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In JoVE (1)

Other Publications (22)

Articles by Ioannis Eleftherianos in JoVE

 JoVE Biology

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

1Department of Biological Sciences, The George Washington University


JoVE 4173

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Other articles by Ioannis Eleftherianos on PubMed

High-throughput Detection of Knockdown Resistance in Myzus Persicae Using Allelic Discriminating Quantitative PCR

The peach-potato aphid Myzus persicae (Sulzer) has developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, termed knockdown resistance (kdr). This reduced sensitivity is caused by two mutations, L1014F (kdr) and M918T (super-kdr), in the para-type voltage-gated sodium channel. We have developed a diagnostic dose bioassay to detect kdr and provide preliminary information on the genotype present. We also developed two allelic discrimination PCR assays to determine precisely the genotypes of the two mutations (L1014F and M918T) in individual M. persicae using fluorescent Taqman MGB probes. In combination with assays for elevated carboxylesterase levels and modified acetylcholinesterase (MACE), this suite of assays allows for rapid high-throughput diagnosis, in individual aphids, of the three main resistance mechanisms of practical importance in the UK.

RNAi Suppression of Recognition Protein Mediated Immune Responses in the Tobacco Hornworm Manduca Sexta Causes Increased Susceptibility to the Insect Pathogen Photorhabdus

Bacterial pathogens either hide from or overcome the immune response of their hosts. Here we show that two different species of insect pathogenic bacteria, Photorhabdus luminescens TT01 and Photorhabdus asymbiotica ATCC43949, were both recognized by the immune system of their host Manduca sexta, as indicated by a rapid increase in the levels of mRNAs encoding three different inducible microbial recognition proteins, Hemolin, Immulectin-2 and peptidoglycan recognition protein. RNA interference (RNAi)-mediated inhibition of expression ("knock-down") of each of these genes at the level of both mRNA and protein was achieved through injection of double-stranded RNA (dsRNA). Knock-down of any one of these genes markedly decreased the ability of the insects to withstand infection when exposed to either species of Photorhabdus, as measured by the rate at which infected insects died. RNAi against Immulectin-2 caused the greatest reduction in host resistance to infection. The decreased resistance to infection was associated with reduced hemolymph phenoloxidase activity. These results show not only that Photorhabdus is recognized by the Manduca sexta immune system but also that the insect's immune system plays an active, but ultimately ineffective, role in countering infection.

Prior Infection of Manduca Sexta with Non-pathogenic Escherichia Coli Elicits Immunity to Pathogenic Photorhabdus Luminescens: Roles of Immune-related Proteins Shown by RNA Interference

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

The Immunoglobulin Family Protein Hemolin Mediates Cellular Immune Responses to Bacteria in the Insect Manduca Sexta

Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.

An Antibiotic Produced by an Insect-pathogenic Bacterium Suppresses Host Defenses Through Phenoloxidase Inhibition

Photorhabdus is a virulent pathogen that kills its insect host by overcoming immune responses. The bacterium also secretes a range of antibiotics to suppress the growth of other invading microorganisms. Here we show that Photorhabdus produces a small-molecule antibiotic (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene (ST) that also acts as an inhibitor of phenoloxidase (PO) in the insect host Manduca sexta. The Photorhabdus gene stlA encodes an enzyme that produces cinnamic acid, a key precursor for production of ST, and a mutation in stlA results in loss of ST production and PO inhibitory activity, which are both restored by genetic complementation of the mutant and also by supplying cinnamic acid. ST is produced both in vitro and in vivo in sufficient quantities to account for PO inhibition and is the only detectable solvent-extractable inhibitor. A Photorhabdus stlA- mutant is significantly less virulent, proliferates slower within the host, and provokes the formation of significantly more melanotic nodules than wild-type bacteria. Virulence of the stlA- mutant is also rescued by supplying cinnamic acid. The proximate cause of the virulence effect, however, is the inhibition of PO, because the effect of the stlA- mutation on virulence is abolished in insects in which PO has been knocked down by RNA interference (RNAi). Thus, ST has a dual function both as a PO inhibitor to counter host immune reactions and also as an antibiotic to exclude microbial competitors from the insect cadaver.

