTGII) contains a typical TG-like homologue, two putative integrin binding motif (RGD and KGD), and five calcium-binding sites; three catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two groups, STGI and STGII, and LvTGII is more closely related to STGII than to STGI. LvTGII mRNA was detected in all tested tissues of L. vannamei, and was highly expressed in haemocytes. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant increase of LvTGI and LvTGII mRNA expression at 6 h followed by a notable decrease at 24 h in LvTGI and a continually increase in LvTGII indicating a complementary effect, which implied that both LvTGs involved in the immune response of shrimp, and LvTGII was more important in the later defense response. The gene silencing of LvTGII in shrimp significantly decreased LvTGII expression and TG activity of haemocytes, and significantly increased clotting time of haemolymph, suggests that the cloned LvTGII is a clotting enzyme involved in haemolymph coagulation of L. vannamei. In conclusion, the cloned LvTGII is a clotting enzyme involved in coagulation of haemolymp and immune response of white shrimp, L. vannamei.

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d’ être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection.

Effect of copper on the immunomodulatory activities of haemocytes in juveniles of the abalone Haliotis rufescens cultivated under hatchery conditions

The effect of short-term exposure to copper on the cellular and physiological response in haemocytes of juvenile abalone Haliotis rufescens cultivated in hatcheries in the north of Chile is evaluated. The animals were exposed for 168hours to concentrations of 2.5μg/L and 10μg/L of Cu+2, which have been reported as representative of coastal zones close to mining activity, particularly copper mining. The haemocytes were collected from the animals and the effect of the metal was quantified through the total haemocyte count (THC), phagocytosis, phenoloxidase activity, and generation of superoxide anions. The results showed that copper exposure decreased THC and phagocytosis significantly, while phenoloxidase activity and superoxide anions production increased significantly with respect to untreated controls. This study suggests a relation between exposure to the trace copper concentrations and the alterations of in vitro cellular and physiological parameters of the abalone's immune system in a dose- and time-dependent manner. This study emphasize the importance of heavy metal contamination on abalone cultures considering that the haemocytes are crucial for the proper function of the immune system and important health indicators to evaluate the effect of ambient stress factors on these organisms.

Effects of RNA interference-mediated knock-down of hypoxia-inducible factor-α on respiratory burst activity of the Pacific oyster Crassostrea gigas hemocytes

In mammals, hypoxia-inducible factor-1 α (HIF-1α) is known to play important roles not only in oxygen homeostasis but also in innate immune responses. In this study, to assess the functional role of HIF-α in respiratory burst activity of Crassostrea gigas hemocytes, oysters were injected with HIF-α- or green fluorescent protein (GFP)-targeted-long double-stranded RNAs (dsRNAs), and at 1, 3, and 7 days post-injection, knock-down of C. gigas HIF-α expression and production of reactive oxygen species (ROS) were analyzed. Expression of HIF-α in mantle, gill, and hemocytes of C. gigas was clearly down-regulated by injection of the HIF-α-targeted-long dsRNA, but was not inhibited by the GFP-targeted-long dsRNA, indicating that HIF-α expression was suppressed through sequence-specific and systemic RNA interference (RNAi). Respiratory burst activity of hemocytes was significantly increased by administration of GFP-targeted-long dsRNA. However, knock-down of HIF-α expression led to significant decrease of chemiluminescence (CL) response of C. gigas hemocytes at 3 and 7 days post-administration of HIF-α-targeted-long dsRNA, indicating the critical role of HIF-α in activation of respiratory burst activity of oyster hemocytes.

Phagocytosis and respiratory burst activity of haemocytes from the ivory snail, Babylonia areolata

