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Description of Heterorhabditis americana n. sp. (Rhabditida, Heterorhabditidae), a new entomopathogenic nematode species isolated in North America

Parasites & Vectors volume 18, Article number: 101 (2025) Cite this article

Abstract

Background

Heterorhabditis are important biological control agents in agriculture. Two Heterorhabditis populations, S8 and S10, were isolated from agricultural soils in the United States of America. Molecular analyses, based on mitochondrial and nuclear genes, showed that these populations are conspecific and represent a novel species of the “Bacteriophora” clade. This species was named Heterorhabditis americana n. sp. and is described in this study.

Methods

To describe H. americana n. sp., we carried out phylogenetic reconstructions using multiple genes, characterized their morphology, conducted self-crossing and cross-hybridization experiments, and isolated and identified their symbiotic bacteria.

Results

Heterorhabditis americana n. sp. is molecularly and morphologically similar to H. georgiana. Morphological differences between the males of H. americana n. sp. and H. georgiana include variations in the excretory pore position, the gubernaculum size, the gubernaculum-to-spicule length ratio, the tail length, and the body diameter. Infective juveniles (IJs) of H. americana n. sp. differ from H. georgiana IJs because H. americana n. sp. IJs have an invisible bacterial cell pouch posterior to the cardia and a small posterior phasmid, whereas H. georgiana IJs have a visible bacterial cell pouch and an inconspicuous phasmid. Hermaphrodites of H. americana n. sp. and H. georgiana are differentiated by the body length, the nerve ring distance from the anterior end, the excretory pore distance from the anterior end, the anal body diameter, and the c′ ratio. Females of H. americana n. sp. can be differentiated from H. georgiana females by the anal body diameter and the c′ ratio. Reproductive isolation was confirmed, as H. americana n. sp. does not produce viable offspring with any of the species of the “Bacteriophora” clade. Heterorhabditis americana n. sp. is associated with the symbiotic bacterium Photorhabdus kleinii.

Conclusions

Based on the observed morphological and morphometric differences, the distinct phylogenetic placement, and the reproductive isolation, the nematode isolates S8 and S10 represent a novel species, which we named Heterorhabditis americana n. sp. This study provides a detailed characterization of this novel species, contributing to enhancing our knowledge of species diversity and evolutionary relationships of the Heterorhabditis genus.

Graphical Abstract

Background

Entomopathogenic nematodes (EPNs) of the genus Heterorhabditis Poinar (1976) are soil-dwelling organisms that parasitize small arthropods, particularly insects [1]. These nematodes maintain an obligate mutualistic relationship with entomopathogenic bacteria (EPB) of the genus Photorhabdus [2, 3]. The nematodes colonize their host through natural openings (mouth, spiracles, or anus), or by directly penetrating the cuticle. Inside the host, the EPNs release their symbiotic bacterial partners [4, 5]. The bacteria then proliferate and release immunosuppressive compounds, lytic enzymes, and toxins, leading to death of the host [6,7,8,9]. The insects tissues serve as a nutrient rich source, in which the nematodes grow and reproduce. Upon resource depletion, the nematode re-establish symbiosis with Photorhabdus bacteria and abandon the depleted insect cadaver in search of a new host [10]. Due to the ability to rapidly kill their host, this lethal symbiotic pair is used for the biocontrol of agricultural pests [11,12,13,14,15].

There is still some controversy regarding the number of valid species of Heterorhabditis [16,17,18,19,20]. Most of the described species are currently considered valid species, while others have been synonymized, reinstated as valid species, or proposed as species inquirendae [16, 17, 19, 21,22,23]. More specifically, Heterorhabditis heliothidis (Khan, Brooks & Hirschmann, 1976) Poinar, Thomas & Hess, 1977, previously synonymized with H. bacteriophora, was proposed as species inquirenda, and H. egyptii Abd-Elgawad & Ameen (2005) and H. hambletoni Pereira (1937) were reinstated as valid species [22]. Moreover, H. brevicaudis, H. gerrardi, H. hawaiiensis, H. pakistanensis, H. somsookae, and H. sonorensis were synonymized [16, 17, 23]. Hence, the genus Heterorhabditis contains at least 22 species that are currently considered valid [17, 22, 24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43]. Two of them, H. egyptii and H. hambletoni, lack molecular data and cannot be included in molecular or phylogenetic studies. An additional species, Heterorhabditis alii, has recently been proposed, although morphological and molecular support for its novel taxonomic status is lacking [44]. It is therefore proposed here as a species inquirenda.

It is important to note that the primary argument supporting the proposals to synonymize several Heterorhabditis species was the small differences observed in the internal transcribed spacer (ITS) sequences [16, 17]. However, due to the limited phylogenetic resolution power of ribosomal genes, additional molecular evidence, ideally from phylogenomic analyses of whole nuclear and/or mitochondrial genome data, should be incorporated to substantiate these synonymization proposals [18, 22, 24, 45]. In support of this, a recent phylogenomic study evaluated the molecular systematics value of multiple commonly used and several novel gene markers to resolve the taxonomy of the genus Heterorhabditis. The most robust and well-resolved phylogenetic relationships were reconstructed using both whole nuclear and mitochondrial genomes [45].

In this study, we characterized a novel entomopathogenic nematode species, Heterorhabditis americana n. sp., based on morphological and molecular evidence. The objectives of this study were (1) to reconstruct phylogenetic relationships using taxonomically relevant genes to confirm the distinct evolutionary lineage of this species, (2) to perform detailed morphological and morphometric analyses, (3) to carry out self-crossing and cross-hybridization experiments to test for the reproductive isolation of H. americana n. sp., and (4) to isolate and characterize the symbiotic bacteria associated with this novel nematode species. Our study contributes to the understanding of entomopathogenic nematode biodiversity and supports the development of sustainable pest management strategies in agriculture.

