Differentiating between scavengers and entomopathogenic nematodes: Which is Oscheius chongmingensis?

Journal of Invertebrate Pathology 167 (2019) 107245

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Differentiating between scavengers and entomopathogenic nematodes: Which is Oscheius chongmingensis? Keyun Zhanga, a b

⁎,1

, Tiffany Baiocchib,1, Dihong Lub, Dennis Z. Changb, Adler R. Dillmanb,

T

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College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Department of Nematology, University of California, Riverside, CA 92521, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Entomopathogenic nematodes Oscheius Scavenger Parasite

Entomopathogenic nematodes (EPNs) continue to be explored for their potential usefulness in biological control and pest management programs. As more insect-associated species of nematodes are discovered and described, it is possible that scavengers and kleptoparasites may be mischaracterized as EPNs. If a nematode species is truly an entomopathogen it should display similar infectivity, as well as behaviors and preferences, to those of established EPN species, such as Steinernema carpocapsae. In this study we evaluated dauers of the putative EPN species Oscheius chongmingensis. We examined virulence, odor preferences as a measure of host-seeking behavior, and features of its bacterial symbiont Serratia nematodiphila. We determined that O. chongmingensis behaves more like a scavenger than an EPN. Not only did O. chongmingensis exhibit very poor pathogenicity in Galleria mellonella (wax moth larvae), it also displayed odor (host-seeking) preferences that are contrary to the well-known EPN S. carpocapsae. We also found that the bacterial symbiont of O. chongmingensis was antagonistic to S. carpocapsae; S. carpocapsae IJs were unable to develop when S. nematodiphila was a primary food source. We conclude that there is insufficient evidence to support the characterization of O. chongmingensis as an EPN; and based on the attributes of its preferences for already-infected or deceased hosts, suggest that this nematode is a scavenger, which may be on an evolutionary trajectory leading to an entomopathogenic lifestyle.

1. Introduction Entomopathogenic nematodes (EPNs) are a distinct guild of insectparasitic nematodes, characterized by their ability to rapidly kill insect hosts, as well as their utilization of a mutualistic associated bacteria, which helps facilitate their parasitic lifestyle. There are more than a hundred species of EPNs described within Steinernema and Heterorhabditis, the two historically well-studied genera of EPNs (Lewis and Clarke, 2012; Stock, 2015). There are also a growing number of rhabditid nematodes, especially Oscheius spp., being described as EPNs; including Oscheius carolinensis (Ye et al., 2010), Oscheius chongmingensis (formerly Heterorhabditoides chongmingensis) (Zhang et al., 2008), Oscheius gingeri (Pervez et al., 2013), and Oscheius onirici (Torrini et al., 2015). The status of a nematode as an EPN marks it as being of potential interest in biological control, as EPNs have been used in biological control against insect pests since their original discovery (Gaugler, 1988; Kaya and Gaugler, 1993; Lu et al., 2016). Amid the increasing number of described EPNs, there is uncertainty as to whether some of

these newly described species actually fit the criteria for being EPNs and whether they have any usefulness in biological control programs (Campos-Herrera et al., 2015; Dillman et al., 2012a; Rae and Sommer, 2011). One reason for the confusion comes from an incomplete initial assessment regarding the hallmark-characteristics of EPNs when evaluating a new species; namely a non-transient—though not necessarily obligate—association with bacteria to facilitate pathogenesis and rapid host death. Another reason for the uncertainty comes from conflating parasitism and scavenging. Free-living bacterivorous nematodes (FLBNs) are scavengers that can compete with EPNs for the resource-rich insect cadavers within the soil environment (Campos-Herrera et al., 2012, 2015; Duncan et al., 2003, 2007). Interactions between EPNs and FLBNs are complex and often result in competition that can potentially impact EPN health or reproduction (Blanco-Pérez et al., 2017, 2019; Campos-Herrera et al., 2015). The impacts on EPN reproduction depend on the species involved in the competition as well as initial-infecting inoculum and bacterial competition (Blanco-Pérez et al., 2017). Adding to the

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Corresponding authors. E-mail addresses: [email protected] (K. Zhang), [email protected] (T. Baiocchi), [email protected] (D. Lu), [email protected] (D.Z. Chang), [email protected] (A.R. Dillman). 1 These authors are equal contribution to this paper. https://doi.org/10.1016/j.jip.2019.107245 Received 10 June 2019; Received in revised form 6 September 2019; Accepted 9 September 2019 Available online 10 September 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.

