Differences in the virulence of Heterorhabditis bacteriophora and Steinernema scarabaei to three white grub species: The relative contribution of the nematodes and their symbiotic bacteria

Because susceptibility of white grub species to entomopathogenic nematodes differs, we compared the virulence of Photorhabdus temperata and Xenorhabdus koppenhoeferi, the symbiotic bacteria of the nematodes Heterorhabditis bacteriophora and Steinernema scarabaei, respectively, to the three white grub species, Popillia japonica, Rhizotrogus majalis, and Cyclocephala borealis. Both bacteria were pathogenic to all three grub species even at 2 cells/grub. However, the median lethal dose at 48 h post injection and median lethal time at 20 cells/grub showed that P. temperata was more virulent than X. koppenhoeferi to C. borealis. Although H. bacteriophora is less pathogenic than S. scarabaei to R. majalis and P. japonica, their symbiotic bacteria did not differ in virulence against these two grub species, and they also showed similar growth patterns both in vitro and inside R. majalis larvae at 20 °C. We then tested the pathogenicity of oral- and intrahemocoel-introduced H. bacteriophora to R. majalis to determine whether nematodes are able to successfully vector the bacteria into the hemolymph. Hemocoel injected H. bacteriophora was pathogenic to R. majalis indicating successful bacterial release, but orally introduced H. bacteriophora were not. Dissection of grubs confirmed that the orally introduced H. bacteriophora were unable to penetrate into the hemolymph through the gut wall. We conclude that the low susceptibility of R. majalis to H. bacteriophora is not due to the symbiotic bacteria but rather to the nematode’s poor ability to penetrate through the gut wall and the cuticle to vector the bacteria into the hemolymph.     

Differential pathogenicity of two entomopathogenic bacteria,Photorhabdus temperata subsp. temperata and Xenorhabdus nematophila against the red flour beetle,Tribolium castaneum

Two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata (Ptt) and Xenorhabdus nematophila (Xn), are symbiotically associated with the nematodes, Heterorhabdis megidis and Steinernema carpocapsae, respectively. There is little information on natural host ranges of the nematodes, but a significant difference in pathogenicity was observed between these two bacteria against the red flour beetle, Tribolium castaneum, in which Ptt exhibited more than six times higher pathogenicity than Xn. The pathogenic difference was not due to their inhibitory effect on phospholipase A₂ activity that is required for expression of immune response of T. castaneum. The culture broths of both bacterial species had insecticidal activities when injected into the hemocoel. When the bacterial culture broths were fractionated into aqueous and organic extracts, most insecticidal activity remained in the aqueous extracts. The aqueous extracts of two bacteria contained proteins which showed different profiles.     

Characterization of Xenorhabdus isolates from La Rioja (Northern Spain) and virulence with and without their symbiotic entomopathogenic nematodes (Nematoda: Steinernematidae)

Eighteen Xenorhabdus isolates associated with Spanish entomopathogenic nematodes of the genus Steinernema were characterized using a polyphasic approach including phenotypic and molecular methods. Two isolates were classified as Xenorhabdus nematophila and were associated with Steinernema carpocapsae. Sixteen isolates were classified as Xenorhabdus bovienii, of which fifteen were associated with Steinernema feltiae and one with Steinernema kraussei. Two X. bovienii Phase II were also isolated, one instable phase isolated from S. feltiae strain Rioja and one stable phase from S. feltiae strain BZ. Four representative bacterial isolates were chosen to study their pathogenicity against Spodoptera littoralis with and without the presence of their nematode host. The four bacterial isolates were pathogenic for S. littoralis leading to septicemia 24 h post-injection and killing around 90% of the insect larvae 36 h post-injection, except for that isolated from S. kraussei. After 48 h of injection, this latter isolate showed a lower final population in the larval hemolymph (10⁷ instead of 10⁸ CFU per larvae) and a lower larval mortality (70% instead of 95-100%). The virulence of the nematode-bacteria complexes against S. littoralis showed similar traits with a significant insect larvae mortality (80-90%) 5 days post-infection except for S. kraussei, although this strain reached similar of larval mortality at 7 days after infection.     

