Variation in pathogenicity of different strains of Xenorhabdus nematophila; Differential immunosuppressive activities and secondary metabolite production

Journal of Invertebrate Pathology 166 (2019) 107221

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Variation in pathogenicity of different strains of Xenorhabdus nematophila; Differential immunosuppressive activities and secondary metabolite production

T

Md. Ariful Hasana, Shabbir Ahmeda, Md. Mahi Imam Mollaha, Dongwoon Leeb, Yonggyun Kima,

⁎

a b

Department of Plant Medicals, Andong National University, Andong 36729, Republic of Korea School of Environmental Ecology and Tourism, Kyungpook National University, Sangju 37224, Republic of Korea

ARTICLE INFO

ABSTRACT

Keywords: Steinernema carpocapsae Xenorhabdus nematophila Eicosanoid Pathogenicity Identification Secondary metabolite Immunity

Xenorhabdus nematophila, an entomopathogenic bacterium, is mutualistic with the nematode Steinernema carpocapsae. The bacterium produces secondary metabolites to inhibit target insect phospholipase A2 (PLA2) and induce immunosuppression, which is required for the pathogenicity of this bacterium-nematode complex. However, it was unclear if immunosuppressive intensity of the bacteria was correlated with their insecticidal potency. We compared six different X. nematophila strains inhibiting the immune responses of the beet armyworm (Spodoptera exigua) to explain their virulence variations. In addition to four known strains obtained from the Korean Agricultural Culture Collection, we identified two new strains (SK1 and SK2) of X. nematophila from two different isolates of S. carpocapsae. Although all six strains were virulent, they showed significant variation in median lethal bacterial dosage (LD50). The LD50 of most strains was 15–30 CFU/larva, however, the LD50 of the SK1 strain was more than two-fold higher against S. exigua larvae. Immunosuppressive activities of the six strains were measured by comparing hemocyte-spreading behavior and nodule formation; the SK1 strain was significantly less potent than other bacterial strains. These suppressed hemocyte behaviors were recovered by adding arachidonic acid (a catalytic product of PLA2) into all six strains. Bacterial culture broth was fractionated with different organic solvents and the ability to inhibit immune response and PLA2 activity were assessed. All organic extracts had immunosuppressive activities and PLA2-inhibitory activities. GC-MS analysis showed that these organic extracts possessed a total of 87 different compounds. There were variations in chemical components among the six bacterial strains. Organic extracts of SK1 strain, which exhibited the lowest virulence, contained the least number of secondary metabolites.

1. Introduction Two genera of entomopathogenic nematodes (EPN), Steinernema and Heterorhabditis, infect and kill a wide range of insects mostly belonging to Lepidoptera, Coleoptera, Diptera, and Hemiptera (Peters, 2013). They exhibit a unique mutualistic relationship with entomopathogenic bacteria in genera Xenorhabdus and Photorhabdus, respectively (Boemare, 2002). Typically, one nematode species hosts one symbiotic bacterial species while one bacterial species can be hosted by more than two symbiotic nematode species (Goodrich-Blair and Clarke, 2007). Thus, species diversity of EPN is greater than their symbiotic bacteria. For example, 61 EPN species belonging to genus Steinernema are known to exhibit symbiotic relationship with 26 species of Xenorhabdus (Sajnaga et al., 2018). EPNs spend most of their developmental stages in target insect hosts ⁎

except infective juveniles (IJs, 3rd instar larvae in dauer stage), which are free-living in soil and seek new hosts (Lacey et al., 2015). After IJs enter insect hemocoel through mouth, anus, and/or spiracles, they release their symbiotic bacteria (Dowds and Peters, 2002). In the hemocoel, the bacteria induce immunosuppression and toxemia using an array of virulence factors including secondary metabolites and toxin proteins to kill target insects (Waterfield et al., 2009; Tobias et al., 2018). In the insect cadaver, bacteria produce exoenzymes, such as proteases and lipases, to digest insect tissues to provide nutrients for host nematodes to develop and reproduce (Richards and GoodrichBlair, 2010). To maintain monoxenic conditions, these bacteria also produce antimicrobials to protect the specific nematode-bacterial complex from other saprotrophic microorganisms, bacteriovorous nematodes, and scavenging insects (Eleftherianos, 2009). Conversely, these nematodes provide benefits to the bacteria by vectoring

Corresponding author. E-mail address: [email protected] (Y. Kim).

https://doi.org/10.1016/j.jip.2019.107221 Received 30 May 2019; Received in revised form 24 July 2019; Accepted 25 July 2019 Available online 26 July 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.

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transmission and providing physical shelter that allow the bacteria to survive outside the insect host (Cao and Goodrich-Blair, 2017). Thus, EPNs devoid of symbiotic bacteria are not pathogenic to insects as they are unable to develop inside target insects (Poinar and Thomas, 1966; Poinar et al., 1977; Han and Ehlers, 2000). Innate insect immunity consists of cellular and humoral responses (Lemaitre and Hoffman, 2007). Upon recognition of pathogens by pattern recognition receptors, signals stimulate immune tissues to release autocrine or paracrine immune mediators including nitric oxide, cytokine, biogenic monoamines, and eicosanoids (Gillespie et al., 1997). There is cross-talk among immune mediators, in which eicosanoid is the most downstream signal to activate cellular and humoral immune responses (Kim et al., 2018a). Eicosanoid signalling begins with activation of phospholipase A2 (PLA2), which catalyzes phospholipids to release precursors for eicosanoid biosynthesis (Dennis, 1994). Subsequent oxygenase reactions can produce various eicosanoids including prostaglandins, leukotrienes, and epoxyeicosatrienoic acids (Stanley, 2000). Kim et al. (2005) have suggested that PLA2 is a common molecular target of Xenorhabdus and Photorhabdus to induce immunosuppression. Inhibition of PLA2 activity occurs at an early stage of infection, within 3 h of inoculation into the insect hemocoel (Kim et al., 2018b). Benzylideneacetone, the first PLA2 inhibitor, was identified from cultured broth of X. nematophila (Ji and Kim, 2004). Subsequently, other PLA2 inhibitors have been identified from different cultured broths of Xenorhabdus and Photorhabdus (Seo et al., 2012; Sadekuzzaman et al., 2017). Furthermore, two bacterial species, different in virulence, exhibited differential intensity of inhibition against PLA2 activity (Ahmed and Kim, 2018). This suggests that bacterial virulence is primarily determined by inhibitory activities against the host insect immune system and immunosuppression is induced by bacterial secondary metabolites. To correlate immunosuppressive activity with bacterial virulence, we compared virulence of six different strains of X. nematophila against two different lepidopteran and coleopteran insect species. To further analyze variation in virulence, we assessed inhibitory activities of these bacteria against the insect immune response and PLA2 activity. Finally, bacterial cultured broths were fractionated by GC-MS to compare composition of secondary compounds produced by these six bacterial species.

