OxyR is required for oxidative stress resistance of the entomopathogenic bacterium Xenorhabdus nematophila and has a minor role during the bacterial interaction with its hosts.

Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae, and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.

Function and Global Regulation of Type III Secretion System and Flagella in Entomopathogenic Nematode Symbiotic Bacteria.

Currently, it is widely accepted that the type III secretion system (T3SS) serves as the transport platform for bacterial virulence factors, while flagella act as propulsion motors. However, there remains a noticeable dearth of comparative studies elucidating the functional disparities between these two mechanisms. Entomopathogenic nematode symbiotic bacteria (ENS), including Xenorhabdus and Photorhabdus, are Gram-negative bacteria transported into insect hosts by Steinernema or Heterorhabdus. Flagella are conserved in ENS, but the T3SS is only encoded in Photorhabdus. There are few reports on the function of flagella and the T3SS in ENS, and it is not known what role they play in the infection of ENS. Here, we clarified the function of the T3SS and flagella in ENS infection based on flagellar inactivation in X. stockiae (flhDC deletion), T3SS inactivation in P. luminescens (sctV deletion), and the heterologous synthesis of the T3SS of P. luminescens in X. stockiae. Consistent with the previous results, the swarming movement of the ENS and the formation of biofilms are dominated by the flagella. Both the T3SS and flagella facilitate ENS invasion and colonization within host cells, with minimal impact on secondary metabolite formation and secretion. Unexpectedly, a proteomic analysis reveals a negative feedback loop between the flagella/T3SS assembly and the type VI secretion system (T6SS). RT-PCR testing demonstrates the T3SS's inhibition of flagellar assembly, while flagellin expression promotes T3SS assembly. Furthermore, T3SS expression stimulates ribosome-associated protein expression.

Antagonistic Efficacy of Symbiotic Bacterium Xenorhabdus sp. SCG against Meloidogyne spp.

The inhabitation and parasitism of root-knot nematodes (RKNs) can be difficult to control, as its symptoms can be easily confused with other plant diseases; hence, identifying and controlling the occurrence of RKNs in plants remains an ongoing challenge. Moreover, there are only a few biological agents for controlling these harmful nematodes. In this study, Xenorhabdus sp. SCG isolated from entomopathogenic nematodes of genus Steinernema was evaluated for nematicidal effects under in vitro and greenhouse conditions. The cell-free filtrates of strain SCG showed nematicidal activity against Meloidogyne species J2s, with mortalities of > 88% at a final concentration of 10%, as well as significant nematicidal activity against the three other genera of plant-parasitic nematodes in a dose-dependent manner. Thymine was isolated as active compounds by assay-guided fractionation and showed high nematicidal activity against M. incognita. Greenhouse experiments suggested that cell-free filtrates of strain SCG efficiently controlled the nematode population in M. incognita-infested tomatoes (Solanum lycopersicum L., cv. Rutgers). In addition, a significant increase in host plant growth was observed after 45 days of treatment. To our knowledge, this is the first to demonstrate the nematicidal activity spectrum of isolated Xenorhabdus species and their application to S. lycopersicum L., cv. Rutgers under greenhouse conditions. Xenorhabdus sp. SCG could be a promising biological nematicidal agent with plant growth-enhancing properties.

The Xenorhabdus nematophila LrhA transcriptional regulator modulates production of γ-keto-N-acyl amides with inhibitory activity against mutualistic host nematode egg hatching.

