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 laumondii 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. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-023-05990-z.


Background
Entomopathogenic nematodes belonging to the families Heterorhabditidae and Steinernematididae are highly effective biocontrol agents against agricultural pests.These nematodes have independently evolved mutual associations with insect pathogenic bacteria of the genera Photorhabdus and Xenorhabdus, respectively [1][2][3][4].At the infective juvenile (IJ) stage, these nematodes, which reside in the soil, actively search for insect hosts [5].When an appropriate host is located, the IJs penetrate the insect body through natural openings or by directly breaking through the cuticle.They then release their bacterial symbionts upon sensing unknown chemical cues in the hemolymph [6,7].The bacteria multiply and produce virulence factors and toxins that kill the infected host [8][9][10].Furthermore, the bacteria secrete exoenzymes that degrade the insect tissues and produce several metabolites essential for nematode growth, development, and reproduction [11,12].The bacteria also produce potent secondary metabolites that act as antibiotics and deter scavenging arthropods.Upon resource depletion, the new generation of nematodes disperses in search of new hosts [9,13].
Heterorhabditis species are generally more virulent than those of Steinernema [14].However, they are less speciose than Steinernema [15,16].Despite this, new valid species of Heterorhabditis are often described and added to the list.The genus Heterorhabditis comprises 21 valid species, including two recently described species, Heterorhabditis ruandica from Rwanda and Heterorhabditis zacatecana from Mexico [15,17].Most of the valid species described so far have been molecularly characterized, except for Heterorhabditis egyptii [18] and Heterorhabditis hambletoni [19], which have only been morphologically characterized.The genus Heterorhabditis is globally distributed, although some species are only reported in certain geographic regions.In India, for instance, three species of Heterorhabditis have been documented so far: Heterorhabditis indica [20,21], Heterorhabditis bacteriophora [22], and Heterorhabditis baujardi [23].Heterorhabditis indica, described by Poinar et al. [20], is the only new species of the genus Heterorhabditis reported from India to date.
In this study, we present the discovery of, and characterize, a new entomopathogenic nematode species, Heterorhabditis casmirica n. sp., and its symbiotic bacteria, recovered from the union territory of Jammu and Kashmir, India.Our study contributes to the characterization of soil biodiversity in general and advances our efforts to understand the biodiversity of an important group of biological control agents, which are essential tools for ecofriendly and sustainable agricultural practices.

Nematode origin
Six populations of nematodes, namely HM, HM8, HP1, HPH, HH1, and HH4, were obtained from soil samples collected in the northwestern part of the union territory of Jammu and Kashmir, India.The samples were collected from soils around the roots of walnut and willow trees in the Anantnag district (Global Positioning System coordinates 33.828914, 75.100091; altitude 1606 m above sea level).Each one of these six populations was isolated from different soil samples.Each soil sample was separated by about 2 km from each other.Nematodes were isolated from soil samples using Corcyra cephalonica as a bait insect.Insects with nematode infestation symptoms were washed with double distilled H 2 O, sterilized with 0.1% NaOCl 2 , and then placed in White traps to recover the new generation of IJs [24].Recovered nematodes were reared using Galleria mellonella larvae as hosts under laboratory conditions [25,26].The IJs were stored in 250-mL tissue culture flasks in a biological oxygen demand incubator at 15 °C [27,28].The new species has been registered at ZooBank under urn:lsid:zoobank.org:pub:BBFC7CC6-7294-4548-AA7F-5CD5293E4103.