2.3. Nematode source and culturing Two unidentified nematode isolates (Hb and GNUS143) were collected in Moonkyung (36°35′40″N/128°11′58″E), Korea. These collected nematodes were multiplied using L5 larvae of S. exigua. Briefly, about 2000 IJs in 500 μL distilled water were topically applied to a Petri dish (9 cm diameter, 3 cm height) containing 5 larvae. Infected larvae were incubated at 25 °C with diet for 72 h. Dead larvae were transferred to White traps (Lee et al., 2000). IJs were harvested from the White traps every day and stored in sterilized distilled water at 10 °C for no more than 21 days before use (Park et al., 1998). 2.4. Molecular diagnosis of nematode isolates Genomic DNA (gDNA) was extracted from 0.5 g of IJs using published methods (Kang et al., 2004). Extracted gDNA was used as a template to amplify the internal transcribed spacer (ITS) region using PCR primers (5′-TTG ATT ACG TCC CTG CCC TTT-3′ and 5′-TTT CAC TCG CCG TTA CTA AGG-3′) reported by Vrain et al. (1992). PCR conditions were: 35 cycles of denaturation at 94 °C for 1 min, annealing at 46 °C for 1 min, and extension at 72 °C for 1 min. A second PCR was performed using the resulting PCR product with M13 universal primer sequence-linked ITS primers at 5′ end and subjected to DNA sequencing with M13 forward and reverse primers by Macrogen (Seoul, Korea). Nucleotide sequence was analyzed using BlastN program of the National Center for Biotechnology Information (NCBI) and aligned with BioEdit 7.2.5. An evolutionary relationship was inferred using the Neighbor-Joining method with MEGA6. Bootstrapping values on branches were obtained with 1000 repetitions. 2.5. Bioassay of nematode isolates Insecticidal activity of nematode isolates was determined by treating filter paper in a Petri dish (9 cm diameter, 3 cm height) containing five larvae of S. exigua or T. molitor with different concentrations of IJs. An experimental unit consisted of two dishes. It was replicated 3 times per nematode concentration. Mortality was assessed every 8 h after nematode inoculation for 3 days at 25 °C. Median lethal concentration (LC50) was estimated based on mortality data at 3 days after treatment. Median lethal time (LT50) was estimated for each IJ concentration treatment. Both LC50 and LT50 values were estimated using EPA Probit program, version 1.5 (U.S. Environmental Protection Agency, Cincinnati, OH, USA).

2. Materials and methods 2.1. Preparation of test insects

2.6. Isolation and identification of symbiotic bacteria

Spodoptera exigua larvae were collected from Welsh onion (Allium fistulsum L.) fields and reared on artificial diet (Goh et al., 1990) under laboratory conditions with constant temperature of 25 ± 2 °C, photoperiod of 16:8h (L:D), and relative humidity of 60 ± 5%. Under these conditions, larvae developed through five instars (L1–L5) before pupation. Test larvae used were at the early L5 stage. Adults were fed with 10% sucrose solution. Tenebrio molitor larvae were provided by Bio Utility, Inc. (Andong, Korea) and larvae with body length of approximately 2 cm were used for pathogenicity test.

Approximately 200 IJ nematodes were topically applied onto L5 S. exigua larvae followed by incubation at 25 °C for 10 h. Hemolymph of S. exigua was collected and streaked onto NBTA medium (nutrient agar supplemented with 25 mg bromothymol blue and 40 mg triphenyltetrazolium chloride per 1 L) to culture bacteria. In addition, approximately 500 IJs were surface-sterilized with 70% ethanol for 3 min and homogenized in sterile distilled water. The homogenate was then streaked onto NBTA medium. Blue colored-colonies on NBTA plates were subsequently single-cultured in tryptic soy broth (TSB) (Difco, Sparks, MD, USA) at 25 °C for 48 h. After washing cultured cells 3 times with sterilized H2O by centrifuging the culture medium at 4000g for 2 min at 4 °C, cells were re-suspended in sterilized 100 mM phosphatebuffered saline (PBS, pH 7.4) for subsequent pathogenicity tests. For biochemical characterization, Gram-staining was performed using the method of Bensen (1990). Catalase, peroxidase, and oxidase activities were examined using the procedure of Schaad (1988). These characteristics were used to determine bacterial genus by comparing them with those of bacteria described in Bergey's Manual (Krieg and Hart, 1984). Acid production characteristics of isolates using different carbon sources were assessed with a colorimetric method using GN

2.2. Chemicals Arachidonic acid (AA: 5,8,11,14-eicosatetraenoic acid) was purchased from Sigma-Aldrich Korea (Seoul, Korea) and dissolved in dimethyl sulfoxide (DMSO). A PLA2 surrogate substrate, 1-hexadecanoyl2-(1-pyrenedecanoyl)-sn-glycerol-3-phosphatidylcholine, was purchased from Molecular Probes (Eugene, OR, USA) and dissolved in high grade ethanol (Sigma-Aldrich Korea). Anticoagulant buffer (ACB, pH 4.5) was prepared with 186 mM NaCl, 17 mM Na2EDTA, and 41 mM citric acid. 2

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microplate (Biolog, Hayward, CA, USA) and compared with characters of different Xenorhabdus spp. Bacterial genomic DNA was extracted using a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, USA). The 16S rRNA region was amplified by PCR using PCR primers (5′-AGA GTT TGA TCC TGG CTC AG-3′ and 5′-GGC TAC CTT GTT ACG ACT T-3′) reported by Eden et al. (1991). PCR was performed with 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 30 s, and extension at 72 °C for 1 min. The resulting PCR product was bidirectionally sequenced. The nucleotide sequence obtained was assessed with the same method used for nematode identification.

(sPLA2 Assay Kit, Cayman, Ann Arbor, MI, USA) was used with diheptanoyl thiophosphatidylcholine as enzyme substrate and Ellman’s reagent [5,5-di-thio-bis-(2-nitrobenzoic acid), DTNB] to create 5-thio-2nitrobenzoic acid, a colored product. DTNB was prepared in 10 mM in 0.4 M Tris buffer (pH 8.0). Enzyme substrate was prepared in 1.66 mM. The assay buffer used 25 mM Tris (pH 7.5) containing 10 mM CaCl2, 100 mM KCl, and 0.3 mM Triton X-100. A reaction mixture (200 μL) contained 10 μL of sPLA2 enzyme (Vatanparast et al., 2018), 10 μL of DTNB, 5 μL of test inhibitor, and 175 μL of substrate. For the control, the same volume of reaction mixture consisted of 10 μL of sPLA2 enzyme, 10 μL of DTNB, 5 μL of DMSO, and 175 μL of substrate. The reaction mixture for the negative control consisted of 10 μL of assay buffer, 10 μL of DTNB, 5 μL of DMSO, and 175 μL of substrate. Cellular PLA2 (cPLA2) activity was measured using a commercial assay kit (cPLA2 assay kit, Cayman) with arachidonyl thiophosphatidylcholine as substrate. The cPLA2 assay used 160 mM Hepes buffer (300 mM NaCl, 20 mM CaCl2, 8 mM Triton X-100, 60% glycerol, and 2 mg/ml BSA, pH 7.4). A reaction mixture (200 μL) consisted of 10 μL of cPLA2 enzyme (Park, 2015; Vatanparast et al., 2018), 10 μL of DTNB, 5 μL of test inhibitor, and 175 μL of substrate. For the control, the same volume of reaction mixture consisted of 10 μL of cPLA2 enzyme, 10 μL of DTNB, 5 μL of DMSO, and 175 μL of substrate. For the negative control, the test inhibitor in the same reaction mixture was replaced with 5 μL of DMSO. Absorbance change of the reaction product at wavelength of 405 nm was measured using a microplate reader (Victor, PerkinElmer, Waltham, MA, USA). Each treatment was replicated with three biologically independent enzyme preparations using different larval samples.

