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Both Metarhizium robertsii ARSEF 2575 (Mr2575) and Metarhizium anisopliae ARSEF 549 (Ma549) infect a range of insects whilst also interacting with plants; however, little is known about the traits that affect the competitive ability of different strains. We examined the interactions between Mr2575 and Ma549 in culture and during co-infection of plants (Arabidopsis thaliana) and insects. Mr2575 outcompetes Ma549 under nutrient-limiting conditions, including root exudates, giving it a priority advantage on Arabidopsis roots. However, during co-infection of Manduca sexta or Drosophila melanogaster, Ma549's higher blastospore production enhanced its competitive ability within the host. In large M. sexta (fifth instar), blastospores facilitate dispersal, suppress host melanization and prevent Mr2575 from spreading from infection sites, reducing conidia production. However, colonization of smaller hosts such as first instar M. sexta and D. melanogaster did not provide Ma549 with a competitive advantage, as conidial production was dependent on retaining control of the cuticle through which conidiating hyphae emerge. Unexpectedly, Ma549 and Mr2575 segregate within hosts, suggesting resource partitioning with Mr2575 predominating in the thoraxes of Drosophila, especially in females, and Ma549 in the abdomen. In fifth instar M. sexta, Mr2575 was most prevalent around spiracles and the front end of segments, despite Ma549 and Mr2575 having similar susceptibility to hypoxia. Dispersing conidia homogeneously into the hemocoel of fifth instar M. sexta eliminated the blastospore production advantage, making Ma549 and Mr2575 equally competitive, with strict partitioning of Mr2575 at the anterior and Ma549 at the posterior ends of segments. As Metarhizium species have multiple roles in natural ecosystems and agroecosystems these discoveries are relevant to understanding their impact on maintaining biodiversity and for exploiting them to enhance food security., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sheng, St. Leger. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plant-associated microbial communities are key to shaping many aspects of plant biology. In this study, we tested whether soil microbial communities and herbivory influence the bacterial community of tomato plants and whether their influence in different plant compartments is driven by microbial spillover between compartments or whether plants are involved in mediating this effect. We grew our plants in soils hosting three different microbial communities and covered (or not) the soil surface to prevent (or allow) passive microbial spillover between compartments, and we exposed them (or not) to herbivory by Manduca sexta. Here we show that the soil-driven effect on aboveground compartments is consistently detected regardless of soil coverage, whereas soil cover influences the herbivore-driven effect on belowground microbiota. Together, our results suggest that the soil microbiota influences aboveground plant and insect microbial communities via changes in plant metabolism and physiology or by sharing microorganisms via xylem sap. In contrast, herbivores influence the belowground plant microbiota via a combination of microbial spillover and changes in plant metabolism. These results demonstrate the important role of plants in linking aboveground and belowground microbiota, and can foster further research on soil microbiota manipulation for sustainable pest management., (© 2024. The Author(s).)

The two leading yeast pathogens of humans, Candida albicans and Cryptococcus neoformans , cause systemic infections in >1.4 million patients worldwide with mortality rates approaching 75%. It is thus imperative to study fungal virulence mechanisms, efficacy of antifungal drugs, and host response pathways. While this is commonly done in mammalian models, which are afflicted by ethical and practical concerns, invertebrate models, such as wax moth larvae and nematodes have been introduced over the last two decades. To complement existing invertebrate host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth Manduca sexta as a novel host model. These caterpillars can be maintained at 37°C, are suitable for injections with defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, including C. albicans, C. neoformans, C. auris , and C. glabrata . Importantly, fungal burden can be assessed daily throughout the course of infection in a single caterpillar's feces and hemolymph. Infected caterpillars can be rescued by treatment with antifungal drugs. Notably, these animals are large enough for weight to provide a reliable and reproducible measure of fungal disease and to facilitate host tissue-specific expression analyses. M. sexta caterpillars combine a suite of parameters that make them suitable for the study of fungal virulence.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii ( Xb-Sj ), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp 1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath ( xbpS1 ) or tail fibre ( xbpH1 ) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii ( Xsz-Sr ), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj . Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr . To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj , suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta , while in competition with the xenorhabdicin-deficient strain ( Xb-Sj:S1 ), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

The use of non-human animal models for infection experiments is important for investigating the infectious processes of human pathogenic bacteria at the molecular level. Mammals, such as mice and rabbits, are also utilized as animal infection models, but large numbers of animals are needed for these experiments, which is costly, and fraught with ethical issues. Various non-mammalian animal infection models have been used to investigate the molecular mechanisms of various human pathogenic bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. This review discusses the desirable characteristics of non-mammalian infection models and describes recent non-mammalian infection models that utilize Caenorhabditis elegans, silkworm, fruit fly, zebrafish, two-spotted cricket, hornworm, and waxworm., (© 2020 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

Xenorhabdus species are symbionts of entomopathogenic nematodes and pathogens of susceptible insects. Nematodes enter insect hosts and perforate the midgut to invade the haemocoel where Xenorhabdus bacteria are released transitioning to their pathogenic stage. During nematode invasion microbes from the insect gut translocate into the haemocoel. Different species of nematodes carrying specific strains of Xenorhabdus can also invade the same insect. Xenorhabdus species thereby compete for nutrients and space with both related strains and non-related gut microbes. While Xenorhabdus species produce diverse antimicrobial compounds in complex media, their functions in insect hosts are not well understood. We show that Xenorhabdus szentirmaii produced ngrA-dependent antibiotics that were active against both gut-derived microbes and Xenorhabdus nematophila whereas antibiotics of X. nematophila were not active against X. szentirmaii. X. nematophila growth was inhibited in co-cultures with wild-type X. szentirmaii in medium that mimics insect haemolymph. An antibiotic-deficient strain of X. szentirmaii was created by inactivating the ngrA gene that encodes the enzyme that attaches the 4' phosphopantetheinyl moiety to non-ribosomal peptide synthetases involved in antibiotic biosynthesis. X. nematophila growth was not inhibited in co-cultures with the ngrA strain. The growth of X. nematophila was suppressed in Manduca sexta co-injected with wild-type X. szentirmaii and X. nematophila. In contrast, growth of X. nematophila was not suppressed in M. sexta co-injected with the ngrA strain. Two unique compounds were detected by MALDI-TOF MS analysis in haemolymph infected with the wild-type but not with the ngrA strain. Finally, killing of M. sexta was delayed in insects infected with the ngrA strain. These findings indicate that in the insect host X. szentirmaii produces ngrA-dependent products involved in both interspecies competition and virulence.

