The Drosophila melanogaster prophenoloxidase system participates in immunity against Zika virus infection.

Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from a wide range of pathogens, making it a convenient genetic model to study various human pathogenic viruses and host antiviral immunity. Here we explore for the first time the contribution of the Drosophila phenoloxidase (PO) system to host survival and defenses against Zika virus (ZIKV) infection by analyzing the role of mutations in the three prophenoloxidase (PPO) genes in female and male flies. We show that only PPO1 and PPO2 genes contribute to host survival and appear to be upregulated following ZIKV infection in Drosophila. Also, we present data suggesting that a complex regulatory system exists between Drosophila PPOs, potentially allowing for a sex-dependent compensation of PPOs by one another or other immune responses such as the Toll, Imd, and JAK/STAT pathways. Furthermore, we show that PPO1 and PPO2 are essential for melanization in the hemolymph and the wound site in flies upon ZIKV infection. Our results reveal an important role played by the melanization pathway in response to ZIKV infection, hence highlighting the importance of this pathway in insect host defense against viral pathogens and potential vector control strategies to alleviate ZIKV outbreaks.

Haemocyte-mediated immunity in insects: Cells, processes and associated components in the fight against pathogens and parasites.

The host defence of insects includes a combination of cellular and humoral responses. The cellular arm of the insect innate immune system includes mechanisms that are directly mediated by haemocytes (e.g., phagocytosis, nodulation and encapsulation). In addition, melanization accompanying coagulation, clot formation and wound healing, nodulation and encapsulation processes leads to the formation of cytotoxic redox-cycling melanin precursors and reactive oxygen and nitrogen species. However, demarcation between cellular and humoral immune reactions as two distinct categories is not straightforward. This is because many humoral factors affect haemocyte functions and haemocytes themselves are an important source of many humoral molecules. There is also a considerable overlap between cellular and humoral immune functions that span from recognition of foreign intruders to clot formation. Here, we review these immune reactions starting with the cellular mechanisms that limit haemolymph loss and participate in wound healing and clot formation and advancing to cellular functions that are critical in restricting pathogen movement and replication. This information is important because it highlights that insect cellular immunity is controlled by a multilayered system, different components of which are activated by different pathogens or during the different stages of the infection.

Dicer-2 Regulates Resistance and Maintains Homeostasis against Zika Virus Infection in Drosophila.

Zika virus (ZIKV) outbreaks pose a massive public health threat in several countries. We have developed an in vivo model to investigate the host-ZIKV interaction in Drosophila We have found that a strain of ZIKV replicates in wild-type flies without reducing their survival ability. We have shown that ZIKV infection triggers RNA interference and that mutating Dicer-2 results in enhanced ZIKV load and increased susceptibility to ZIKV infection. Using a flavivirus-specific Ab, we have found that ZIKV is localized in the gut and fat body cells of the infected wild-type flies and results in their perturbed homeostasis. In addition, Dicer-2 mutants display severely reduced insulin activity, which could contribute toward the increased mortality of these flies. Our work establishes the suitability of Drosophila as the model system to study host-ZIKV dynamics, which is expected to greatly advance our understanding of the molecular and physiological processes that determine the outcome of this disease.

Participation of the Serine Protease Jonah66Ci in the Drosophila Antinematode Immune Response.

Serine proteases and serine protease homologs form the second largest gene family in the Drosophila melanogaster genome. Certain genes in the Jonah multigene family encoding serine proteases have been implicated in the fly antiviral immune response. Here, we report the involvement of Jonah66Ci in the Drosophila immune defense against Steinernema carpocapsae nematode infection. We find that Drosophila Jonah66Ci is upregulated in response to symbiotic (carrying the mutualistic bacterium Xenorhabdus nematophila) or axenic (lacking Xenorhabdus) Steinernema nematodes and is expressed exclusively in the gut of Drosophila larvae. Inactivation of Jonah66Ci provides a survival advantage to larvae against axenic nematodes and results in differential expression of Toll and Imd pathway effector genes, specifically in the gut. Also, inactivation of Jonah66Ci increases the numbers of enteroendocrine and mitotic cells in the gut of uninfected larvae, and infection with Steinernema nematodes reduces their numbers, whereas the numbers of intestinal stem cells are unaffected by nematode infection. Jonah66Ci knockdown further reduces nitric oxide levels in response to infection with symbiotic Steinernema nematodes. Finally, we show that Jonah66Ci knockdown does not alter the feeding rates of uninfected Drosophila larvae; however, infection with axenic Steinernema nematodes lowers larval feeding. In conclusion, we report that Jonah66Ci participates in maintaining homeostasis of certain physiological processes in Drosophila larvae in the context of Steinernema nematode infection. Similar findings will take us a step further toward understanding the molecular and physiological mechanisms that take place during parasitic nematode infection in insects.

