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.

Multigene Sequence-Based and Phenotypic Characterization Reveals the Occurrence of a Novel Entomopathogenic Nematode Species, Steinernema anantnagense n. sp.

Three entomopathogenic nematode populations were isolated from agricultural fields in the Anantnag district of Jammu and Kashmir (India). Sequences of multiple gene regions and phenotypic features show that they are conspecific and represent a novel species. Molecular and morphological features provided evidence for placing the new species into the "Kushidai" clade. Within this clade, analysis of sequence data of the internal transcribed spacer (ITS) gene, the D2D3 region of the 28S rRNA gene, the mitochondrial cytochrome oxidase I (mtCOI) gene, and the mitochondrial 12S (mt12S) gene depicted the novel species as a distinctive entity closely related to Steinernema akhursti, S. kushidai, and S. populi. Phylogenetic analyses also show that the new species is a sister species to S. akhursti, and these two species are closely related to S. kushidai and S. populi. Additionally, the new species does not mate or produce fertile progeny with any of the closely related species, reinforcing its uniqueness from a biological species concept standpoint. The new species is further characterized by the third-stage infective juveniles with almost straight bodies (0.7-0.8 mm length), poorly developed stoma and pharynx, and conoid-elongate tail (49-66 µm) with hyaline posterior part. Adult females are characterized by short and conoid tails bearing a short mucron in the first generation and long conoid tails with thin mucron in the second generation. Adult males have ventrally curved spicules in both generations. Moreover, the first-generation male has rounded manubrium, fusiform gubernaculum, conoid and slightly ventrally curved tails with minute mucron, and the second generation has rhomboid manubrium anteriorly ventrad bent, and tails with long and robust mucron. The morphological, morphometrical, molecular, and phylogenetic analyses support the new species status of this nematode, which is hereby described as Steinernema anantnagense n. sp. The bacterial symbiont associated with S. anantnagense n. sp. represents a novel species, closely related to Xenorhabdus japonica. These findings shed light on the diversity of entomopathogenic nematodes and their symbiotic bacteria, providing valuable information for future studies in this field.

Xenorhabdus aichiensis sp. nov., Xenorhabdus anantnagensis sp. nov., and Xenorhabdus yunnanensis sp. nov., Isolated from Steinernema Entomopathogenic Nematodes.

Three bacterial strains, XENO-2T, XENO-7T, and XENO-10T, isolated from Steinernema entomopathogenic nematodes, were found to represent novel Xenorhabdus species. In this study, we describe these new species by whole-genome and whole-proteome phylogenomic reconstructions, by calculating sequence identity scores using core genome sequences, and by phenotypic characterization. Phylogenomic reconstructions using ribosomal and house-keeping genes, and whole-genome and whole-proteome sequences show that XENO-2T and XENO-10T are closely related to Xenorhabdus japonica DSM 16522T and that XENO-7T is closely related to Xenorhabdus bovienii subsp. africana XENO-1T and to X. bovienii subsp. bovienii T228T. The dDDH values between XENO-2T and XENO-10T and between XENO-2T and X. japonica DSM 16522T are 56.4 and 51.8%, respectively. The dDDH value between XENO-10T and X. japonica DSM 16522T is 53.4%. The dDDH values between XENO-7T and X. bovienii subsp. africana XENO-1T and between XENO-7T and X. bovienii subsp. bovienii T228T are 63.6 and 69.4%, respectively. These dDDH values are below the 70% divergence threshold for prokaryotic species delineation. The newly described species are highly pathogenic to G. mellonella larvae, grow at pH between 5 and 9 (optimum 5-7), at salt concentrations of 1-3% (optimum 1-2%), and temperatures between 20 and 37 °C (optimum 28-30 °C). Biochemical tests such as lysine decarboxylase, ornithine decarboxylase, urease, gelatinase, citrate utilization, indole and acetoin production, and cytochrome oxidase tests allow to differentiate the novel species from their more closely related species. Considering these genetic and phenotypic divergencies, we propose the following new species: Xenorhabdus aichiensis sp. nov. with XENO-7T (= CCM 9233T = CCOS 2024T) as the type strain, Xenorhabdus anantnagensis sp. nov., with XENO-2T (= CCM 9237T = CCOS 2023T) as the type strain, and Xenorhabdus yunnanensis sp. nov., with XENO-10T (= CCM 9322T = CCOS 2071T) as the type strain. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of entomopathogenic bacteria associated with insect parasitic nematodes.

Morphological and molecular profiling of an entomopathogenic nematode Steinernema feltiae: Unlocking its biocontrol potential against vegetable insect pests.

A population of entomopathogenic nematodes, belonging to the Feltiae-clade and labelled J13, was discovered in the agricultural soils of the hilly regions of the Union territory of Jammu and Kashmir, India. Based on morphological, morphometric, and molecular analyses, the nematodes were identified as Steinernema feltiae. The J13 nematode isolate was tested in a laboratory assay for its pathogenicity against six major pests of vegetable crops: Pieris brassicae, Plutella xylostella, Helicoverpa armigera, Agrotis iplison, Trichoplusia ni, and Exelastis atomosa. The morphology of the isolated nematode closely matched the original description, except for the adult females, which had prominent epiptygmata instead of the weakly developed, double-flapped epiptygmata described in the original report. Analysis of the internal transcribed spacer and large subunit rRNA data from the J13 nematodes showed 100% similarity to sequences of the type population, indicating that they are conspecific. The virulence assays revealed that the nematode caused 100% mortality in the tested insect pests within 4872 hours, even at the lowest concentration of 50 infective juveniles per insect. The calculated median lethal concentration varied among the pests, with the lowest number of infective juveniles needed to achieve 50% larval killing being 117 for P. xylostella, 181.74 for P. brassicae, 226.35 for H. armigera, and 202.07 for T. ni at 24 hours post-inoculation. These findings suggest that S. feltiae isolated during the present investigation, may be a viable option for the biocontrol of these insect pests in Kashmir valley, India.