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Attempts were made to evaluate the purified bioactive compounds of Xenorhabdus nematophila against Meloidogyne incognita. In order to extract the purified compounds, a solid-supported liquid-liquid extraction system with a flow rate (1 mL/min) was used to purify bioactive molecules. Compounds were individually collected concentrated and evaluated against M. incognita. Among 25 fractions the L19 fraction, exhibited 98% inhibition in egg hatching and mortality of juveniles. The biomolecules were identified through Liquid Chromatography- Mass Spectroscopy (LC-MS) technique. To decipher the mode of action of compounds, molecular docking studies were performed with potential protein targets such as acetylcholinesterase, β-1,4-endoglucanase, glutathione S-transferase-1, cytochrome c oxidase, G-protein coupled receptor and Fatty acid and retinol-binding proteins of M. incognita. The results revealed that among eight compounds from the L19 fraction, malonate and pidopidon exhibited greater binding affinity towards the selected protein targets of M. incognita. In vitro studies with malonate and pidopidon against M. incognita showcased a 99% reduction in egg hatching and juvenile mortality. Moreover, greenhouse experiments revealed that malonate compounds not only reduced 94% of the M. incognita population but also enhanced the plant growth parameters in tomato by 60%. Hence the present study stands novel in exploiting the nematicidal compounds from X. nematophila giving limelight to explore pidopidon and malonate as novel nematicidal compounds for the management of M. incognita. Graphical abstract:The nematicidal efficacy of entomopathogenic nematodes (EPNs) associated with their symbiotic bacteria Xenorhabdus nematophila against Meloidogyne incognita. In this study we used different concentration of biomolecules to inhibit egg hatching and juvenile mortality. Molecular docking was carried out using different protein to understand the effect of biomolecules. [Display omitted] • Bioactive molecules from Xenorhabdus nematophilus were extracted and purified using a solid-supported liquid-liquid extraction system at 1 mL/min, resulting in 25 fractions screened for egg hatching and juvenile mortality. • LCMS analysis revealed Malonate, and Pidopidon, as major compounds. • The pure compounds revealed inhibition in egg hatching and juvenile mortality. [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes (EPNs) are widely used as a biological control agent in pest management. The purpose of this study was to determine the effectiveness and penetration rates of certain EPNs. Three different EPN species were utilized in the study: Heterorhabditis bacteriophora HBH hybrid strain, Steinernema carpocapsae TUR-S4 isolate, Steinernema feltiae TUR-S3 and S-Bilecik isolates. These isolates were applied at different doses (50, 100, 150, 200, 250, 300, 350, and 400 Infective Juveniles (IJs)/Petri) on larvae of Tenebrio molitor and adults of Rhyzopertha dominica. Additionally, the penetration rates of the mentioned EPN species on T. molitor larvae and R. dominica adults were determined. As a result, the highest mortality rate observed in R. dominica adults was 93.33% when treated with the HBH isolate at the dose of 400 IJs/Petri dishes. For T. molitor larvae, the highest mortality rate was 100% at doses of 8.85 IJs/cm2, 10.60 IJs/cm2, 12.40 IJs/cm2, 14.15 IJs/cm2 for HBH and TUR-S3, 10.60 IJs/cm2, 12.40 IJs/cm2, 14.15 IJs/cm2 for TUR-S4, and 7.10 IJs/cm2, 8.85 IJs/cm2, 10.60 IJs/cm2, 12.40 IJs/cm2, 14.15 IJs/cm2 for S-Bilecik. When examining the penetration rates, the highest rate of 23.33% was observed in R. dominica adults treated with HBH, while for T. molitor , this rate was 22.67% in individuals treated with HBH. These findings underscore the importance of continued investigation into the potential of these EPNs for sustainable pest management strategies. • Entomopathogenic nematodes (EPNs) play a crucial role as biological control agents in controlling agricultural pests. • The HBH isolate of Heterorhabditis bacteriophora showed high mortality rates of 93.33% in Rhyzopertha dominica adults. • Specific doses for each EPN species resulted in a 100% mortality rate for Tenebrio molitor larvae. • The HBH isolate demonstrated the highest penetration rates, with 23.33% observed in R. dominica and 22.67% in T. molitor. [ABSTRACT FROM AUTHOR]

Abstract In this work, we analysed the efficacy of different commercial bio-insecticides (Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora and Bacillus thuringiensis) by valuating the mortality induced on two insect models, Galleria mellonella (Lepidoptera) and Sarcophaga africa (Diptera) after exposure to different temperatures (10, 20 and 30 °C). Moreover, we investigated the effects of temperature on the basal humoral immunity of the two target insects; particularly, phenoloxidase (PO) and lysozyme activity. Our results show that G. mellonella is susceptible to all bio-insecticides at all the examined temperatures, except when infected at 10 °C with S. carpocapsae and at 30 °C with S. feltiae and B. thuringiensis. S. africa is more susceptible at 30 °C to all bioinsecticides; whereas, when infected at 10 and 20 °C, H. bacteriophora is the most efficient. Temperature modulates PO activity of both G. mellonella and S. africa , otherwise variations in lysozyme activity is observed only in G. mellonella. Except for a possible correlation between the increased lysozyme activity and the delayed Bt efficacy recorded on G. mellonella at 30 °C, a different resistance to bio-insecticides at different temperatures does not seem to be associated to variations of the host basal immunity, probably due to immunoevasive and immunodepressive strategies of these entomopathogens. Graphical abstract fx1 Highlights • Entomopathogens efficacy was evaluated on insect models at different temperatures. • The lethality of the pathogens to their hosts changed in relation to temperature. • Temperature modulated phenoloxidase and lysozyme activity mainly in Lepidoptera. [ABSTRACT FROM AUTHOR]