Mounting an immune response consumes resources, which should lead to increased feeding. However, activating the immune system reduces feeding (i.e. illness-induced anorexia) in both vertebrates and invertebrates, suggesting that it may be beneficial. We suggest that illness-induced anorexia may be an adaptive response to conflicts between immune defense and food detoxification. We found that activating an immune response in the caterpillar Manduca sexta increased its susceptibility to the toxin permethrin. Conversely, a sublethal dose of permethrin reduced resistance to the bacterium Serratia marcescens , demonstrating a negative interaction between detoxification and immune defense. Immune system activation and toxin challenge each depleted the amount of glutathione in the hemolymph. Increasing glutathione concentration in the hemolymph increased survival for both toxin- and immune+toxin-challenged groups. The results of this rescue experiment suggest that decreased glutathione availability, such as occurs during an immune response, impairs detoxification. We also found that the expression of some detoxification genes were not upregulated during a combined immune-toxin challenge, although they were when animals received a toxin challenge alone. These results suggest that immune defense reduces food detoxification capacity. Illness-induced anorexia may protect animals by decreasing exposure to food toxins when detoxification is impaired., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Many animals are inhabited by microbial symbionts that influence their hosts' development, physiology, ecological interactions, and evolutionary diversification. However, firm evidence for the existence and functional importance of resident microbiomes in larval Lepidoptera (caterpillars) is lacking, despite the fact that these insects are enormously diverse, major agricultural pests, and dominant herbivores in many ecosystems. Using 16S rRNA gene sequencing and quantitative PCR, we characterized the gut microbiomes of wild leaf-feeding caterpillars in the United States and Costa Rica, representing 124 species from 15 families. Compared with other insects and vertebrates assayed using the same methods, the microbes that we detected in caterpillar guts were unusually low-density and variable among individuals. Furthermore, the abundance and composition of leaf-associated microbes were reflected in the feces of caterpillars consuming the same plants. Thus, microbes ingested with food are present (although possibly dead or dormant) in the caterpillar gut, but host-specific, resident symbionts are largely absent. To test whether transient microbes might still contribute to feeding and development, we conducted an experiment on field-collected caterpillars of the model species Manduca sexta Antibiotic suppression of gut bacterial activity did not significantly affect caterpillar weight gain, development, or survival. The high pH, simple gut structure, and fast transit times that typify caterpillar digestive physiology may prevent microbial colonization. Moreover, host-encoded digestive and detoxification mechanisms likely render microbes unnecessary for caterpillar herbivory. Caterpillars illustrate the potential ecological and evolutionary benefits of independence from symbionts, a lifestyle that may be widespread among animals., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2017 American Society for Microbiology.)

The Toll pathway is one of the most important signaling pathways regulating insect innate immunity. Spatzle is a key protein that functions as a Toll receptor ligand to trigger Toll-dependent expression of immunity-related genes. In this study, a novel spatzle gene (ApSPZ) from the Chinese oak silkworm Antheraea pernyi was identified. The ApSPZ cDNA is 1065 nucleotides with an open reading frame (ORF) of 777 bp encoding a protein of 258 amino acids. The protein has an estimated molecular weight of 29.71 kDa and an isoelectric point (PI) of 8.53. ApSPZ is a nuclear and secretory protein with no conserved domains or membrane helices and shares 40% amino acid identity with SPZ from Manduca sexta. Phylogenetic analysis indicated that ApSPZ might be a new member of the Spatzle type 1 family, which belongs to the Spatzle superfamily. The expression patterns of several genes involved in the Toll pathway were examined at different developmental stages and various tissues in 5th instar larvae. The examined targets included A. pernyi spatzle, GNBP, MyD88, Tolloid, cactus and dorsalA. The RT-PCR results showed that these genes were predominantly expressed in immune-responsive fat body tissue, indicating that the genes play a crucial role in A. pernyi innate immunity. Moreover, A. pernyi infection with the fungus Nosema pernyi and the gram-positive bacterium Enterococcus pernyi, but not the gram-negative bacterium Escherichia coli, activated the Toll signaling pathway. These results represent the first study of the Toll pathway in A. pernyi, which provides insight into the A. pernyi innate immune system.