2.7. Bioassay of symbiotic bacteria To determine insecticidal activity of symbiotic bacterium, bacterial suspension was prepared with PBS. For time course of bacterial pathogenicity, 105 colony-forming unit (CFU) was injected into each larva using a 5-μL Hamilton microsyringe (Hamilton, Reno, NV, USA) after surface-sterilizing test insect with 70% ethanol. Mortality was monitored for 72 h after bacterial injection. For dose-mortality assay, different bacterial concentrations (0, 102, 103, 104, 105, 106, and 107 CFU/ larva) were used for treatment as described above. Mortality was measured at 24 h after bacterial injection. Each treatment consisted of three replicates and each replicate used 10 larvae per bacterial concentration. Median lethal dosage (LD50) and LT50 values were estimated for each bacterial isolate as described above. 2.8. Influence of bacteria-cultured broth on hemocyte-spreading behavior

2.11. Fractionation of bacterial culture broth using organic solvents

Bacteria were cultured in TSB for 24 h at 28 °C. After removing bacterial cells by centrifugation at 800g for 20 min, the supernatant was filtered (0.22 μm pore size) to remove remaining bacterial cells. The filtrate was used for the hemocyte-spreading behavior assay. About 250 μL of hemolymph was collected from L5 larvae by cutting a proleg and was mixed with 750 μL of ACB. After centrifuging the hemocyte suspension at 800g for 5 min, 800 μL of the supernatant was removed and 250 μL of TC100 insect culture medium (Hyclone, Daegu, Korea) was added to the pellet to make hemocyte suspension. For hemocytespreading behavior assay, a reaction mixture was prepared by mixing 9 μL of cell suspension with 1 μL of bacterial filtrate. A rescue experiment was performed using AA (10 μg/μL in DMSO), 8 μL cell suspension was mixed with 1 μL of bacterial filtrate and 1 μL of AA. After incubating the mixture at room temperature for 40 min under darkness, hemocytes were observed under a phase contrast microscope (Olympus S730, Tokyo, Japan) at 800× magnification. Spreading hemocytes were recognized by cytoplasmic extension. Each treatment consisted of three replications with independently prepared hemocyte suspension. In each replication, 100 hemocytes were randomly chosen and assessed for hemocyte-spreading behavior.

Each bacterial strain was cultured in 1 L of TSB at 28 °C for 48 h to maximize the production of secondary metabolites (Seo et al., 2010). The cultured broth was centrifuged at 10,000g for 20 min at 4 °C to obtain supernatant for subsequent fractionation. First, the same volume (1 L) of hexane was mixed with the supernatant and separated into organic and aqueous fractions. The aqueous phase was combined with the same volume of ethyl acetate. These processes were sequentially used for chloroform and butanol organic solvents. Resulting organic extracts [hexane extract (HEX), ethyl acetate extract (EAX), chloroform extract (CX), and butanol extract (BX)] were dried with a rotary evaporator (Eyela N-1110, Rikakikai, Tokyo, Japan) at 20–40 °C depending on the organic solvent. Dried materials were weighed and resuspended in 5 mL of methanol. 2.12. Thin layer chromatography (TLC) of organic extracts TLC was performed to analyze organic extracts of bacterial culture broth. Each organic extract was spotted at the bottom of a silica gel plate (20 × 20 cm; Merck, Darmstadt, Germany) and then placed in a shallow pool of a mixture of chloroform, methanol, and acetic acid (7:2.5:0.5, v/v) as an eluent in a development chamber. It was then allowed to run by capillary action until the solvent reached the top end of the plate. The silica gel plate was removed and dried. Separated components were then stained with a mixture (19:1, g/g) of sea sand (Merck) and iodine (Duksan, Ansan, Korea).

2.9. Nodulation assay Three-day-old L5 S. exigua larvae were used for a hemocyte nodule formation assay in response to injection of 4 × 104 bacterial cells/larva. Bacteria were heat-killed for the treatment at 90 °C for 10 min. After plating heat-treated bacteria onto TSB medium plate followed by culturing at 28 °C for 24 h, there was no bacterial growth after heat treatment. At 8 h after immune challenge, nodules were counted by dissecting larvae under a stereomicroscope (Stemi SV 11, Zeiss, Jena, Germany) at 50× magnification. AA treatment used 10 μg per larva along with bacterial injection. Each treatment consisted of three replicates, five larvae per replicate.

2.13. Gas chromatography-mass spectrometry (GC-MS) of organic extracts GC-MS analysis was performed using a GC (7890B, Agilent Technologies, Santa Clara, CA, USA) coupled with MS (5977A Network, Agilent Technologies). The GC had an HP 5 MS column (non-polar column, Agilent Technologies) with internal diameter of 30 m × 250 μm and film thickness of 0.25 μm. Carrier gas was helium, flowing at a rate of 1 mL/min. Injector temperature was set at 200 °C. Injection mode was split mode with split ratio 10:1. Initial oven

2.10. Measurement of PLA2 activity To measure secretory PLA2 (sPLA2) activity, a commercial assay kit 3

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temperature was set at 100 °C for 3 min. It was then raised to 300 °C at 5 °C/min. This oven temperature was held for 10 min. Total running time was 53 min. Mass spectra were recorded in EI mode at 70 eV with scanning range of 33–550 m/z. Purified samples were respectively identified by COMPARING mass spectra of compounds with those in the database of NIST11 and literature data (http://nistmassspeclibrary. com). 2.14. Statistical analysis All data for continuous variables were subjected to one-way analysis of variance (ANOVA) using PROG GLM in SAS program (SAS Institute, 1989). Mortality data were subjected to arcsine transformation and used for ANOVA. Means were compared with the least significant difference (LSD) test at Type I error of 0.05. 3. Results 3.1. Identification of two S. carpocapsae strains Two nematode isolates showing entomopathogenic behavior were collected in Korea (Figs. S1A, S1A′). In insect cadavers (Figs. S1B, S1B′), different developmental stages of nematodes were observed for both isolates (Figs. S1C-G, S1C′-G′). To identify nematode species, ITS regions were amplified and sequenced (Figs. S2A, S2B): 987 bp from Isolate 1 and 984 bp from Isolate 2. They were compared with a known ITS region sequence of C. elegans. Results confirmed that these sequences contained 5.8S rRNA, ITS-1, and ITS-2 regions as well as partial sequences of 18S and 28S rRNAs. They were deposited at GenBank with accession numbers of MK530238 and MK530239, respectively. These sequences showed high similarities (> 90%) with known ITS sequences of several Steinernema carpocapsae strains (Table S1). To analyze the relationship of these isolates with other known Steinernema spp., a phylogenetic tree was constructed using ITS sequences (Fig. S2C). Both isolates clustered with S. carpocapsae. Molecular identification was further supported by their morphometric characters showing similarity to S. carpocapsae (Table S2). The two identified S. carpocapsae strains were pathogenic to lepidopteran (S. exigua) and coleopteran (T. molitor) larvae (Fig. 1). With increasing numbers of IJs inoculated, mortality of both target insects increased. S. exigua larvae were more susceptible to the nematodes than were T. molitor larvae. Estimates of LC50 and LT50 were lower for S. exigua than for T. molitor (Table 1). However, there was little difference between the two nematode isolates in their insecticidal activities against the two target insects.

