Entomopathogenic Nematodes Heterorhabditis Megidis Research Articles

Immunosuppression mechanism of entomopathogenic bacteria against Galleria mellonella larvae

Abstract Entomopathogenic nematodes (EPNs) and their symbiotic bacteria play a great role in pest bio-control and therefore have been used widely in agricultural production. However, immunologic mechanisms on how they kill their hosts remain little known. Here we investigated the response of cellular immunity to infection by Enterobacter gergoviae LY2W, which is entomopathogenic bacterium of EPN Heterorhabditis megidis 90. Hemocytes started distortion and disintegration after 12 h with a decrease of hemolymph by 34.9%. More interestingly, hemocyte density decreased dramatically to less than 35% of the control at 12 h before larva death. Moreover, the expression of genes coding diptericin and drosomycin responsible for the signaling regulation of humoral immunity indicated that LY2W cells promoted the signaling regulation of humoral immunity mainly by activating Toll and Imd pathways. Therefore, immunosuppression of entomopathogenic bacteria against G. mellonella depended mainly upon disordering the signaling regulation of humoral immunity, destroying hemocyte, and decreasing hemolymph.

Diffusion of the maize root signal (E)-β-caryophyllene in soils of different textures and the effects on the migration of the entomopathogenic nematode Heterorhabditis megidis

Maize roots respond to feeding by larvae of the beetle Diabrotica virgifera virgifera by releasing (E)-β-caryophyllene (Eβc). This insect-induced root volatile attracts entomopathogenic nematodes (EPN) and thereby helps to protect the roots against herbivore damage. Previous studies suggest that diffusion of Eβc occurs through the gaseous rather than the aqueous phase in sand and its diffusion is best at low levels of humidity. However, it remains largely unknown how Eβc diffuses in typical natural and agricultural soils. To fully understand the function and efficiency of root-produced Eβc as a belowground signal it is important to know how it spreads in real soils and how soil properties affect its diffusion. Using gas chromatography-mass spectrometry analyses, the diffusion of two doses of synthetic Eβc (200 ng and 20,000 ng) injected in sand was compared with the diffusion of Eβc injected in clay, clay-loam and sandy-loam soils, at 3 moisture levels (5, 10 and 20% water), and at two distances (5 and 10cm) from the Eβc injection point. The diffusion of the compound was measured with a Solid Phase Micro Extraction (SPME) fiber every 30minutes over a period of 9 hours. We found that, in contrast to the pattern observed for pure sand, Eβc diffused best when humidity was high in the three agricultural soils. In subsequent experiments we used glass-trays to create two types of mesocosms to assess the effect of synthetic or root-produced Eβc on the dispersal of the EPN Heterorhabditis megidis and its infection of sentinel hosts in the trays. The presence of synthetic Eβc did not affect the ability of H. megidis to infect the sentinel host. However, under the test conditions, Eβc released from maize roots influenced the migration behaviour of H. megidis depending on soil type. The results suggest that D. virgifera-damaged maize plants may recruit H. megidis more efficiently in clay loam soils than in other types of soil. These new insights into the diffusion dynamics and attraction efficiency of the root-produced signal Eβc may help efforts to develop novel strategies for the sustainable management of the maize pest D. virgifera.

Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella

The entomopathogenic nematode Heterorhabditis spp. is considered a promising agent in the biocontrol of injurious insects of agriculture. However, different symbiotic bacteria associated with the nematode usually have different specificity and virulence toward their own host. In this study, two symbiotic bacteria, LY2W and NK, were isolated from the intestinal canals of two entomopathogenic nematode Heterorhabditis megidis 90 (PDSj1 and PDSj2) from Galleria mellonela, separately. To determine their species classification, we carried out some investigations on morphology, culture, biochemistry, especially 16S rDNA sequence analyses. As a result, both of them belong to Enterobacter spp., showing the closest relatedness with Enterobacter gergoviae (LY2W) and Enterobacter cloacae (NK), respectively. Moreover, the toxicity to Galleria mellonella was examined using both the metabolites and washed cells (primary and secondary) of these two strains. The results indicated both metabolites and cells of the primary-type bacteria could cause high mortalities (up to 97%) to Galleria mellonella, while those of the primary-type bacteria only killed 20%. These findings would provide new symbiotic bacteria and further references for biological control of the agricultural pest.