Previous research has documented patchy distributions of entomopathogenic nematodes (EPNs) in the field, but more uniform distributions of EPNs could increase their biocontrol services in agroecosystems. Although laboratory studies have identified variation in foraging behavior of EPN species, less data is available on factors influencing their dispersal and resulting distribution patterns in the field. We examined the effect of biotic environmental factors including habitat type and plant cover, soil management and associated arthropod composition on the short- and long-term dispersal potential of Steinernema carpocapsae ALL and Heterorhabditis bacteriophora GPS11 emerging from nematode-infected host cadavers in the field. Based on bait trapping in the soil, both EPN species showed equivalent potential to disperse up to 2 m, actively or passively, in both grass and cultivated potatoes. Arthropod abundance, assessed by pitfall traps and soil core sampling, was equal in the two habitats. S. carpocapsae was detected in larger numbers in the water in pitfall traps, whereas H. bacteriophora numbers were positively correlated with mites in soil core samples. We documented aggregation of H. bacteriophora populations over the two years of the study in vegetable plots, which is consistent with earlier studies. Spatial distributions after dispersing from a grassy border into the adjacent cultivated field plots were more aggregated for H. bacteriophora than for S. carpocapsae . In most cases, no significant differences in the dispersal and spatial distribution of generally low population densities of H. bacteriophora were observed in plots with different soil management regimes, the exception being significantly lower aggregation in the first year of the study in plots with reduced tillage, cover crops, and no herbicide or insecticide treatments. [ABSTRACT FROM AUTHOR]

The role of adipokinetic hormone (AKH) in the firebug Pyrrhocoris apterus adults infected by the entomopathogenic nematode (EPN) Steinernema carpocapsae was examined in this study. It was found that co-application of EPN and AKH enhanced firebug mortality about 2.5 times within 24 h (from 20 to 51% in EPN vs. EPN + AKH treatments), and resulted in metabolism intensification, as carbon dioxide production in firebugs increased about 2.1 and 1.6 times compared to control- and EPN-treated insects, respectively. Accordingly, firebugs with reduced expression of AKH receptors showed a significantly lower mortality (by 1.6 to 2.9-folds), and lower general metabolism after EPN + AKH treatments. In addition, EPN application increased Akh gene expression in the corpora cardiaca (1.6 times), AKH level in the corpora cardiaca (1.3 times) and haemolymph (1.7 times), and lipid and carbohydrate amounts in the haemolymph. Thus, the outcomes of the present study demonstrate involvement of AKH into the anti-stress reaction elicited by the nematobacterial infection. The exact mechanism by which AKH acts is unknown, but results suggested that the increase of metabolism and nutrient amounts in haemolymph might play a role. [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes (EPN) are globally important inundative biological control agents. Their widespread use makes environmental risk assessment important, but very few comprehensive post-application risk assessments have been conducted for EPN. We apply a rigorous risk analysis procedure to the use of EPN applied in a forest ecosystem to suppress the large pine weevil ( Hylobius abietis ). In this synthesis, we provide a quantitative evaluation of five risk categories: (a) establishment, (b) dispersal, (c) host range, (d) direct non-target effects and (e) indirect non-target effects. A low level of risk was identified (35–51 out of a possible total of 125). Species exotic to the clear-fell forest ecosystem ( Steinernema carpocapsae and Heterorhabditis downesi ) were accorded a lower overall risk status than native species and strains ( Steinernema feltiae ), largely as a result of their shorter persistence in the target environment. We conclude that EPN are a low risk viable alternative control for pine weevil compared to the higher risk conventional control using pyrethroid or neonicotinoid insecticides. [ABSTRACT FROM AUTHOR]

The efficiency of entomopathogenic fungi, Beauveria bassiana and Entomophthora sp. as well as the nematode, Steinernema carpocapsae , against 3rd, 5th instar nymphs and adults of the desert locust, Schistocerca gregaria (Forskal) had been studied under laboratory conditions. Fungi at the concentrations 2.3 × 10 5 , 7 × 10 7 , 3.9 × 10 9 and 4 × 10 5 , 6.7 × 10 7 , 2.2 × 10 9 spores/ml and nematode at 5000, 2500, 1250 IJS/ml, respectively were applied on the desert locust by two different treatment methods; spray and soil application. The obtained results revealed that all the locust nymphs and adults were susceptible to fungi and nematode with high mortality rate records. The nematode killed approximately 100% of the tested locust individuals within 3–5 days postinfection and was found more effective than fungi in less time. On the other hand entomopathogenic fungi and nematode were found to be more effective when applied on the soil surface rather than spray treatment. This was common at any concentration used. Based on mortality percentages, all tested fungi and nematode had high potentials for biocontrol agents against S. gregaria . [ABSTRACT FROM AUTHOR]