Fig. 1. Virulence of Steinernema carpocapsae Isolate 1 (A) and Isolate 2 (B) against larvae of two target insects, Spodoptera exigua and Tenebrio molitor at 25 °C. Infective juveniles (IJs) at different dosages were applied to a Petri dish containing five host insect larvae. Each experimental unit consisted of two dishes. Each dosage was replicated three times. Nematode dosage was calculated by dividing the applied IJ number by five larvae to obtain IJs per larva. Mortality was recorded 72 h after IJ treatment. Asterisks above standard deviation bars indicate significant difference between two species at Type I error = 0.05 (LSD test). In contrast, ‘NS’ stands for no-significant difference.

3.2. Identification of symbiotic bacteria of S. carpocapsae isolates To isolate symbiotic bacteria from these S. carpocapsae strains, surface-sterilized IJs and hemolymph of S. exigua infected with IJs were used as bacterial sources. Using blue colonies SK1 and SK2 on NBTA medium from Isolate 1 and Isolate 2, respectively, 16S rRNA sequences were assessed for both strains (Figs. S3A, S3B). Blast analysis of DNA sequences revealed high sequence similarities (> 99%) with Xenorhabdus nematophila. Phylogenetic analysis showed that these two strains (SK1 and SK2) clustered with X. nematophila (Fig. S3C). The 16S rRNA sequences of SK1 and SK2 were deposited at GenBank with accession numbers of MK893998 and MK893999, respectively. To support the molecular identification of X. nematophila, biochemical characteristics of these bacteria were assessed. Results showed that they belonged to Xenorhabdus genus (Table S3). Carbon utility measured with Biolog microbial identification indicated that these isolates shared highest homologies with X. nematophila (Table S4).

3.3. Comparative analysis of insecticidal activities of six different X. nematophila strains Insecticidal activities of six different X. nematophila strains including SK1 and SK2 were investigated by injecting live bacteria into hemocoels of S. exigua and T. molitor larvae (Fig. 2). All six strains were virulent to S. exigua and T. molitor larvae. Their virulence was determined by dosage and time elapsed after bacterial infection. Compared to the other five strains, SK1 was much less virulent, requiring significantly higher bacterial dosages than the other five strains to kill target insects (Fig. 2A). To compare these bacterial strains with respect to speed to kill, approximately 27 h was required for SK1 to kill half of test larvae of T. molitor while the other five strains killed the host in 14–16 h. SK1 also took longer time to kill S. exigua larvae (Fig. 2B). 4

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Table 1 Median lethal concentration (LC50) of S. carpocapsae isolates against two insect targets. Nematode isolate

Target insect

N

LC50 (IJs/larva)

Slope ± SE

LT50 (h)1

Slope ± SE

Isolate 1

S. exigua T. molitor

30 30

59.0 (38.3–90.9) 118.0 (80.4–173.2)

1.66 ± 0.10 1.80 ± 0.09

34.8 (29.5–41.0) 41.0 (35.0–48.1)

4.43 ± 0.04 4.58 ± 0.04

Isolate 2

S. exigua T. molitor

30 30

68.9 (46.0–103.5) 141.6 (94.9–203.5)

1.69 ± 0.09 1.56 ± 0.09

38.6 (33.0–45.0) 41.8 (35.7–49.0)

4.77 ± 0.04 4.59 ± 0.04

1

Treatment dosage at 700 IJs per larva.

3.4. Comparative analysis of host immunosuppressive activity of six different X. nematophila strains

addition of arachidonic acid (AA, a PLA2 catalytic product for eicosanoid biosynthesis). In the nodulation assay, approximately 60 nodules were formed in response to bacterial challenge in the control (Fig. 3B). However, injection of bacterial culture broth significantly suppressed such nodule formation in response to bacterial challenge. The bacterial broth derived from SK1 significantly (P < 0.05) suppressed nodule formation, but was less potent than those of the other five strains. AA addition significantly (P < 0.05) rescued the suppressed nodulation for all strains.

Variations in insecticidal activities of six different X. nematophila strains might be induced by differential immunosuppressive activities of bacterial culture broths. To test this hypothesis, bacterial culture broths were compared for inhibition against hemocyte-spreading behavior (Fig. 3A). Approximately 95% of hemocytes exhibited spreading behavior in the control. However, when hemocytes were incubated with 10% cultured broth (filtered with 0.22 μm) of each of the six strains, hemocyte-spreading behavior was significantly suppressed. Particularly, the bacterial broth derived from SK1 was significantly (P < 0.05) potent to suppress the cellular immune response, but less potent than the other five strains. Suppressed hemocyte-spreading behavior was significantly (P < 0.05) rescued for all six strains by

3.5. Comparative analysis of secondary metabolites of six different X. nematophila strains Inhibitory activities of bacterial culture broths suggested that these different X. nematophila strains might synthesize and release secondary

Fig. 2. Variation in bacterial virulence among six strains of Xenorhabdus nematophila against Spodoptera exigua and Tenebrio molitor larvae at 25 °C. In addition to two new strains, SK1 and SK2 of X. nematophila, four known strains are ‘M’ for X. nematophila Mexico, ‘F’ for X. nematophila France, ‘12145’ for X. nematophila ATCC12145, and ‘K1′ for X. nematophila K1. For LD50 estimation, different bacterial dosages were micro-injected to insect hemocoels. Each treatment was replicated three times with 10 larvae per replication. Mortality was measured 24 h after bacterial injection. For LT50 estimation, a bacterial dosage (105 CFU/larva) was injected and mortality was monitored every 8 h. Different letters above standard deviation indicate significant difference among means at Type I error = 0.05 (LSD test). 5

