Evaluation and entomopathogenicity of gut bacteria associated with dauer juveniles of Oscheius chongmingensis (Nematoda: Rhabditidae)

Abstract The nematodes of genus Oscheius are insect parasites with a potential role as biological control agents. The composition of gut microbiota and its potential assistant role in the complex pathogenic mechanism of nematodes have been poorly illustrated. In this study, the intestinal bacteria associated with dauer juveniles of the nematode Oscheius chongmingensis Tumian were classified by 16S rDNA high‐throughput sequencing. The raw reads were assigned to 845 operational taxonomic units (OTUs) after quality filtering. The results showed that the genus Ochrobactrum, with a proportion of 59.82%, was the most abundant genus, followed by 7.13% Bacillus, 4.7% Albidiferax, 4.26% Acinetobacter, and 3.09% Rhodococcus. The two dominant bacteria, Ochrobactrum and Bacillus, were further isolated by culturing on NBTA and LB medium respectively, and then identified as Ochrobactrum tritici and Bacillus cereus by morphological and 16S rDNA sequence analysis. Furthermore, the entomopathogenicity of these two bacterial species was studied. The results showed that O. tritici caused 93.33% mortality within 144 hr in the 4th‐instar larvae of Galleria mellonella treated with 2 × 109 CFU/ml, whereas B. cereus showed 100% mortality at a concentration of 3.3 × 107 CFU/ml within 48 hr. These findings, especially the presence of O. tritici, which had not been found in other nematode species in the genus Oscheius, indicate that the associated nematode O. chongmingensis may have particular utility as a biocontrol agent.

The dauer juvenile of entomopathogenic nematodes is the only infective juvenile stage (IJs) that invades insect hosts, and releases bacteria to produce toxins (Dillman et al., 2012). Furthermore, entomopathogenic nematodes are highly virulent due to the symbiotic bacteria found in IJs. In addition, entomopathogenic bacteria are promising sources of antimicrobial, insecticidal and nematicidal compounds, which might become potential biopesticides. Some nematodes of Oscheius sp. have been found to associate with insect pathogenic bacteria. However, the diversity of bacteria associated with nematodes of Oschieus sp. differs from the specific bacterial symbionts of the genera Heterorhabditis and Steinernema.
Notably, S. nematodiphila was associated with the nematode species O. chongmingensis, which was collected from the soil of Chongming Island in the southeastern area of Shanghai, China . In the present work, the same nematode species, the Oscheius chongmingensis Tumian strain, isolated from an alfalfa field in the city of Hailar, Inner Mongolia, China (Liu, Mráček, Zhang, & Půža, 2012), the presence of the same or different associated bacterial genera in its intestine was assessed. In previous studies, dauer juveniles of O. chongmingensis Tumian showed pathogenicity to Galleria mellonella and Tenebrio molitor (Cao, Liu, Xie, Cao, & Li, 2007), and this nematode strain also led to 90% mortality of the longhorn beetle, Batocera lineolata (Coleoptera: Cerambycidae), which attacks walnut trees in walnut fields (Liu & Wei, 2015). Hence, O. chongmingensis Tumian has been identified as a potential biocontrol agent.
Therefore, in the present study, the intestinal bacteria associated with the dauer juveniles of O. chongmingensis Tumian were investigated and the effects of two associated bacteria on pathogenicity were analyzed. The results will benefit further study of the infection mechanism of this nematode against insect pests.

| Nematode culture and DNA extraction
Galleria mellonella larvae were reared in the Entomology and Nematology Laboratory, Department of Entomology, China Agricultural University (CAU), using artificial diets (Pu & Liu, 2009 All operations were carried out under a sterile laminar flow hood.
Purified DNA extracts were stored at −20°C until PCR amplification.