Manduca sextais a lepidopteran model widely used to study insect physiological processes, including innate immunity. In this study, we explored the proteomes of cell-free hemolymph from larvae injected with a sterile buffer (C for control) or a mixture of bacteria (I for induced). Of the 654 proteins identified, 70 showed 1.67 to >200-fold abundance increases after the immune challenge; 51 decreased to 0-60% of the control levels. While there was no strong parallel between plasma protein levels and their transcript levels in hemocytes or fat body, the mRNA level changes (i.e.I/C ratios of normalized read numbers) in the tissues concurred with their protein level changes (i.e.I/C ratios of normalized spectral counts) with correlation coefficients of 0.44 and 0.57, respectively. Better correlations support that fat body contributes a more significant portion of the plasma proteins involved in various aspects of innate immunity. Consistently, ratios of mRNA and protein levels were better correlated for immunity-related proteins than unrelated ones. There is a set of proteins whose apparent molecular masses differ considerably from the calculatedMr's, suggestive of posttranslational modifications. In addition, some lowMrproteins were detected in the range of 80 to >300 kDa on a reducing SDS-polyacrylamide gel, indicating the existence of highMrcovalent complexes. We identified 30 serine proteases and their homologs, 11 of which are known members of an extracellular immune signaling network. Along with our quantitative transcriptome data, the protein identification, inducibility, and association provide leads toward a focused exploration of humoral immunity inM. sexta., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Dwindling resources might be expected to induce a gradual decline in immune function. However, food limitation has complex and seemingly paradoxical effects on the immune system. Examining these changes from an immune system network perspective may help illuminate the purpose of these fluctuations. We found that food limitation lowered long-term (i.e. lipid) and short-term (i.e. sugars) energy stores in the caterpillar Manduca sexta. Food limitation also: altered immune gene expression, changed the activity of key immune enzymes, depressed the concentration of a major antioxidant (glutathione), reduced resistance to oxidative stress, reduced resistance to bacteria (Gram-positive and -negative bacteria) but appeared to have less effect on resistance to a fungus. These results provide evidence that food limitation led to a restructuring of the immune system network. In severely food-limited caterpillars, some immune functions were enhanced. As resources dwindled within the caterpillar, the immune response shifted its emphasis away from inducible immune defenses (i.e. those responses that are activated during an immune challenge) and increased emphasis on constitutive defenses (i.e. immune components that are produced consistently). We also found changes suggesting that the activation threshold for some immune responses (e.g. phenoloxidase) was lowered. Changes in the configuration of the immune system network will lead to different immunological strengths and vulnerabilities for the organism., (© 2016. Published by The Company of Biologists Ltd.)

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called "nutritional virulence" strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway.

To exert their toxic effect, Bacillus thuringiensis Cry1Ab toxin undergoes a sequential binding mechanism with different larval gut proteins including glycosyl-phosphatidyl-inositol anchored proteins like aminopeptidase-N (APN) or alkaline-phosphatase (ALP) and a transmembrane cadherin to form pre-pore structures that insert into the membrane. Cadherin binding induces oligomerization of the toxin by facilitating removal of the N-terminal region, while APN/ALP binding helps in oligomer membrane insertion. Cry1AbMod toxin was engineered to lack N-terminal region of the toxin and shown to counter resistance linked to cadherin mutations. In this manuscript we determined the toxicity of Cry1AbMod to Manduca sexta larvae silenced in the expression of cadherin, ALP or APN receptors. As previously reported Cry1Ab toxicity relied principally in ALP and cadherin in comparison to APN. Our data shows that Cry1AbMod counters resistance associated with low cadherin expression but was not effective against ALP silenced larvae. These results show that Cry1AbMod could be effective against resistance insects linked to mutations on binding molecules involved in toxin oligomerization but not against resistant insects linked to mutations on binding molecules involved in oligomer membrane insertion., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) produced by the nitric oxide synthase (NOS) enzyme is a reactive oxygen molecule widely considered as important participant in the immune system of different organisms to confront microbial infections. In insects the NO molecule has also been implicated in immune response against microbial pathogens. Bacillus thuringiensis (Bt) is an insect-pathogenic bacterium that produces insecticidal proteins such as Cry toxins. These proteins kill insects because they form pores in the larval-midgut cells. Here we show that intoxication of Manduca sexta larvae with Cry1Ab activates expression of NOS with a corresponding increase in NO. This effect is not observed with a non-toxic mutant toxin Cry1Ab-E129K that is affected in pore formation. The increased production of NO triggered by intoxication with LC50 dose of Cry1Ab toxin is not associated with higher expression of antimicrobial peptides. NO participates in Cry1Ab toxicity since inhibition of NOS by selective l-NAME inhibitor prevented NO production and resulted in reduced mortality of the larvae. The fact that mortality was not completely abolished by L-NAME indicates that other processes participate in toxin action and induction of NO production upon Cry1Ab toxin administration accounts only for a part of the toxicity of this protein to M. sexta larvae., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Research on how insect immunity changes with age as insects develop within an instar, or larval developmental stage, is limited and contradictory. Insects within an instar are preparing for the next developmental stage, which may involve changes in morphology or habitat. Immunity may also vary accordingly. To determine how immunity varies in the fifth instar, we tested humoral immune responses, antimicrobial peptide activity, and phenoloxidase activity using the tobacco hornworm, Manduca sexta. We determined that while M. sexta have more robust antimicrobial peptide and phenoloxidase responses at the beginning of their fifth instar, this did not translate into better survival of bacterial infection or lower bacterial load in the hemolymph. We also determined that M. sexta injected with bacteria early in the fifth instar experience lower growth rates and longer development times than caterpillars of the same age injected with sham. This could indicate a shift in energy allocation from growth and development to metabolically costly immune responses. Because of the importance of insects as pests and pollinators, understanding how immunity varies throughout development is critical.