Entomopathogenic nematodes, especially Steinernema carpocapsae , can provide good control of the larvae of the black cutworm, Agrotis ipsilon (Hufnagel), a cosmopolitan pest of golf course turfgrass. However, these biocontrol agents do not meet the high expectations for level and consistency of control by golf course managers on the affected high-profile areas. We explored three different approaches to further improve nematode performance. Combinations of S. carpocapsae with two other nematode species, S. feltiae or Heterorhabditis bacteriophora , did not improve control rates compared to S. carpocapsae alone. Syringing (i.e., light irrigation applied twice daily during hot weather) improved S. carpocapsae efficacy by on average 8% and also significantly reduced control variability (mean 78%, range 75–87%) at 109 nematodes per ha. The beneficial effect of syringing on S. carpocapsae performance can in part be ascribed to the improved persistence of the IJs in the syringing regime. The most effective approach to improve S. carpocapsae efficacy was through splitting applications into two applications at half rate applied 3 days apart. This improved efficacy by 13% on average while significantly reducing control variability (mean 84%, range 80–92%) at a rate of 109 nematodes per ha. At the generally recommended rate of 2.5 × 109 S. carpocapsae per ha, split applications and syringing should consistently provide black cutworm control of 90% or higher and could make this approach competitive with synthetic insecticide use. • Steinernema carpocapsae provides good control of black cutworm larvae in turfgrass. • We explored three different approaches to further improve nematode performance. • Combinations of S. carpocapsae with other nematode species did not improve control. • Syringing (light irrigation during hot weather) improved S. carpocapsae efficacy by 8%. • Split applications (twice half rate applied 3 days apart) improved efficacy by 13%. [ABSTRACT FROM AUTHOR]

[Display omitted] • By IJs concentration established Steinernema in PTM increased. • Sex ratio of S. carpocapsae in pre-pupa was affected by IJ concentration. • An inverse relationship was recorded between penetration rate and IJs concentration. • No correlation was observed between the invasion efficiencies (IE) and LC50 values of EPNs. • Despite higher Steinernema IE into pupae, mortality was lower compared to pre-pupa. Virulence and invasion efficiency of the three entomopathogenic nematodes, Heterorhabditis bacteriophora , Steinernema carpocapsae and S. feltiae against the potato tuber moth (PTM), Phthorimaea operculella was evaluated. Also evaluated were the sex ratio of Steinernema spp. and host stages to determine if 1) the developmental stage of the host affects sex ratio of nematodes; 2) infective juveniles (IJs) concentration affects sex ratio in host developmental stages and 3) the establishment of IJs is affected by developmental stages of host. The PTM pre-pupa and pupa were exposed to IJs in filter substrate petri dish bioassays. By increasing the IJs concentrations, the number of established Steinernema spp. in both PTM stages increased and only decreased at the highest concentration. No reduction in established nematode numbers at the highest concentration was observed for H. bacteriophora. Sex ratio of S. carpocapsae in pre-pupa was affected by IJ concentration. PTM was more susceptible to Steinernema spp. than H. bacteriophora. Pre-pupa were more susceptible to S. feltiae but S. carpocapsae recorded as the most virulent EPN on pupa. Invasion efficiencies were similar for Steinernema and considerably higher than for H. bacteriophora. Despite a higher invasion efficiency of Steinernema into pupae, mortality was lower compared to pre-pupa No correlation was recorded between the invasion efficiencies of the EPNs and mortalities of PTM. The results showed that the invasion efficiency is not appropriate criterion to reflect the virulence of studied EPNs. Compared to H. bacteriophora both tested Steinernema spp. were good candidates for further studies as biocontrol agents of PTM. [ABSTRACT FROM AUTHOR]

Highlights: [•] Field trials to control pine weevil using EPN and EPF were conducted. [•] Treatments including EPN more efficacious than those not. [•] Effects of EPF and EPN were additive. [•] Native Beauveria sp. in three trials was identified as Beauveria caledonica in one. [•] Applied fungi can reach this cryptic pest at depths of up to 18cm. [ABSTRACT FROM AUTHOR]

Highlights: [•] Steinernema carpocapsae responds to electrical fields (without current) and to magnetic fields. [•] We believe this is the first report of nematode directional movement in response to a magnetic field. [•] The findings may have implications for foraging behavior. [ABSTRACT FROM AUTHOR]

Abstract: Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. These symbiotic complexes are virulent against the insect host. Many protease genes were shown previously to be induced during parasitism, including one predicted to encode an aspartic protease, which was cloned and analyzed in this study. A cDNA encoding Sc-ASP155 was cloned based on the EST fragment. The full-length cDNA of Sc-ASP155 consists of 955 nucleotides with multiple domains, including a signal peptide (aa1–15), a pro-peptide region (aa16–45), and a typical catalytic aspartic domain (aa71–230). The putative 230 amino acid residues have a calculated molecular mass of 23,812Da and a theoretical pI of 5.01. Sc-ASP155 blastp analysis showed 40–62% amino acid sequence identity to aspartic proteases from parasitic and free-living nematodes. Expression analysis showed that the sc-asp155 gene was up-regulated during the initial parasitic stage, especially in L3 gut and 6h induced nematodes. Sequence comparison revealed that Sc-ASP155 was a member of an aspartic protease family and phylogenetic analysis indicated that Sc-ASP155 was clustered with Sc-ASP113. In situ hybridization showed that sc-asp155 was expressed in subventral cells. Additionally, we determined that sc-asp155 is a single-copy gene in S. carpocapsae. Homology modeling showed that Sc-ASP155 adopts a typical aspartic protease structure. The up-regulated Sc-ASP155 expression revealed that this protease could play a role in the parasitic process. In this study, we have cloned the gene and determined the expression of the pepsin-like aspartic protease Sc-ASP155 in S. carpocapsae. [Copyright &y& Elsevier]

