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.     

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.     

Genetic and Proteomic Characterization of rpoB Mutations and Their Effect on Nematicidal Activity in Photorhabdus luminescens LN2

Rifampin resistant (Rif(R)) mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The Rif(R) mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs) of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 Rif(R) mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing Rif(R) mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 Rif(R) mutants. The rpoB (P564L) mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s). The non-nematicidal-producing Rif(R) mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and Rif(R) mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS), and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively) were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregualted dsbA). These results indicate that the rpoB mutations greatly influence the symbiotic association between the symbionts and their entomopathogenic nematode hosts.     

Closely coupled evolutionary history of ecto‐ and endosymbionts from two distantly related animal phyla

The level of integration between associated partners can range from ectosymbioses to extracellular and intracellular endosymbioses, and this range has been assumed to reflect a continuum from less intimate to evolutionarily highly stable associations. In this study, we examined the specificity and evolutionary history of marine symbioses in a group of closely related sulphur‐oxidizing bacteria, called Candidatus Thiosymbion, that have established ecto‐ and endosymbioses with two distantly related animal phyla, Nematoda and Annelida. Intriguingly, in the ectosymbiotic associations of stilbonematine nematodes, we observed a high degree of congruence between symbiont and host phylogenies, based on their ribosomal RNA (rRNA) genes. In contrast, for the endosymbioses of gutless phallodriline annelids (oligochaetes), we found only a weak congruence between symbiont and host phylogenies, based on analyses of symbiont 16S rRNA genes and six host genetic markers. The much higher degree of congruence between nematodes and their ectosymbionts compared to those of annelids and their endosymbionts was confirmed by cophylogenetic analyses. These revealed 15 significant codivergence events between stilbonematine nematodes and their ectosymbionts, but only one event between gutless phallodrilines and their endosymbionts. Phylogenetic analyses of 16S rRNA gene sequences from 50 Cand. Thiosymbion species revealed seven well‐supported clades that contained both stilbonematine ectosymbionts and phallodriline endosymbionts. This closely coupled evolutionary history of marine ecto‐ and endosymbionts suggests that switches between symbiotic lifestyles and between the two host phyla occurred multiple times during the evolution of the Cand. Thiosymbion clade, and highlights the remarkable flexibility of these symbiotic bacteria.     

Differences between the pathogenic processes induced by Steinernema and Heterorhabditis (Nemata: Rhabditida) in Pseudaletia unipuncta (Insecta: Lepidoptera)

Larvae of Pseudaletia unipuncta are moderately susceptible to infections caused by entomopathogenic nematodes, being a desirable host to study pathogenic processes caused by Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema glaseri and their associated bacteria. The ability of the infective stage of these nematodes to invade hosts is quite different. S. carpocapsae invades the highest number of insects and presents the highest penetration rate, followed by H. bacteriophora. Regression analysis between the number of insects parasitized and the number of IJs counted per insect, over time, showed a high correlation for S. carpocapsae whereas for H. bacteriophora it was low. Dose-response was most evident at a concentration below 100 IJs per insect on H. bacteriophora, whereas on S. carpocapsae it was found for doses ranging from 100 to 2,000 IJs. Student's t test analysis of dose-response showed parallel, yet unequal, slopes for both strains of H. bacteriophora, whereas distinct regressions were obtained for S. carpocapsae and S. glaseri, thus, evidencing each species develop a distinct pathogenic process. Insects injected with Photorhabdus luminescens died within 50 h after injection, whereas those treated with X. nematophila died much later. Moreover, the mortality in insects exposed to H. bacteriophora complex and injected with P. luminescens was close, but insects injected with bacteria died faster. Insect mortality in treatments with complexes S. carpocapsae and S. glaseri was significantly higher than that which was observed in insects injected with symbiotic bacteria.     

<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).     

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。     

The entomopathogenic bacterial endosymbionts Xenorhabdus and Photorhabdus: convergent lifestyles from divergent genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.     

昆虫病原线虫与其共生细菌共生关系的研究进展

昆虫病原斯氏和异小杆线虫与共生细菌的共生关系是这类线虫作为害虫生物防治因子的基础。从线虫共生细菌的信息、营养、抗菌和病原作用,以及线虫对共生细菌的保护和媒介作用综述昆虫病原线虫与其共生细菌的共生关系;描述这一共生关系的影响因子;同时,讨论了未来的研究方向和应用前景。     

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.     

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.     

不同昆虫寄主对昆虫病原线虫共生菌的敏感性比较

用菜青虫、棉铃虫、甜菜夜蛾、玉米螟、粘虫、黄粉虫等 6种昆虫对 1 0株昆虫病原线虫共生菌进行了敏感性测定。结果表明 :供试菌株对 6种昆虫都有胃毒活性 ,不同菌株对同一种昆虫的毒力差别较大 ,同一菌株对不同昆虫差别也很大。 1 0株菌在 1 2 0h对菜青虫的校正死亡率和体重抑制率均最高 ,显然是最敏感的寄主。在 1 0株共生菌中 ,XenorhabdusnematophilaHB3 1 0 5 9菌株的胃毒活性最高。     

