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The development of an assay to determine insecticidal properties for either biological and conventional plant protection products plays an important role on the early screening of potential pathogens or derived toxins candidates. The standard methods for the evaluation it has been by bioassay, especially determination of LD50 or LC 50 requiring the use of relatively large numbers of insects and toxin tests. There are several problems connected with these bioassays: availability of insects and in the right life stage, mass producing the candidate species, preparation, reproducibly and costs relative to intensive manpower. These aspects are really important especially when bio-insectides should be tested against new target eg. invasive species in a new country. We present the results of the use of alternative systems in a screening study of Bacillus thuringensis (Bt) strain as potential pathogen for the Red Palm weevil, Rhynchophorous ferrugineus. We propose the use of different techniques such as growth inhibition, effect of pathogen on total number of hemocytes, ratio of selected hemocytes, espression of HSP 70 protein. In particular this protein is involved in the biochemical pathways response in stressed animals. Tests were carried out against the potential less sensitive stage to have a new predictive parameter for screening the reaction of the host to the pathogen. The preliminary results obtained with the proposed tests allows to enlarge the information of the potential pathogenicity of the entomopathogen bacteria and highlight the potential of this set of tests as screening methods to select new potential entomopathogens or bioinsecticides.

Negli echinodermi la difesa dell’organismo dalle invasioni microbiche avviene tramite meccanismi cellulari e umorali dell’immunità innata. Una componente fondamentale dei fattori umorali è costituita dai peptidi antimicrobici (AMPs) molto conservati con un ampio spettro di attività antimicrobica contro batteri, virus e funghi. Solitamente gli AMPs sono costituiti da meno di 100 amminoacidi, sono cationici e formano strutture anfipatiche. In questo lavoro dimostriamo che i celomociti dell’Echinoideo Paracentrotus lividus e dell’Oloturoideo Holothuria tubulosa contengono sostanze con attività antimicrobica verso diversi ceppi batterici patogeni umani. In entrambe le specie, nel precipitato acido del supernatante del lisato dei celomociti è presente una frazione peptidica a basso peso molecolare (Low Molecular Peptides. LMP) con massa compresa tra i 3 e i 5 kDa che mostra attività antibatterica, valutata come concentrazione minima inibente (MIC). In particolare, in P. lividus la frazione peptidica con massa molecolare inferiore a 5 kDa è stata valutata contro un gruppo di batteri di riferimento, Gram-positivi e Gram-negativi e contro funghi patogeni dell’uomo: Staphylococcus. aureus ATCC 29213, ATCC 25923, ATCC 43866, Staphylococcus. epidermis DSM 3269, 1457, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231 e Candida tropicalis ATCC 13803. Il 5-CC risulta attivo contro tutti i ceppi microbici testati in vitro, con valori di MIC compresi fra 15.8 mg ml-1e 253.7 mg ml-1. Inoltre, nella specie Paracentrotus lividus, sono stati caratterizzati gli AMPs presenti nella frazione peptidica, attraverso l’utilizzo di un RP-HPLC/nESI-MSMS. I risultati hanno individuato tre principali peptidi (Paracentrina I, II, III) con una taglia molecolare rispettivamente di 1251.7, 2088.1 e 2292.2 d. L’analisi MSMS ha evidenziato che i peptidi comprendono rispettivamente le sequenze peptidiche 9-19, 12-31 e 24-41 della β-timosina di P. lividus (AN AJ439718). La β-timosina è uno dei peptidi antibatterici presenti negli invertebrati. In H. tubulosa dal precipitato acido del supernatante del lisato dei celomociti è stata isolata una frazione di 3 kDa che ha mostrato attività verso diversi ceppi batterici: Enterococcus fecalis, Pseudomonas aeruginosa, Escherichia coli e Staphylococcus aureus con valori di MIC compresi fra 16,5 µg ml-1e 22.5 µg ml-1. Inoltre in entrambe le specie, le LMPs sono capaci di inibire l’attività dei biofilm da stafilococco, come dimostrato da osservazioni al microscopio fluorescenza e confocale. Ulteriori studi sono necessari per caratterizzare meglio queste componenti antimicrobiche, sia da un punto di vista biochimico che molecolare, sia per valutarne la localizzazione cellulare e tissutale e la modulazione dell’espressione genica in presenza di microorganismi. In conclusione i celomociti degli echinodermi possono rappresentare un’importante fonte di agenti antimicrobici utili per lo sviluppo di nuove strategie per il trattamento dei biofilm.

