Stages of infection during the tripartite interaction between Xenorhabdus nematophila, its nematode vector, and insect hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Two Distinct Hemolytic Activities in Xenorhabdus nematophila Are Active against Immunocompetent Insect Cells

Xenorhabdus spp. and Photorhabdus spp. are major insect bacterial pathogens symbiotically associated with nematodes. These bacteria are transported by their nematode hosts into the hemocoel of the insect prey, where they proliferate within hemolymph. In this work we report that wild strains belonging to different species of both genera are able to produce hemolysin activity on blood agar plates. Using a hemocyte monolayer bioassay, cytolytic activity against immunocompetent cells from the hemolymph of Spodoptera littoralis (Lepidoptera: Noctuidae) was found only in supernatants of Xenorhabdus; none was detected in supernatants of various strains of Photorhabdus. During in vitro bacterial growth of Xenorhabdus nematophila F1, two successive bursts of cytolytic activity were detected. The first extracellular cytolytic activity occurred when bacterial cells reached the stationary phase. It also displayed a hemolytic activity on sheep red blood cells, and it was heat labile. Among insect hemocyte types, granulocytes were the preferred target. Lysis of hemocytes by necrosis was preceded by a dramatic vacuolization of the cells. In contrast the second burst of cytolytic activity occurred late during stationary phase and caused hemolysis of rabbit red blood cells, and insect plasmatocytes were the preferred target. This second activity is heat resistant and produced shrinkage and necrosis of hemocytes. Insertional inactivation of flhD gene in X. nematophila leads to the loss of hemolysis activity on sheep red blood cells and an attenuated virulence phenotype in S. littoralis (A. Givaudan and A. Lanois, J. Bacteriol. 182:107–115, 2000). This mutant was unable to produce the early cytolytic activity, but it always displayed the late cytolytic effect, preferably active on plasmatocytes. Thus, X. nematophila produced two independent cytolytic activities against different insect cell targets known for their major role in cellular immunity.     

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.     

Txp40, a Ubiquitous Insecticidal Toxin Protein from Xenorhabdus and Photorhabdus Bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

Isolation and Entomotoxic Properties of the Xenorhabdus nematophilus F1 Lecithinase

Xenorhabdus spp. and Photorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar. Substrate specificity studies showed that Photorhabdus spp. exhibited a broad lipase activity, while most of the Xenorhabdus spp. secreted a specific lecithinase. Xenorhabdus spp. occur spontaneously in two variants, phase I and phase II. Only the phase I variants of Xenorhabdus nematophilus and Xenorhabdus bovienii strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth). Five enzymatic isomers responsible for this activity were separated from the supernatant of a X. nematophilus F1 culture in two chromatographic steps, cation-exchange chromatography and C₁₈ reverse-phase chromatography. The substrate specificity of the X. nematophilus F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated. The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae. None of the isomers exhibited toxicity with the insects tested, Locusta migratoria, Galleria mellonella, Spodoptera littoralis, and Manduca sexta. The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.     

Characterization of entomopathogenic nematodes and symbiotic bacteria active against Spodoptera frugiperda (Lepidoptera: Noctuidae) and contribution of bacterial urease to the insecticidal effect

Entomopathogenic nematodes carrying symbiotic bacteria represent one of the best non-chemical strategies for insect control. Infective juveniles of Heterorhabditidae and Steinernematidae nematodes actively seek the host in the soil, penetrating through insect’s openings to reach the hemocoel where symbiotic bacteria in the genera Photorhabdus or Xenorhabdus, respectively, are released. The bacteria replicate and produce virulence factors that rapidly kill the insect host, providing nutrients for the nematodes development and reproduction within the insect cadaver. More studies are necessary to better understand the factors implicated in the nematode-bacteria association, particularly focusing the bacterial symbionts, the final effectors of the insect death. Our group has shown that ureases are lethal to some groups of insects and may contribute to the entomopathogenic properties of the symbiotic bacteria.The fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is one of the major insect pests in corn (Zea mays) crops in Brazil, with infestations resulting in reduction up to 39% yield and losses amounting US$ 500 million annually. Native strains of entomopathogenic nematodes active against S. frugiperda represent a promising alternative to the intensive use of chemical insecticides to control fall armyworm population in corn plantations.In this study we screened soil nematodes collected in the south region of Brazil for pathogenicity against S. frugiperda. Symbiotic bacteria associated with these nematodes were isolated and characterized. We also evaluated urease production by the symbiotic bacteria in vitro and along the course of infection in S. frugiperda and demonstrated that urease production correlated positively to their entomopathogenicity.     