Transcript analysis reveals the involvement of NF-κB transcription factors for the activation of TGF-ß signaling in nematode-infected Drosophila.

The common fruit fly Drosophila melanogaster is a powerful model for studying signaling pathway regulation. Conserved signaling pathways underlying physiological processes signify evolutionary relationship between organisms and the nature of the mechanisms they control. This study explores the cross-talk between the well-characterized nuclear factor kappa B (NF-κB) innate immune signaling pathways and transforming growth factor beta (TGF-ß) signaling pathway in response to parasitic nematode infection in Drosophila. To understand the link between signaling pathways, we followed on our previous studies by performing a transcript-level analysis of different TGF-ß signaling components following infection of immune-compromised Drosophila adult flies with the nematode parasites Heterorhabditis gerrardi and H. bacteriophora. Our findings demonstrate the requirement of NF-κB transcription factors for activation of TGF-ß signaling pathway in Drosophila in the context of parasitic nematode infection. We observe significant decrease in transcript level of glass bottom boat (gbb) and screw (scw), components of the bone morphogenic protein (BMP) branch, as well as Activinß (actß) which is a component of the Activin branch of the TGF-ß signaling pathway. These results are observed only in H. gerrardi nematode-infected flies compared to uninfected control. Also, this significant decrease in transcript level is found only for extracellular ligands. Future research examining the mechanisms regulating the interaction of these signaling pathways could provide further insight into Drosophila anti-nematode immune function against infection with potent parasitic nematodes.

The peptidoglycan recognition protein PGRP-LE regulates the Drosophila immune response against the pathogen Photorhabdus.

Photorhabdus bacteria are potent pathogens of insects and humans. To elucidate the infection strategies Photorhabdus employs to subvert the host innate immune response, it is critical to use model organisms that permit the genetic dissection of the dynamics involved in host-pathogen interactions. Here, we employed the fruit fly Drosophila melanogaster to interrogate the role of the immune deficiency (Imd) pathway receptor peptidoglycan recognition protein LE (PGRP-LE) in the regulation of the fly's response to the insect pathogen Photorhabdus luminescens and the insect/human pathogen P. asymbiotica. We show that PGRP-LE is upregulated in response to injection of Photorhabdus bacteria in background control flies, and that loss-of-function PGRP-LE mutant flies are more sensitive specifically to P. luminescens infection and harbor a higher bacterial burden of this species compared to background controls. Also, our results indicate that the absence of functional PGRP-LE alters the transcriptional pathway activity of Imd and Jnk signaling upon infection with P. asymbiotica, while infection with P. luminescens modifies the activity of Jak/Stat signaling. These findings denote the participation of the PGRP-LE receptor in the response of D. melanogaster to Photorhabdus challenge and contribute to a better understanding of pathogen detection and host immune regulation against virulent microbial invaders.

Thioester-Containing Proteins 2 and 4 Affect the Metabolic Activity and Inflammation Response in Drosophila.

Drosophila melanogaster is an outstanding model for studying host antipathogen defense. Although substantial progress has been made in understanding how metabolism and immunity are interrelated in flies, little information has been obtained on the molecular players that regulate metabolism and inflammation in Drosophila during pathogenic infection. Recently, we reported that the inactivation of thioester-containing protein 2 (Tep2) and Tep4 promotes survival and decreases the bacterial burden in flies upon infection with the virulent pathogens Photorhabdus luminescens and Photorhabdus asymbiotica Here, we investigated physiological and pathological defects in tep mutant flies in response to Photorhabdus challenge. We find that tep2 and tep4 loss-of-function mutant flies contain increased levels of carbohydrates and triglycerides in the presence or absence of Photorhabdus infection. We also report that Photorhabdus infection leads to higher levels of nitric oxide and reduced transcript levels of the apical caspase-encoding gene Dronc in tep2 and tep4 mutants. We show that Tep2 and Tep4 are upregulated mainly in the fat body rather than the gut in Photorhabdus-infected wild-type flies and that tep mutants contain decreased numbers of Photorhabdus bacteria in both tissue types. We propose that the inactivation of Tep2 or Tep4 in adult Drosophila flies results in lower levels of inflammation and increased energy reserves in response to Photorhabdus, which could confer a survival-protective effect during the initial hours of infection.

