Generation of Reactive Oxygen Species in Different Fractions of the Coelomocytes of Holothurian Eupentacta fraudatrix in Response to the Thermostable Toxin of Yersinia pseudotuberculosis in Vitro

Pure fraction (92%-95%) of phagocytes (FP) and a mixture of amoebocytes(62%) and morula cells (38 %) FPMC of the holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirota) were obtained by using ficoll verographine step gradient. Basal production of reactive oxygen species (ROS) in FP quantified by using reduction of nitroblue tetrazolium (NBT) was more than twice that in FPMC. Thermostable toxin of Yersinia pseudotuberculosis (TST) at different concentrations ( 0.2; 0.5; 2.5 μg/ml, but not 0.1 μg/ml) stimulated NBT reduction in FPMC after 24 h incubation. In FP, TST at concentrations of 0.1 and 0.2 μg/ml inhibited and at concentrations of 0.5 and 2.5 μg/ml stimulated NBT reduction after 24 h incubation. Maximal effect was observed in FP and FPMC at TST concentrations of 0.5 and 0.2 μg/ml, respectively. Addition of catalase (0.7 μg/ml) to the cells treated with TST (2.5 μg/ml) was followed by a decrease in NBT reduction compared to that under toxin treatment alone. TST stimulated superoxide dismutase activity in concentration dependent manner (maximum at 0.5 μg/ml concentration in FP) after 24 h treatment, and this stimulation was prevented by a commercial catalase. Plant lectin concanavalin A stimulated NBT reduction more than 5 fold in FPMC compared to the control. With addition of TST, lectin stimulated ROS to lesser extent than that with lectin alone. When catalase, TST, and lectin were added into the FPMC simultaneously, ROS increase was similar to that under lectin treatment alone. On the whole, data obtained indicated that ROS generation in holothurian coelomocytes especially occurs in both stimulated and not stimulated phagocytes, and that changes in ROS production by these cells may be one of the mechanisms of antibacterial protection of holothurians.

Active bleb formation is abated in Lytechinus variegatus red spherule coelomocytes after disruption of acto-myosin contractility

Red spherule coelomocytes are immune cells in the sea urchin Lytechinus variegatus that have been characterizedas motile O2 transport cells. Video microscopy of living red spherule coelomocytes reveals a constitutive, dynamicarray of cellular morphologies and movements. Cells continuously send out and retract membrane blebs all over thecell surface as part of their normal cellular physiology. Disruption of microtubules by perfusion with either nocodazoleor taxol had no effect on bleb formation or motility. Perfusion with cytochalasin B abated bleb formation andrevealed cells that exhibited multiple small spheres attached by short membrane extensions. Attenuation of blebbingand intracellular organelle motility were restored by washing out with cytochalasin B. Treatment with phalloidinalso abated bleb formation and revealed a smooth, spherical cellular morphology. The effects of phalloidin werecompletely reversible after washout. Red spherule coelomocytes treated with blebbistatin rounded up with anirreversible retraction of blebs into surface blebs that were greatly reduced in size, number and motility. Normal cellsurface bleb formation and intracellular organelle motility were not restored after washout of the drug. These resultsindicate that the acto-myosin contractile mechanism contributes to the dynamics of constitutive cell surface membraneblebbing in invertebrate immune cells.

Chlorine substitution-dependent toxicities of polychlorinated biphenyls to the earthworm Eisenia fetida in soil

Polychlorinated biphenyls(PCBs)with different chlorine substitution patterns often coexist in e-waste-processing sites.However,the single and combined toxicity of PCBs to soil organisms and the influence of chlorine substitution patterns remain largely unknown.Herein,we evaluated the distinct in vivo toxicity of PCB28(a trichlorinated PCB),PCB52(a tetrachlorinated PCB),PCB101(a pentachlorinated PCB),and their mixture to earthworm Eisenia fetida in soil,and looked into the underlining mechanisms in an in vitro test using coelomocytes.After a 28-days exposure,all PCBs(up to 10 mg/kg)were not fatal to earthworms,but could induce intestinal histopathological changes and microbial community alterations in the drilosphere system,along with a significant weight loss.Notably,pentachlorinated PCBs with a low bioaccumulation ability showed greater inhibitory effects on the growth of earthworm than lowly chlorinated PCBs,suggesting that bioaccumulation was not the main determinant of chlorine substitution-dependent toxicity.Furthermore,in vitro assays showed that the highly chlorinated PCBs induced a high-percentage apoptosis of eleocytes in the coelomocytes and significantly activated antioxidant enzymes,indicating that the distinct cellular vulnerability to lowly/highly chlorinated PCBs was the main contributor to the PCBs toxicity.These findings emphasize the specific advantage of using earthworms in the control of lowly chlorinated PCBs in soil due to their high tolerance and accumulation ability.

Sea urchin coelomocyte arylsulfatase: a modulator of the echinoderm clotting pathway

Sea urchin petaloid coelomocytes efectuate the coting pathway by undergoing a rapid and dymamic cellular transfomation that leads to cellular adhesion and wounds closure.We have identified high levels of activity of aylsulfatase(Ars)associated with coelomocytes of the sea urchin ytechimu varieganus (Lamarck,1816).Ars activity was extracted from clotted coelomocytes with EDTA and showed high levels of activity up to a 1:100 dilution Clot fomation from isolated coelomic fuid was significantly inhbited by the ARS inhibitor,p-nito-phenyl phosphate.Ars activity was collected by 80%ethanol precipitation,a diagnostic test previously used in Ars isolation Cellular extraction studies in the presence and absence of the non-ionic detergent Tniton X-100 indicated that some Ars activity was present intacellularly.possibly in intacellular membrame-bound compart-ments,however the majonity of Ars activity was extracted from the extracellular coelomocyte membrane.Poly-clonal anti-sea urchin embryo Ars antibodies recognized a single protein band with an approximate molecular weight of 75 kDa on westem blots.Immumofluorescence using the anti-sea urchin Ars antibody revealed an in-tacellular and extacelular staining of Ars in both petalloid and filopodial coelomocytes.Taken together,these data indicate that coelomocyte Ars might be involved in cell-to-cell croslinking of suface sulfated polysaccha-nides vital for clot fomation.