Nematode morphological and morphometric characterization, light and scanning electron microscopy
Hermaphroditic females, males and amphimictic females were obtained by dissecting G. mellonella cadavers in Ringer's solution 4 and 6 days after infestation, respectively [26,28].The IJs were collected from White traps after emerging from the G. mellonella cadavers.The nematodes were then killed with hot water, fixed in TAF solution (2 mL triethanolamine, 7 mL of 40% commercial formaldehyde solution, and 91 mL distilled water), transferred to anhydrous glycerin, and mounted on permanent glass slides with additional layers of paraffin wax to prevent flattening during microscopy [29,30].Morphological measurements (in micrometers) were taken using Nikon DS-L2 image acquisition software on a phasecontrast microscope (Nikon Eclipse 80i).Twenty specimens at each developmental stage were measured.Light microscopy (LM) and scanning electron microscopy (SEM) photographs were obtained using various nematological techniques detailed by Abolafia [31].In brief, nematodes fixed in 4% formalin solution were processed to anhydrous glycerin using Siddiqi's method with lactophenol-glycerin solutions [32].Subsequently, the nematodes were permanently mounted on glass microscope slides using the glycerin-paraffin method [33,34].The LM photographs were captured using a Nikon Eclipse 80i microscope (Olympus, Tokyo, Japan) with differential interference contrast optics and a Nikon Digital Sight DS-U1 camera.For SEM, nematodes preserved in glycerin were removed from permanent microscope slides by removing the cover glass, rehydrated in distilled water, dehydrated in a graded ethanol-acetone series, critically point dried with liquid CO 2 , mounted on SEM stubs with copper tape, coated with gold in a sputter coater, and finally observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany) [35].The LM and SEM micrographs, obtained at different magnifications for each structure, were processed and combined using Adobe Photoshop Creative Suite (Microsoft, Redmond, WA).
Comparisons were made between all the valid described species of Heterorhabditis based on morphological, morphometric and molecular characters, using the keys published by Machado et al. [17].Demanian indices and other ratios were calculated following the method outlined by de Man [36].The stoma morphology was described using the terminology provided by De Ley et al. [37], the spicule and gubernaculum morphology was described using the terminology established by Abolafia and Peña-Santiago [38] and the terminology for pharynx follows the proposals of Bird and Bird [39] and Baldwin and Perry [40].

Self-crossing and cross-hybridization experiments
Self-crossing and cross-hybridization experiments were carried out on lipid agar plates following the methodology described by Dix et al. [41].Heterorhabditis casmirica n. sp.isolates HM, HM8, HP1, HPH, HH1, and HH4 were crossed with each other and allowed to interact with Indian populations of H. bacteriophora (P4, P5 and KAS), H. indica (TH7, TH8 and TH9) and H. baujardi (HeTD4) nematodes.Control experiments were also conducted by self-crossing all the nematode species/strains.In each experiment, 20 second-generation males and 20 secondgeneration virgin females of each species were placed on 35-mm-diameter lipid agar plates and incubated at 25 °C.Progeny production was observed daily for 7 consecutive days.The experiments were conducted twice under the same conditions.

