Entomopathogenic Nematode Heterorhabditis Bacteriophora Research Articles

Effects of tannin-rich host plants on the infection and establishment of the entomopathogenic nematode Heterorhabditis bacteriophora

Parasitized animals can self-medicate. As ingested plant phenolics, mainly tannins, reduce strongyle nematode infections in mammalian herbivores. We investigated the effect of plant extracts known to be anthelmintic in vertebrate herbivores on the recovery of the parasitic entomopathogenic nematode Heterorhabditis bacteriophora infecting African cotton leafworm (Spodoptera littoralis). Nematode infective juveniles (IJs) were exposed to 0, 300, 900, 1200, 2400ppm of Pistacia lentiscus L. (lentisk), Inula viscosa L. (strong-smelling inula), Quercus calliprinos Decne. (common oak) and Ceratonia siliqua L. (carob) extracts on growth medium (in vitro assay). In control treatments, 50–80% of IJs resumed development to J4, young and developed adult hermaphrodites, whereas all extracts, except for C. siliqua at 300ppm, impaired IJ exsheathment and development. The highest concentration of I. viscosa extract (2400ppm) had the strongest effect, killing 95% of exposed nematodes. Surviving nematodes did not recover, remaining at the IJ stage. Over the whole cycle, I. viscosa extract inhibited recovery to 25% or less, and did not allow full development to adulthood, whereas 65% of IJs in the control treatment recovered and resumed development, 12% reaching complete maturation within 72h of incubation. When herbivorous S. littoralis larvae were fed with different plant extracts in vivo, I. viscosa had the strongest effect at concentrations above 300ppm, with 90% of insect-invading IJs not developing to parasitic stages, whereas in the control treatment, 85% of IJs resumed development. Exposure to C. siliqua extract also inhibited exsheathment and development of 75% of the IJs. Half of those that resumed development reached full maturation. P. lentiscus and Q. calliprinos extracts also inhibited development of 50% IJs. Our results suggest that H. bacteriophora can be used to study herbal medication against parasites in animals.

Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis.

The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont.

Non‐target effects of insecticides, entomopathogenic fungi and nematodes applied against western corn rootworm larvae in maize

AbstractField studies were conducted in southern Hungary over two field seasons to assess potential non‐target effects of entomopathogenic nematodes (Heterorhabditis bacteriophora), entomopathogenic fungi (Metarhizium anisopliae), clothianidin‐coated seeds and tefluthrin granules applied into the soil against larvae of the chrysomelid Diabrotica virgifera virgifera, one of the major North American and European maize pests. From field gauze cages set up over groups of maize plants, 1944 specimens of non‐target ground‐dwelling or emerging arthropods from 22 taxa were collected. The most abundant taxa belonged to the Diptera, Araneae and Hemiptera. Multivariate statistics revealed significant effects of soil conditions, year and treatments on non‐target species composition. Soil parameters explained 10.5% of the overall variability in species composition. The application of biological control agents or insecticides for the control of Diabrotica larvae, together only slightly but significantly influenced non‐target species composition, explaining 5.6% of the data variability. When pairwise comparisons were conducted, no significant effects on non‐target compositions were detected for the entomopathogenic nematode, the entomopathogenic fungus, tefluthrin or clothianidin. However, clothianidin treatments tended to reduce densities of the beneficial arthropod taxa Coccinellidae, Hymenoptera, Araneae, and particularly Staphylinidae and Chrysopidae, whilst tefluthrin tended to reduce the Coleopteran families. No such trends were apparent for the two biological control agent treatments. Reasons for the small treatment effects on non‐targets may be the generally poor arthropod diversity in soils of intensive field crops such as maize, as well as the application of all agents into relatively narrow soil areas close to maize plants.

