Dauer juvenile recovery transcriptome of two contrasting EMS mutants of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode Heterorhabditis bacteriophora, symbiotically associated with enterobacteria of the genus Photorhabdus, is a biological control agent against many insect pests. Dauer Juveniles (DJ) of this nematode are produced in industrial-scale bioreactors up to 100 m3 in liquid culture processes lasting approximately 11 days. A high DJ yield (> 200,000 DJ·mL-1) determines the success of the process. To start the mass production, a DJ inoculum proceeding from a previous monoxenic culture is added to pre-cultured (24 h) Photorhabdus bacteria. Within minutes after contact with the bacteria, DJ are expected to perceive signals that trigger their further development (DJ recovery) to reproductive hermaphrodites. A rapid, synchronized, and high DJ recovery is a key factor for an efficient culture process. In case of low percentage of DJ recovery, the final DJ yield is drastically reduced, and the amount of non-desired stages (males and non-fertilized females) hinders the DJ harvest. In a preliminary work, a huge DJ recovery phenotypic variability in H. bacteriophora ethyl methanesulphonate (EMS) mutants was determined. In the present study, two EMS-mutant lines (M31 and M88) with high and low recovery phenotypes were analyzed concerning their differences in gene expression during the first hours of contact with Photorhabdus supernatant containing food signals triggering recovery. A snapshot (RNA-seq analysis) of their transcriptome was captured at 0.5, 1, 3 and 6 h after exposure. Transcripts (3060) with significant regulation changes were identified in the two lines. To analyze the RNA-seq data over time, we (1) divided the expression profiles into clusters of similar regulation, (2) identified over and under-represented gene ontology categories for each cluster, (3) identified Caenorhabditis elegans homologous genes with recovery-related function, and (4) combined the information with available single nucleotide polymorphism (SNP) data. We observed that the expression dynamics of the contrasting mutants (M31 and M88) differ the most within the first 3 h after Photorhabdus supernatant exposure, and during this time, genes related to changes in the DJ cuticle and molting are more active in the high-recovery line (M31). Comparing the gene expression of DJ exposed to the insect food signal in the haemolymph, genes related to host immunosuppressive factors were not found in DJ upon bacterial supernatant exposure. No link between the position of SNPs associated with high recovery and changes in gene expression was determined for genes with high differential expression. Concerning specific transcripts, nine H. bacteriophora gene models with differential expression are provided as candidate genes for further studies.

Compatibility of Entomopathogenic Nematodes with Chemical Insecticides for the Control of Drosophila suzukii (Diptera: Drosophilidae).

The spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is a pest that reduces the productivity of small fruits. Entomopathogenic nematodes (EPNs) and chemical insecticides can suppress this pest, but the compatibility of the two approaches together requires further examination. This laboratory study evaluated the compatibility of Steinernema brazilense IBCBn 06, S. carpocapsae IBCBn 02, Heterorhabditis amazonensis IBCBn 24, and H. bacteriophora HB with ten chemical insecticides registered for managing D. suzukii pupae. In the first study, most insecticides at the recommended rate did not reduce the viability (% of living infective juveniles (IJs)) of S. braziliense and both Heterorhabditis species. The viability of S. carpocapsae was lowered by exposure to spinetoram, malathion, abamectin, azadirachtin, deltamethrin, lambda-cyhalothrin, malathion, and spinetoram after 48 h. During infectivity bioassays, phosmet was compatible with all the EPNs, causing minimal changes in infectivity (% pupal mortality) and efficiency relative to EPN-only controls, whereas lambda-cyhalothrin generally reduced infectivity of EPNs on D. suzukii pupae the most, with a 53, 75, 57, and 13% reduction in infectivity efficiency among H. bacteriophora, H. amazonensis, S. carpocapsae, and S. brazilense, respectively. The second study compared pupal mortality caused by the two most compatible nematode species and five insecticides in various combinations. Both Heterorhabditis species caused 78-79% mortality among D. suzukii pupae when used alone, and were tested in combination with spinetoram, malathion, azadirachtin, phosmet, or novaluron at a one-quarter rate. Notably, H. bacteriophora caused 79% mortality on D. suzukii pupae when used alone, and 89% mortality when combined with spinetoram, showing an additive effect. Novaluron drastically reduced the number of progeny IJs when combined with H. amazonensis by 270 IJs and H. bacteriophora by 218. Any adult flies that emerged from EPN-insecticide-treated pupae had a shorter lifespan than from untreated pupae. The combined use of Heterorhabditis and compatible chemical insecticides was promising, except for novaluron.

Biocontrol potential of entomopathogenic nematodes against the grey maize weevil Tanymecus dilaticollis (Coleoptera: Curculionidae) adults.