Nematode molecular characterization and phylogenetic relationships
Genomic DNA was extracted from individual hermaphroditic females isolated from insect cadavers infested with H. casmirica n. sp.HM, HM8, HP1, HPH, HH1, or HH4, as described [42].Briefly, individual virgin females were washed separately with Ringer's solution and then washed in phosphate-buffered saline (pH 7.2).Virgin females were then individually transferred to sterile polymerase chain reaction (PCR) tubes (0.2 mL) containing 20 μL extraction buffer (17.6 μL nuclease-free distilled H 2 O, 2 μL of 5X PCR buffer, 0.2 μL 1% Tween, and 0.2 μL proteinase K).Samples were frozen at −20 °C for 60 min or overnight and then immediately incubated in a PCR thermocycler at 65 °C for 1.2 h, followed by incubation at 95 °C for 10 min.The lysates were cooled on ice and centrifuged at 6500 g for 3 min.The resulting supernatants were used as DNA templates to amplify different taxonomically relevant gene markers.A fragment of ribosomal rRNA (rRNA) containing the internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2) was amplified using primers 18S (5′-TTG ATT ACG TCC CTG CCC TTT-3′) (forward) and 28S (5′-TTT CAC TCG CCG TTA CTA AGG-3′) (reverse) [43].A fragment of rRNA containing the D2-D3 regions of the 28S rRNA was amplified using primers D2F (5′-CCT TAG TAA CGG CGA GTG AAA-3′) (forward) and 536 (5′-CAG CTA TCC TGA GGA AAC -3′) (reverse) [44].The 12S mitochondrial gene was amplified using primers 505F (5′-GTT CCA GAA TAA TCG GCT AGAC-3′) (forward) and 506R (5′-TCT ACT TTA CTA CAA CTT ACT CCCC-3′) (reverse) [44] and the mitochondrially encoded cytochrome oxidase subunit I gene (MT-COI) was amplified using primers HCF (5′-TTA CAT GAT ACT TAT TAT G-3′) (forward) and HCF (5′-CTG ATA ACT GTG ACC AAA TAC ATA -3′) (reverse) [45].The PCR reactions consisted of 2 µL of DNA extract, 12.5 µL of DreamTaq Green PCR Master Mix (Thermo Scientific, USA), 0.75 µL of each forward and reverse primer at 10 µM and 9 µL of nuclease-free distilled H 2 O.The PCR reactions were performed using a thermocycler (Applied Biosystems Veriti 96-Well Thermal Cycler) with the following settings: (i) for ITS, D2-D3 and 12S-one cycle of 3 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min 30 s at 72 °C, followed by a single final elongation step at 72 °C for 20 min; (ii) for the MT-COI gene-one cycle of 3 min at 94 °C followed by 38 cycles of 10 s at 94 °C, 30 s at 40 °C, 60 s at 72 °C, followed by a single final elongation step at 72 °C for 10 min [46].PCR was followed by electrophoresis (45 min, 100 V) of 5 μL of PCR products in a 1% Trisboric acid-ethylenediaminetetraacetic acid-buffered agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA).PCR products were purified using the FastGene Gel/PCR extraction kit (Nippon Genetics, Japan) and sequenced using reverse and forward primers by Sanger sequencing (Bioserve, Hyderabad, India).The obtained sequences were manually curated, trimmed and deposited at the National Center for Biotechnology Information (NCBI) under the accession numbers given in Additional file 1: Table S4.To complete this data set and to obtain genomic sequences of nematodes that belong to all the valid described species of the genus Heterorhabditis, we searched the database of the NCBI by using the Basic Local Alignment Search Tool and the accession numbers of the sequences obtained previously [17,47].The resulting sequences were used to reconstruct phylogenetic relationships by the maximum likelihood method based on the following nucleotide substitution models: Tamura-Nei (TN93+G+I) (MT-COI) and Kimura 2-parameter (K2+G) (D2-D3) (ITS).To select the best substitution models, best-fit nucleotide substitution model analyses were carried out in MEGA 11 [48][49][50][51].Sequences were aligned with MUS-CLE (v3.8.31) [52].The trees with the highest log likelihood are shown.The percentage of trees in which the associated taxa clustered is shown next to the branches.Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BIONJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach, and selecting the topology with a superior log-likelihood value.In some cases, a discrete gamma distribution (+G) was used to model evolutionary rate differences between sites, and the rate variation model allowed for some sites to be evolutionarily invariable (+I).The trees are drawn to scale, with branch lengths measured in the number of substitutions per site.Graphical representation and edition of the phylogenetic trees were performed with Interactive Tree of Life v3.5.1 [53,54].

Symbiotic relationships
The Photorhabdus entomopathogenic bacteria associated with the different H. casmirica n. sp.nematode populations were isolated as described previously [55,56].
Briefly, larvae of G. mellonella (Lepidoptera: Pyralidae) were exposed to 100 nematode IJs.Three to 4 days later, insect cadavers were surface sterilized and cut open with a surgical blade.Bacteria-digested internal organs were spread onto Luria-Bertani (LB) agar plates and incubated at 28 °C for 24-48 h.Photorhabdus-like colonies were then streaked on fresh LB agar plates until monocultures were obtained.A single primary form colony was then selected and used for further experiments.Bacteria primary forms were determined by examining colony morphology, colony texture, pigment production, and bioluminescence.The strains were further subcultured and maintained on LB agar plates at 28 °C.An initial molecular characterization, using 16S rRNA gene sequences, was carried out to determine the taxonomic affiliation of the obtained bacterial cultures as described previously [3,4,17,56].Phylogenetic reconstruction and sequence comparisons based on whole genome sequences were carried out to confirm the taxonomic affiliation of the obtained bacterial cultures as described previously [3,55,56].Briefly, genomic DNA was extracted and purified using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Switzerland) following the manufacturer's instructions.The resulting DNA was used for library preparation using the TruSeq DNA PCR-Free LT Library Prep (FC-121-3003) kit.Indexed libraries were then pooled at equimolar concentrations and sequenced [2 × 150 base pairs (bp)] on an Illumina HiSeq 3000.Raw Illumina reads were quality trimmed using Trimmomatic 0.39 [57].The resulting reads were assembled with SPAdes 3.14.1 (k-mer sizes of 31, 51, 71, 91, and 111 bp) [58].Scaffolds with a mean read depth smaller than 20% of the median read depth of the longer scaffolds (≥ 5000 bp) as well as scaffolds that were shorter than 200 bp were removed.The final assemblies were polished using Pilon 1.22 [59].Phylogenetic relationships were reconstructed based on the assembled genomes and the genome sequences of all valid published species of the genus [3,55,56].For this, core genome alignments were created using Roary 3.6.2[60].Based on this alignment, a maximum likelihood tree was constructed using Fasttree 2.1.10based on the Jukes-Cantor plus CAT nucleotide evolution model [61].

