A Nematode Isolate, Oscheius Tipulae, Exhibiting a Wide Entomopathogenic Spectrum and its Application to Control Dipteran Insect Pests.

An entomopathogenic nematode, Oscheius tipulae, was isolated from a soil sample. The identification of this species was supported by morphological and molecular markers. The nematode isolate exhibited pathogenicity against different target insects including lepidopteran, coleopteran, and dipteran insects. The virulence of this nematode was similar to that of a well-known entomopathogenic nematode, Steinernema carpocapsae, against the same insect targets. A comparative metagenomics analysis of these two nematode species predicted the existence of a combined total of 272 bacterial species in their intestines, of which 51 bacterial species were shared between the two nematode species. In particular, the common gut bacteria included several entomopathogenic bacteria including Xenorhabdus nematophila, which is known as a symbiotic bacterium to S. carpocapsae. The nematode virulence of O. tipulae to insects was enhanced by an addition of dexamethasone but suppressed by an addition of arachidonic acid, suggesting that the immune defenses of the target insects against the nematode infection is mediated by eicosanoids, which would be manipulated by the symbiotic bacteria of the nematode. Unlike S. carpocapsae, O. tipulae showed high virulence against dipteran insects including fruit flies, onion flies, and mosquitoes. O. tipulae showed particularly high control efficacies against the onion maggot, Delia platura, infesting the Welsh onion in the rhizosphere in both pot and field assays.

Exploring the effects of entomopathogenic nematode symbiotic bacteria and their cell free filtrates on the tomato leafminer Tuta absoluta and its predator Nesidiocoris tenuis.

The use of biocontrol agents, such as predators and entomopathogenic nematodes, is a promising approach for the effective control of the tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidaean), an oligophagous insect feeding mainly on Solanaceae species and a major pest of field- and greenhouse-grown tomatoes globally. In this context, the effects of two entomopathogenic nematode species Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae), as well as their respective bacterial symbionts, Xenorhabdus nematophila and Photorhabdus luminescens (Enterobacterales: Morganelaceae), which were applied as bacterial cell suspensions and as crude cell-free liquid filtrates on T. absoluta larvae, were investigated. The results showed that of all treatments, the nematodes S. carpocapsae and H. bacteriophora were the most effective, causing up to 98 % mortality of T. absoluta larvae. Regarding bacteria and their filtrates, the bacterium X. nematophila was the most effective (69 % mortality in young larvae), while P. luminescens and both bacterial filtrates showed similar potency (ca. 48-55 % mortality in young larvae). To achieve a holistic approach of controlling this important pest, the impact of these factors on the beneficial predator Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) was also studied. The results demonstrated that although nematodes and especially S. carpocapsae, caused significant mortality on N. tenuis (87 %), the bacterial cell suspensions of X. nematophila and P. luminescens and crude cell-free liquid filtrates had minimum impact on this beneficial predator (∼11-30 % mortality).

The Activin Branch Ligand Daw Regulates the Drosophila melanogaster Immune Response and Lipid Metabolism against the Heterorhabditis bacteriophora Serine Carboxypeptidase.

Despite impressive advances in the broad field of innate immunity, our understanding of the molecules and signaling pathways that control the host immune response to nematode infection remains incomplete. We have shown recently that Transforming Growth Factor-ß (TGF-ß) signaling in the fruit fly Drosophila melanogaster is activated by nematode infection and certain TGF-ß superfamily members regulate the D. melanogaster anti-nematode immune response. Here, we investigate the effect of an entomopathogenic nematode infection factor on host TGF-ß pathway regulation and immune function. We find that Heterorhabditis bacteriophora serine carboxypeptidase activates the Activin branch in D. melanogaster adults and the immune deficiency pathway in Activin-deficient flies, it affects hemocyte numbers and survival in flies deficient for Activin signaling, and causes increased intestinal steatosis in Activin-deficient flies. Thus, insights into the D. melanogaster signaling pathways and metabolic processes interacting with H. bacteriophora pathogenicity factors will be applicable to entomopathogenic nematode infection of important agricultural insect pests and vectors of disease.

OxyR is required for oxidative stress resistance of the entomopathogenic bacterium Xenorhabdus nematophila and has a minor role during the bacterial interaction with its hosts.

Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae, and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.

Effect of male parental gamma irradiation on host suitability of its F1 progeny of a lepidopteran tropical pest, Spodoptera litura (Fabr.) towards development and virulence of entomopathogenic nematodes, Steinernema thermophilum.

The suitability of F1 progeny insect larvae of the irradiated male parent, Spodoptera litura (Fabr.) for infective juveniles (IJs) of entomopathogenic nematodes (EPN), Steinernema thermophilum was assessed to comprehend the feasibility of combining EPNs with nuclear pest control tactic. As compared to the control, the IJs induced faster host mortality with reduced proliferation in F1 host larvae. IJs derived from F1 host larvae exhibited almost similar proliferation capacity on normal hosts as in control. Further, the molecular basis of EPNs induced mortality in F1 host larvae was evaluated. Dual stress of EPN infection and irradiation induced downregulation of the relative mRNA expression of antimicrobial genes and upregulated expression of antioxidative genes. A pronounced effect of EPNs in association with irradiation stress was apparent on host mortality. Radiation induced sterile F1 insect larvae of S. litura acted as a reasonably suitable host for EPNs and also provided the environment for developing viable EPNs for their potential use as biocontrol agents.

Metabolic disruptions in Biomphalaria glabrata induced by Heterorhabditis bacteriophora HP88: Implications for entomopathogenic nematodes in biological control.

Research on the use of entomopathogenic nematodes (EPNs) as a potential tool for the biological control of invertebrates has been growing in recent years, including studies involving snails with One Health importance. In this study, the effect of exposure time (24 or 48 h) of Heterorhabditis bacteriophora HP88 on the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the concentration of total proteins, uric acid, and urea in the hemolymph of Biomphalaria glabrata, were investigated. The concentrations of these metabolic markers were measured weekly until the end of the third week after exposure. Along with a significant reduction in total protein levels, a significant increase (p < 0.01) in uric acid and urea contents in the hemolymph of B. glabrata exposed to H. bacteriophora was observed. The accumulation of urea in these mollusks could lead to deleterious effects due to its high toxicity, inducing significant cell damage. Variations in transaminase activities were also observed, with snails exposed to EPNs showing significantly higher values (p < 0.01) than individuals in the control group, both for ALT and AST. These results indicate that experimental exposure to infective juveniles of H. bacteriophora causes significant alterations in the metabolic pattern of B. glabrata, compromising the maintenance of its homeostasis. Finally, exposure for 48 h caused more damage to the planorbid in question compared to snails exposed for 24 h, suggesting that the exposure time may influence the intensity of the host's response.

The sky is not the limit: Successful foliar application of Steinernema spp. entomopathogenic nematodes to control Lepidopteran caterpillars.

Entomopathogenic nematodes (EPNs) are ubiquitous soil-thriving organisms that use chemical cues to seek and infect soil-dwelling arthropods, yielding various levels of biological control. Going beyond soil application, scientists and practitioners started exploring the option of applying EPNs onto the foliage of crops in attempts to manage leaf-dwelling insect pests as well. Despite some success, particularly with protective formulations, it remains uncertain whether EPNs could indeed survive the phyllospheric environment, and successfully control foliar insect pests. In this context, we tested the potential of commercially produced Steinernema feltiae and S. carpocapsae, two of the most commonly used EPNs in the field of biological control, in controlling Lepidopteran foliar pests of economic importance, i.e. Tuta absoluta and Spodoptera spp. caterpillars as models. We first tested the survival and efficacy of both EPN species against the Lepidopteran caterpillars when applied onto tomato, sweet pepper and lettuce leaves, under controlled conditions and in commercial greenhouse conditions, respectively. Subsequently, we explored the behavioural responses of the EPNs to environmental cues typically encountered in the phyllosphere, and analysed plant volatile organic compounds (VOCs). Our results show that both S. feltiae and S. carpocapsae successfully survived and infected the foliar caterpillars, reaching similar level of control to a standard chemical pesticide in commercial practices. Remarkably, both EPN species survived and remained effective up to four days in the phyllosphere, and needed only a few hours to successfully penetrate the caterpillars. Interestingly, S. feltiae was attracted to VOCs from tomato plants, and tended to prefer those from caterpillar-induced plants, suggesting that the nematodes may actively forage toward its host, although it has never been exposed to leaf-borne volatiles during its evolution. The present study shows the high potential of steinernematids in managing major foliar pests in greenhouses and in becoming a key player in foliar biological control. In particular, the discovery that EPNs use foliar VOCs to locate caterpillar hosts opens up new opportunities in terms of application techniques and affordable effective doses.