The grey maize weevil, Tanymecus dilaticollis, is a polyphagous species, which is among the most important pests of maize in Southeastern Europe. The efficacy of commercial products with two species of entomopathogenic nematodes (EPNs), Steinernema carpocapsae and Heterorhabditis bacteriophora, was investigated against adults of the grey maize weevil under laboratory conditions. Nemastar®, containing S. carpocapsae was more effective on T. dilaticollis adults than Nematop® containing H. bacteriophora, when applied uniformly to the surface of the soil, on Petri dishes containing T. dilaticollis adults. Results showed that S. carpocapsae rates of 83-333 infective juveniles/adult caused > 94% mortality in T. dilaticollis adults, whereas H. bacteriophora caused 27-61%, adult mortality, after exposure of insects to the commercial products of EPNs for 15 days. The infection rates of EPNs increased with concentration applied and ranged from 70-83% and 19-64% for Nemastar® and Nematop®, respectively. Subsequent field and semi-field tests were conducted with Nemastar® (application rate of 50 million S. carpocapsae per 100 m2) in maize crops with biological (mycoinsecticide Naturalis®, biofungicides and fertilizers) and chemical seed treatment (Gaucho® FS 600; active ingredient: imidacloprid) in Knezha, Bulgaria. Nematodes were found only in the dead specimens, in open plots and cages sprayed with the commercial nematode product. Nematode sprayings contributed for higher maize yields in the open maize plots in the fields with different seed treatments. We suggest that the use of powder formulation of S. carpocapsae in combination with biologically treated maize seeds can contribute to minimize the use of chemical insecticides against the grey maize weevil. The results obtained can be used as a base to further tests to ascertain the efficacy of EPNs products before they can be recommended for use in the integrated approach to T. dilaticollis management.

Antifungal Effect of Metabolites from Bacterial Symbionts of Entomopathogenic Nematodes on Fusarium Head Blight of Wheat.

Fungal diseases such as Fusarium head blight (FHB) are significant biotic stressors, negatively affecting wheat production and quality. This study explored the antifungal activity of the metabolites produced by the bacterial symbionts of entomopathogenic nematodes (EPNs) against FHB-causing Fusarium sp. Fusarium graminearum. To achieve this, the symbiotic bacteria of nine EPN isolates from the EPN collection at the Agricultural Research Council-Small Grains (ARC-SG) were isolated from the cadavers of Galleria mellonella (Lepidoptera: Pyralidae) larvae after infection with EPNs. Broth cultures (crude) and their supernatants (filtered and autoclaved) of each bacterial isolate were used as bacterial metabolite treatments to test their inhibitory effect on the mycelial growth and spore germination of F. graminearum. Mycelial growth inhibition rates varied among both bacterial isolates and treatments. Crude metabolite treatments proved to be more effective than filtered and autoclaved metabolite treatments, with an overall inhibition rate of 75.25% compared to 23.93% and 13.32%, respectively. From the crude metabolite treatments, the Xenorhabdus khoisanae SGI 197 bacterial isolate from Steinernema beitlechemi SGI 197 had the highest mean inhibition rate of 96.25%, followed by Photorhabdus luminescens SGI 170 bacteria isolated from Heterorhabditis bacteriophora SGI 170 with a 95.79% mean inhibition rate. The filtered metabolite treatments of all bacterial isolates were tested for their inhibitory activity against Fusarium graminearum spore germination. Mean spore germination inhibition rates from Xenorhabdus spp. bacterial isolates were higher (83.91 to 96.29%) than those from Photorhabdus spp. (6.05 to 14.74%). The results obtained from this study suggest that EPN symbiotic bacterial metabolites have potential use as biological control agents of FHB. Although field efficacy against FHB was not studied, the significant inhibition of mycelial growth and spore germination suggest that the application of these metabolites at the flowering stage may provide protection to plants against infection with or spread of F. graminearum. These metabolites have the potential to be employed as part of integrated pest management (IPM) to inhibit/delay conidia germination until the anthesis (flowering stage) of wheat seedlings has passed.

Movers and shakers: Do nematodes that move more invade more?

Entomopathogenic nematodes (EPNs) are roundworms that parasitize insects with the aid of symbiotic bacteria. These nematodes have been used both as model organisms and for biological control of pests. The specialized third stage of an EPN, known as an infective juvenile (IJ) must forage to find a host with strategies varying from species to species (cruising, ambushing, and intermediate). Some IJs move more than others to find a host, despite an increased risk of predation and desiccation. This hints at potential underlying benefits (e.g., increased invasion) for EPNs that move more. We assessed whether EPNs that moved farther down a soil column also exhibit higher levels of invasion when compared to nematodes that remained at or near their point of origin. We found that movers in the cruisier and intermediate species: Steinernema riobrave, Heterorhabditis bacteriophora, and H. indica had higher invasion rates compared to their counterparts that did not move. S. carpocapsae, an ambusher, did not exhibit invasion differences between EPNs that moved versus those that did not. For the three cruiser/intermediate EPNs we tested, our results support our hypothesis that EPNs that tend to move more enjoy related benefits such as increased invasion potential. Further studies are required to explore other parameters that may interact with movement. The results of this study can potentially be used to develop EPN strains that move more and invade more, and thus can potentially be more effective biological control agents.

