Mild thermal stress affects Steinernema carpocapsae infective juvenile survival but not protein content.

Steinernema nematodes and their Xenorhabdus symbionts are a malleable model system to study mutualistic relations. One of the advantages they possess is their ability to be disassociated under in vitro rearing conditions. Various in vitro methods have been developed to produce symbiont colonized and aposymbiotic (symbiont-free) nematodes. Until now, there has been no investigation on how in vitro rearing conditions may have an impact on the storage ability and the protein content of the infective juvenile at different storage temperatures. Thus, in this study, we investigated how infective juvenile longevity and protein content are impacted when the nematodes were reared with two in vitro methods (lipid and liver kidney agar) considering colonized and uncolonized nematodes, and under two different temperatures: 15 °C and 20 °C (mild stress). Infective juveniles reared in vitro (with or without their symbionts) had lower 8-week survival rates. No in vitro reared, colonized IJs survived to the desired 16-week time point. Survival of infective juveniles stored under mild stress temperature (20 °C) was lower than that observed at 15 °C. However, when comparing the interaction between rearing condition and storage temperature, there were not significant differences. With respect to protein content, in vivo, colonized infective juveniles maintained a static protein content over time, suggesting symbiont colonization may influence protein metabolism and/or turnover in infective juveniles.

Effects of cpxR on the growth characteristics and antibiotic production of Xenorhabdus nematophila.

CpxR is a global response regulator that negatively influences the antimicrobial activities of Xenorhabdus nematophila. Herein, the wildtype and ΔcpxR mutant of X. nematophila were cultured in a 5-l and 70-l bioreactor. The kinetic analysis showed that ΔcpxR significantly increased the cell biomass and antibiotic activity. The maximum dry cell weight (DCW) and antibiotic activity of ΔcpxR were 20.77 ± 1.56 g L-1 and 492.0 ± 31.2 U ml-1 and increased by 17.28 and 97.33% compared to the wildtype respectively. Xenocoumacin 1 (Xcn1), a major antimicrobial compound, was increased 3.07-fold, but nematophin was decreased by 48.7%. In 70-l bioreactor, DCW was increased by 18.97%, while antibiotic activity and Xcn1 were decreased by 27.71% and 11.0% compared to that in 5-l bioreactor respectively. Notably, pH had remarkable effects on the cell biomass and antibiotic activity of ΔcpxR, where ΔcpxR was sensitive to alkaline pH conditions. The optimal cell growth and antibiotic activity of ΔcpxR occurred at pH 7.0, while Xcn1 was increased 5.45- and 3.87-fold relative to that at pH 5.5 and 8.5 respectively. These findings confirmed that ΔcpxR considerably increased the biomass of X. nematophila at a late stage of fermentation. In addition, ΔcpxR significantly promoted the biosynthesis of Xcns but decreased the production of nematophin.

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

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

Characterization of the pixB gene in Xenorhabdus nematophila and discovery of a new gene family.

Xenorhabdus nematophila are Gram-negative bacteria that engage in mutualistic associations with entomopathogenic nematodes. To reproduce, the nematodes invade insects and release X. nematophila into the haemolymph where it functions as an insect pathogen. In complex medium, X. nematophila cells produce two distinct types of intracellular crystalline inclusions, one composed of the methionine-rich PixA protein and the other composed of the PixB protein. Here we show that PixB crystalline inclusions were neither apparent in X. nematophila cells grown in medium that mimics insect haemolymph (Grace's medium) nor in cells grown directly in the insect haemocoel. The identified pixB gene was regulated by a conserved σ70 promoter while the pixA promoter was less well conserved. Expression of pixA and pixB under biological conditions was analysed using GFP promoter reporters. Microplate fluorescence detection and flow cytometry analyses revealed that pixB was expressed at high levels in Grace's medium and in insect haemolymph and at lower levels in complex medium, while pixA was expressed at lower levels under all conditions. Although pixB was highly expressed in Grace's medium, PixB crystalline inclusions were not present, suggesting that under biological conditions PixB production may be controlled post-transcriptionally. Although a pixB-minus strain was constructed, the function of PixB remains unresolved. The pixB gene was present in few Xenorhabdus species and pixB-type genes were identified in some Proteobacteria and Gram-positive species, while pixA was only present in Xenorhabdus species. Two conserved sequences were identified in PixB-type proteins that characterize this previously unrecognized gene family.