Volatile organic compounds released from entomopathogenic nematode-infected insect cadavers for the biocontrol of Meloidogyne incognita.

BACKGROUND: Root-knot nematodes (RKNs), Meloidogyne spp., are one of the most destructive polyphagous plant-parasitic nematodes. They pose a serious threat to global food security and are difficult to control. Entomopathogenic nematodes (EPNs) show promise in controlling RKNs. However, it remains unclear whether the volatile organic compounds (VOCs) emitted from EPN-infected cadavers can control RKNs. RESULTS: We investigated the fumigation activity of VOCs released from cadavers infected by five different species of EPNs on RKNs in Petri dishes, and found that VOCs released from Steinernema feltiae (SN strain) and S. carpocapsae (All strain) infected cadavers had a significant lethal effect on second-stage juveniles (J2s) of Meloidogyne incognita. The VOCs released from the cadavers infected with S. feltiae were analyzed using SPME-GC/MS. Dimethyl disulfide (DMDS), tetradecane, pentadecane, and butylated hydroxytoluene (BHT), were selected for a validation experiment with pure compounds. The DMDS compound had significant nematicidal activity and repelled J2s. DMDS also inhibited egg hatching and the invasion of tomato roots by J2s. In a pot experiment, the addition of S. feltiae-infected cadavers and cadavers wrapped with a 400-mesh nylon net also significantly reduced the population of RKNs in tomato roots after 7 days. The number of root knots and eggs was reduced by 58% and 74.34%, respectively, compared to the control. CONCLUSION: These results suggested that the VOCs emitted by the EPN-infected cadavers affected various developmental stages of M. incognita and thus have the potential to be used in controlling RKNs through multiple methods. © 2024 Society of Chemical Industry.

Biocontrol potential of six Heterorhabditis bacteriophora strains isolated in the Azores Archipelago.

Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm-2, a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.

PHASMARHABDITIS CALIFORNICA (NEMATODA: RHABDITIDAE) HAS REDUCED ESTABLISHMENT SUCCESS AND PROGENY PRODUCTION IN THE PRESENCE OF PRISTIONCHUS ENTOMOPHAGUS (NEMATODA: DIPLOGASTRIDAE).

Phasmarhabditis (syn. Pellioditis) californica is a facultative parasite that has been marketed as a popular biocontrol agent against pestiferous slugs in England, Scotland, and Wales. The necromenic nematode Pristionchus entomophagus has also been recovered from slugs infected with Ph. californica. In this study, we experimentally investigated the outcome of single and mixed applications of Pr. entomophagus and Ph. californica on the slug Deroceras reticulatum (Müller). Host mortality was comparable for single and mixed applications of Ph. californica, with time to death significantly shorter in both treatment groups compared with controls. However, trials with Pr. entomophagus alone did not cause any significant host mortality relative to controls. Compared with the single Ph. californica applications, mixed applications resulted in 67% fewer infective juveniles establishing in the host, and subsequently far fewer infective juveniles were recovered in the next generation. In contrast, the establishment rate and progeny production in Pr. entomophagus were not impacted by the presence of Ph. californica (i.e., mixed applications). Hence, the presence of Pr. entomophagus had a deleterious effect on the establishment success and progeny production of Ph. californica. Our findings reveal an asymmetrical, antagonistic interaction between Ph. californica and Pr. entomophagus and highlight the importance of understanding the ecological relationships between co-occurring species. A decrease in parasite establishment success and progeny production has the potential to directly impact the persistence, sustainability, and efficacy of Ph. californica as a biological control agent.

Long-term suppression of turfgrass insect pests with native persistent entomopathogenic nematodes.