Highly Potent Extracts from Pea (Pisum sativum) and Maize (Zea mays) Roots Can Be Used to Induce Quiescence in Entomopathogenic Nematodes.

Root exudates can play an important role in plant-nematode interactions. Recent studies have shown that the root cap exudates obtained from several plant species trigger a state of dormancy or quiescence in various genera of nematodes. This phenomenon is not only of fundamental ecological interest, but also has application potential if the plant-produced compound(s) could be used to control harmful nematodes or help to prolong the shelf-life of beneficial entomopathogenic nematodes (EPNs). The identification of the compound(s) involved in quiescence induction has proven to be a major challenge and requires large amounts of active material. Here, we present a high-throughput method to obtain bioactive root extracts from flash-frozen root caps of green pea and maize. The root cap extract obtained via this method was considerably more potent in inducing quiescence than exudate obtained by a previously used method, and consistently induced quiescence in the EPN Heterorhabditis megidis, even after a 30-fold dilution. Extracts obtained from the rest of the root were equally effective in inducing quiescence. Infective juveniles (IJs) of H. megidis exposed to these extracts readily recovered from their quiescent state as soon as they were placed in moist soil, and they were at least as infectious as the IJs that had been stored in water. Excessive exposure of IJs to air interfered with the triggering of quiescence. The implications of these results and the next steps towards identification of the quiescence-inducing compound(s) are discussed from the perspective of applying EPN against soil-dwelling insect pests.

The role of root architecture in foraging behavior of entomopathogenic nematodes

As obligate parasites, entomopathogenic nematodes (EPN) rely on insect hosts to complete their development. In insect pest management, EPN infectiousness has varied a lot. A better understanding of their host-finding behavior in the rhizosphere is therefore crucial to enhance EPN potential in biological control. As previously demonstrated, roots can be used as a pathway to insect hosts by EPN, but this interaction and its impact on EPN foraging remain poorly documented. Three artificial model-roots with different degrees of complexity and connectivity were designed to investigate the impact of root architecture on foraging behavior of the EPN Heterorhabditis megidis. Insect baits were placed at the bottom of each model-root that was subsequently buried in moist sand. After injection of the EPN, the number of EPN-infected baits as well as the number of mature nematodes inside each individual carcass was recorded. The influence of insect-induced root volatiles was also evaluated by spiking the baits with a synthetic version of a natural insect-induced root cue. The ecological relevance of the results was tested in soil with two maize genotypes each exhibiting broadly different root architectures. H. megidi performed better in presence of model-roots. Foraging performances of H. megidis declined with the increasing model-root complexity. Adding the synthetic root volatile dramatically changed this pattern and favored the EPN on the most complex model-roots. H. megidis also moved in the vicinity of maize roots to find the insect baits in soil, and natural root architecture also tended to shape H. megidis foraging behavior. This study adds to the scarce body of literature characterizing physical and chemical interactions between EPN and roots. The present data illustrate that root architecture not only modifies plant quality but also shapes upper trophic levels’ ecology.

Oral toxicity of Photorhabdus culture media on gene expression of the adult sweetpotato whitefly, Bemisia tabaci

The oral toxicity of culture media of the symbiotic bacteria, Photorhabdus temperata, mutually associated with entomopathogenic nematode Heterorhabditis megidis and Photorhabdus luminescens ssp. laumondii (TT01) mutually associated with Heterorhabditis bacteriophora, were investigated in the adults of Bemisia tabaci. The oral ingestion of sucrose diet solutions (20%) containing bacteria-free supernatant of the culture media from symbiotic bacteria gradually increased mortalities and was completely lethal at 60h after the treatments, whereas the mortalities of the controls, sucrose solutions with or without media that uncultured with bacteria, were less than 17% up to 84h of incubation. The effects of oral ingestion of symbiont culture media were demonstrated on the expression rates of several genes of B. tabaci using quantitative real-time RT-PCR analysis. Genes associated with immunity (knottin) and nervous system (acetylcholine receptor, acetylcholine esterase and sodium channel) were up-regulated while genes involved in metabolism (cytochromep450 and carboxylesterase) were down-regulated, but genes involved in development (ecdysone receptor), reproduction (vitellogenin) and stress (hsp70, hsp90 and shsp) did not change transcription rates. Our results provide information for the understanding of the mechanism of symbiont pathogenic factors for the manipulation of host physiology at the transcription level.