Results and discussion
Six populations of Heterorhabditis nematodes (HM, HM8, HP1, HPH, HH1, and HH4) were isolated from agricultural soils in Kashmir, India.Initial molecular and    morphological characterization showed that they are genetically identical, morphologically very similar, and represent a novel species closely related to H. bacteriophora.The nematode population HM was chosen as the type material to describe this newly discovered species.

Hermaphroditic females
Hermaphroditic female body C-shaped when heat relaxed, body robust, always containing many juveniles, in some specimens a few eggs were visible.Cuticle almost smooth, about 0.8 to 1.6 µm thick.Lateral fields and phasmids not distinguishable under LM.Anterior end tapering anteriorly.Labial region with six prominent lips, each with a terminal conoid labial papilla.Cephalic papillae not observed with LM.Amphidial apertures pore-like.Stoma rhabditoid type, 1.1-1.7 times the lip region width, with a short cheilostom with a hardly visible refringent rounded cheilorhabdia, gymnostom with refringent bar-like rhabdia, well-developed, and funnelshaped stegostom surrounded by the pharyngeal collar and bearing minute rhabdia.Pharynx with sub-cylindrical procorpus, slightly swollen metacorpus, robust isthmus, and poorly developed, spheroid basal bulb with inconspicuous valves.Nerve ring surrounding the isthmus, at 55-74% of neck length.Excretory pore at basal bulb level or intestine level, at 94-120% of neck length.Cardia conoid.Reproductive system didelphic-amphidelphic.Ovaries well developed, reflexed.Oviducts poorly differentiated.Uteri with numerous embryonated eggs.Vagina short.Vulva a transverse slit, with smooth top and scarcely prominent lips, close to mid-body.Rectum slender, about 0.9-1.4times the anal body diameter.Anal region swelling posteriorly.Tail conoid with narrower pointed terminus, lacking a mucron.Phasmids inconspicuous.

Amphimictic females
Body arcuate with general morphology similar to that of hermaphroditic females.Body tapering toward anterior end; labial papillae acute and prominent.Reproductive system didelphic-amphidelphic with ovaries well developed, reflexed, oviducts and uteri poorly visible, vagina very short, and vulva small with a transverse slit opening.Rectum slightly longer than that of hermaphroditic females, about 1.7-1.9times longer than the anal body diameter.Anal lips usually prominent.Tail conoid longer than that of hermaphroditic females, with acute tip lacking a mucron.Phasmids very small, located at 50-62% of tail length.

Males
Body curved ventrally (open C-shape) or sometimes straight when heat relaxed.Anterior end truncate.Lip region with six scarcely separated lips and six conoid liplets at oral margin; six labial papillae at liplet tips and four cephalic papillae at the base of the dorsal and ventral lips.Amphidial aperture pore-like, just posterior to the lateral lips.Stoma 0.8-1.4times the lip region width, with short cheilostom and hardly visible refringent rounded cheilorhabdia, short gymnostom with refringent bar-like rhabdia, and long, funnel-shaped stegostom surrounded by the pharyngeal collar and bearing minute rhabdia.Pharynx with subcylindrical procorpus, scarcely swollen metacorpus, isthmus robust and slightly narrower than metacorpus, and basal bulb poorly developed, spheroid, with poorly developed valvular apparatus.Nerve ring located surrounding isthmus, at 55-69% of neck length.Excretory pore located at basal bulb or intestine level, at 99-107% of neck length.Cardia conoid, protruding into intestine.Intestine without differentiation although with narrower walls at anterior end.Reproductive system monorchid, with testis anteriorly reflexed and vas deferens well developed.Spicules well developed, separate, with small, almost quadrangular manubrium with very refringent dorsal and ventral walls, frequently smaller at the left spicule, calamus developed, and almost straight lamina with acute tip, poorly developed dorsal hump, and ventral velum slightly developed.Gubernaculum robust,