Entomopathogenic nematodes (EPNs) can control several important turfgrass insect pests including white grubs, weevils, cutworms, and sod webworms. But most of the research has focused on inundative releases in a biopesticide strategy using EPN strains that may have lost some of their ability to persist effectively over years of lab maintenance and / or selection for virulence and efficient mass-production. Our study examined the potential of fresh field isolate mixes of endemic EPNs to provide multi-year suppression of turfgrass insect pests. In early June 2020, we applied isolate mixes from golf courses of the EPNs Steinernema carpocapsae, Heterorhabditis bacteriophora, and their combination to plots straddling fairway and rough on two golf courses in central New Jersey, USA. Populations of EPNs and insect pests were sampled on the fairway and rough side of the plots from just before EPN application until October 2022. EPN populations increased initially in plots treated with the respective species. Steinernema carpocapsae densities stayed high for most of the experiment. Heterorhabditis bacteriophora densities decreased after 6 months and stabilized at lower levels. Several insect pests were reduced across the entire experimental period. In the fairway, the combination treatment reduced annual bluegrass weevil larvae (59 % reduction) and adults (74 %); S. carpocapsae reduced only adults (42 %). White grubs were reduced by H. bacteriophora (67 %) and the combination (63 %). Black turfgrass ataenius adults were reduced in all EPN treatments (43-62 %) in rough and fairway. Sod webworm larvae were reduced by S. carpocapsae in the fairway (75 %) and the rough (100 %) and by H. bacteriophora in the rough (75 %). Cutworm larvae were reduced in the fairway by S. carpocapsae (88 %) and the combination (75 %). Overall, our observations suggest that inoculative applications of fresh field isolate mixes of endemic EPNs may be a feasible approach to long-term suppression of insect pests in turfgrass but may require periodic reapplications.

Heterorhabditis indica (Nematoda: Rhabditida) a possible new biological control agent against the vector of Chagas disease.

Chagas disease is a zoonosis caused by the protozoan Trypanosoma cruzi and transmitted through the feces of triatomines, mainly in Latin America. Since the 1950s, chemical insecticides have been the primary method for controlling these triatomines, yet resistance has emerged, prompting the exploration of alternative approaches. The objective of this research was to test the capacity of the entomopathogenic nematodes Heterorhabditis indica and its symbiotic bacteria Photorhabdus luminescens, to produce mortality of Triatoma dimidiata a key vector of T. cruzi in Mexico under laboratory conditions. Two bioassays were conducted. In the first bioassay, the experimental unit was a 250 ml plastic jar with 100 g of sterile soil and three adult T. dimidiata. Three nematode quantities were tested: 2250, 4500, and 9000 nematodes per 100 g of sterile soil (n/100 g) per jar, with 3 replicates for each concentration and 1 control per concentration (1 jar with 100 g of sterile soil and 3 T. dimidiata without nematodes). The experimental unit of the second bioassay was a 500 ml plastic jar with 100 g of sterile soil and 4 adult T. dimidiata. This bioassay included 5, 50, 500, and 5000 n/100 g of sterile soil per jar, with 3 replicates of each quantity and 1 control per quantity. Data were analyzed using Kaplan-Meyer survival analysis. Electron microscopy was used to assess the presence of nematodes and tissue damage in T. dimidiata. The results of the first bioassay demonstrated that the nematode induced an accumulated average mortality ranging from 55.5 % (2250 n/100 g) to 100 % (4500 and 9000 n/100 g) within 144 h. In the second bioassay, the 5000 n/100 g concentration yielded 87.5 % mortality at 86 h, but a concentration as small as 500 n/100 g caused 75 % mortality from 84 h onwards. Survival analysis indicated higher T. dimidiata mortality with increased nematode quantities, with significant differences between the 4500, 5000, and 9000 n/100 g and controls. Electron microscopy revealed the presence of nematodes and its presumably symbiotic bacteria in the digestive system of T. dimidiata. Based on these analyses, we assert that the H. indica and P. luminescens complex causes mortality in adult T. dimidiata under laboratory conditions.

Integrated approaches for Solenopsis invicta (Hymenoptera: Formicidae) management: insights from laboratory studies with entomopathogenic nematodes and insecticides.