Efficacy of commercially available predators, nematodes and fungal entomopathogens for augmentative control of Drosophila suzukii

AbstractThe recent arrival of Drosophila suzukii, an invasive pest of soft‐skinned fruit with a wide host range, has resulted in increased production costs for growers and the need for additional insecticide applications each growing season. There are few effective organic insecticides for D. suzukii, and insecticide use in conventional farms may be disruptive to natural enemies, suggesting a need for effective biological control to combat D. suzukii. Commercially available natural enemies were evaluated for their potential use in augmentative releases, including: the predators Orius insidiosus and Dalotia coriaria; the entomopathogenic fungi Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus; and the entomopathogenic nematodes Heterorhabditis bacteriophora, Steinernema feltiae and S. carpocapsae. This suite of natural enemies was chosen to target D. suzukii adults as well as larvae in hanging or dropped fruit. Of the cultured fungal strains tested, only M. anisopliae significantly decreased D. suzukii survival, but it had low residual activity and no effect on D. suzukii fecundity. O. insidiosus decreased D. suzukii survival in simple laboratory arenas but not on potted blueberries or bagged blueberry branches outdoors. D. coriaria did not decrease D. suzukii survival in infested blueberries in simple laboratory arenas. The nematodes tested showed low infection rates and were not able to affect D. suzukii survival. Although this suite of natural enemies showed limited ability to suppress D. suzukii under the tested conditions, these and related natural enemies are present as part of the endemic natural enemy community in agricultural fields, where they may contribute to D. suzukii suppression.

Attractant Role of Bacterial Bioluminescence of Photorhabdusluminescenson a Galleria mellonella Model

Though the intricate relationship between the entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterial counterpart Photorhabdus luminescensis generally known; the role of bioluminescence produced by the bacterial symbiont is yet to be identified. The objective of this study was to determine if bacterial luminosity plays a crucial role in attraction of larval insect hosts. This study focused on bacterial bioluminescence produced from both in vitro and in vivo culturing of the bacterial symbiont. The obtained results portrays that the average distance between Galleria mellonellalarvae and the bacterial light source (P. luminescens)decreased in a linear fashion as a function of increasing intensities of luminosity; thereby supporting the hypothesis that bioluminescence offers a symbiotic role to attract insect host larvae.

Efficacy of the entomopathogenic nematode Heterorhabditis bacteriophora using different application methods in the presence or absence of a natural enemy

Various application methods for the entomopathogenic nematode Heterorhabditis bacteriophora were evaluated under laboratory and field conditions. Four different methods of applying the infective juveniles (IJs) of the nematode to soil were assessed including (1) insect cadavers (referred to as nematode-infected cadavers), (2) subsurface injection, (3) spraying, and (4) drip irrigation. In the laboratory experiment, except for the control with no nematodes, all treatments showed more than 95% insect mortality of the bait insect (Galleria mellonella) with no significant differences among treatments. The same experimental setup was conducted with the introduction of mites, Sancassania polyphyllae (Acari: Acaridae), which are natural enemies of entomopathogenic nematodes. The treatment groups with mites and the control group without mites showed more than 87% insect mortality and no significant differences were observed. The nematode-infected cadaver application method was further evaluated by using cadavers of different ages containing H. bacteriophora IJs in the presence of mites. Larval mortality of the bait insect was significantly lower when 3- or 6-day-old nematode-insect cadavers were used in the presence of mites. Different application methods were also tested in a corn field. No significant differences were observed among the application methods. Our results indicate that the different application methods had no significant effect on larval mortality, but the presence of mites had a negative effect on the cadaver application when the cadavers were 3 days old but had no significant effect when the cadavers were 6 and 9 days old.