Infective sheathed juveniles (third-stage juvenile ensheathed in cuticle of second-stage juvenile)
Body straight when heat relaxed.Sheath (second-stage cuticle) present.Cuticle with longitudinal ridges except for the anterior part of the body, with annuli at the lip region and with tessellate pattern posterior to the lip region.Lip region lacking differentiated lips, bearing six labial papillae and cephalic papillae not visible.Amphidial aperture pore-like, having a cuticular dimple-like structure at its anterior part.Oral opening triradiate, closed.Stoma tubular, about twice as wide as the lip region.Pharynx slender, with corpus subcylindrical, isthmus narrower and slender, and basal bulb pyriform without developed valves.Nerve ring surrounding the isthmus, at 64-76% of neck length.Excretory pore at isthmus level, at 81-94% of neck length.Hemizonid clearly visible.Cardia conoid, surrounded by the intestinal tissue.Bacterial pouch not visible.Lateral fields not well differentiated from cuticle.Rectum narrow, not clearly discernible.Anus not well developed.Tail conoid-elongate with finely rounded terminus, without mucron.Terminal hyaline part 30-45% of tail length.Phasmids not visible.

Infective non-sheathed juveniles (third-stage juvenile)
Body with habitus straight when heat relaxed.Cuticle with transversal striae (annuli).Lateral field with two prominent longitudinal ridges.Lip region rounded, lacking differentiated lips, and labial and cephalic papillae not visible.Amphidial apertures oval.Oral opening rounded, closed, bearing a small dorsal tooth.Stoma, pharynx, nerve ring and excretory pore location similar to the sheathed stage.Hemizonid well developed.Cardia
The males of H. casmirica n. sp.can be distinguished from those of H. bacteriophora based on the neck length (106-118 vs. 99-105 µm), b ratio (6.4-8.2 vs. 9.1), c′ ratio (1.1-1.6 vs. 1.8),D% (99-107 vs 117 µm), spicules with a rectangular manubrium with strongly refringent walls (vs rectangular with scarcely refringent walls), gubernaculum more than a half of the spicule length (vs.shorter) and GP1 at the level of the manubrium (vs.more anterior in the type population).In comparison to male H. beicherriana, differences include body size (0.6-0.9 vs. 0.9-1.2mm), maximum body diameter (24-48 vs. 51-73 μm), the distance from the anterior end to the excretory pore (102-120 vs. 130-157 μm), the distance from the anterior end to the nerve ring (58-80 vs. 81-108 μm), the tail length (16-32 vs. 32-45 μm), D% (99-107 vs. 102-120 µm), GP1 at spicule level (vs.more anterior), the shape of the spicule manubrium (quadrangular vs. oblongate) and gubernaculum (more than half of the spicule length vs. similar length).Compared to males of H. egyptii, differences lie in the c ratio (24-35 vs. 19.5).When compared to males of H. georgiana, differences lie in the position of the excretory pore (at bulb or intestine level vs. posterior to the basal bulb only), spicules with rectangular manubrium with strongly refringent walls (vs rectangular with scarcely refringent walls) and gubernaculum (more than a half of the spicule length vs. a half of the spicule length).Compared to males of H. ruandica, differences include the shape of the spicule manubrium (well developed, quadrangular and with strongly refringent walls vs. poorly developed, triangular and not refringent), the shape of the gubernaculum manubrium (hook-like vs. straight) and gubernaculum (more than a half of the spicule length vs. a half ).Compared to males of H. zacatecana, differences include the shape of the spicule manubrium (quadrangular with strongly refringent walls vs. rounded and not refringent), bursa with GP1-GP2 distance shorter (less than the corresponding body diameter vs. slightly longer), GP2-GP3 slightly separated (vs.very closed), spicule manubrium (with angular anterior end vs. with rounded anterior end), the shape of the gubernaculum manubrium (hook-like vs. slightly curved) and gubernaculum more than a half of the spicule length (vs.shorter).Lastly, differences from males of H. hambletoni include the distance from the anterior end to the nerve ring (58-80 vs. 80-90 μm).With respect to the males of all of the other species, H. casmirica n. sp. has a different spicule morphology (manubrium with thick and refringent walls and lacking a dorsal hump vs. thin walls and a small dorsal hump) and gubernaculum with a hook-like manubrium (vs.straight).