Abstract: Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. During invasion, this nematode is able to express many proteases, including aspartic proteases. Genes encoding these aspartic proteases have been identified in the EST, and aspartic protease has been found in excretory–secretory products. The total protease was shown to digest blood hemoglobin in a zymogram gel. When the protein was partially purified by pepstatin affinity chromatography, it was observed to have high activity against both hemoglobin and the synthetic substrate Phe-Ala-Ala-Phe-(4NO2)-Phe-Val-Leu (4-pyridylmethyl) ester. The protein was confirmed by mass spectrometry and was found to be encoded by the gene sc-asp113. A cDNA encoding aspartic protease was cloned based on the EST fragment, which was constructed in our lab. The full-length cDNA of Sc-ASP113 consists of 1257 nucleotides encoding a protein with multiple domains, including a signal peptide (aa 1–15), a propeptide region (aa 16–45), and a typical catalytic aspartic domain (aa 68–416). The cleavage site of the signal peptide is predicted to be between Ala15 and Ala16. The putative 418 amino acid residues have a calculated molecular mass of 44,742Da and a theoretical pI of 5.14. BLAST analysis showed 33–56% amino acid sequence identity to aspartic proteases from parasitic and free living nematodes. Expression analysis showed that the sc-asp113 gene was up-regulated during the initial parasitic stage, especially during L3 inside the gut. In vitro, we showed that treatment with insect homogenate for 6h is sufficient to induce the expression of this protease in treated infective juveniles. Sequence comparison and evolutionary analysis revealed that Sc-ASP113 is a member of the aspartic protease family with the potential for tissue degradation. Phylogenetic analysis indicates that Sc-ASP113 branched between Haemonchus contortus and Steinernema feltiae proteases. Homology modeling showed that Sc-ASP113 adopts a typical aspartic protease structure. The up-regulation of Sc-ASP113 expression indicates that this protease could play a role in the parasitic process. To facilitate the exploration of this protease as a virulence factor, here we describe the purification of the protease and its molecular characterization in S. carpocapsae. [Copyright &y& Elsevier]

Abstract: The nematode Steinernema carpocapsae infects and kills many pest insects in agro-ecosystems and is commonly used in biocontrol of these pests. Growth of the nematodes prior to distribution for biocontrol commonly results in deterioration of traits that are essential for nematode persistence in field applications. To better understand the mechanisms underlying trait deterioration of the efficacy of natural parasitism in entomopathogenic nematodes, we explored the maintenance of fitness related traits including reproductive capacity, heat tolerance, virulence to insects and ‘tail standing’ (formerly called nictation) among laboratory-cultured lines derived from natural, randomly mating populations of S. carpocapsae. Laboratory cultured nematode lines with fitness-related trait values below wild-type levels regained wild-type levels of reproductive and heat tolerance traits when outcrossed with a non-deteriorated line, while virulence and ‘tail standing’ did not deteriorate in our experiments. Crossbreeding two trait-deteriorated lines with each other also resulted in restoration of trait means to wild-type levels in most crossbred lines. Our results implicate inbreeding depression as the primary cause of trait deterioration in the laboratory cultured S. carpocapsae. We further suggest the possibility of creating inbred lines purged of deleterious alleles as founders in commercial nematode growth. [Copyright &y& Elsevier]

Abstract: Steinernema carpocapsae is a parasitic nematode that is high virulent to insects. The parasitic juvenile reaches the insect haemocoelium by passing through mid-gut barriers and develops there. During invasion, the nematode was predicted to express a large set of proteases, including metalloproteases, one of which was sequenced and expressed in this work. A 1583-nucleotide cDNA encoding a putative metalloprotease containing a 28-aa signal peptide, a 79-aa propeptide and a 311-aa mature protease with a predicted molecular mass of 35.2kDa and a theoretical pI of 5.9 was cloned from the parasitic stage of the nematode. Sequence analyses predicted signature sequences of the astacin metalloprotease family, an astacin domain, a zinc-binding motif and a methionine turn motif; therefore, this protein was identified as an astacin and designated as Sc-AST. The astacin domain of Sc-AST has an amino acid sequence homology of 46% to prototypical astacin from Astacus astacus and 82% to Caenorhabditis elegans NAS-8. Like NAS-8 of C. elegans, Sc-AST has a C-terminal ShK toxin domain. Recombinant Sc-AST was produced in an Escherichia coli system and was purified by affinity chromatography. Maldi-MS/MS analysis of purified recombinant protein matched the Sc-AST sequence with a significance score of 499. Sc-AST was produced in the correct folding conformation, showed activities against gelatin and azocasein substrates and was inhibited by divalent metal-chelating agents. Sc-AST presented an optimum pH of 7.5 and temperature of 37°C and K m, V max and k cat values of 1.86mM, 0.281μM/min and 27.9s−1, respectively. Expression analyses indicated that Sc-AST is up-regulated in the parasitic stage and is strongly induced in vitro by insect tissues, thus suggesting that it plays a role in the parasitic process. [ABSTRACT FROM AUTHOR]