High Levels of the Xenorhabdus nematophila Transcription Factor Lrp Promote Mutualism with the Steinernema carpocapsae Nematode Host.

Xenorhabdus nematophila bacteria are mutualistic symbionts of Steinernema carpocapsae nematodes and pathogens of insects. The X. nematophila global regulator Lrp controls the expression of many genes involved in both mutualism and pathogenic activities, suggesting a role in the transition between the two host organisms. We previously reported that natural populations of X. nematophila exhibit various levels of Lrp expression and that cells expressing relatively low levels of Lrp are optimized for virulence in the insect Manduca sexta The adaptive advantage of the high-Lrp-expressing state was not established. Here we used strains engineered to express constitutively high or low levels of Lrp to test the model in which high-Lrp-expressing cells are adapted for mutualistic activities with the nematode host. We demonstrate that high-Lrp cells form more robust biofilms in laboratory media than do low-Lrp cells, which may reflect adherence to host tissues. Also, our data showed that nematodes cultivated with high-Lrp strains are more frequently colonized than are those associated with low-Lrp strains. Taken together, these data support the idea that high-Lrp cells have an advantage in tissue adherence and colonization initiation. Furthermore, our data show that high-Lrp-expressing strains better support nematode reproduction than do their low-Lrp counterparts under both in vitro and in vivo conditions. Our data indicate that heterogeneity of Lrp expression in X. nematophila populations provides diverse cell populations adapted to both pathogenic (low-Lrp) and mutualistic (high-Lrp) states.IMPORTANCE Host-associated bacteria experience fluctuating conditions during both residence within an individual host and transmission between hosts. For bacteria that engage in evolutionarily stable, long-term relationships with particular hosts, these fluctuations provide selective pressure for the emergence of adaptive regulatory mechanisms. Here we present evidence that the bacterium Xenorhabdus nematophila uses various levels of the transcription factor Lrp to optimize its association with its two animal hosts, nematodes and insects, with which it behaves as a mutualist and a pathogen, respectively. Building on our previous finding that relatively low cellular levels of Lrp are optimal for pathogenesis, we demonstrate that, conversely, high levels of Lrp promote mutualistic activities with the Steinernema carpocapsae nematode host. These data suggest that X. nematophila has evolved to utilize phenotypic variation between high- and low-Lrp-expression states to optimize its alternating behaviors as a mutualist and a pathogen.

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

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

Effect of inoculum age and physical parameters on in vitro culture of the entomopathogenic nematode Steinernema feltiae.

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae have a symbiotic association with bacteria which makes them virulent against insects. EPNs have been mass produced using in vivo and in vitro methods, including both solid and liquid fermentation. This study assessed the effect of nematode inoculum age on the production of Steinernema feltiae in liquid, solid and biphasic processes. Several physical parameters were also assessed: the effect of medium viscosity, flask size and aeration speed on the recovery and yield of infective juveniles (IJs). Inoculum age treatments included inoculum liquid cultures that were 7, 14, 21 and 28 days old. Nematodes from the same inoculum were added to one liquid medium (liquid culture), one solid medium with bacteria previously grown in sponge (solid culture) and a variation of the solid medium (a biphasic culture), in which the bacteria were first grown in liquid and, then, soaked into the sponges, with the purpose of providing a more homogeneous bacterial culture before nematode inoculation. Experiments were conducted in Erlenmeyer flasks. Eight treatments were established involving combinations of three variables: two media (with and without 0.2% agar), two flask sizes (250 and 150 ml) and two agitation speeds (180 and 280 rpm). The study showed increases in nematode yield for liquid cultures, but not for solid or biphasic cultures, with the advance of the inoculum age up to 28 days of growth. Furthermore, the addition of 0.2% agar to the liquid medium and increasing the aeration rate by using larger flasks with higher agitation speed may increase nematode recovery and final yield. The experiments were conducted using shake flasks but the results may also be applicable for bioreactors.

Development and population dynamics of Steinernema yirgalemense (Rhabditida: Steinernematidae) and growth characteristics of its associated Xenorhabdus indica symbiont in liquid culture.