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Secondary metabolites included in the organic extracts were detected in TLC (Fig. S4) and GC (Fig. S5). Their chemical identities were predicted from GC-MS. A total of 87 compounds were obtained from extracts of six bacterial strains (Table 2). Seven compounds were commonly detected in all six bacterial strains while 37 compounds were specifically detected in only one of six bacterial strains. The number of the detected compounds was low in bacterial culture broth of SK1, the least potent strain. 4. Discussion Immunosuppression is closely associated with insecticidal activity in two entomopathogenic bacteria genera, Xenorhabdus and Photorhabdus (Ahmed and Kim, 2018). These bacteria can inhibit PLA2 with their secondary metabolites to prevent eicosanoid biosynthesis and induce immunosuppression (Eom et al., 2014). We further investigated the relationship between insecticidal activity and immunosuppression among different strains within X. nematophila in order to remove the heterogeneous genetic background occurring in different species. We used six strains of X. nematophila, of which two strains were newly identified in this study. Two bacterial strains were isolated from two different local isolates of S. carpocapsae. They were identified using morphological and molecular markers. Interestingly, ITS sequences of these two nematode isolates were different, with Isolate 1 being remotely clustered with another S. carpocapsae group containing Isolate 2. These results suggest that the two local populations have genetic distance within species. Although these two X. nematophila strains, derived from genetically distinct hosts, exhibited highly homologous 16S rRNA sequences, they also exhibited differences in insecticidal activity by more than two-fold. SK1 was quite different in insecticidal activity compared to five other X. nematophila strains, including SK2. Our results suggest that there is variation in virulence among different strains of X. nematophila. Insecticidal activity of X. nematophila includes septicemia and toxemia (McQuade and Stock, 2018). X. nematophila inhibits the host insect immune response by suppressing PLA2 activity (Kim et al., 2005). Suppressed PLA2 could not effectively hydrolyze phospholipids to release eicosanoid biosynthesis precursor(s). Considering that eicosanoids have immune mediation function (Kim et al., 2018a), larvae infected by X. nematophila suffer severe immunosuppression that can lead to septicemia. Regarding toxemia, several toxin proteins in X. nematophila are known, including Xpt (Morgan et al., 2001), XnGroEL (Kumari et al., 2014), Txp40 (Brown et al., 2006), XaxAB (Vigneux et al., 2007), and a 12 kDa protein (Hemalatha et al., 2018). Yang et al. (2017) reported another X. nematophila protein, PirAB (Photorhabdus insect-related proteins), in Photorhabdus luminescens TT01. They showed that this binary protein had hemocoel insecticidal activity against Galleria mellonella larvae at a low LD50 (1.6 mg/larva). Thus, variation in insecticidal activity among the six strains of X. nematophila might have contributed to their different immunosuppressive and toxin-producing activities. Strain-level variations have been explored through genome analysis using 10 strains of X. bovienii (Murfin et al., 2015). It was found that non-ribosomal peptide synthetase (NRPS) gene clusters and insecticidal toxin component gene exhibited heterogeneity, suggesting variation in virulence factors (Murfin et al., 2015). X. bovienii is symbiotic with at least nine Steinernema species. The species included different strains (Bisch et al., 2015). X. bovienii CS03 has no insecticidal activity against Spodoptera littoralis at a dosage of 103 cfu per insect while other X. bovienii strains have significant virulence at the same dosage. According to Bucher’s definition (Bucher, 1960, 1973), a bacterium is considered entomopathogenic if it is able to kill an insect host at a dosage smaller than 105 cells. X. bovienii CS03 never colonized the insect hemocoel due to its high susceptibility to antimicrobial peptides (Bisch et al., 2015). When the genome of Xb CS03 was sequenced and compared with the genome of a virulent strain, X. bovienii SS-2004 (Xb SS-2004), it was revealed that Xb CS03 strain contained more

Fig. 3. Immunosuppressive activities of bacterial culture broths of six different Xenorhabdus nematophila strains by inhibiting eicosanoid biosynthesis in Spodoptera exigua. In addition to two new strains of SK1 and SK2 of X. nematophila, four known strains are ‘M’ for X. nematophila Mexico, ‘F’ for X. nematophila France, ‘12145′ for X. nematophila ATCC12145, and ‘K1′ for X. nematophila K1. (A) Inhibitory activities of bacterial culture broth filtrates (‘BF’) against hemocyte-spreading behavior of S. exigua. Each treatment was replicated three times with independently prepared hemocyte suspension. (B) Inhibitory activities of BFs against hemocyte nodule formation of S. exigua. Heat-killed bacteria (4 × 104 cells/larva) were injected to L5 larvae. Injection volume was 5 μL. For BF treatment, 4 μL of bacterial suspension and 1 μL of test BF were used. For AA rescue experiment, 1 μL of AA (10 μg/μL) was added to 3 μL of bacterial suspension and 1 μL of test BF. Each treatment consisted of three replicates and each replication used five larvae. Different letters above standard deviation bars in each bacterial strain indicate significant difference among means at Type I error = 0.05 (LSD test).

metabolites exhibiting immunosuppressive activities. When the bacterial culture broths were fractionated with different organic solvents, all organic extracts possessed inhibitory activities against immune-associated characters (Fig. 4). Compared to the control, nodule formation was significantly (P < 0.05) suppressed in larvae treated with different organic extracts. Ethyl acetate and butanol extracts appeared to suppress more potently than the other two organic extracts for all six bacterial strains (Fig. 4A). K1 strain in hexane extract, SK2 and K1 strains in ethyl acetate extract, F strain in chloroform extract, and 12,145 strain in butanol extract exhibited relatively high inhibitory activities. These organic extracts were also inhibitory against both PLA2 activities (Fig. 4B, 4C). The highest inhibitory activity was observed in hexane extract of SK2 strain against sPLA2. 6

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Fig. 4. Immunosuppressive factors of organic extracts of bacterial culture broths of six different X. nematophila (Xn) strains. In addition to two new strains of SK1 and SK2 of X. nematophila, four known strains are ‘M’ for X. nematophila Mexico, ‘F’ for X. nematophila France, ‘12145′ for X. nematophila ATCC12145, and ‘K1′ for X. nematophila K1. (A) Inhibitory activities of bacterial extracts against hemocyte nodule formation. Each bacterial extract (10 µg/larva) was injected into L5 larvae of S. exigua along with heat-killed Xn (4 × 104 cells). Control (‘CON’) used methanol instead of bacterial extract. Each treatment used 15 larvae. (B) Inhibitory activities of bacterial extracts on hemolymph sPLA2 enzyme activity of L5 larvae of S. exigua. (C) Inhibitory activities of bacterial extracts on fat body cPLA2 enzyme activity of L5 larvae of S. exigua. Different letters above standard deviation bars indicate significant differences among means at Type I error = 0.05 (LSD test).

