Antagonism between entomopathogenic fungi and bacterial symbionts of entomopathogenic nematodes

Mutual effects between the symbiotic bacteria of entomopathogenic nematodes, Photorhabdus luminescens and Xenorhabdus poinarii, and entomopathogenic fungi were investigated in vitro. A dual culture assay on nutrient agar supplemented with bromothymol blue and triphenyltetrazolium chloride (NBTA) medium revealed that P. luminescens is antagonistic to Metarhizium anisopliae, Beauveria bassiana, B. brongniartii and Paecilomyces fumosoroseus by inhibiting their growth and conidial production; the fungal growth was not inhibited by X. poinarii. In a second laboratory experiment, crude extract produced by M. anisopliae was tested for its activity against P. luminescens and X. poinarii. Crude extract from M. anisopliae was antibacterial to P. luminescens and X. poinarii at 1000 g/ml and inhibited their growth on NBTA, but had no effect at 100 or 10 g/ml. The influence of the crude extract of M. anisopliae on the dispersal of infective juveniles (IJs) of Heterorhabditis megidis and Steinernema glaseri was assayed on Sabouraud Dextrose Agar (SDA) plates. Results showed that the crude extract of M. anisopliae had no toxic effects even at highest concentration (1000 g/ml).     

Production technology for entomopathogenic nematodes and their bacterial symbionts

Entomopathogenic nematodes (genera Steinernema and Heterorhabditis) kill insects with the aid of mutualistic bacteria. The nematode–bacteria complex is mass produced for use as biopesticides using in vivo or in vitro methods, i.e., solid or liquid fermentation. In vivo production (culture in live insect hosts) is low technology, has low startup costs, and resulting nematode quality is high, yet cost efficiency is low. In vitro solid culture, i.e., growing the nematodes and bacteria on crumbled polyurethane foam, offers an intermediate level of technology and costs. In vivo production and solid culture may be improved through innovations in mechanization and streamlining. In vitro liquid culture is the most cost-efficient production method but requires the largest startup capital and nematode quality may be reduced. Liquid culture may be improved through progress in media development, nematode recovery, and bioreactor design. A variety of formulations is available to facilitate nematode storage and application. Journal of Industrial Microbiology & Biotechnology (2002) 28, 137–146 DOI: 10.1038/sj/jim/7000230 Received 16 August 2001/ Accepted in revised form 10 November 2001     

A survival-reproduction trade-off in entomopathogenic nematodes mediated by their bacterial symbionts

In this work, we investigate the investment of entomopathogenic Steinernema nematodes (Rhabditidae) in their symbiotic association with Xenorhabdus bacteria (Enterobacteriaceae). Their life cycle comprises two phases: (1) a free stage in the soil, where infective juveniles (IJs) of the nematode carry bacteria in a digestive vesicle and search for insect hosts, and (2) a parasitic stage into the insect where bacterial multiplication, nematode reproduction, and production of new IJs occur. Previous studies clearly showed benefits to the association for the nematode during the parasitic stage, but preliminary data suggest the existence of costs to the association for the nematode in free stage. IJs deprived from their bacteria indeed survive longer than symbiotic ones. Here we show that those bacteria-linked costs and benefits lead to a trade-off between fitness traits of the symbiotic nematodes. Indeed IJs mortality positively correlates with their parasitic success in the insect host for symbiotic IJs and not for aposymbiotic ones. Moreover mortality and parasitic success both positively correlate with the number of bacteria carried per IJ, indicating that the trade-off is induced by symbiosis. Finally, the trade-off intensity depends on parental effects and, more generally, is greater under restrictive environmental conditions.     

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.     

Diversity and distribution of entomopathogenic nematodes (Nematoda: Steinernematidae, Heterorhabditidae) and their bacterial symbionts (gamma-Proteobacteria: Enterobacteriaceae) in Jordan

Until now, only a few systematic surveys of entomopathogenic nematodes (EPN) have been conducted in Middle Eastern countries. Many of the recovered EPN species in this region have shown to own distinctive qualities that enable their survival in unique environments, such as high temperatures and low moisture levels tolerance. These new species and strains, with unique environmental tolerances, are more suitable for their consideration in pest management programs in xerophytic regions. With this background in mind, we recently conducted a survey of EPN in Jordan. This study records for the first time the diversity and distribution of these nematodes and their bacterial symbionts in this country. Jordan’s three geographic regions: (1) the highlands, (2) Jordan valley and (3) the desert region were sampled. Within each region, natural habitats and agricultural regions characteristic to each region were considered for sampling purposes. Four EPN species including three Steinernema and one Heterorhabditis were recovered. Nematodes were identified using a combination of molecular markers and classic morphological diagnostic tools. Bacterial symbionts were identified by analysis of 16S rRNA sequences. Abiotic characteristics such as soil type, soil pH, and elevation were also recorded. We herein report the diversity of EPN species in Jordan and discuss their potential in Biocontrol and IPM programs for this country.     

