Bacterial bioluminescence is an important regulator of multitrophic interactions in the soil.

Enormous efforts have been made to understand the functions of bioluminescence; however, its relevance in soil ecosystems has barely been investigated. In addition, our understanding of the biological relevance of bioluminescence is hampered by the scarcity of tools to genetically manipulate this trait. Using the symbionts of entomopathogenic nematodes, Photorhabdus bacteria, we show that bioluminescence plays important regulatory roles in multitrophic interactions in the soil. Through genetic modifications and exploiting natural variability, we provide direct evidence for the multifunctional nature of bioluminescence. It regulates abiotic and biotic stress resistance, impacts other trophic levels, including nematodes, insects, and plants, and contributes to symbiosis. Our study contributes to understanding the factors that have driven the evolution and maintenance of this trait in belowground ecosystems.

Molluscicidal property of symbiotic bacteria associated with entomopathogenic nematodes against Indoplanorbis exustus and Radix rubiginosa, the intermediate hosts of trematode parasites.

Indoplanorbis exustus and Radix rubiginosa act as intermediate hosts for veterinary and medical trematode parasites. Snail control is a strategy used to decrease the number of snails and interrupt the life cycle of parasites. The objective of this study was to evaluate the efficacy of Xenorhabdus and Photorhabdus extracts against I. exustus and R. rubiginosa in the laboratory. Ethyl acetate extracts of selected symbiotic bacteria were tested for their molluscicidal activities according to World Health Organization guidelines. Additionally, pathological changes in the snails were observed after treatment with the LC50 values under a light microscope. Indoplanorbis exustus and R. rubiginosa were susceptible to all ethyl acetate extracts of symbiotic bacteria. The lowest LC50 and LC90 at 24 h for I. exustus after exposure to Photorhabdus laumondii subsp. laumondii (bALN18.2_TH) extracts were 81.66 and 151.02 ppm, respectively. Similarly, the lowest LC50 and LC90 at 24 h for R. rubiginosa after exposure to Photorhabdus luminescence subsp. akhurstii (bAPY3.5_TH) extracts were 49.21 and 147.66 ppm, respectively. Photorhabdus species had more substantial molluscicidal effects than Xenorhabdus on these snails. The ethyl acetate extracts of these bacteria are effective when contacting the epithelial cells and foot muscle of the snails. To our knowledge, this is the first report on using Xenorhabdus and Photorhabdus extracts to evaluate molluscicidal activities. These symbiotic bacteria, Xenorhabdus and Photorhabdus, may be useful for controlling snail intermediate hosts.

Analysis of the immune transcriptome of the invasive pest spotted wing drosophila infected by Steinernema carpocapsae.

Drosophila suzukii is a pest of global concern due to its great impact on several crops. The entomopathogenic nematode Steinernema carpocapsae was highly virulent to the larvae of the fly although some immune mechanisms were triggered along the infection course. Thus, to understand the gene activation profile we performed a comparative transcriptome of D. suzukii larvae infected with S. carpocapsae and Xenorhabdus nematophila to map the differentially expressed genes involved in the defence response. The analysis exposed the induction of genes involved in the humoral response such as the antimicrobial peptides and pattern-recognition receptors while there was a suppression of the cellular defence. Besides, genes involved in melanisation, and clot formation were downregulated hindering the encapsulation response and wound healing. After the infection, larvae were in a stress condition with an enrichment of metabolic and transport functionalities. Concerning the stress response, we observed variations of the heat-shock proteins, detoxification, and peroxidase enzymes. These findings set a genetical comprehensive knowledge of the host-pathogen relation of D. suzukii challenged with S. carpocapsae which could support further comparative studies with entomopathogenic nematodes.

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).

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.

Ascarosides and Symbiotic Bacteria of Entomopathogenic Nematodes Regulate Host Immune Response in Galleria mellonella Larvae.