| PCR amplification and highthroughput sequencing
DNA fragments of the bacterial 16S rDNA gene, targeting the hypervariable region V3-V4, were amplified using the primer pairs 336F (5′-GTACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GTGGACTACHVGGGTWTCTAAT-3′). PCR amplification was carried out in triplicate 50 μl reactions containing 30 ng of genomic DNA, 0.3 μl of Pyrobest DNA Polymerase (2.5 U/μl, TaKaRa), 2 μl of each barcoded fusion primer (10 μM), and 4 μl of dNTPs (2.5 mM), in the appropriate 10 × Pyrobest Buffer and ddH 2 O. Negative control samples were treated similarly with the exclusion of template DNA. The following thermal program was used for amplification: an initial denaturation at 95°C for 5 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 40 s, with a final extension step at 72°C for 10 min. The triplicate PCR products were pooled and purified from 2.0% agarose gels. The amplicon products were quantified by the Qubit fluorescence quantitative system and then combined in an equimolar ratio in a single tube. Paired-end sequencing was performed on the Illumina platform (MiSeq) (Allwegene Technology Co., Ltd., Beijing, China).

| Analysis of data processing and bacterial diversity
The data were processed by removing low-quality reads using Trimmomatic software and Readfq (version 6.0) and then splicing the paired reads into a sequence based on the PE data by FLASH (version 1.2.10) and Pear software. Sequences with lengths shorter than 200 bp or with maxhomop >10 were removed using the Mothur pipeline (Schloss, Westcott, Ryabin, & Hall, 2009). Sequences with ambiguous bases, primer mismatches, errors in barcodes, and chimerism were filtered using Usearch (version 8.0.1623) (Edgar, Haas, Clemente, & Quince, 2011). The remaining high-quality sequences were aligned to a reference alignment derived from the 16S rDNA gene database (DeSantis, Hugenholtz, Larsen, & Rojas, 2006).
To improve the accuracy of the annotation, sequences were also searched against the NCBI nt database using BLASTn (Altschul, Gish, Miller, & Myers, 1990). Taxonomic information was assigned by the NCBI database according to the highest scoring sequence. The diversity index and species richness estimate were calculated using the Mothur pipeline. The α-diversity was represented by rarefaction curves plotting the cumulative number of OTUs at a 3% distance level. Diversity was measured by counting the number of observed OTUs using the Shannon index, Chao1 index, phylogenetic diversity and observed number of species as described previously (Magurran, 2004;Chao, 1984). The estimators were calculated by subsampling the smallest number of sequences from each sample. Statistical analysis was performed in R (version 3.11) (R Development Core Team, 2014). The dominant taxonomic unit, species richness and relative abundances in each sample were determined from rank-abundance curves. Beta diversity and species composition analysis were constructed by using the QIIME pipeline and R software.

| Isolation of dominant bacteria from nematodes
Associated bacteria were obtained from the infective stages of O. chongmingensis Tumian by two methods. Dauer juveniles of nematodes were surface-sterilized by immersion in 1% (w/v) NaClO (sodium hypochlorite solution) for 30 min and then in 75% alcohol for 1 min; after washing three times in sterile water, they were streaked onto nutrient agar NBTA (peptone, 10 g; beef extract, 5 g; NaCl, 5 g; agar, 18 g; water, 1 L, 0.0025% bromothymol blue, 0.004% triphenyltetrazolium chloride; pH 7.0-7.2) and LB (tryptone, 10 g; NaCl, 10 g; yeast extract, 5 g; water, 1 L; pH 7.0-7.2). In addition, associated bacteria were isolated from the hemolymph of dead G. mellonella larvae infected with the nematode species. The dead larvae were surface-sterilized by dipping into 75% ethanol for 1 min and placed in a sterile petri dish to dry. Sterile scissors were used to dissect the 3 rd segment from the head of each larva. A sterile loop was used to touch the hemolymph and streak it on the nutrient agar media. The plates were sealed and incubated in the dark at 28°C for 48 hr. Developing bacterial colonies that differed in morphology or in color were transferred to NBTA or LB plates (Akhurst, 1980). Before and after each test, cultures were streaked onto NBTA and LB to confirm that there had been no changes from one form to the other.
These colonies were added to LB nutrient broth and shaken for 48 hr (150 rpm) at 28°C in the dark. Subsequently, the bacterial suspension was centrifuged at 4,000 g for 8 min and the supernatant decanted. Sterile water was added to the pellets and mixed thoroughly to obtain a concentrated suspension of the bacterial symbionts. The total number of bacteria in the suspension was measured with a spectrophotometer with a wavelength at 600 nm. The cell number in the suspension was estimated by counting colonies on culture plates of different concentration gradients and measuring the OD 600 value. Some of the colonies were sampled for molecular analysis.