Life cycle
Heterorhabditis casmirica n. sp. is a highly pathogenic nematode species that can be easily raised on G. mellonella larvae at a temperature ranging from 18 to 24 °C.The life cycle of this new species is comparable to that of other Heterorhabditis species.When G. mellonella larvae are exposed to 50-100 IJs, they die within 36-48 h and appear bright reddish after 48-72 h.First-and secondgeneration adults of H. casmirica n. sp.can be found in the insect cadavers 5-6 and 7-9 days after infection, respectively.The pre-infective juveniles left the host body, matured for a few days, and then migrated to the water traps after 15-21 days.

Type host and locality
The specific host for H. casmirica n. sp. is currently unknown as these nematodes were isolated from soil samples using the insect baiting technique [24,77,78].Heterorhabditis casmirica n. sp.populations were collected from soil samples in the union territory of Jammu and Kashmir, located in the northwest region of India, and specifically in the Himalayan Pir Panjal region.

Type material
The type material for H. casmirica n. sp.(holotype male, 15 hermaphroditic female paratypes, 15 male paratypes, 15 amphimictic female paratypes and 19 J3, all belonging to the HM population) were deposited in the National Nematode Collection of India, Indian Agricultural Research Institute, New Delhi.Nematode cultures are maintained at the Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, India.

Etymology
The specific name "casmirica" is derived from the Kashmir division (Casmiria in Latin), the geographical region where the nematodes used to describe the new species were collected.

Cross-hybridization experiments
No viable offspring were observed when H. casmirica n. sp.nematodes of the HM strain were allowed to interact with Indian populations of H. bacteriophora, H. indica, and H. baujardi.However, fertile progenies were observed when six different populations of H. casmirica n. sp.nematodes were allowed to interact, indicating that these populations are conspecific but reproductively isolated from closely related species, including H. bacteriophora, H. indica, and H. baujardi.Fertile progeny was also observed when all the nematode strains self-fertilized.

Nematode molecular characterization
The six populations of H. casmirica n. sp. were molecularly characterized based on the sequences of various genetic regions, including the ITS region of the rRNA (NCBI accession numbers OQ517936-OQ517941), the D2-D3 expansion segments of 28S rRNA (NCBI accession numbers OQ517947-OQ517952), mitochondrial 12S rRNA (NCBI accession numbers OQ517975-OQ517980), and MT-COI (NCBI accession numbers OQ517969-OQ517974  S2).All these three species share 100% similarity in the D2-D3 rRNA sequences flanked by primers D2A and D3B (Additional file 1: Table S3).Currently, very few mitochondrial 12S rRNA gene sequences are publicly available for molecular comparisons and phylogenetic analysis.Nevertheless, the sequences obtained in this study were deposited in the NCBI database for future taxonomic studies.

Nematode phylogenetic reconstructions
Phylogenetic analyses based on different genetic markers show that H. casmirica n. sp.belongs to the "bacteriophora" clade, which is currently composed of H. bacteriophora, H. beicherriana, H. georgiana, H. ruandica, and H. zacatecana (Figs.7,8,9).MT-COI is particularly useful for the differentiation of all of these closely related species, and clearly shows that H. casmirica n. sp. and H. bacteriophora, its more closely related species, form two independent subclusters (Fig. 7).However, sequences of the ITS and D2-D3 regions of the rRNA gene, although allowing for the differentiation of certain species (Figs.7,8), provide lower phylogenetic resolving power than the MT-COI gene, as reported by Dhakal et al. [46] and Machado et al. [17].Hence, MT-COI is particularly useful for the molecular discrimination of closely related species of the genus Heterorhabditis.