Insects protect themselves through their immune systems. Entomopathogenic nematodes and their bacterial symbionts are widely used for the biocontrol of economically important pests. Ascarosides are pheromones that regulate nematode behaviors, such as aggregation, avoidance, mating, dispersal, and dauer recovery and formation. However, whether ascarosides influence the immune response of insects remains unexplored. In this study, we co-injected ascarosides and symbiotic Photorhabdus luminescens subsp. kayaii H06 bacteria derived from Heterorhabditis bacteriophora H06 into the last instar larvae of Galleria mellonella. We recorded larval mortality and analyzed the expressions of AMPs, ROS/RNS, and LPSs. Our results revealed a process in which ascarosides, acting as enhancers of the symbiotic bacteria, co-induced G. mellonella immunity by significantly increasing oxidative stress responses and secreting AMPs (gallerimycin, gloverin, and cecropin). This led to a reduction in color intensity and the symbiotic bacteria load, ultimately resulting in delayed host mortality compared to either ascarosides or symbiotic bacteria. These findings demonstrate the cross-kingdom regulation of insects and symbiotic bacteria by nematode pheromones. Furthermore, our results suggest that G. mellonella larvae may employ nematode pheromones secreted by IJs to modulate insect immunity during early infection, particularly in the presence of symbiotic bacteria, for enhancing resistance to invasive bacteria in the hemolymph.

Preliminary Screening on Antibacterial Crude Secondary Metabolites Extracted from Bacterial Symbionts and Identification of Functional Bioactive Compounds by FTIR, HPLC and Gas Chromatography-Mass Spectrometry.

Secondary metabolites, bioactive compounds produced by living organisms, can unveil symbiotic relationships in nature. In this study, soilborne entomopathogenic nematodes associated with symbiotic bacteria (Xenorhabdus stockiae and Photorhabdus luminescens) were extracted from solvent supernatant containing secondary metabolites, demonstrating significant inhibitory effects against E. coli, S. aureus, B. subtilus, P. mirabilis, E. faecalis, and P. stutzeri. The characterization of these secondary metabolites by Fourier transforms infrared spectroscopy revealed amine groups of proteins, hydroxyl and carboxyl groups of polyphenols, hydroxyl groups of polysaccharides, and carboxyl groups of organic acids. Furthermore, the obtained crude extracts were analyzed by high-performance liquid chromatography for the basic identification of potential bioactive peptides. Gas chromatography-mass spectrometry analysis of ethyl acetate extracts from Xenorhabdus stockiae identified major compounds including nonanoic acid derivatives, proline, paromycin, octodecanal derivatives, trioxa-5-aza-1-silabicyclo, 4-octadecenal, methyl ester, oleic acid, and 1,2-benzenedicarboxylicacid. Additional extraction from Photorhabdus luminescens yielded functional compounds such as indole-3-acetic acid, phthalic acid, 1-tetradecanol, nemorosonol, 1-eicosanol, and unsaturated fatty acids. These findings support the potential development of novel natural antimicrobial agents for future pathogen suppression.

Laboratory Investigations on the Potential Efficacy of Biological Control Agents on Two Thrips Species, Onion Thrips (Thrips tabaci Lindeman) and Western Flower Thrips (Frankliniella occidentalis (Pergande)).

Thrips biocontrol research in greenhouse crops has focused primarily on western flower thrips (WFT; Frankliniella occidentalis). However, recent outbreaks of onion thrips (OT; Thrips tabaci) in Ontario, Canada, demonstrate that biocontrol-based IPM programs for WFT do not control OT sufficiently to prevent crop losses. A lack of comparative studies makes it difficult to determine which program components for WFT are failing for OT. We conducted several laboratory trials examining the extent to which commercial biocontrol products kill OT compared to WFT. These included phytoseiid mites (Amblyseius swirskii, Neoseiulus cucumeris, Amblydromalus limonicus, Iphiseius degenerans), a large generalist predator (Orius insidiosus), an entomopathogenic fungus (Beauveria bassiana strain GHA), and entomopathogenic nematodes (Steinernema feltiae, S. carpocapsae, Heterorhabditis bacteriophora). In no-choice trials, A. swirskii and O. insidiosus consumed more OT than WFT (first instars and adults, respectively). In choice trials, A. swirskii, N. cucumeris, and O. insidiosus consumed more OT than WFT. Steinernema feltiae caused higher mortality in OT than WFT. There was no difference in mortality between thrips species exposed to other biocontrol agents. This suggests available tools have the potential to manage OT as well as WFT. Possible explanations why this potential is not realized in commercial settings are explored.