The Imaginal Disc Growth Factors 2 and 3 participate in the Drosophila response to nematode infection.

The Drosophila imaginal disc growth factors (IDGFs) induce the proliferation of imaginal disc cells and terminate cell proliferation at the end of larval development. However, the participation of Idgf-encoding genes in other physiological processes of Drosophila including the immune response to infection is not fully understood. Here, we show the contribution of Idgf2 and Idgf3 in the Drosophila response to infection with Steinernema carpocapsae nematodes carrying or lacking their mutualistic Xenorhabdus nematophila bacteria (symbiotic or axenic nematodes, respectively). We find that Idgf2 and Idgf3 are upregulated in Drosophila larvae infected with symbiotic or axenic Steinernema and inactivation of Idgf2 confers a survival advantage to Drosophila larvae against axenic nematodes. Inactivation of Idgf2 induces the Imd and Jak/Stat pathways, whereas inactivation of Idgf3 induces the Imd, Toll and Jak/Stat pathways. We also show that inactivation of the Imd pathway receptor PGRP-LE upregulates Idgf2 against Steinernema nematode infection. Finally, we demonstrate that inactivation of Idgf3 induces the recruitment of larval haemocytes in response to Steinernema. Our results indicate that Idgf2 and Idgf3 might be involved in different yet crucial immune functions in the Drosophila antinematode immune response. Similar findings will promote the development of new targets for species-specific pest control strategies.

Identification of candidate infection genes from the model entomopathogenic nematode Heterorhabditis bacteriophora.

BACKGROUND: Despite important progress in the field of innate immunity, our understanding of host immune responses to parasitic nematode infections lags behind that of responses to microbes. A limiting factor has been the obligate requirement for a vertebrate host which has hindered investigation of the parasitic nematode infective process. The nematode parasite Heterorhabditis bacteriophora offers great potential as a model to genetically dissect the process of infection. With its mutualistic Photorhabdus luminescens bacteria, H. bacteriophora invades multiple species of insects, which it kills and exploits as a food source for the development of several nematode generations. The ability to culture the life cycle of H. bacteriophora on plates growing the bacterial symbiont makes it a very exciting model of parasitic infection that can be used to unlock the molecular events occurring during infection of a host that are inaccessible using vertebrate hosts. RESULTS: To profile the transcriptional response of an infective nematode during the early stage of infection, we performed next generation RNA sequencing on H. bacteriophora IJs incubated in Manduca sexta hemolymph plasma for 9 h. A subset of up-regulated and down-regulated genes were validated using qRT-PCR. Comparative analysis of the transcriptome with untreated controls found a number of differentially expressed genes (DEGs) which cover a number of different functional categories. A subset of DEGs is conserved across Clade V parasitic nematodes revealing an array of candidate parasitic genes. CONCLUSIONS: Our analysis reveals transcriptional changes in the regulation of a large number of genes, most of which have not been shown previously to play a role in the process of infection. A significant proportion of these genes are unique to parasitic nematodes, suggesting the identification of a group of parasitism factors within nematodes. Future studies using these candidates may provide functional insight into the process of nematode parasitism and also the molecular evolution of parasitism within nematodes.

The Global Transcription Factor Lrp Is both Essential for and Inhibitory to Xenorhabdus nematophila Insecticidal Activity.