A Nematode Symbiont Sheds Light on Invertebrate Immunity

Photorhabdus bacteria live in a 'symbiosis of pathogens' with nematodes that invade and kill insects. Recent work has begun to use the power of the model insect Drosophila to dissect the molecular basis of the invertebrate immune response to the combined insult of the worms and their symbiotic bacterial pathogens. By using RNA interference, it is now also possible to dissect this complex tripartite interaction in a range of both model and non-model hosts.

ATP-sensitive Potassium Channels Mediate Survival During Infection in Mammals and Insects

Specific homeostatic mechanisms confer stability in innate immune responses, preventing injury or death from infection. Here we identify, from a screen of N-ethyl-N-nitrosourea-mutagenized mice, a mutation causing both profound susceptibility to infection by mouse cytomegalovirus and approximately 20,000-fold sensitization to lipopolysaccharide (LPS), poly(I.C) and immunostimulatory (CpG) DNA. The LPS hypersensitivity phenotype is not suppressed by mutations in Myd88, Trif, Tnf, Tnfrsf1a, Ifnb, Ifng or Stat1, genes contributing to LPS responses, and results from an abnormality extrinsic to hematopoietic cells. The phenotype is due to a null allele of Kcnj8, encoding Kir6.1, a protein that combines with SUR2 to form an ATP-sensitive potassium channel (K(ATP)) expressed in coronary artery smooth muscle and endothelial cells. In Drosophila melanogaster, suppression of dSUR by RNA interference similarly causes hypersensitivity to infection by flock house virus. Thus, K(ATP) evolved to serve a homeostatic function during infection, and in mammals it prevents coronary artery vasoconstriction induced by cytokines dependent on TLR and/or MDA5 immunoreceptors.

Inheritance of L1014F and M918T Sodium Channel Mutations Associated with Pyrethroid Resistance in Myzus Persicae

Two amino acid substitutions (L1014F and M918T) in the voltage-gated sodium channel confer target-site resistance to pyrethroid insecticides in the peach potato aphid, Myzus persicae. Pyrethroid-resistant and -susceptible M. persicae clones with various combinations of these mutations were crossed under laboratory conditions, and the genotypes of aphid progeny were analysed by direct DNA sequencing of the IIS4-S6 region of the sodium channel gene. Segregation patterns showed that in aphids heterozygous for both L1014F and M918T, both mutations were present in the same resistance allele. Despite these mutations appearing largely recessive in other pest species, such aphids exhibited strong resistance to pyrethroids in leaf-dip bioassays. These results have important implications for the spread and management of pyrethroid resistance in field populations.

Rapid Virulence Annotation (RVA): Identification of Virulence Factors Using a Bacterial Genome Library and Multiple Invertebrate Hosts

Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.

The Yersinia Pseudotuberculosis and Yersinia Pestis Toxin Complex is Active Against Cultured Mammalian Cells

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

A Single Locus from the Entomopathogenic Bacterium Photorhabdus Luminescens Inhibits Activated Manduca Sexta Phenoloxidase

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo. Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli. The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.

Novel Antibiotic Compounds Produced by the Insect Pathogenic Bacterium Photorhabdus

Phototorhabdus is an insect pathogenic enterobacterium which maintains a mutualistic interaction with heterorhabditid nematodes. While the bacteria live in the nematode gut, the nematodes live in the soil and infect insect larvae, releasing their symbiotic bacteria into the insect blood. Here the bacteria reproduce and kill the insect by septicaemia. The nematodes then feed on the bacterial biomass and undergo several rounds of reproduction before emerging from the cadaver carrying their bacterial symbionts. Photorhabdus secretes a versatile armory of antimicrobial molecules into the insect corpse. These biocides exert a range of antimicrobial killing activities and serve a dual function. They minimize competition from non-symbiotic bacteria and prevent microbial putrefaction of the nematode-infected insect cadaver. The goal of this review is to describe current knowledge of the molecular mechanisms involved in the production of bacteriocins by Photorhabdus. Recent important advances in identifying novel potent antibiotic compounds from Photorhabdus and elucidating their complex mode of action in relation to pathogenicity and symbiosis associations are also discussed. The last part of this review focuses on the potential role Photorhabdus antibiotics may play in contributing to the discovery of novel pharmaceutical and agrochemical products. The present article is a short review of recent patents on Photohabdus.