Systemic root signalling in a belowground, volatile‐mediated tritrophic interaction

Plants attacked by leaf herbivores release volatile organic compounds (VOCs) both locally from the wounded site and systemically from non-attacked tissues. These volatiles serve as attractants for predators and parasitoids. This phenomenon is well described for plant leaves, but systemic induction of VOCs in the roots has remained unstudied. We assessed the spatial and temporal activation of the synthesis and release of (E)-β-caryophyllene (EβC) in maize roots upon feeding by larvae of Diabrotica virgifera virgifera, as well as the importance of systemically produced EβC for the attraction of the entomopathogenic nematode Heterorhabditis megidis. The production of EβC was found to be significantly stronger at the site of attack than in non-attacked tissues. A weak, but significant, increase in transcriptional activity of the EβC synthase gene tps23 and a corresponding increase in EβC content were observed in the roots above the feeding site and in adjacent roots, demonstrating for the first time that herbivory triggers systemic production of a volatile within root systems. In belowground olfactometers, the nematodes were significantly more attracted towards local feeding sites than systemically induced roots. The possible advantages and disadvantages of systemic volatile signalling in roots are discussed.

Belowground Chemical Signaling in Maize: When Simplicity Rhymes with Efficiency

Maize roots respond to feeding by larvae of the beetle Diabrotica virgifera virgifera by releasing (E)-beta-caryophyllene. This sesquiterpene, which is not found in healthy maize roots, attracts the entomopathogenic nematode Heterorhabditis megidis. In sharp contrast to the emission of virtually only this single compound by damaged roots, maize leaves emit a blend of numerous volatile organic compounds in response to herbivory. To try to explain this difference between roots and leaves, we studied the diffusion properties of various maize volatiles in sand and soil. The best diffusing compounds were found to be terpenes. Only one other sesquiterpene known for maize, alpha-copaene, diffused better than (E)-beta-caryophyllene, but biosynthesis of the former is far more costly for the plant than the latter. The diffusion of (E)-beta-caryophyllene occurs through the gaseous rather than the aqueous phase, as it was found to diffuse faster and further at low moisture level. However, a water layer is needed to prevent complete loss through vertical diffusion, as was found for totally dry sand. Hence, it appears that maize has adapted to emit a readily diffusing and cost-effective belowground signal from its insect-damaged roots.

Simultaneous feeding by aboveground and belowground herbivores attenuates plant‐mediated attraction of their respective natural enemies

Herbivore-damaged plants emit volatile organic compounds that attract natural enemies of the herbivores. This form of indirect plant defence occurs aboveground as well as belowground, but it remains unclear how simultaneous feeding by different herbivores attacking leaves and roots may affect the production of the respective defence signals. We employed a setup that combines trapping of volatile organic signals and simultaneous measurements of the attractiveness of these signals to above and belowground natural enemies. Young maize plants were infested with either the foliar herbivore Spodoptera littoralis, the root herbivore Diabrotica virgifera virgifera, or with both these important pest insects. The parasitic wasp Cotesia marginiventris and the entomopathogenic nematode Heterorhabditis megidis were strongly attracted if their respective host was feeding on a plant, but this attraction was significantly reduced if both herbivores were on a plant. The emission of the principal root attractant was indeed reduced due to double infestation, but this was not evident for the leaf volatiles. The parasitoid showed an ability to learn the differences in odour emissions and increased its response to the odour of a doubly infested plant after experiencing this odour during an encounter with hosts. This first study to measure effects of belowground herbivory on aboveground tritrophic signalling and vice-versa reemphasizes the important role of plants in bridging interactions between spatially distinct components of the ecosystem.

Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53

Abstract The white grub, Hoplia philanthus Fuessly (Coleoptera: Scarabaeidae), is a major pest of turf and ornamental plants in Belgium. Previously, the combination of lethal concentration of the entomopathogenic nematodes Heterorhabditis megidis or Steinernema glaseri with the entomopathogenic fungus Metarhizium anisopliae (strain CLO 53) caused additive or synergistic mortality to third-instar H. philanthus in the laboratory and greenhouse. In this present study, we examined this interaction under field conditions and compared a combination of a commercial formulation of Heterorhabditis bacteriophora (Nema-green ® ) and M. anisopliae . Controls were M. anisopliae , chlorpyrifos (Dursban 5 Granules) and H. bacteriophora . Field applications (surface or subsurface) were made against a mixed population of second/third-instar H. philanthus at a sport field and lawn infested in the province of West-Flanders. In both trials, the combination of M. anisopliae with H. bacteriophora at 5 × 10 12 conidia/ha +2.5 × 10 9 infective juveniles/ha resulted in additive or synergistic effects, causing more than 95% grub mortality when the nematodes was applied 4 weeks after the application of fungus. However, application of nematode, chlorpyrifos or fungus alone provided 39–66%, 42–60% (surface) and 33–76%, 82–100% or 37–65%, (subsurface) control of H. philanthus . We concluded that the pathogen combinations we tested are compatible elements of integrated pest management and are likely to improve control of H. philanthus larvae and perhaps other insect pests beyond what is expected from single application of the pathogen.

Interaction between Metarhizium anisopliae CLO 53 and entomopathogenic nematodes for the control of Hoplia philanthus

Abstract The interaction between the entomopathogenic fungus Metarhizium anisopliae CLO 53 and the entomopathogenic nematodes Heterorhabditis megidis and Steinernema glaseri against third-instar Hoplia philanthus was studied in a series of laboratory and greenhouse experiments. Insect larvae were exposed to various concentrations of M. anisopliae and nematodes and larval mortality was assessed weekly. Nematodes were added simultaneously or 2, 3, and 4 weeks after application of M. anisopliae . Throughout the experiments, the combined application of M. anisopliae with nematodes increased larval mortality either in an additive or a synergistic way. To achieve stronger synergistic effects, larvae had to be exposed to M. anisopliae for at least 3 or 4 weeks before the addition of nematodes. We observed this interaction between M. anisopliae and both nematode species. The nematode reproduction in insect larvae exposed to the M. anisopliae – H. megidis combination was not significantly higher than in larvae exposed to only H. megidis . The combination of higher concentrations of M. anisopliae with H. megidis resulted in an antagonistic effect on nematode reproduction. In the greenhouse trial, third-instar H. philanthus were placed in 3-L pots with perennial ryegrass ( Lolium perenne ) treated with M. anisopliae , the nematode H. megidis or S. glaseri , or the combination of fungus and nematode. Combinations of M. anisopliae and nematodes generated a strong synergistic effect only at higher concentrations (2 × 10 12 and 2 × 10 13 conidia/ha). This effect was observed when M. anisopliae was applied first and followed by the nematodes 4 weeks later. The combined use of M. anisopliae with nematodes may offer an integrated approach to increase the efficacy of entomopathogenic nematodes for H. philanthus control and perhaps other insect pests.

Interactions between host plants, the subterranean burrower bug, Cyrtomenus bergi, and the entomopathogenic nematode Heterorhabditis megidis

AbstractInteractions between seedlings of Zea mays L. as a host plant, the subterranean burrower bug, Cyrtomenus bergi Froeschner, as a pest, and Heterorhabditis megidis Poinar, an entomopathogenic nematode, as a biological control agent, were studied, with a particular emphasis on the host finding behaviour of the nematode. For the trials a sand-filled Y-tube olfactometer was used. In the Y-arms, a bug, a maize seedling, a combination of both, or sand only was offered. Six experiments were carried out with differently treated Y-arms. Nematodes were significantly attracted to maize as well as to the combination of maize and bug, but not to C. bergi alone or to sand only. The influence of the feeding strategy of C. bergi on the host finding behaviour of H. megidis is discussed.

Density-dependent fecundity and infective juvenile production in the entomopathogenic nematode, Heterorhabditis megidis.