Methods

Nematode origin and rearing

Nematodes were isolated from soil samples by the insect baiting [46] and the White trap methods [47]. Soil samples to isolate H. americana n. sp. S8 were collected in the southeastern part of the state of Nebraska (decimal degree coordinates 40.8341, −96.6686), and the soil samples to isolate H. americana n. sp. S10 were collected in the state of South Dakota (decimal degree coordinates 43.9925, −96.7284).

Morphological characterization

Hermaphrodites, amphimictic males, and amphimictic females were obtained by dissecting Galleria mellonella insects in Ringer’s solution [24]. Infective juveniles (IJs) were collected from White traps upon their emergence from G. mellonella cadavers [47]. Nematodes were fixed in 4% formaldehyde at 80 °C, then transferred to anhydrous glycerin, and mounted on permanent glass slides with extra paraffin wax layers to preserve their three-dimensional shape, as detailed by Grisse [48]. Morphological measurements were performed on an Olympus BX51 microscope (Tokyo, Japan) equipped with integrated software. Fifteen specimens at each developmental stage were morphologically characterized.

Light microscopy (LM) and scanning electron microscopy (SEM)

Specimens for light microscopy (LM) and scanning electron microscopy (SEM) were prepared following the protocol of Abolafia [49]. Briefly, nematodes fixed in 4% formalin were transferred to anhydrous glycerin using the Siddiqi’s lactophenol-glycerin method [50]. The nematode specimens were then permanently mounted on glass slides following the glycerin-paraffin method [50, 51]. LM images were captured on a Nikon Eclipse 80i microscope (Olympus, Tokyo, Japan) equipped with differential interference contrast (DIC) optics and a Nikon Digital Sight DS-U1 camera. For SEM, nematodes stored in glycerin were rehydrated in distilled water (dH2O), dehydrated through a graded ethanol-acetone series, dried at a critical point using liquid CO2, mounted on SEM stubs with copper tape, coated with gold in a sputter coater, and imaged with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany) [52]. Images from LM and SEM were processed and merged using Adobe® Photoshop® CS (Microsoft Corporation, Redmond, WA, USA). Morphological characteristics of all valid Heterorhabditis species were compiled from original publications [17, 22, 24, 25, 27,28,29,30,31,32,33,34,35,36,37,38,39, 41, 42, 53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74]. Demanian indices and other morphological ratios were calculated according to de Man [75]. Stoma morphology was described following De Ley [76], and spicule and the gubernaculum structures were described following the terminology of Abolafia and Peña-Santiago [77].

Self-crossing and cross-hybridization experiments

Self-crossing and cross-hybridization tests were conducted on lipid agar plates [78]. Given that H. americana n. sp. is morphologically and molecularly more similar to the species of the “Bacteriophora” clade, the following nematode species and isolates were included in these experiments: H. americana n. sp. S8 and S10, H. bacteriophora Brecon, H. beicherriana Cherry, H. casmirica HM, H. georgiana Kesha, H. ruandica Rw14_N-C4a, and H. zacatecana MEX-39. For each crossing type, 40 males and 40 virgin females were placed on 35 mm lipid agar plates and incubated at 25 ± 2 °C. The experiments followed a factorial design, with three replicates for each crossing type. Progeny production was recorded daily over 15 days. Each experiment was conducted three times under similar conditions.

Nematode molecular characterization

Genomic DNA (gDNA) was extracted from approximately 10,000–20,000 IJs using the Norgen’s Genomic DNA Isolation Kit (Norgen Biotek Corp., Thorold, ON, Canada), following the manufacturer’s guidelines. The genomic regions amplified via polymerase chain reaction (PCR) included the mitochondrial cytochrome c oxidase I (cox-1) gene, the D2–D3 expansion segments of the 28S rRNA gene and the ITS region of the rRNA gene. The sequences of the primers used for PCR were evaluated in previous studies and are listed in Table S1 [16, 79,80,81,82]. PCR conditions are described in detail in our previous studies [22, 24, 83]. The PCR products were separated by electrophoresis (45 min at 100 V) on a 1% TBA (Tris–boric acid–ethylenediaminetetraacetic acid [EDTA]) buffered agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA). Following electrophoresis, PCR products were purified using the FastGene Gel/PCR Extraction Kits (Nippon Genetics Co., Japan) and sequenced bidirectionally by Sanger sequencing (Microsynth AG, Balgach, Switzerland). Resulting sequences were manually curated, trimmed, and submitted to the National Center for Biotechnology Information (NCBI) database. Accession numbers are listed in Table S2.

Intra-individual genetic diversity

To evaluate intra-individual genetic diversity, DNA was extracted from single virgin females. These females were individually washed with Ringer’s solution, and then with phosphate-buffered saline solution (PBS, pH 7.2), and transferred to sterile 0.5 ml Eppendorf tubes containing 20 μl of extraction buffer (17.7 μl nuclease-free dH2O, 2 μl 10× PCR buffer, 0.2 μl 1% Tween 20, and 0.1 μl proteinase K). Samples were frozen at −20 °C for 24 h, then incubated at 65 °C in a water bath for 1.2 h, followed by a 95 °C incubation for 10 min. Lysates were cooled on ice and centrifuged at 6000×g for 2 min. PCR and sequencing were carried out as described above.

Phylogenetic relationships

Gene sequences used for the phylogenetic reconstructions were retrieved from the NCBI database using previously reported accession numbers [16, 22, 24] (Table S2). Phylogenetic relationships were reconstructed using the maximum likelihood method, based on the following nucleotide substitution models: Kimura 2-parameter (K2+G+I) for cox-1 gene, Kimura 2-parameter (K2+G) for D2–D3 rRNA gene, and Tamura 3-parameter (TN92) for ITS rRNA gene. The best substitution models were determined through model-fit analysis in MEGA 7 [84,85,86,87]. Sequences were aligned using MUSCLE (v3.8.31) [88]. Phylogenetic trees with the highest log likelihood values, showing percentage clustering of taxa at branch points are shown. Initial trees for heuristic searches were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with a superior log likelihood value. Evolutionary rate variations among sites were modeled with a discrete gamma distribution (+G), and some positions were treated as invariant (+I). The phylogenetic trees were graphically represented and edited using Interactive Tree of Life (v3.5.1) [89, 90].

