Nematode Heterorhabditis Research Articles

Forecasting Western Corn Rootworm (Diabrotica virgifera virgifera LeConte) Density and Non-Chemical Control of Larvae: A Practical Review

The western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte; Chrysomelidae) is one of the most significant maize pests in Europe, with farmers spending a substantial amount (approximately 140 EUR) on its control. In the context of climate change, WCRs could pose an even greater threat to EU maize production, particularly as the European Union continues to withdraw an increasing number of effective yet environmentally harmful active agents. Biological control methods have now emerged to the forefront in creating sustainable agriculture. In this review, we carried out an extensive literature analysis on methods for forecasting WCRs and evaluated the practical applicability of the latest non-chemical control methods targeting its larvae. Effective forecasting is essential for successful pest management, enabling informed planning and the selection of the most suitable control methods. Several traditional predicting techniques remain in use today, but recent advancements have introduced modern electronic forecasting units combined with sensor-equipped pheromone and colour traps, as well as thermal sum calculations. Research has demonstrated that crop rotation is one of the most effective methods for controlling WCR larvae. Biological agents, such as entomopathogenic fungi (Beauveria bossiana and Mettarrhyzum anasoplia), entomopathogenic nematodes (Heterorhabditis bacteriophora), and botanical insecticides such as azadirachtin can significantly reduce larval populations and root damage, thereby maintaining infestation levels below the economic threshold. Genetically modified maize plants that produce specific toxins, along with conventional breeding efforts to increase root system regeneration, are also promising tools for the sustainable management of this pest. This review summarizes the solutions for prediction of western corn rootworm infestations and non-chemical control of its larvae. Accurate forecasting methods provide a clear picture of infestation levels in a given area, enabling precisely targeted control measures. In all cases, the control should be directed primarily against the larvae, thereby reducing root damage and reducing the size of the emerging imago population. This review demonstrates that biological control methods targeting larvae can be as effective as pesticides, supporting sustainable pest management.

Damage to the host cadaver, simulating the effects of scavenging, differentially affects fitness of entomopathogenic nematode species

Insect cadavers infected by entomopathogenic nematodes (EPN) are defended against scavengers by chemical and other means. Despite these defences, the cadaver may be bitten before being rejected. In this study, we investigated the effect of damage to the cadaver cuticle on the fitness of nematodes (Heterorhabditis downesi or Steinernema feltiae) developing inside. We first quantified the severity of scavenger damage to EPN-infected Galleria mellonella Linnaeus cadavers in the field, and separately, with crickets (Gryllus bimaculatus De Geer) in the laboratory. In field and laboratory, EPN-infected cadavers suffered less damage than freeze-killed controls, and damage consisted mainly of small lesions to the cuticle. In further experiments, scavenging damage was simulated shortly after death of infected cadavers by piercing the cuticle 0, 1, 3 or 5 times and incubating in moist (100% relative humidity (RH)) or dry (60-70% RH) conditions. The greater the level of damage, the greater the loss of moisture from the cadaver (estimated by weight loss), and this was exacerbated in dry conditions. The number of Infective Juveniles (IJs) emerging from H. downesi-infected cadavers was significantly reduced by damage, especially in dry conditions. In addition, emerging IJs were progressively smaller with increasing damage. For this species, number of IJs was negatively correlated with moisture loss, indicated that the reduction in fitness was mediated by desiccation. For S. feltiae, damage impacted IJ number to a lesser extent and size was not affected. The reduction in numbers was not explained by moisture loss, indicating that for S. feltiae, some factor other than desiccation (perhaps competition with opportunistic microbes) impacts the nematodes when the cuticle is damaged. The greater vulnerability of H. downesi than S. feltiae to scavenger damage to the host cadaver may be due to its longer developmental time in the host resulting in longer exposure to damaging conditions. In conclusion, damage simulating biting by scavengers impacts the fitness of EPN, with the effect depending on nematode species, environmental conditions and the extent of damage. These findings have implications for the success of field application of EPN in infected cadavers.