Biochemical and physiological characteristics of Xenorhabdus species,symbiotically associated with entomopathogenic nematodes including Steinernema kushidai and their pathogenicity against Spodoptera litura (Lepidoptera: Noctuidae)

The symbiotic bacterium strain, SK-1 isolated from Steinernema kushidai, a new species of entomopathogenic nematode, was compared with other strains of Xenorhabdus species. Like other Xenorhabdus nematophilus strains, this new strain is gram-negative, facultatively anaerobic, peritrichously flagellated rod and negative for catalase and nitrate reduction. It can be distinguished from the other Xenorhabdus spp. by differences in reactions to phenylalanine deaminase, no acid production from myo-inositol and utilizations of inosine, dl-malate, formate and methanol. Intra-haemocoelic injection of actual cells or liquid culture supernatant into sixth instar larvae of Spodoptera litura for either Phase I or II variants were not pathogenic. Other strains of X. nematophilus showed pathogenicity for whole cell injections. The supernatants of strain D-1 and ATCC 19061, which are symbionts of Steinernema carpocapsae were pathogenic, however pathogenicity decreased and then terminated by increases in temperature.     

Phenotypic variation and host interactions of Xenorhabdus bovienii SS-2004, the entomopathogenic symbiont of Steinernema jollieti nematodes

Xenorhabdus bovienii (SS-2004) bacteria reside in the intestine of the infective-juvenile (IJ) stage of the entomopathogenic nematode, Steinernema jollieti. The recent sequencing of the X. bovienii genome facilitates its use as a model to understand host - symbiont interactions. To provide a biological foundation for such studies, we characterized X. bovienii in vitro and host interaction phenotypes. Within the nematode host X. bovienii was contained within a membrane bound envelope that also enclosed the nematode-derived intravesicular structure. Steinernema jollieti nematodes cultivated on mixed lawns of X. bovienii expressing green or DsRed fluorescent proteins were predominantly colonized by one or the other strain, suggesting the colonizing population is founded by a few cells. Xenorhabdus bovienii exhibits phenotypic variation between orange-pigmented primary form and cream-pigmented secondary form. Each form can colonize IJ nematodes when cultured in vitro on agar. However, IJs did not develop or emerge from Galleria mellonella insects infected with secondary form. Unlike primary-form infected insects that were soft and flexible, secondary-form infected insects retained a rigid exoskeleton structure. Xenorhabdus bovienii primary and secondary form isolates are virulent towards Manduca sexta and several other insects. However, primary form stocks present attenuated virulence, suggesting that X. bovienii, like Xenorhabdus nematophila may undergo virulence modulation.     

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.     

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。     

Phenotypic characterization of the Xenorhabdus bacterial symbiont of a Texas strain of the entomopathogenic nematode Steinernema riobrave, and characterization of the Xenorhabdus bovienii bacterial symbiont of a Newfoundland strain of Steinernema feltiae

Two bacterial symbionts of entomopathogenic nematodes, one of which originated from Texas, U.S.A., and the other from Newfoundland, Canada, were characterized phenotypically. These strains belonged to the genus Xenorhabdus. The Newfoundland (NF) strain was shown to be X. bovienii but the Texas (TX) strain was not identified at the species level. Four additional cultures of Xenorhabdus were included in the study. These were a strain of X. bovienii (Ume?), which was from a nematode of European origin, and strains of X. nematophilus, X. beddingii, and X. poinarii. The tests used in this study indicated identical properties for the NF (North American) and Ume? (European) strains of X. bovienii. These could be differentiated from the other strains studied by their failure to grow at 34 degrees C and resistance to low concentrations of a mixture of amoxilline and clavulanic acid. The Xenorhabdus TX strain could be differentiated from the other strains studied by its failure to grow at 10 degrees C. Of the tests done, approximately 30 were useful in distinguishing between the strains and species studied.     

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.     

Molecular interactions between bacterial symbionts and their hosts

Symbiotic bacteria are important in animal hosts, but have been largely overlooked as they have proved difficult to culture in the laboratory. Approaches such as comparative genomics and real-time PCR have provided insights into the molecular mechanisms that underpin symbiont-host interactions. Studies on the heritable symbionts of insects have yielded valuable information about how bacteria infect host cells, avoid immune responses, and manipulate host physiology. Furthermore, some symbionts use many of the same mechanisms as pathogens to infect hosts and evade immune responses. Here we discuss what is currently known about the interactions between bacterial symbionts and their hosts.     