The KdpD/KdpE Two-component System of Photorhabdus Asymbiotica Promotes Bacterial Survival Within M. Sexta Hemocytes

Many bacteria persist within phagocytes, deploying complex sets of tightly regulated virulence factors to manipulate and survive within host cells. So far, no single factor has been identified that is sufficient to allow intracellular persistence of an otherwise non-pathogenic bacterium. Here we report that the two-component KdpD/KdpE sensor kinase/response regulator of the insect and human pathogen Photorhabdus asymbiotica (Pa) is sufficient to allow a harmless laboratory strain of E. coli to resist phagocytic killing and persist within insect hemocytes, ultimately killing the insect. Screening of a cosmid library of Pa in E. coli by injection into the moth Manduca sexta, previously identified three overlapping clones which caused the insect to cease feeding and subsequently die. Transposon mutagenesis revealed a cosmid encoded kdp high affinity potassium pump regulon was responsible for this phenotype. Gentamycin protection assays and confocal microscopy revealed the cosmid clones were persisting inside insect hemocytes far longer than control bacteria. Cloning and expression of PakdpD/kdpE alone into E. coli recapitulated the phenotype. Bioassay results and transcriptional analysis of various E. coli kdp mutants harboring the Pa kdp genes confirmed that Pa KdpD/KdpE was able to induce the E. coli kdp pump structural genes in response to exposure to insect hemocytes but not blood plasma alone. The finding that Pa KdpD/KdpE can facilitate resistance of E. coli to phagocytic killing suggests a central role for potassium in this process, supporting previous work implicating potassium sensing in virulence of other bacteria and also in the normal process of protease killing of engulfed bacteria by neutrophils.

Dissecting the Immune Response to the Entomopathogen Photorhabdus

Bacterial pathogens either hide from or modulate the host's immune response to ensure their survival. Photorhabdus is a potent insect pathogenic bacterium that uses entomopathogenic nematodes as vectors in a system that represents a useful tool for probing the molecular basis of immunity. During the course of infection, Photorhabdus multiplies rapidly within the insect, producing a range of toxins that inhibit phagocytosis of the invading bacteria and eventually kill the insect host. Photorhabdus bacteria have recently been established as a tool for investigating immune recognition and defense mechanisms in model hosts such as Manduca and Drosophila. Such studies pave the way for investigations of gene interactions between pathogen virulence factors and host immune genes, which ultimately could lead to an understanding of how some Photorhabdus species have made the leap to becoming human pathogens.

Role and Importance of Phenoloxidase in Insect Hemostasis

In response to microbial infection, insects mount several defense reactions including the induction of proteolytic cascades that lead to localized melanization and coagulation. Melanization requires the activation of prophenoloxidase (proPO) to its active form phenoloxidase (PO), a key enzyme that leads to the formation of melanin at wound sites and around intruding microorganisms in the hemolymph. Clotting is critical in limiting hemolymph loss and initiating wound healing following injury; it quickly acts to form a solid barrier against infection by immobilizing microorganisms and promoting their killing. Recent advances in Drosophila and other insects imply a possible link between PO and the coagulation system, although the exact molecular mechanisms controlling this interaction appear to be complex and are still not well defined. The development of hemolymph experimental techniques in Drosophila larvae together with proteomic analysis have further led to the identification of proPO as a cross-linking component that is involved in the hardening and melanization of clots. However, clot PO activity varies between insect species and life stages, depending on physiological and ecological conditions. Here we review our current knowledge of the association between PO and coagulation and discuss the implications of the previous findings on insect innate immunity and hemostasis.

RNA Interference in Lepidoptera: an Overview of Successful and Unsuccessful Studies and Implications for Experimental Design

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

A Serine Proteinase Homologue, SPH-3, Plays a Central Role in Insect Immunity

Numerous vertebrate and invertebrate genes encode serine proteinase homologues (SPHs) similar to members of the serine proteinase family, but lacking one or more residues of the catalytic triad. These SPH proteins are thought to play a role in immunity, but their precise functions are poorly understood. In this study, we show that SPH-3 (an insect non-clip domain-containing SPH) is of central importance in the immune response of a model lepidopteran, Manduca sexta. We examine M. sexta infection with a virulent, insect-specific, Gram-negative bacterium Photorhabdus luminescens. RNA interference suppression of bacteria-induced SPH-3 synthesis severely compromises the insect's ability to defend itself against infection by preventing the transcription of multiple antimicrobial effector genes, but, surprisingly, not the transcription of immune recognition genes. Upregulation of the gene encoding prophenoloxidase and the activity of the phenoloxidase enzyme are among the antimicrobial responses that are severely attenuated on SPH-3 knockdown. These findings suggest the existence of two largely independent signaling pathways controlling immune recognition by the fat body, one governing effector gene transcription, and the other regulating genes encoding pattern recognition proteins.