Sodium Hypochlorite: Optimization of Application as an Exsheathing Agent for Nematode Larvae to Minimize the Risk of Reduced Viability

Objective: Nematode larvae exsheathing is a crucial procedure employed in experimental studies such as nematode viability assays, evaluating nematodes' resistance to anthelmintics, and evaluating the efficacy of anthelmintic medications. The exsheathment procedure allows for quantifying the direct impact on larvae when the protective sheath is removed. Sodium hypochlorite (NaOCl) is a commonly used artificial exsheathment medium. However, concerns have been raised about its potential impact on the viability of exsheathed larvae. A previous study utilized Strongyloides papillosus larvae, a nematode species without a protective sheath, to investigate the effects of NaOCl on parasite viability. The findings revealed that there are specific concentration (C) and exposure-time (T) thresholds (0.3% > C > 0.2%; 10 min > T > 5 min) beyond which viability decreases, and excessive exposure can cause significant damage. The current study aims to determine the optimal concentration of NaOCl that can induce exsheathment in ensheathed-type larvae without compromising their viability. Methods: Third-stage strongylid larvae of donkey origin were utilized for this study, with chemical concentrations of 3%, 0.3%, 0.2%, 0.1%, or 0.05%. Larval motility served as the criterion for evaluating viability. Results: NaOCl can exsheath larvae at any of the five concentration levels evaluated, depending on the duration of exposure to the compound. A concentration of 0.2% is considered optimal, inducing exsheathment as early as 2 minutes. Extended exposure for up to 10 minutes did not impact larval viability. A 3% solution causes larval damage. Conclusions: Regarding the effect of NaOCl on nematode larvae, the results validate the earlier findings with Strongyloides papillosus and those by other authors employing nematodes such as Haemonchus contortus and Cooperia curticei, and the entomopathogenic nematode Heterorhabditis bacteriophora.

Morphological and Morphometric Identification of Steinernema abbasi (Nematoda: Steinernematidae) from Düzce, Türkiye

Interest in the use of nematodes as biological pest control agents has grown exponentially over the past two decades. Entomopathogenic nematodes (EPNs) (Steinernema and Heterorhabditis) have been particularly successful in managing pest problems in agriculture and horticulture. One of the most important requirements in biological control programmes is the accurate identification of pests and any beneficial organisms with biocontrol potential. The aim of this study was to identify an unknown entomopathogenic nematode that was collected from a hazelnut orchard in Düzce, Türkiye. Our study reports methods for the identification of Steinernema abbasi, based on morphological and morphometric characterization. The unknown entomopathogenic nematode, Strain 1, was identified as S. abbasi.

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.

Characterisation and toxins analysis of symbiotic bacteria Photorhabdus luminescens isolated from Heterorhabditis indica ICRI-18 strain

Entomopathogenic nematodes are proven alternatives to pesticides in crop protection as they belong to the Heterorhabditidae and Steinernematidae families. The role of entomopathogenic nematodes has expanded considerably in the recent past for their efficacy as biopesticide which is influenced by nematode species and strain. Members of the genus Heterorhabditis have been found to harbor a symbiotic bacteria Photorhabdus sp. When the nematode is ingested by the insect, Photorhabdus sp. is released into the host haemocoel where they multiply rapidly and kill the insect. The main objective of our research was to examine the insect killing toxin property of symbiotic bacteria isolated from the entomopathogenic nematodes Heterorhabditis indica. The antagonistic potential of insecticidal toxin from Photorhabdus sp. was investigated. The results prove that toxic components produced by these bacteria could kill pests either through ingestion or injection. The broad-spectrum antimicrobial property produced by Photorhabdus sp. was analysed by conducting both antibiogram and antibiosis studies.

Can we manage alien invasive insects without altering native soil faunal communities? A field trial on Popillia japonica

Biological and integrated pest control are key assets to environmentally friendly management of cropland. Use of entomopathogens against target pests is common, yet the effects of such released organisms on native, non-target invertebrates are currently poorly known. This is particularly true for relatively inconspicuous components of agroecosystems such as soil biological biota. Popillia japonica, a polyphagous alien invasive scarab beetle native to Japan, is now present in Europe where huge efforts are being conducted to control the pest and slow its spread. In particular, entomopathogenic nematodes and fungi have been used for larval control. Here we test the effects of those agents on both P. japonica larvae and non-target soil biota by comparing soil arthropod assemblages before and after treatment of field plots in an irrigated perennial meadow.Application of an indigenous strain of the nematode Heterorhabditis bacteriophora resulted in significant reduction in numbers of P. japonica larvae. In contrast, an indigenous strain of the entomopathogenic fungus Metarhizium robertsii failed to provide significant control. We found no evident effect on non-target edaphic arthropods under either treatment in comparison to untreated control plots. Some taxa, namely Collembola and Acarina, showed a disproportionate increase in treated plots, suggesting that some changes may occur in the long term, at least in particular arthropod groups. Our results suggest the selected strain of H. bacteriophora is an excellent candidate for managing P. japonica larvae with minimal adverse impact on non-target species and ecosystem functioning.