Morphological and molecular relationships between H. casmirica n. sp. and specimens of H. bacteriophora present in India
At the morphological level, H. casmirica n. sp.differs from previously reported Indian isolates of H. bacteriophora [22,30] (Additional file 1: Table S1).In particular, we observed that the males differ in spicule manubrium with strongly refringent walls (vs with scarcely refringent walls), gubernaculum more than a half of the spicule length (vs.shorter) and GP1 at manubrium level (vs.more anterior in the type population).The amphimictic females differ in smaller phasmids (vs.inconspicuous).The IJs differ in the distance from the anterior end to the nerve ring (79-94 vs. 48-74 µm), presence of bacterial sac (invisible vs. visible in the ventricular portion of the intestine), and size of phasmids (very small at posterior part of tail vs. inconspicuous) (Additional file 1: Table S1).
At the molecular level, H. casmirica n. sp.differs in 17 nucleotide positions in the MT-COI gene from several     Notably, the Indian populations DH7, DH8, CH21, P5 and P6 share 100% similarity with the type population of H. bacteriophora across all the gene markers used, and hence corroborate the conclusions of previous studies [22,30].The phylogenetic study further confirms the distinctiveness of the Indian populations of H. bacteriophora from H. casmirica n. sp. and establishes their similitude with the type population of H. bacteriophora (Figs.7,8,9).

Symbiotic relationships
Phylogenetic reconstructions based on core genome sequences and sequence comparisons show that the bacterial symbionts isolated from H. casmirica n. sp. are very similar and belong to the subspecies Photorhabdus laumondii subsp.clarkei (Fig. 10).The digital DNA-DNA hybridization (dDDH) scores between BOJ47 T , the type strain of the species P. laumondii subsp.clarkei, and strains HH4, HPH, and HP1, isolated from H. casmirica   S5.The scale bar shows the number of substitutions per site n.sp.HH4, HPH and HP1, are 94.3%, which is above the 70 and 79% thresholds that delimit prokaryotic species and subspecies, and confirms that they are conspecific [79].

A side note on the nomenclature of Heterorhabditis marelatus
The term "marelatus" was created by combining the Latin words "mare" meaning sea and "latus" meaning side in an attempt to translate the type locality "seaside" into Latin [70].Hence, marelatus was formed as a noun, not as an adjective.Sudhaus [80] changed the specific epithet of the species Heterorhabditis marelatus to "marelata."This change was perhaps motivated by the fact that the genus noun Heterorhabditis is feminine and that, in Latin, the specific epithet should agree in gender with the genus.However, nouns in Latin do not vary according to gender, and therefore we propose that the correct term is "marelatus." Hence, we propose that the original species nomenclature, Heterorhabditis marelatus, should be maintained.

Conclusions
Six populations of Heterorhabditis nematodes were identified in the present study that exhibited clear distinctions in their morphology, morphometric and molecular characteristics, as well as reproductive isolation and phylogenetic separation from all known Heterorhabditis species.We propose the name Heterorhabditis casmirica n. sp. for this new species, which is the second new Heterorhabditis entomopathogenic nematode species reported from the Indian subcontinent.Our results highlight the importance of using both classical taxonomy and molecular markers (MT-COI, ITS, small subunit, and large subunit) to accurately describe new Heterorhabditis species and their bacterial symbionts.The discovery of H. casmirica n. sp. and its associated bacterial symbiont expands our understanding of the biodiversity and distribution of these biocontrol agents and underscores their potential in the development of new biocontrol strategies against insect pests.

Fig. 2 a
Fig. 2 a-j Heterorhabditis casmirica n. sp.(light microscopy images).a, d, g Anterior end of hermaphroditic female, amphimictic female and male, respectively.b, e, h Neck region of hermaphroditic female, amphimictic female and male, respectively (arrowhead indicates the excretory pore).c, f Posterior end of hermaphroditic female and amphimictic female, respectively.i, j Posterior end of male at spicule and bursa levels, respectively [arrowhead indicates the genital papillae (GP)]

Fig. 4 a
Fig. 4 a-h Heterorhabditis casmirica n. sp.(light microscopy images).a, e Entire body of J2 and J3, respectively.b, f Neck region of J2 and J3, respectively (arrowhead indicates the excretory pore).c, g Posterior end of J2 and J3, respectively (arrowhead indicates the anus).d, h Cuticle of J2 and J3, respectively (arrowheads indicate the lateral field)

a
Dashes indicate that the data are not provided in the original publication b Calculated from the drawings provided in the original publication c Calculated from other measurements provided in the original publication

a
Dashes indicate that the data are not provided in the original publication b Calculated from the drawings provided in the original publication

a
Dashes indicate that the data are not provided in the original publication b Calculated from the drawings provided in the original publication c Calculated from other measurements provided in the original publication