BACKGROUND: In agricultural pest management, especially in combatting the invasive red imported fire ant (RIFA, Solenopsis invicta), significant challenges emerge as a consequence of the constraints of solely depending on chemical insecticides or entomopathogenic nematodes (EPNs). The utilization of chemical insecticides carries environmental and ecological hazards, whereas EPNs, when applied independently, might not offer the immediate effectiveness necessary for adequate RIFA suppression. Acknowledging these hurdles, our study investigates a synergistic method that integrates EPNs with chemical insecticides, aiming to fulfill the urgent demand for more efficient and environmentally friendly pest control solutions. RESULTS: Our evaluation focused on the interaction between the highly pathogenic Steinernema riobrave 7-12 EPN strain and prevalent insecticides, specifically beta-cypermethrin and a mixture of bifenthrin and clothianidin, applied at highly diluted recommended concentrations. The findings revealed a notable increase in RIFA mortality rates when EPNs and these insecticides were used together, outperforming the results achieved with each method individually. Remarkably, this enhanced efficacy was especially evident at lower concentrations of the bifenthrin-clothianidin mixture, indicating a valuable approach to minimizing reliance on chemical insecticides in agriculture. Furthermore, the high survival rates of EPNs alongside the tested insecticides indicate their compatibility and potential for sustained use in integrated pest management programs. CONCLUSION: Our research underscores the effectiveness of merging EPNs with chemical insecticides as a powerful and sustainable strategy for RIFA management. This combined approach not only meets the immediate challenges of pest control in agricultural settings, but also supports wider environmental objectives by reducing the dependency on chemical insecticides. © 2024 Society of Chemical Industry.

Toxicological impacts of microplastics on virulence, reproduction and physiological process of entomopathogenic nematodes.

Microplastics have emerged as significant and concerning pollutants within soil ecosystems. Among the soil biota, entomopathogenic nematodes (EPNs) are lethal parasites of arthropods, and are considered among the most effective biological agents against pests. Infective juveniles (IJs) of EPNs, as they navigate the soil matrix scavenging for arthropod hosts to infect, they could potentially encounter microplastics. Howver, the impact of microplastics on EPNs has not been fully elucidated yet. We addressed this gap by subjecting Steinernema feltiae EPNs to polystyrene microplastics (PS-MPs) with various sizes, concentrations, and exposure durations. After confirming PS-MP ingestion by S. feltiae using fluorescent dyes, we found that the PS-MPs reduced the survival, reproduction, and pathogenicity of the tested EPNs, with effects intensifying for smaller PS-MPs (0.1-1 µm) at higher concentrations (105 µg/L). Furthermore, exposure to PS-MPs triggered oxidative stress in S. feltiae, leading to increased reactive oxygen species levels, compromised mitochondrial membrane potential, and increased antioxidative enzyme activity. Furthermore, transcriptome analyses revealed PS-MP-induced suppression of mitochondrial function and oxidative phosphorylation pathways. In conclusion, we show that ingestion of PS-MPs by EPNs can compromise their fitness, due to multple toxicity effects. Our results bear far-reaching consequences, as the presence of microplastics in soil ecosystems could undermine the ecological role of EPNs in regulating pest populations.

Behavioral and molecular response of the insect parasitic nematode Steinernema carpocapsae to plant volatiles.

Entomopathogenic nematodes (EPNs) use the chemical cues emitted by insects and insect-damaged plants to locate their hosts. Steinernema carpocapsae, a species of EPN, is an established biocontrol agent used against insect pests. Despite its promising potential, the molecular mechanisms underlying its ability to detect plant volatiles remain poorly understood. In this study, we investigated the response of S. carpocapsae infective juveniles (IJs) to 8 different plant volatiles. Among these, carvone was found to be the most attractive volatile compound. To understand the molecular basis of the response of IJs to carvone, we used RNA-Seq technology to identify gene expression changes in response to carvone treatment. Transcriptome analysis revealed 721 differentially expressed genes (DEGs) between carvone-treated and control groups, with 403 genes being significantly upregulated and 318 genes downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the responsive DEGs to carvone attraction were mainly involved in locomotion, localization, behavior, response to stimulus, and olfactory transduction. We also identified four upregulated genes of chemoreceptor and response to stimulus that were involved in the response of IJs to carvone attraction. Our results provide insights into the potential transcriptional mechanisms underlying the response of S. carpocapsae to carvone, which can be utilized to develop environmentally friendly strategies for attracting EPNs.