Entomopathogenic nematodes have become a valuable addition to the range of biological control agents available for insect control. An endemic nematode, Steinernema yirgalemense, has been found to be effective against a wide range of key insect pests. The next step would be the mass production this nematode for commercial application. This requires the establishment of monoxenic cultures of both the nematode and the symbiotic bacterium Xenorhabdus indica. First-stage juveniles of S. yirgalemense were obtained from eggs, while X. indica was isolated from nematode-infected wax moth larvae. The population density of the various life stages of S. yirgalemense during the developmental phase in liquid culture was determined. The recovery of infective juveniles (IJs) to the third-stage feeding juveniles, was 67 ± 10%, reaching a maximum population density of 75,000 IJs ml- 1 on day 13 after inoculation. Adult density increased after 8 days, with the maximum female density being 4600 ml- 1 on day 15, whereas the maximum male density was 4300 ml- 1 on day 12. Growth curves for X. indica showed that the exponential phase was reached 15 h after inoculation to the liquid medium. The stationary phase was reached after 42 h, with an average of 51 × 107 colony-forming units ml- 1. Virulence tests showed a significant difference in insect mortality between in vitro- and in vivo-produced nematodes. The success obtained with the production of S. yirgalemense in liquid culture can serve as the first step in the optimizing and upscaling of the commercial production of nematodes in fermenters.

Role of secondary metabolites in establishment of the mutualistic partnership between Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

Xenorhabdus nematophila engages in a mutualistic partnership with the nematode Steinernema carpocapsae, which invades insects, migrates through the gut, and penetrates into the hemocoel (body cavity). We showed previously that during invasion of Manduca sexta, the gut microbe Staphylococcus saprophyticus appeared transiently in the hemocoel, while Enterococcus faecalis proliferated as X. nematophila became dominant. X. nematophila produces diverse secondary metabolites, including the major water-soluble antimicrobial xenocoumacin. Here, we study the role of X. nematophila antimicrobials in interspecies competition under biologically relevant conditions using strains lacking either xenocoumacin (ΔxcnKL strain), xenocoumacin and the newly discovered antibiotic F (ΔxcnKL:F strain), or all ngrA-derived secondary metabolites (ngrA strain). Competition experiments were performed in Grace's insect medium, which is based on lepidopteran hemolymph. S. saprophyticus was eliminated when inoculated into growing cultures of either the ΔxcnKL strain or ΔxcnKL:F strain but grew in the presence of the ngrA strain, indicating that ngrA-derived antimicrobials, excluding xenocoumacin or antibiotic F, were required to eliminate the competitor. In contrast, S. saprophyticus was eliminated when coinjected into M. sexta with either the ΔxcnKL or ngrA strain, indicating that ngrA-derived antimicrobials were not required to eliminate the competitor in vivo. E. faecalis growth was facilitated when coinjected with either of the mutant strains. Furthermore, nematode reproduction in M. sexta naturally infected with infective juveniles colonized with the ngrA strain was markedly reduced relative to the level of reproduction when infective juveniles were colonized with the wild-type strain. These findings provide new insights into interspecies competition in a host environment and suggest that ngrA-derived compounds serve as signals for in vivo nematode reproduction.

Microbial population dynamics in the hemolymph of Manduca sexta infected with Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

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.

Inhibitory effect of Xenorhabdus nematophila TB on plant pathogens Phytophthora capsici and Botrytis cinerea in vitro and in planta.

Entomopathogenic bacteria Xenorhabdus spp. produce secondary metabolites with potential antimicrobial activity for use in agricultural productions. This study evaluated the inhibitory effect of X. nematophila TB culture on plant pathogens Botrytis cinerea and Phytophthora capsici. The cell-free filtrate of TB culture showed strong inhibitory effects (>90%) on mycelial growth of both pathogens. The methanol-extracted bioactive compounds (methanol extract) of TB culture also had strong inhibitory effects on mycelial growth and spore germinations of both pathogens. The methanol extract (1000 µg/mL) and cell-free filtrate both showed strong therapeutic and protective effects (>70%) on grey mold both in detached tomato fruits and plants, and leaf scorch in pepper plants. This study demonstrates X. nematophila TB produces antimicrobial metabolites of strong activity on plant pathogens, with great potential for controlling tomato grey mold and pepper leaf scorch and being used in integrated disease control to reduce chemical application.

Extracellular novel metalloprotease from Xenorhabdus indica and its potential as an insecticidal agent.

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at 28°C in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in 67.9 ± 0.64% mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

Cabanillasin, a new antifungal metabolite, produced by entomopathogenic Xenorhabdus cabanillasii JM26.