Abstract: The main insect pest in Brazilian corn is fall armyworm, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae). Entomopathogenic nematodes (EPNs) can be used to control this pest, and can be applied together with various insecticides. Thus, the objective of this work was to evaluate the efficacy of mixtures of EPNs and insecticides to control S. frugiperda in corn crops. In laboratory bioassays three species of EPNs were tested (Heterorhabditis indica, Steinernema carpocapsae and Steinernema glaseri) together with 18 registered insecticides to control S. frugiperda in corn. Efficacy of association between insecticides and EPNs on S. frugiperda larvae was evaluated against the insect''s third instar, 2 and 4 days after applications in laboratory. Experiments in the field were performed in two consecutive years, with located application of H. indica and S. carpocapsae (250 IJs/cm2) mixed with chlorpyrifos (0.3 L/ha) and lufenuron (0.15 L/ha) on the corn husk. In laboratory, after two days exposure the interaction between chlorpyrifos and H. indica was synergistic, while interaction with cypermethrin, spinosad, methoxyfenozide and deltamethrin + triazofos was additive, as was interaction between lufenuron, chlorpyrifos and cypermethrin with S. carpocapsae. In contrast, the interaction between chlorpyrifos (Vexter™ and Lorsban™) and lufenuron with S. glaseri was synergistic. In the field, the best treatment was the mixture of H. indica with lufenuron (0.15 L/ha), with 62.5% and 57.5% larval mortality in the two evaluation years in the field, respectively. [Copyright &y& Elsevier]

Abstract: The fall armyworm, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is considered the main key pest of corn crops in Brazil. Entomopathogenic nematodes (EPNs) may be used to control this pest, applied together with other different entomopathogen agents or phytosanity products in the spraying mixture. Thus, the objective of work was to evaluate the compatibility of EPNs with different insecticides used of S. frugiperda control in laboratory conditions. Three species of EPNs (Heterorhabditis indica, Steinernema carpocapsae and Steinernema glaseri) and 18 insecticides registered to control of S. frugiperda in corn crops were tested. Compatibility of the insecticides with EPNs was evaluated by observing mortality and infectivity of infecting juveniles (IJs) 48h after immersion in solution of the insecticide formulations. Among all insecticides tested, Lorsban™ (chlorpyrifos), Decis™ (deltamethrin), Match™ (lufenuron), Deltaphos™ (deltramethrin + triazophos), Dimilin™ (diflubenzuron), Stallion™ (gamacyhalothrin), Karate Zeon™ (lambdacyhalothrin) Tracer™ (spinosad), Vexter™ (chlorpyrifos), Galgotrin™ (cypermethrin), Certero™ (triflumuron), and Talcord™ (permethrin) were compatible (class 1) with the three nematode species tested under laboratory conditions. [Copyright &y& Elsevier]

Abstract: Steinernema carpocapsae is an insect parasitic nematode widely used in pest control programs. The efficacy of this nematode in controlling insects has been found to be related to the pathogenicity of the infective stage. In order to study the parasitic mechanisms exhibited by this parasite, a cDNA library of the induced S. carpocapsae parasitic phase was generated. A total of 2500 clones were sequenced and 2180 high-quality ESTs were obtained from this library. Cluster analysis generated a total of 1592 unique sequences including 1393 singletons. About 63% of the unique sequences had significant hits (e ≤1e−05) to the non-redundant protein database. The remaining sequences most likely represent putative novel protein coding genes. Comparative analysis identified 377 homologs in C. elegans, 431 in C. briggsae and 75 in other nematodes. Classification of the predicted proteins revealed involvement in diverse cellular, metabolic and extracellular functions. One hundred and nineteen clusters were predicted to encode putative secreted proteins such as proteases, proteases inhibitors, lectins, saposin-like proteins, acetylcholinesterase, anti-oxidants, and heat-shock proteins, which can possibly have host interactions. This dataset provides a basis for genomic studies towards a better understanding of the events that occur in the parasitic process of this entomopathogenic nematode, including invasion of the insect haemocoelium, adaptations to insect innate immunity and stress responses, and production of virulence factors. The identification of key genes in the parasitic process provides useful tools for the improvement of S. carpocapsae as a biological agent. [Copyright &y& Elsevier]

Abstract: A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory–secretory products of parasitic stage Steinernema carpocapsae. The purified protease had an apparent molecular mass of 30kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K m , V max and k cat values of 409μM/min, 0.389μM/min and 24.9s−1, respectively. The protease was most active at pH 8.0 and 35°C, and its proteolytic activity was almost completely reduced after incubation at 75°C for 30min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions. [Copyright &y& Elsevier]

Abstract: Wireworms, the subterranean larval stage of click beetles (Coleoptera: Elateridae), are an important pests of potatoes throughout the world. Laboratory assays were done to identify virulent strains of entomopathogenic nematodes (EPN) and fungi against wireworm, Agriotes lineatus (L.) (Coleoptera: Elateridae). A fungus, Metarhizium anisopliae (Metsch), Sorokin strains V1002 and LRC181A, caused 90 and 100% mortality of A. lineatus, 3 weeks post-inoculation. Other M. anisopliae strains caused mortality ranging between 10 and 70%, whereas strains of Beauveria bassiana (Balsamo) Vuillemin and Paecilomyces fumosoroseus (Wize) were non-pathogenic to A. lineatus. The EPN, Heterorhabditis bacteriophora Poinar strain UWS1, caused significantly greater mortality (67%) to A. lineatus than other EPN species (0–50%), 3 weeks post-inoculation. The organophosphate insecticide Mocap 10G gave 100% control. The present results suggest that M. anisopliae strain V1002 has considerable potential for the control of the wireworm tested. [Copyright &y& Elsevier]

