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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 -
Pathogens (Basel, Switzerland) Aug 2023Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the...
Vector-borne diseases pose a severe threat to human and animal health. L. (Diptera: Culicidae) is a widespread mosquito species and serves as a vector for the transmission of infectious diseases such as West Nile disease and Lymphatic Filariasis. Synthetic insecticides have been the prime control method for many years to suppress populations. However, recently, the use of insecticides has begun to be questioned due to the detrimental impact on human health and the natural environment. Therefore, many authorities urge the development of eco-friendly control methods that are nontoxic to humans. The bacterial associates [ and spp. (Enterobacterales: Morganellaceae)] of entomopathogenic nematodes (EPNs) ( spp. and spp.) (Rhabditida: Heterorhabditidae and Steinernematidae) are one of the green approaches to combat a variety of insect pests. In the present study, the mosquitocidal activity of the cell-free supernatants and cell suspension (4 × 10 cells mL) of four different symbiotic bacteria (, , , and subsp. ) was assessed against different development stages of (The 1st/2nd and 3rd/4th instar larvae and pupa) under laboratory conditions. The bacterial symbionts were able to kill all the development stages with varying levels of mortality. The 1st/2nd instar larvae exhibited the highest susceptibility to the cell-free supernatants and cell suspensions of symbiotic bacteria and the efficacy of the cell-free supernatants and cell suspensions gradually declined with increasing phases of growth. The highest effectiveness was achieved by the KCS-4S strain inducing 95% mortality to the 1st/2nd instar larvae. The results indicate that tested bacterial symbionts have great potential as an eco-friendly alternative to insecticides.
PubMed: 37764903
DOI: 10.3390/pathogens12091095 -
Antibiotics (Basel, Switzerland) Sep 2023Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug...
Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen and the gallinaceous bird pathogen ) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that and are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.
PubMed: 37760758
DOI: 10.3390/antibiotics12091462 -
MicroPublication Biology 2023The fruit fly is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The...
The fruit fly is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The Gram-negative bacterium is an insect-specific pathogen that forms a mutualistic relationship with the entomopathogenic nematode . Here we find that oral infection of larvae with moderately reduces their survival ability while the bacteria replicate efficiently in the infected insects. This information will contribute towards understanding host gut immunity against potent bacterial pathogens.
PubMed: 37711508
DOI: 10.17912/micropub.biology.000938 -
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