Efficacy of two entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae for control of the leopard moth borer Zeuzera pyrina (Lepidoptera: Cossidae) larvae under laboratory conditions

The biological traits of the entomopathogenic nematodes (EPNs), Steinernema carpocapsae and Heterorhabditis bacteriophora, against the larvae of the leopard moth, Zeuzera pyrina were evaluated in the laboratory. The traits included pathogenicity, penetration potential as well as foraging behaviour. Plate assays were performed using a range of EPN concentrations (5, 10, 20, 50 and 100 infective juveniles (IJs) per larva). The LC50 values for S. carpocapsae and H. bacteriophora were 6.4 and 8.4 IJs larva−1 after 72 h. Both EPN species caused high mortality in branch experiments. Significantly higher mortality rates occurred in the larger larvae after exposure to S. carpocapsae. Both EPN species successfully penetrated the Z. pyrina larvae as well as larvae of Galleria mellonella L. (Lepidoptera: Galleridae).The proportional response of H. bacteriophora to host-associated cues was strongly higher than S. carpocapsae in Petri dishes containing agar 1, 12 and 24 h after EPN application. These results highlight the efficiency of EPNs for the control of Z. pyrina larvae. However, due to the cryptic habitat of Z. pyrina larvae in their galleries in the trees, field trails need to be conducted to further evaluate this potential.

Interactions between entomopathogenic nematodes and imidacloprid for rose sawfly control

Rose sawfly, Arge ochropus (Gmelin), is one of the most important pests of ornamental plants such as roses and wild rose bushes in Northern Iran. We investigated the interactions between the insecticides imidacloprid and the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae as control agents of fifth-instar larvae in the laboratory. The larvae were very susceptible to S. carpocapsae (LC50: 21 infective juvenile per larva) and H. bacteriophora (LC50: 32). Combinations of two imidacloprid rates (LC30 and LC50) and four rates of each nematode species (LC25–LC75) were tested. Combinations with the lower imidacloprid rate except for that with the highest H. bacteriophora rate caused higher mortality than both respective single-agent treatments. In combination with the higher imidacloprid rate, only one combination with H. bacteriophora and two combinations with S. carpocapsae caused higher mortality than both respective single-agent treatments. Interactions were generally stronger at the lower imidacloprid rate and were stronger for S. carpocapsae (synergistic in seven combinations, additive in one) than for H. bacteriophora (synergistic in two, additive in six). Synergistic imidacloprid-S. carpocapsae combinations could be a useful tool for the control of A. ochropus larvae that would simultaneously control other common pests susceptible to imidacloprid.

Activated and inactivated immune responses in Caenorhabditis elegans against Photorhabdus luminescens TT01.

The Gram-negative bacterium Photorhabdus luminescens which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora, has a broad insecticidal and nematicidal activity. The virulence of P. luminescens toward the non-mutualistic nematode Caenorhabditis elegans has not been described. We showed that when fed on P. luminescens, the intestinal cells of C. elegans worms become delicate and some crystal-like structure was developed within the intestinal lumen. Next, we examined the requirement of the p38 mitogen-activated protein kinase (MAPK) and insulin/IGF-1 signaling pathway against P. luminescens. Depletion of pmk-1 by RNAi enhances susceptibility to P. luminescens, and numerous downstream targets regulated by the p38 MAPK pathway were induced when fed on P. luminescens. On the other hand, knockdown of daf-16 has no effects on C. elegans lifespan, but knockdown of daf-2 dramatically increased resistance to P. luminescens in a daf-16-dependent manner. We also revealed one of the daf-2 ligands ins-7 was induced and ins-7 deletion mutant survived longer when fed on P. luminescens. These results suggest the p38 MAPK pathway is activated and required for the host defense against P. luminescens. Insulin/IGF-1 signaling pathway is inactivated by P. luminescens through the overexpression of insulin-like gene.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-274) contains supplementary material, which is available to authorized users.