Abstract: Natural entomopathogenic nematode (EPN) populations were surveyed in La Rioja (Northern Spain) during two consecutive years (2003–2005) to study their diversity, occurrence, and life characteristics under different agricultural management representing natural areas and perennial and annual crops from organic and conventional systems. Native EPN species and strains were identified using morphological and molecular characteristics. Virulence, infection cycle length and reproductive potential were assessed using Galleria mellonella larvae. The EPN occurrence was evaluated through abundance, recovery frequency, larval mortality percentage and EPN population density. EPNs were also related to selected soil physical and chemical variables as well as to some soil pollutants such as heavy metals and organochlorine pesticide residues. Only two steinernematids species were identified: Steinernema feltiae was observed throughout all seasons from natural and agricultural areas and Steinernema carpocapsae in summer and autumn of 2004 from perennial crops only. The virulence of native strains was lower than other previously isolated Spanish strain from natural areas or crop field edges. EPN abundance and recovery frequency indicated that habitat type might influence EPNs occurrence stronger than seasonality with the intensity of agricultural management inversely affecting their distribution. Moreover, clay, P2O5, Zn, Cu and hexachlorobenzene contents negatively correlating with EPN population density. We consider that agricultural management should be taken into account if EPNs are going to be used as biological control agents. [Copyright &y& Elsevier]

Abstract: The dauer juvenile (DJ) stage of the insect parasitic nematode Steinernema carpocapsae is the only stage in the life cycle which is capable of surviving outside its host and it is adapted for tolerating environmental stresses and for host finding. We have isolated 45 unique expressed sequence tags (ESTs) that are up-regulated in response to desiccation in S. carpocapsae DJs. The majority of these ESTs were co-expressed in response to desiccation and osmotic stress and were generally not induced in response to heat and cold stress. Thirty-two ESTs showed similarity to known sequences. Among these were sequences which encode putative signalling molecules or transcription factors, sequences which detoxify reactive oxygen species, two C-type lectin sequences, ESTs which encode membrane-associated proteins and seven distinct late embryogenic abundant (LEA) sequences. We also isolated 13 novel ESTs. These data show that the molecular response to desiccation stress in entomopathogenic nematode DJs is complex and parallels many of the adaptive changes which occur in drought tolerant plants during exposure to desiccation and osmotic stress. A notable feature of the desiccation response of plants is the number and diversity of hydrophilic LEA proteins synthesised in response to desiccation. All of the LEA sequences detected in animals to date, including those reported in this study, belong to LEA3 group. We show that S. carpocapsae expresses several novel sequences which encode putative hydrophilic and natively unfolded proteins. It is likely that these novel and putative proteins play an important role in desiccation tolerance, possibly by carrying out analogous roles in nematodes to those carried out by the other LEA protein classes in plants. [Copyright &y& Elsevier]

Abstract: 1. The effect of cold (5°C) and warm (35°C) storage on desiccation tolerance of cold-adapted Steinernema feltiae, intermediate S. carpocapsae and warm-adapted S. riobrave was evaluated at 5 and 35°C. 2. Desiccation survival of all three species at 5°C was enhanced by both the cold and warm storage. At 35°C, desiccation survival of S. feltiae and S. riobrave was enhanced by only warm storage and of S. carpocapsae by only cold storage. 3. When desiccated at 5°C, both warm and cold stored S. carpocapsae and S. feltiae, and the cold stored S. riobrave maintained significantly higher original virulence against Galleria mellonella larvae than cohorts stored at culture temperature. When desiccated at 35°C, both warm and cold stored S. riobrave, and warm stored S. carpocapsae and S. feltiae maintained significantly higher original virulence than cohorts stored at culture temperature. 4. After 3h exposure to ultra violet (UV) radiation stress at 340nm, the warm and cold stored S. carpocapsae and S. riobrave maintained significantly higher original virulence than cohorts stored at culture temperature. The virulence of UV stressed S. feltiae was unaffected by storage temperature. 5. Trehalose content of both warm and cold stored S. feltiae, cold stored S. carpocapsae and warm stored S. riobrave was positively correlated with their desiccation survival at 5 and 35°C. Trehalose content was also positively correlated with the virulence of both warm and cold stored S. carpocapsae, and warm stored S. riobrave following exposure to UV stress. [Copyright &y& Elsevier]