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14 days at room temperature (20° ± 3 °C). S. carpocapsae (strain All) was also reared using standard techniques on waxworms with IJs being harvested from White traps (Kaya and Stock, 1997; McMullen and Stock, 2014; White, 1927). C. elegans (strain N2) was cultured as previously described (Brenner, 1974), and dauers were collected via starvation (Karp, 2018). Briefly, C. elegans was grown on NGM plates with OP50 (Escherichia coli) bacteria as a food substrate. To obtain dauers, plates were inoculated with C. elegans and left undisturbed until all the food had been consumed (usually 7–10 days), after which spots of sterile tap water were placed on the lid to facilitate collection of dauers. A few days later the dauers were collected from the lids, rinsed three times in tap water and stored at room temperature (20 ± 3 °C) for use in experiments within 2 weeks. To ensure that the population of collected C. elegans was primarily made of up dauers, a sample of the population was tested with 1% SDS as previously described protocol (Nika et al., 2016). Populations of C. elegans dauers collected contained approximately 80–83% dauers (with the remaining 17–20% being other juvenile stages). For Pristionchus pacificus (PS312), a silica sand with agar method was employed to culture and collected its dauers. In detail, 9-cm diameter plastic Petri plates with 2% agar were prepared by sprinkling autoclaved silica sand on the surface of each plate. Ten last-instar G. mellonella were placed into each of the prepared plates along with 500 P. pacificus dauers. Plates were incubated at room temperature 20 ± 3 °C for approximately 14–16 days at which point dauers could be seen emerging from the cadaver and gathering on the walls of the plate. These dauers were washed off the plate and rinsed 3 times in clean tap water before being quantified to determine the yield of dauers. To ensure that the population of collected P. pacificus was primarily made of up dauers, a sample of the population was tested with 1% SDS (Nika et al., 2016). Populations of P. pacificus collected contained approximately 88–92% dauers, with 8–12% being other juvenile stages. The collected P. pacificus was then used within 2 weeks for infection experiments. Bacterial strains: Phase I and Phase II X. nematophila (HBG800), as well as S. nematodiphila (0503SBS1), were cultured on Luria-Bertani (LB) medium containing 1.0% tryptone, 0.5% yeast extract, 0.5% NaCl, and 0.01% SP (Sodium pyruvate). Bacteria grown on LA (Lipid Agar) was prepared as previously described (Vivas and Goodrich-Blair, 2001), as was NBTA (nutrient agar, 0.0025% bromothymol blue, 0.004% triphenyltetrazolium chloride medium, and 0.01% SP) as needed for various experiments. Bacteria were grown for 24 h at 28 °C regardless of media type; and liquid cultures vials of media were shaken at 200 rpm.

complexity of these interactions, is the fact that EPN species produce infective juveniles (IJs) that may act either as true entomopathogens (i.e. infecting healthy, live hosts) (Dillman et al., 2012a) or as facultative scavengers (i.e. colonizing an already-dead host) (Puza and Mracek, 2010; San-Blas and Gowen, 2008). In some cases, EPN IJs are more attracted to already-dead or infected hosts than to naïve hosts (Baiocchi et al., 2017), and it has even been observed that EPNs can utilize scavenging tactics as a method of survival (San-Blas and Gowen, 2008). However, the lifestyles of EPNs and FLBNs are distinct and can be differentiated. EPNs display active host-seeking behaviors and show a preference for live insect host volatiles and even individual host-associated odors; to which many EPNs respond in a species-specific manner (Dillman et al., 2012b). Additionally, EPNs and other parasitic nematodes use a variety of species-specific cues (including temperature (Castelletto et al., 2014), tactile (Lewis et al., 1992), and chemosensory cues (Chaisson and Hallem, 2012; Dillman et al., 2012b)) to locate and infect suitable hosts. Furthermore, EPNs often utilize entomopathogenic bacterial symbionts, which are transmitted over many generations as juvenile EPNs re-associate specifically with their symbiont(s) in the process of developing to the IJ stage. Once an IJ finds and invades a host, this bacteria is quickly and efficiently released inside the insect host to facilitate pathogenicity and rapid insect death (Lewis et al., 2015). FLBNs on the other hand include species such as Caenorhabditis elegans, Caenorhabditis briggsae, a variety of Oscheius spp. as well as nematodes from other genera. FLBNs are typically characterized by their lack of specific associations—or only transient associations—with animals (including insects), fungi or plants (Dillman et al., 2012a). However, some species of Oscheius have been found to have close associations with dead insects, either as saprophagous (Lam and Webster, 1971) or necromenic (Sudhaus, 2008) nematodes; or even as potential EPNs (Lam and Webster, 1971; Torrini et al., 2015; Zhang et al., 2008). In addition, some FLBNs such as Oscheius tipulae, O. onirici and Acrobeloids spp. have been found to naturally co-occur with EPNs in certain regions of the world and have been isolated in conjunction with EPNs from Galleria mellonella (waxworm) cadavers retrieved from baited traps in the soil (Campos-Herrera et al., 2019; Campos-Herrera et al., 2015; Jaffuel et al., 2018). Although some FLBNs may only be associated with insect cadavers, certain Oscheius spp. have exhibited virulence against G. mellonella. However, there are indications that the virulence may be much lower than that of a well-established EPNs such as Steinernema carpocapsae (Torrini et al., 2015). Differentiating between EPNs, scavengers, and kleptoparasites is important, as more species of nematodes are discovered and described. This is a relatively new area of research, with much remaining to be discovered. For this study, our goal was to re-evaluate O. chongmingensis and determine whether it is an EPN or a scavenger. We expected that if O. chongmingensis is not an EPN, its infectivity and host-odor preferences should differ from that of well-characterized EPNs such as S. carpocapsae. To evaluate the potential differences between O. chongmingensis and S. carpocapsae we examined infectivity, behavioral responses and competition interactions between these two species.