Symbiotic relationships

The Photorhabdus entomopathogenic bacteria associated with H. americana n. sp. S8 and H. americana n. sp. S10 nematodes were isolated following our established protocols [91, 92]. To this end, G. mellonella larvae (Lepidoptera: Pyralidae) were infested with 100 IJ nematodes. After 3–4 days, the insect cadavers were surface-sterilized and dissected with a surgical blade. Internal insect tissues were spread onto Luria–Bertani (LB) agar plates and incubated at 28 °C for 1–2 days. Photorhabdus-like colonies were subcultured to obtain monocultures. A single colony was selected and used for further experiments. Molecular identification was carried out based on whole-genome-based phylogenetic reconstructions and sequence similarity values [93]. Whole genomes were obtained as described [94]. Whole-genome sequence similarities were assessed through the digital DNA-DNA hybridization (dDDH) method using the recommended formula 2 of the genome-to-genome distance calculator (GGDC) [95,96,97,98].

Results and discussion

Order: Rhabditida Chitwood, 1933

Infraorder: Strongylida Weinland, 1858

Family: Heterorhabditidae Poinar, 1976

Genus: Heterorhabditis Poinar, 1976

Heterorhabditis americana Machado, Abolafia, Robles, Ruiz-Cuenca, Bhat, Shokoohi, Půža, Zhang, Erb, Robert & Hibbard n. sp.

Type strain: Heterorhabditis americana n. sp. S8 is designated as the type strain of the species. An additional population, S10, of this species has been molecularly characterized in this study.

Type specimens: A slide containing the male holotype (Mh-am-UL2025) and three slides with paratypes representing each developmental stage—hermaphroditic females (H-am-UL2025), males (M-am-UL2025), females (F-am-UL2025), and IJs (IJ-am-UL2025)—were deposited in the Nematology Collection at the University of Limpopo, South Africa. In addition, slides containing the following paratypes were deposited in the Nematology Collection at the University of Jaen, Spain: six hermaphroditic females (USA002-01 and USA002-02), four females (USA002-03), four males (USA002-04), four J2 juveniles (USA002-05), and four J3 juveniles (USA002-05).

Type-host: The type host is unknown as the nematodes of this genus can be hosted by different insect species. Heterorhabditis americana n. sp. S8 and H. americana n. sp. S10 were isolated from soil samples using Galleria larvae as baits.

Type-locality: Heterorhabditis americana n. sp. S8 was isolated from soil samples collected in the southeastern part of Nebraska (decimal degrees coordinates 40.8341, −96.6686), and H. americana n. sp. S10 was isolated from soil samples collected in South Dakota (decimal degrees coordinates 43.9925, −96.7284).

Site in host: whole internal body.

Representative DNA sequences: Representative sequences of H. americana n. sp. S8 were deposited in the GenBank database under the accession numbers PQ483104 (ITS), OK646639 (D2–D3), PQ483125 and PQ341163 (cytochrome c oxidase subunit I, cox-1), PQ367560 (NADH dehydrogenase subunit 4, nad-4), PQ367701 (fanconi-associated nuclease 1, fan-1), and PQ367748 (serine/threonine-protein phosphatase 4 regulatory subunit 1, ppfr-1). Representative sequences of H. americana n. sp. S10 include PQ483105 (ITS), MW817559 (D2–D3), PQ483126 and PQ341161 (cytochrome c oxidase subunit I, cox-1), PQ367558 (NADH dehydrogenase subunit 4, nad-4), PQ367699 (fanconi-associated nuclease 1, fan-1), and PQ367746 (serine/threonine-protein phosphatase 4 regulatory subunit 1, ppfr-1). Additional sequences are provided in previous literature [45].

ZooBank registration: To comply with the regulations set out in Article 8.5 of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN), details of the new species have been submitted to ZooBank. The Life Science Identifier (LSID) of the article is urn:lsid:zoobank.org:pub:36EBE371-1A95-4267-BE3C-91E264B27EA3. The LSID for the new name H. americana is urn:lsid:zoobank.org:act:0B2179A2-EE62-4D33-AB75-30DDFE8C7D66.

Etymology: The specific name of this nematode species is derived from the continent where the specimens used for its description were collected (America).

Description

Morphological and morphometric characteristics of H. americana n. sp. are presented in Figs. 1, 2, 3, 4, 5, 6, 7, 8, and 9 and in Tables 1, 2, 3, 4, and 5. Values for H. americana n. sp. are presented in bold.

Fig. 1
figure 1

Line drawings of H. americana n. sp. S8. A–E Neck regions of a hermaphroditic female, a female, a male, a J2 infective juvenile, and a J3 infective juvenile, respectively. F–H Lip regions of a hermaphroditic female, a female, and a male, respectively. I A hermaphroditic female. J An infective juvenile. K An adult male. L An adult female. M–P Posterior ends of a hermaphroditic female, a female, a male, and a J2 infective juvenile, respectively

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Fig. 2
figure 2

Light microscope (LM) photographs of H. americana n. sp. S8 hermaphroditic females and dioecious adults. A, B Pharyngeal regions of a hermaphroditic female and an adult female, respectively. C–E Lip regions of a hermaphroditic female, an adult female, and an adult male, respectively. F, G Posterior ends of a hermaphroditic female and a female adult, respectively. H, I Entire bodies of a hermaphroditic female and a female adult, respectively

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Fig. 3
figure 3

Light microscope (LM) photographs of H. americana n. sp. S8 males. A Entire body. B Posterior end at bursa level (arrows pointing at the genital papillae). C–F Posterior end at spicules level (arrow pointing at the harpoon-like terminus of the gubernaculum)