ATP-sensitive Potassium Channel (K(ATP))-dependent Regulation of Cardiotropic Viral Infections

The effects of the cellular environment on innate immunity remain poorly characterized. Here, we show that in Drosophila ATP-sensitive potassium channels (K(ATP)) mediate resistance to a cardiotropic RNA virus, Flock House virus (FHV). FHV viral load in the heart rapidly increases in K(ATP) mutant flies, leading to increased viremia and accelerated death. The effect of K(ATP) channels is dependent on the RNA interference genes Dcr-2, AGO2, and r2d2, indicating that an activity associated with this potassium channel participates in this antiviral pathway in Drosophila. Flies treated with the K(ATP) agonist drug pinacidil are protected against FHV infection, thus demonstrating the importance of this regulation of innate immunity by the cellular environment in the heart. In mice, the Coxsackievirus B3 replicates to higher titers in the hearts of mayday mutant animals, which are deficient in the Kir6.1 subunit of K(ATP) channels, than in controls. Together, our data suggest that K(ATP) channel deregulation can have a critical impact on innate antiviral immunity in the heart.

Drosophila Immunity Research on the Move

Drosophila has been established as useful model for infectious diseases because it allows large numbers of whole animals to be studied and provides powerful genetic tools and conservation with signaling and pathogenesis mechanisms in vertebrates. During the past twenty years, significant progress has been made on the characterization of innate immune responses against various pathogenic organisms in flies (Fig. 1). In this year's Drosophila Research Conference, which was held in San Diego (March 30-April 3) and sponsored by the Genetics Society of America, the immunity and pathogenesis session comprised seven platform presentations and 34 posters that highlighted the latest advances in Drosophila infection and immunity field. The presented work covered a wide range of studies from immune signaling pathways and the molecular basis of humoral and cellular immune mechanisms to the role of endosymbionts in fly immune function and effects of immune priming. Here, we give an overview of the presented work and we explain how these findings will open new avenues in Drosophila immunity research.

Insect Immune Responses to Nematode Parasites

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites.

A Novel Method for Infecting Drosophila Adult Flies with Insect Pathogenic Nematodes

Drosophila has been established as an excellent genetic and genomic model to investigate host-pathogen interactions and innate immune defense mechanisms. To date, most information on the Drosophila immune response derives from studies that involve bacterial, fungal or viral pathogens. However, immune reactions to insect parasitic nematodes are still not well characterized. The nematodes Heterorhabditis bacteriophora live in symbiosis with the entomopathogenic bacteria Photorhabdus luminescens, and they are able to invade and kill insects. Interestingly, Heterorhabditis nematodes are viable in the absence of Photorhabdus. Techniques for infecting Drosophila larvae with these nematodes have been previously reported. Here, we have developed a method for infecting Drosophila adult flies with Heterorhabditis nematodes carrying (symbiotic worms) or lacking (axenic worms) their associated bacteria. The protocol we present can be readily adapted for studying parasitic strategies of other insect nematodes using Drosophila as the host infection model.

Molecular Mechanisms of Aging and Immune System Regulation in Drosophila

Aging is a complex process that involves the accumulation of deleterious changes resulting in overall decline in several vital functions, leading to the progressive deterioration in physiological condition of the organism and eventually causing disease and death. The immune system is the most important host-defense mechanism in humans and is also highly conserved in insects. Extensive research in vertebrates has concluded that aging of the immune function results in increased susceptibility to infectious disease and chronic inflammation. Over the years, interest has grown in studying the molecular interaction between aging and the immune response to pathogenic infections. The fruit fly Drosophila melanogaster is an excellent model system for dissecting the genetic and genomic basis of important biological processes, such as aging and the innate immune system, and deciphering parallel mechanisms in vertebrate animals. Here, we review the recent advances in the identification of key players modulating the relationship between molecular aging networks and immune signal transduction pathways in the fly. Understanding the details of the molecular events involved in aging and immune system regulation will potentially lead to the development of strategies for decreasing the impact of age-related diseases, thus improving human health and life span.

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