pseudogenes than Xb SS-2004, especially genes involved in host invasion and exploitation (toxins, invasins, or extracellular enzymes) (Bisch et al., 2016). These reports led us to further explore virulence factors in the culture media of six strains of X. nematophila. A total of 87 compounds were predicted from organic extracts of bacterial culture broth. These compounds probably include non-bacterial metabolites from bacterial medium components or other culture plasticware. For example, 9 compounds were present in all six strains of X. nematophila. Among these common compounds, bis-(2-ethylhexyl) phthalate might have originated from plasticware because several phthalates are used for plasticizers. However, a large number of compounds were present in specific bacterial strains. Thirty-seven compounds were detected in only one of six bacterial strains. These results suggest that a large number of the identified compounds originate from bacterial metabolites. Putative bacterial origins include hydrocarbons and fatty acids that are presumably derived from bacterial cell wall components. However, a number of small compounds are amino acid derivatives that are likely to be produced by a number of secondary metabolite biosynthetic gene clusters (BGCs) predominantly manufactured by NRPS or polyketide synthase (PKS) (Cai et al., 2016). For example, P. luminescens TTO1 genome encodes 23 predicted BGCs, making up 6.5% of the genome (Bode, 2009). In X. nematophila, watersoluble peptide antimicrobial compounds xenocoumacin 1 and xenocoumacin 2 are produced by NRPS-PKS (Park et al., 2009). Two

compounds identified in this study are dipeptides (cyclic GL and cyclic LF), possibly produced from NRPS of X. nematophila. Organic extracts from the six bacterial culture broths showed immunosuppressive and PLA2-inhibitory activities. Their immunosuppressive activities measured by hemocyte nodulation were highly correlated with insecticidal activities. Nodulation requires mediation of eicosanoids that are produced by catalytic activity of PLA2 (Park et al., 2015). Here, the PLA2 gene expression is inducible to bacterial infection via Toll/IMD signaling pathways (Sajjadian et al., 2019). This supports the hypothesis that the inhibitory intensity against target insect PLA2 enzyme activity by Xenorhabdus and Photorhabdus is functionally related to bacterial pathogenicity (Ahmed and Kim, 2018). Variations in immunosuppression among different strains might have originated from regulatory variations and phenotypic variations in addition to genetic variation in BGCs. Phenotypic variation occurs when an isogenic population exhibits two or more distinct phenotypes. Repetitive laboratory culturing of X. nematophila can lead to phenotypic variation from primary to secondary forms (Forst and Clarke, 2002). Global transcriptional factor Lrp can regulate phenotypic variation in X. nematophila, leading to attenuation of virulence and immunosuppression in insect hosts (Hussa et al., 2015). Thus, any variation in Lrp expression among six X. nematophila strains may lead to differential pathogenicity by altering expression of virulence genes, thus affecting immunosuppression.

X. nematophila

X. nematophila

X. nematophila

X. nematophila

Fig. 4. (continued) 8

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Table 2 (continued)

Table 2 Eighty-seven compounds identified in organic extracts of bacterial culture broth of six different X. nematophila strains. RT (min)

2.246 2.447 2.459 2.471 2.677 3.628 4.153 4.607 4.926 5.027 5.464 6.621 6.768 7.173 7.772 7.791 8.652 9.372 9.915 10.133 11.355 11.373 11.615 11.762 12.294 12.489 12.583 12.672 12.762 13.929 14.036 14.596 14.602 14.785 15.853 16.196 16.969 17.589 17.772 18.309 18.745 18.245 18.929 18.935 19.077 19.242 19.277 20.340 20.671 20.984 21.102 21.308 21.551 21.686 21.828 22.145 22.157 22.885

Compounds

Ethanol, 2,2-diethoxy1-Butanamine, N-butyl4-Ethylamino-n-butylamine Leucine Butanoic acid, butyl ester Pyrazine, 3-ethyl-2,5-dimethylPhenylethyl Alcohol 4H-Pyran-4-one, 2,3-dihydro-3,5dihydroxy-6-methylHexanoic acid, 5-oxo-, ethyl ester Octanoic acid 1-Decene Benzeneacetic acid Butane, 1,1-dibutoxy5-Thiazoleethanol, 4-methylNonadecane 1,2-Ethanediol, 1-phenyll-Leucine, N-methoxycarbonyl-, methyl ester n-Decanoic acid 2-Tetradecene, (E)Tetradecane Phthalimide o-Cyanobenzoic acid Dodecane, 2-methylTetradecane, 3-methylCyclopentadecane Pentadecane N-Butyryl-DL-homoserine lactone Phenol, 2,4-bis(1,1dimethylethyl)2(1H)-Quinolinone, 4,8-dimethyl Pentadecane, 2-methylDodecanoic acid Cetene Z-8-Hexadecene Hexadecane 2-Bromo dodecane Hexadecane, 2-methylHeptadecane 1H-Thieno[3,4-d]imidazole-4propanoic acid, hexahydro-2-oxoPyrrolo[1,2-a]pyrazine-1,4-dione, hexahydroHeptadecane, 2-methylCyclo-(glycyl-l-leucyl) 4-Mercaptophenol 1-Octadecene E-15-Heptadecenal Octadecane Phenol, 3,5-dimethoxyHexadecane, 2,6,10,14tetramethyl2,6-Dichlorobenzenesulfonyl chloride Cyclotetradecane Hexadecanenitrile E-8-Methyl-9-tetradecen-1-ol acetate 7,9-Di-tert-butyl-1-oxaspiro(4,5) deca-6,9-diene-2,8-dione Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)Diethyldithiophosphinic acid Benzenepropanoic acid, 3,5-bis (1,1-dimethylethyl)-4-hydroxy-, methyl ester 1,2-Benzenedicarboxylic acid, butyl 2-methylpropyl ester 2-Mercaptobenzothiazole Cyclohexadecane, 1,2-diethyl-

RT (min)

Compounds

X. nematophila strains1 SK1

SK2

M

F

12,145

K1

− − − − + − − +

+ + − + + + − −

− + + − + − − +

− + − − + − + +

− + − − + − − +

− + − − + − − +

− − − + − − + − −

+ − + − + − + + −

− + − − − + − − −

− − − − − + − − −

− − − − − + − − −

− + − − − + − − +

− − − − − − − − + − +

− + + + − − + + + + +

− − + − + − − − + + +

− + + + − − − − + + +

+ − + + − − − − − + +

− − + + − + − − + + +

+ − − + − + − − + −

− + − + − + − + −

+ − − − + + − − + +

+ − − + − + − + + −

− − + + − + − − − −

+ + − − + + − + + −

+

+

−

−

−

+

− + − + − + + −

+ + − − + + + +

− − − − − + + −

− + − − − + + −

− − + − + + + −

− − − − + + + −

−

−

−

−

−

+

− − −

− + −

+ − −

− − +

− − −

− − −

+

−

−

−

−

−

+

+

+

+

+

+

+ +

− −

+ +

+ +

+ +

+ +

−

+

−

−

−

−

− +

− +

− −

+ −

+ +

+ −

22.891 23.009 23.032 23.164 24.260 24.419 24.496 24.850 24.886 25.328 25.411 26.509 26.609 24.886 26.645 27.377 28.881 28.912 29.16 29.302 31.061 31.078 32.153 32.159 32.212 32.914 32.95 35.488

