A pathogenic parasite interferes with phagocytosis of insect immunocompetent cells.

Phagocytosis activity of hemocytes of the host Galleria mellonella (Lepidoptera) was modulated by the infection of the entomopathogenic nematode Steinernema feltiae (Rahbditida) and was found to be correlated with the opsonization of bacteria by hemolymph factors. The presence of nematodes resulted in a significative decrease in phagocytosis of bacteria by host hemocytes, both in in vivo and in in vitro assays. Host interacting proteins (HIPs), which appear to function as opsonic factors and are essential to perform immune responses, were removed by S. feltiae from host hemolymph, by means of its epicuticle binding properties. Host humoral factors sequestered by the parasite have been identified by monodimensional and 2D electrophoretic analysis. The data suggest that S. feltiae, living in association with symbiontic bacteria (Xenorhabdus nematophilus), develop an immune suppressive strategy to support its bacteria, which diminished the effectiveness of immunological surveillance by the host.

Parasite suppression of the oxidations of eumelanin precursors in Drosophila melanogaster.

In insects, eukaryotic endoparasites encounter a series of innate immune effector responses mediated in large part by circulating blood cells (hemocytes) that rapidly form multilayer capsules around foreign organisms. Critical components of the encapsulation response are chemical and enzyme-catalyzed oxidations involving phenolic and catecholic substrates that lead to synthesis of eumelanin. These responses are initiated immediately upon infection and are very site-specific, provoking no undesirable systemic responses in the host. In this study, we were interested to learn if the principal oxidation pathways leading to the synthesis of eumelanin in larvae of Drosophila melanogaster were targets for inhibition by immune suppressive factors (ISF) derived from a virulent strain of the endoparasitic wasp Leptopilina boulardi. Comparative in vitro assays monitored by sensitive electrochemical detection methods showed that ISF derived from female reproductive tissues significantly diminished the oxidations of the two diphenol eumelanin precursors, dopamine and 5,6-dihydroxyindole (DHI). The oxidations of the monophenol tyrosine, and two other related diphenols, dopa and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), were not significantly inhibited by ISF. The data suggest that melanogenesis represents at least one of the host responses suppressed by L. boulardi ISF, and that the oxidation pathways selectively targeted for inhibition are those synthesizing decarboxylated pigment precursors derived from DHI. These observations, together with previous reports of adverse effects of ISF on the ability of hemocytes to adhere to foreign surfaces, suggest a multifaceted approach by the parasitoid to circumvent the innate immune response of D. melanogaster.

Production and utilization of hydrogen peroxide associated with melanogenesis and tyrosinase-mediated oxidations of DOPA and dopamine.

The synthesis and involvement of H(2)O(2) during the early stages of melanogenesis involving the oxidations of DOPA and dopamine (diphenolase activity) were established by two sensitive and specific electrochemical detection systems. Catalase-treated reaction mixtures showed diminished rates of H(2)O(2) production during the autoxidation and tyrosinase-mediated oxidation of both diphenols. Inhibition studies with the radical scavenger resveratrol revealed the involvement in these reactions of additional reactive intermediate of oxygen (ROI), one of which appears to be superoxide anion. There was no evidence to suggest that H(2)O(2) or any other ROI was produced during the tyrosinase-mediated conversion of tyrosine to DOPA (monophenolase activity). Establishing by electrochemical methods the endogenous production H(2)O(2) in real time confirms recent reports, based in large part on the use of exogenous H(2)O(2), that tyrosinase can manifest both catalase and peroxidase activities. The detection of ROI in tyrosinase-mediated in vitro reactions provides evidence for sequential univalent reductions of O(2), most likely occurring at the enzyme active site copper. Collectively, these observations focus attention on the possible involvement of peroxidase-H(2)O(2) systems and related ROI-mediated reactions in promoting melanocytotoxic and melanoprotective processes.

Melanization immune responses in mosquito vectors.

The production and deposition of melanin pigments on invading pathogens and parasites represents a unique, innate immune response in the phylum Arthropoda. This immune response has started to receive considerable attention because of the potential to exploit this mechanism to control mosquito-borne diseases. In this article, we summarize knowledge about this complex biochemistry, the use of melanin biosynthesis in diverse physiological processes and the gaps in knowledge that must be addressed if this immune process is to be manipulated in genetic-based control strategies.