2.2. Virulence assays To assess virulence of O. chongmingensis dauers and S. carpocapsae IJs, four conditions were evaluated: infectivity in soil, infectivity using the standard filter-paper method, infectivity using a method without filter paper, and infectivity using agar/sand virulence assays. Virulence assays in soil were conducted using a modified protocol based on previously described assays (Dolinski et al., 2006). Briefly, 6-cm Petri dishes were filled with 20 g autoclaved soil (soil provided by the UCR Greenhouse) at 10% moisture by weight. 100 S. carpocapsae IJs or O. chongmingensis dauers were suspended in sterile tap water and transferred in a volume of 250 μL to each Petri dish. For control runs, 250 μL of sterile tap water was added to soil. Each plate received an additional 2 mL of sterile tap water, before a single G. mellonella (last-instar larvae) was placed into the dish, one G. mellonella per plate. Ten plates were prepared for each condition: O. chongmingensis, S. carpocapsae (positive control), and water as a negative control. Plates were incubated in the dark at room temperature (20 ± 3 °C), and larval mortality was monitored and recorded daily for 5–8 days. For the filter paper method, the procedure was done similarly to previously described work (Kaya and Stock, 1997). A 70 mm diameter filter paper was placed in the bottom of a 6-cm diameter plastic Petri

2. Materials and methods 2.1. Nematodes and associated bacterial strains O. chongmingensis DZ0503CMFT were propagated in the last-instar larvae of G. mellonella using a modified protocol where the waxworms were placed in medium Petri dishes (6 cm) and exposed to approximately 50 O. chongmingensis dauers per waxworm larvae in a total volume of 250 μL of tap water, with no filter paper substrate (Kaya and Stock, 1997; McMullen and Stock, 2014). Thus, the waxworms were in standing water. IJs were harvested from cadavers using White traps (White, 1927). We also reared O. chongmingensis by incubating IJs on plates with a bacterial lawn of Serratia nematodiphila 0503SBS1 for 2

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plate and five G. mellonella (last-instar larvae) were added to the dish followed by 500 dauers or IJS suspended in 250 µL of sterilized tap water. To negative control plates, 250 µL of sterilized tap water was applied. Six plates (5 waxworms each) were prepared for each condition: O. chongmingensis, S. carpocapsae (positive control), and water as a negative control. Plates were incubated in the dark at room temperature, 20 ± 3 °C, and larval mortality was monitored and recorded daily for 5–8 days. Additionally, we prepared an infection set up that had been used to culture the O. chongmingensis over the last few years in the Zhang lab. This set-up is similar to the filter paper set-up, but without the presence of filter paper. This “without filter paper” virulence assay, where there is standing water, was intended to demonstrate that the set-up provides an environment that reduces the health of the host and can allow for non-EPN species to colonize the insect host. A 6-cm Petri dish is prepared by adding five G. mellonella (last-instar larvae) per plate and 100 IJs or dauers in a volume of 250 µL (in sterile tap water); and water alone (250 µL) was applied as a control. Plates were incubated in the dark at room temperature, 20 ± 3 °C, and larval mortality was monitored and recorded daily for 5–8 days. For each condition six plates of five G. mellonella larvae each were prepared and evaluated. For agar/sand virulence assays 9-cm plates were filled with autoclaved 2% agar. Prior to the assay, plates were sprinkled with autoclaved silica sand. To each plate, 100 IJs or dauers suspended in sterile tap water were applied in a volume of 250 µL; water alone (250 µL) was applied as a control. Then, 10 G. mellonella (last instar larvae), were applied to each plate. Plates were incubated in the dark at room temperature and were monitored daily for larval death. Three plates for each condition were prepared and evaluated. For all four of the abovementioned virulence assays the entire set up was repeated in triplicate.