Virulence of entomopathogenic nematodes and their symbiotic bacteria, under laboratory conditions, aiming controlling Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) on sugarcane / Virulência de nematoides entomopatogênicos e suas bactérias simbióticas, sob condições de laboratório, visando controlar Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) na cana-de-açúcar

Sugarcane crops Saccharum spp. (Poales: Poaceae) produces different derivatives to the world: sugar, ethanol and bioenergy. Despite the application of pesticides, insect pests still cause economic losses, among these the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) causing direct and indirect damage to the plant. This study assess the virulence of three entomopathogenic nematodes (EPNs) species and their symbiont bacteria against the pink sugarcane mealybug, under laboratory conditions. Fourteen treatments represented by control (distilled water), Heterorhabditis bacteriophora Poinar, 1976 (HB EN01) (Rhabditida: Heterorhabditidae), Steinernema rarum (Doucet, 1986) (PAM25) and Steinernema carpocapsae Weiser, 1955 (All) (Rhabditida: Steinermatidae) at concentrations of 25, 50, 75 and 100 infective juveniles (IJs)/insect, and the standard chemical product, thiamethoxam, were assayed. In a second experiment, the bacteria Photorhabdus luminescens (Thomas and Poinar, 1979), Xenorhabdus szentirmaii Lengyel, 2005 and Xenorhabdus nematophila (Poinar and Thomas, 1965) (Enterobacterales: Morganellaceae) at 3.0 x 109 cells/ml were assessed for each treatment. Ten replications were stablished, each one counting ten females/mealybugs inside a 10 cm Petri dish, amounting 100 individuals/treatment. All treatments were kept under stable conditions (25±1 ºC, H 70±10%, in the dark). All nematodes species infected S. sacchari. Steinerma rarum (PAM25) provided the highest mortality against the pink sugarcane mealybug (79.25%), followed by H. bacteriophora (HB EN01) (58.25%) and S. carpocapsae (All) (42.50%) (P<0.001). The mortality rate caused by X. szentirmaii, P. luminescens and X. nematophila were 40, 45 and 20%, respectively. Steinerma rarum (PAM25) has conditions to be a potential agent to be incorporate into the integrated pest management in sugarcane.

A cultura da cana-de-açúcar Saccharum spp. (Poales: Poaceae) produz diferentes derivados para o mundo: açúcar, etanol e bioenergia. Apesar da aplicação de pesticidas, os insetos-praga ainda causam prejuízos econômicos, dentre eles a cochonilha rosada da cana-de-açúcar Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) causando danos diretos e indiretos à planta. Este estudo avaliou a virulência de três espécies de nematoides entomopatogênicos (NEPs) e suas bactérias simbiontes contra a cochonilha rosada da cana-de-açúcar, em condições de laboratório. Quatorze tratamentos representados pelo controle (água destilada), Heterorhabditis bacteriophora Poinar, 1976 (HB EN01) (Rhabditida: Heterorhabditidae), Steinernema rarum (Doucet, 1986) (PAM25) e Steinernema carpocapsae Weiser, 1955 (All) (Rhabditida: Steinermatidae) nas concentrações de 25, 50, 75 e 100 juvenis infectantes (JIs)/inseto, e o produto químico padrão, tiametoxam, foram testados. Em um segundo experimento, a bactéria Photorhabdus luminescens (Thomas e Poinar, 1979), Xenorhabdus szentirmaii Lengyel, 2005 e Xenorhabdus nematophila (Poinar e Thomas, 1965) (Enterobacterales: Morganellaceae) em 3,0 x 109 células/ml foram avaliadas para cada tratamento. Dez repetições foram estabelecidas, cada uma contendo dez fêmeas/cochonilhas dentro de uma placa de Petri de 10 cm, totalizando 100 indivíduos/tratamento. Todos os tratamentos foram mantidos em condições estáveis (25±1 ºC, U 70±10%, no escuro). Todas as espécies de nematoides infectaram S. sacchari. Steinerma rarum (PAM25) proporcionou a maior mortalidade contra a cochonilha rosada da cana-de-açúcar (79,25%), seguida por H. bacteriophora (HB EN01) (58,25%) e S. carpocapsae (All) (42,50%) (P<0,001). As taxas de mortalidade causada por X. szentirmaii, P. luminescens e X. nematophila foram de 40, 45 e 20%, respectivamente. Steinerma rarum (PAM25) tem condições de ser um agente potencial a ser incorporado ao manejo integrado de pragas da cana-de-açúcar.