Haemocytes from the ivory snail, Babylonia areolata phagocytized Saccharomyces cerevisiae and Vibrio parahaemolyticus after 30 min. Haemocytes phagocytized V. parahaemolyticus at a greater rate than they phagocytized S. cerevisiae. The phagocytic rate (PP) of V. parahaemolyticus by granulocytes to was a little higher than that of S. cerevisiae. The phagocytic index (PI) of V. parahaemolyticus by granulocytes was significantly higher than that of S. cerevisiae. The same was true of hyalinocytes. The PP of granulocytes was significantly higher than that of hyalinocytes for each pathogen. No difference in PI was observed in granulocytes and hyalinocytes. Two defense mechanisms of B. areolata were quantified using flow cytometry. Haemocyte phagocytosis was quantified using fluorescent microbeads and respiratory burst activity was measured using H2O2 increases detected by 2′, 7′-dichlorofluorescein diacetate. Both phagocytosis and respiratory burst activity of the haemocytes increased over time. After 90 min the phagocytic rate no longer increased. In the case of respiratory burst, the greatest increase in fluorescence occurred between 30 and 120 min, no further increase was seen after 120 min. These results showed unequivocally that a native (unstimulated) haemocyte oxidative burst was active in B. areolata. The aim of this study was to further the knowledge of immunology in gastropods.

Mono- and Polynuclear Copper(II) Complexes of Alloferons 1 with Point Mutations (H6A) and (H12A): Stability Structure and Cytotoxicity

Mononuclear and polynuclear copper(II) complexes of the alloferons 1 (Allo1) with point mutations (H6A) H(1)GVSGA(6)GQH(9)GVH(12)G-COOH (Allo6A) and (H12A) H(1)GVSGH(6)GQH(9)GVA(12)G-COOH (Allo12A) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 3:1 was obtained. At physiological pH 7.4 and a 1:1 metal-to-ligand molar ratio, the Allo6A and Allo12A peptides form CuL complexes with the 4N {NH2, N(Im)-H(1),2N(Im)} binding mode. The amine nitrogen donor and the imidazole nitrogen atoms (H(9)H(12) or H(6)H(9)) can be considered to be independent metal-binding sites in the species formed for the systems studied. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2:1 and 3:1 molar ratios dominate in solution, respectively. The induction of apoptosis in vivo in Tenebrio molitor cells by the ligands and their copper(II) complexes at pH 7.4 was studied. The biological results show that copper(II) ions in vivo did not cause any apparent apoptotic features. The most active was the Cu(II)-Allo12A complex formed at pH 7.4 with a {NH2, N(Im)-H(1),N(Im)-H(6),N(Im)-H(9)} binding site. It exhibited 123% higher of caspase activity in hemocytes than the native peptide, Allo1.

Effects of the dinoflagellate Alexandrium minutum and its toxin (saxitoxin) on the functional activity and gene expression of Crassostrea gigas hemocytes

Blood cells or hemocytes are the major component of the cellular immune responses in bivalves. Recent evidences have demonstrated that the immune functions of these cells can be impaired by harmful algae and their toxins. In this study, we compared the in vitro effect of the toxic dinoflagellate Alexandrium minutum and its purified paralytic toxin, saxitoxin (STX) on the oyster Crassostrea gigas hemocytes. Oyster hemocytes were incubated with A. minutum or STX, and their size, complexity, viability, phagocytic activity and reactive oxygen species (ROS) production were analyzed through flow cytometry. Also, expression of eleven defense-related genes was evaluated through real-time quantitative PCR. Cell incubation with both, the noxious microalga and STX did not alter hemocyte viability neither complexity. However, their phagocytic capacity and ROS production were significantly affected by both treatments. On the other hand, only A. minutum exposure resulted in increase of hemocyte size. Transcript levels of the gene encoding the cytokine Interleukin-17 was significantly higher (4.5-fold) after hemocytes were exposed to A. minutum, which contrasted with a decreased (7-fold) number of transcripts, when hemocytes were treated with STX. The purified toxin also led to lower transcript levels of a cytochrome P450 gene, the CYP356A1 (3.5-fold). On the other hand, hemocytes incubated with STX presented higher mRNA levels of antimicrobial peptide defensin-2 (7-fold) and heat shock protein Hsp70 (2-fold) genes. Altogether, these results suggest that the noxious alga A. minutum and STX can indeed negatively affect immunocompetence of C. gigas hemocytes, although in distinct manners. These differences could be due to a lower STX bioavailability in living microalgal cultures or alternatively to the presence of allelopathic compounds produced by living microalgae. This is the first report comparing the transcription of defense-related genes in oysters exposed to a harmful alga and its purified toxin.

Integrin αPS3/βν-mediated Phagocytosis of Apoptotic Cells and Bacteria in Drosophila

Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named α and β. Integrin βν, a β-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an α-subunit that forms a complex and cooperates with βν. Examinations of RNAi-treated animals suggested that αPS3, but not any of four other α-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of αPS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both αPS3 and βν was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of αPS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between αPS3 and βν. These results collectively indicated that integrin αPS3/βν serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes.