Enterococcus faecalis is a commensal and pathogen of humans and insects. In Manduca sexta, E. faecalis is an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigate E. faecalis factors required for commensalism, we identified E. faecalis genes that are upregulated in the gut of M. sexta using recombinase-based in vivo expression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designated pglA. A pglA deletion mutant was impaired in both pathogenesis and gut persistence in M. sexta and produced enhanced biofilms compared with the wild type in an in vitro polystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants for E. faecalis commensal and pathogenic interactions with M. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to prevent E. faecalis infections., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Xenorhabdus nematophila engages in a mutualistic association with the nematode Steinernema carpocapsae. The nematode invades and traverses the gut of susceptible insects. X. nematophila is released in the insect blood (hemolymph), where it suppresses host immune responses and functions as a pathogen. X. nematophila produces diverse antimicrobials in laboratory cultures. The natural competitors that X. nematophila encounters in the hemolymph and the role of antimicrobials in interspecies competition in the host are poorly understood. We show that gut microbes translocate into the hemolymph when the nematode penetrates the insect intestine. During natural infection, Staphylococcus saprophyticus was initially present and subsequently disappeared from the hemolymph, while Enterococcus faecalis proliferated. S. saprophyticus was sensitive to X. nematophila antibiotics and was eliminated from the hemolymph when coinjected with X. nematophila. In contrast, E. faecalis was relatively resistant to X. nematophila antibiotics. When injected by itself, E. faecalis persisted (~10(3) CFU/ml), but when coinjected with X. nematophila, it proliferated to ~10(9) CFU/ml. Injection of E. faecalis into the insect caused the upregulation of an insect antimicrobial peptide, while the transcript levels were suppressed when E. faecalis was coinjected with X. nematophila. Its relative antibiotic resistance together with suppression of the host immune system by X. nematophila may account for the growth of E. faecalis. At higher injected levels (10(6) CFU/insect), E. faecalis could kill insects, suggesting that it may contribute to virulence in an X. nematophila infection. These findings provide new insights into the competitive events that occur early in infection after S. carpocapsae invades the host hemocoel., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The tobacco hornworm, Manduca sexta, has been used as a biochemical model for studying insect physiological processes. While the transcriptomes of its fat body, hemocytes, midgut, and antennae have been examined in several studies, limited information is available for proteins in tissues, cells, or body fluids of this insect. In keeping pace with the M. sexta genome project, we launched a pilot study to identify differences in the peptidome of cell-free hemolymph samples from larvae injected with buffer or a mixture of bacteria. At 24 h after injection, plasma was collected and treated with 50% acetonitrile to precipitate large proteins. The supernatants, containing peptides (<25 kDa) and other stable proteins (>25 kDa), were digested with trypsin and analyzed by nano-liquid chromatography and nano-electrospray tandem mass spectrometry (nanoLC-MS/MS) on an LTQ Orbitrap XL mass spectrometer. Known M. sexta cDNA sequences and gene transcripts from the draft genome were translated in silico to generate a database of polypeptides (i.e. peptides and proteins) in this species. By searching the database, we identified 268 hemolymph polypeptides, 50 of which showed 1.67-200 fold abundance increases after the immune challenge, as judged by significant changes in normalized spectral counts between the control and induced plasma. These included a total of 33 antimicrobial peptides (attacins, cecropins, defensins, diapausins, gallerimycin, gloverin, lebocins, lysozymes), pattern recognition receptors, and proteinase inhibitors. Although there was no strong parallel (correlation coefficients: -0.13, 0.11, 0.39 and 0.62) between plasma peptide levels and their transcript levels in control or induced hemocytes or fat body, we observed the mRNA level changes in hemocytes and fat body concurred with their peptide level changes with correlation coefficients of 0.67 and 0.76, respectively. These data suggest that fat body contributed a significant portion of the plasma polypeptides involved in various aspects of innate immunity after the bacterial injection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Six novel linear peptides, named "rhabdopeptides", have been identified in the entomopathogenic bacterium Xenorhabdus nematophila after the discovery of the corresponding rdp gene cluster by using a promoter trap strategy for the detection of insect-inducible genes. The structures of these rhabdopeptides were deduced from labeling experiments combined with detailed MS analysis. Detailed analysis of an rdp mutant revealed that these compounds participate in virulence towards insects and are produced upon bacterial infection of a suitable insect host. Furthermore, two additional rhabdopeptide derivatives produced by Xenorhabdus cabanillasii were isolated, these showed activity against insect hemocytes thereby confirming the virulence of this novel class of compounds., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species(1-5), as well as the wealth of information available regarding the insect's immune system(6-8), and the pending genome sequence(9) make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection. The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter(10). The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking(11) and oral toxicity assays(12). Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula. The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides(7); the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR(13). The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes(6). To analyze these responses, injected insects can be dissected and visualized by microscopy(13, 14).

The physiology of a newly recognized Serratia species, termed South African Caenorhabditis briggsae Isolate (SCBI), which is both a nematode mutualist and an insect pathogen, was investigated and compared to that of Serratia marcescens Db11, a broad-host-range pathogen. The two Serratia strains had comparable levels of virulence for Manduca sexta and similar cytotoxic activity patterns, but motility and lipase and hemolytic activities differed significantly between them.