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Fig. 4
figure 4

Light microscope (LM) micrographs of H. americana n. sp. S8 third-stage (J3) juveniles ensheathed in the J2 cuticle. A Entire body. B Pharyngeal region. C Anterior end. D Tail region. E Cuticle

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Fig. 5
figure 5

Light microscope (LM) micrographs of H. americana n. sp. S8 third-stage (J3) juveniles. A Entire body. B Pharyngeal region. C Anterior end. D Cuticle. E–F Tail region

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Fig. 6
figure 6

Scanning electron microscope (SEM) photographs of H. americana n. sp. S8 hermaphroditic females. A–C Anterior ends in sub-ventral, lateral, and frontal views, respectively (white arrows pointing at the amphids). D Excretory pore (white arrow). E Vulva covered by vaginal plug (arrow). F Entire body. G–H Posterior ends in sub-ventral and ventral views, respectively

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Fig. 7
figure 7

Scanning electron microscope (SEM) photographs of H. americana n. sp. S8 dioecious adults. A, B Anterior ends of a female and a male, respectively, in frontal view, (white arrows pointing at the amphids). C, D Excretory pores of a male in ventral view and a female in sub-ventral view. E, F Posterior end of a male adult in lateral view (arrows pointing at the genital papillae). G Entire body of a male. H Cuticle of a female adult. I Tail of a female adult in sub-ventral view (white arrow pointing at the anus, black arrow pointing at the phasmid). J, K Posterior end of a male adult in ventral view

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Fig. 8
figure 8

Scanning electron microscope (SEM) photographs of H. americana n. sp. S8 third-stage (J3) juveniles ensheathed in the J2 cuticle. A Pharyngeal region in lateral view. B, C, E Anterior end in sub-ventral, lateral and frontal views, respectively (arrows pointing at the amphids). D Entire body. F Excretory pore (arrow). G Cuticle at midbody. H, I Tail in ventral and lateral views, respectively (arrows pointing at the phasmids). J Left phasmid (arrow)

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Fig. 9
figure 9

Scanning electron microscope (SEM) photographs of H. americana n. sp. S8 third-stage juvenile (J3). A–C Lip region in ventral, sublateral, and frontal views, respectively (arrows pointing at the amphids). D Entire body. E, F Excretory pore at lateral and ventral views, respectively. F Lateral field. H Cuticle at anterior end in lateral view. I, J Posterior end in lateral and ventral views, respectively (arrows pointing at the phasmid). K Lateral field at anus level

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Table 1 Morphometric values of infective juveniles (IJs) and adult generations of H. americana n. sp. nematodes
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Table 2 Comparative morphometric values of Heterorhabditis adult males
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Table 3 Comparative morphometric values of Heterorhabditis hermaphrodite females
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Table 4 Comparative morphometric values of Heterorhabditis adult females
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Table 5 Comparative morphometric values of Heterorhabditis infective juveniles
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Hermaphroditic females

Hermaphroditic female C-shaped after heat relaxation, body robust, always with many juveniles inside, in some specimens few eggs visible. Cuticle smooth under light microscope, about 1 µm thick. Anterior end tapering anteriorly, labial region with six prominent lips, each with a terminal labial papilla. Cephalic papillae not visible under LM. Pore-like amphidial apertures. Rhabditoid stoma, measuring 0.7–0.9 times the width of the lip region. Stoma with short cheilostom, with barely visible, refringent, and rounded cheilorhabdia. Gymnostom well-developed, with refringent bar-like rhabdia, and funnel-shaped stegostom surrounded by the pharyngeal collar, bearing minute rhabdia. Pharynx with slightly swollen metacorpus, subcylindrical procorpus, robust isthmus, and weakly developed, spheroid basal bulb with inconspicuous valves. Nerve ring at 75–77% of neck length, surrounding the isthmus. Excretory pore at 91–113% of neck length, at basal bulb level. Cardia conoid. Reproductive system didelphic-amphidelphic. Ovaries reflexed and well-developed. Oviducts poorly differentiated. Uteri with numerous embryonated eggs. Vagina short. Vulva a transverse slit, with smooth top and prominent lips, located on a slightly protruding area, close to midbody. Rectum slender, about twice the anal body diameter. Anal region swelling posteriorly. Tail conoid with pointed terminus, lacking mucro. Phasmids inconspicuous.

Amphimictic females

General morphology similar to hermaphroditic females. Body arcuate, tapering towards the anterior end. Labial papillae more acute and prominent than the papillae of hermaphrodites. Reproductive system didelphic-amphidelphic. Well-developed ovaries, reflexed. Oviducts and uteri hardly visible. Very short vagina, and vulva small having transverse slit opening. Rectum shorter than hermaphroditic female rectum, measuring about 1.7–2.2 times the diameter of the anal body. Prominent anal lips. Tail conoid with acute tip, lacking mucro. Phasmids not visible.