Virulence of entomopathogenic nematodes and their symbiotic bacteria, under laboratory conditions, aiming controlling Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) on sugarcane

Abstract Sugarcane crops Saccharum spp. (Poales: Poaceae) produces different derivatives to the world: sugar, ethanol and bioenergy. Despite the application of pesticides, insect pests still cause economic losses, among these the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) causing direct and indirect damage to the plant. This study assess the virulence of three entomopathogenic nematodes (EPNs) species and their symbiont bacteria against the pink sugarcane mealybug, under laboratory conditions. Fourteen treatments represented by control (distilled water), Heterorhabditis bacteriophora Poinar, 1976 (HB EN01) (Rhabditida: Heterorhabditidae), Steinernema rarum (Doucet, 1986) (PAM25) and Steinernema carpocapsae Weiser, 1955 (All) (Rhabditida: Steinermatidae) at concentrations of 25, 50, 75 and 100 infective juveniles (IJs)/insect, and the standard chemical product, thiamethoxam, were assayed. In a second experiment, the bacteria Photorhabdus luminescens (Thomas and Poinar, 1979), Xenorhabdus szentirmaii Lengyel, 2005 and Xenorhabdus nematophila (Poinar and Thomas, 1965) (Enterobacterales: Morganellaceae) at 3.0 x 109 cells/ml were assessed for each treatment. Ten replications were stablished, each one counting ten females/mealybugs inside a 10 cm Petri dish, amounting 100 individuals/treatment. All treatments were kept under stable conditions (25±1 ºC, H 70±10%, in the dark). All nematodes species infected S. sacchari. Steinerma rarum (PAM25) provided the highest mortality against the pink sugarcane mealybug (79.25%), followed by H. bacteriophora (HB EN01) (58.25%) and S. carpocapsae (All) (42.50%) (P 0.001). The mortality rate caused by X. szentirmaii, P. luminescens and X. nematophila were 40, 45 and 20%, respectively. Steinerma rarum (PAM25) has conditions to be a potential agent to be incorporate into the integrated pest management in sugarcane.

Resumo A cultura da cana-de-açúcar Saccharum spp. (Poales: Poaceae) produz diferentes derivados para o mundo: açúcar, etanol e bioenergia. Apesar da aplicação de pesticidas, os insetos-praga ainda causam prejuízos econômicos, dentre eles a cochonilha rosada da cana-de-açúcar Saccharicoccus sacchari (Cockerell, 1895) (Hemiptera: Pseudococcidae) causando danos diretos e indiretos à planta. Este estudo avaliou a virulência de três espécies de nematoides entomopatogênicos (NEPs) e suas bactérias simbiontes contra a cochonilha rosada da cana-de-açúcar, em condições de laboratório. Quatorze tratamentos representados pelo controle (água destilada), Heterorhabditis bacteriophora Poinar, 1976 (HB EN01) (Rhabditida: Heterorhabditidae), Steinernema rarum (Doucet, 1986) (PAM25) e Steinernema carpocapsae Weiser, 1955 (All) (Rhabditida: Steinermatidae) nas concentrações de 25, 50, 75 e 100 juvenis infectantes (JIs)/inseto, e o produto químico padrão, tiametoxam, foram testados. Em um segundo experimento, a bactéria Photorhabdus luminescens (Thomas e Poinar, 1979), Xenorhabdus szentirmaii Lengyel, 2005 e Xenorhabdus nematophila (Poinar e Thomas, 1965) (Enterobacterales: Morganellaceae) em 3,0 x 109 células/ml foram avaliadas para cada tratamento. Dez repetições foram estabelecidas, cada uma contendo dez fêmeas/cochonilhas dentro de uma placa de Petri de 10 cm, totalizando 100 indivíduos/tratamento. Todos os tratamentos foram mantidos em condições estáveis (25±1 ºC, U 70±10%, no escuro). Todas as espécies de nematoides infectaram S. sacchari. Steinerma rarum (PAM25) proporcionou a maior mortalidade contra a cochonilha rosada da cana-de-açúcar (79,25%), seguida por H. bacteriophora (HB EN01) (58,25%) e S. carpocapsae (All) (42,50%) (P 0,001). As taxas de mortalidade causada por X. szentirmaii, P. luminescens e X. nematophila foram de 40, 45 e 20%, respectivamente. Steinerma rarum (PAM25) tem condições de ser um agente potencial a ser incorporado ao manejo integrado de pragas da cana-de-açúcar.

Isolation, identification of entomopathogenic nematodes with insights into their distribution in the Syrian coast regions and virulence against Tuta absoluta.