Comparison of virulence of hybridized entomopathogenic nematode Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) strains and their parents

Yuksek biyolojik mucadele potansiyeline sahip olan Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae) turunun hedef alinan zararli bocek uzerindeki etkinligi farkli iklim ozelliklerine sahip olan bolgelerde degisiklik gostermektedir. Bu calismada; Antalya (H.b. 6), Canakkale (H.b. 876), Kirklareli (H.b. 17), Izmir (H.b. HIZ), Sanliurfa (H.b. HSU) ve Adana (H.b. 10) illerinden izole edilmis olan 6 farkli H. bacteriophora izolati kullanilmistir. Bu izolatlarin etkinlikleri Galleria mellonella (Lepidoptera: Pyralidae)’ nin son donem larvalari uzerinde 6 farkli uygulama dozunda belirlenmistir. En dusuk etkinlik degerine sahip olan izolatin erkek ve disi bireyleri ile denemelerde kullanilan diger tum irklarin erkek ve disi bireyleri in vitro ortam kosullarinda hibritlenerek 10 farkli irk elde edilmistir. Hibridizasyon sonucunda elde edilen irklarin G. mellonella larvalari uzerindeki etkinlikleri ebeveyn irklar ile ayni sekilde belirlenmistir. Calisma sonunda ebeveyn ve hibrit irklara ait olan etkinlik verileri karsilastirilmis ve elde edilen 10 yeni hibrit irkin %70’ inin ebeveynlerine gore yuksek, %30’ unun ise ebeveynlerine gore daha dusuk degerde etkinlik gosterdigi tespit edilmistir. Ayrica hibrit irklarin bocek uzerindeki etkinlik degeri uzerine erkek ve disi bireylerin farkli etkiler gosterdigi de tespit edilmistir. Ebeveynlerine gore yuksek etkinlik degerine sahip olan hibrit irklarin etkinlik degerlerinin %57.1’ inin erkek, %42.9’unun ise disi bireylerin etkisi altinda kaldigi bulunmustur

First report of pathogenicity of entomopathogenic nematodes of the genus Heterorhabditis on partially engorged females of Dermacentor nitens (Acari: Ixodidae)

The aim of this study was to evaluate the effect of different concentrations of the entomopathogenic nematodes (EPNs) Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 on the reproductive biology of partially engorged females of Dermacentor nitens. Four groups were formed, with each group containing 10 females and exposed to concentrations of 0, 75, 300, and 1200 nematodes for each female. This procedure was performed separately for each nematode. The following biological parameters were evaluated: egg mass weight, egg production index, hatching percentage, and percentage of control. H. bacteriophora HP88 at the two highest concentrations (300 and 1200 EPNs/female) caused a reduction (p<0.05) on the egg mass and egg production index. Was noted a significant reduction (p<0.05) in the percentage of hatched in all the treated groups. For H. indica LPP1, all treatments resulted in decreased (p<0.05) values for all the parameters. The percentages of controls obtained at concentrations of 75, 300, and 1200 EPNs/female were 56.3, 89.3, and 98.8 and 77.5, 77.1, and 95.9 for H. bacteriophora HP88 and H. indica LPP1, respectively. Therefore, it is concluded that these nematodes showed pathogenicity toward partially engorged females of D. nitens, thereby negatively affecting the reproductive biology of this tick.

Entomopathogenic nematodes associated with essential oil of Lippia sidoides for control of Rhipicephalus microplus (Acari: Ixodidae)

In vitro effect of the association of the entomopathogenic nematodes Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 with the essential oil of Lippia sidoides on engorged females of Rhipicephalus microplus was assessed. Engorged females with homogeneous weights (p >0.05) were divided into six groups of ten ticks each (each female = an experimental unit). In the treated groups, the ticks were exposed to the oil (40 μl/ml=4%) and nematodes (300 entomopathogenic nematodes (EPNs/tick) separately and also to the nematodes together with the oil, while in the control group, the females were immersed in Tween 80 (3%). All the treatments caused a significant reduction in the egg mass weight (p <0.05). The hatching percentage of larvae was reduced (p <0.05) in the groups treated with H. bacteriophora HP88, H. indica LPP1, and H. bacteriophora HP88 associated with the oil, but in the group treated only with the oil, the hatching of percentage of larvae rate was similar to that of the control group (p >0.05). The percentage of control in the group exposed only to the oil was 50.7%, while in the other treated groups, the percentage of control was greater than 95 %, reaching 100 %in the group treated with H. indica LPP1 associated with the oil. The results of the L. sidoides essential oil in the concentrations tested in association with both H. bacteriophora HP88 and H. indica LPP1 indicate that these combinations can be used to control R. microplus.