Morphofunctional study of hemocytes from lions-paw scallop Nodipecten subnodosus

Hemocytes play an important role in internal defense in mollusk bivalves; they are generally divided into granular and hyaline types, where the granules possess molecules to combat foreign particles. We investigated the morphology, staining reactions, and immune-related activities of hemocytes from the pectinid lions-paw scallop Nodipecten subnodosus. We showed that, in contrast to the conditions in most bivalves and similar to other pectinids, no granular cells were observed; only very few semi-granular cells were present and the large majority of cells were hyalinocytes and blast-like cells. The cytoplasm in many hyalinocytes showed vesicles of various sizes, but none with the staining characteristics of typical granules. When hemolymph was removed from these scallops, the plasma did not clot and the hemocytes rapidly adhered to one another in suspension. When living hemocytes were placed on a glass surface, they clustered into groups of various sizes; within minutes, pseudopods extended from the cells and outward migration began. Many cells spread to form extensive networks of flat cells, and other undifferentiated cells did not spread and kept a rounded morphology. Some hemocytes were immunologically active because they phagocytize Escherichia coli bacteria. Hemocytes expressed the carbohydrates residues N-acetyl-d-glucosamine, α-d-glucose, α-d-mannose, and α-L-fucose moieties, and cytochemical assays showed activity of acid phosphatase, specific esterase, and non-specific esterase, enzymes commonly associated with lysosomes and immunologic activity. These observations serve as a useful tool for further investigation of morphofunctional studies and physiological status of scallops.

Allograft inflammatory factor-1 stimulates hemocyte immune activation by enhancing phagocytosis and expression of inflammatory cytokines in Crassostrea gigas

Allograft inflammatory factor-1 (AIF-1) is a calcium-binding cytokine associated with immune cell activation and inflammatory response. Presently, we have identified and characterized an AIF-1 in a marine bivalve mollusk, Crassostrea gigas, and designated it as CgAIF-1. The full-length CgAIF-1 cDNA is 794 bp, encoding a protein of 149 amino acids with two conserved EF hand Ca2+-binding motifs. CgAIF-1 is constitutively expressed in various tissues with enriched expression in hemocytes. Moreover, CgAIF-1 transcription is induced by multiple Pathogen-Associated Molecular Patterns (PAMPs), including poly (I: C), LPS, PGN, HKLM and HKVA, but is limited by 1,3-β-glucan. Furthermore, recombinant CgAIF-1 can specifically stimulate phagocytic ability of granulocytes, but not of intermediate cells and hyalinocytes. CgAIF-1 also enhances mRNA levels of MIF, TNF and IL-17. These results provide the first functional evidence that CgAIF-1 is involved in hemocyte activation in C. gigas, revealing conserved functions of AIF-1 in host defense from mollusks to mammals.

Nitrite Stress on Density of Hemocyte and Phenoloxidase Activity in Hemolymph of <i>Charybdis japonica</i>

In order to discuss the effects of nitrite on the density of hemocyte (DHC) and phenoloxidase (PO) activity in hemolymph of Charybdis japonica, the crabs were exposed to six nitrite-N groups (0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg L-1 ambient nitrite-N concentrations). The DHC and PO activity was examined on days 1, 3, 5, 10 and 15 following the stress. Results showed that the two indexes appeared to have a decline trend after an initial ascent. After 1 day treatment, the DHC of five treatments were higher than that of the control group respectively, while the PO activity of 8.0 and 10.0 mg L-1 nitrite-N exposed group decreased compared with control. With time elapsed, the DHC of lower concentration nitrite-N exposed group increased faster, while the PO activity of higher concentration nitrite-N exposed group rised faster than other treatments. After 15-day stress, all parameters tested were lower than those of control group barring the DHC of 2.0 and 4.0 mg L-1 nitrite-N exposed group and the PO activity of 2.0, 4.0, and 6.0 mg L-1 nitrite-N exposed group.