Since the early 1980s, fungi have emerged as a major cause of human disease. Fungal infections are associated with high levels of morbidity and mortality, and are now recognized as an important public health problem. Gram-negative bacterial strains of genus Xenorhabdus are known to form symbiotic associations with soil-dwelling nematodes of the Steinernematidae family. We describe here the discovery of a new antifungal metabolite, cabanillasin, produced by Xenorhabdus cabanillasii. We purified this molecule by cation-exchange chromatography and reverse-phase chromatography. We then determined the chemical structure of cabanillasin by homo- and heteronuclear NMR and MS-MS. Cabanillasin was found to be active against yeasts and filamentous fungi involved in opportunistic infections.

Phospholipase A2 inhibitors in bacterial culture broth enhance pathogenicity of a fungus Nomuraea rileyi.

An entomopathogenic fungus, Nomuraea rileyi, was isolated and its identity was confirmed by its internal transcribed spacer DNA sequence. The isolated N. rileyi exhibited a specific pathogenicity to lepidopteran species. This study was focused on enhancing the fungal pathogenicity by using immunosuppressive agents. In response to infection of N. rileyi, Spodoptera exigua larvae significantly induced catalytic activity of phospholipase A(2) (PLA(2)) in three immune-associated tissues, namely hemocytes, fat body, and hemolymph plasma. Furthermore, the infected S. exigua larvae induced transcription of several antimicrobial peptide (AMP) genes. Two entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata subsp. temperata (Ptt), possessed specific PLA(2)-inhibitory activities and their culture broths significantly inhibited the enzyme activities in hemocytes, fat body, and plasma of S. exigua. In addition, the bacterial metabolites inhibited transcription of AMP genes in S. exigua that would normally respond to the immune challenge by N. rileyi. The immunosuppressive effect of Xn or Ptt bacterial broth resulted in significant enhancement of the fungal pathogenicity against late instar larvae of S. exigua and Plutella xylostella. The effect of such a mixture was confirmed by field assay against two lepidopteran species. These results suggest that the bacterial and fungal mixture can be applied to develop a novel biopesticide to control lepidopteran species.

Phospholipase A2 inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata.

The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A(2) (PLA(2)), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA(2) inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA(2)-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA(2) inhibitors, three of which (benzylideneacetone [BZA], proline-tyrosine [PY], and acetylated phenylalanine-glycine-valine [FGV]) were reported in a previous study. Four other compounds (indole, oxindole, cis-cyclo-PY, and p-hydroxyphenyl propionic acid) were identified and shown to significantly inhibit PLA(2). X. nematophila culture broth contained these seven compounds, while P. temperata subsp. temperata culture broth contained three compounds (BZA, acetylated FGV, and cis-cyclo-PY). BZA was detected in the largest amount among these PLA(2) compounds in both bacterial culture broths. All seven bacterial metabolites also showed significant inhibitory activities against immune responses, such as phenoloxidase activity and hemocytic nodulation; BZA was the most potent. Finally, this study characterized these seven compounds for their insecticidal activities against the diamondback moth, Plutella xylostella. Even though these compounds showed relatively low toxicities to larvae, they significantly enhanced the pathogenicity of Bacillus thuringiensis. This study reports bacterial-origin PLA(2) inhibitors, which would be applicable for developing novel insecticides.

Role of Mrx fimbriae of Xenorhabdus nematophila in competitive colonization of the nematode host.

Xenorhabdus nematophila engages in mutualistic associations with the infective juvenile (IJ) stage of specific entomopathogenic nematodes. Mannose-resistant (Mrx) chaperone-usher-type fimbriae are produced when the bacteria are grown on nutrient broth agar (NB agar). The role of Mrx fimbriae in the colonization of the nematode host has remained unresolved. We show that X. nematophila grown on LB agar produced flagella rather than fimbriae. IJs propagated on X. nematophila grown on LB agar were colonized to the same extent as those propagated on NB agar. Further, progeny IJs were normally colonized by mrx mutant strains that lacked fimbriae both when bacteria were grown on NB agar and when coinjected into the insect host with aposymbiotic nematodes. The mrx strains were not competitively defective for colonization when grown in the presence of wild-type cells on NB agar. In addition, a phenotypic variant strain that lacked fimbriae colonized as well as the wild-type strain. In contrast, the mrx strains displayed a competitive colonization defect in vivo. IJ progeny obtained from insects injected with comixtures of nematodes carrying either the wild-type or the mrx strain were colonized almost exclusively with the wild-type strain. Likewise, when insects were coinjected with aposymbiotic IJs together with a comixture of the wild-type and mrx strains, the resulting IJ progeny were predominantly colonized with the wild-type strain. These results revealed that Mrx fimbriae confer a competitive advantage during colonization in vivo and provide new insights into the role of chaperone-usher fimbriae in the life cycle of X. nematophila.