Interspecific competition between entomopathogenic nematodes (Steinernema) is modified by their bacterial symbionts (Xenorhabdus)

Background  

Symbioses between invertebrates and prokaryotes are biological systems of particular interest in order to study the evolution of mutualism. The symbioses between the entomopathogenic nematodes Steinernema and their bacterial symbiont Xenorhabdus are very tractable model systems. Previous studies demonstrated (i) a highly specialized relationship between each strain of nematodes and its naturally associated bacterial strain and (ii) that mutualism plays a role in several important life history traits of each partner such as access to insect host resources, dispersal and protection against various biotic and abiotic factors. The goal of the present study was to address the question of the impact of Xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different Steinernema species. For this, we monitored experimental interspecific competition between (i) two nematode species: S. carpocapsae and S. scapterisci and (ii) their respective symbionts: X. nematophila and X. innexi within an experimental insect-host (Galleria mellonella). Three conditions of competition between nematodes were tested: (i) infection of insects with aposymbiotic IJs (i.e. without symbiont) of both species (ii) infection of insects with aposymbiotic IJs of both species in presence of variable proportion of their two Xenorhabdus symbionts and (iii) infection of insects with symbiotic IJs (i.e. naturally associated with their symbionts) of both species.     

Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila

The symbiotic interaction between Steinernema carpocapsae and Xenorhabdus nematophila was investigated by comparing the reproduction, morphology, longevity, behavior, and efficacy of the infective juvenile (IJ) from nematodes reared on mutant or wild-type bacterium. Nematodes reared on the mutant X. nematophila HGB151, in which an insertion of the bacterial gene, rpoS, eliminates the retention of the bacterium in the intestinal vesicle of the nematode, produced IJs without their symbiotic bacterium. Nematodes reared on the wild-type bacterium (HGB007) produced IJs with their symbiotic bacterium. One or the other bacterial strain injected into Galleria mellonella larvae followed by exposing the larvae to IJs that were initially symbiotic bacterium free produced progeny IJs with or without their Xenorhabdus-symbiotic bacterium. The two bacterial strains were not significantly different in their effect on IJ production, sex ratio, or IJ morphology. IJ longevity in storage was not influenced by the presence or absence of the bacterial symbiont at 5 and 15 °C, but IJs without their bacterium had greater longevity than IJs with their bacterium at 25 and 30 °C, suggesting that there was a negative cost to the nematode for maintaining the bacterial symbiont at these temperatures. IJs with or without their symbiotic bacterium were equally infectious to Spodoptera exigua larvae in laboratory and greenhouse and across a range of soil moistures, but the absence of the bacterial symbiont inhibited nematodes from producing IJ progeny within the host cadavers. In some situations, such as where no establishment of an alien entomopathogenic nematode is desired in the environment, the use of S. carpocapsae IJs without their symbiotic bacterium may be used to control some soil insect pests.     

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic ^1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis ⁴. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects ⁵. For transmission between insect hosts, the bacteria colonize the intestine of the nematode''s infective juvenile stage ^6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects ^9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems ⁹.We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila¹⁴. Similar methods have been used to investigate other nematode-bacterium associations ^9,15-18and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization ^14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues ^14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication ²²or grinding ²³, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes ^21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization ^17,18, and is less laborious than other methods, including sonication ^22,25-27and individual nematode dissection ^28,29.     