Description of the transcriptomes of immune response-activated hemocytes from the mosquito vectors Aedes aegypti and Armigeres subalbatus.

Mosquito-borne diseases, including dengue, malaria, and lymphatic filariasis, exact a devastating toll on global health and economics, killing or debilitating millions every year (54). Mosquito innate immune responses are at the forefront of concerted research efforts aimed at defining potential target genes that could be manipulated to engineer pathogen resistance in vector populations. We aimed to describe the pivotal role that circulating blood cells (called hemocytes) play in immunity by generating a total of 11,952 Aedes aegypti and 12,790 Armigeres subalbatus expressed sequence tag (EST) sequences from immune response-activated hemocyte libraries. These ESTs collapsed into 2,686 and 2,107 EST clusters, respectively. The clusters were used to adapt the web-based interface for annotating bacterial genomes called A Systematic Annotation Package for Community Analysis of Genomes (ASAP) for analysis of ESTs. Each cluster was categorically characterized and annotated in ASAP based on sequence similarity to five sequence databases. The sequence data and annotations can be viewed in ASAP at https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm. The data presented here represent the results of the first high-throughput in vivo analysis of the transcriptome of immunocytes from an invertebrate. Among the sequences are those for numerous immunity-related genes, many of which parallel those employed in vertebrate innate immunity, that have never been described for these mosquitoes. The sequences and annotations presented in this paper have been submitted to GenBank under accession numbers AY 431103 to AY 433788 (Aedes aegypti) and AY 439334 to AY 441440 (Armigeres subalbatus).

Interactions of iron with reactive intermediates of oxygen and nitrogen.

Iron not only functions as a cofactor for various enzymes, but it is also a source of potentially cytotoxic molecules produced through interactions with certain reactive intermediates of oxygen (ROI) and nitrogen (RNI). Protection from such iron-mediated damage results in large part from homeostatic mechanisms that regulate the sequestration of iron. Perturbations in iron homeostasis can result in an array of adverse cellular manifestations including oxidative and nitrosative stress, enhanced production of free radicals, macromolecular damage, and cell death. This brief review focuses on some of the potentially adverse reactions of iron with ROI and RNI.

Dexamethasone inhibition of the cellular immune response of Drosophila melanogaster against a parasitoid.

Host larvae of Drosophila melanogaster injected with the eicosanoid biosynthesis inhibitor, dexamethasone, prior to parasitization by the wasp Leptopilina boulardi, exhibited significantly reduced rates of melanotic encapsulation in comparison with control and saline-injected larvae. The results of this investigation suggest that prostaglandins and other eicosanoids are involved as cell-signaling molecules in the hemocytic encapsulation reaction of D. melanogaster larvae.

Resistance of the insect cell line IPLB-LdFB to salsolinol-induced apoptosis.

Apoptosis is a form of cell death that is manifested in Parkinson's disease (PD) and certain other neurodegenerative disorders. Metabolites of salsolinol (SAL), an intraneuronal, dopamine-derived tetrahydroisoquinoline (TIQ), have been shown to induce apoptosis in human dopaminergic neuroblastoma cells, implicating these molecules as causative or contributory factors in the selective killing of nigrostriatal dopaminergic neurons, a cardinal manifestation of Parkinson's disease. Since insects employ dopamine and related catecholamines in a variety of processes including cuticular sclerotization and cellular immune reactions, it was of interest to know how insect cells metabolized exogenous SAL. Propidium iodide staining combined with flow cytometry showed that IPLB-LdFB cells from Lymantria dispar exhibited no significant (P < 0.05) increase in apoptosis when incubated for 48 h with concentrations of SAL ranging from 10 microM to 1 mM. A significant increase in apoptosis (P < 0.05) was observed in cell cultures containing the highest concentration of SAL tested (5 mM), but only 12.4% of the cells manifested this form of cell death. High pressure liquid chromatography with electrochemical detection (HPLC-ED) was used to document the production of two potentially cytotoxic quinonoids generated during the autoxidation of SAL, a reaction that was found to be significantly (P < 0.05) enhanced by peroxidase. The resistance of IPLB-LdFB cells to SAL-induced apoptosis is attributed to the ability of these insect cells to metabolize and/or detoxify such dopamine-derived catecholic TIQs. Thus, the biochemical pathways employed by insect cells in these processes may be of considerable interest to individuals investigating certain neurodegenerative disorders.