Xenorhabdus nematophila is a symbiotic Gammaproteobacterium that produces diverse natural products that facilitate mutualistic and pathogenic interactions in their nematode and insect hosts, respectively. The interplay between X. nematophila secondary metabolism and symbiosis stage is tuned by various global regulators. An example of such a regulator is the LysR-type protein transcription factor LrhA, which regulates amino acid metabolism and is necessary for virulence in insects and normal nematode progeny production. Here, we utilized comparative metabolomics and molecular networking to identify small molecule factors regulated by LrhA and characterized a rare γ-ketoacid (GKA) and two new N-acyl amides, GKA-Arg (1) and GKA-Pro (2) which harbor a γ-keto acyl appendage. A lrhA null mutant produced elevated levels of compound 1 and reduced levels of compound 2 relative to wild type. N-acyl amides 1 and 2 were shown to be selective agonists for the human G-protein-coupled receptors (GPCRs) C3AR1 and CHRM2, respectively. The CHRM2 agonist 2 deleteriously affected the hatch rate and length of Steinernema nematodes. This work further highlights the utility of exploiting regulators of host-bacteria interactions for the identification of the bioactive small molecule signals that they control. IMPORTANCE: Xenorhabdus bacteria are of interest due to their symbiotic relationship with Steinernema nematodes and their ability to produce a variety of natural bioactive compounds. Despite their importance, the regulatory hierarchy connecting specific natural products and their regulators is poorly understood. In this study, comparative metabolomic profiling was utilized to identify the secondary metabolites modulated by the X. nematophila global regulator LrhA. This analysis led to the discovery of three metabolites, including an N-acyl amide that inhibited the egg hatching rate and length of Steinernema carpocapsae nematodes. These findings support the notion that X. nematophila LrhA influences the symbiosis between X. nematophila and S. carpocapsae through N-acyl amide signaling. A deeper understanding of the regulatory hierarchy of these natural products could contribute to a better comprehension of the symbiotic relationship between X. nematophila and S. carpocapsae.

Morphological, molecular and ecological characterization of a native isolate of Steinernema feltiae (Rhabditida: Steinernematidae) from southern Chile.

BACKGROUND: Steinernema feltiae is an entomopathogenic nematode used in biological control programs with a global distribution. Populations of this species show phenotypic plasticity derived from local adaptation and vary in different traits, such as location and host penetration. The aim of this work was to describe a Chilean isolate of this nematode species, using integrative approaches. METHODS: Nematode morphological and morphometric studies were conducted along with molecular analysis of nuclear genes. The symbiotic bacterium was also identified by sequencing the 16S rRNA gene. Some ecological characteristics were described, including the temperature requirements for the nematode life cycle and the effect of soil water content for optimal reproduction. RESULTS: Morphometric characterization revealed a large intra-specific variability. The isolate identity was also corroborated with the analysis of nuclear genes. Based on the 16S gene, its symbiont bacteria, Xenorhabdus bovienii, was identified. The lowest, optimal and highest temperatures found to limit the infestation and reproduction on Galleria mellonella were 10, 20 and 30 °C, respectively; the emergence from the host larvae occurred approximately 10 days after inoculation. Differences were observed in offspring, and 120 infective juveniles (IJ)/larva was the most prolific dose at 20 °C. The soil water content did not affect the number of IJ invaders, penetration efficacy and IJ emergence time or offspring per larva, but it caused a delay in achieving full mortality at the permanent wilting point with respect to saturation and field capacity. CONCLUSIONS: For the first time, a Chilean isolate of S. feltiae is described in detail considering morphological, molecular and ecological aspects. The isolate was shown to be efficient in soil containing water, with optimal temperatures ranging from 15 to 25 °C for host infestation and production of an abundant offspring; these characteristics would allow its potential use as control agents in a wide geographical area of the country.

Symbiosis, virulence and natural-product biosynthesis in entomopathogenic bacteria are regulated by a small RNA.

Photorhabdus and Xenorhabdus species have mutualistic associations with nematodes and an entomopathogenic stage1,2 in their life cycles. In both stages, numerous specialized metabolites are produced that have roles in symbiosis and virulence3,4. Although regulators have been implicated in the regulation of these specialized metabolites3,4, how small regulatory RNAs (sRNAs) are involved in this process is not clear. Here, we show that the Hfq-dependent sRNA, ArcZ, is required for specialized metabolite production in Photorhabdus and Xenorhabdus. We discovered that ArcZ directly base-pairs with the mRNA encoding HexA, which represses the expression of specialized metabolite gene clusters. In addition to specialized metabolite genes, we show that the ArcZ regulon affects approximately 15% of all transcripts in Photorhabdus and Xenorhabdus. Thus, the ArcZ sRNA is crucial for specialized metabolite production in Photorhabdus and Xenorhabdus species and could become a useful tool for metabolic engineering and identification of commercially relevant natural products.