35.789 Total

1-Nonadecene Eicosane Octadecane, 1-chloroCyclic octaatomic sulfur 9-Eicosyne Oleanitrile 2-Benzimidazolethiol Heptadecanenitrile Nonadecanenitrile Heptadecanoic acid, 16-methyl-, methyl ester 2,5-Piperazinedione, 3-methyl-6(phenylmethyl)1-Docosene 9-Tricosene, (Z)1-Eicosene Docosane 2,5-Piperazinedione, 3-benzyl-6isopropylCyclo-(l-leucyl-l-phenylalanyl) Benzyl butyl phthalate 9-Octadecenamide, (Z)Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(phenylmethyl)3-Benzylidene-hexahydro-pyrrolo [1,2-a]pyrazin-1,4-dione Hexanoic acid, 2-ethyl-, hexadecyl ester Bis(2-ethylhexyl) phthalate Diisooctyl phthalate Di-n-octyl phthalate Cyclotetracosane 1-Hexacosene 4-(cis-6-Methoxymethyl-3,4dimethyl-3-cyclohexenyl)-trans-3buten-2-one 2,4dinitrophenylhydrazone Cyclotrisiloxane, hexamethyl87

X. nematophila strains1 SK1

SK2

M

F

12,145

K1

+ + − − − + − − − −

− + − − + + − + − −

+ + − + − + − − − +

+ + − + − + + − + −

+ − − + − + − − − −

+ + + + − + + − − +

+

+

−

−

−

−

+ − − − −

− − − − −

− + + + +

+ − − + +

+ − − − +

+ − + + +

+ − + +

− + − +

− − − +

+ − − +

+ − + +

+ − − +

−

−

−

−

−

+

−

+

−

−

−

−

+ + − − − −

+ − + + − −

+ − − − − −

+ − − − − −

+ − − − + +

+ − − − − −

+ 30

− 44

− 33

+ 37

+ 33

+ 43

1

In addition to two new strains of SK1 and SK2 of X. nematophila, four known strains are ‘M’ for X. nematophila Mexico, ‘F’ for X. nematophila France, ‘12145’ for X. nematophila ATCC12145, and ‘K1′ for X. nematophila K1.

Acknowledgments This work was supported by a grant (No. 2017R1A2B3009815) of National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning (MSIP), Republic of Korea. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107221. References Ahmed, S., Kim, Y., 2018. Differential immunosuppression by inhibiting PLA2 affects virulence of Xenorhabdus hominickii and Photorhabdus temperata temperata. J. Invertebr. Pathol. 157, 136–146. Bensen, H.J., 1990. Microbiological applications, fifth ed. Wm. C. Brown Publishers, IA, pp. 376. Bisch, G., Pagès, S., McMullen II, J.G., Stock, S.P., Duvic, B., Givaudan, A., Gaudriault, S., 2015. Xenorhabdus bovienii CS03, the bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran insects. J. Invertebr. Path. 124, 15–22. Bisch, G., Ogier, J.C., Médigue, C., Rouy, Z., Vincent, S., Tailliez, P., Givaudan, A., Gaudriault, S., 2016. Comparative genomics between two Xenorhabdus bovienii strains highlights differential evolutionary scenarios within an entomopathogenic bacterial species. Genome Biol. Evol. 8, 148–160. Bode, H.B., 2009. Entomopathogenic bacteria as a source of secondary metabolites. Curr.

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Md. A. Hasan, et al. Opin. Chem. Biol. 13, 224–230. Boemare, N.E., 2002. Biology, taxonomy and systematics of Photorhabdus and Xenorhabdus. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New York, NY, pp. 35–56. Brown, S.E., Cao, A.T., Dobson, P., Hines, E.R., Akhurst, R.J., East, P.D., 2006. Txp40, a Ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria. Appl. Environ. Microbiol. 72, 1653–1662. Bucher, G.E., 1960. Potential bacterial pathogens of insects and their characteristics. J. Insect Pathol. 2, 172–195. Bucher, G.E., 1973. Definition and identification of insect pathogens. Ann. N. Y. Acad. Sci. 217, 8–17. Cai, X., Nowak, S., Wesche, F., Bischoff, I., Kaiser, M., Fürst, R., Bode, H.B., 2016. Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design. Nat. Chem. 9, 379–386. Cao, M., Goodrich-Blair, H., 2017. Ready or not: microbial adaptive responses in dynamic symbiosis environments. J. Bacteriol. 199, e00883–e916. Dennis, E.A., 1994. Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 269, 13057–13060. Dowds, B.C.A., Peters, A., 2002. Virulence mechanisms. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New York, NY, pp. 79–98. Eden, P.A., Schmidt, T.M., Blakemore, R.P., Pace, N.R., 1991. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNAspecific DNA. Int. J. Syst. Evol. Microbiol. 41, 324–325. Eleftherianos, I.G., 2009. Novel antibiotic compounds produced by the insect pathogenic bacterium Photorhabdus. Recent Pat. Antiinfect. Drug Discov. 4, 81–89. Eom, S., Park, Y., Kim, Y., 2014. Sequential immunosuppressive activities of bacterial secondary metabolites from the entomopathogenic bacterium Xenorhabdus nematophila. J. Microbiol. 52, 161–168. Forst, S., Clarke, D., 2002. Bacteria-nematode symbiosis. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI, New York, NY, pp. 57–77. Gillespie, J.P., Trenczek, T., Kanost, M.R., 1997. Biological mediators of insect immunity. Annu. Rev. Entomol. 42, 611–643. Goh, H.G., Lee, S.G., Lee, B.P., Choi, K.M., Kim, J.H., 1990. Simple mass-rearing of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), on an artificial diet. Kor. J. Appl. Entomol. 29, 180–183. Goodrich-Blair, H., Clarke, D.J., 2007. Mutualism and pathogenesis in Xenorhabdus and Photorhabdus: two roads to the same destination. Mol. Microbiol. 64, 260–268. Han, R., Ehlers, R.U., 2000. Pathogenicity, development, and reproduction of Heterorhabditis bacteriophora and Steinernema carpocapsae under axenic in vivo conditions. J. Invertebr. Pathol. 75, 55–58. Hemalatha, D., Prabhu, S., Rani, W.B., Anandham, R., 2018. Isolation and characterization of toxins from Xenorhabdus nematophilus against Ferrisia virgata (Ckll.) on tuberose, Polianthes tuberosa. Toxicon 146, 42–49. Hussa, E.A., Casanova-Torres, Á.M., Goodrich-Blair, H., 2015. The global transcription factor Lrp controls virulence modulation in Xenorhabdus nematophila. J. Bacteriol. 197, 3015–3025. Ji, D., Kim, Y., 2004. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits the expression of an antibacterial peptide, cecropin, of the beet armyworm, Spodoptera exigua. J. Insect Physiol. 50, 489–496. Kang, J.S., Shin, S.C., Choi, K.S., Lee, S.H., Lee, S.G., Moon, I.S., 2004. Development of an efficient PCR-based diagnosis protocol for the identification of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae). Nematology 6, 279–285. Kim, Y., Ji, D., Cho, S., Park, Y., 2005. Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression. J. Invertebr. Pathol. 89, 258–264. Kim, Y., Ahmed, S., Stanley, D., An, C., 2018a. Eicosanoid-mediated immunity in insects. Dev. Comp. Immunol. 83, 130–143. Kim, H., Choi, D., Jung, J., Kim, Y., 2018b. Eicosanoid mediation of immune responses at early bacterial infection stage and its inhibition by Photorhabdus temperata subsp. temperata, an entomopathogenic bacterium. Arch. Insect Biochem. Physiol. 99, e21502. Krieg, N.R., Hart, J.G., 1984. Bergey's manual of systematic bacteriology, vol. 1, pp. 506–512. Kumari, P., Kant, S., Zaman, S., Mahapatro, G.K., Banerjee, N., Sarin, N.B., 2014. A novel insecticidal GroEL protein from Xenorhabdus nematophila confers insect resistance in tobacco. Transgenic Res. 23, 99–107. Lacey, L.A., Grzywacz, D., Shapiro-Ilan, D.I., Frutos, R., Brownbridge, M., Goettel, M.S., 2015. Insect pathogens as biological control agents: back to the future. J. Invertebr. Pathol. 132, 1–41. Lee, S., Kim, Y., Han, S., 2000. An improved collecting method of the infective juveniles of the entomopathogenic nematode, Steinernema carpocapsae Weiser. Korean J. Soil Zool. 5, 97–100.

