Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae)

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.     

Nematodes, bacteria, and flies: a tripartite model for nematode parasitism

More than a quarter of the world's population is infected with nematode parasites, and more than a hundred species of nematodes are parasites of humans [1-3]. Despite extensive morbidity and mortality caused by nematode parasites, the biological mechanisms of host-parasite interactions are poorly understood, largely because of the lack of genetically tractable model systems. We have demonstrated that the insect parasitic nematode Heterorhabditis bacteriophora, its bacterial symbiont Photorhabdus luminescens, and the fruit fly Drosophila melanogaster constitute a tripartite model for nematode parasitism and parasitic infection. We find that infective juveniles (IJs) of Heterorhabditis, which contain Photorhabdus in their gut, can infect and kill Drosophila larvae. We show that infection activates an immune response in Drosophila that results in the temporally dynamic expression of a subset of antimicrobial peptide (AMP) genes, and that this immune response is induced specifically by Photorhabdus. We also investigated the cellular and molecular mechanisms underlying IJ recovery, the developmental process that occurs in parasitic nematodes upon host invasion and that is necessary for successful parasitism. We find that the chemosensory neurons and signaling pathways that control dauer recovery in Caenorhabditis elegans also control IJ recovery in Heterorhabditis, suggesting conservation of these developmental processes across free-living and parasitic nematodes.     

The exbD gene of Photorhabdus temperata is required for full virulence in insects and symbiosis with the nematode Heterorhabditis

Photorhabdus are bacteria found colonizing the gut of a specialized stage of the nematode Heterorhabditis, called the infective juvenile (IJ). The IJ is a free-living stage of the nematode that seeks out and infects insect larvae. Once inside the insect the IJ release Photorhabdus into the haemolymph where the bacteria rapidly proliferate, killing the insect within 48-72 h. The nematodes grow and reproduce in the insect cadaver by feeding on the Photorhabdus biomass. In this study we use Photorhabdus temperata K122 to show that genes involved in iron acquisition play a key role during the course of the tripartite bacteria-nematode-insect interaction. We show that a strain carrying a mutation in a gene with homology to exbD, encoding a component of the TonB complex, is unable to grow well in conditions where iron is not freely available. In addition, this mutant, BMM417, requires a longer time to kill the insect larvae than the wild-type bacteria and this defect in pathogenicity is complemented by the co-injection of iron. Moreover, the increase in LT(50) observed with BMM417 is correlated with a significantly slower in vivo growth rate suggesting that iron is limiting in the insect. We also show that BMM417 is unable to support the growth and development of the Heterorhabditis nematode. Addition of exogenous iron to the growth media restores nematode growth and development on BMM417, suggesting that aspects of iron metaboism in Photorhabdus are important during the symbiosis with the nematode.     

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.     

昆虫病原斯氏线虫SY-5对重组Cip晶体蛋白的营养利用（英文稿）

【目的】初生型Photorhabdus luminescens细菌产生两种胞内晶体蛋白CipA和CipB,为其共生的昆虫病原异小杆线虫提供营养。探索非共生的斯氏线虫对Cip蛋白的营养利用情况。【方法】在已构建重组Cip蛋白大肠杆菌表达体系的基础上,建立重组菌细胞与无菌斯氏SY-5线虫共培养系统,检测线虫的生长发育情况。【结果】Cip蛋白对目标线虫生长有显著支持作用:发育为成虫的比例达到65%-82%,雌虫的怀卵率为80%-95%,平均怀卵量为30-50粒,并显著降低各虫态的死亡率。【结论】Cip蛋白不仅为共生的异小杆线虫提供营养,亦能为斯氏线虫所利用。     

昆虫病原线虫共生细菌共生性的分子生物学研究进展

嗜线虫致病杆菌属Xenorhabdus和发光杆菌属Photorhabdus细菌隶属肠杆菌科Enterobacteriaceae,对多种害虫致病能力强,分别与斯氏属Steinernema和异小杆属Heterorhabditis昆虫病原线虫互惠共生。该两属共生细菌既存在对昆虫寄主的病原性,又存在与线虫寄主的共生性。共生细菌与其线虫寄主的共生性主要表现以下4方面：（1）细菌产生食物信号诱导滞育不取食的感染期线虫恢复;（2）细菌为线虫生长与繁殖提供营养;（3）细菌能于感染期线虫的肠道定殖与生长;（4）细菌产生杀线虫毒素杀死非共生线虫。本文综述了共生菌以上4方面的共生性及其相关的分子机制。     

Isolation of insect pathogenic bacteria, Providencia rettgeri, from Heterorhabditis spp.