Antifungal activity of Xenorhabdus spp. and Photorhabdus spp. against the soybean pathogenic Sclerotinia sclerotiorum.

The fungus, Sclerotinia sclerotiorum, causes white mold disease and infects a broad spectrum of host plants (> 500), including soybean with yield losses of up to 70%. Biological control is a potential alternative for management of this severe plant pathogen, and relative to chemical fungicides, provides broad benefits to the environment, farmers and consumers. The symbiotic bacteria of entomopathogenic nematodes, Xenorhabdus spp. and Photorhabdus spp., are characterized by the production of antimicrobial compounds, which could serve as potential sources for new bio-fungicides. The objectives of this study were to assess cell-free supernatants (CFS) of 16 strains of these bacteria cultures on S. sclerotiorum mycelium growth; assess the volatiles of X. szentirmaii cultures on the fungus mycelium and sclerotium inhibition; and evaluate the X. szentirmaii cultures as well as their CFS on the protection of soybean seeds against the white mold disease. Among the 16 strains, the CFS of X. szentirmaii showed the highest fungicidal effect on growth of S. sclerotiorum. The CFS of X. szentirmaii inhibited > 98% of fungus growth from mycelium and sclerotia, whereas the volatiles generated by the bacterium culture inhibited to 100% of fungus growth and 100% of sclerotia production. The bacterial culture diluted to 33% in water and coated on soybean seeds inhibited S. sclerotiorum and protected soybean plants, allowing 78.3% of seed germination and 56.6% of plant development. Our findings indicate potential for a safe and novel control method for S. sclerotiorum in soybean. Moreover, this is the first study to indicate that volatile organic compounds from Xenorhabdus spp. can be used in plant disease suppression.

R-type bacteriocins of Xenorhabdus bovienii determine the outcome of interspecies competition in a natural host environment.

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, xbp1, 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.

Xenorhabdus nematophila bacteria shift from mutualistic to virulent Lrp-dependent phenotypes within the receptacles of Steinernema carpocapsae insect-infective stage nematodes.

Xenorhabdus nematophila bacteria are mutualists of Steinernema carpocapsae nematodes and pathogens of insects. Xenorhabdus nematophila exhibits phenotypic variation between insect virulence (V) and the mutualistic (M) support of nematode reproduction and colonization initiation in the infective juvenile (IJ) stage nematode that carries X. nematophila between insect hosts. The V and M phenotypes occur reciprocally depending on levels of the transcription factor Lrp: high-Lrp expressors are M+V- while low-Lrp expressors are V+M-. We report here that variable (wild type) or fixed high-Lrp expressors also are optimized, relative to low- or no-Lrp expressors, for colonization of additional nematode stages: juvenile, adult and pre-transmission infective juvenile (IJ). In contrast, we found that after the bacterial population had undergone outgrowth in mature IJs, the advantage for colonization shifted to low-Lrp expressors: fixed low-Lrp expressors (M-V+) and wild type (M+V+) exhibited higher average bacterial CFU per IJ than did high-Lrp (M+V-) or no-Lrp (M-V-) strains. Further, the bacterial population becomes increasingly low-Lrp expressing, based on expression of an Lrp-dependent fluorescent reporter, as IJs age. These data support a model that virulent X. nematophila have a selective advantage and accumulate in aging IJs in advance of exposure to insect hosts in which this phenotype is necessary.

Osmolality as a Novel Mechanism Explaining Diet Effects on the Outcome of Infection with a Blood Parasite.