| Morphology and identification of bacterial isolates
Cultural properties such as colony size, shape, and color were observed after 48 hr incubation at 28°C on nutrient agars (NBTA and LB), and then, the bacteria were gram stained.
Pure isolates of two strains, NMA-1 and NMA-2, were used for identification following 16S rDNA gene sequencing. For DNA extraction, bacterial isolates were grown individually in 5 ml of LB broth at 28°C for 24 hr. DNA was extracted following the procedure described previously (Krsek & Wellington, 1999) with some modifications. The sample was suspended in 100 μl 1 × TE (pH 8.0) and 100 μl of lysozyme solution (50 mg·mL −1 ) was added. The tube was placed in a 37 o C water bath for 20 min. Subsequently, 100 μl of 10% SDS solution (10% w/v SDS) and 5 μl of proteinase K (20 mg·mL −1 ) were added, and the contents of the tube were gently mixed and placed in a 37 o C water bath for 30 min. Then, 100 μl of NaClO solution (5 M) was added, and the mixture was gently whirled for 1 min; the supernatant was treated with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1) successively to remove proteins and other impurities.
Thereafter, the DNA was precipitated with 90 μl of isopropanol for 10 min at room temperature and then pelleted by centrifugation at 12,000 g and 4°C for 10 min. The precipitate was washed with 75% ethanol and dried in a laminar flow cabinet for 1 hr prior to resuspension with 50 μl of sterile water. DNA extracts were stored at −20°C until further analysis.
The gene sequences were amplified using three different primer sets: two targeting the bacterial 16S rDNA gene and a second pair targeting a different gene, recA (Table 1). The primer sets for bacteria were 27f/1492r and S1/A1 (Yang, 2008) (Table 1), while the primer set for the Brucella spp. recA gene was recA-f/recA-r (Scholz, Pfeffer, Witte, & Neubauer, 2008). PCR amplification was performed in a thermal cycler (Bio-Gener) using approximately 1.0 μl of bacterial DNA, 2.5 μl of 10 × PCR buffer, 2.0 μl of 2.5 mM dNTPs, 1.0 μl of each primer (10 μM), 0.3 μl of Taq DNA polymerase, and 17.2 μl of ddH 2 O. Thermal cycling conditions were as follows: 3 min at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 55°C for 27f/1492r and recA-f/recA-r or 1 min at 63°C for S1/A1, 1.5 min at 72°C, and a final step at 72°C for 10 min. The PCR products were sequenced by BGI (Beijing, China).

| Sequence and phylogenetic relationship analysis of bacteria
For phylogenetic analysis, all nucleotide sequences obtained were compared with NCBI database sequences using BLASTn. The selected sequences were aligned using ClustalW, and neighbor-joining trees of the homologous sequences were constructed using the maximum likelihood method with 1,000 bootstrap replications in the MEGA 7.0 program (Kumar, Steche, & Tamura, 2016).

| Physiological and biochemical characteristics of bacterial isolates
Physiological and biochemical tests were performed at 28°C according to Bergey's Manual of Determinative Bacteriology (Buchanan & Gibbons, 1986) and Common Bacterial System Identification Manual (Dong, Cai, Lu, Xie, & Liu, 2001).
In addition, 100 μl of serial dilutions was streaked onto LB agar plates to count the number of colonies, and the OD 600 values of gradient dilutions were measured by an ultraviolet-visible light detector (LabTech) to make a standard curve. G. mellonella larvae were used as the test insect for assessing the pathogenicity of the two dominant bacteria associated with O. chongmingensis Tumian. A cell suspension (50 μl) was injected into 4 th -instar G. mellonella larvae using a 50 μl Anting microsyringe (Shanghai, China). Sterilized water was used as a control. Thirty larvae were used per concentration, and three replications were performed. Insect mortality was observed every 24 hr after injection.