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 11days. 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 (24h) 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 6h 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 3h 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.

Beware glowing cadavers: bioluminescence of nematode symbiont Photorhabdus protects nematode-infected host cadavers from nocturnal scavengers

Photorhabdus spp. are the only known terrestrial bioluminescent bacteria. We show that the bioluminescence produced by these bacteria reduces scavenging activity on the insect cadavers they colonize. Photorhabdus spp. are the symbiont of the insect pathogenic nematodes Heterorhabditis spp. Together they kill insects and colonize the cadaver. The function of their bioluminescence has been the subject of debate, but here for the first time we demonstrate an ecological benefit. In our experiments, fewer Photorhabdus temperata -infected cadavers than uninfected cadavers were scavenged, but only in dark conditions where their bioluminescence would be visible. This was the case both in the field and in laboratory experiments with Lehmannia valentiana slugs (the primary scavengers found in our field tests). We also show that L. valentiana is innately deterred from scavenging on uninfected cadavers in proximity to light imitating the bioluminescence of Photorhabdus, indicating that luminescence can be a deterrent independent of chemical cues. We propose a multimodal defence where bioluminescence works together with the chemical defences also produced by Photorhabdus to deter scavengers, such as slugs, from feeding on the host cadaver, with the potential for aposematism.

Assessment of infection of Stomoxys calcitrans larvae by entomopathogenic nematodes Heterorhabditis amazonensis NEPT11.

This study aimed to evaluate the virulence of Heterorhabditis amazonenses NEPT11 against larvae of Stomoxys calcitrans. Groups of 10 third-instar fly larvae were deposited in Petri dishes, to which were added 50, 100 and 200 EPNs/larva in 4ml of distilled water. The volume of the control group was the same as the treated group, but without EPNs. Larval mortality was observed daily, until larvae died or adults emerged. The Petri dishes were kept on laboratory shelves at 27 ± 1 °C and 70 ± 10% RH. The experiment was replicated six times. A regression analysis revealed quadratic behavior with increasing concentrations, indicating that the concentration of 200 EPNs/larva (48%) was the most efficient among the tested concentrations, while concentrations of 50 and 100 EPNs/larva killed 26.6 and 40% of larvae, respectively. In general, none of the treatments resulted in a mortality rate of more than 50%, but all the treated groups exhibited a higher mortality than that of the control group. It is concluded that the EPN H. amazonensis NEPT11 shows a promising potential to control third-instar larvae of S. calcitrans. However, further studies are needed in different situations to better understand the activity of this organism against the immature stages of the stable fly.

Dos inseticidas ao controle biológico de pragas na cana-de-açúcar: uma experiência de meio século na Venezuela

Biological control has been established as a unique measure for managing borers of the genus Diatraea and the candelilla Aeneolamia spp., both of which are key pests of sugarcane in Venezuela. Optimal results have been achieved with the use of parasitoids and entomopathogens. Systematic releases of the Amazonian fly Lydella minense from the mid-20th century and the establishment of the braconid wasp Cotesia flavipes from 1987 led to effective control of borers. In the case of Aeneolamia varia, a pest formerly controlled with frequent insecticide applications, an efficient solution was the introduction of the entomopathogenic fungus Metarhizium anisopliae in 1977. This measure has been established as efficient in controlling adult Aeneolamia, though nymphs were less controlled. This issue was effectively addressed in 2004 with the introduction of the entomopathogenic nematode Heterorhabditis bacteriophora, which has been widely used as a biological measure to avoid the application of insecticides. To illustrate this, we can consider the case of Azucarera Río Turbio. Despite experiencing a reduction in cultivated areas in recent harvests due to various factors, this establishment has witnessed a significant decline in Diatraea infestations over the past 58 years. Infestation rates have decreased from 15% to 3.18%, resulting in a saving of 170,552.62 metric tons valued at USD 49,196,045. The cost of implementing biological control measures amounts to USD 1,187,189, translating into a favorable benefit-cost ratio of USD 41.44. Before the adoption of biological control methods, Aeneolamia infestations were exclusively controlled using chemical insecticides applied to both the foliage for adults and the soil for nymphs. Concurrently, since then, entomopathogenic fungus M. anisopliae, produced by national companies, has been deployed across 87,000 hectares between 1986 and 1990. Moreover, since 2004, the use of entomopathogenic nematodes to control Aeneolamia nymphs has been integrated into pest management practices.