Recent research has suggested that the outcome of host-parasite interactions is dependent on the diet of the host, but most previous studies have focused on "top-down" mechanisms, i.e., how the host's diet improves the host immune response to drive down the parasite population and improve host fitness. In contrast, the direct impacts of host nutrition on parasite fitness and the mechanisms underpinning these effects are relatively unexplored. Here, using a model host-pathogen system (Spodoptera littoralis caterpillars and Xenorhabdus nematophila, an extracellular bacterial blood parasite), we explore the effects of host dietary macronutrient balance on pathogen growth rates both in vivo and in vitro, allowing us to compare pathogen growth rates both in the presence and absence of the host immune response. In vivo, high dietary protein resulted in lower rates of bacterial establishment, slower bacterial growth, higher host survival, and slower speed of host death; in contrast, the energy content and amount of carbohydrate in the diet explained little variation in any measure of pathogen or host fitness. In vitro, we show that these effects are largely driven by the impact of host dietary protein on host hemolymph (blood) osmolality (i.e., its concentration of solutes), with bacterial growth being slower in protein-rich, high-osmolality hemolymphs, highlighting a novel "bottom-up" mechanism by which host diet can impact both pathogen and host fitness.

The hipBAXn operon from Xenorhabdus nematophila functions as a bonafide toxin-antitoxin module.

Here, for the first time, we have investigated the hipBAXn toxin-antitoxin (TA) module from entomopathogenic bacterium Xenorhabdus nematophila. It is a type II TA module that consists of HipAXn toxin and HipBXn antitoxin protein and located in the complementary strand of chromosome under XNC1_operon 0810 locus tag. For functional analysis, hipAXn toxin, hipBXn antitoxin, and an operon having both genes were cloned in pBAD/His C vector and transformed in Escherichia coli cells. The expression profiles and endogenous toxicity assay were performed in these cells. To determine the active amino acid residues responsible for the toxicity of HipAXn toxin, site-directed mutagenesis (SDM) was performed. SDM results showed that amino acid residues S149, D306, and D329 in HipAXn toxin protein were significantly essential for its toxicity. For transcriptional analysis, the 157 bp upstream region of the hipBAXn TA module was identified as a promoter with bioinformatics tools. Further, the LacZ reporter construct with promoter region was prepared and LacZ assays as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed under different stress conditions. Electrophoretic mobility shift assay (EMSA) was also performed with recombinant HipAXn toxin, HipBXn antitoxin protein, and 157 bp promoter region. Results showed that the hipBAXn TA module is a well-regulated system in which the upregulation of gene expression was also found compulsive in different SOS conditions. KEY POINTS: •Functional characterization of hipBA Xn TA module from Xenorhabdus nematophila. •hipBA Xn TA module is a functional type II TA module. •Transcriptional characterization of hipBA Xn TA module. •hipBA Xn TA module is a well regulated TA module. Graphical abstract.

Morphological adjustment in free-living Steinernema feltiae infective juveniles to increasing concentration of Nemafric-BL phytonematicide.

Third-stage larvae (L3) of Steinernema feltiae exist as free-living infective juveniles (IJ), with suspended development activities. In contrast, parasitic stages (L1, L2, L4, adult) have mutualistic relations with Xenorhabdus species bacteria, along with unique morphological changes and development inside the cadaver of host insects and/or plant-parasitic nematodes. Commercial IJ strains are tolerant to cucurbitacin-containing phytonematicides, but we have scant information on how morphological adjustments in IJ are achieved. In this study, we investigated the nature of morphological adjustments in commercial S. feltiae IJ strains to Nemafric-BL phytonematicide, which contains cucurbitacin B as active ingredient. Post-72 h exposure to phytonematicide concentration, IJ specimens were fixed on mounting slides. Length (body, excretory pore to anterior end, pharynx, rectum, stoma, tail), diameter (head width, neck base, mid-body, anal body), cuticle thickness and De Man ratios were measured with a computer software programme attached to Omax light microscope. Morphometric data against increasing phytonematicide concentration exhibited either density-dependent quadratic, linear or neutral relations. Increase in body length at low phytonematicide concentration was accompanied by decrease in tail length and pharynx length during muscle contraction when IJ were still alive. After death at high phytonematicide concentration, the opposite morphometric effects ensued due to muscle relaxation. The observed changes in morphometric structures were explained on the basis of morphological adjustments that modulated volumes of pseudocoelom cavity in IJ. The modulation is intended to maintain hydrostatic pressure within permissible upper limits in order to avoid structural damage to internal organs embedded in the pseudocoelom fluids.