Lemaitre, B., Hoffmann, J., 2007. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25, 697–743. McQuade, R., Stock, S.P., 2018. Secretion systems and secreted proteins in Gram-negative entomopathogenic bacteria: their roles in insect virulence and beyond. Insects 9, 68. Morgan, J.A.W., Sergeant, M., Ellis, D., Ousley, M., Jarrett, P., 2001. Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296. Appl. Environ. Microbiol. 67, 2062–2069. Murfin, K.E., Whooley, A.C., Klassen, J.L., Goodrich-Blair, H., 2015. Comparison of Xenorhabdus bovienii bacterial strain genomes reveals diversity in symbiotic functions. BMC Genomics 16, 889. Park, J., 2015. Elucidation of a prostaglandin biosynthesis pathway using novel phospholipase A2 and cyclooxygenase. Master Degree Thesis. Andong National University, Andong, Korea. Park, Y., Kim, Y., Yi, Y., Han, S., 1998. Optimal storage conditions of the entomopathogenic nematode, Steinernema carpocapsae. Korean J. Appl. Entomol. 3, 10–16. Park, D., Ciezki, K., Van Der Hoeven, R., Singh, S., Reimer, D., Bode, H.B., Forst, S., 2009. Genetic analysis of xenocoumacin antibiotic production in the mutualistic bacterium Xenorhabdus nematophila. Mol. Microbiol. 75, 938–949. Park, Y., Sunil, K., Rahul, K., Stanley, D., Kim, Y., 2015. A novel calcium-independent cellular PLA2 acts in insect immunity and larval growth. Insect Biochem. Mol. Biol. 66, 13–23. Peters, A., 2013. Application and commercialization of nematodes. Appl. Microbiol. Biotechnol. 97, 6181–6188. Poinar, G.O.J., Thomas, G.M., 1966. Significance of Achromobacter nematophilus (Achromobactericeae Eubacteriales) in the development of the nematode, DD-136 (Neoaplectana sp. Steinernematidae). Parasitology 56, 385–390. Poinar, G.O.J., Thomas, G.M., Hess, R., 1977. Characteristics of the specific bacterium associated with Heterorhabditis bacteriophora (Heterorhabditidae: Rhabditida). Nematologica 23, 97–102. Richards, G.R., Goodrich-Blair, H., 2010. Examination of Xenorhabdus nematophila lipases in pathogenic and mutualistic host interactions reveals a role for xlpA in nematode progeny production. Appl. Environ. Microbiol. 76, 221–229. Sadekuzzaman, M., Park, Y., Lee, S., Kim, K., Jung, J.K., Kim, Y., 2017. An entomopathogenic bacterium, Xenorhabdus hominickii ANU101, produces oxindole and suppresses host insect immune response by inhibiting eicosanoid biosynthesis. J. Invertebr. Pathol. 145, 13–22. Sajjadian, S.M., Vatanparast, M., Kim, Y., 2019. Toll/IMD signal pathways mediate cellular immune responses via induction of intracellular PLA2 expression. Arch. Insect Biochem. Physiol. 101, e21559. Sajnaga, E., Kazimierczak, W., Skowronek, M., Lis, M., Skrzypek, T., Waśko, A., 2018. Steinernema poinari (Nematoda: Steinernematidae): a new symbiotic host of entomopathogenic bacteria Xenorhabdus bovienii. Arch. Microbiol. 200, 1307–1316. SAS Institute, Inc., 1989. SAS/STAT Users Guide, Release 6, third ed. SAS Institute, Cary, NC. Schaad, N.W., 1988. Laboratory Guide to Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN. Seo, S., Jang, H., Kim, K., Kim, Y., 2010. Comparative analysis of immunosuppressive metabolites synthesized by an entomopathogenic bacterium, Photorhabdus temperata ssp. temperata, to select economic bacterial culture media. Korean J. Appl. Entomol. 49, 409–416. Seo, S., Lee, S., Hong, Y., Kim, Y., 2012. Phospholipase A2 inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata. Appl. Environ. Microbiol. 78, 3816–3823. Stanley, D.W., 2000. Eicosanoids in Invertebrate Signal Transduction Systems. Princeton University Press, Princeton, NJ. Tobias, N.J., Shi, Y.M., Bode, H.B., 2018. Refining the natural product repertoire in entomopathogenic bacteria. Trends Microbiol. 26, 833–840. Vatanparast, M., Ahmed, S., Herrero, S., Kim, Y., 2018. A non-venomous sPLA2 of a lepidopteran insect: its physiological functions in development and immunity. Dev. Comp. Immunol. 89, 83–92. Vigneux, F., Zumbihl, R., Jubelin, G., Ribeiro, C., Poncet, J., Baghdiguian, S., Givaudan, A., Brehelin, M., 2007. The xaxAB genes encoding a new apoptotic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens. J. Biol. Chem. 282, 9571–9580. Vrain, T.C., Wakarchuk, D.A., Levesque, A.C., Hamilton, R.I., 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fund. Appl. Nematol. 15, 563–574. Waterfield, N.R., Ciche, T., Clarke, D., 2009. Photorhabdus and a host of hosts. Annu. Rev. Microbiol. 63, 557–574. Yang, Q., Zhang, J., Li, T., Liu, S., Song, P., Nangong, Z., Wang, Q., 2017. PirAB protein from Xenorhabdus nematophila HB310 exhibits a binary toxin with insecticidal activity and cytotoxicity in Galleria mellonella. J. Invertebr. Pathol. 148, 43–50.

10