Mapping of the entomocidal fragment of Spodoptera -specific Bacillus thuringiensis toxin CryIC

 Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI poteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins. Received: 15 April 1996/Accepted: 10 July 1996     

Sequential immunosuppressive activities of bacterial secondary metabolites from the entomopahogenic bacterium Xenorhabdus nematophila

The entomopathogenic bacterium Xenorhabdus nematophila secretes at least eight bacterial metabolites that play crucial roles suppressing target insect immune responses by inhibiting eicosanoid biosynthesis. We analyzed sequential changes in bacterial metabolite production during bacterial growth and analyzed their individual immunosuppressive activities against the insect host, Spodoptera exigua. X. nematophila exhibited a typical bacterial growth pattern in both insect host and culture medium, and eight metabolites were secreted at different time points. At the early growth phase (6–12 h), Ac-FGV and PHPP were detected in significant amounts in the culture broth. At this early phase, both Ac-FGV (18 μg/ml) and oxindole (110 μg/ml) levels significantly inhibited phenoloxidase and phospholipase A₂ activities in S. exigua hemolymph. At the late growth phase (12–36 h), all eight metabolites were detected at significant levels (10–140 μg/ml) in the culture broth and were sufficient to induce hemocyte toxicity. These results suggest that X. nematophila sequentially produces immunosuppressive metabolites that might sequentially and cooperatively inhibit different steps of insect immune responses.     

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.     

Long-Term Persistence of Yersinia pseudotuberculosis in Entomopathogenic Nematodes

Entomopathogenic nematodes (EPNs) are small worms whose ecological behaviour consists to invade, kill insects and feed on their cadavers thanks to a species-specific symbiotic bacterium belonging to any of the genera Xenorhabdus or Photorhabdus hosted in the gastro-intestinal tract of EPNs. The symbiont provides a number of biological functions that are essential for its EPN host including the production of entomotoxins, of enzymes able to degrade the insect constitutive macromolecules and of antimicrobial compounds able to prevent the growth of competitors in the insect cadaver. The question addressed in this study was to investigate whether a mammalian pathogen taxonomically related to Xenorhabdus was able to substitute for or “hijack” the symbiotic relationship associating Xenorhabdus and Steinernema EPNs. To deal with this question, a laboratory experimental model was developed consisting in Galleria mellonella insect larvae, Steinernema EPNs with or without their natural Xenorhabdus symbiont and Yersinia pseudotuberculosis brought artificially either in the gut of EPNs or in the haemocoel of the insect larva prior to infection. The developed model demonstrated the capacity of EPNs to act as an efficient reservoir ensuring exponential multiplication, maintenance and dissemination of Y. pseudotuberculosis.     

Benzylideneacetone and other phenylethylamide bacterial metabolites induce apoptosis to kill insects

Xenorhabdus and Photorhabdus are entomopathogenic bacteria that can induce immunosuppression against target insects by suppressing eicosanoid biosynthesis, leading to fatal septicemia. These bacteria can synthesize and release secondary metabolites such as benzylideneacetone (BZA) and other phenylethylamide compounds that can inhibit phospholipase A₂ (PLA₂) and shut down eicosanoid biosynthesis. However, insecticidal activities of these bacterial metabolites remain unclear. Thus, the objective of this study was to assess cytotoxicities of BZA and seven other bacterial metabolites to insect cells. These eight bacterial metabolites exhibited significant cytotoxicities against an insect cell line Sf9 at micromolar range. Especially, BZA and cPY were highly potent at low micromolar range. When these eight bacterial metabolites were injected to hemocoels of Spodoptera exigua larvae, they significantly decreased total count of hemocytes. In Sf9 cell line and hemocytes, these bacterial metabolites induced cell membrane blebbings, apoptotic vesicles, and genomic DNA fragmentation. Terminal deoxyribonucleotidyl transferase nick end translation assay showed that these bacterial metabolites caused significant DNA breakages in cells in a dose-dependent manner. However, a pan caspase inhibitor treatment significantly rescued the cell death induced by these bacterial metabolites. Cytotoxicities of these bacterial metabolites were highly correlated with their insecticidal activities. These results indicate that the insecticidal activities of the bacterial metabolites may be induced by their apoptotic activities against hemocytes and other insect cells. Taken together, these results suggest that phenylethylamide compounds might have potential as novel insecticides.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, and the pending genome sequence⁹ make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter¹⁰. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking¹¹ and oral toxicity assays¹². Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides⁷; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR¹³. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes⁶. To analyze these responses, injected insects can be dissected and visualized by microscopy^{13, 14}.     

Previously unrecognized stages of species‐specific colonization in the mutualism between Xenorhabdus bacteria and Steinernema nematodes

The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate and species‐specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species‐specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal‐intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species‐specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by theirSteinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.