Nematodes of the genus Heterorhabditis carry bacteria of the genus Photorhabdus into insects including pests of horticultural crops. The bacteria kill the insect and provide conditions which allow for the growth and development of the nematodes. It is reported here that the majority of Heterorhabditis spp. strains tested contained a second bacterial species which was identified as Providencia rettgeri. Injection of the bacteria into waxmoth larvae showed that P. rettgeri was at least as pathogenic as Photorhabdus sp. K122. Both had LD₅₀ values of less than one bacterial cell/larva, but P. rettgeri killed the insects at a considerably faster rate than K122 at both 28°C and 9°C. Since Photorhabdus kills very slowly at low temperatures, it appeared that P. rettgeri might be a better pest control agent under these conditions. However, P. rettgeri was not pathogenic when carried into insect larvae by the nematode, indicating that the nematode suppressed either its release or pathogenicity. It will be necessary to find ways of bypassing or inhibiting this suppression for P. rettgeri to fulfil its potential in pest control.     

Identification of symbiotic bacteria (Photorhabdus and Xenorhabdus) from the entomopathogenic nematodes Heterorhabditis marelatus and Steinernema oregonense based on 16S rDNA sequence

Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts.     

Insecticidal Toxic Proteins Produced by Photorhabdus luminescens akhurstii,a Symbiont of Heterorhabditis indica

We describe the isolation and characterization of an insect pathogenic bacterium from the entomopathogenic nematode Heterorhabditis indica (Karnataka strain), an isolate from the southern regions of India. The strain has been identified and characterized by phenotypic, biochemical tests and PCR-RFLP analysis of the 16S rRNA gene as Photorhabdus luminescens subsp. akhurstii. The insecticidal toxin complex produced by this bacterium has been purified through a series of steps including ultrafiltration, anion exchange chromatography, and gel filtration chromatography. The toxin consists of two protein complexes of approximately 1,000 kD and was active against the larvae of Spodoptera litura and Galleria mellonella.     

Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination

Photorhabdus and Xenorhabdus bacteria colonize the intestines of the infective soil-dwelling stage of entomophagous nematodes, Heterorhabditis and Steinernema, respectively. These nematodes infect susceptible insect larvae and release the bacteria into the insect blood. The bacteria kill the insect larvae and convert the cadaver into a food source suitable for nematode growth and development. After several rounds of reproduction the nematodes are recolonized by the bacteria before emerging from the insect cadaver into the soil to search for a new host. Photorhabdus and Xenorhabdus bacteria therefore engage in both pathogenic and mutualistic interactions with different invertebrate hosts as obligate components of their life cycle. In this review we aim to describe current knowledge of the molecular mechanisms utilized by Photorhabdus and Xenorhabdus to control their host-dependent interactions. Recent work has established that there is a trade-off between pathogenicity and mutualism in both these species of bacteria suggesting that the transition between these interactions must be under regulatory control. Despite the superficial similarity between the life cycles of these bacteria, it is now apparent that the molecular components of the regulatory networks controlling pathogenicity and mutualism in Photorhabdus and Xenorhabdus are very different.     

Postembryonic RNAi in <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis>: a nematode insect parasite and host for insect pathogenic symbionts

Background  

Heterorhabditis bacteriophora is applied throughout the world for the biological control of insects and is an animal model to study interspecies interactions, e.g. mutualism, parasitism and vector-borne disease. H. bacteriophora nematodes are mutually associated with the insect pathogen, Photorhabdus luminescens. The developmentally arrested infective juvenile (IJ) stage nematode (vector) specifically transmits Photorhabdus luminescens bacteria (pathogen) in its gut mucosa to the haemocoel of insects (host). The nematode vector and pathogen alone are not known to cause insect disease. RNA interference is an excellent reverse genetic tool to study gene function in C. elegans, and it would be useful in H. bacteriophora to exploit the H. bacteriophora genome project, currently in progress.     

携带cip基因的酿酒酵母对自由生活线虫Panagrellus redivivus生长繁殖的影响

Photorhabdus luminescens细菌与昆虫病原异小杆属Heterorhabditis线虫专性共生。初生型共生细菌产生两种胞内晶体蛋白CipA and CipB,为共生线虫提供营养。为探索Cip蛋白是否对自由生活的全齿复活线虫Panagrellus redivivus具有类似的营养功能,建立了Cip蛋白的重组酿酒酵母表达体系,并用于饲喂无菌的P. redivivus线虫J1幼虫。重组酿酒酵母表达的Cip蛋白能为线虫所利用,表现为营养支持作用,体现为线虫生长发育速度的加快以及繁殖能力的提高,说明Cip蛋白能为此种自由生活线虫提供营养来源。     

Photorhabdus temperata subsp. stackebrandtii subsp. nov. (Enterobacteriales: Enterobacteriaceae)

The bacterial symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora strain GPS11 was characterized by 16S rRNA gene sequence and physiological traits. The phylogenetic tree built upon 16S rRNA gene sequences clustered the GPS11 bacterial isolate with Photorhabdus temperata strains which have been previously isolated from Heterorhabditis species. The phylogenetic tree further identified four subgroups in P. temperata, and the relationships among these subgroups were confirmed by gyrase subunit B (gyrB) gene sequence analysis. The subgroup containing the GPS11 bacterial isolate differs from other subgroups in sequences of 16S rRNA and gyrB gene, physiological traits, nematode host species, and geographic origin. Therefore, the subgroup comprising the GPS11 bacterial isolate is proposed here as a new subspecies: Photorhabdus temperata subsp. stackebrandtii subsp. nov. (type strain GPS11). The type strain has been deposited in ATCC and DSMZ collections.     