Males

Body curved ventrally (open C-shaped), sometimes straight after heat relaxation. Anterior end truncate. Lip region with six narrowly separated lips, having six conoid liplets at oral margin. Liplet tips with six labial papillae. Lip region with four cephalic papillae, located at the base of the dorsal and ventral lips. Pore-like amphidial aperture, posterior to the lateral lips. Stoma 0.7–1.0 times the width of the lip region, with short cheilostom. Stoma with refringent rounded cheilorhabdia, barely visible. Short gymnostom with refringent bar-like rhabdia, and long, funnel-shaped stegostom surrounded by the pharyngeal collar, bearing small rhabdia. Pharynx with slightly swollen metacorpus, subcylindrical procorpus, isthmus slightly narrower than metacorpus, robust, and basal bulb spheroid and poorly developed, with poorly developed valvular apparatus. Nerve ring surrounding isthmus, located at 63–73% of neck length. Excretory pore at bulb level, located at 103–113% of neck length. Cardia conoid, protruding into intestine. Intestine poorly differentiated, although with narrower walls at anterior end. Reproductive system monorchic, with testis reflexed anteriorly. Well-developed vas deferens. Spicules well-developed, separate, with small almost quadrangular manubrium, calamus developed, and robust lamina with acute tip, prominent dorsal hump, in some specimens, hump not developed. Velum poorly developed ventrally. Gubernaculum robust, straight or slightly curved ventrally, with a size 35–47% of spicule length, with serrated dorsal margin and harpoon-like tip. Tail ventrally curved posteriorly, flanked by the bursa, conoid, with acute tip. Bursa peloderan bearing nine pairs of bursal papillae. The genital papillae arrangement is 1+2/3+3: three precloacal genital papillae, GP1 located 11–22 µm anterior to GP2 and GP3; GP2 and GP3 are equal in length; GP4–9 are post-cloacal; GP4–6 form a group located posterior to cloaca; GP4 is shorter than GP5 and GP6; GP5 and GP6 are of equal length; GP7–9 form a group close to the tail end; GP7 is slightly curved outward; GP8 is shorter than GP7 and GP9.

Infective sheathed juveniles (J3 stage covered by the J2 stage cuticle)

Body straight after heat relaxation. Second-stage cuticle present. Cuticle with longitudinal ridges, except at the anterior part of body, posterior to the lip region, with tessellate pattern. Lateral fields not differentiated from the cuticular ridges. Lips not differentiated, with six labial papillae. Cephalic papillae not visible. Pore-like amphidial aperture, showing cuticular dimple-like structures at anterior part. Oral opening triradiate, closed. Stoma tubular, about twice the lip region width. Nerve ring at 60–70% of neck length, surrounding the isthmus. Excretory pore located 70–90% of neck length, at basal bulb level. Well-visible hemizonid. Pharynx slender, with corpus subcylindrical, isthmus narrower and basal bulb pyriform without developed valves. Cardia conoid, surrounded by the intestinal tissue. Bacterial pouch not visible. Rectum narrow, about 1.5 times the anal body diameter. Anus poorly developed. Tail conoid-elongate with sharp terminus. Tail without mucro. Terminal hyaline part 34–55% of tail length. Phasmids very small, located at middle length of tail.

Infective non-sheathed juveniles (J3 stage)

Body straight to slightly curved ventrally after heat relaxation. Cuticle with transverse striae (annuli). Lateral fields with two ridges. Rounded lip region, lacking differentiated lips. Labial and cephalic papillae not visible. Oral opening closed, rounded, with a small dorsal tooth. Amphidial apertures oval. Stoma tubular, measuring 1.8 to 2.0 times the width of the lip region. Pharynx, nerve ring and excretory pore located at a similar position. Well-developed hemizonid. Cardia conoid, surrounded by intestinal tissue. Rectum narrow and poorly visible. Anus closed. Tail conoid with acute tip, and without mucro. Terminal hyaline part present, very short. Phasmids very small, located at posterior part of tail.

Diagnosis of Heterorhabditis americana n. sp.

Heterorhabditis americana n. sp. is characterized by a distinct combination of morphological and morphometric characters in males, hermaphroditic females, and IJs (Table 1). In males, the key diagnostic characteristics include a truncated lip region, tail lengths ranging from 18–29 µm, and spicule lengths between 36–48 µm. The ratio of spicule length relative to anal body diameter (SW%) ranges from 168–219, and the ratio of gubernaculum to spicule length (GS%) ranges between 35–47. Males have nine pairs of genital papillae, with three pairs present in the terminal group of the bursa. Hermaphroditic females have pore-like amphids, prominent lips with unique patterns, and non-prominent anal lips. The IJs are characterized by a body length of 0.47–0.57 mm, an excretory pore situated 90–97 µm from the anterior end, a neck length of 90–124 µm, a tail length of 64–100 µm, and a lateral field with 11 ridges (Table 1).

Morphological relationships of Heterorhabditis americana n. sp. with closely related species

Morphologically, H. americana n. sp. is similar to H. bacteriophora, H. beicherriana, H. casmirica, H. egyptii, H. georgiana, H. ruandica, and H. zacatecana. Various morphological and morphometric traits differ between H. americana n. sp. and their closely related species (Tables 2, 3, 4, 5). Males of H. americana n. sp. differ from the males of H. bacteriophora in the distance from the excretory pore to the anterior end (109–114 vs. 114–130 μm), tail length (18–29 vs. 28–38 µm), and GS% (35–47% vs. 50%). Compared to H. beicherriana males, differences include spicule manubrium shape (quadrangular vs. oblong), body size (0.69–0.88 vs. 0.89–1.19 mm), body diameter (33–43 vs. 51–73 μm), excretory pore to anterior end distance (109–114 vs. 130–157 μm), nerve ring to anterior end distance (64–78 vs. 81–108 μm), neck length (101–113 vs. 116–143 μm), tail length (18–29 vs. 32–45 μm), gubernaculum length (13–20 vs. 22–27 µm), and the GS% (35–47 vs. 48–59). Differences with H. casmirica males include spicule manubrium shape (rectangular with scarcely refringent walls vs. rectangular with strongly refringent walls), GP1 positioning (more anterior vs. at spicule level), and a lower GS% (35–47% vs. 45–63%). In contrast to H. egyptii, the new species has a longer excretory pore to anterior end distance (109–114 vs. 80–97 μm), and higher D% values (111–125 vs. 84–91). Differences with H. georgiana include excretory pore position (at the bulb level vs. posterior to the basal bulb), nerve ring to anterior end distance (64–78 vs. 72–93 μm), tail length (18–29 vs. 29–41 µm), mid-body diameter (36–43 vs. 43–55 μm), gubernaculum length (13–20 v 20–28 µm) and GS% (35–47% vs. 51–64%). Differences with H. ruandica males include the spicule manubrium shape (well developed, quadrangular with strongly refringent walls vs. poorly developed, triangular, and not refringent), gubernaculum manubrium shape (harpoon-like vs. straight tip), mid-body diameter (33–43 vs. 40–51 µm), excretory pore to anterior end distance (109–114 vs. 61–109 µm) and E% (111–125 vs. 61–97). Lastly, compared to H. zacatecana, differences include a quadrangular spicule manubrium with strongly refringent walls (vs. rounded and not refringent), angular anterior end of the spicule manubrium (vs. rounded), hook-like gubernaculum manubrium (vs. slightly curved), longer distance from nerve ring to anterior end (109–114 vs. 77–109 µm), and longer neck length (101–113 vs. 71–108 µm (Table 2).