In invertebrates, cellular and humoral components are evolved to maintain their homeostais and integrity. Both these components respond to different non-self such as microorganisms, vertebrate erythrocytes and foreign proteins. The aim of this research was to study the possible modulating effects of Bacillus thuringiensis (Bt) in a commercial and vegetative form on the immune response of the echinoderm, sea cucumber Holothuria tubulosa. Bt is one of the most used biopesticides worldwide, covering the 90% of the organic market. Sicily is a region vocated to biological control and agricultural area are often closed to the coastal area. Investigate the effect of biopesticides on other non target organisms is important in a more exhaustive environmental risk assessment. Moreover correlative studies indicate that the immune surveillance is affected by both Bt forms in vivo challenge. In particularly we have evaluated the phagocitosys vs Saccharomyces cerevisiae, hemagglutination using rabbit erythrocytes and coelomocyte cytotoxic activity against rabbit erythrocytes and K562 tumor target cells. These results can contribute to improve the knowledge about the use of Bt like biological insecticidal on non target organisms.

Blood cells freshly collected from sea bass (Dicentrarchus labrax) were exposed in vitro to different concentrations of cadmium (Cd) and copper (Cu) at 10-7 M, 10-5 M, 10-3 M, and exam- ined for neutral red retention capacity and for cell vitality with MTT assay. A relationship between heavy metal exposure and alteration in responses of blood cells in a dose-time-dependent was found. Our results showed that fish blood cells may constitute an interesting biological model for experimen- tal and applied toxicology, especially in the case of environmental pollution.

To gain further insight into the role of cortisol in Wsh innate immune responses, we cloned and sequenced a 2592 bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton’s mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL chemiluminescence response (CL) has been related to increased respiratory burst of phagocytes stimulated with zymosan, PCLs, preincubated in vitro with cortisol at various concentrations, were assayed for their CL response. Dose-dependent cortisol inhibitory eVects, and signiWcant competitive activity of a low concentration of mifepristone (RU486), a glucocorticoid-receptor blocker, supported that cortisol–GR interaction was involved in modulating CL response via a genomic pathway. Results also indicated that cortisol could be eVective through an additional not-genomic way, and showed that high doses of RU486 exerted an inhibitory eVect on PCL chemiluminescence activity.

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.

A tumor necrosis factor-alpha (TNFalpha)-like gene from Ciona intestinalis (CiTNF alpha-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster Eiger A and B TNF-like sequences were distinctly separated from the chordate TNFs. Thus, the ascidian TNFalpha-like cytokine was upregulated by in vivo LPS challenge supporting its pro-inflammatory role. In the pharynx, increased expression levels were found following analysis by real-time polymerase chain reaction, whereas in situ hybridization assay showed positive hemocytes both in the tissue and in circulating hemocytes. Finally, Western blot with monoclonal antibodies disclosed human TNFalpha epitopes in a 15-kDa protein component of the hemolymph serum and in a 43-kDa protein contained in the hemocyte lysate supernatant prepared in the presence of detergents. Both soluble and hemocyte-bound CiTNF alpha-like protein therefore appeared to be modulated by the LPS challenge.

Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1a-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4 h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60 kDa band identifiable as 1a-chain-Ci-type IX-Col. Observations of body wall sections (immunohistochemistry method) supported the role of hemocytes and showed that epidermis expressed Ci-type IX-Col 1a-chain in the time course of the inflammatory reaction (within 24 h). Transcript and protein were mainly found in the epidermis that outlined the proximal side of the tunic matrix (at 24 h after LPS injection), in cells associated with the epidermis at 4 and 192 h. In conclusion, the C. intestinalis inflammatory response to LPS challenge appeared to be composed of a complex reaction set, and for the first time we showed in ascidians a granulation tissue with FACIT-collagen production that could participate in inflammation and wound healing. Like in vertebrates, C. intestinalis acute inflammatory reactions result in a regulated pattern of tissue repair with collagen expression during remodelling. Ci-type IX-Col could be involved in a network of non-fibril-forming collagens that participates in the organization of extracellular matrix and defense responses.