Hyalophora gloveri gloverin is a glycine-rich and heat stable antimicrobial protein with activity mainly against Escherichia coli. However, Spodoptera exigua gloverin is active against a Gram-positive bacterium but inactive against E. coli. In this study, we investigated expression profile, binding ability and antimicrobial activity of Manduca sexta gloverin (MsGlv). Msglv transcript was detected in several tissues of naïve larvae with higher levels in the midgut and testis. Expression of Msglv mRNA in larvae was up-regulated by active Spätzle-C108 and peptidoglycans (PGs) of E. coli and Staphylococcus aureus, and the activation was blocked by pre-injection of antibody to M. sexta Toll, suggesting that Msglv expression is regulated by the Toll-Spätzle pathway. Recombinant MsGlv bound to the O-specific antigen and outer core carbohydrate of lipopolysaccharide (LPS), Gram-positive lipoteichoic acid (LTA) and PG, and laminarin, but not to E. coli PG or mannan. MsGlv was active against Bacillus cereus, Saccharomyces cerevisiae and Cryptococcus neoformans, but was almost inactive against E. coli and S. aureus. Our results suggest that gloverins are active against some bacteria and fungi., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Gene silencing by RNA interference (RNAi) can be a useful reverse genetics tool in eukaryotes. However, some species appear refractory to RNAi. To study the role of the differential expression of RNAi proteins in RNAi, we isolated partial dicer-2, argonaute-2 translin, vasa intronic gene (VIG) and tudor staphylococcus/micrococcal nuclease (TSN) genes from the tobacco hornworm, Manduca sexta, a well-studied insect model which we have found to be variably sensitive to RNAi. We found that the RNAi gene, translin, was expressed at minimal levels in M. sexta tissue and that there is a specific, dose-dependent upregulation of dicer-2 and argonaute-2 expression in response to injection with dsRNA, but no upregulation of the other genes tested. Upregulation of gene expression was rapid and transient. In order to prolong the upregulation we introduced multiple doses of dsRNA, resulting in multiple peaks of dicer-2 gene expression. Our results have implications for the design of RNAi experiments and may help to explain differences in the sensitivity of eukaryotic organisms to RNAi., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Our previous research showed that immulectin-2 (IML-2), a C-type lectin from the tobacco hornworn, Manduca sexta, is a pattern recognition receptor (PRR) that can bind to pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PG) and β-1,3-glucan, and IML-2 plays an important role in cellular encapsulation, melanization, phagocytosis, and prophenoloxidase (proPO) activation. Unlike most mammalian C-type lectins that contain a single carbohydrate-recognition domain (CRD), IML-2 is composed of tandem CRDs, and the C-terminal CRD2 contains an extended loop, which is not present in most C-type CRDs. We hypothesize that the extended loop may participate in ligand binding, encapsulation, melanization, phagocytosis and/or proPO activation in M. sexta. To test this hypothesis, two deletion mutant proteins (IML-2Δ220-244 and IML-2Δ220-257), in which the extended loop of the CRD2 was partially or completely deleted, were expressed and purified. By comparing the characteristics of recombinant IML-2, IML-2Δ220-244 and IML-2Δ220-257, we found that deletion of the extended loop in CRD2 impaired the ability of IML-2 to bind microbial PAMPs and to stimulate proPO activation, indicating that the extended loop of IML-2 plays an important role in ligand binding and biological functions.

The polysaccharide capsule of Streptococcus pneumoniae is one of the most important virulence factors responsible for human infections and in mouse infection models as well. Larvae of Manduca sexta were used as an alternative animal model in order to test the impact of the pneumococcal capsule on virulence in the insect host. The unencapsulated S. pneumoniae strain R6 was able to cause disease and induce killing in the larvae, and similar results were obtained with related commensal species. However, using the same dose of S. pneumoniae, encapsulated strains including the type 2 D39 strain, the progenitor of R6, and genetically unrelated S. pneumoniae strains of serotype 2, 4, 6B, 23F and 19A, all had increased virulence potential compared to the R6 strain. Between 20 and 70% of the larvae were affected after 96 h compared to 12% observed with R6. Two type 6B S. pneumoniae strains were more virulent compared to the other strains. S. pneumoniae R6 transformants producing the type 6B capsule showed a similar elevated disease potential, confirming the contribution of the pneumococcal polysaccharide capsule to virulence in M. sexta., (Copyright © 2012 S. Karger AG, Basel.)

The Toxin Complex (TC) is a large multi-subunit toxin first characterized in the insect pathogens Photorhabdus and Xenorhabdus, but now seen in a range of pathogens, including those of humans. These complexes comprise three protein subunits, A, B and C which in the Xenorhabdus toxin are found in a 4:1:1 stoichiometry. Some TCs have been demonstrated to exhibit oral toxicity to insects and have the potential to be developed as a pest control technology. The lack of recognisable signal sequences in the three large component proteins hinders an understanding of their mode of secretion. Nevertheless, we have shown the Photorhabdus luminescens (Pl) Tcd complex has been shown to associate with the bacteria's surface, although some strains can also release it into the surrounding milieu. The large number of tc gene homologues in Pl make study of the export process difficult and as such we have developed and validated a heterologous Escherichia coli expression model to study the release of these important toxins. In addition to this model, we have used comparative genomics between a strain that releases high levels of Tcd into the supernatant and one that retains the toxin on its surface, to identify a protein responsible for enhancing secretion and release of these toxins. This protein is a putative lipase (Pdl1) which is regulated by a small tightly linked antagonist protein (Orf53). The identification of homologues of these in other bacteria, linked to other virulence factor operons, such as type VI secretion systems, suggests that these genes represent a general and widespread mechanism for enhancing toxin release in gram negative pathogens.

The Bt toxin is widely used in agriculture both as a spray and in transgenic plants, yet its precise mode of action against lepidopterans is poorly understood. The study by Mason et al. [mBio 2(3):e00065-11, 2011] revealed that Bt toxin enables an inhabitant of the midgut, Enterococcus faecalis, to enter the hemolymph of larvae and cause septicemia, leading to death.

An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene.

Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.