In the entomopathogenic bacterium Xenorhabdus nematophila, cell-to-cell variation in the abundance of the Lrp transcription factor leads to virulence modulation; low Lrp levels are associated with a virulent phenotype and suppression of antimicrobial peptides (AMPs) in Manduca sexta insects, while cells that lack lrp or express high Lrp levels are virulence attenuated and elicit AMP expression. To better understand the basis of these phenotypes, we examined X. nematophila strains expressing fixed Lrp levels. Unlike the lrp-null mutant, the high-lrp strain is fully virulent in Drosophila melanogaster, suggesting that these two strains have distinct underlying causes of virulence attenuation in M. sexta Indeed, the lrp-null mutant was defective in cytotoxicity against M. sexta hemocytes relative to that in the high-lrp and low-lrp strains. Further, supernatant derived from the lrp-null mutant but not from the high-lrp strain was defective in inhibiting weight gain when fed to 1st instar M. sexta These data suggest that contributors to the lrp-null mutant virulence attenuation phenotype are the lack of Lrp-dependent cytotoxic and extracellular oral growth inhibitory activities, which may be particularly important for virulence in D. melanogaster In contrast, the high-Lrp strain was sensitive to the antimicrobial peptide cecropin, had a transient survival defect in M. sexta, and had reduced extracellular levels of insecticidal activity, measured by injection of supernatant into 4th instar M. sexta Thus, high-lrp strain virulence attenuation may be explained by its hypersensitivity to M. sexta host immunity and its inability to secrete one or more insecticidal factors.IMPORTANCE Adaptation of a bacterial pathogen to host environments can be achieved through the coordinated regulation of virulence factors that can optimize success under prevailing conditions. In the insect pathogen Xenorhabdus nematophila, the global transcription factor Lrp is necessary for virulence when injected into Manduca sexta or Drosophila melanogaster insect hosts. However, high levels of Lrp, either naturally occurring or artificially induced, cause attenuation of X. nematophila virulence in M. sexta but not D. melanogaster Here, we present evidence suggesting that the underlying cause of high-Lrp-dependent virulence attenuation in M. sexta is hypersensitivity to host immune responses and decreased insecticidal activity and that high-Lrp virulence phenotypes are insect host specific. This knowledge suggests that X. nematophila faces varied challenges depending on the type of insect host it infects and that its success in these environments depends on Lrp-dependent control of a multifactorial virulence repertoire.

Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria.

BACKGROUND: Symbiotic interactions between microbes and animals are common in nature. Symbiotic organisms are particularly common in insects and, in some cases, they may protect their hosts from pathogenic infections. Wolbachia and Spiroplasma endosymbionts naturally inhabit various insects including Drosophila melanogaster fruit flies. Therefore, this symbiotic association is considered an excellent model to investigate whether endosymbiotic bacteria participate in host immune processes against certain pathogens. Here we have investigated whether the presence of Wolbachia alone or together with Spiroplasma endosymbionts in D. melanogaster adult flies affects the immune response against the virulent insect pathogen Photorhabdus luminescens and against non-pathogenic Escherichia coli bacteria. RESULTS: We found that D. melanogaster flies carrying no endosymbionts, those carrying both Wolbachia and Spiroplasma, and those containing Wolbachia only had similar survival rates after infection with P. luminescens or Escherichia coli bacteria. However, flies carrying both endosymbionts or Wolbachia only contained higher numbers of E. coli cells at early time-points post infection than flies without endosymbiotic bacteria. Interestingly, flies containing Wolbachia only had lower titers of this endosymbiont upon infection with the pathogen P. luminescens than uninfected flies of the same strain. We further found that the presence of Wolbachia and Spiroplasma in D. melanogaster up-regulated certain immune-related genes upon infection with P. luminescens or E. coli bacteria, but it failed to alter the phagocytic ability of the flies toward E. coli inactive bioparticles. CONCLUSION: Our results suggest that the presence of Wolbachia and Spiroplasma in D. melanogaster can modulate immune signaling against infection by certain insect pathogenic and non-pathogenic bacteria. Results from such studies are important for understanding the molecular basis of the interactions between endosymbiotic bacteria of insects and exogenous microbes.

The insulin receptor substrate Chico regulates antibacterial immune function in Drosophila.