Manipulation of pH shift to enhance the growth and antibiotic activity of Xenorhabdus nematophila.

To evaluate the effects of pH control strategy on cell growth and the production of antibiotic (cyclo(2-Me-BABA-Gly)) by Xenorhabdus nematophila and enhance the antibiotic activity. The effects of uncontrolled- (different initial pH) and controlled-pH (different constant pH and pH-shift) operations on cell growth and antibiotic activity of X. nematophila YL00I were examined. Experiments showed that the optimal initial pH for cell growth and antibiotic production of X. nematophila YL001 occurred at 7.0. Under different constant pH, a pH level of 7.5 was found to be optimal for biomass and antibiotic activity at 23.71 g/L and 100.0 U/mL, respectively. Based on the kinetic information relating to the different constant pH effects on the fermentation of X. nematophila YL001, a two-stage pH control strategy in which pH 6.5 was maintained for the first 24 h, and then switched to 7.5 after 24 h, was established to improve biomass production and antibiotic activity. By applying this pH-shift strategy, the maximal antibiotic activity and productivity were significantly improved and reaching 185.0 U/mL and 4.41 U/mL/h, respectively, compared to values obtained from constant pH operation (100.0 U/mL and 1.39 U/mL/h).

Virulence and pathogen multiplication: a serial passage experiment in the hypervirulent bacterial insect-pathogen Xenorhabdus nematophila.

The trade-off hypothesis proposes that the evolution of pathogens' virulence is shaped by a link between virulence and contagiousness. This link is often assumed to come from the fact that pathogens are contagious only if they can reach high parasitic load in the infected host. In this paper we present an experimental test of the hypothesis that selection on fast replication can affect virulence. In a serial passage experiment, we selected 80 lines of the bacterial insect-pathogen Xenorhabdus nematophila to multiply fast in an artificial culture medium. This selection resulted in shortened lag phase in our selected bacteria. We then injected these bacteria into insects and observed an increase in virulence. This could be taken as a sign that virulence in Xenorhabdus is linked to fast multiplication. But we found, among the selected lineages, either no link or a positive correlation between lag duration and virulence: the most virulent bacteria were the last to start multiplying. We then surveyed phenotypes that are under the control of the flhDC super regulon, which has been shown to be involved in Xenorhabdus virulence. We found that, in one treatment, the flhDC regulon has evolved rapidly, but that the changes we observed were not connected to virulence. All together, these results indicate that virulence is, in Xenorhabdus as in many other pathogens, a multifactorial trait. Being able to grow fast is one way to be virulent. But other ways exist which renders the evolution of virulence hard to predict.

Effects of constant and shifting dissolved oxygen concentration on the growth and antibiotic activity of Xenorhabdus nematophila.

To evaluate the effects of dissolved oxygen (DO) control strategy on cell growth and the production of antibiotic (cyclo(2-Me-BABA-Gly)) by Xenorhabdus nematophila. The effects of different agitation speeds and DO concentrations on cell growth and antibiotic activity of X. nematophila YL001 were examined. Experiments showed that higher agitation speeds and DO concentrations at earlier fermentation stage were favorable for cell growth and antibiotic production. At mid- and later-stage, properly decreasing DO concentration can strengthen cell growth and antibiotic production. Based on the kinetic information about the effects of agitation speeds and DO concentrations on the fermentation, the two-stage DO control strategy in which DO concentration was controlled to 70% in the first 18 h, and then switched to 50% after 18 h, was established to improve the biomass and antibiotic activity. By applying this DO-shift strategy in X. nematophila YL001 fermentation, maximal antibiotic activity and biomass reached 252.0+/-6.10 U/mL and 30.04+/-2.50 g/L, respectively, thus was 18.99% and 15.36% more than in the cultures at constantly 50% DO.

Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida).

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 x 10(3) DJ per milliliter for S. carpocapsae and 1 to 8 x 10(3) DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 x 10(3) DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3-6 x 10(3) DJs per milliliter in order to obtain >10(3) parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 x 10(3) DJs per milliliter to reach >2 x 10(3) parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 x 10(3) and 105 x 10(3) per mililiter for S. feltiae.