The entomopathogenic nematode Heterorhabditis megidis may undergo several rounds of reproduction within a single host. Infective juveniles (IJs) are formed within each generation during a process referred to as endotokia matricida, which involves the progressive consumption of the parent hermaphrodite or female by the developing IJs prior to emergence from the host cadaver. The present study examines the extent to which within-host population dynamics exhibit density-dependent variation. Particular attention is paid to the effect of infection density on the relative production of IJs and 'normal', non-infective offspring within each generation and on the emergence of the IJs from the host. Fecundity was found to be negatively density dependent across generations. However, at high infection density the first generation hermaphrodites invested relatively more in IJs at the expense of producing non-infective offspring. It is suggested that this pattern resulted from an adaptive, phenotypically plastic allocation of reproductive investment between offspring types in response to increased competition. The F1 and F2 IJs were also shown to emerge from the host in relatively discrete pulses.

Effect of Timber Condition on Parasitization of Pine Weevil ( Hylobius abietis L.) Larvae by Entomopathogenic Nematodes under Laboratory Conditions

The large pine weevil ( Hylobius abietis L.) is one of the most important pests in coniferous reforestation in Europe. Larvae develop in the stumps of recently felled trees; the emerging adults feed on the bark of seedlings and may kill them. The ability of the entomopathogenic nematodes Heterorhabditis megidis and Steinernema carpocapsae to invade pine weevil larvae in Sitka spruce ( Picea sitchensis ) buried in moist sand was evaluated. Overall, four times as many H. megidis as S. carpocapsae invaded pine weevil larvae. The two species of nematode differed in their response to timber condition. The number of S. carpocapsae invading pine weevil larvae was twice as high in billets inoculated with the wood-rotting fungus Phlebiopsis gigantea as in fresh timber, while the number of H. megidis invading was reduced by 25%. Invasion into non-feeding insects (larvae of the wax moth Galleria mellonella ) contained in timber disks was also affected by timber quality, indicating that nematode behaviour was affected directly by the physical or chemical condition of the timber, though trophically mediated effects may also have been involved.

Influences of host size and host species on the infectivity and development of Heterorhabditis megidis (strain NLH-E 87.3)

The infectivity, time to first emergence of infective juveniles (IJs), total number of IJs per insect and IJs body length of the entomopathogenic nematode Heterorhabditis megidis (strain NLH-E87.3) after development in larvae of two insect hosts, Galleria mellonella (greater wax moth) and Otiorhynchus sulcatus (vine weevil) was studied. At a dose of 30 IJs, larvae of G. mellonella show to be significantly more susceptible than O. sulcatus larvae. At a dose of one IJ, vine weevil larvae were more susceptible. The number of invading infective juveniles (IJs) increased with host size while the host mortality at a dose of one IJ decreased with the increase of host size. Time to first emergence was longer at a dose of one IJ per larva and increased with the increase of host size in both insect species. Reproduction of IJs differed between host species, host sizes and doses of nematodes. Generally, the IJs body size increased with an increasing host size. The longest infective juveniles were produced at the lowest IJ doses. Results are discussed in relation to the influence of different host species and their different sizes on the performance of H. megidis (strain NLH-E87.3) as a biological control agent.

The Effect of Day of Emergence from the Insect Cadaver on the Behavior and Environmental Tolerances of Infective Juveniles of the Entomopathogenic Nematode Heterorhabditis megidis (Strain UK211)

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are obligate parasites of insect larvae. Inside the host they develop into sexually mature adult stages and complete their life cycle. Two or 3 adult nematode generations can occur in the insect host. The increase in nematode population density in the insect cadaver, together with limiting nutrient conditions, result in the formation of IJs. These IJs emerge into the soil to search for a new host. It typically takes 7-8 days for all IJs to emerge from a parasitized insect. We have investigated the effect of the day of emergence of IJs from insect cadavers on the environmental tolerance and behavior of the EPN Heterorhabditis megidis strain UK211. The IJs that emerge early display good initial host-finding ability and increased temperature tolerance but disperse poorly and have poor tolerance to desiccation. Conversely, the IJs that emerge later display poor initial host-finding ability and poor temperature tolerance but they disperse well and possess increased desiccation tolerance. These phenotypic differences are likely to facilitate early-emerging IJs in locating and infecting hosts in the vicinity of the cadaver, whereas IJs that emerge late are adapted to disperse away from their natal cadaver. We hypothesize that adaptive phenotypic plasticity rather than allelic variability may provide the genetic basis for the different physiological and behavioral phenotypes of the early- and late-emerging IJs.