BACKGROUND: Molecular and genetic studies in model organisms have recently revealed a dynamic interplay between immunity and ageing mechanisms. In the fruit fly Drosophila melanogaster, inhibition of the insulin/insulin-like growth factor signaling pathway prolongs lifespan, and mutations in the insulin receptor substrate Chico extend the survival of mutant flies against certain bacterial pathogens. Here we investigated the immune phenotypes, immune signaling activation and immune function of chico mutant adult flies against the virulent insect pathogen Photorhabdus luminescens as well as to non-pathogenic Escherichia coli bacteria. RESULTS: We found that D. melanogaster chico loss-of-function mutant flies were equally able to survive infection by P. luminescens or E. coli compared to their background controls, but they contained fewer numbers of bacterial cells at most time-points after the infection. Analysis of immune signaling pathway activation in flies infected with the pathogenic or the non-pathogenic bacteria showed reduced transcript levels of antimicrobial peptide genes in the chico mutants than in controls. Evaluation of immune function in infected flies revealed increased phenoloxidase activity and melanization response to P. luminescens and E. coli together with reduced phagocytosis of bacteria in the chico mutants. Changes in the antibacterial immune function in the chico mutants was not due to altered metabolic activity. CONCLUSIONS: Our results indicate a novel role for chico in the regulation of the antibacterial immune function in D. melanogaster. Similar studies will further contribute to a better understanding of the interconnection between ageing and immunity and lead to the identification and characterization of the molecular host components that modulate both important biological processes.

Drosophila anti-nematode and antibacterial immune regulators revealed by RNA-Seq.

BACKGROUND: Drosophila melanogaster activates a variety of immune responses against microbial infections. However, information on the Drosophila immune response to entomopathogenic nematode infections is currently limited. The nematode Heterorhabditis bacteriophora is an insect parasite that forms a mutualistic relationship with the gram-negative bacteria Photorhabdus luminescens. Following infection, the nematodes release the bacteria that quickly multiply within the insect and produce several toxins that eventually kill the host. Although we currently know that the insect immune system interacts with Photorhabdus, information on interaction with the nematode vector is scarce. RESULTS: Here we have used next generation RNA-sequencing to analyze the transcriptional profile of wild-type adult flies infected by axenic Heterorhabditis nematodes (lacking Photorhabdus bacteria), symbiotic Heterorhabditis nematodes (carrying Photorhabdus bacteria), and Photorhabdus bacteria alone. We have obtained approximately 54 million reads from the different infection treatments. Bioinformatic analysis shows that infection with Photorhabdus alters the transcription of a large number of Drosophila genes involved in translational repression as well in response to stress. However, Heterorhabditis infection alters the transcription of several genes that participate in lipidhomeostasis and metabolism, stress responses, DNA/protein synthesis and neuronal functions. We have also identified genes in the fly with potential roles in nematode recognition, anti-nematode activity and nociception. CONCLUSIONS: These findings provide fundamental information on the molecular events that take place in Drosophila upon infection with the two pathogens, either separately or together. Such large-scale transcriptomic analyses set the stage for future functional studies aimed at identifying the exact role of key factors in the Drosophila immune response against nematode-bacteria complexes.

Nitric oxide levels regulate the immune response of Drosophila melanogaster reference laboratory strains to bacterial infections.

Studies on the innate immune response against microbial infections in Drosophila melanogaster involve mutant strains and their reference strains that act as experimental controls. We used five standard D. melanogaster laboratory reference strains (Oregon R, w1118, Canton-S, Cinnabar Brown, and Yellow White [YW]) and investigated their response against two pathogenic bacteria (Photorhabdus luminescens and Enterococcus faecalis) and two nonpathogenic bacteria (Escherichia coli and Micrococcus luteus). We detected high sensitivity among YW flies to bacterial infections and increased bacterial growth compared to the other strains. We also found variation in the transcription of certain antimicrobial peptide genes among strains, with Oregon and YW infected flies showing the highest and lowest gene transcription levels in most cases. We show that Oregon and w1118 flies possess more circulating hemocytes and higher levels of phenoloxidase activity than the other strains upon infection with the nonpathogenic bacteria. We further observed reduced fat accumulation in YW flies infected with the pathogenic bacteria, which suggests a possible decline in physiological condition. Finally, we found that nitrite levels are significantly lower in infected and uninfected YW flies compared to w1118 flies and that nitric oxide synthase mutant flies in YW background are more susceptible to bacterial infection compared to mutants in w1118 background. Therefore, increased sensitivity of YW flies to bacterial infections can be partly attributed to lower levels of nitric oxide. Such studies will significantly contribute toward a better understanding of the genetic variation between D. melanogaster reference strains.

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.

Cross-talk between immunity and behavior: insights from entomopathogenic fungi and their insect hosts.