Activation of an immune response in Litopenaeus vannamei by oral immunization with phagocytosis activating protein (PAP) DNA

The phagocytosis activating protein (PAP) gene has been reported to stimulate the phagocytic activity of shrimp hemocytes and to protect shrimp from several pathogens. In this study oral administration of the chitosan-PAP gene to shrimp was investigated for its ability to induce immunity. The PAP gene was cooperated into a phMGFP plasmid, named PAP-phMGFP. Chitosan-PAP-phMGFP nanoparticles were formed by mixing a low molecular weight chitosan (50 kDa) and a high molecular weight chitosan (150 kDa) with various ratios of PAP-phMGFP. The optimal ratio of chitosan PAP-phMGFP nanoparticles was first determined by transfection into Chinese Hamster Ovary (CHO) cells before being used for oral immunization in shrimp. The chitosan-PAP-phMGFP nanoparticles at a ratio of 2:1 with the low molecular weight chitosan were optimum for transfecting the CHO cells. The shrimp were then fed with 25, 50, 100 and 150 μg/shrimp/day of chitosan-PAP-phMGFP (2:1) nanoparticles then challenged by the white spot syndrome virus (WSSV). Shrimp fed with 50 μg of chitosan-PAP-phMGFP nanoparticles per day for 7 consecutive days, produced the highest relative percent survival (RPS) (94.45 ± 9.86%). The presence of PAP-phMGFP was detected in every shrimp tissue including the hemolymph, lymphoid organ, heart, hepatopancreas, intestine and muscle. The folds increase of the PAP gene expression increased significantly together with an increase of the phagocytic activity in the immunized shrimp. The stability of the PAP-phMGFP in the immunized shrimp hemolymph was detected by determination of the expression of the GFP at various days after immunization ceased. GFP expression was detected until the 15th day but not at the 30th day after immunization ceased. A quantitative analysis of the WSSV copies in shrimp heart tissue was significantly reduced in the immunized shrimp. In addition, chitosan-PAP-phMGFP nanoparticles protected shrimp against WSSV, Yellow head virus (YHV) and Vibrio harveyi with RPS values of 83.34 ± 7.86%, 55.56 ± 15.72% and 53.91 ± 5.52%, respectively. This study therefore confirms the role of the PAP gene in shrimp immunity and may lead to the development of a way to prevent microbial diseases of shrimp at an industrial level by appropriate feeding of a chitosan/DNA complex.

Phosphorylation is required for myosin regulatory light chain (PmMRLC) to control yellow head virus infection in shrimp hemocytes

The cellular signal-transduction process is largely controlled by protein phosphorylation. Shrimp infected with yellow head virus show dramatic changes in their hemocyte phosphoproteomic patterns, and aberrant activation of phosphorylation-based signaling networks has been implicated in a number of diseases. In this study, we focused on phosphorylation of Penaeus monodon myosin regulatory light chain (PmMRLC) that is induced at an early hour post YHV infection and is concomitant with cellular actin remodeling. In shrimp cell cultures, this phosphorylation was inhibited by the myosin light chain kinase (MLCK) inhibitors ML-7 and ML-9, suggesting that PmMLC phosphorylation is MLCK pathway-dependent. Blocking PmMRLC phosphorylation resulted in increased replication of YHV and reduction of phagocytic activities of shrimp hemocytes called semigranular cells (SGC) and granular cells (GC). Injection of MLCK inhibitors prior to YHV challenge resulted in dose-dependent elevation in quantity of YHV-positive GC and cytoplasmic YHV protein, coincident with high shrimp mortality. Altogether, we demonstrated that PmMRLC phosphorylation increases after YHV infection in shrimp and that inhibition of the phosphorylation leads to increased YHV replication, reduced hemocyte phagocytic activity (probably through actin remodeling) and subsequent shrimp death. Thus, further studies on the MLCK activation pathway may lead to new strategies in development and implementation of therapy for YHV infections in shrimp.

Gα73Β is a downstream effector of JAK/STAT signalling and a regulator of Rho1 inDrosophilahaematopoiesis

JAK/STAT signalling regulates many essential developmental processes including cell proliferation and haematopoiesis, whereas its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. Here we show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, although Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. We show that Gα73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, we also show that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. Our findings link JAK/STAT transcriptional outputs, Gα73B activity and Rho1-dependent cytoskeletal rearrangements and cell motility, therefore connecting a pathway associated with cancer with a marker indicative of invasiveness. As such, we suggest a mechanism by which JAK/STAT pathway signalling may promote metastasis.

Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles

Malaria is a global public health problem, especially in sub-Saharan Africa, where the mosquito Anopheles gambiae Giles serves as the major vector for the protozoan Plasmodium falciparum Welch. One determinant of malaria vector competence is the mosquito's immune system. Hemocytes are a critical component as they produce soluble immune factors that either support or prevent malaria parasite development. However, despite their importance in vector competence, understanding of their basic biology is just developing. Applying novel technologies to the study of mosquito hemocytes, we investigated the effect of blood meal on hemocyte population dynamics, DNA replication and cell cycle progression. In contrast to prevailing published work, the data presented here demonstrate that hemocytes in adult mosquitoes continue to undergo low basal levels of replication. In addition, blood ingestion caused significant changes in hemocytes within 24 h. Hemocytes displayed an increase in cell number, size, granularity and Ras-MAPK signaling as well as altered cell surface moieties. As these changes are well-known markers of immune cell activation in mammals and Drosophila melanogaster Meigen, we further investigated whether a blood meal changes the expression of hemocyte-derived immune factors. Indeed, hemocytes 24 h post-blood meal displayed higher levels of critical components of the complement and melanization immune reactions in mosquitoes. Taken together, this study demonstrates that the normal physiological process of a blood meal activates the innate immune response in mosquitoes. This process is likely in part regulated by Ras-MAPK signaling, highlighting a novel mechanistic link between blood feeding and immunity.

<i>In vitro</i> Studies on Cellular Mediated Immune Response in Haemocytes of Crab— <i>Episesarma tetragonum</i>

Studying the biological interaction with the invertebrate immune system is attractive for the advancement of a basic knowledge on invertebrate and vertebrate general immunity, because it offers various possible alternatives for disease management. The invertebrate immune system involves encapsulation, nodule formation and phagocytosis process by haemocytes (cellular mediated). In the present study, we investigated the morphology of the haemocytes of Episesarma tetragonum , their role in cellular mediated immune responses such as encapsulation, phagocytosis and viability assays. Encapsulation response of haemocytes is tested with the CM cellulose, Sepharose CL-6B and DEAE Sepharose 6CLB beads. DEAE Sepharose 6CLB beads showed (80%) good cellular response. Phagocytic activity of haemocytes showed pathogen engulfing activity against the microbial patterns (N-acteyl muramic acid (NAM), N-acetyl glutamic acid (NAG), lipopolysaccharide, β-1,3-glucans, chitins, laminarins, zymosan and peptidoglycans) present on the surface of the pathogens. Phagocytic activities of the haemocytes were decreased when increasing the incubation time. Viability of haemocytes was decreased when the time intervals of absorption of tryphan blue were increased. The present investigation enlightens the role of hemocytes in the cellular mediated immune responses of crustacean crab including all invertebrates.

Nucleolar activity of haemocytes in the adult firebug as an alternative animal model

Firebug haemocytes are a newly developed alternative animal model, but data on their physiology are deficient. We examined the nucleolar activity, which reflects the intensity of cellular proteosynthesis, using toluidine blue and AgNOR stain technology. Prohaemocytes, granulocytes and plasmatocytes contain on an average two nucleoli, whereas spherulocytes have on average six. The number of compact nucleoli, which are the attribute of intensive proteosynthetic activity, was significantly higher in prohaemocytes. Our data support the hypothesis that prohaemocytes have a physiology similar to mammalian haematopoietic stem cells.

Flow Cytometric Analysis of In Vitro Cytotoxicity of Cadmium in Haemocytes from the Tiger Shrimp, Penaeus monodon

This study investigated the toxic effects of cadmium on viability, reactive oxygen species (ROS) production and non-specific esterase activity of Penaeus monodon haemocytes in vitro, using a flow cytometric assay. After 6h in vitro exposure with 10(-9)-10(-3)M Cd(2+), cell viability, ROS production and esterase activity of haemocytes from P. monodon were determined. Results showed that at the lowest exposures (10(-9)-10(-6)M), Cd(2+) induced no effect on cell viability, ROS production and esterase activity. At a higher level (10(-5)M) of exposure, production of ROS was stimulated while Cd(2+) had no effect on cell viability and esterase activity. At the two highest concentrations (10(-4) and 10(-3)M), Cd(2+) caused increased ROS production, cell death and inhibited esterase activity. These results showed a relationship between Cd(2+) exposure dose and its cytotoxicity on shrimp haemocytes. Cadmium was cytotoxic and immunotoxic for P. monodon haemocytes in vitro when the dose reached 10(-4)M. The study also suggested that flow cytometry could be used as a tool for cytotoxic research of aquatic contamination on shrimp.

