Probiotic based-diet effect on the immune response and induced stress in irradiated mass reared Ceratitis capitata males (Diptera: Tephritidae) destined for the release in the sterile insect technique programs.

Ceratitis capitata (medfly) is one of the most devastating crop pests worldwide. The Sterile Insect Technique (SIT) is a control method that is based on the mass rearing of males, their sterilization, and release in the field. However, the effectiveness of the technique depends on the quality of the released males and their fitness. We previously isolated and selected a probiotic bacteria (Enterobacter sp.), from wild-caught medflies, according to criteria that improved biological quality traits of reared medfly males.We firstly evaluated the impact of the irradiation on the expression of different immune and stress genes in the medfly sterile males. Expression was measured at differents time points ranging from 0 to 168 h after irradiation to capture the response of genes with distinct temporal expression patterns. Then, we supplemented the larval diet with previously isolated Enterobacter sp.strain, live and autoclaved at various concentrations to see whether the probiotic treatments affect, through their protective role, the gene expression level, and quality traits. The irradiation had significant effect on the genes attacin, cecropin, PGPR-LC, hsp23, and hsp70 level expression. The expression of attacin and PGPR-LC was up-regulated while that of cecropin was down-regulated. Hsp genes showed decreased levels between 0 and 18 h to peak at 72 h. However, the supplementation of the probiotic strain, either live or autoclaved, was statistically significant only for attacingene. However, significant interaction time x probiotic was noticed for attacin, cecropin, hsp23 and hsp70. The probiotic treatments also improved the quality control parameters like pupal weight. From this work we can conclude that a consortium of parabiotics (autoclaved probiotics) treatment will be recommended in insectaries considering both the beneficial effects on mass reared insects and its general safety for insectary workers and for environment.

Selection of Ceratitis capitata (Diptera: Tephritidae) specific recombinant monoclonal phage display antibodies for prey detection analysis.

Several recombinant antibodies against the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), one of the most important pests in agriculture worldwide, were selected for the first time from a commercial phage display library of human scFv antibodies. The specificity and sensitivity of the selected recombinant antibodies were compared with that of a rabbit polyclonal serum raised in parallel using a wide range of arthropod species as controls. The selected recombinant monoclonal antibodies had a similar or greater specificity when compared with classical monoclonal antibodies. The selected recombinant antibodies were successfully used to detect the target antigen in the gut of predators and the scFv antibodies were sequenced and compared. These results demonstrate the potential for recombinant scFv antibodies to be used as an alternative to the classical monoclonal antibodies or even molecular probes in the post-mortem analysis studies of generalist predators.

Hydrogen peroxide is produced by E. coli challenged haemocytes and regulates phagocytosis, in the medfly Ceratitis capitata. The active role of superoxide dismutase.

Hydrogen peroxide (H(2)O(2)) participates as a second messenger in cell signaling. In this paper, the role of H(2)O(2) was investigated, in Escherichia coli phagocytosis by the haemocytes of the medfly Ceratitis capitata. Block of H(2)O(2) synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (NEM) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and H(2)O(2). Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H(2)O(2) synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with ß integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced H(2)O(2) in the differential phosphorylation of MAP kinases.

A beta integrin subunit regulates bacterial phagocytosis in medfly haemocytes.

We have recently reported that the activation of focal adhesion kinase (FAK) and its downstream targets upon pathogen challenge regulate phagocytosis in medfly haemocytes. The goal of this study was to further explore the signalling pathway underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate the haemocyte surface receptor, through which the extracellular signals in response to bacteria are transmitted intracellularly. The presented data demonstrate the expression of a beta integrin subunit in the surface of medfly haemocytes that transmits signals upon pathogen triggering to FAK and its downstream targets, Src, MAP kinases and Elk-1-like protein, for the engulfment of pathogen. Interestingly LPS is not internalized through integrins.

Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes.

Phagocytosis, melanization and nodulation in insects depend on phenoloxidase (PO) activity. In this report, we demonstrated that these three processes appear to be also dependent on dopa decarboxylase (Ddc) activity. Using flow cytometry, RNA interference, immunoprecipitation and immunofluorescence, we demonstrated the constitutive expression of Ddc and its strong association with the haemocyte surface, in the medfly Ceratitis capitata. In addition, we showed that Escherichia coli phagocytosis is markedly blocked by small interfering RNA (siRNA) for Ddc, antibodies against Ddc, as well as by inhibitors of Ddc activity, namely carbidopa and benzerazide, convincingly revealing the involvement of Ddc activity in phagocytosis. By contrast, latex beads and lipopolysaccharide (LPS) did not require Ddc activity for their uptake. It was also shown that nodulation and melanization processes depend on Ddc activation, because antibodies against Ddc and inhibitors of Ddc activity prevent haemocyte aggregation and melanization in the presence of excess E. coli. Therefore, phagocytosis, melanization and nodulation depend on haemocyte-surface-associated PO and Ddc. These three unrelated mechanisms are based on tyrosine metabolism and share a number of substrates and enzymes; however, they appear to be distinct. Phagocytosis and nodulation depend on dopamine-derived metabolite(s), not including the eumelanin pathway, whereas melanization depends exclusively on the eumelanin pathway. It must also be underlined that melanization is not a prerequisite for phagocytosis or nodulation. To our knowledge, the involvement of Ddc, as well as dopa and its metabolites, are novel aspects in the phagocytosis of medfly haemocytes.

The role of N-beta-alanyldopamine synthase in the innate immune response of two insects.

Insects trigger a multifaceted innate immune response to fight microbial infections. We show that in the yellow mealworm, Tenebrio molitor, septic injuries induce the synthesis of N-beta-alanyldopamine (NBAD), which is known as the main sclerotization precursor of insect brown cuticles. We demonstrate that NBAD synthase is induced in the epidermis of the mealworm and of the Medfly, Ceratitis capitata, by infection with Escherichia coli. Our results indicate that synthesis of NBAD seems to be a novel component of the overall innate immune response in insects.

Distinct signalling pathways promote phagocytosis of bacteria, latex beads and lipopolysaccharide in medfly haemocytes.

In insects, phagocytosis is an important innate immune response against pathogens and parasites, and several signal transduction pathways regulate this process. The focal adhesion kinase (FAK)/Src and mitogen activated protein kinase (MAPK) pathways are of central importance because their activation upon pathogen challenge regulates phagocytosis via haemocyte secretion and activation of the prophenoloxidase (proPO) cascade. The goal of this study was to explore further the mechanisms underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate that (1) phagocytosis of bacteria (both Gram-negative and Gram-positive) is dependent on RGD-binding receptors, FAK/Src and MAPKs, (2) latex bead phagocytosis is RGD-binding-receptor-independent and dependent on FAK/Src and MAPKs, (3) lipopolysaccharide internalization is RGD-binding-receptor-independent and FAK/Src-independent but MAPK-dependent and (4) in unchallenged haemocytes in suspension, FAK, Src and extracellular signal-regulated kinase (ERK) signalling molecules participating in phagocytosis show both a functional and a physical association. Overall, this study has furthered knowledge of FAK/Src and MAPK signalling pathways in insect haemocyte immunity and has demonstrated that distinct signalling pathways regulate the phagocytic activity of biotic and abiotic components in insect haemocytes. Evidently, the basic phagocytic signalling pathways among insects and mammals appear to have remained unchanged during evolution.