| Basic data processing and statistics
16S rRNA gene sequencing of the dauer juvenile nematode samples was performed by paired-end sequencing. After the quality check, unqualified sequences shorter than 200 bp were removed.
According to the quality criteria, approximately 97.47% of the raw sequences were used for subsequent analysis (Table 2). Next, filtering processes were performed to remove edundant, chimeras and undesirable sequences, generating 51,651 clean reads (91.69%) for dauer juveniles. After rarefying the reads to the smallest number, 51,651 bacterial sequences from nematodes were retained.

| Diversity and richness of bacterial species
The Chao1 index was used to analyze OTU richness and species richness at the 0.03 dissimilarity level. The shannon index, phylogenetic diversity (PD, whole tree) and observed number of species were used to assess the diversity of the bacterial communities from nematodes.
Similar results were obtained (Table 3). The Shannon index of the nematode sample was 3.60, and the value of Good's coverage was 0.99. In addition, the value of the PD whole tree was 65.2 (Table 3).

These results suggested that dauer juveniles of O. chongmingensis
Tumian had high bacterial diversity.

| Analysis of bacterial community structure and predominant species
The 845 OTUs from the nematode sample were assigned to cor-

| Morphological characteristics of bacteria
Two bacterial species were isolated from surface-sterilized nematodes and from the hemolymph of G. mellonella larvae infected by the nematodes. After growth on NBTA plates for 48 hr, the NMA-1 strain was obtained and distinguished as gram-negative (Figure 3a).
During incubation on NBTA, the pink colonies of NMA-1 were opaque, mucoid, smooth, protuberant and rapidly confluent. The gram-positive NMA-2 strain was obtained on LB nutrient agar after 24 hr (Figure 3b). The colonies of the NMA-2 strain were large (approximately 15 mm in diameter), and white waxy with a rough surface and a flat, irregular shape.

| 16S rDNA sequence and phylogeny of two dominant bacteria
The associated bacteria were confirmed by molecular analysis based

F I G U R E 1 The composition and relative abundances of bacterial phyla associated with the dauer juveniles of Oscheius chongmingensis
Tumian. Only OTUs with at least 20 sequences are represented a confidence level of 88%). Alternatively, NJ trees ( Figure 5) based on the S1/A1 sequence showed that all 17 B. cereus isolates belonged to a single cluster, including strain NMA-2 (with 86% bootstrap support).

| Physiological characteristics of two dominant bacteria
Phenotypic characteristics are shown in Table 4. Both strains assimilated D-glucose, sucrose, D-fructose, and D-trehalose but not mannitol or lactose, and they produced urease, lecithinase and amylase. Furthermore, both strains hydrolyzed starch. Strain NMA-1 produced indole, but did not hydrolyze gelatin, while strain NMA-2 did both.

| Pathogenicity of associated bacteria to G. mellonella larvae
To assess the ability of the nematode symbiotic bacteria to grow and survive within the host larval hemocoel and to test their pathogenicity, 50 μl of different bacterial suspensions was injected into  Figure 7; a significant difference in insect mortality (p < 0.05) was observed between bacterial species. After 24 hr treatment, low insect mortalities of 0%-6.67% were observed for O. tritici, while 90% mortality caused by B. cereus was detected. Up to 120 hr after injection, larvae injected with the highest concentration (2.0 × 10 10 CFU/ml) showed 90% mortality (Figure 7a). However, the highest mortality of larvae was 100% at 48 hr after injection of B. cereus NMA-2 (3.3 × 10 7 CFU/ml) (Figure 7b). Since B. cereus NMA-2 was more pathogenic than O. tritici NMA-1 after culture for 48 hr, it was possible that these bacteria had different characteristics and different effects on nematode reproduction and pathogenicity.