Abstract: Korean entomopathogenic nematodes (EPN), Steinernema carpocapsae Pocheon strain, Steinernema sp. GSNUS-4 strain, Steinernema sp. GSNUS-14 strain, Steinernema sp. GSNUS-16 strain, Heterorhabditis sp. GSNUH-2 strain, and Heterorhabditis sp. GSNUH-3 strain were evaluated for the biological control of pellucid zygaenid, Pryeria sinica Moore (Lepidoptera: Zygaenidae), a leaf-feeding insect pest of Euonymus japonica Thunberg. In addition, persistence of S. carpocapsae Pocheon strain on E. japonica foliage was checked. Nematode species, strain, and concentration influenced infectivity of Korean EPNs against 3rd instar of P. sinica larvae. S. carpocapsae Pocheon strain was the most effective nematode. Larval mortality by S. carpocapsae Pocheon strain in the exposure experiments was low in both light and dark conditions representing 36.7% at 100 infective juveniles/larva in each dark and light condition. Mortality by fenitrothion, however, was 100% 3 days later. Infectivity of S. carpocapsae Pocheon strain was not different between non-exposed foliage and exposed foliage to sunlight for 30 min. Mortality of 4th instar of P. sinica larvae was very low for both S. carpocapsae Pocheon alone and S carpocapsae Pocheon with spreading agent. Application time of nematodes, shading, and leaf position on the tree influenced persistence of S. carpocapsae Pocheon strain on E. japonica foliage. Nematode survival on foliage was positively correlated with shading level and higher at the lowest level than the middle or upper level of the tree. [Copyright &y& Elsevier]

Abstract: The large pine weevil (Hylobius abietis) is a major pest of young conifer and broadleaf transplants on restock sites in the UK. The objectives of this study were two-fold. The first was to test the effectiveness of two methods of application of the entomopathogenic nematode Steinernema carpocapsae in infecting the various life stages of the weevil in Sitka spruce (Picea sitchensis) stumps. The second objective was to investigate the importance of distribution and development of each of the weevil life stages in order to improve our understanding of factors influencing infection by S. carpocapsae. The method of nematode application on 24 July 1997, as well as the distribution and life stage of H. abietis within the stump and root system of Sitka spruce, were all shown to affect nematode efficacy as a field-based biological control agent. In contrast, the time of felling of the trees from July to September 1995 to create these stumps did not result in different rates of nematode infection of H. abietis in the stumps 2 years post-felling, but did influence development of the pest. H. abietis larvae were more widely distributed throughout the stump and root system than pupae, callow adults and pre-emergent adults that tended to be in the upper regions of the soil and close to the stump bole. Callow adults were highly susceptible to nematode infection, but larvae and older adults were not. Pupae had similar infection to larvae possibly due to late nematode application, as laboratory studies showed similar infection rates to callow adults. The results showed that S. carpocapsae tended to remain near the point of application, with the incidence of H. abietis infection declining with increasing distance from the stump and with increasing soil depth. The distribution of the most susceptible stages was, therefore, optimal for maximum nematode infection. At the time of the trial, susceptible H. abietis life stages comprised only 14–20% of the H. abietis population and the low level of control achieved (5.2–18.3%) reflects the differential susceptibility of these stages. The short duration of the callow adult and pupal stages, combined with the variation in development rates, indicated a fairly narrow application window in which to maximise infection rates. Timing of nematode treatment to proceed or better coincide with the susceptible stages of the lifecycle, multiple applications and/or increased dose rates of nematodes would probably have increased the overall level of control. Of the two application techniques used the subsurface drench was more efficient than the surface spray application, the former targeting a greater proportion of the susceptible life stages of H. abietis. [Copyright &y& Elsevier]

The effect of thermal acclimation on trehalose accumulation and the acquisition of thermotolerance was studied in three species of entomopathogenic nematodes adapted to either cold or warm temperatures. All three Steinernema species accumulated trehalose when acclimated at either 5 or 35°C, but the amount of trehalose accumulation differed by species and temperature. The trehalose content of the cold adapted Steinernema feltiae increased by 350 and 182%, of intermediate Steinernema carpocapsae by 146 and 122% and of warm adapted Steinernema riobrave by 30 and 87% over the initial level (18.25, 27.24 and 23.97 μg trehalose/mg dry weight, respectively) during acclimation at 5 and 35°C, respectively. Warm and cold acclimation enhanced heat (40°C for 8 h) and freezing (−20°C for 4 h) tolerance of S. carpocapsae and the enhanced tolerance was positively correlated with the increased trehalose levels. Warm and cold acclimation also enhanced heat but not freezing tolerance of S. feltiae and the enhanced heat tolerance was positively correlated with the increased trehalose levels. In contrast, warm and cold acclimation enhanced the freezing but not heat tolerance of S. riobrave, and increased freezing tolerance of only warm acclimated S. riobrave was positively correlated with the increased trehalose levels. The effect of acclimation on maintenance of original virulence by either heat or freeze stressed nematodes against the wax moth Galleria mellonella larvae was temperature dependent and differed among species. During freezing stress, both cold and warm acclimated S. carpocapsae (84%) and during heat stress, only warm acclimated S. carpocapsae (95%) maintained significantly higher original virulence than the non-acclimated (36 and 47%, respectively) nematodes. Both cold and warm acclimated S. feltiae maintained significantly higher original virulence (69%) than the non-acclimated S. feltiae (0%) during heat but not freezing stress. In contrast, both warm and cold acclimated S. riobrave maintained significantly higher virulence (41%) than the non-acclimated (14%) nematodes during freezing, but not during heat stress. Our data indicate that trehalose accumulation is not only a cold associated phenomenon but is a general response of nematodes to thermal stress. However, the extent of enhanced thermal stress tolerance conferred by the accumulated trehalose differs with nematode species. [Copyright &y& Elsevier]