A Mediator subunit imparts robustness to a polyphenism decision.

Polyphenism is a type of developmental plasticity that translates continuous environmental variability into discontinuous phenotypes. Such discontinuity likely requires a switch between alternative gene-regulatory networks, a principle that has been borne out by mechanisms found to promote morph-specific gene expression. However, whether robustness is required to execute a polyphenism decision has awaited testing at the molecular level. Here, we used a nematode model for polyphenism, Pristionchus pacificus, to identify the molecular regulatory factors that ensure the development of alternative forms. This species has a dimorphism in its adult feeding structures, specifically teeth, which are a morphological novelty that allows predation on other nematodes. Through a forward genetic screen, we determined that a duplicate homolog of the Mediator subunit MDT-15/MED15, P. pacificus MDT-15.1, is necessary for the polyphenism and the robustness of the resulting phenotypes. This transcriptional coregulator, which has a conserved role in metabolic responses to nutritional stress, coordinates these processes with its effects on this diet-induced polyphenism. Moreover, this MED15 homolog genetically interacts with two nuclear receptors, NHR-1 and NHR-40, to achieve dimorphism: Single and double mutants for these three factors result in morphologies that together produce a continuum of forms between the extremes of the polyphenism. In summary, we have identified a molecular regulator that confers discontinuity to a morphological polyphenism, while also identifying a role for MED15 as a plasticity effector.

Entomopathogenic potential of bacteria associated with soil-borne nematodes and insect immune responses to their infection.

Soil-borne nematodes establish close associations with several bacterial species. Whether they confer benefits to their hosts has been investigated in only a few nematode-bacteria systems. Their ecological function, therefore, remains poorly understood. In this study, we isolated several bacterial species from rhabditid nematodes, molecularly identified them, evaluated their entomopathogenic potential on Galleria mellonella larvae, and measured immune responses of G. mellonella larvae to their infection. Bacteria were isolated from Acrobeloides sp., A. bodenheimeri, Heterorhabditis bacteriophora, Oscheius tipulae, and Pristionchus maupasi nematodes. They were identified as Acinetobacter sp., Alcaligenes sp., Bacillus cereus, Enterobacter sp., Kaistia sp., Lysinibacillus fusiformis, Morganella morganii subsp. morganii, Klebsiella quasipneumoniae subsp. quasipneumoniae, and Pseudomonas aeruginosa. All bacterial strains were found to be highly entomopathogenic as they killed at least 53.33% G. mellonella larvae within 72h post-infection, at a dose of 106 CFU/larvae. Among them, Lysinibacillus fusiformis, Enterobacter sp., Acinetobacter sp., and K. quasipneumoniae subsp. quasipneumoniae were the most entomopathogenic bacteria. Insects strongly responded to bacterial infection. However, their responses were apparently little effective to counteract bacterial infection. Our study, therefore, shows that bacteria associated with soil-borne nematodes have entomopathogenic capacities. From an applied perspective, our study motivates more research to determine the potential of these bacterial strains as biocontrol agents in environmentally friendly and sustainable agriculture.

Role of adipokinetic hormone in the Colorado potato beetle, Leptinotarsa decemlineata infected with the entomopathogenic nematode Steinernema carpocapsae.

The effects of the entomopathogenic nematode Steinernema carpocapsae on the Colorado potato beetle (CPB) Leptinotarsa decemlineata and the involvement of adipokinetic hormone (AKH) in the responsive reactions were examined in this study. It was observed that nematode application doubled the amount of AKH (Peram-CAH-I and Peram-CAH-II) in the central nervous system of L. decemlineata, indicating mobilization of anti-stress reactions in the body. Furthermore, the external co-application of Peram-CAH-II with the nematode significantly increased beetle mortality (5.6 and 1.8 times, 1 and 2 days after application, respectively). The mechanism underlying this phenomenon was investigated. As the effect on gut characteristics was equivocal, it was assumed that the nematodes profited from the observed mobilization of metabolites from the fat body into the Peram-CAH-II-induced hemolymph. This phenomenon supplied nematodes with a more nutrient-dense substrate on which they propagated. Furthermore, Peram-CAH-II lowered vitellogenin expression in the fat body, particularly in males, thus limiting the anti-pathogen defense capacity of the protein. However, there could be other possible mechanisms underpinning this chain of events. The findings could be theoretically intriguing but could also aid in developing real insect pest control methods in the future.