Bacterial community profile after the lethal infection of Steinernema-Xenorhabdus pairs into soil-reared Tenebrio molitor larvae.

The host microbiota may have an impact on pathogens. This is often studied in laboratory-reared hosts but rarely in individuals whose microbiota looks like that of wild animals. In this study, we modified the gut microbiota of the insect Tenebrio molitor by rearing larvae in soil sampled from the field. We showed by high throughput sequencing methods that this treatment modifies the gut microbiota so that it is more diversified than that of laboratory-reared insects, and closely resembled the one of soil-dwelling insects. To describe what the entomopathogenic bacterial symbiont Xenorhabdus (Enterobacteriaceae), vectored by the soil-dwelling nematode Steinernema, might experience in natural conditions, we studied the infestation of the soil-reared T. molitor larvae with three Steinernema-Xenorhabdus pairs. We performed the infestation at 18°C, which delays the emergence of new infective juveniles (IJs), the soil-dwelling nematode forms, but which is a temperature compatible with natural infestation. We analyzed by high throughput sequencing methods the composition of the bacterial community within the insect cadavers before the first emergences of IJs. These bacterial communities were generally characterized by one or two non-symbiont taxa. Even for highly lethal Steinernema-Xenorhabdus pairs, the symbiont does not dominate the bacterial community within the insect cadaver.

Functional interaction of bacterial virulence factors of Xenorhabdus nematophila with a calcium-independent cytosolic PLA2 of Spodoptera exigua.

Phospholipase A2 (PLA2) hydrolyzes the ester bond of phospholipids (PLs) at sn-2 and releases free fatty acids and lysophospholipids that are subsequently changed into various signal molecules to mediate various physiological processes. Numerous PLA2s are known in various biological systems and can be divided into at least 16 groups. Although different PLA2s recently have been annotated from several insect species, physiological roles are known for only a few genes. Two calcium-independent PLA2s (Se-iPLA2A and Se-iPLA2B) are known in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). We generated and purified a recombinant Se-iPLA2B (rSe-iPLA2B) using a bacterial expression system and analyzed the enzyme kinetics. rSe-iPLA2B exhibited catalytic activities against both arachidonyl (AA)-PL and non-AA-PL substrates. It was highly susceptible to iPLA2-specific inhibitor, but insensitive to inhibitors specific to secretory PLA2s or calcium-dependent cytosolic PLA2s. Increasing calcium concentrations prevented enzyme activity, and culture medium of an entomopathogenic bacterium, Xenorhabdus nematophila, or its organic extracts significantly inhibited enzyme activity. Binding assays of rSe-iPLA2B with known secondary metabolites identified from X. nematophila indicated that benzylideneacetone was the most potent inhibitor with a high binding affinity at 0.2 µM against rSe-iPLA2B. Furthermore, rSe-iPLA2B catalyzed the release of fatty acids from PLs extracted from S. exigua fat body, suggesting its physiological role in maintaining PL integrity. All these catalytic activities indicate that Se-iPLA2B has the typical biochemical properties of other iPLA2s. Its high binding affinity to secondary metabolites of X. nematophila suggests that it is a molecular target of X. nematophila, an entomopathogen.

Selection of Bacterial Mutants in Late Infections: When Vector Transmission Trades Off against Growth Advantage in Stationary Phase.