Mutualistic association of Photorhabdus asymbiotica with Japanese heterorhabditid entomopathogenic nematodes

Gram-negative bacteria, Photorhabdus luminescens and P. temperata, form a mutualistic association with entomopathogenic heterorhabditid nematodes while P. asymbiotica is known as an opportunistic human pathogen that causes disseminated bacteremic spread on two continents, the United States and Australia. In the course of our phylogenetic study of Photorhabdus bacteria associated with Japanese Heterorhabditis nematodes, we found two Photorhabdus isolates (Photorhabdus sp. Cbkj163 and OnIr40) whose partial 16S rRNA gene sequence showed high similarities to clinical isolates of this pathogen from Heterorhabditis indica. The phylogenetic study, based upon the gyrase subunit B gene sequences of the two isolates, revealed clustering with these clinical isolates of P. asymbiotica from both the United States and Australia but not with other Photorhabdus bacteria associated with nematodes. The two bacterial isolates were also found to share microbiological and biochemical characteristics with clinical and entomopathogenic Photorhabdus strains. Moreover, not only the two novel Photorhabdus isolates but also an Australian clinical isolate of P. asymbiotica formed mutualistic association with H. indica isolates. These data suggest that the bacteria isolated from H. indica CbKj163 and OnIr40 are a novel subspecies of P. asymbiotica, and that some clinical isolates of P. asymbiotica could have originated from bacteria associated with entomopathogenic nematodes.     

<Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> subsp. <Emphasis Type="Italic">kleinii</Emphasis> subsp. nov. (Enterobacteriales: Enterobacteriaceae)

Association between bacteria Photorhabdus and their nematode hosts Heterorhabditis represents one of the emerging models in symbiosis studies. In this study, we isolated the bacterial symbionts of the nematode Heterorhabditis georgiana. Using gyrB sequences for phylogenetic analysis, these strains were shown to be part of the species of Photorhbdus luminescens but with clear separation from currently recognized subspecies. Physiological properties and DNA–DNA hybridization profiles also supported the phylogenetic relationship of these strains. Therefore, a new subspecies, Photorhabdus luminescens subsp. kleinii subsp. nov., is proposed with the type strain KMD37^T (=DSM 23513 =ATCC =NRRL B-59419).     

Molecular differentiation and phylogeny of entomopathogenic nematodes (rhabditida: heterorhabditidae) based on ND4 gene sequences of Mitochondrial DNA.

We determined partial ND4 gene sequences of mitochondrial DNA from 15 heterorhabditid nematode isolates, representing 5 species collected from different regions of the world, by using polymerase chain reaction (PCR) and direct-sequencing of PCR products. Aligned nucleotide as well as amino acid sequences were used to differentiate nematode species by comparing sequence divergence and to infer phylogeny of the nematodes by using maximum parsimony and likelihood methods. Robustness of our phylogenetic trees was checked by bootstrap tests. The 15 nematode isolates can be divided into 7 haplotypes based on DNA sequences. On a larger scale, the sequence divergence revealed 4 distinct groups corresponding to 4 described species. No sequence divergence was detected from 5 isolates of Heterorhabditis bacteriophora or between Heterorhabditis marelatus to Heterorhabditis hepialius. Our sequence data yielded phylogenetic trees with identical topologies when different tree-building methods were used. Most relationships were also confirmed by using amino acid sequences in maximum parsimony analysis. Our molecular phylogeny of Heterorhabditis species support an existing taxonomy that is based largely on morphology and the sequence divergence of the ND4 gene permits species identification.     

Cell Invasion and Matricide during Photorhabdus luminescens Transmission by Heterorhabditis bacteriophora Nematodes

Many animals and plants have symbiotic relationships with beneficial bacteria. Experimentally tractable models are necessary to understand the processes involved in the selective transmission of symbiotic bacteria. One such model is the transmission of the insect-pathogenic bacterial symbionts Photorhabdus spp. by Heterorhabditis bacteriophora infective juvenile (IJ)-stage nematodes. By observing egg-laying behavior and IJ development, it was determined that IJs develop exclusively via intrauterine hatching and matricide (i.e., endotokia matricida). By transiently exposing nematodes to fluorescently labeled symbionts, it was determined that symbionts infect the maternal intestine as a biofilm and then invade and breach the rectal gland epithelium, becoming available to the IJ offspring developing in the pseudocoelom. Cell- and stage-specific infection occurs again in the pre-IJ pharyngeal intestinal valve cells, which helps symbionts to persist as IJs develop and move to a new host. Synchronous with nematode development are changes in symbiont and host behavior (e.g., adherence versus invasion). Thus, Photorhabdus symbionts are maternally transmitted by an elaborate infectious process involving multiple selective steps in order to achieve symbiont-specific transmission.