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Parasites & Vectors Oct 2023Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural...
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.
Topics: Female; Animals; Rhabditoidea; Phylogeny; Nematoda; Photorhabdus; Whole Genome Sequencing
PubMed: 37880744
DOI: 10.1186/s13071-023-05990-z -
World Journal of Microbiology &... Nov 2023The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and...
The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, invading host insects. They carry cells of their bacterial symbiont Photorhabdus spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the Photorhabdus cells into the insect's haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called "recovery". For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with Photorhabdus before DJs are added. In absence of the insect's food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from H. bacteriophora were isolated and identified as P. laumondii, P. kayaii and P. thracensis. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.
Topics: Animals; Photorhabdus; Rhabditoidea; Nematoda; Insecta; Culture Media; Symbiosis
PubMed: 37953398
DOI: 10.1007/s11274-023-03803-0 -
Journal of Applied Microbiology Jun 2024This study aimed to overproduce industrially relevant and safe bio-compound trans-cinnamic acid (tCA) from Photorhabdus luminescens with deletion strategies and...
AIM
This study aimed to overproduce industrially relevant and safe bio-compound trans-cinnamic acid (tCA) from Photorhabdus luminescens with deletion strategies and homologous expression strategies that had not been applied before for tCA production.
METHODS AND RESULTS
The overproduction of the industrially relevant compound tCA was successfully performed in Photorhabdus luminescens by deleting stlB (TTO1ΔstlB) encoding a cinnamic acid CoA ligase in the isopropylstilbene pathway and the hcaE insertion (knockout) mutation (hcaE:: cat) in the phenylpropionate catabolic pathway, responsible for tCA degradation. A double mutant of both stlB deletion and hcaE insertion mutation (TTO1DM ΔstlB-hcaE:: cat) was also generated. These deletion strategies and the phenylalanine ammonium lyase-producing (PI-PAL from Photorhabdus luminescens) plasmid, pBAD30C, carrying stlA (homologous expression mutants) are utilized together in the same strain using different media, a variety of cultivation conditions, and efficient anion exchange resin (Amberlite IRA402) for enhanced tCA synthesis. At the end of the 120-hour shake flask cultivation, the maximum tCA production was recorded as 1281 mg L-1 in the TTO1pBAD30C mutant cultivated in TB medium, with the IRA402 resin keeping 793 mg L-1 and the remaining 488 mg L-1 found in the supernatant.
CONCLUSION AND IMPACT OF THE STUDY
tCA production was successfully achieved with homologous expression, coupled with deletion and insertion strategies. 1281 mg L-1is the highest tCA concentration that achieved by bacterial tCA production in flask cultivation, according to our knowledge. IRA402 resin adsorbers seem useful for enhancing tCA acquisition in bacterial cultures. Mutations on the hcaE and stlB genes clearly increased the amount of tCA. P. luminescens is an effective bacterial candidate to produce tCA in industrial applications with the implemented strategies.
PubMed: 38906846
DOI: 10.1093/jambio/lxae149 -
Journal of Invertebrate Pathology Jul 2024Aedes-transmitted arboviral infections such as Dengue, Yellow Fever, Zika and Chikungunya are increasing public health problems. Xenorhabdus and Photorhabdus bacteria...
Aedes-transmitted arboviral infections such as Dengue, Yellow Fever, Zika and Chikungunya are increasing public health problems. Xenorhabdus and Photorhabdus bacteria are promising sources of effective compounds with important biological activities. This study investigated the effects of cell-free supernatants of X. szentirmaii, X. cabanillasii and P. kayaii against Ae. aegypti eggs and larvae and identified the bioactive larvicidal compound in X. szentirmaii using The EasyPACId method. Among the three tested bacterial species, X. cabanillasii exhibited the highest (96%) egg hatching inhibition and larvicidal activity (100% mortality), whereas P. kayaii was the least effective species in our study. EasyPACId method revealed that bioactive larvicidal compound in the bacterial supernatant was fabclavine. Fabclavines obtained from promoter exchange mutants of different bacterial species such as X. cabanillasii, X. budapestensis, X. indica, X. szentirmaii, X. hominckii and X. stockiae were effective against mosquito larvae. Results show that these bacterial metabolites have potential to be used in integrated pest management (IPM) programmes of mosquitoes.
Topics: Animals; Aedes; Photorhabdus; Larva; Xenorhabdus; Ovum; Mosquito Control; Mosquito Vectors; Pest Control, Biological; Insecticides
PubMed: 38734162
DOI: 10.1016/j.jip.2024.108126 -
Angewandte Chemie (International Ed. in... Jan 2024The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer...
The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural β-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by β-lactones, the predominant class of ClpP inhibitors.
Topics: Staphylococcus aureus; Catalytic Domain; Dipeptides; Virulence; Endopeptidase Clp
PubMed: 38029352
DOI: 10.1002/anie.202314028 -
Bio-protocol Jul 2023The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs)...
The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.
PubMed: 37449040
DOI: 10.21769/BioProtoc.4709 -
World Journal of Microbiology &... Mar 2024The entomopathogenic nematode Heterorhabditis bacteriophora, symbiotically associated with enterobacteria of the genus Photorhabdus, is a biological control agent...