Unlabelled: A dynamic homeostasis is maintained between the host and native bacteria of the gastrointestinal tract in animals, but migration of bacteria from the gut to other organs can lead to disease or death. Enterococcus faecalis is a commensal of the gastrointestinal tract; however, Enterococcus spp. are increasingly frequent causes of nosocomial infections with a high mortality rate. We investigated the commensal-to-pathogen switch undergone by E. faecalis OG1RF in the lepidopteran model host Manduca sexta associated with its location in the host. E. faecalis persists in the harsh midgut environment of M. sexta larvae without causing apparent illness, but injection of E. faecalis directly into the larval hemocoel is followed by rapid death. Additionally, oral ingestion of E. faecalis in the presence of Bacillus thuringiensis insecticidal toxin, a pore-forming toxin that targets the midgut epithelium, induces an elevated mortality rate. We show that the loss of gut integrity due to B. thuringiensis toxin correlates with the translocation of E. faecalis from the gastrointestinal tract into the hemolymph. Upon gaining access to the hemolymph, E. faecalis induces an innate immune response, illustrated by hemocyte aggregation, in larvae prior to death. The degree of hemocyte aggregation is dependent upon the route of E. faecalis entry. Our data demonstrate the efficacy of the M. sexta larval model system in investigating E. faecalis-induced sepsis and clarifies controversies in the field regarding the events leading to larval death following B. thuringiensis toxin exposure., Importance: This study advances our knowledge of Enterococcus faecalis-induced sepsis following translocation from the gut and provides a model for mammalian diseases in which the spatial distribution of bacteria determines disease outcomes. We demonstrate that E. faecalis is a commensal in the gut of Manduca sexta and a pathogen in the hemocoel, resulting in a robust immune response and rapid death, a process we refer to as the "commensal-to-pathogen" switch. While controversy remains regarding Bacillus thuringiensis toxin-induced killing, our laboratory previously found that under some conditions, the midgut microbiota is essential for B. thuringiensis toxin killing of Lymantria dispar (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196-15199, 2006; B. Raymond, et al., Environ. Microbiol. 11:2556-2563, 2009; P. R. Johnston, and N. Crickmore, Appl. Environ. Microbiol. 75:5094-5099, 2009). We and others have demonstrated that the role of the midgut microbiota in B. thuringiensis toxin killing is dependent upon the lepidopteran species and formulation of B. thuringiensis toxin (N. A. Broderick, K. F. Raffa, and J. Handelsman, Proc. Natl. Acad. Sci. U. S. A. 103:15196-15199, 2006; N. A. Broderick, et al., BMC Biol. 7:11, 2009). This work reconciles much of the apparently contradictory previous data and reveals that the M. sexta-E. faecalis system provides a model for mammalian sepsis.

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.

Background: Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt) indicating that oligomer formation is a key step in toxicity of Cry toxins., Methodology/principal Findings: The DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt) probably by competition of binding with toxin receptors., Conclusions/significance: We show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.

The Cry1Ab δ-endotoxin V171C mutant protein exhibits a 25-fold increase in toxicity against Lymantria dispar, which correlates with a faster rate of partitioning into the midgut membrane and slightly decreased protein stability. This is an insect-specific mechanism; similar results were not observed in Manduca sexta, another Cry1Ab δ-endotoxin-susceptible insect.

As a comparison to a similar study on Photorhabdus strains, 15 Xenorhabdus bacterial strains and secondary phenotypic variants of two strains were screened for proteolytic activity by five detection methods. Although the number and intensity of proteolytic activities were different, every strain was positive for proteolytic activity by several tests. Zymography following native PAGE detected two groups of activities with different substrate affinities and a higher and lower electrophoretic mobility that were distinguished as activity 1 and 2, respectively. Zymography following SDS-PAGE resolved three activities, which were provisionally named proteases A, B, and C. Only protease B, an ∼55-kDa enzyme, was produced by every strain. This enzyme exhibited higher affinity to the gelatin substrate than to the casein substrate. Of the chromogenic substrates used, three were hydrolyzed: furylacryloyl-Ala-Leu-Val-Tyr (Fua-ALVY), Fua-LGPA (LGPA is Leu-Gly-Pro-Ala) (a substrate for collagen peptidases), and succinyl-Ala-Ala-Pro-Phe-thiobenzyl (Succ-AAPF-SBzl). All but the Fua-LGPA-ase activity seemed to be from secreted enzymes. According to their substrate preference profiles and inhibitor sensitivities, at least six such proteolytic enzymes could be distinguished in the culture medium of Xenorhabdus strains. The proteolytic enzyme that was secreted the earliest, protease B and the Succ-AAPF-SBzl-hydrolyzing enzyme, appeared from the early logarithmic phase of growth. Protease B could also be detected in the hemolymph of Xenorhabdus-infected Galleria mellonella larvae from 15 h postinfection. The purified protease B hydrolyzed in vitro seven proteins in the hemolymph of Manduca sexta that were also cleaved by PrtA peptidase from Photorhabdus. The N-terminal sequence of protease B showed similarity to a 55-kDa serralysin type metalloprotease in Xenorhabdus nematophila, which had been identified as an orthologue of Photorhabdus PrtA peptidase.

Leucine-rich repeat containing proteins are involved in immune response in many capacities. In insects, these include Toll-like receptors and the Anopheles gambiae proteins APL1 and LRIM1. Here we describe the identification and characterization of leureptin, a novel extracellular protein with 13 leucine-rich repeats from hemolymph of the insect Manduca sexta. After injection of bacteria, leureptin mRNA level increased in fat body, but protein levels in plasma decreased, an indication that leureptin is consumed during the immune response. Leureptin bound to bacterial lipopolysaccharide (LPS). Microscopy using leureptin antiserum showed that leureptin associates with hemocytes after injection of bacteria, an indication that leureptin is involved in hemocyte responses to bacterial infection. Sequence database searches suggest similar proteins are present in other Lepidopteran species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Summary: Photorhabdus sp. are entomopathogenic bacteria which, upon experimental infection, interact with the insect immune system, but little is known about the roles of their symbiotic nematode partners Heterorhabditis sp. in natural infections. Here, we investigated the respective contributions of nematodes and bacteria by examining humoral and cellular immune reactions of the model lepidopteran insect Manduca sexta against Heterorhabditis carrying Photorhabdus, nematodes free of bacteria (axenic nematodes) and bacteria alone. Insect mortality was slower following infection with axenic nematodes than when insects were infected with nematodes containing Photorhabdus, or the bacteria alone. Nematodes elicited host immune responses to a lesser extent than bacteria. Transcription of certain recognition and antibacterial genes was lower when insects were naturally infected with nematodes carrying no bacteria compared to insects that received bacteria, either with or without nematodes. Axenic nematodes also did not elicit such high levels of phenoloxidase activity and haemocyte aggregates as did treatments involving Photorhabdus. By contrast, the phagocytic capability of host haemocytes was decreased by both axenic and bacteria-associated nematodes, but not by Photorhabdus alone. These results imply that both bacteria and nematodes contribute separately to the pathogenic modulation of host immune responses during natural infections by the mutualistic Heterorhabdus-Photorhabdus complex.