Mass production of entomopathogenic nematodes for plant protection

Entomopathogenic nematodes of the genera Heterorhabditis and Steinernema are commercially used to control pest insects. They are symbiotically associated with bacteria of the genera Photorhabdus and Xenorhabdus, respectively, which are the major food source for the nematodes. The biology of the nematode-bacterium complex is described, a historical review of the development of in vitro cultivation techniques is given and the current use in agriculture is summarised. Cultures of the complex are pre-incubated with the symbiotic bacteria before the nematodes are inoculated. Whereas the inoculum preparation and preservation of bacterial stocks follow standard rules, nematodes need special treatment. Media development is mainly directed towards cost reduction, as the bacteria are able to metabolise a variety of protein sources to provide optimal conditions for nematode reproduction. The process technology is described, discussing the influence of bioreactor design and process parameters required to obtain high nematode yields. As two organisms are grown in one vessel and one of them is a multicellular organism, the population dynamics and symbiotic interactions need to be understood in order to improve process management. Major problems can originate from the delayed or slow development of the nematode inoculum and from phase variants of the symbiotic bacteria that have negative effects on nematode development and reproduction. Recent scientific progress has helped to understand the biological and technical parameters that influence the process, thus enabling transfer to an industrial scale. As a consequence, costs for nematode-based products could be significantly reduced.     

When mutualists are pathogens: an experimental study of the symbioses between Steinernema (entomopathogenic nematodes) and Xenorhabdus (bacteria)

In this paper, we investigate the level of specialization of the symbiotic association between an entomopathogenic nematode (Steinernema carpocapsae) and its mutualistic native bacterium (Xenorhabdus nematophila). We made experimental combinations on an insect host where nematodes were associated with non-native symbionts belonging to the same species as the native symbiont, to the same genus or even to a different genus of bacteria. All non-native strains are mutualistically associated with congeneric entomopathogenic nematode species in nature. We show that some of the non-native bacterial strains are pathogenic for S. carpocapsae. When the phylogenetic relationships between the bacterial strains was evaluated, we found a clear negative correlation between the effect a bacterium has on nematode fitness and its phylogenetic distance to the native bacteria of this nematode. Moreover, only symbionts that were phylogenetically closely related to the native bacterial strain were transmitted. These results suggest that co-evolution between the partners has led to a high level of specialization in this mutualism, which effectively prevents horizontal transmission. The pathogenicity of some non-native bacterial strains against S. carpocapsae could result from the incapacity of the nematode to resist specific virulence factors produced by these bacteria.     

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.     

Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae

Photorhabdus luminescens (Enterobacteriaceae) is a symbiont of entomopathogenic nematodes Heterorhabditis spp. (Nematoda: Rhabditida) used for biological control of insect pests. For industrial mass production, the nematodes are produced in liquid media, pre-incubated with their bacterial symbiont, which provides nutrients essential for the nematode's development and reproduction. Particularly under in vitro conditions, P. luminescens produces phase variants, which do not allow normal nematode development. The phase variants were distinguished based on dye absorption, pigmentation, production of antibiotic substances, occurrence of crystalline inclusion proteins and bioluminescence. To understand the significance of the phase shift for the symbiotic interaction between the bacterium and the nematode, feeding experiments tested the effect of homologous and heterologous P. luminescens phase variants isolated from a Chinese Heterorhabditis bacteriophora (HO6), the Heterorhabditis megidis type strain from Ohio (HNA) and the type strain of Heterorhabditis indica (LN2) on the in vivo and in vitro development and reproduction of the nematode species H. bacteriophora (strain HO6) and another rhabditid and entomopathogenic nematode, Steinernema carpocapsae (A24). In axenically cultured insect larvae (Galleria mellonella) and in vitro in liquid media, H. bacteriophora produced offspring on phase I of its homologous symbiont and on the heterologous symbiont of H. megidis, but not on the two corresponding phase II variants. In solid media, nematode yields were much lower on phase II than on phase I variants. On the heterologous phase I symbiont isolated from H. indica the development of H. bacteriophora was not beyond the fourth juvenile stage of the nematode in any of the media tested, but further progressed on phase II with even a small amount of offspring recorded in solid media. Infective juveniles of S. carpocapsae did not develop beyond the J3 stage on all phase I P. luminescens. They died in phase I P. luminescens isolated from H. bacteriophora. Development to adults was recorded for S. carpocapsae on all phase II symbionts and offspring were produced in all media except in liquid. It is concluded that a lack of essential nutrients or the production of toxins is not responsible for the negative impact of homologous phase II symbiont cells on the development and reproduction of H. bacteriophora. The infective juveniles of H. bacteriophora retained cells of the homologous phase I symbiont, but not phase II cells and cells from heterologous symbionts, indicating that the transmission of the symbiont by the infective juvenile is selective for phase I cells and the homologous bacterial associate.     

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.     

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 luminescens LN2转座突变菌株的研究

发光杆菌属Photorhabdus细菌与异小杆属Heterorhabditis昆虫病原线虫的共生关系是这类生物杀虫剂产业化生产和田间应用的基础。本文采用Tn5转座方法构建了共生细菌P. luminescens LN2突变体库;从中筛选出一个对其共生线虫H. indica LN2的生长繁殖有显著促进作用的突变菌株（LN2-M2716）;测定了该突变菌株的菌落特征、对大蜡螟Galleria mellonella及非特异共生线虫H. bacteriophora H06的毒性、对线虫产量的影响。结果显示,LN2-M2716菌株在菌落形态、色素分泌、过氧化氢酶反应、荧光、食物信息作用以及对大蜡螟毒力等方面与野生型菌株差异不明显;但对非特异共生线虫H. bacteriophora H06的毒性及对特异共生线虫H. indica LN2生长繁殖的促进作用方面均明显高于野生型菌株。论文结果为构建支持线虫高产的菌株提供了关键技术。