Long term effects of carbaryl exposure on antiviral immune responses in Xenopus laevis.

Water pollutants associated with agriculture may contribute to the increased prevalence of infectious diseases caused by ranaviruses. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable experimental platform for evaluating the effects of common waterborne pollutants, such as the insecticide carbaryl. Following 3 weeks of exposure to 10 ppb carbaryl, X. laevis tadpoles exhibited a marked increase in mortality and accelerated development. Exposure at lower concentrations (0.1 and 1.0 ppb) was not toxic, but it impaired tadpole innate antiviral immune responses, as evidenced by significantly decreased TNF-α, IL-1ß, IFN-I, and IFN-III gene expression. The defect in IFN-I and IL-1ß gene expression levels persisted after metamorphosis in froglets, whereas only IFN-I gene expression in response to FV3 was attenuated when carbaryl exposure was performed at the adult stage. These findings suggest that the agriculture-associated carbaryl exposure at low but ecologically-relevant concentrations has the potential to induce long term alterations in host-pathogen interactions and antiviral immunity.

Molecular cloning, characterization and expression analysis of cathepsin O in silkworm Bombyx mori related to bacterial response.

Cathepsins are the main members of the cysteine family and play important roles in immune response in vertebrates. The Cathepsin O of Bombyx mori (BmCathepsin O) was cloned from the hemocytes by the rapid amplification of cDNA ends (RACE). The genomic DNA was 6131bp long with a total of six exons and five introns. Its pre-mRNA was spliced to generate two spliceosomes. By comparisons with other reported cathepsins O, it was concluded that the identity between them ranged from 29 to 39%. Expression analysis indicated that BmCathepsin O was specific-expressed in hemocytes, and highly expressed at the 4th molting and metamorphosis stages. Immunofluorescence assay and qRT-PCR showed that BmCathepsin O was expressed in granulocytes and plasmatocytes. Interestingly, BmCathepsin O was significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo, which suggested that BmCathepsin O may be regulated by 20E. Moreover, activation of BmCathepsin O was also observed in hemocytes challenged by Escherichia coli, indicating its potential involvement in the innate immune system of silkworm, B. mori. In summary, our studies provide a new insight into the functional features of Cathepsin O.

Metamorphosis and collagen-IV-fragments stimulate innate immune response in the greater wax moth, Galleria mellonella.

A novel link between development and immunity in insects is introduced. Transiently enhanced expression of lysozyme, gallerimycin and the insect metalloproteinase inhibitor was discovered at the onset of metamorphosis of the greater wax moth, Galleria mellonella. Relative quantification of mRNAs encoding for these antimicrobial peptides using real-time PCR documents their induced expression during transformation of last instar larvae into prepupae and upon injection of either recombinant interstitial collagenase (MMP-1) or small-sized fragments of collagen type IV. The latter were also found to stimulate both nuclear import of c-Rel-proteins in the fat body, implicating activation of Toll or Imd-related signaling pathways, and subsequent synthesis of antimicrobial peptides. Obtained results implicate that degradation of collagen-IV by either microbial metalloproteinases associated with invading pathogens or endogenous matrix metalloproteinases contributing to degradation of extracellular matrix during metamorphosis stimulate innate immune responses.

Cytotoxic activity of blowfly Calliphora vicina hemocytes.

The cytotoxic activity of hemocytes isolated from larvae of the blowfly Calliphora vicina was tested using human myelogenous leukemia K562 cells as target. Both single cell and cytotoxicity assays demonstrated that the hemocytes recognize the K562 cells as nonself, firmly attach to their surface and induce target destruction in a manner resembling the effect of mammalian cytotoxic lymphocytes. The cytotoxic activity increased dramatically in the course of larval metamorphosis and was considerably higher shortly before the onset of pupariation, compared to the activity of human peripheral blood or mouse spleen lymphocytes. In insects, the cytotoxic hemocytes may take part in defense against eukaryotic parasites and in the elimination of aberrant self cells, as well as in developmental processes such as metamorphosis.

Regulation of antibacterial protein synthesis following infection and during metamorphosis of Manduca sexta.

Larvae of the tobacco hornworm, Manduca sexta, respond to intrahemocoelic injection of bacteria or bacterial cell wall peptidoglycan with induced synthesis of a suite of antibacterial proteins. Previous studies have demonstrated peptidoglycan regulation of the synthesis of these antibacterial proteins. In addition to eliciting enhanced synthesis of antibacterial proteins, peptidoglycan fragments also elicit a "malaise syndrome" characterized by decreased feeding and growth, delayed metamorphosis, and altered excretion. We speculate that these symptoms may be components of a mechanism to flush out and sterilize the midgut lumen, one of the primary sources of bacterial infection in insects. Studies of naive larvae have demonstrated the accumulation of lysozyme in the differentiating pupal midgut epithelium and release of lysozyme into the pupal midgut lumen after the larval midgut epithelium has been sloughed off. These observations have been extended by the identification of potent bactericidal activity against E. coli and immunoreactive hemolin, together with lysozyme, in the lumen of the newly differentiated pupal midgut.