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.

Although the importance of peptidoglycan recognition proteins (PGRPs) in detecting bacteria and promoting immunity is well recognized in Drosophila melanogaster and other insect species, such a role has not yet been experimentally established for PGRPs in the tobacco hornworm, Manduca sexta. In this study, we purified M. sexta PGRP1 from the baculovirus-insect cell expression system, tested its association with peptidoglycans and intact bacteria, and explored its possible link with the prophenoloxidase activation system in larval hemolymph. Sequence comparison suggested that PGRP1 is not an amidase and lacks residues for interacting with the carboxyl group of meso-diaminopimelic acid-peptidoglycans (DAP-PGs). M. sexta PGRP1 gene was constitutively expressed at a low level in fat body, and the mRNA concentration became much higher after an injection of Escherichia coli. Consistently, the protein concentration in larval plasma increased in a time-dependent manner after the immune challenge. Purified recombinant PGRP1 specifically bound to soluble DAP-PG of E. coli but not to soluble Lys-type PG of Staphylococcus aureus. In addition, this recognition protein completely bound to insoluble PGs from Micrococcus luteus, Bacillus megaterium and Bacillus subtilis, whereas its association with the bacterial cells was low even though their peptidoglycans are exposed on the cell surface. After PGRP1 had been added to plasma of naïve larvae in the absence of microbial elicitor, there was a concentration-dependent increase in prophenoloxidase activation. Phenoloxidase activity, as usual, increased after the plasma was incubated with peptidoglycans or bacterial cells. These increases became more prominent when insoluble M. luteus or B. megaterium PG or soluble E. coli PG and PGRP1 were both present. Statistic analysis suggested a synergistic effect caused by interaction between PGRP1 and these PGs. Taken together, these results indicated that PGRP1 is a member of the M. sexta prophenoloxidase activation system, which recognizes peptidoglycans from certain bacteria and initiates the host defense response. The unexplained difference between the purified PGs and intact bacteria clearly reflects our general lack of understanding of PGRP1-mediated recognition and how it leads to proPO activation., ((c) 2010 Elsevier Ltd. All rights reserved.)

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.

Injecting the insect pathogenic bacterium Photorhabdus luminescens into the blood system of the model lepidopteran insect Manduca sexta induces nitric oxide synthase (NOS) expression in the fat body and blood cells (haemocytes), whereas following oral ingestion of bacteria NOS expression is limited to the gut. We used RNA interference to knock-down expression of NOS throughout the insect. Preventing NOS induction in this way adversely affected the survival of orally infected insects and caused a significant increase in the number of bacteria crossing into the haemolymph. By contrast, knock-down of NOS had no effect on the mortality rate of insects infected with P. luminescens by injection. Pharmacological inhibition of NOS decreased both nitric oxide (NO) levels in the gut wall and survival of orally infected insects, whereas elevation of gut wall NO using an NO donor increased survival of NOS silenced caterpillars. Together, our results imply that induced synthesis of NO is important in mediating insect immune defence against the pathogen by inhibiting transfer of bacteria across the gut wall.

It was recently proposed that gut bacteria are required for the insecticidal activity of the Bacillus thuringiensis-based insecticide, DiPel, toward the lepidopterans Manduca sexta, Pieris rapae, Vanessa cardui, and Lymantria dispar. Using a similar methodology, it was found that gut bacteria were not required for the toxicity of DiPel or Cry1Ac or for the synergism of an otherwise sublethal concentration of Cry1Ac toward M. sexta. The toxicities of DiPel and of B. thuringiensis HD73 Cry(-) spore/Cry1Ac synergism were attenuated by continuously exposing larvae to antibiotics before bioassays. Attenuation could be eliminated by exposing larvae to antibiotics only during the first instar without altering larval sterility. Prior antibiotic exposure did not attenuate Cry1Ac toxicity. The presence of enterococci in larval guts slowed mortality resulting from DiPel exposure and halved Cry1Ac toxicity but had little effect on B. thuringiensis HD73 Cry(-) spore/Cry1Ac synergism. B. thuringiensis Cry(-) cells killed larvae after intrahemocoelic inoculation of M. sexta, Galleria mellonella, and Spodoptera litura and grew rapidly in plasma from M. sexta, S. litura, and Tenebrio molitor. These findings suggest that gut bacteria are not required for B. thuringiensis insecticidal activity toward M. sexta but that B. thuringiensis lethality is reduced in larvae that are continuously exposed to antibiotics before bioassay.

We identified Xenorhabdus nematophila transposon mutants with defects in lipase activity. One of the mutations, in yigL, a conserved gene of unknown function, resulted in attenuated virulence against Manduca sexta insects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.

Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway.