Monitoring the Photorhabdus spp. bacterial load in Heterorhabditis bacteriophora dauer juveniles over different storage times and temperatures: A molecular approach

Biological control products based on the entomopathogenic nematode Heterorhabditis bacteriophora can vary in virulence (quality). The influence of their symbiotic bacteria Photorhabdus spp. inside the infective dauer juvenile (DJ) on DJ quality has not received much attention in the past. The presence of the bacteria in the DJ is crucial for its biocontrol potential. This investigation provides a method to quantify the bacterial load inside the DJ based on a qPCR technique. Information from the genome of Photorhabdus laumondii strain DE2 was used to identify single copy genes with no homology to any other bacterial accessions. One gene (hereby named CG2) was selected for primers design and for further qPCR experiments. Cross-amplification tests with P. thracensis and P. kayaii, also symbionts of H. bacteriophora, were positive, whereas no amplicons were produced for P. temperata or Xenorhabdus nematophila. We tested our qPCR system in DJ populations carrying defined proportions of bacteria-free (axenic) vs bacteria-carrying nematodes. With an increasing proportion of axenic DJ in a population, virulence declined, and the virulence was proportional to the amount of bacterial DNA detected in the population by qPCR. Along liquid storage over long time, virulence also decreased, and this factor correlated with the reduction of bacterial DNA on the respective DJ population. We observed that stored DJ kept virulent up to 90 days and thereafter the virulence as well as the amount of bacterial DNA drastically decreased. Storage temperature also influenced the bacterial survival. Inside formulated DJ, the loss of bacterial DNA on the DJ population was accelerated under storage temperatures below 7.5 °C, suggesting that reproduction of the bacterial cells takes place when growth temperature is favorable. The role of bacterial survival inside stored DJ can now be adequately addressed using this molecular quality-control technique.

Genotypic markers associated with cold storage survival of the entomopathogenic nematode Heterorhabditis bacteriophora

Summary Limited shelf life is a major constraint to successful commercialisation of entomopathogenic nematodes (EPN), and to extend shelf life, dauer juveniles (DJ) are formulated and stored at low temperatures (4-8°C). We evaluated the cold storage potential of strains of Heterorhabditis bacteriophora formulated in diatomaceous earth at storage temperatures between 5 and 9°C. When assessing DJ decline to reach 75% survival (MT75) in the formulation for the respective temperatures, H. bacteriophora strain HB4 had the highest survival of 25 days at 9°C, while strain D2D6 survived longest at 8°C for 28 days. A set of 22 H. bacteriophora wild type inbred lines was then phenotyped for cold tolerance in water under oxidative stress in 70 mM H2O2 at 2°C. The MT50 (time to 50% survival) ranged from 11 to 23 days. The phenotypic data were correlated with the respective genotypic data, identifying four single nucleotide polymorphic (SNP) markers associated with cold tolerance. The survival of two lines (PT11 and IR11) with opposite extreme cold tolerance pheno- and genotypes was evaluated in diatomaceous earth formulation at 2°C with the cold tolerant IR11 surviving 3 days longer than PT11. Our study yields a set of valuable SNP markers employable in rapid genotyping of cold tolerance and tracking this trait during the breeding process.