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 m in liquid culture processes lasting approximately 11 days. A high DJ yield (> 200,000 DJ·mL) determines the success of the process. To start the mass production, a DJ inoculum proceeding from a previous monoxenic culture is added to pre-cultured (24 h) Photorhabdus bacteria. Within minutes after contact with the bacteria, DJ are expected to perceive signals that trigger their further development (DJ recovery) to reproductive hermaphrodites. A rapid, synchronized, and high DJ recovery is a key factor for an efficient culture process. In case of low percentage of DJ recovery, the final DJ yield is drastically reduced, and the amount of non-desired stages (males and non-fertilized females) hinders the DJ harvest. In a preliminary work, a huge DJ recovery phenotypic variability in H. bacteriophora ethyl methanesulphonate (EMS) mutants was determined. In the present study, two EMS-mutant lines (M31 and M88) with high and low recovery phenotypes were analyzed concerning their differences in gene expression during the first hours of contact with Photorhabdus supernatant containing food signals triggering recovery. A snapshot (RNA-seq analysis) of their transcriptome was captured at 0.5, 1, 3 and 6 h after exposure. Transcripts (3060) with significant regulation changes were identified in the two lines. To analyze the RNA-seq data over time, we (1) divided the expression profiles into clusters of similar regulation, (2) identified over and under-represented gene ontology categories for each cluster, (3) identified Caenorhabditis elegans homologous genes with recovery-related function, and (4) combined the information with available single nucleotide polymorphism (SNP) data. We observed that the expression dynamics of the contrasting mutants (M31 and M88) differ the most within the first 3 h after Photorhabdus supernatant exposure, and during this time, genes related to changes in the DJ cuticle and molting are more active in the high-recovery line (M31). Comparing the gene expression of DJ exposed to the insect food signal in the haemolymph, genes related to host immunosuppressive factors were not found in DJ upon bacterial supernatant exposure. No link between the position of SNPs associated with high recovery and changes in gene expression was determined for genes with high differential expression. Concerning specific transcripts, nine H. bacteriophora gene models with differential expression are provided as candidate genes for further studies.
Topics: Female; Male; Animals; Ethyl Methanesulfonate; Transcriptome; Caenorhabditis elegans; Biological Control Agents; Bioreactors
PubMed: 38451353
DOI: 10.1007/s11274-024-03902-6 -
Molecules (Basel, Switzerland) Jun 2024Secondary metabolites, bioactive compounds produced by living organisms, can unveil symbiotic relationships in nature. In this study, soilborne entomopathogenic...
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 ( and ) were extracted from solvent supernatant containing secondary metabolites, demonstrating significant inhibitory effects against , , , , , and . 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 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 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.
Topics: Chromatography, High Pressure Liquid; Anti-Bacterial Agents; Spectroscopy, Fourier Transform Infrared; Gas Chromatography-Mass Spectrometry; Symbiosis; Secondary Metabolism; Photorhabdus; Xenorhabdus; Microbial Sensitivity Tests; Animals
PubMed: 38930979
DOI: 10.3390/molecules29122914 -
Acta Tropica Aug 2024Chagas disease is a zoonosis caused by the protozoan Trypanosoma cruzi and transmitted through the feces of triatomines, mainly in Latin America. Since the 1950s,...
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.
Topics: Animals; Chagas Disease; Photorhabdus; Triatoma; Mexico; Survival Analysis; Rhabditida; Biological Control Agents; Pest Control, Biological; Rhabditoidea; Disease Vectors; Trypanosoma cruzi
PubMed: 38801912
DOI: 10.1016/j.actatropica.2024.107262 -
Archives of Insect Biochemistry and... Jan 2024Phospholipase A (PLA ) catalyzes phospholipids at the sn-2 position to release free fatty acids, including arachidonic acid (AA) or its precursor. The free AA is then...
Phospholipase A (PLA ) catalyzes phospholipids at the sn-2 position to release free fatty acids, including arachidonic acid (AA) or its precursor. The free AA is then oxygenated into different eicosanoids, which mediate the diverse physiological processes in insects. Any inhibition of the PLA catalysis would give rise to serious malfunctioning in insect growth and development. An onion moth, Acrolepiopsis sapporensis, encodes four different PLA genes (As-PLA A-As-PLA D), in which As-PLA A is dominantly expressed at all developmental stages and in different larval tissues. RNA interference of the As-PLA A expression significantly reduced the PLA activity of A. sapporensis, which suffered from immunosuppression. A recombinant As-PLA A protein was purified from a bacterial expression system, which exhibited a typical Michaelis-Menten kinetics and hence susceptible to a specific inhibitor to sPLA and dithiothreitol. A total of 19 bacterial metabolites derived from Xenorhabdus and Photorhabdus were screened against the recombinant As-PLA A. Five potent metabolites were highly inhibitory and followed a competitive enzyme inhibition. These five inhibitors suppressed the immune responses of A. sapporensis by inhibiting hemocyte-spreading behavior and phenoloxidase activity. However, an addition of AA could significantly rescue the immunosuppression induced by the selected inhibitors. These studies suggest that the recombinant As-PLA A protein can be applied for high-throughput screening of insect immunosuppressive compounds.
Topics: Animals; Spodoptera; Phospholipases A2, Secretory; Eicosanoids; Larva; Insecta; Arachidonic Acid
PubMed: 38288493
DOI: 10.1002/arch.22081