Bacterial infections are often composed of cells with distinct phenotypes that can be produced by genetic or epigenetic mechanisms. This phenotypic heterogeneity has proved to be important in many pathogens, because it can alter both pathogenicity and transmission. We studied how and why it can emerge during infection in the bacterium Xenorhabdus nematophila, a pathogen that kills insects and multiplies in the cadaver before being transmitted by the soil nematode vector Steinernema carpocapsae We found that phenotypic variants cluster in three groups, one of which is composed of lrp defective mutants. These mutants, together with variants of another group, have in common that they maintain high survival during late stationary phase. This probably explains why they increase in frequency: variants of X. nematophila with a growth advantage in stationary phase (GASP) are under strong positive selection both in prolonged culture and in late infections. We also found that the within-host advantage of these variants seems to trade off against transmission by nematode vectors: the variants that reach the highest load in insects are those that are the least transmitted.IMPORTANCE Pathogens can evolve inside their host, and the importance of this mutation-fueled process is increasingly recognized. A disease outcome may indeed depend in part on pathogen adaptations that emerge during infection. It is therefore important to document these adaptations and the conditions that drive them. In our study, we took advantage of the possibility to monitor within-host evolution in the insect pathogen X. nematophila We demonstrated that selection occurring in aged infection favors lrp defective mutants, because these metabolic mutants benefit from a growth advantage in stationary phase (GASP). We also demonstrated that these mutants have reduced virulence and impaired transmission, modifying the infection outcome. Beyond the specific case of X. nematophila, we propose that metabolic mutants are to be found in other bacterial pathogens that stay for many generations inside their host.

Spodoptera frugiperda transcriptional response to infestation by Steinernema carpocapsae.

Steinernema carpocapsae is an entomopathogenic nematode (EPN) used in biological control of agricultural pest insects. It enters the hemocoel of its host via the intestinal tract and releases its symbiotic bacterium Xenorhabdus nematophila. In order to improve our knowledge about the physiological responses of its different hosts, we examined the transcriptional responses to EPN infestation of the fat body, the hemocytes and the midgut in the lepidopteran pest Spodoptera frugiperda. The tissues poorly respond to the infestation at an early time post-infestation of 8 h with only 5 genes differentially expressed in the fat body of the caterpillars. Strong transcriptional responses are observed at a later time point of 15 h post-infestation in all three tissues. Few genes are differentially expressed in the midgut but tissue-specific panels of induced metalloprotease inhibitors, immune receptors and antimicrobial peptides together with several uncharacterized genes are up-regulated in the fat body and the hemocytes. Among the most up-regulated genes, we identified new potential immune effectors, unique to Lepidoptera, which show homology with bacterial genes of unknown function. Altogether, these results pave the way for further functional studies of the responsive genes' involvement in the interaction with the EPN.

Xenorhabdus khoisanae SB10 produces Lys-rich PAX lipopeptides and a Xenocoumacin in its antimicrobial complex.

BACKGROUND: Xenorhabdus spp. live in close symbiosis with nematodes of the Steinernema genus. Steinernema nematodes infect an insect larva and release their symbionts into the haemocoel of the insect. Once released into the haemocoel, the bacteria produce bioactive compounds to create a semi-exclusive environment by inhibiting the growth of bacteria, yeasts and molds. The antimicrobial compounds thus far identified are xenocoumacins, xenortides, xenorhabdins, indole derivatives, xenoamicins, bicornutin and a number of antimicrobial peptides. The latter may be linear peptides such as the bacteriocins xenocin and xenorhabdicin, rhabdopeptides and cabanillasin, or cyclic, such as PAX lipopeptides, taxlllaids, xenobactin and szentiamide. Thus far, production of antimicrobial compounds have been reported for Xenorhabdus nematophila, Xenorhabdus budapestensis, Xenorhabdus cabanillasii, Xenorhabdus kozodoii, Xenorhabdus szentirmaii, Xenorhabdus doucetiae, Xenorhabdus mauleonii, Xenorhabdus indica and Xenorhabdus bovienii. Here we describe, for the first time, PAX lipopeptides and xenocoumacin 2 produced by Xenorhabdus khoisanae. These compounds were identified using ultraperformance liquid chromatography, linked to high resolution electrospray ionisation mass spectrometry and tandem mass spectrometry. RESULTS: Cell-free supernatants of X. khoisanae SB10 were heat stable and active against Bacillus subtilis subsp. subtilis, Escherichia coli and Candida albicans. Five lysine-rich lipopeptides from the PAX group were identified in HPLC fractions, with PAX1' and PAX7 present in the highest concentrations. Three novel PAX7 peptides with putative enoyl modifications and two linear analogues of PAX1' were also detected. A small antibiotic compound, yellow in colour and λmax of 314 nm, was recovered from the HPLC fractions and identified as xenocoumacin 2. The PAX lipopeptides and xenocoumacin 2 correlated with the genes and gene clusters in the genome of X. khoisanae SB10. CONCLUSION: With UPLC-MS and MSe analyses of compounds in the antimicrobial complex of X. khoisanae SB10, a number of PAX peptides and a xenocoumacin were identified. The combination of pure PAX1' peptide with xenocoumacin 2 resulted in high antimicrobial activity. Many of the fractions did, however, contain labile compounds and some fractions were difficult to resolve. It is thus possible that strain SB10 may produce more antimicrobial compounds than reported here, as suggested by the APE Ec biosynthetic complex. Further research is required to develop these broad-spectrum antimicrobial compounds into drugs that may be used in the fight against microbial infections.