Insects are one of the most successful animals in nature, and entomopathogenic fungi play a significant role in the natural epizootic control of insect populations in many ecosystems. The interaction between insects and entomopathogenic fungi has continuously coevolved over hundreds of millions of years. Many components of the insect innate immune responses against fungal infection are conserved across phyla. Additionally, behavioral responses, which include avoidance, grooming, and/or modulation of body temperature, have been recognized as important mechanisms for opposing fungal pathogens. In an effort to investigate possible cross-talk and mediating mechanisms between these fundamental biological processes, recent studies have integrated and/or explored immune and behavioral responses. Current information indicates that during discrete stages of fungal infection, several insect behavioral and immune responses are altered simultaneously, suggesting important connections between the two systems. This review synthesizes recent advances in our understanding of the physiological and molecular aspects influencing cross-talk between behavioral and innate immune antifungal reactions, including chemical perception and olfactory pathways.

Wolbachia endosymbionts in Drosophila regulate the resistance to Zika virus infection in a sex dependent manner.

Drosophila melanogaster has been used extensively for dissecting the genetic and functional bases of host innate antiviral immunity and virus-induced pathology. Previous studies have shown that the presence of Wolbachia endosymbionts in D. melanogaster confers resistance to infection by certain viral pathogens. Zika virus is an important vector-borne pathogen that has recently expanded its range due to the wide geographical distribution of the mosquito vector. Here, we describe the effect of Wolbachia on the immune response of D. melanogaster adult flies following Zika virus infection. First, we show that the presence of Wolbachia endosymbionts promotes the longevity of uninfected D. melanogaster wild type adults and increases the survival response of flies following Zika virus injection. We find that the latter effect is more pronounced in females rather than in males. Then, we show that the presence of Wolbachia regulates Zika virus replication during Zika virus infection of female flies. In addition, we demonstrate that the antimicrobial peptide-encoding gene Drosocin and the sole Jun N-terminal kinase-specific MAPK phosphatase Puckered are upregulated in female adult flies, whereas the immune and stress response gene TotM is upregulated in male individuals. Finally, we find that the activity of RNA interference and Toll signaling remain unaffected in Zika virus-infected female and male adults containing Wolbachia compared to flies lacking the endosymbionts. Our results reveal that Wolbachia endosymbionts in D. melanogaster affect innate immune signaling activity in a sex-specific manner, which in turn influences host resistance to Zika virus infection. This information contributes to a better understanding of the complex interrelationship between insects, their endosymbiotic bacteria, and viral infection. Interpreting these processes will help us design more effective approaches for controlling insect vectors of infectious disease.

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.

A Simple Protocol for Isolating Hemolymph from Single Drosophila melanogaster Adult Flies.

Drosophila melanogaster is an excellent model for dissecting innate immune signaling and functions. Humoral and cellular immune mechanisms in the fly take place in the hemolymph, where host defense components are secreted and act in response to microbial invaders. Studying hemolymph factors is critical for understanding the regulation of the host's antimicrobial immune system. Therefore, methods for extracting the fly hemolymph efficiently and in sufficient quantities are essential for isolating and characterizing immune proteins and peptides. Here, we describe a novel and simple hemolymph isolation protocol for single D. melanogaster male and female adults. This procedure substantially improves the already used technique and allows fly immunologists to explore innate immune hemolymph activity in D. melanogaster individuals.

Functional role of the TGF-ß signaling in the Drosophila immune response.

TGF-ß signaling pathways are present in diverse animal species, which indicates their evolutionary importance in modulating several conserved biological processes and maintaining host homeostasis by adjusting the activity of innate immune mechanisms. Drosophila melanogaster utilizes two related but separable cascades of the canonical TGF-ß signaling pathway: The Bone Morphogenetic Protein and Activin branches. Recent studies have produced significant information on the immune role of TGF-ß signaling in the fruit fly model during response against certain bacterial pathogens. Results from further investigations have generated novel insights into the role of Drosophila TGF-ß signaling molecules as immune regulators opposing infection against nematode parasites and their mutualistic bacterial partners. This knowledge has revealed a previously unknown layer of the host innate immune system. Here we summarize these recent breakthroughs focusing on the participation of TGF-ß signaling factors in various Drosophila immune processes in relation to infection with potent bacteria and nematode parasites. The presented information provides important clues indicating directions for future research into the design of novel strategies for the effective control of infectious diseases caused by bacterial pathogens and parasitic nematodes.