Two hypotheses on the synthesis of the protectants glycerol and trehalose of the infective juveniles (IJs) of Steinernema carpocapsae during osmotic dehydration were tested and utilised to evaluate the function and importance of glycerol on survival of the nematodes during osmotic dehydration. This was achieved by comparing the changes in survival, morphology, behaviour and levels of glycerol, trehalose and permeated compounds of the IJs dehydrated in seven hypertonic solutions at two temperature regimes: (1) 5 °C for 15 days; and (2) 23 °C for 1 day followed by 5 °C for another 14 days. The results substantiate both hypotheses tested: (1) the permeability of the IJs to various compounds, such as sucrose or ethylene glycol, when they are dehydrated in hypertonic solutions of these compounds; and (2) suppression of the synthesis of protectant glycerol but not trehalose when IJs are dehydrated at low temperature. The results also showed that: (1) although trehalose was the preferred dehydration protectant, glycerol played an important role in rapidly balancing the osmotic pressure when IJs were exposed in hypertonic solutions; (2) the presence of glycerol was essential for the IJs to survive and function properly even under moderate osmotic dehydration, especially when IJs were dehydrated in salt solutions; and (3) some exogenous compounds permeated into IJs during osmotic dehydration such as ethylene glycol, may function in the same way as glycerol and significantly improve the survival and function of the IJs. The results indicate that each of the protectants glycerol and trehalose has a specific function and neither is replaceable by the other. [Copyright &y& Elsevier]

• S. carpocapsae IJs moved in higher rates toward R. ferrugineus larvae than to its recorded chewing sounds. • Higher number of genes were downregulated in IJs exposed to insect and speaker compared to non-exposed IJs. • Differential transcriptional regulation of olfactory transduction signaling pathways exerts the host-cue. • Neuropeptides and neuromodulators were found differentially regulated in response to host cue in S. carpocapsae. As the larvae of the date palm pest, the red palm weevil, Rhynchophorus ferrugineus, feeds on the host tissue, they emit a distinctive sound which can be recorded outside of the infected tree. We evaluated the response of infective juveniles (IJs) of the entomopathogenic nematodes Steinernema carpocapsae to the R. ferrugineus larvae and it's sound source, separately. In the presence of the insect larvae, 50.2 % of total IJs moved toward those larvae. Recorded insect larvae sound emitted by the speaker resulted in 7% of total IJs near the sound source. RNA-Seq data indicated that more genes were downregulated in S. carpocapsae IJs exposed to insect and speaker compared to non-stimulated IJs. IJs exposed to insect exhibited more up-regulated genes than IJs exposed to speaker. Enriched pathways and biological processes in IJs were similar for both stimuli. The inhibition of locomotion, regulation of neurotransmitter secretion, response to biotic stimulus, and cellular response to chemical stimuli were enriched with unique GO terms for speaker treatment. The regulation of localization, sodium ion transmembrane transport, regulation of response to stress and response to organic substances were the GO categories enriched unique to insect. The host-parasitic interaction was regulated by the differential expression of Ras/MAP kinase, TGF-beta signaling, insulin signaling, AMPK signaling, PPAR signaling pathways and many developmental pathways. More prominent R. ferrugineus host localization by S. carpocapsae was primarily due to the differential transcriptional regulation of olfactory signal transduction, FOXO-family proteins, calcium signaling, WNT and mTOR signaling pathway. The neural basis for the nematode attraction to insect host is based on the chemosensation and the mechanosensation. Many neuropeptides and neuromodulators are involved in regulating the foraging behavior of S. carpocapsae. The results of this study provide new insights into the molecular mechanisms that allow these nematodes to seek insect hosts. Our finding, especially the molecular ones suggest that chemical cues emitted by the active insect host are stimulants of nematodes attraction. Whereas the sound emitted by the insect has minor effects on the nematode behavior. [ABSTRACT FROM AUTHOR]

The Steinernema carpocapsae - Xenorhabdus nematophila association is a nematobacterial complex used in biological control of insect crop pests. The infection success of this dual pathogen strongly depends on its interactions with the host's immune system. Here, we used the lepidopteran pest Spodoptera frugiperda to analyze the respective impact of each partner in the induction of its immune responses. First, we used previously obtained RNAseq data to construct the immunome of S. frugiperda and analyze its induction. We then selected representative genes to study by RT-qPCR their induction kinetics and specificity after independent injections of each partner. We showed that both X. nematophila and S. carpocapsae participate in the induction of stable immune responses to the complex. While X. nematophila mainly induces genes classically involved in antibacterial responses, S. carpocapsae induces lectins and genes involved in melanization and encapsulation. We discuss putative relationships between these differential inductions and the pathogen immunosuppressive strategies. • We describe a comprehensive immunome of the lepidopteran pest Spodoptera frugiperda. • We show the induction of the different immune pathways by the nematobacterial complex S. carpocapsae – X. nematophila. • Each pathogen partner induced a strong stable response of the immunome. • The bacterial partner induces the expression of genes involved in antibacterial response. • The nematode partner induces the expression of genes involved in melanization and encapsulation. [ABSTRACT FROM AUTHOR]

Nematodes are known to parasitise all major invertebrate groups in soils, and it has been assumed that their host finding relies on attraction to chemical cues. We studied movement of three species of insect-parasitic nematodes, Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis megidis in response to chemical and vibrational cues. All species showed strong, significant taxes towards the vibrations. We also show that in soils, the utility of chemical cues as attractants is substantially reduced by the presence of organic matter. [Copyright &y& Elsevier]