The effect of temperature conditioning (9°C and 20°C) on the proteome of entomopathogenic nematode infective juveniles.

Entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis are parasites which kill and reproduce within insects. While both have life cycles centred around their developmentally arrested, nonfeeding and stress tolerant infective juvenile (IJ) stage, they are relatively distantly related. These IJs are promising biocontrol agents, and their shelf life and stress tolerance may be enhanced by storage at low temperatures. The purpose of this study was to investigate how the proteome of the IJs of two distantly related EPN species is affected by storage at 9°C (for up to 9 weeks) and 20°C (for up to 6 weeks), using label-free quantitative proteomics. Overall, more proteins were detected in S. carpocapsae (2422) than in H. megidis (1582). The S. carpocapsae proteome was strongly affected by temperature, while the H. megidis proteome was affected by both time and temperature. The proteins which increased in abundance to the greatest extent in S. carpocapsae IJs after conditioning at 9°C were chaperone proteins, and proteins related to stress. The proteins which increased in abundance the most after storage at 20°C were proteins related to the cytoskeleton, cell signalling, proteases and their inhibitors, which may have roles in infection. The proteins which decreased in abundance to the greatest extent in S. carpocapsae after both 9°C and 20°C storage were those associated with metabolism, stress and the cytoskeleton. After storage at both temperatures, the proteins increased to the greatest extent in H. megidis IJs were those associated with the cytoskeleton, cell signalling and carbon metabolism, and the proteins decreased in abundance to the greatest extent were heat shock and ribosomal proteins, and those associated with metabolism. As the longest-lived stage of the EPN life cycle, IJs may be affected by proteostatic stress, caused by the accumulation of misfolded proteins and toxic aggregates. The substantial increase of chaperone proteins in S. carpocapsae, and to a greater extent at 9°C, and the general decrease in ribosomal and chaperone proteins in H. megidis may represent species-specific proteostasis mechanisms. Similarly, organisms accumulate reactive oxygen species (ROS) over time and both species exhibited a gradual increase in proteins which enhance ROS tolerance, such as catalase. The species-specific responses of the proteome in response to storage temperature, and over time, may reflect the phylogenetic distance and/or different ecological strategies.

Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), isolate HP88, induces reproductive and physiological alterations in Biomphalaria glabrata (Gastropoda: Planorbidae): an alternative for biological control of schistosomiasis.

Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) that is mutually associated with Photorhabdus luminescens, utilized globally for biological control of numerous organisms. Freshwater snails of the species Biomphalaria glabrata have been incriminated as the main intermediate hosts of Schistosoma mansoni in Brazil, but virtually nothing is known about the susceptibility of these gastropod to EPNs. Information in this respect is relevant for control of these intermediate hosts, and thus of the helminthiases they transmit. This paper for the first time reports the susceptibility of B. glabrata to infective juveniles of H. bacteriophora (isolate HP88) under laboratory conditions. For that purpose, six groups were formed: three Control groups (not exposed) and three Treated groups, in which the snails were exposed to 300 juveniles infecting the nematode over three weeks. The entire experiment was conducted in triplicate, using a total of 270 snails. Significant physiological alterations in B. glabrata were observed in response to the infection by H. bacteriophora HP88, characterized by decreased levels of hemolymphatic glucose as well as reduced contents of glycogen stored in the host's digestive gland. In parallel, the hemolymphatic activity of lactate dehydrogenase increased in the infected snails, indicating that the infection induces breakdown of carbohydrate homeostasis in B. glabrata. Additionally, all the reproductive parameters analyzed were reduced as a consequence of the infection. The results indicate the occurrence of the phenomenon of parasitic castration in the B. glabrata/H. bacteriophora HP88 interface, probably due to the depletion of galactogen in the parasitized organism. Although the infection did not cause lethality in the population of infected snails, H. bacteriophora HP88 compromised the reproductive performance of B. glabrata, suggesting its applicability in programs for biological control of this planorbid.