The occurrence and distribution of entomopathogenic nematodes (EPNs) in the Syrian coast regions remain relatively uncharted. To address this gap in our knowledge, an extensive survey of these ecosystems was essential. This study aims to isolate and identify EPNs from diverse ecosystems within the coastal regions. The distribution of EPNs in cultivated and natural environments was analyzed according to habitat, altitude, and sampling season factors. Between 2017 and 2020, EPNs were recovered from 27 out of 821 soil samples (3.28%) and collected from 24 out of 375 sampling sites (6.4%). Based on morphological, morphometric, and molecular (ITS) characteristics, four EPN species were identified: Heterorhabditis indica (51.85%), representing the first report of its occurrence in the coastal regions, H. bacteriophora (33.33%), H. pakistanense (7.4%), which is also reported for the first time in Syria, and Steinernema affine (7.4%). There were statistical differences in the abundance and recovery frequency of EPNs in each type of habitat. Additionally, there were statistical differences in the altitude and sampling season recovery frequency. Co-inertia analysis revealed correlation between the distribution and occurrence of EPNs in vegetation habitats, altitude, and sampling seasons, as well as some soil characteristics. H. indica and H. bacteriophora were associated with citrus orchards, low-altitude ranges, moderate organic matter, and acidic soil. More specifically, H. indica isolates were correlated with olive orchards, vegetable fields, autumn season, and clay, sandy, and sandy loam soils. Meanwhile, H. bacteriophora isolates were correlated with tobacco fields, grasslands, alkaline pH, spring season, silty loam, and clay loam soils. H. pakistanense was linked to pear orchards, vineyards, moderate pH, and low organic matter. S. affine occurred in walnut orchards, silty soil, higher altitudes, and winter season. The virulence levels of three native EPN isolates (S. affine, H. indica and H. bacteriophora) were evaluated against 3rd and 4th instar larvae (outside and inside mines) and pupae of T. absoluta, a destructive pest in Syria. All three native EPN species exhibited ability to infect and kill the insect, with observed significant differences in their virulence. This study provides an understanding of EPN occurrence, distribution, and their potential for application in sustainable pest control strategies in Syria.

Natural occurrence of entomopathogenic nematodes (Steinernema and Heterorhabditis) and Pristionchus nematodes in black truffle soils from Spain.

The European truffle beetle Leiodes cinnamomeus is the most important pest in black truffle (Tuber melanosporum) plantations. Current control methods against it are inefficient, so entomopathogenic nematodes (EPNs) could play an important role in their population regulation due to their efficacy against many soil-dwelling insect pests. A survey of EPNs and Pristionchus nematodes was conducted in truffle soils of Spain, considering environmental and physical-chemical soil factors. A total of 164 soil samples were collected from forests, productive plantations and null-low productive plantations, representing three distinct black truffle-growing habitat types. EPNs were isolated from seven soil samples (4.3%); four nematodes were identified as Steinernema feltiae and three as Heterorhabditis bacteriophora. Both species were sampled in three types of soil texture (loam, sandy loam or sandy clay loam), characterized by alkaline pH (7.5 to 8.5) and high organic matter (2.1-11.04%). The presence of these EPNs was influenced by habitat type and organic matter content. Pristionchus nematodes were isolated from truffle soil, around truffle fruit bodies and under the elytra of L. cinnamomeus, with Pristionchus maupasi being the most commonly identified species. No significant associations were found between environmental and soil factors and the occurrence of Pristionchus nematodes. These nematodes were found in alkaline soils (pH 7.75 to 8.7), across all seven sampled soil textures, with variable organic matter content (0.73%-5.92%). The ecological trends and the presence of Pristionchus may affect the occurrence of EPNs and their prospective use as biological control agents against L. cinnamomeus in black truffle plantations.

A sustainable grower-based method for entomopathogenic nematode production.

Entomopathogenic nematodes in the genera Steinernema and Heterorhabditis, produced through in vitro or in vivo methods, are effective insect biological control agents. In vivo production yields good-quality nematodes, but the costs associated with obtaining insects and labor make this production system have a low economy of scale. Conceivably, if growers can produce their own nematodes, then the cost could be reduced. Grower-based production systems described to-date are not sustainable because they rely on outside sources to obtain or calibrate inoculum. Here, we describe a self-sufficient grower-based system where the grower can produce in-house nematodes after obtaining the initial inoculum from a reliable source. We validated our approach in 2 experiments comparing in vivo nematode production from standard White traps and a grower-based approach using polyacrylamide gel. For both tested species, Steinernema carpocapsae (Weiser) and Heterorhabditis bacteriophora Poinar, the grower-based approach produced equal to or more nematodes than the standard method. For example, when comparing the average yield of S. carpocapsae-infective juveniles per Galleria mellonella cadaver (n = 30), the standard White trap method produced 159,114 ± 9,669, whereas the grower-based approach produced 244,029 ± 16,241. The sustainable system described herein has promise for wide adoption by growers.

