Entomopathogenic nematodes (EPNs), assisted by their mutualistic bacterial symbionts, are obligate insect pathogens in nature, representing a highly-specified tripartite system for studying bacterium–nematode–insect interactions (Dillman et al., 2012, Dillman and Sternberg, 2012). Additionally, Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus sp. exhibit high genetic diversity (Askary, 2010). However, no genome of H. bacteriophora or its symbiont has been reported in China to date, hindering evolutionary studies of nematode–bacterial interactions as well as the development of effective control strategies against insect pests.
A new EPN strain was found from a bayberry orchard soil in Qujing, Yunnan, in 2025. Nematodes were isolated via insect baiting method (Bedding and Akhurst, 1975) and extracted using White trap (White, 1927). Through 18S, ITS, 28S and COI sequences and morphological identification, it was determined to be Heterorhabditis bacteriophora, named as strain QJ1. Genomic DNA (gDNA) was extracted from about 100,000 surface-sterilized infective juveniles (IJs) using a Takara genomic DNA extracting kit for whole-genome sequencing. Symbiotic bacteria were isolated from IJs, identified as Photorhabdus laumondii by 16S rRNA sequencing, and gDNA was extracted from bacteria grown on Luria-Bertani (LB) medium.
DNA libraries (350 bp) were constructed and sequenced on an Illumina NextSeq 6000 platform (Novogene, China). The genomes were assembled using SPAdes v3.15.5 (Labroo et al., 2021), and contigs with coverage ≤ 7 and length ≤ 1,000 bp were subsequently removed. The nematode genome was annotated using BRAKER v3.0.8 (Hoff et al., 2019) with RNAseq data (NCBI accession SRR6294671) as extrinsic evidence, while the bacterial genome was annotated using Prokka v1.12 (Seemann, 2014). The completeness of the nematode genome was evaluated with BUSCO v5.5.0 using the nematoda_odb10 database, and that of the bacterial genome using the enterobacterales_odb10 database (Simão et al., 2015). OrthoFinder v1.1.4 (Emms and Kelly, 2019) with default settings was used to identify orthologous groups by clustering the proteome of publicly available H. bacteriophora and its symbiont strain TT01 (Duchaud et al., 2003, Bai et al., 2013). Putative natural-product biosynthetic gene clusters (BGCs) of the bacterium were predicted by antiSMASH 8.0 (Blin et al., 2025) with default parameters. The assembled genome of H. bacteriophora QJ1 consists of 13,128 contigs totaling 65.7 Mb and contains 14,574 protein-coding genes. The assembled genome has a 75.6% completeness analyzed using BUSCO (S:74.7%, D:0.9%, F:6.7%; Table 1). Although the BUSCO completeness score of QJ1 is lower than that of the type strain TT01 (C:89.3% [S:49.2%, D:40.1%]), the proportion of duplicated BUSCOs in QJ1 is substantially lower, suggesting reduced assembly redundancy and a more collapsed haploid representation. In addition, compared with TT01, QJ1 contains 2,663 (20.3%) strain-specific genes.
Genome assembly statistics of Heterorhabditis bacteriophora strain QJ1 and its symbiosis bacterium Photorhabdus laumondii
| Assembly | H. bacteriophora | P. laumondii |
|---|---|---|
| Number of contig | 13,128 | 128 |
| BUSCO (%) | 75.6 | 99.5 |
| Length of largest contig (kb) | 75 | 253 |
| Total length (Mb) | 65.7 | 5.1 |
| GC content (%) | 33.04 | 42.51 |
| N50 (kb) | 7.6 | 69.7 |
| Number of protein coding gene | 14,574 | 4,387 |
The genome size of P. laumondii from QJ1 is 5.1 Mb (GC content = 42.5%) with a BUSCO completeness of 99.5% (S:99.3%, D:0.2%, F:0.2%;), and it encodes 4,387 protein-coding genes (Table 1). Among these, 10.7% of the genes are non-orthologous to P. laumondii TT01. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that both P. laumondii QJ1 and TT01 have 36 genes involved in bacterial secretion systems (Type I, II, III and VI), of which two are QJ1-specific. In addition, we found that QJ1 contains 7 strain-specific genes that have domains potentially involved in Type VI secretion systems (T6SS).
A comparative analysis of BGCs was performed between P. laumondii QJ1 and TT01. A total of 28 BGCs were identified in QJ1, while 26 BGCs were detected in TT01. The two strains shared 21 core BGC types. Notably, QJ1 possessed three BGC types (azole-containing-RiPP, amglyccycl, and arylpolyene) that are absent in TT01.
The genomic sequencing data, genome assemblies, and annotations of H. bacteriophora and P. laumondii have been deposited in the NCBI database under BioProject accession number PRJNA1417294.
This research was supported by the National Natural Science Foundation of China 32200495; Frontier Cross-Disciplinary Innovation Team Construction Project of the Institute of Future Agriculture, Northwest A&F University.
Y.M. and Z.W. designed the experiments. All authors performed the experiments. Y.M. analysed the data. Y.M., Z.W., and Z.H. wrote and revised the manuscript.
The authors declare no competing interests.