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.

Harnessing nature's arsenal: Ochrobactrum bacteria metabolites in the battle against root- knot nematode - Insights from in vitro and molecular docking studies.

Agricultural Productivity and plant health are threatened by the root-knot nematode. The use of biocontrol agents reduces the need for chemical nematicides and improves the general health of agricultural ecosystems by offering a more environmentally friendly and sustainable method of managing nematode infestations. Plant-parasitic nematodes can be efficiently managed with the use of entomopathogenic nematodes (EPNs), which are widely used biocontrol agents. This study focused on the nematicidal activity of the secondary metabolites present in the bacteria Ochrobactrum sp. identified in the EPN, Heterorhabditisindica against Root-Knot Nematode (Meloidogyne incognita). Its effect on egg hatching and survival of juveniles of root- knot nematode (RKN) was examined. The ethyl acetate component of the cell-free culture (CFC) filtrate of the Ochrobactrum sp. bacteria was tested at four different concentrations (25 %, 50 %, 75 % and 100 %) along with broth and distilled water as control. The bioactive compounds of Ochrobactrum sp. bacteria showed the highest suppression of M. incognita egg hatching (100 %) and juvenile mortality (100 %) at 100 % concentration within 24 h of incubation. In this study, unique metabolite compounds were identified through the Gas Chromatography- Mass Spectrometry (GC-MS) analysis, which were found to have anti- nematicidal activity. In light of this, molecular docking studies were conducted to determine the impact of biomolecules from Ochrobactrum sp. using significant proteins of M. incognita, such as calreticulin, sterol carrier protein 2, flavin-containing monooxygenase, pectate lyase, candidate secreted effector, oesophageal gland cell secretory protein and venom allergen-like protein. The results also showed that the biomolecules from Ochrobactrum sp. had a significant inhibitory effect on the different protein targets of M. incognita. 3-Epimacronine and Heraclenin were found to inhibit most of the chosen target protein. Among the targets, the docking analysis revealed that Heraclenin exhibited the highest binding affinity of -8.6 Kcal/mol with the target flavin- containing monooxygenase. Further, the in vitro evaluation of 3- Epimacronine confirmed their nematicidal activity against M. incognita at different concentrations. In light of this, the present study has raised awareness of the unique biomolecules of the bacterial symbiont Ochrobactrum sp. isolated from H. indica that have nematicidal properties.

Genome Sequence Analysis of Native Xenorhabdus Strains Isolated from Entomopathogenic Nematodes in Argentina.

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

Entomopathogenic pseudomonads can share an insect host with entomopathogenic nematodes and their mutualistic bacteria.

A promising strategy to overcome limitations in biological control of insect pests is the combined application of entomopathogenic pseudomonads (EPPs) and nematodes (EPNs) associated with mutualistic bacteria (NABs). Yet, little is known about interspecies interactions such as competition, coexistence, or even cooperation between these entomopathogens when they infect the same insect host. We investigated the dynamics of bacteria-bacteria interactions between the EPP Pseudomonas protegens CHA0 and the NAB Xenorhabdus bovienii SM5 isolated from the EPN Steinernema feltiae RS5. Bacterial populations were assessed over time in experimental systems of increasing complexity. In vitro, SM5 was outcompeted when CHA0 reached a certain cell density, resulting in the collapse of the SM5 population. In contrast, both bacteria were able to coexist upon haemolymph-injection into Galleria mellonella larvae, as found for three further EPP-NAB combinations. Finally, both bacteria were administered by natural infection routes i.e. orally for CHA0 and nematode-vectored for SM5 resulting in the addition of RS5 to the system. This did not alter bacterial coexistence nor did the presence of the EPP affect nematode reproductive success or progeny virulence. CHA0 benefited from RS5, probably by exploiting access routes formed by the nematodes penetrating the larval gut epithelium. Our results indicate that EPPs are able to share an insect host with EPNs and their mutualistic bacteria without major negative effects on the reproduction of any of the three entomopathogens or the fitness of the nematodes. This suggests that their combination is a promising strategy for biological insect pest control.