Temperature effects on Korean entomopathogenic nematodes, Steinernema glaseri and S. longicaudum, and their symbiotic bacteria

We investigated the temperature effects on the virulence, development, reproduction, and motility of two Korean isolates of entomopathogenic nematodes, Steinernema glaseri Dongrae strain and S. longicaudum Nonsan strain. In addition, we studied the growth and virulence of their respective symbiotic bacterium, Xenorhabdus poinarii for S. glaseri and Xenorhabdus sp. for S. longicaudum, in an insect host at different temperatures. Insects infected with the nematode-bacterium complex or the symbiotic bacterium was placed at 13 degrees C, 18 degrees C, 24 degrees C, 30 degrees C, or 35 degrees C in the dark and the various parameters were monitored. Both nematode species caused mortality at all temperatures tested, with higher mortalities occurring at temperatures between 24 degrees C and 30 degrees C. However, S. longicaudum was better adapted to cold temperatures and caused higher mortality at 18 degrees C than S. glaseri. Both nematode species developed to adult at all temperatures, but no progeny production occurred at 13 degrees C or 35 degrees C. For S. glaseri, nematode progeny production was best at inocula levels above 20 infective juveniles/host at 24 degrees C and 30 degrees C, but for S. longicaudum, progeny production was generally better at 24 degrees C. Steinernema glaseri showed the greatest motility at 30 degrees C, whereas S. longicaudum showed good motility at 24 degrees C and 30 degrees C. Both bacterial species grew at all tested temperatures, but Xenorhabdus sp. was more virulent at low temperatures (13 degrees C and 18 degrees C) than X poinarii.     

Morphology and ultrastructure of the bacterial receptacle in Steinernema nematodes (Nematoda: Steinernematidae)

Infective juveniles of entomopathogenic nematodes in the genus Steinernema harbor symbiotic bacteria, Xenorhabdus spp., in a discrete structure located in the anterior portion of the intestine known as the 'bacterial receptacle' (formerly known as the bacterial or intestinal vesicle). The receptacle itself is a structured environment in which the bacteria are spatially restricted. Inside this receptacle, bacterial symbionts are protected from the environment and grow to fill the receptacle. Until now, no comparative study across different Steinernema spp. has been undertaken to investigate if morphological variation in this structure exists at the interspecific level. In this study, we examined the bacterial receptacles of 25 Steinernema spp. representatives of the currently accepted five evolutionary clades. Our observations confirmed the bacterial receptacle is a modification of the two most anterior cells of the ventricular portion of the intestine. Size of the bacterial receptacle varied across the examined species. Steinernema monticolum (clade II) had the largest receptacle of all examined species (average: 46×17 μm) and S. rarum (no clade affiliation) was noted as the species with the smallest observed receptacle (average: 8×5 μm). At the morphological level, species can be grouped into two categories based on the presence or absence of vesicle within the receptacle. The receptacles of all examined species harbored an intravesicular structure (IVS) with variable morphology. All examined taxa members of the 'feltiae' (clade III) and 'intermedium' (clade II) clades were characterized by having a vesicle. This structure was also observed in S. diaprepesi (clade V), S. riobrave (clade IV) and S. monticolum (clade I).     

Interaction between Steinernema carpocapsae with the arthropode fauna and natural populations of entomopathogenic nematodes in orchard biotope

The introduction of a novel entomopathogenis nematode Steinernema carpocapsae strain "agriotos" (Rhabditida: Steinernematidae) into the soil of an orchard resulted in the reduction (up to 50 %) of total amount of phytophagous insects. No negative effect on the groups of beneficial arthropods, caused by the nematode, has been found. Recommended optimal application rate is 500 thousand invasive nematode larvae per 1 m2 of the soil. Increase or decrease of the application rate resulted in the rise of the abundance of phytophagous insects. This fact proved the existence of regulating factors determining optimal ratios of the amounts of parasites at micro- and macro-levels. Activation of native populations of entomopathogenic nematodes in soil surface layer has been observed after the introduction of the novel parasite species.     

Antimicrobial activity of Xenorhabdus sp. RIO (Enterobacteriaceae), symbiont of the entomopathogenic nematode,Steinernema riobrave (Rhabditida: Steinernematidae)

Galleria mellonella larvae infected with Steinernema riobrave soon showed (after 24 h) the typical growth of its Xenorhabdus sp. RIO symbiont and, in parallel, the growth of another Gram negative bacterial species in the body cavity. A population of Entercoccus sp. in the nematode infected larvae collapsed to zero by 96 h. The level of antibiotic and antimycotic activity followed a pattern similar to that of the growth curve to stationary phase of the Xenorhabdus sp. RIO symbiont, over a period of 168 h. The antimycotic activity was composed of exo- and endochitinases as well as other proteinaceous and some small molecule compounds. The changing pH, relatively high growth rate of Xenorhabdus sp. RIO compared with that of other Gram negative bacterial species and of collapse of the Enterococcus sp. population enabled Xenorhabdus sp. RIO to out-compete other species.     

Isolation and molecular characterization of five entomopathogenic nematode species and their bacterial symbionts from eastern Australia

BioControl - Entomopathogenic nematodes (EPNs) are used in biological control of pest insects but their potential may be limited by strain availability from different bioregions and effectiveness...