Pheno- and genotyping in vitro dauer juvenile recovery in the nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is an effective biological-control agent of insect pests. The dauer juveniles (DJs) seek for, infect insects, and release cells of the carried symbiotic bacterium of the genus Photorhabdus. Inside the host, the DJs perceive signals from the insect's haemolymph that trigger the exit from the arrested stage and the further development to mature adults. This developmental step is called DJ recovery. In commercial production, a high and synchronous DJ recovery determines the success of liquid-culture mass production. To enhance the understanding about genetic components regulating DJ recovery, more than 160 mutant- and 25 wild type inbred lines (WT ILs) were characterized for DJ recovery induced by cell-free bacterial supernatant. The mutant lines exhibited a broader DJ recovery range than WT ILs (4.6-67.2% vs 1.6-35.7%). A subset of mutant lines presented high variability of virulence against mealworm (Tenebrio molitor) (from 22 to 78% mortality) and mean time survival under oxidative stress (70 mM H2O2; from 10 to 151 h). Genotyping by sequencing of 96 mutant lines resulted in more than 150 single nucleotide polymorphisms (SNPs), of which four results are strongly associated with the DJ recovery trait. The present results are the basis for future approaches in improving DJ recovery by breeding under in vitro liquid-culture mass production in H. bacteriophora. This generated platform of EMS-mutants is as well a versatile tool for the investigation of many further traits of interest in EPNs. KEYPOINTS: • Exposure to bacterial supernatants of Photorhabdus laumondii induces the recovery of Heterorhabditis bacteriophora dauer juveniles (DJs). Both, the bacteria and the nematode partner, influence this response. However, the complete identity of its regulators is not known. • We dissected the genetic component of DJ recovery regulation in H. bacteriophora nematodes by generating a large array of EMS mutant lines and characterizing their recovery pheno- and genotypes. • We determined sets of mutants with contrasting DJ recovery and genotyped a subset of the EMS-mutant lines via genotyping by sequencing (GBS) and identified SNPs with significant correlation to the recovery trait.

Drosophila melanogaster Imd signaling interacts with insulin signaling and alters feeding rate upon parasitic nematode infection.

Significant progress has been made in recent years on exploring immunometabolism, a field that integrates two processes essential for maintaining tissue and organismal homeostasis, immunity and metabolism. The nematode parasite Heterorhabditis gerrardi, its mutualistic bacteria Photorhabdus asymbiotica, and the fruit fly Drosophila melanogaster constitute a unique system to investigate the molecular basis of host immunometabolic response to nematode-bacterial complexes. In this study, we explored the contribution of the two major immune signaling pathways, Toll and Imd, to sugar metabolism in D. melanogaster larvae during infection with H. gerrardi nematodes. We infected Toll or Imd signaling loss-of-function mutant larvae with H. gerrardi nematodes and assessed larval survival ability, feeding rate, and sugar metabolism. We found no significant differences in the survival ability or levels of sugar metabolites in any of the mutant larvae when responding to H. gerrardi infection. However, we found that the Imd mutant larvae have higher feeding rate than controls during the early stages of infection. In addition, feeding rates are lower in Imd mutants relative to the control larvae as the infection progresses. We further showed that Dilp2 and Dilp3 gene expression increases in Imd mutants compared to controls early in the infection, but their expression levels decrease at later times. These findings indicate that Imd signaling activity regulates the feeding rate and Dilp2 and Dilp3 expression in D. melanogaster larvae infected with H. gerrardi. Results from this study facilitate our understanding of the link between host innate immunity and sugar metabolism in the context of infectious diseases caused by parasitic nematodes.

Efficacy of entomopathogenic fungi, nematodes and spinetoram combinations for integrated management of Thrips tabaci.

BACKGROUND: Two consecutive field trials using a blend of entomopathogens in combination with a new chemistry insecticide were conducted to determine treatment effects on onion thrips (Thrips tabaci Lindeman) populations, crop damage, plant development, crop yield and impact on natural enemies. Products were tested in an onion cropping system and included the insect pathogenic fungus Beauveria bassiana (isolate WG-11), an entomopathogenic nematode Heterorhabditis bacteriophora (strain VS) and the new-chemistry chemical insecticide spinetoram. RESULTS: In all treatments, a significant decrease in thrips per plant population was detected in both trials. Overall, dual application of entomopathogens and insecticide was more effective than singly applied treatments. The lowest number of thrips larvae (1.96 and 3.85) and adults (0.00 and 0.00) were recorded when treated with dual application of B. bassiana and spinetoram at 7 days post application (DPA) after the second spray application in 2017and 2018, respectively. Damage on onion plants was considerably decreased in all treatments relative to the control. The lowest damage was observed on onion plants treated with B. bassiana + spinetoram at 7 DPA after the second spray application during both years. A significant decrease in the number of natural enemies (beetles, spiders, mites, lacewings, ants and bugs) on onion plants was recorded during both years. Insect pathogens when applied alone and in combination with each other considerably protected arthropod natural enemies compared to insecticide application applied alone. Significant increase in plant agronomic traits was observed compared to the control. Among all the treatments, B. bassiana + spinetoram produced maximum leaf length, leaf weight, total leaves, neck diameter, bulb diameter, number of rings per bulb, bulb weight, dry matter and plant yield following the 2017and 2018 applications, respectively. CONCLUSION: The findings of the study reveal the potential of using insect pathogens and insecticide for control of T. tabaci. However, combinations containing spinetoram are harmful to nontarget organisms, whereas biological control agents help in protecting biodiversity in onion agroecosystems. © 2023 Society of Chemical Industry.