New Insights into the Colonization and Release Processes of Xenorhabdus nematophila and the Morphology and Ultrastructure of the Bacterial Receptacle of Its Nematode Host, Steinernema carpocapsae

We present results from epifluorescence, differential interference contrast, and transmission electron microscopy showing that Xenorhabdus nematophila colonizes a receptacle in the anterior intestine of the infective juvenile (IJ) stage of Steinernema carpocapsae. This region is connected to the esophagus at the esophagointestinal junction. The process by which X. nematophila leaves this bacterial receptacle had not been analyzed previously. In this study we monitored the movement of green fluorescent protein-labeled bacteria during the release process. Our observations revealed that Xenorhabdus colonizes the distal region of the receptacle and that exposure to insect hemolymph stimulated forward movement of the bacteria to the esophagointestinal junction. Continued exposure to hemolymph caused a narrow passage in the distal receptacle to widen, allowing movement of Xenorhabdus down the intestine and out the anus. Efficient release of both the wild type and a nonmotile strain was evident in most of the IJs incubated in hemolymph, whereas only a few IJs incubated in nutrient-rich broth released bacterial cells. Incubation of IJs in hemolymph treated with agents that induce nematode paralysis dramatically inhibited the release process. These results suggest that bacterial motility is not required for movement out of the distal region of the receptacle and that hemolymph-induced esophageal pumping provides a force for the release of X. nematophila out of the receptacle and into the intestinal lumen.     

In-silico analysis of genomic distribution and functional association of hipBA toxin-antitoxin (TA) homologs in entomopathogen Xenorhabdus nematophila

Bacteria have a particular strategy to invade the host immune system by forming an undetectable dormant state that may resuscitate and cause disease even after inhabiting for years in a host body. Several mechanisms are known to be responsible for bacterial dormancy, among them the hipBA toxin-antitoxin (TA) system which was initially identified in Escherichia coli. Here we explore the genomic distribution and functional association of hipBA TA homologs from an entomopathogenic bacterium Xenorhabdus nematophila. This bacterium is a symbiotic model with the nematode Steinernema carpocapsae. We found that HipA toxin homologs are more closely related than HipB antitoxins and have satisfactory adenine (for HipA homologs) and nucleic acid (for HipB homologs) ligand partners with a typical TA interaction network that may promote the X. nematophila towards a stringent response to form the dormant state. Such homologs distribution is an inclusion in the current TA repertoire of X. nematophila.     

Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen that forms a symbiotic association with the nematode, Steinernema carpocapsae. Xenorhabdus is carried into the insect host by the nematode, is released into the hemolymph and participates in killing the insect. The bacteria grow to high concentrations supporting the development of the nematode in the hemolymph. OmpR is a global regulatory protein involved in the regulation of porin genes, motility, acid tolerance and virulence in several enteric bacteria. To study the role of ompR in the lifecyle of Xenorhabdus, an ompR -minus strain was constructed. The ompR strain produced markedly reduced levels of the porin protein, OpnP and was both hypermotile and exhibited a hyperhemolysis phenotype. Inactivation of flhDC, the master regulator for flagella synthesis, eliminated hemolysin production in the ompR strain, suggesting that ompR regulates hemolysin production via flhDC. The ompR mutant strain was virulent towards insect hosts. However, when nematodes were grown on a mixture of the wild-type and the ompR strain, only the wild-type strain was recovered indicating that ompR is required for competitive symbiotic interaction with the nematode. The role of ompR in the symbiosis between the bacterium and the nematode is under investigation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA₁, oppA₂, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

Evaluation of persistence and effectiveness of bacterial cell suspensions and culture filtrates against M. incognita on brinjal

Keeping in view the staid health and ecological apprehensions coupled with the use of pesticides, entomopathogenic nematodes have the potential to supersede pesticides for the management of various pests. Brinjal plants are the most seriously affected by Meloidogyne incognita. The main objective of this study was to evaluate the persistence effectiveness of bacterial cell suspensions (Xenorhabdus and Photorhabdus spp.) and their culture filtrates in soil up to 7, 14 and 21?days and their response against M. incognita as a source of biological control for nematode management. In a life cycle study, Xenorhabdus and Photorhabdus spp., isolated from Steinernema asiaticum and Heterorhabditis bacteriophora, were proved more effective in influencing the life cycle of RKNs. It was found that all the treatments of bacterial cell suspensions and their culture filtrates at all persistent times proved effective in reducing the number of females and egg masses as compared to control. It delayed penetration of nematode juveniles (J2) into host roots. It was concluded that persistence effectiveness of bacteria and their metabolites decreased in soil with time.     