In the case of hermaphroditic females, H. americana n. sp. differs from H. bacteriophora in body length (1.92–3.19 vs. 3.63–4.39 mm), excretory pore to anterior end distance (148–169 vs. 189–217 μm), neck length (148–183 vs. 189–205 μm), tail length (54–76 vs. 81–93 µm), anal body diameter (23–29 vs. 40–53 μm), V% value (48–51 vs. 41–47), and D% value (111–125 vs. 106) values. Compared to H. beicherriana, differences include anterior end to excretory pore distance (148–169 vs. 165–297 μm), anterior end to nerve ring distance (113–134 vs. 135–243 μm), neck length (148–183 vs. 192–343 μm), tail length (54–76 vs. 68–130 μm), anal body diameter (23–29 vs. 51–92 μm), V% value (48–51 vs. 41–47), and D% value (111–125 vs. 76–94) values. With H. casmirica, key differences include excretory pore position (at bulb level vs. more posterior), tail length (72–114 vs. 54–76 μm), and demanian ratios. Differences with H. egyptii, include tail length (54–76 vs. 83–115 μm) and anal body diameter (23–29 vs. 33–51 μm). Compared to H. georgiana, they differ in body size (1.92–3.19 vs. 3.23–4.93 mm), nerve ring to anterior end distance (113–134 vs. 143–217 μm), distance from anterior end to excretory pore (148–169 vs. 200–277 μm), and anal body diameter (23–29 vs. 42.6 µm). Compared to H. ruandica, differences include the distance from the excretory pore to the anterior end (148–169 vs. 106–153 µm), distance from the anterior end to the nerve ring (113–134 vs. 78–108 µm), anal body diameter (23–29 vs. 29–51 µm), b ratio (13–18 vs. 21–27), and E% value (111–125 vs. 67–103). Compared to H. zacatecana, H. americana n. sp. has a shorter body length (1.92–3.19 vs. 4.41–6.18 mm), shorter maximum body diameter (140–221 vs. 235–385 µm), shorter neck length (148–183 vs. 174–231 µm), shorter anal body diameter (23–29 vs. 34–58 μm), lower b ratio (13–18 vs. 20–34), lower c ratio (30–49 vs. 52–90), and higher D% value (111–125 vs. 55–95) (Table 3).

In the case of amphimictic females, H. americana n. sp. differs from H. bacteriophora in body length (1.72–2.43 vs. 3.18–3.85 mm), excretory pore to anterior end distance (101–136 vs. 174–214 μm), neck length (132–160 vs. 155–183 μm), tail length (53–73 vs. 71–93 μm), a ratio (14–16 vs. 21.4), b ratio (12–16 vs. 18.8), and D% (84–102 vs. 114). Compared to H. beicherriana, the new species differs in the anal body diameter (21–26 vs. 35–81 μm) and c′ ratio (2.3–3.1 vs. 1.6–2.4). Compared to H. georgiana, differences include the anal body diameter (21–26 vs. 42 μm) and c′ ratio (2.3–3.1 vs. 1.5). Compared to H. casmirica, the main differences are the excretory pore position (at bulb level vs. more posterior), c ratio (28–40 vs. 16–31), and D% value (76–90 vs. 99–116) values. Compared to H. egyptii, key differences include the body diameter (116–169 vs. 56–84 μm), distance from excretory pore to the anterior end (101–136 vs. 69–106 μm), distance from nerve ring to the anterior end (97–112 vs. 69–94 μm), neck length (132–160 vs. 106–125 μm), and D% value (84–102 vs. 78). Compared to H. ruandica, the new species can be distinguished by differences in the mid-body diameter (116–169 vs. 68–83 µm), nerve ring to anterior end distance (97–112 vs. 69–97 µm), neck length (132–160 vs. 107–132 µm), and c ratio (28–40 vs. 16–24 µm). Differences with H. zacatecana are in the mid-body diameter (116–169 vs. 160–228 µm), nerve ring to anterior end distance (97–112 vs. 71–96 µm), anal body diameter (21–26 vs. 31–41 µm), and b ratio (12–16 vs. 16–21 µm) (Table 4).

Heterorhabditis americana n. sp. IJs can be distinguished from H. bacteriophora IJs by a higher c′ ratio (6.5–9.4 vs. 6.0), lack of a visible bacterial sac (invisible in H. americana vs. visible in the ventricular part of the intestine in H. bacteriophora), and very small phasmids at the posterior tail compared to inconspicuous phasmids in H. bacteriophora. Compared to H. beicherriana, H. americana differs in the shape of amphidial apertures (oval vs. inconspicuous), the bacterial sac (invisible vs. visible), and size of posterior tail phasmids (very small vs. inconspicuous), and the excretory pore to anterior end distance (90–101 vs. 100–122 μm), and the nerve ring to anterior end distance (64–87 vs. 85–106 μm). Compared to H. casmirica, IJs of H. americana n. sp. can be differentiated by the position of excretory pore, which is located at basal bulb level in H. americana sp. n. rather than at the isthmus level. Compared to H. georgiana, the IJs of H. americana n. sp. lack a visible bacterial sac near the cardia and have very small phasmids at the posterior part of the tail (vs. inconspicuous ones). Differences with H. egyptii include a longer tail (64–100 vs. 53–75 µm). Additionally, H. americana n. sp. IJs can be differentiated from H. ruandica by a longer anterior end to excretory pore distance (90–97 vs. 70–89 µm), greater tail length (64–100 vs. 49–64 µm), and the presence of a smaller cephalic tooth (small vs. large). The new species differs from to H. zacatecana by longer tail (64–100 vs. 52–63 μm), and higher a ratio (24–29 vs. 19–24). Further morphological differences between males, hermaphroditic females, amphimictic females, and IJs of H. americana n. sp. compared to other Heterorhabditis species, are outlined in Tables 2, 3, 4, 5.