Table 6
Pairwise distances (in base pairs) of the mitochondrially encoded cytochrome C oxidase subunit I gene (MT-COI) regions between Heterorhabditis casmirica n. sp and other species of Heterorhabditis Data for H. casmirica n. sp. are in italic. .Data below the diagonal indicate percentage similarity.Data above the diagonal indicate the total difference between the characters Species (MT-COI)

Fig. 7
Fig. 7 Maximum-likelihood phylogenetic tree between the newly described Heterorhabditis casmirica n. sp. and described species of Heterorhabditis based on nucleotide sequences of MT-COI flanked by primers HCF and HCR.Numbers at nodes represent bootstrap values based on 100 replications.Bars represent average nucleotide substitutions per sequence position.National Center for Biotechnology Information (NCBI) accession numbers of the nucleotide sequences used for the analyses are shown in Additional file 1: TableS4.The scale bar shows the number of substitutions per site

Fig. 9
Fig. 9 Maximum-likelihood phylogenetic tree reconstructed from the nucleotide sequences of the D2-D3 expansion segments of the 28S rRNA (D2-D3), flanked by primers D2A and D2B.Accession numbers of the nucleotide sequences used for the analyses are shown in Additional file 1: Table S4.Numbers at nodes represent bootstrap values based on 100 replications.Bars represent average nucleotide substitutions per sequence position

Fig. 10
Fig. 10 Phylogenetic reconstruction based on core genome sequences of Photorhabdus bacterial strains; 2,227,040 nucleotide positions (2216 core genes) were used in the analysis.Numbers at the nodes represent Shimodaira-Hasegawa-like branch supports.Bar represents average nucleotide substitutions per sequence position.NCBI accession numbers of the genome sequences used for the reconstruction are shown in Additional file 1: TableS5.The scale bar shows the number of substitutions per site

Table 1
Morphometrics of the infective juvenile (IJ) and adult generations of Heterorhabditis casmirica n. sp.(population HM)All data, with the exception of n, ratios and percentages, are given in micrometers, and are shown as the mean ± SD (range) a Dashes indicate that these characters are absent in these generations

Table 2
Comparative morphometrics of Heterorhabditis IJs All data, with the exception of ratios and percentages, are given in micrometers, and are shown as the mean ± SD (range).Data for H. casmirica n. sp. are in italic.For abbreviations, see Table1

Table 3
Comparative morphometrics of Heterorhabditis adult malesAll data, with the exception of ratios and percentages, are given in micrometers, and are shown as the mean ± SD (range).Data for H. casmirica n. sp. are in italic.For abbreviations, see Table1

Table 4
Comparative morphometrics of Heterorhabditis hermaphroditic femalesAll data, with the exception of ratios and percentages, are given in micrometers, and are shown as the mean ± SD (range).Data for H. casmirica n. sp. are in italic.For abbreviations, see Table1

Table 5
Comparative morphometrics of Heterorhabditis amphimictic females All data, with the exception of ratios and percentages, are given in micrometers, and are shown as the mean ± SD (range).Data for H. casmirica n. sp. are in italic.For abbreviations, see Table1 conoid, surrounded by intestinal tissue.Rectum narrow and hardly visible.Anus closed.Tail conoid with refringent acute tip without mucron.Phasmids very small, located at posterior part of tail.
).The ITS region of H. casmirica n. sp. is 771 bp in length, with ITS1 comprising 389 bp, 5.8S comprising 154 bp, and ITS2 comprising 228 bp.The MT-COI region flanked by primers HCF and HCR of H. casmirica n. sp.shows sequence similarity scores ranging from 75 to 94% with other Heterorhabditis species, and differs in 17-57 nucleotide positions (Table 6).Considering this genetic region, H. casmirica n. sp. is closely related to H. bacteriophora, H. ruandica, and H. zacatecana (Table 6).Heterorhabditis bacteriophora and H. ruandica both share 94% similarity with H. casmirica n. sp. and differ in 17 nucleotide positions.Heterorhabditis zacatecana shares 93% similarity with H. casmirica n. sp., and differs in 21 nucleotide positions.Fewer differences between H. casmirica n. sp. and its more closely related species were observed in the rRNA gene sequences.When compared with H. casmirica n. sp., H. bacteriophora and H. zacatecana both share 99.7% similarity and differ in two nucleotide positions, while H. ruandica shares 99.5% similarity and differs in four nucleotide positions in the ITS rRNA sequences flanked by primers TW81 and AB28 (Additional file 1: Table