A multigene approach for assessing evolutionary relationships of Xenorhabdus spp. (γ-Proteobacteria), the bacterial symbionts of entomopathogenic Steinernema nematodes

Xenorhabdus spp., are gram-negative bacterial symbionts of entomopathogenic nematodes in the genus Steinernema. A specialized and intimate relationship exists between nematode and bacteria, affecting many of their life history traits, such as nutrition, dispersal, host-finding, foraging and defense from biotic and abiotic factors. Xenorhabdus currently comprises more than 20 species isolated from Steinernema spp. with diverse host range, host foraging behavior, reproductive modes and environmental tolerance. Xenorhabdus phylogenies have historically been based on 16s rDNA sequence analyses, and only recently has data from housekeeping genes been employed. The prevalence of lateral gene transfer among bacteria calls for a wider perspective when considering their phylogeny. With the increasing number of Xenorhabdus species and strains, various perspectives need to be considered for investigating the evolutionary history of these nematode bacterial symbionts, In this study, we reconstruct the evolutionary histories of 30 species of Xenorhabdus considering the traditional 16s rDNA gene region as well as the housekeeping genes recA and serC. Datasets were analyzed individually and then combined, using a variety of phylogenetic criteria.     

Symbiont-mediated competition: Xenorhabdus bovienii confer an advantage to their nematode host Steinernema affine by killing competitor Steinernema feltiae

Bacterial symbionts can affect several biotic interactions of their hosts, including their competition with other species. Nematodes in the genus Steinernema utilize Xenorhabdus bacterial symbionts for insect host killing and nutritional bioconversion. Here, we establish that the Xenorhabdus bovienii bacterial symbiont (Xb-Sa-78) of Steinernema affine nematodes can impact competition between S. affine and S. feltiae by a novel mechanism, directly attacking its nematode competitor. Through co-injection and natural infection assays we demonstrate the causal role of Xb-Sa-78 in the superiority of S. affine over S. feltiae nematodes during competition. Survival assays revealed that Xb-Sa-78 bacteria kill reproductive life stages of S. feltiae. Microscopy and timed infection assays indicate that Xb-Sa-78 bacteria colonize S. feltiae nematode intestines, which alters morphology of the intestine. These data suggest that Xb-Sa-78 may be an intestinal pathogen of the non-native S. feltiae nematode, although it is a nonharmful colonizer of the native nematode host, S. affine. Screening additional X. bovienii isolates revealed that intestinal infection and killing of S. feltiae is conserved among isolates from nematodes closely related to S. affine, although the underlying killing mechanisms may vary. Together, these data demonstrate that bacterial symbionts can modulate competition between their hosts, and reinforce specificity in mutualistic interactions.     

Attraction of four entomopathogenic nematodes to four white grub species

To better understand the differences in the efficacy of entomopathogenic nematode species against white grub species, we are studying the various steps of the infection process of entomopathogenic nematodes into different white grub species using nematode species/strains with particular promise as white grub control agents. In this study we compared the attraction of the entomopathogenic nematodes Steinernema scarabaei (AMK001 strain), Steinernema glaseri (NC1 strain), Heterorhabditis zealandica (X1 strain), and Heterorhabditis bacteriophora (GPS11 strain) to third-instars of the scarabs Popillia japonica, Anomala orientalis, Cyclocephala borealis, and Rhizotrogus majalis, and late-instar greater wax moth, Galleria mellonella, larvae. Individual larvae were confined at the bottom of 5.5 cm vertical sand columns, nematodes added to the sand surface after 24 h, and nematodes extracted after another 24 h. Nematode attraction to hosts was strongly affected by nematode species but the effect of insect species varied with nematode species. S. glaseri had a high innate dispersal rate (i.e., in absence of insects) and was strongly attracted to insects without significant differences among insect species. S. scarabaei had a very low innate dispersal rate so that even a strong relative response to insects resulted in low absolute dispersal rates toward insects. S. scarabaei tended to be most attracted to G. mellonella and least attracted to C. borealis. H. zealandica had a high innate dispersal rate but only responded weakly to insects without significant differences among species. H. bacteriophora had limited innate dispersal and only weakly responded to insects with G. mellonella tending to be the most attractive and C. borealis the least attractive insect. It has to be noted that we cannot exclude that the use of different rearing hosts (A. orientalis and P. japonica larvae for S. scarabaei, G. mellonella larvae for the other nematodes) might have had an impact on the nematodes dispersal and relative attraction behavior. This study indicates that host attractiveness and nematode dispersal rates may contribute but do not play a major role in the variability in white grub susceptibility and/or nematode virulence.     