Male morphology

Since H. americana n. sp. is morphologically very similar to H. beicherriana and H. georgiana, the males of three additional isolates, H. beicherriana M6, H. georgiana (Kesha, topotype), and H. georgiana Hbb, were studied. Heterorhabditis americana n. sp., H. beicherriana, and H. georgiana differ particularly in the arrangement of the bursal papillae and the morphology of the spicules (Fig. 10). With respect to the bursal papillae, H. americana n. sp. and H. georgiana have similar distance between GP1 and GP2–GP3, while H. beicherriana has shorter distance between these papillae. Regarding the spicules, all the three species possess manubria of similar shape (rectangular, anteriorly open, and posteriorly thickened outward). However, the lamina lacks a dorsal hump in both H. americana and H. beicherriana, whereas H. georgiana has as a well-developed dorsal hump (Fig. 10).

Fig. 10
figure 10

Male tails of Heterorhabditis beicherriana and H. georgiana. A H. georgiana (population Kesha, topotype). B, C H. georgiana (population Hbb). D–F H. beicherriana (population M6)

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Cross-hybridization experiments

No progeny was observed when H. americana n. sp. S8 and H. americana n. sp. S10 were paired interspecifically with the following nematode isolates: H. bacteriophora (Brecon, type), H. beicherriana (Cherry, type), H. casmirica (HM, type), H. georgiana (Kesha, type), H. ruandica (Rw14_N-C4a, type), or H. zacatecana (MEX-39, type). In contrast, progeny was observed when these isolates were paired intraspecifically. These results show that H. americana n. sp. is reproductively isolated from the other Heterorhabditis species evaluated in this study. This reproductive isolation supports the classification of H. americana n. sp. as a distinct and novel species.

Nematode molecular characterization and phylogenetic relationships

Phylogenetic reconstructions based on mitochondrial cytochrome oxidase subunit 1 (cox-1) gene sequences and the ITS region of the rRNA gene show that H. americana n. sp. is closely related to H. georgiana and belongs to the “Bacteriophora” clade (Figs. 11 and 12). This clade includes H. bacteriophora, H. beicherriana, H. casmirica, H. georgiana, H. ruandica, and H. zacatecana (Figs. 11 and 12). The sequences of the D2–D3 expansion segments of the 28S rRNA gene, however, are of limited phylogenetic value, as closely related species are not phylogenetically resolved using this gene marker (Fig. S1). Sequence similarity scores further support a closer phylogenetic relationship between H. americana n. sp. and H. georgiana (Figs. S2–S7). The cox-1 gene sequences of these two species are 96.7% identical, differing by 11 nucleotides. The ITS sequences are 99.8% identical, differing by one nucleotide, and the D2–D3 sequences show no nucleotide differences (Figs. S2–S7). Comparatively lower sequence similarity scores were observed between H. americana n. sp. and all the other species of the “Bacteriophora” clade (Figs. S2–S7). No intra-individual variability was detected in any of the markers analyzed in this study.

Fig. 11
figure 11

Maximum-likelihood phylogenetic tree reconstructed from the sequences of the mitochondrial cytochrome c oxidase I (cox-1) gene. Phylogenetic analyses included 342 nucleotide positions, flanked by primers HCF and HCR. Numbers at nodes represent bootstrap values based on 100 replications. Bars represent average nucleotide substitutions per sequence position. Accession numbers of the nucleotide sequences used for the reconstructions are presented in Table S2

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Fig. 12
figure 12

Maximum-likelihood phylogenetic tree reconstructed from sequences of the internal transcribed spacer (ITS) region of the rRNA gene. Phylogenetic analyses included 796 nucleotide positions, flanked by primers TW81 and AB28. Numbers at nodes represent bootstrap values based on 100 replications. Bars represent average nucleotide substitutions per sequence position. Accession numbers of the nucleotide sequences used for the analyses are presented in Table S2

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Symbiotic relationships

Based on phylogenomic reconstructions using whole-genome sequences and on the sequence similarity values, H. americana n. sp. S8 and H. americana n. sp. S10 nematodes maintain symbiotic relationships with Photorhabdus kleinii (Fig. 13). Digital DNA–DNA hybridization (dDDH) values between S8-52 and S10-54, which are the symbiotic bacterial strains isolated from H. americana n. sp. S8 and H. americana n. sp. S10, respectively, are 100%, between S8-52 and P. kleinii DSM 23513T are 94%, and between S10-54 and P. kleinii DSM 23513T are also 94% (Fig. S8). When dDDH values between two strains are higher than 79%, the two strains are considered conspecific [97, 98].

Fig. 13
figure 13

Phylogenetic reconstruction based on core genome sequences of the different Photorhabdus species. Bar represents average nucleotide substitutions per sequence position. Numbers at the nodes represent SH-like branch supports. Accession numbers of the genome sequences used for the reconstruction are presented in Table S3

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Authors’ note on the recent proposal of Heterorhabditis alii as a novel species

Recently, Shamseldean et al. [44] proposed Heterorhabditis alii Shamseldean, Abo-Shady, El-Awady & Heikal, 2024 as a novel species. However, the morphology and the morphometry of the studied specimens closely align with the type population of H. indica Poinar, Karunakar & David, 1992. SEM photographs of juveniles show transverse ridges on the ventral side, which are likely artifacts of the specimen preparation process. Unfortunately, no photographs of males were provided. On the other hand, the ITS sequence provided (NCBI accession OP555450) is of low quality and appears to be chimeric. Nevertheless, in the phylogenetic tree, H. alii clusters with H. indica and H. hawaiiensis Gardner, Stock & Kaya, 1994 (currently considered a junior synonym of H. indica). Based on these findings, H. alii is declared species inquirenda. Additional morphological and molecular support should be provided by the original descriptors to further support its novel species status.

