Vincenzo Arizza

Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis

Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes (~50%), granulocytes (~35%), prohemocytes (~8%), oenocytes (~4%) and spherulocytes (~3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply (~12%) and then remained at a low level, while the number of other circulating cells was almost unchanged.

In vitro anti-biofilm activity of Boswellia spp. oleogum resin essential oils.

Aims:  To evaluate the anti‐biofilm activity of the commercially available essential oils from two Boswellia species.

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system.

The phenoloxidase (PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by means of 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identified by a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatant from hemocyte populations separated on a discontinuous Percoll density gradient. In Styela plicata, the enzyme appeared to be contained in morula cells only. In Ciona intestinalis, PO activity was shown in univacuolar refractile granulocyte and granular hemocyte. In Phallusia mammillata both compartment cell and granular hemocytes were positive. Enzymatic assay following electrophoretic analysis on polyacrylamide gel electrophoresis (PAGE) or SDS-PAGE indicated that hemocyte lysate presented orthodiphenoloxidase (catecholase) activity. The enzymes from the three species differed in molecular size, activating substances and trypsin sensitivity.

Antimicrobial and antistaphylococcal biofilm activity from the sea urchin Paracentrotus lividus

Aims:  Staphylococcal biofilm‐associated infections are resistant to conventional antibiotics. Consequently, new agents are needed to treat them. With this aim, we focused on the effector cells (coelomocytes) of the sea urchin Paracentrotus lividus immune system.

Tributyltin affects phagocytic activity of Ciona intestinalis hemocytes

Organotin compounds have been used in marine anti-fouling paints as biocides. Because tunicates are vulnerable to these compounds in their natural habitats, we used Ciona intestinalis to establish an assay for phagocytosis in vitro of yeast by hemocytes after exposure to different concentrations (0.0015, 0.015, 0.15 and 1.5 microM) of four organotin compounds: tributyltin (TBT), triphenyltin (TPT), dibutyltin (DBT) and diphenyltin (DPT). To evaluate the phagocytic activity, we used a method based on fluorescence excitation of yeast pre-treated with eosin-Y. The percentage of phagocytosis decreased from 45.1 +/- 3.49 to 22.4 +/- 5.14 at 1.5 microM of TBT (P < 0.001); it was significantly reduced in presence of the ionophore A23187. TPT, DPT and DBT did not show significant effects on phagocytosis. Because the effect of TBT was irreversible, differences between the inhibitory mechanisms of ionophore and TBT are suggested. These results indicate that for future analyses, tunicates should become excellent models for dissecting events such as phagocytosis that are associated with immunosuppression after exposure to xenobiotics.

Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and anti-staphylococcal biofilm agents

The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.

CYTOTOXIC ACTIVITY OF Ciona intestinalis (Tunicata) HEMOCYTES: PROPERTIES OF THE IN VITRO REACTION AGAINST ERYTHROCYTE TARGETS

Hemocytes (effectors) of Ciona intestinalis showed a natural cytotoxic capacity (HCA) when assayed in vitro against erythrocytes (targets). Cytotoxic cells lysed, to a variable extent, rabbit (RE), human (A, B, O), guinea pig, and sheep (SE) erythrocytes. Hemocyte cytotoxic activity (HCA) assayed against SE is a calcium-dependent reaction, occurs rapidly (15-30 min), at 25-37 degrees C over a wide range of pH (5.4-8.0). Assays were carried out using: 1) the medium in which hemocytes were maintained, 2) the soluble portion of hemocyte lysates, and 3) debris prepared from hemocyte lysates. Results suggest that HCA is a cell-mediated process that requires effector-target cell contacts. Anti-SE (calcium-dependent) and anti-RE (calcium-independent) agglutinins were also found in the reaction medium, probably released by hemocytes as a consequence of the in vitro experiments. The occurrence of HCA was independent of any allogeneic reaction between mixed hemocytes. Various levels of cytotoxic activity reveal hemocyte specificity.

Cloning and expression of a type IX-like collagen in tissues of the ascidian Ciona intestinalis

Collagens are highly preserved proteins in invertebrates and vertebrates. To identify the collagens in urochordates, the total RNA extracted from the pharynx of the ascidian Ciona intestinalis was hybridized with a heterologous probe specific for the echinoderm Paracentrotus lividus fibrillar type I-like larval collagen. Using this probe, two main bands (i.e. 6 and 2.8 kb mRNA) were observed on Northern blot hybridization. The cDNA library prepared from poly(A)+RNA extracted from pharyngeal tissue was screened and a cDNA that specifies a type IX-like collagen was identified. This molecule presents a conceptual open reading frame for a protein containing 734 amino acids. In particular, we showed a 1 alpha chain type IX-like collagen characterized by three short triple-helical domains interspersed with four non-triple-helical sequences, with structural features of fibril-associated collagens with interrupted triple-helices (FACIT) collagens. Northern blot hybridizations indicate a 2.8 kb transcript size. Sequence comparison indicated homology (47.64%, 48.95%) between the type IX-like collagen of C. intestinalis and mouse and human type IX collagen. In situ hybridization of tunic and pharynx tissues shows the presence of transcripts in connective tissue cells.

Inhibitory activity of sphingomyelin on hemolytic activity of coelomic fluid of Holothuriapolii (echinodermata)

The hemolytic activity of coelomic fluid from Holothuria polii is specifically inhibited by sphingomyelin. This phospholipid is the constituent of the membrane which probably interacts with the hemolysin thereby leading to the lysis.

Biological Responses of Rhynchophorus ferrugineus (Coleoptera: Curculionidae) to Steinernema carpocapsae (Nematoda: Steinernematidae)

ABSTRACT Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera: Curculionidae) is becoming a serious problem in Mediterranean areas where it is well-adapted, and now is present even in the United States (California). The infestations are primarily in urban areas where chemical control is not advisable and million of Euros are spent to control it. The effects of the entomopathogenic nematode Steinernema carpocapsae (Nematoda: Steinernematidae) on mortality, growth, as well as the immune activity of R. ferrugineus larvae, were investigated. R. ferrugineus mortality exhibited a positive trend with the dosage and duration of exposure to S. carpocapsae. The median lethal dose and median lethal time, important to optimize the treatments, were calculated. S. carpocapsae also had a detrimental effect on R. ferrugineus weight. In vivo and in vitro effects of S. carpocapsae on the phagocytic responses of R. ferrugineus hemocytes also were recorded. S. carpocapsae was not encapsulated by R. ferrugineus hemocytes. After 24 h, the number of hemocytes recorded in treated larvae was reduced. To investigate the defensive abilities of R. ferrugineus humoral and cellular immune systems, specifically against the bacterium Xenorhabdus nematophila (Enterobacteraceae), the minimum inhibitory concentration that inhibits bacterial growth was measured. This is the first time that this technique is applied to entomopathogenic bacteria.