Entomopathogenic nematodes for biological control of Psylliodes chrysocephala (Coleoptera: Chrysomelidae) in oilseed rape.

Winter oilseed rape (Brassica napus) is one of the largest crops in Europe and the cabbage stem flea beetle Psylliodes chrysocephala is one of its major pests. Since the ban of neonicotinoids for seed treatment, farmers apply pyrethroids in autumn to control the cabbage stem flea beetle. Current studies show that the insect develops resistance to this group of chemicals. Biological control with entomopathogenic nematodes (EPNs) represents a possible, environmentally friendly alternative control measure. In the present work, we considered three strategies to control the cabbage stem flea beetle: applying the nematodes against the first larval stage in the soil, against the second and third larval stages inside the plant or against the adult beetles. In laboratory experiments, we found the third larval instar to be the most susceptible stage and the adult beetle the less susceptible one. Steinernema feltiae and the cold active SDT1-IL1 Heterorhabditis bacteriophora strain, with a reduction potential of 89 and 76 %, respectively, proved to be the most virulent EPNs against P. chrysocephala in pot experiments at 15 °C. Moreover, we performed four field trials to test the efficacy of H. bacteriophora and S. feltiae against the larvae. The highest reduction in the field trials was 45% and 39%, obtained with SDT1-IL1 and a mixture of H. bacteriophora and S.feltiae, respectively. The present study provides preliminary information about the potential of EPNs to control P. chrysocephala and represents a start point for the development of a competitive and sustainable alternative to pyrethroids.

HETERORHABDITIS BACTERIOPHORA NEMATODES ARE SENSITIVE TO THE BACTERIAL PATHOGEN PHOTORHABDUS ASYMBIOTICA.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora infects a wide range of insect hosts with the aid of its mutualistic bacteria Photorhabdus luminescens. While the mutualistic relationship between H. bacteriophora and P. luminescens and the infectivity of the nematode-bacteria complex have been characterized, how nematode fitness is affected by entomopathogenic bacteria existing in association with other EPN species remains poorly understood. In this study, the survival of H. bacteriophora infective juveniles containing or lacking P. luminescens was tested against the entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus asymbiotica as well as the non-pathogenic Escherichia coli. While X. nematophila and E. coli did not significantly affect the survival of H. bacteriophora, P. asymbiotica exerted a significant effect on nematode survival, particularly on those lacking P. luminescens. These results imply that P. asymbiotica encodes factors that are pathogenic to EPNs. Future efforts will focus on the identification of the bacterial molecular components that induce these effects. This study makes an important contribution to a growing body of research aimed at exploiting the full potential of nematode-bacterial complexes for eliminating noxious insect pests and treating infectious diseases caused by parasitic nematodes.

Evaluation of Entomopathogenic Nematodes against Common Wireworm Species in Potato Cultivation.

Wireworms (Coleoptera: Elateridae) are common insect pests that attack a wide range of economically important crops including potatoes. The control of wireworms is of prime importance in potato production due to the potential damage of the larvae to tuber quantity and quality. Chemical insecticides, the main control strategy against wireworms, generally fail to provide satisfactory control due to the lack of available chemicals and the soil-dwelling habits of the larvae. In the last decades, new eco-friendly concepts have emerged in the sustainable control of wireworms, one of which is entomopathogenic nematodes (EPNs). EPNs are soil-inhabitant organisms and represent an ecological approach to controlling a great variety of soil-dwelling insect pests. In this study, the susceptibility of Agriotes sputator Linnaeus and A. rufipalpis Brullé larvae, the most common wireworm species in potato cultivation in Türkiye, to native EPN strains [Steinernema carpocapsae (Sc_BL22), S. feltiae (Sf_BL24 and Sf_KAY4), and Heterorhabditis bacteriophora (Hb_KAY10 and Hb_AF12)] were evaluated at two temperatures (25 and 30 °C) in pot experiments. Heterorhabditis bacteriophora Hb_AF12 was the most effective strain at 30 °C six days post-inoculation and caused 37.5% mortality to A. rufipalpis larvae. Agriotes sputator larvae were more susceptible to tested EPNs at the same exposure time, and 50% mortality was achieved by two EPNs species, Hb_AF12 and Sc_BL22. All EPN species/strains induced mortality over 70% to both wireworm species at both temperatures at 100 IJs/cm2, 18 days post-treatment. The results suggest that tested EPN species/strains have great potential in the control of A. sputator and A. rufipalpis larvae.