Interaction of the Koinobiont ParasitoidMicroplitis rufiventrisof the Cotton Leafworm,Spodoptera littoralis, with Two Entomopathogenic Rhabditids,Heterorhabditis bacteriophoraandSteinernema carpocapsae

Entomopathogenic nematodes are generally considered beneficial organisms. However, they can affect beneficial insects such as parasitoids. The interaction between the entomopathogenic nematodes Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Steinernema carpocapsae Weiser, and the parasitoid Microplitis rufiventris Kokujev (Hymenoptera: Braconidae) was investigated in the laboratory. In non-parasitized hosts, Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) larvae exposed to H. bacteriophora showed a higher percent mortality than those exposed to S. carpocapsae. Both nematodes were able to invade and propagate in non-parasitized S. littoralis larvae and those parasitized by M. rufiventris. Both nematode species reproduced in Microplitis-parasitized hosts, but there was a higher number of nematodes in non-parasitized larvae. S. carpocapsae yielded higher numbers of infective juveniles than H. bacteriophora. Generally, the number of nematodes harvested increased as their host's size increased. The interaction between the nematodes and parasitoid favored the nematodes when the nematodes were inoculated during the parasitoid egg stage or the young parasitoid larvae, thus giving the nematodes a better chance to grow and reproduce, resulting in the death of the parasitoid larvae. Conversely, when the nematodes were inoculated during the late larval instar of the parasitoid, the competition partially favored the wasp, thus giving approximately 50% of the wasps a better chance to develop, emerge, and reproduce, providing evidence that both nematodes and wasps could reproduce in the same host. Egg maturation of female wasps derived from nematode-infected hosts was not significantly different than those from control hosts. The combined application of nematodes and parasitoids may be beneficial if the detrimental effects of the nematodes on the parasitoid could be avoided by precisely timing the application strategies. It is clear that Microplitis larvae and the nematodes share the host larva and engage in a trophic interaction with each other. Intraguild predation is briefly discussed.

Efficacy of soil treatments of entomopathogenic nematodes against the larvae, pupae and adults of Tuta absoluta and their interaction with the insecticides used against this insect

The efficacy of soil treatments of three native entomopathogenic nematodes (Steinernema carpocapsae, S. feltiae and Heterorhabditis bacteriophora) against Tuta absoluta larvae, pupae and adults was determined under laboratory conditions. The effect of three insecticides commonly used against T. absoluta, in the survival, infectivity and reproduction of these nematode strains was also evaluated. When dropped into soil to pupate, soil application of nematodes resulted in a high mortality of larvae: 100, 52.3 and 96.7 % efficacy for S. carpocapsae, S. feltiae and H. bacteriophora respectively. No mortality of pupae was observed and mortality of adults emerging from soil was 79.1 % for S. carpocapsae and 0.5 % for S. feltiae. The insecticides tested had a negligible effect on nematode survival, infectivity and reproduction. No sublethal effects were observed. Infective juveniles that survived to insecticide exposition were able to infect Galleria larvae with no significant differences from the control. The Galleria larvae affected by the three insecticides tested served as suitable hosts for the infection and reproduction of the nematodes. These results suggest that larvae of T. absoluta, falling from leaves following insecticide application, could be suitable hosts for nematodes, thereby increasing their concentration and persistence in the soil.