Long-term effects and persistence of Steinernema scarabaei applied for suppression of Anomala orientalis (Coleoptera: Scarabaeidae)

The entomopathogenic nematode, Steinernema scarabaei, is adapted to scarab larvae as hosts and has already shown exceptional potential for inundative control of these pests. To determine the long-term effects of S. scarabaei application on scarab populations and the nematode’s persistence, S. scarabaei was applied in mid-September at rates from 0.06 to 2.5 × 10⁹ infective juveniles (IJs)/ha to turfgrass plots seeded with oriental beetle, Anomala orientalis, larvae. Scarab and nematode populations were monitored for 3–4 years thereafter. S. scarabaei provided excellent A. orientalis control (77–100%) within 1 month of application at rates of 0.25–2.5 × 10⁹ (IJs)/ha and particularly in the following spring at rates of 0.1–2.5 × 10⁹ (IJs)/ha (86–100%). S. scarabaei provided significant control in the next A. orientalis generation in two out of 10 treatments in fall (i.e., 13 months after application) and six out of 10 treatments in the following spring. Thereafter, significant control was only observed occasionally. S. scarabaei numbers were highly variable, and few significant differences among treatments were observed. S. scarabaei recovery from the treated plots was generally more consistent through the first spring after application and became more variable thereafter, but S. scarabaei was recovered for up to 4 years in the experimental plots. Endemic populations of Heterorhabditis bacteriophora and Steinernema carpocapsae, regularly recovered from the experimental plots and often in higher numbers than S. scarabaei, had no significant effect on A. orientalis densities but were able to coexist with S. scarabaei. Our observations suggest that, once current problems with its mass production can be overcome, S. scarabaei could be augmented periodically in areas with recurrent scarab infestations to provide long-term suppression.     

Steinernema scarabaei, an entomopathogenic nematode for control of the European chafer

A new entomopathogenic nematode species, Steinernema scarabaei, was evaluated for efficacy against two white grub species, the European chafer, Rhizotrogus majalis, and the Japanese beetle, Popillia japonica, in laboratory, greenhouse, and field trials. In laboratory assays, S. scarabaei caused greater mortality than Heterorhabditis bacteriophora. S. scarabaei was highly virulent with an LC₅₀ of 5.5–6.0 and 5.7 infective juveniles (IJs) per third-instar larva in R. majalis and P. japonica, respectively. In a greenhouse trial, S. scarabaei provided greater mortality of R. majalis at all application rates (0.156–1.25 × 10⁹ IJs/ha) than Steinernema glaseri and H. bacteriophora (both at 1.25 × 10⁹ IJs/ha). Combination of imidacloprid and S. scarabaei resulted in an antagonistic interaction. In a fall field trial, S. scarabaei provided 88 and 75% control of R. majalis at 2.5 × 10⁹ and 10⁹ IJs/ha, respectively, and 54% control of P. japonica at 10⁹ IJs/ha; H. bacteriophora had no effect on mortality of either white grub species. In a spring field trial, unusually cool temperatures impeded nematode activity. Against R. majalis, S. scarabaei provided moderate control (56–59%), whereas Heterorhabditis marelatus provided no control. Mortality of P. japonica was moderate (49–66%) in both S. scarabaei and H. marelatus treatments. Overwinter persistence of S. scarabaei activity was demonstrated in a spring assay of soil from fall treated plots in which nematode infection was absent from control plots and present in treated plots.     

Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida)

The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10¹⁰ bacterial cells ml⁻¹, a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10⁹ and 10⁸ cells ml⁻¹. Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT₅₀ = 17.1 h) than for Steinernema carpocapsae (RT₅₀ = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect’s food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.     

Neoaplectana species: Specificity of association with bacteria of the genus Xenorhabdus

Each of five Neoaplectana (Nematoda: Steinernematidae) species was cultured monoxenically with various Xenorhabdus (Eubacteriales: Enterobacteriaceae) isolates. The nematodes were usually able to reproduce when cultured with the bacterial symbiont of any one of the five Neoaplectana spp. but never with Xenorhabdus luminescens, symbiotic with Heterorhabditis spp., or with the Xenorhabdus sp. isolated from an undescribed steinernematid species. Only Neoaplectana bibionis could be cultured with the Xenorhabdus symbiont of Steinernema kraussei. A high proportion of infectives were able to carry within their intestine X. nematophilus isolated from other strains of the same nematode species; a small proportion of infectives were able to carry X. nematophilus isolated from other nematode species.     

Genetic and phenotypic characterizations of <Emphasis Type="Italic">Xenorhabdus</Emphasis> species (Enterobacteriales: Enterobacteriaceae) isolated from steinernematid nematodes (Rhabditida: Steinernematidae) in Japan

The bacterial species of the genus Xenorhabdus in the family Enterobacteriaceae have a mutualistic association with steinernematid entomopathogenic nematodes (EPNs), which have been used as biological control agents against soil insect pests. In this study we present the genetic and phenotypic characterizations of the Xenorhabdus species isolated from steinernematid nematodes in Japan. The 18 Japanese Xenorhabdus isolates were classified into five bacterial species based on 16S ribosomal RNA (16S rRNA) gene sequences: Xenorhabdus bovienii, Xenorhabdus hominickii, Xenorhabdus indica, Xenorhabdus ishibashii, and Xenorhabdus japonica. There was no genetic variation between the 16S RNA sequences among the three X. ishibashii isolates, 0–0.1% variation among the five X. hominickii isolates, and 0–0.5% among the eight X. bovienii isolates. Phenotypic characterization demonstrated that representative isolates of the five bacterial species shared common characteristics of the genus Xenorhabdus, and only X. hominickii isolates produced indole. Symbiotic association and co-speciation of Xenorhabdus bacteria with Steinernema nematodes from Japan are discussed.     

The bacterium associated with the entomopathogenic nematode Steinernema abbasi (Nematoda: Steinernematidae) isolated from Taiwan

A symbiotic bacterium of the entomopathogenic nematode, Steinernema abbasi, isolated from Taiwan, determined to be a species of Xenorhabdus based on its physiological and biochemical characteristics has been determined to be similar to Xenorhabdus indica of S. abbasi Oman isolate as based on sequence analyses of 16S rDNA.     

Effect of insect host age and diet on the fitness of the entomopathogenic nematode-bacteria mutualism

Insect host age and diet were evaluated as potential factors that could affect the fitness of the entomopathogenic nematode-bacterium mutualistic partnership. Two nematode species were considered: Steinernema carpocapsae and Heterorhabditis sonorensis, together with their symbionts Xenorhabdus nematophila and Photorhabdus luminescens, respectively. The tobacco hornworm, Manduca sexta, was used as the insect host. Insect developmental stage was a factor that impacted nematode virulence. Non-wandering 5th instar M. sexta were found to be more susceptible to nematode infection compared to wandering 5th instars. This was more noticeable for S. carpocapsae than for H. sonorensis. The nutritional status of the host also had an effect on the fitness of the two nematode species tested. In general, insects fed with the reduced diet content were less susceptible to nematode parasitism. The least observed mortality (0.5 %) was in those M. sexta larvae exposed to the low H. sonorensis dose. Host diet also had an effect on the production of IJ progeny in the insect cadavers. For both nematode species tested, the highest yield of emerging IJs was observed from those insect hosts fed with the low nutrient diet and exposed to the highest nematode inoculum. However, for both nematode species tested, the nutritional status of the host did not significantly affect time of emergence of IJ progeny or the reassociation with their bacterial symbionts (expressed as cfu/IJ). This is the first study on the effect of insect host physiology on both EPN and their symbiotic bacteria fitness.