Metarhabditis amsactae: A potential biopesticide isolated from Punjab (India) with potent insecticidal activity and immunomodulatory effects against Galleria mellonella (Lepidoptera: Pyralidae).

A survey was undertaken to isolate entomopathogenic nematodes from Amritsar district of Punjab, India. Out of 20 soil samples collected, two were found positive for the presence of nematodes. 18S and ITS rDNA gene sequencing revealed their identity as Metarhabditis amsactae. To assess its biocontrol potential, Galleria mellonella larvae were treated with concentrations of 20, 40, 80 and 160 IJs/L (infective juveniles/larva) and mortality was recorded from 24 h up to 96 h of nematode exposure. Distilled water without nematodes was used as an untreated control. M. amsactae showed potent larvicidal activity against G. mellonella that was found to be concentration and time dependent. Nematode infection caused 93.33 % larval mortality at 80 IJs/L after 72 h of treatment. 100 % mortality was observed after 96 h. No mortality was observed in control. To evaluate the immunomodulatory effects of M. amsactae, G. mellonella larvae were infected with 100 IJs/L and activities of antioxidant and detoxifying enzymes viz., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), phenol oxidase (PO), glutathione-S-transferase (GST) and acetylcholine esterase (AChE) were appraised after 12, 24, 36 and 48 h of nematode exposure. Malondialdehyde content was also determined. The results obtained demonstrated a significant elevation in all the enzyme activities at all time intervals in treated larvae when compared with untreated control. MDA levels were also enhanced in response to nematode infection. Thus, the present study revealed high insecticidal potential and immunomodulatory effects of M. amsactae on G. mellonella that should be further explored on other insect pests as well.

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.

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.

Entomopathogens in the integrated management of forest insects: from science to practice.

The most important aim of the integrated management of forest insect pests remains the prevention of insect outbreaks, which are a consequence of the interaction of many factors in forest ecosystems, including species composition, age and health of the forest, soil type, the presence of natural enemies, and climatic factors. Integrated pest management until now has been achieved using measures aimed at shaping the functioning of stands in a changing environment. The aim of this review is to summarize research on the use of entomopathogens (microorganisms and nematodes) in the management of forest insect pests and to identify the principal knowledge gaps. We briefly describe the main research directions on the use of pathogens and nematodes to control insect pests and discuss limitations affecting their implementation. Research on entomopathogens for the biocontrol of forest insects has provided a wealth of knowledge that can be used effectively to reduce insect populations. Despite this, few entomopathogens are currently used in integrated pest management in forestry. They are applied in inoculation or inundation biocontrol strategies. While the use of entomopathogens in forest pest management shows great promise, practical implementation remains a distant goal. Consequently, sustainable reduction of forest pests, mainly native species, will be largely based on conservation biological control, which aims to modify the environment to favor the activity of natural enemies that regulate pest populations. This type of biocontrol can be supported by a range of silvicultural measures to increase the resilience of stands to insect infestations. © 2023 Society of Chemical Industry.

α-Terpineol affects social immunity, increasing the pathogenicity of entomopathogenic nematodes to subterranean termites (Isoptera).