Direct and Indirect Plant Defenses are not Suppressed by Endosymbionts of a Specialist Root Herbivore

Insect endosymbionts influence many important metabolic and developmental processes of their host. It has been speculated that they may also help to manipulate and suppress plant defenses to the benefit of herbivores. Recently, endosymbionts of the root herbivore Diabrotica virgifera virgifera have been reported to suppress the induction of defensive transcripts in maize roots, which may explain the finding of another study that once attacked plants become more susceptible to subsequent D. v. virgifera attack. To test this hypothesis, we cured D. v. virgifera from its major endosymbiont Wolbachia and tested whether endosymbiont-free individuals elicit different defense responses in maize roots. The presence of Wolbachia did not alter the induction of defense marker genes and resistance in a susceptible maize hybrid and a resistant line. Furthermore, attacked maize plants emitted the same amount of (E)-β-caryophyllene, a volatile signal that serves as foraging cue for both entomopathogenic nematodes and D. v. virgifera. Finally, the effectiveness of the entomopathogenic nematode Heterorhabditis bacteriophora to infest D. v. virgifera was not changed by curing the larvae from their endosymbionts. These results show that the defense mechanisms of maize are not affected by Wolbachia. Consequently, D. v. virgifera does not seem to derive any plant-defense mediated benefits from its major endosymbiont.

Survival and Infectivity of the Insect-Parasitic Nematode Heterorhabditis bacteriophora Poinar in Solutions Containing Four Different Turfgrass Soil Surfactants.

This laboratory study examined viability and infectivity of the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora Poinar in solutions containing four different turfgrass soil surfactants: Revolution (Aquatrols Corp., Paulsboro, NJ), Aqueduct (Aquatrols Corp., Paulsboro, NJ), Cascade Plus (Precision Laboratories Inc., Waukegan, IL) and OARS (Aqua-Aid Inc., Rocky Mount, NC). Infective juvenile H. bacteriophora were added to solutions containing each of the four surfactants, and nematode viability and infectivity were monitored over time. In one of two trials, nematode survival in solutions containing the surfactants Aqueduct and Cascade Plus was consistently higher compared to the water control and solutions containing Revolution or OARS. Surfactants had no significant influence on nematode infectivity in either trial. Findings indicate that most of the common turfgrass soil surfactants examined should be compatible with EPNs and that some may potentially enhance nematode survival. Results also imply that tank-mixing of EPNs with most turfgrass soil surfactants should not pose a significant risk to the nematodes. The influence of soil surfactants on EPN performance remains to be examined in the field.

Genetic improvement of the biological control nematode Heterorhabditis bacteriophora (Rhabditidomorpha: Heterorhabditidae): heterosis effect enhances desiccation but not heat tolerance

The entomopathogenic nematode Heterorhabditis bacteriophora is commercially used in biological control of soil dwelling insect pests. It reproduces by autogamy (hermaphrodites) enabling the production of inbred lines, but also by amphimixis (through mating of male and female) which allows cross-breeding. When H. bacteriophora is produced in liquid culture, copulation of male and female is prevented and reproduction is solely by self-fertilisation of hermaphrodites. When reared in insects, crosses are possible resulting in heterozygous offspring. Heat and desiccation tolerance of these nematodes have been successfully improved by selective breeding. Trait deterioration was prevented by producing homozygous inbreds through consecutive reproduction in liquid culture, the method also used for commercial mass production. In this study, we investigated possible heterosis effects in desiccation and heat tolerance after cross-breeding of homozygous inbred lines of H. bacteriophora. Increased desiccation tolerance of the heterozygous progeny in comparison to homozygous inbred lines was recorded indicating that heterosis is a possible means for further improvement of this trait. In contrast, the heat tolerance of the heterozygous offspring was lower than that of the homozygous population. The results provide evidence for the tremendous potential of classical genetics to improve beneficial traits of a biological control agent and carry domestication of H. bacteriophora a significant step forward.