Entomopathogenic nematodes isolated from agricultural areas of Thailand and their activity against the larvae of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae).

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have been considered to be effective biological control agents for several insects. In this study, we isolated and identified EPNs from soil samples in agricultural areas of northern Thailand and evaluated their efficacy for controlling larvae of three mosquito vector species, Aedes aegypti, Ae. albopictus and Culex quinquefasciatus. A total of 51 of 1,000 soil samples were positive (5.1% prevalence) for EPNs, which were identified through sequencing of the rDNA and ITS to 37 Steinernema isolates (3.7%) and 14 Heterorhabditis isolates (1.4%). For the bioassay, the larvae of mosquitoes were exposed to Steinernema surkhetense (eALN6.3_TH), Steinernema lamjungense (eALN11.5_TH), Heterorhabditis indica (eACM14.2_TH) and Heterorhabditis bacteriophora (eALN18.2_TH). Heterorhabditis bacteriophora showed the highest efficacy against Ae. aegypti and Cx. quinquefasciatus. At 96 h after exposure, the mortality rates were 60.0 and 91.7%, respectively. The EPNs were observed in the dead mosquito larvae, which were mostly found in the thorax followed by the head and abdomen. Some EPNs were dead with melanization, and some were able to survive in the cavity of mosquito larvae. Our results show the low prevalence of EPN in agricultural areas of Thailand. Moreover, H. bacteriophora may be considered an alternative biocontrol agent for managing and controlling these vector mosquitoes.

From the laboratory to the field: efficacy of entomopathogenic nematodes to control the cattle tick.

BACKGROUND: The control of ticks is challenged by the resistance of tick populations to chemical acaricides. In this study, we evaluated, under laboratory conditions, the efficacy of Heterorhabditis bacteriophora against Rhipicephalus (Boophilus) microplus engorged females with varying body weights (150, 200, 250, 300 or 350 mg per female) or from eight different geographical populations. We also determined the efficacy of H. bacteriophora for tick control under field conditions. RESULTS: R. microplus engorged females with varying body weights exposed to 150 juveniles of H. bacteriophora resulted in a high control efficacy (97.5% to 98.4%). Tests with females from different geographical populations comprised eight tick strains treated with H. bacteriophora and their respective control groups. The biological parameters of females exposed to nematode treatments did not differ significantly and resulted in 89% to 99% of control efficacy. Trials conducted under field conditions were performed in field plots with Megathyrsus maximus grass. Treatment groups received eight cadavers of Tenebrio molitor fully colonized with H. bacteriophora at 1 week prior to the release of female ticks, whereas control groups were untreated. On the first day of the experiment, six engorged females were distributed in each plot. On day 42 and day 63, the apical portion of the grasses with R. microplus larvae were collected and quantified. The population of R. microplus larvae was reduced up to 73.1% in plots treated with H. bacteriophora at day 63 after treatment. CONCLUSION: R. microplus engorged females with varying body weights or from different geographical populations were highly susceptible to H. bacteriophora. The field test demonstrated the efficacy of H. bacteriophora in reducing R. microplus larvae in infested pastures. © 2022 Society of Chemical Industry.

Effect of Host Size on Susceptibility of Melanotus Communis (Coleoptera: Elateridae) Wireworms to Entomopathogens.

Wireworms, the soil-borne larvae of click beetles (Coleoptera: Elateridae), are important crop pests throughout the world. In the eastern U.S., Melanotus communis larvae attack grain, root/ tuber, and vegetable crops. Our objectives were to characterize the pathogenicity and virulence of fungal and nematode entomopathogens on M. communis wireworms, and determine if wireworm size affected virulence. Pathogens tested included five entomopathogenic nematodes, Steinernema carpocapsae (All strain), S. feltiae (SN strain), S. riobrave (355 strain), Heterorhabditis bacteriophora (VS strain), and H. indica (HiHom1 strain); and two entomopathogenic fungi, Beauveria bassiana (GHA strain) and Cordyceps javanica (WF-GA17 strain). None of the pathogens tested caused >15% mortality at 7 or 14 days post-inoculation. Mortality was highest in S. carpocapsae (All strain); the other entomopathogens did not cause higher mortality than the untreated control. Overall, smaller wireworms were more susceptible than larger wireworms. Our results suggested that M. communis wireworms have defenses that limit the ability of the entomopathogens we tested to infect the wireworms. Conceivably, other entomopathogen strains or species may be more effective. Natural populations of entomopathogens may contribute to wireworm population reduction, but further studies are warranted before entomopathogens can be used for M. communis management.

RNA-Sequencing of Heterorhabditis nematodes to identify factors involved in symbiosis with Photorhabdus bacteria.

BACKGROUND: Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria. RESULTS: A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified. CONCLUSIONS: The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.