Biocontrol of subterranean termites is largely impeded by their social immune responses. Studies on biocontrol agents combined with natural insecticides and their possible effects on the immune defense mechanisms of termites are limited. In this study, we investigated the effects of a combined biocontrol strategy using a plant-derived insect ATPase inhibitor, α-terpineol, with the entomopathogenic nematodes (EPNs) Steinernema carpocapsae against the subterranean termite Coptotermes formosanus Shiraki. Survival assays showed that even a low lethal concentration of α-terpineol significantly increased the EPNs-induced virulence in C. formosanus. α-terpineol treatment majorly inhibited the activity of Na+- K+- ATPase, which disturbed the EPNs-induced enhancement of locomotor activity and grooming behavior in termites treated with the combined strategy. Furthermore, the combination treatment had a synergistic inhibitory effect on innate immune responses in C. formosanus, which were measured as changes in the expression of immune-related genes and activities of immune system enzymes. In conclusion, α-terpineol can weaken the immune defense of termites against EPNs at low lethal concentrations, and is a suitable non-synthetic insecticide to prove the biocontrol efficiency of EPNs on C. formosanus. This study provides a theoretical basis and technical reference for a novel biocontrol strategy that promises to overcome the problems of host immune defense in termites.

Stringent in-silico identification of putative G-protein-coupled receptors (GPCRs) of the entomopathogenic nematode Heterorhabditis bacteriophora.

The infective juveniles (IJs) of entomopathogenic nematode (EPN) Heterorhabditis bacteriophora find and infect their host insects in heterogeneous soil ecosystems by sensing a universal host cue (CO2) or insect/plant-derived odorants, which bind to various sensory receptors, including G protein-coupled receptors (GPCRs). Nematode chemosensory GPCRs (NemChRs) bind to a diverse set of ligands, including odor molecules. However, there is a lack of information on the NemChRs in EPNs. Here we identified 21 GPCRs in the H. bacteriophora genome sequence in a triphasic manner, combining various transmembrane detectors and GPCR predictors based on different algorithms, and considering inherent properties of GPCRs. The pipeline was validated by reciprocal BLAST, InterProscan, GPCR-CA, and NCBI CDD search. Functional classification of predicted GPCRs using Pfam revealed the presence of four NemChRs. Additionally, GPCRs were classified into various families based on the reciprocal BLAST approach into a frizzled type, a secretin type, and 19 rhodopsin types of GPCRs. Gi/o is the most abundant kind of G-protein, having a coupling specificity to all the fetched GPCRs. As the 21 GPCRs identified are expected to play a crucial role in the host-seeking behavior, these might be targeted to develop novel insect-pest management strategies by tweaking EPN IJ behavior, or to design novel anthelminthic drugs. Our new and stringent GPCR detection pipeline may also be used to identify GPCRs from the genome sequence of other organisms.

In Vivo Production, Development and Storage of Oscheius myriophila (Nematoda: Rhabditida) in Galleria mellonella (Lepidoptera: Pyralidae).

Entomopathogenic nematodes have been used in biological control for some time and are an alternative for the control of insect pests, but during their implementation, situations have arisen that can be improved. These vary with each species and include their production and storage. Oscheius myriophila, an entomopathogenic nematode (EPN), was monitored for its performance when produced in vivo, as well as its development using Galleria mellonella larvae, using the MC5-2014 strain isolated from soil samples in the municipality of Tepalcingo, Morelos, México. For a study with native strains of EPNs, a wide range of tests must be conducted because the required conditions can be very specific. In vivo production was quantified at initial infective juvenile (IJ) inocula of 50, 100 and 500, and we obtained the same production for the three inocula. The life cycle of the EPNs lasted 12 days, and two generations were observed in which adults were found at days 5 and 9. Both evaluations were performed at a temperature of 27 °C in G. mellonella larvae. In addition, the temperatures of 8, 12, 20 and 24 °C were evaluated for their storage, and we observed that the EPNs can be kept for at least 6 months, maintaining a survival rate of 58.67% and a good infective capacity at a temperature of 12 °C, remaining above 60%.

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

BACKGROUND: Nematodes 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. METHODS: Six 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. RESULTS: The 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 luminescence subsp. clarkei symbiotic bacteria. CONCLUSIONS: The 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.