Conclusions

Given the morphological and morphometric differences, the distinct phylogenetic placement, and the reproductive isolation, the nematode isolates S8 and S10 represent a novel species, which we named H. americana n. sp.

Availability of data and materials

The gene sequences obtained in this study were deposited in the GenBank database under the accession numbers given in Additional file 1: Tables S2 and S3. Data supporting the conclusions of this article are included within the article. The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

GP:

Genital papilla

IJs:

Infective juveniles

ITS:

Internal transcribed spacer

LB:

Luria–Bertani

LM:

Light microscopy

NCBI:

National Center for Biotechnology Information

SEM:

Scanning electron microscopy

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Acknowledgements

The authors thank the Institute of Biology of the University of Neuchatel (Switzerland), the University of Limpopo (South Africa), and the Swiss National Science Foundation (SNSF) for their support. We are grateful to Ralf-Udo Ehlers and Carlos Molina (e-nema GmbH, Schwentinental, Germany), David I. Shapiro-Ilan (USDA-ARS, SEA), Larry Duncan (Institute of Food and Agricultural Science, University of Florida, FL, USA), Antoinette P. Malan (Department of Conservation Ecology and Entomology, Stellenbosch University, Matieland, Stellenbosch, South Africa), and Hugues Baimey (University of Parakou, Parakou, Benin) for providing nematodes. SEM pictures were obtained with the assistance of technical staff (Amparo Martínez-Morales) and equipment of the Centro de Instrumentación Científico-Técnica (CICT) of the University of Jaén (Spain).

Funding

The work of RARM was supported by the Swiss National Science Foundation (Grant No. 186094 to RARM). The work of AHB was supported by a Swiss Government Excellence Scholarship (Grant No. 2021.0463 to AHB). The work of ME and XZ was supported by the Swiss National Science Foundation (Grant No. 155781 to ME). The work of CAMR was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant No. ERC-2019-STG949595) and the Swiss National Science Foundation (Grant No. 310030_189071). Light microscopy and scanning electron microscopy studies were performed thanks to the financial support of the Research Support Plan “POAIUJA 2021/2022: EI_RNM02_2021” of the University of Jaén, Spain.

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Author notes

  1. Joaquín Abolafia, María-Cristina Robles, Alba N. Ruiz-Cuenca, Aashaq Hussain Bhat, and Ebrahim Shokoohi contributed equally to this work.

Authors and Affiliations

  1. Experimental Biology Research Group, Institute of Biology, Faculty of Sciences, University of Neuchâtel, Neuchâtel, Switzerland

    Ricardo A. R. Machado & Aashaq Hussain Bhat

  2. Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus ‘Las Lagunillas’, Jaén, Spain

    Joaquín Abolafia, María-Cristina Robles & Alba N. Ruiz-Cuenca

  3. University Centre for Research and Development and Department of Bioscience, Chandigarh University, Mohali, 140413, Punjab, India

    Aashaq Hussain Bhat

  4. Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Sovenga, 0727, South Africa

    Ebrahim Shokoohi

  5. Institute of Entomology, Biology Centre of the Czech Academy of Sciences, CAS, 37005, České Budějovice, Czech Republic

    Vladimír Půža

  6. Faculty of Agriculture and Technology, University of South Bohemia, 37005, České Budějovice, Czech Republic

    Vladimír Půža

  7. Institute of Plant Sciences, University of Bern, Bern, Switzerland

    Xi Zhang, Matthias Erb & Christelle A. M. Robert

  8. Plant Genetics Research Unit, United States Department of Agriculture (USDA)-Agricultural Research Service, Columbia, MO, USA

    Bruce Hibbard

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  1. Ricardo A. R. Machado
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Contributions

Conceptualization: Ricardo A. R. Machado. Formal analysis: Ricardo A. R. Machado, Joaquín Abolafia, Ebrahim Shokoohi, Aashaq Hussain Bhat. Funding acquisition: Ricardo A. R. Machado, Joaquín Abolafia, Ebrahim Shokoohi. Investigation: Ricardo A. R. Machado, Joaquín Abolafia, María-Cristina Robles, Alba N. Ruiz-Cuenca, Ebrahim Shokoohi, Aashaq Hussain Bhat, Xi Zhang, Vladimír Půža. Methodology: Ricardo A. R. Machado, Joaquín Abolafia, Ebrahim Shokoohi. Aashaq Hussain Bhat Project administration: Ricardo A. R. Machado. Resources: Ricardo A. R. Machado, Matthias Erb, Christelle A. M. Robert, Bruce Hibbard. Supervision: Ricardo A. R. Machado, Joaquín Abolafia. Visualization: Ricardo A. R. Machado, Joaquín Abolafia, María-Cristina Robles, Alba N. Ruiz-Cuenca, Ebrahim Shokoohi. Writing—original draft: Ricardo A. R. Machado, Joaquín Abolafia, Aashaq Hussain Bhat. Writing—review & editing: Ricardo A. R. Machado, Joaquín Abolafia, Ebrahim Shokoohi, Aashaq Hussain Bhat, Vladimír Půža, Christelle A. M. Robert, Bruce Hibbard. All authors approved the final version of the manuscript.

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Machado, R.A.R., Abolafia, J., Robles, MC. et al. Description of Heterorhabditis americana n. sp. (Rhabditida, Heterorhabditidae), a new entomopathogenic nematode species isolated in North America. Parasites Vectors 18, 101 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13071-025-06702-5

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Parasites & Vectors

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