Immune gene transcription in Drosophila adult flies infected by entomopathogenic nematodes and their mutualistic bacteria

Despite impressive advances in the broad field of insect innate immunity, our understanding of the molecular basis of insect immune responses to nematode infections remains incomplete. Here we have investigated the transcriptional induction of immune pathway genes in the fruit fly Drosophila melanogaster upon infection with the entomopathogenic (or insect pathogenic) nematodes Heterorhabditis bacteriophora and their mutualistic bacteria Photorhabdus luminescens, either collectively or separately. We show that in most cases, infection of wild-type adult flies with Heterorhabditis nematodes carrying or lacking mutualistic Photorhabdus bacteria results in the up-regulation of genes in the Toll, Imd, JAK/STAT, JNK and TGF-beta pathways. We also find that direct injection of Photorhabdus bacteria into flies fails to induce the transcription of antimicrobial peptide genes and stress-related genes in Drosophila. These results suggest that Heterorhabditis nematodes and their associated Photorhabdus bacteria employ distinct strategies to evade the Drosophila immune response and establish infection.

Effects of NeemAzal-U on survival, host infestation and reproduction of entomopathogenic and plant-parasitic nematodes: Heterorhabditis bacteriophora and Meloidogyne incognita

To determine the effects of neem active ingredients (azadirachtin) on the entomopathogenic nematode Heterorhabditis bacteriophora and the plant-parasitic nematode Meloidogyne incognita, the nematodes were separately treated with different concentrations of NeemAzal-U (i.e., 0.06%, 0.3% and 0.6%) and its blank formulation. Direct exposure of both nematode species to NeemAzal-U for 14 days caused 14% (control mortality = 6.5%) and 44.5% (control mortality = 14.3% ) mortality of H. bacteriophora and M. incognita, respectively. Heterorhabditis bacteriophora kept either in different NeemAzal-U solutions or water did not differ in their parasitisation efficiency of wax moth larvae Galleria mellonella. However, infection rate of M. incognita decreased with increasing neem concentration and a reduction of gall formation of up to 88% was recorded with the highest neem concentration. The number of infective juveniles (IJs) of H. bacteriophora produced per Galleria larva was not influenced by neem treatments of IJs used for host infection. In contrast, the highest neem concentration reduced hatching (85%) and increased mortality (68%) of M. incognita juveniles compared with the water control. It can be concluded that NeemAzal-U can be applied together with ento-mopathogenic nematodes for pest control without detrimental impact on the beneficial nematodes. Furthermore, NeemAzal-U can contribute to the control of M. incognita but only if high concentrations are used.

Evaluation of entomopathogenic fungi and a nematode against the soil‐dwelling stages of the crane fly Tipula paludosa

Larvae of the crane fly Tipula paludosa (Diptera: Nematocera) are economically important pests of grasslands, nurseries and organically grown crops. This study was conducted to evaluate several entomopathogenic fungal strains, the entomopathogenic nematode Heterorhabditis bacteriophora and the organophosphate insecticide Dursban WG (75% chlorpyrifos) against larval stages of T. paludosa under laboratory and greenhouse conditions. Metarhizium robertsii strain V1005 was the most virulent, causing 100% larval mortality 4 weeks post-inoculation. Eight other M. robertsii strains and M. brunneum (ARSEF 3297) caused mortality ranging between 0 and 60%, whereas strains of Beauveria bassiana, Isaria fumosorosea and Lecanicillium longisporum were non-pathogenic to T. paludosa. In greenhouse tests, medium (2 × 10(14) conidia ha(-1) ) and high (2 × 10(16) conidia ha(-1) ) concentrations of V1005 gave higher larval mortalities (90 and 100%) than lower (2 × 10(13) conidia ha(-1) ) concentrations (52 and 78%) at 4 and 8 weeks after treatment respectively. Dursban WG gave 100% larval mortality 4 weeks after treatment; H. bacteriophora UWS1 caused 28 and 65% mortality 4 and 8 weeks after treatment respectively. This study shows that M. robertsii V1005 has considerable potential for the control of T. paludosa, thereby reducing the use of chemical insecticides.