| D ISCUSS I ON
In the present work, we identified and analyzed the gut bacterial community of dauer juveniles of O. chongmingensis Tumian. We found gut microbiota with high diversity and two dominant bacterial OTUs. Comparison of the microbial composition revealed that F I G U R E 4 Phylogenetic relationships of the strain NMA-1 and other closely related Ochrobactrum species in a neighbor-joining tree based on analysis of the recA sequence. Bootstrap values are 1,000 replications and above 50% are shown at the branch points by MEGA 7.0 F I G U R E 5 Phylogenetic tree of strain NMA-2 and other related Bacillus species in a neighbor-joining tree based on analysis of the S1/A1 sequence data. Bootstrap values are 1,000 replications and above 50% are shown at the branch points by MEGA 7.0  (Jelveh & Cunha, 1999). Some species of Ochrobactrum can biodegrade polycyclic aromatic hydrocarbons (Arulazhagan & Vasudevan, 2011). Some endophytic bacteria belonging to Ochrobactrum sp.
have been isolated from plants, and these strains exhibit maximum antagonistic activity and possess higher chitinase and glucanase activity (Zhao, Xu, Chang, & Li, 2016;Zang, Zhao, Liu, & Liang, 2014;Sowndhararajan, Marimuthu, & Manian, 2013). Metabolites produced by bacterial species of Ochrobactrum inhibit the growth of a wide range of bacteria and fungi (Han, Peng, & Yi, 2011;Lebuhn, Achouak, Schloter, & Berge, 2000). Only one report on one species in the genus, O. anthropi, has previously associated this genus with a nematode, Steinernema scapterisci (Aguillera, Hodge, Stall, & Smart, 1993 The other most abundant genus detected in this study was Acinetobacter. One species of Acinetobacter, A. baumannii, is an opportunistic pathogen of great concern in nosocomial pneumonias and especially as an invader of burn wounds (Livermore & Woodford, 2006 also has more than one species of associated bacteria. The bacterium Providencia vermicola promotes was one of the bacteria that contributed to promoting nematode culture, in addition to Flavobacterium sp. Another species, Alcaligenes faecalis, however, is unfavorable for nematode reproduction and pathogenicity (Park et al., 2011). In addition, a slug-parasitic nematode, Phasmarhabditis hermaphrodita, has been F I G U R E 6 Death symptoms of Galleria mellonella larvae caused by injection of cell suspensions of Ochrobactrum tritici NMA-1 and Bacillus cereus NMA-2, sterile water as the control associated with multiple bacteria as well. Finally, the core bacterium Moraxella osloensis is the optimum species that can improve nematode production and pathogenicity (Wilson, Glen, George, & Pearce, 1995;Wilson, Glen, Pearce, & Rodgers, 1995).
Further studies are needed to analyze the function of O. tritici, B. cereus or other intestinal bacteria associated with O. chongmingensis Tumian to explore the mechanism of nematode infection pathogenicity to invertebrate pests.

ACK N OWLED G EM ENTS
This research was supported by the China National Key Technology Program (No. 2014BAD23B01 andNo. 2014BAD16B07).

CO N FLI C T O F I NTE R E S T S
The authors declare no conflicts of interest.

AUTH O R S CO NTR I B UTI O N
Qi-zhi Liu conceived and designed the experiments, confirmed the analyzed data and revised manuscript; Jun-rui Fu performed the experiments, analyzed the data, drafted the manuscript and amended the revised manuscript according to the language company's comments.

E TH I C S S TATEM ENT
None required.

DATA ACCE SS I B I LIT Y
All data are provided in full in the results section of this paper apart from the DNA sequences of Ochrobactrum tritici NMA-1 and Bacillus cereus NMA-2 genes which are available at https://submit.ncbi.nlm.