Development of two haemocyte culture systems (in attachment and in suspension) for shrimp immunity studies

For studying shrimp immunity, in vitro haemocyte cultures are essential. Despite various reported attempts, well-described and reproducible culture techniques are lacking. The current work aimed to establish two in vitro haemocyte culture systems for Penaeus (Litopenaeus) vannamei. Haemocyte suspensions were either seeded in conventional Nunc® Nunclon™Δ Surface 24-well cell culture plates with glass cover slips (haemocytes in attachment) or in Nunc® Hydrocell Surface 24-well cell culture plates (haemocytes in suspension). The culture medium was based on L-15 (Leibovitz), and was further supplemented with L-glutathione and protease inhibitors in an attempt to improve haemocyte survival. Parameters such as number of living adherent and non-adherent single cells, number and average diameter of clusters and survival of cells inside clusters were evaluated. Additionally, live-cell imaging videos were recorded. It was found that haemocytes cultured for 1h on glass coverslips in Nunc® Nunclon™Δ Surface plates could be separated in two cell fractions: adherent or non-adherent. Shrimp haemocytes cultured in Nunc® Hydrocell Surface plates remained in suspension and over time formed cell clusters which melanised. L-glutathione supplementation clearly improved haemocyte survival up to 48h and delayed clustering and melanisation; addition of protease inhibitors did not. To validate the system, the phagocytic and antibacterial activities of adherent haemocytes towards Vibrio campbellii were evaluated. After 1h of co-culture, 11.5±0.14% of haemocytes showed phagocytosis with an average of 2.4±0.1 bacteria internalised per haemocyte. Furthermore, haemocytes clearly demonstrated an antibacterial activity. It was concluded that these systems were reproducible and could keep haemocytes functionally active during the time required for the study of innate immune processes. Consequently, these techniques represent powerful tools for studying a variety of cell-mediated and humoral immune responses of shrimp in vitro.

Norepinephrine regulates prophenoloxidase system-related parameters and gene expressions via α- and β-adrenergic receptors in Litopenaeus vannamei

The total (THC) and differential haemocyte counts (DHC), phenoloxidase (PO) activity, and prophenoloxidase (proPO) system-related genes were investigated in haemocytes of Litopenaeus vannamei that received saline, norepinephrine (NE), and NE co-treated with various adrenergic receptor (AR) antagonists both in vivo and in vitro. Results showed that semi-granular and granular cells of shrimp which received NE, NE + phentolamine (Phe), NE + prazosin (Pra), NE + propanolol (Pro) and NE + metoprolol (Met) significantly decreased, while the PO activity of the shrimp received NE + Phe in vivo was significant higher than all the other treatments. PO activities of haemocytes exposed to saline, Pra + NE, and Met + NE were significantly higher than those of haemocytes exposed to NE, Phe + NE, and Pro + NE in vitro. Similar phenomena in lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP), proPO-I, proPO-II, serine proteinases (SP), and peroxinectin (PE) messenger (m)RNA expressions of haemocytes exposed to saline, NE, and NE co-treated with various AR antagonists were observed both in vivo and in vitro. No significant differences were observed for LGBP and proPO-II mRNA expressions between haemocytes treated with saline and Pra + NE, for proPO-I mRNA expression between haemocytes treated with saline and Met + NE; or for SP and PE mRNA expressions among haemocytes treated with saline, Pra + NE, and Met + NE. These results suggest that stress-induced NE may promote the migration of circulating granulocytes to the site of the injection and the existing proPO mRNA translation which had been stored in granulocytes. NE downregulated the LGBP, proPO-I, proPO-II, SP, and PE gene transcription by haemocytes via α1-, β1-, α1-, α1- and β1-, and α1- and β1-ARs, respectively, which subsequently decreased the PO activity by α1- and β1-ARs in haemocytes of L. vannamei.