Partners in crime: symbiont-assisted resource acquisition in Steinernema entomopathogenic nematodes.

Entomopathogenic nematodes in the genus Steinernema (Nematoda: Steinernematidae) have a mutualistic relationship with Xenorhabdus bacteria (Gram-negative Enterobacteriaceae). This partnership however, is pathogenic to a wide range of insect species. Because of their potent insecticidal ability, they have successfully been implemented in biological control and integrated pest management programs worldwide. Steinernema-Xenorhabdus-insect partnerships are extremely diverse and represent a model system in ecology and evolution to investigate symbioses between invertebrates and microbes. The reproductive fitness of the nematode-bacterium partnership is tightly associated, and maintenance of their virulence is critical to the conversion of the insect host as a suitable environment where this partnership can be perpetuated.

Diet modulates the relationship between immune gene expression and functional immune responses.

Nutrition is vital to health and the availability of resources has long been acknowledged as a key factor in the ability to fight off parasites, as investing in the immune system is costly. Resources have typically been considered as something of a "black box", with the quantity of available food being used as a proxy for resource limitation. However, food is a complex mixture of macro- and micronutrients, the precise balance of which determines an animal's fitness. Here we use a state-space modelling approach, the Geometric Framework for Nutrition (GFN), to assess for the first time, how the balance and amount of nutrients affects an animal's ability to mount an immune response to a pathogenic infection. Spodoptera littoralis caterpillars were assigned to one of 20 diets that varied in the ratio of macronutrients (protein and carbohydrate) and their calorie content to cover a large region of nutrient space. Caterpillars were then handled or injected with either live or dead Xenorhabdus nematophila bacterial cells. The expression of nine genes (5 immune, 4 non-immune) was measured 20 h post immune challenge. For two of the immune genes (PPO and Lysozyme) we also measured the relevant functional immune response in the hemolymph. Gene expression and functional immune responses were then mapped against nutritional intake. The expression of all immune genes was up-regulated by injection with dead bacteria, but only those in the IMD pathway (Moricin and Relish) were substantially up-regulated by both dead and live bacterial challenge. Functional immune responses increased with the protein content of the diet but the expression of immune genes was much less predictable. Our results indicate that diet does play an important role in the ability of an animal to mount an adequate immune response, with the availability of protein being the most important predictor of the functional (physiological) immune response. Importantly, however, immune gene expression responds quite differently to functional immunity and we would caution against using gene expression as a proxy for immune investment, as it is unlikely to be reliable indicator of the immune response, except under specific dietary conditions.

The first report of Xenorhabdus indica from Steinernema pakistanense: co-phylogenetic study suggests co-speciation between X. indica and its steinernematid nematodes.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode-bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the 'bicornutum' group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.