Effects of Silver, Gold, and Platinum Nanoparticles on Selected Nematode Trophic Groups

Abstract As trophic organisms, nematodes play an essential role in the soil environment: they mineralize nutrients into plant-available forms, are a food source for other soil organisms, and feed on pathogenic organisms and plant pests, therefore regulating populations of soil microorganisms. The plant-parasitic nematodes are important pests of crops. Nanoparticles (NP) are increasingly used in agriculture and other production sectors. They are present in the soil, not necessarily in trace amounts, and can affect various soil organisms, including nematodes. In this article, the effects of silver (Ag), gold (Au), and platinum (Pt) nanoparticles on the mortality and reproduction activity of selected nematode species from two trophic groups: entomopathogenic nematodes (EPN) (Heterorhabditis bacteriophora and Steinernema feltiae) and plant parasitic nematodes (PPN) (Xiphinema diversicaudatum, Ditylenchus dipsaci, Heterodera schachtii) were studied under laboratory conditions. All nanoparticles decreased the nematode population to an extent depending on the nematode species, nanoparticle type, and exposure time. AgNP had the greatest nematicidal effect, except for AuNP, which reduced the population of H. schachtii the most. The greatest sensitivity to AgNP was observed in X. diversicaudatum (100% mortality), D. dipsaci (90% mortality), and 37 to 13% mortality in other species. Effect of AuNP and PtNP on entomopathogenic nematodes was comparable to the control, not treated with nanoparticles. AuNP and PtNP similarly influenced nematode mortality. However, the effect of nanoparticles on new generations of entomopathogenic nematodes developing in host larvae Galleria mellonella was inconclusive. All nanoparticles decreased the number of larvae of S. feltiae and increased the number of H. bacteriophora migrating outside the cadavers compared to the control.

Alterations in the metabolism of Pseudosuccinea columella (Mollusca: Gastropoda) caused by Heterorhabditis bacteriophora HP88 (Rhabditida: Heterorhabditidae)

The gastropod Pseudosuccinea columella participates in the dissemination of Fasciola hepatica in the environment, acting as the main intermediate host of this parasite in Brazil. The present study sought to elucidate the possible pathogenic effects of the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora on P. columella, by evaluating the influence of infection on alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the concentrations of total protein, uric acid, and urea in the snail's hemolymph. For this, the snails were exposed to EPNs for 24 and 48 h, and for each exposure time, 20 snails were dissected after 7, 14 and 21 days for hemolymph collection. The primary findings suggest a significant proteolysis alongside elevated levels of uric acid and urea in P. columella infected individuals. These findings reveal that H. bacteriophora HP88 infection induced serious changes in the snail's metabolism, triggering important deleterious effects.

Efficiency of temperature and storage duration on some morphological measurements and reproductive capacity of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae)’s Turkish HBH hybrid strain

Entomopathogenic nematodes (EPNs) are successfully used in the biological control of agricultural insect pests. This study aims to determine the body length of hermaphrodite individuals, egg diameter and reproductive capacity obtained from Infective Juveniles (IJs) stored at different temperatures and durations. Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae)’s Hybrid Strain HBH was used in the study. IJs stored at 15, 25 and 35°C for 7, 14 and 21 days were inoculated onto Galleria mellonella L., 1758 (Lepidoptera: Pyralidae) last instar larvae at a dose of 100 IJs. On the 2nd day of infection, hermaphrodite individuals and eggs were obtained by dissecting the larvae. The reproductive capacity was determined 10-12 days after infection. The study was conducted in Bursa Uludağ University, Faculty of Agriculture, Plant Protection Department, Nematology Laboratory in 2023. In conclusion, the longest hermaphrodite individuals and egg diameter were obtained as 6207.22 µm and 55.65 µm, respectively from the IJs stored for 7 days at 15°C. The highest reproductive capacity was also observed as 167.500 IJs per G. mellonella larva in IJs stored under the same conditions with respect to temperature and time. This study is important for assessing the morphological effects of different temperature values and storage durations on EPNs.

Taxonomic and molecular characterization of a new entomopathogenic nematode species, Heterorhabditis casmirica n. sp., and whole genome sequencing of its associated bacterial symbiont

BackgroundNematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural insect pests. This study describes a new species of Heterorhabditis.MethodsSix Heterorhabditis nematode populations were recovered from agricultural soils in Jammu and Kashmir, India. An initial examination using mitochondrial and nuclear genes showed that they belong to a new species. To describe this new species, a variety of analyses were conducted, including reconstructing phylogenetic relationships based on multiple genes, characterizing the nematodes at the morphological and morphometric levels, performing self-crossing and cross-hybridization experiments, and isolating and characterizing their symbiotic bacteria.ResultsThe newly discovered species, Heterorhabditis casmirica n. sp., shares 94% mitochondrial cytochrome C oxidase subunit I gene (COI) sequence identity with Heterorhabditis bacteriophora and Heterorhabditis ruandica, and 93% with Heterorhabditis zacatecana. Morphologically, it differs from H. bacteriophora in its infective juvenile phasmids (present vs. inconspicuous) and bacterial pouch visibility in the ventricular portion of the intestine (invisible vs. visible); genital papilla 1 (GP1) position (at manubrium level vs. more anterior), and in its b ratio (body length/neck length), c ratio (tail length/bulb width), and D% [(excretory pore/neck length) × 100]. Other morphological differences include anterior end to the nerve ring distance (77–100 vs. 121–130 μm), V% [(anterior end of vulva/body length) × 100] (46–57 vs. 41–47) in hermaphroditic females; rectum size (slightly longer than the anal body diameter vs. about three times longer), phasmids (smaller vs. inconspicuous), body length (0.13–2.0 vs. 0.32–0.39 mm), body diameter (73–150 vs. 160–220 μm), anterior end to the excretory pore distance (135–157 vs. 174–214 μm), and demanian ratios in amphimictic females. Morphological differences with H. ruandica and H. zacatecana were also observed. Furthermore, H. casmirica n. sp. did not mate or produce fertile progeny with other Heterorhabditis nematodes reported from India. It was also discovered that H. casmirica n. sp. is associated with 'Photorhabdus laumondii subsp. clarkei symbiotic bacteria.ConclusionsThe discovery of H. casmirica n. sp. provides novel insights into the diversity and evolution of Heterorhabditis nematodes and their symbiotic bacteria. This new species adds to the catalog of entomopathogenic nematodes in India.Graphical

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.

Precision application of the entomopathogenic nematode Heterorhabditis bacteriophora as a biological control agent through the Nemabot

One of the most important development trends of robots in agriculture is to enable highly precise applications that minimize amounts of chemical components that are harmful to the environment. Precision agriculture is fundamental and inevitable worldwide because it provides more yield to an increasing population, while at the same time reducing inputs. The purpose of this study was to apply entomopathogenic nematodes, which are insect parasites used as biological control agents, through Nemabot. A robotic system that can move in the X-Y-Z coordinate plane has an agitating mechanism for suspension based on water and entomopathogenic nematodes and can perform precise dosing with a peristaltic pump designed and produced as a prototype. The experimental results of the robot application on the exact point, volume, amount, and uniformity of dosing show that the proposed method can effectively solve the problem of applying entomopathogenic nematodes, which are economically more expensive than pesticides. The main contribution of this paper is the proposal of a method to solve the problem of applying the agents precisely. This is the first experiment in which biological control products were applied using a robotic system. A patent application (PCT/TR2019/050768) was made, and the patentability claims were approved and officially registered (TR2018 14310B).

Pathogenicity of Heterorhabditis bacteriophora in Ultraviolet Light–Irradiated Agar Suspension on Spodoptera frugiperda (Lepidoptera: Noctuidae) Larvae

Abstract Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is an economically important pest of variety of crops, including maize (Zea mays L.), rice (Oryza sativa L.), and cotton (Gossypium spp.). Conventional chemical insecticides are usually used for management of this pest; however, a viable alternative is the use of microbial agents or their biological products, such as entomopathogenic nematodes. Their efficacy, however, is affected by abiotic conditions including ultraviolet (UV) light and desiccation. It is therefore necessary to develop formulations that preserve or extend the viability of the agent or product while facilitating ease of application. This laboratory study was conducted to determine the pathogenicity of the nematode Heterorhabditis bacteriophora (Poinar) against S. frugiperda third-instar larvae. Infective juveniles (IJs) of the nematode were tested at a concentration of 350 IJs/ml in two substrates (water and 0.1% [w/v] agar suspension) with four periods of exposure (0, 5, 10 and 15 min) to UV radiation at 253.7 nm. Each of the eight treatments included 40 larvae, with each treatment replicated three times in a completely randomized design (two factors of type of medium and UV exposure time). We determined that the 0.1% (w/v) agar suspension, compared with the water substrate, protected the IJs from harmful UV light and thus improved the survival and pathogenicity of the IJs against third-instar S. frugiperda.