The gammaproteobacterium Xenorhabdus nematophila is a blood pathogen of insects that requires the CpxRA signal transduction system for full virulence (E. E. Herbert et al., Appl. Environ. Microbiol. 73:7826-7836, 2007). We show here that the DeltacpxR1 mutant has altered localization, growth, and immune suppressive activities relative to its wild-type parent during infection of Manduca sexta insects. In contrast to wild-type X. nematophila, which were recovered throughout infection, DeltacpxR1 cells did not accumulate in hemolymph until after insect death. In vivo imaging of fluorescently labeled bacteria within live insects showed that DeltacpxR1 displayed delayed accumulation and also occasionally were present in isolated nodes rather than systemically throughout the insect as was wild-type X. nematophila. In addition, in contrast to its wild-type parent, the DeltacpxR1 mutant elicited transcription of an insect antimicrobial peptide, cecropin. Relative to phosphate-buffered saline-injected insects, cecropin transcript was induced 21-fold more in insects injected with DeltacpxR1 and 2-fold more in insects injected with wild-type X. nematophila. These data suggest that the DeltacpxR1 mutant has a defect in immune suppression or has an increased propensity to activate M. sexta immunity. CpxR regulates, directly or indirectly, genes known or predicted to be involved in virulence (E. E. Herbert et al., Appl. Environ. Microbiol. 73:7826-7836, 2007), including lrhA, encoding a transcription factor necessary for X. nematophila virulence, motility, and lipase production (G. R. Richards et al., J. Bacteriol. 190:4870-4879, 2008). CpxR positively regulates lrhA transcript, and we have shown that altered regulation of lrhA in the DeltacpxR1 mutant causes this strain's virulence defect. The DeltacpxR1 mutant expressing lrhA from a constitutive lac promoter showed wild-type virulence in M. sexta. These data suggest that CpxR contributes to X. nematophila virulence through the regulation of lrhA, immune suppression, and growth in Insecta.

Insect hemocytes (blood cells) are a central part of the insect's cellular response to bacterial pathogens, and these specialist cells can both recognize and engulf bacteria. During this process, hemocytes undergo poorly characterized changes in adhesiveness. Previously, a peptide termed plasmatocyte-spreading peptide (PSP), which induces the adhesion and spreading of plasmatocytes on foreign surfaces, has been identified in lepidopteran insects. Here, we investigate the function of this peptide in the moth Manduca sexta using RNA interference (RNAi) to prevent expression of the precursor protein proPSP. We show that infection with the insect-specific bacterial pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli induces proPSP mRNA transcription in the insect fat body but not in hemocytes; subsequently, proPSP protein can be detected in cell-free hemolymph. We used RNAi to silence this upregulation of proPSP and found that the knock-down insects succumbed faster to infection with P. luminescens, but not E. coli. RNAi-treated insects infected with E. coli showed a reduction in the number of circulating hemocytes and higher bacterial growth in hemolymph as well as a reduction in overall cellular immune function compared with infected controls. Interestingly, RNAi-mediated depletion of proPSP adversely affected the formation of melanotic nodules but had no additional effect on other cellular responses when insects were infected with P. luminescens, indicating that this pathogen employs mechanisms that suppress key cellular immune functions in M. sexta. Our results provide evidence for the central role of PSP in M. sexta cellular defenses against bacterial infections.

Serralysins are generally thought to function as pathogenicity factors of bacteria, but so far no hard evidence of this (e.g., specific substrate proteins that are sensitive to the cleavage by these proteases) has been found. We have looked for substrate proteins to a serralysin-type proteinase, PrtA, in a natural host-pathogen molecular interaction system involving Manduca sexta and Photorhabdus luminescens. The exposure in vitro of hemolymph to PrtA digestion resulted in selective cleavage of 16 proteins, provisionally termed PAT (PrtA target) proteins. We could obtain sequence information for nine of these PrtA sensitive proteins, and by searching databases, we could identify six of them. Each has immune-related function involving every aspect of the immune defense: beta-1,3 glucan recognition protein 2 (immune recognition), hemocyte aggregation inhibitor protein (HAIP), serine proteinase homolog 3, six serpin-1 variants, including serpin-1I (immune signaling and regulation), and scolexins A and B (coagulation cascade effector function). The functions of the identified PrtA substrate proteins shed new light on a possible participation of a serralysin in the virulence mechanism of a pathogen. Provided these proteins are targets of PrtA in vivo, this might represent, among others, a complex suppressive role on the innate immune response via interference with both the recognition and the elimination of the pathogen during the first, infective stage of the host-pathogen interaction. Our results also raise the possibility that the natural substrate proteins of serralysins of vertebrate pathogens might be found among the components of the innate immune system.

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.

Background: Toxin complex (Tc) proteins termed TcaABC, TcdAB, and TccABC with insecticidal activity are present in a variety of bacteria including the yersiniae., Results: The tc gene sequences of thirteen Yersinia strains were compared, revealing a high degree of gene order conservation, but also remarkable differences with respect to pseudogenes, sequence variability and gene duplications. Outside the tc pathogenicity island (tc-PAIYe) of Y. enterocolitica strain W22703, a pseudogene (tccC2'/3') encoding proteins with homology to TccC and similarity to tyrosine phosphatases at its C-terminus was identified. PCR analysis revealed the presence of the tc-PAIYe and of tccC2'/3'-homologues in all biotype 2-5 strains tested, and their absence in most representatives of biotypes 1A and 1B. Phylogenetic analysis of 39 TccC sequences indicates the presence of the tc-PAIYe in an ancestor of Yersinia. Oral uptake experiments with Manduca sexta revealed a higher larvae lethality of Yersinia strains harbouring the tc-PAIYe in comparison to strains lacking this island. Following subcutaneous infection of Galleria mellonella larvae with five non-human pathogenic Yersinia spp. and four Y. enterocolitica strains, we observed a remarkable variability of their insecticidal activity ranging from 20% (Y. kristensenii) to 90% (Y. enterocolitica strain 2594) dead larvae after five days. Strain W22703 and its tcaA deletion mutant did not exhibit a significantly different toxicity towards G. mellonella. These data confirm a role of TcaA upon oral uptake only, and suggest the presence of further insecticidal determinants in Yersinia strains formerly unknown to kill insects., Conclusion: This study investigated the tc gene distribution among yersiniae and the phylogenetic relationship between TccC proteins, thus contributing novel aspects to the current discussion about the evolution of insecticidal toxins in the genus Yersinia. The toxic potential of several Yersinia spp. towards M. sexta and G. mellonella demonstrated here for the first time points to insects as a natural reservoir for yersiniae.