Glucocorticoid hormone receptors (GR), members of the nuclear hormone receptor superfamily, are ligand-dependent transcription factors expressed in various tissues by binding to specific DNA sequences. Since glucocorticoids have a role in maintaining the homeostatic status in fish, we previously cloned and sequenced a GR (DlGR1) of adult Dicentrarchus labrax; we also showed mRNA expression (in situ hybridization) and tissue immunohistochemical localization of DlGR1 in several organs. This work has now been extended to the examination of the expression, tissue distribution, and cytolocalization of DlGR1 in larval developmental stages by similar methods to those used for the adult organs. The riboprobe included the DlGR1 cDNA transcriptional activation domain (1.0–1,300 nucleotide sequence) showing no significant similarity with a known second GR cDNA sequence of sea bass. The antibody was specific for an opportunely selected peptide sequence of the DlGR1 transcriptional domain. In histological sections of brain, head kidney, gills, liver, anterior intestine, and spleen cells, the riboprobe was mainly located in the cell nucleus. The antibody identified DlGR1 in the head kidney, gills, liver, and anterior intestine, mainly located in the cytosol. These results are in agreement with the receptor location in adult tissues. The greater presence of both the transcript and protein of DlGR1 in the late developmental stages suggests an increasing expression of this receptor. The cytolocalization (nuclear-cytosolic) and presumptive roles of DlGR1-containing tissues are discussed.

This study shows that high methylmercury concentrations are cytotoxic for Styela plicata hemocytes, whereas sublethal concentrations affect immunocyte responses. Moreover, hemocytes exposed to the xenobiotic present a significantly enhanced phenoloxidase activity as revealed in the hemocyte lysate supernatant compared with the control. Although the cytotoxic activity of S. plicata hemocytes toward rabbit erythrocytes is a PO-dependent cell-target reaction due to quinone products, it was significantly decreased by suitable methylmercury concentrations in the medium. The same xenobiotic concentrations decreased the hemocyte phagocytic activity toward yeast. In both the responses cell-target contacts could be affected by methylmercury, whereas the releasing capacity appeared to be unchanged, as indicated by hemocyte PO-release in the medium. Finally, changes in hemocyte shape and spreading capacity were shown. On the basis of the present results, Styela plicata hemocyte responses could be an additional immunotoxicology test using a microplate method that reveals cell morphological changes and spreading capacity. Copyright © 2007 John Wiley & Sons, Ltd.

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days posthatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of larvae (from 20 days post-hatching) and juveniles and in parenchymal tissue of juveniles. DlFBL mRNA and protein were detected in the intestinal epithelium and in hepatocytes. An amplification product from degenerate primers indicates that lectin isotypes with DlFBL epitopes are expressed in eggs and embryos. Whether the lectin fraction isolated from eggs and embryos includes DlFBL of maternal origin remains unclear.

To gain further insight into the role of cortisol in Wsh innate immune responses, we cloned and sequenced a 2592 bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton’s mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL chemiluminescence response (CL) has been related to increased respiratory burst of phagocytes stimulated with zymosan, PCLs, preincubated in vitro with cortisol at various concentrations, were assayed for their CL response. Dose-dependent cortisol inhibitory eVects, and signiWcant competitive activity of a low concentration of mifepristone (RU486), a glucocorticoid-receptor blocker, supported that cortisol–GR interaction was involved in modulating CL response via a genomic pathway. Results also indicated that cortisol could be eVective through an additional not-genomic way, and showed that high doses of RU486 exerted an inhibitory eVect on PCL chemiluminescence activity. © 2006 Elsevier Inc. All rights reserved.

Studies on inducible ascidian lectins may shed light on the evolutionary emergence of cytokine functions. Here, we show that the levels of opsonins, with IL1alpha-epitopes, increase in Ciona intestinalis hemolymph as a response to an inflammatory stimulus and, in particular, to intratunic injection of lipopolysaccharide (LPS). The inflammatory agent promptly (within 4 h) enhances Ca(2+)-independent serum hemagglutinating and opsonizing activities, which are both inhibited by D-galactose and D-galactosides (alpha-lactose, N-acetyl-D-lactosamine, thio-digalactoside), suggesting that anti-rabbit erythrocyte lectins with galectin properties are involved as opsonins. Inducible galectin molecules contain interleukin-1alpha (IL1alpha) epitopes, and their activities are specifically inhibited by anti-human recombinant IL1alpha antibody. Analysis by SDS-polyacrylamide gel electrophoresis has revealed that the density of the bands of several serum proteins increases within 4 h after LPS injection, correlated with the enhanced serum activity. Moreover, Western blot patterns demonstrate that several serum proteins (59, 37, 30, 23, 15 kDa) cross-react with the antibody as early as 4 h post-injection. Although we have not been able to establish whether, in adition to galectins, various types of D-galactose-specific lectins are contained in the serum, we show, for the first time in invertebrates, that galectin molecules with opsonic properties can be enhanced in response to a non-specific inflammatory stimulus, and that their release can be further stimulated by LPS. Finally, we reveal that multiple galectins share human IL1alpha epitopes, probably because of steric configuration and the oligomerization process.