batch and day-to-day variability; a minimum of 18 assay plates were scored for all tests. 2.4. Reproductive output of O. chongmingensis For these assays the above-mentioned agar and sand method was employed to determine reproductive output of O. chongmingensis infecting live waxworms. In addition to testing live waxworms, we also tested freeze-killed waxworms and waxworms infected with S. carpocapsae IJs. The freeze-killed larvae were prepared by placing them at −20 °C 3–4 days, then thawed 24 h before exposing 10 of the waxworm cadavers to 100 O. chongmingensis dauers. The Sc-infected waxworms were prepared by exposing them to 100 IJs of S. carpocapsae (per waxworm) four days before exposing them to 100 O. chongmingensis dauers. Waxworms killed by S. carpocapsae infection were soft and did not change body color within 24-h, which made it easy to distinguish from waxworms that had died due to other causes. As mentioned above, 9-cm plastic Petri dishes were prepared by filling with 2% agar and then sprinkling 1 g of autoclaved silica sand onto the top of the solid agar from each plate. Then, 10 hosts (live, freeze-killed, or infected) were placed on the plates, followed by 100 O. chongmingensis dauers, suspended in 20 µL of sterilized tap water. These arenas were incubated at room temperature for 14–16 days, at which point IJs were emerging from the waxworm cadavers and gathering on the wall and lid of plates. These IJs were collected from walls and lids of plates by rinsing with tap water and counted to calculate the reproductive output of O. chongmingensis from these infections. Each experiment was repeated in triplicate. O. chongmingensis dauers were easily distinguished from S. carpocapsae IJs due to differences in color and morphology (see Supplementary Fig. S1)

2.3. Odor-preference and host-seeking behavior – Chemotaxis assays

2.5. Development and interspecific-competition between O. chongmingensis and S. carpocapsae

Host-seeking behavior assays were performed as previously described, on chemotaxis assay plates (Bargmann et al., 1993; Dillman et al., 2012b; Hallem et al., 2011). Four-day post-infection cadavers with both O. chongmingensis- and S. carpocapsae-infected G. mellonella were prepared as described above using the standing water method for O. chongmingensis infections and filter paper method for Sc. Freezekilled G. mellonella, larvae were placed in −20 °C refrigerator and frozen for 3 d, then left in room temperature to thaw for 1 d before being used for assays. Five larvae of uninfected, freeze-killed, or 4-day infected G. mellonella were placed in a 50-mL Hamilton gastight syringe and tested against control syringes which were filled with room air, or against another syringe containing uninfected, or freeze-killed, or infected waxworms for competitive assays. Syringes were depressed using a KD Scientific pump (Model KDS 220 Catalog number: 780220NLSU) at a rate of 0.5 mL/min for 5 min to flush clean air from the Nalgene PVC (1/8″ diameter) tubing that connected the syringes to the lid of the testing arena. Experiments were run at the same rate for the duration of the assay, approximately 60 min. Gases from the syringes were delivered to opposite sides of the assay plate through holes drilled into the plate lids directly above the centers of the scoring regions (Baiocchi et al., 2017). For each trial, a pellet containing approximately 100–500 clean IJs or dauers were transferred in a volume of approximately 6 µL onto the center of the assay plate. Assay plates were placed undisturbed on a vibration reducing platform and were scored after 1 h. Methods for counting the nematodes were done as previously described (Baiocchi et al., 2017; Kin et al., 2019). A chemotaxis index (CI) was calculated as CI = (number of worms in host circle – number of worms in control circle)/(number of worms in both circles). Experiments consisted of nine technical replicates (nematodes from the same population) and were only counted if more than seven worms in total moved into the scoring circles. For all chemotaxis assays, at least two replicates (each with nine plates run in parallel) of experiments were performed using separate batches of worms on different days to account for batch-to-

S. nematodiphila 0503SBS1 and either primary or secondary phase X. nematophila (HGB800) were cultivated in LB liquid culture overnight, then spread onto lipid agar (LA) 6-cm plates. These were cultured for 24 h to produce healthy bacterial lawns for subsequent testing. For growth experiments, 500 IJs of either O. chongmingensis or S. carpocapsae were added into bacterial lawns alone, and in competition assays 500 IJs of each were added to the lawns to determine the effects of competition. The number of adult nematodes was counted and recorded every 24 h starting at 2 days (48 h) after inoculation of plates with the IJs. Adults of O. chongmingensis are slender and continually moving, whereas those of S. carpocapsae are robust and rhabdoid (see supplementary Fig. S2), usually curl up and remain motionless, and are easily differentiated from each other using a microscope. Each experiment was repeated in triplicate. For competition assays between S. carpocapsae and O. chongmingensis, two conditions were set: Same starting time and O. chongmingensis delayed start. In the same-starting-time condition, 500 S. carpocapsae IJs and 500 O. chongmingensis dauers were placed on the plate together at the same time and the growth (and presence of adults) was monitored as explained above. For O. chongmingensis delayed start condition, the prepared bacteria plates were first seeded with 500 S. carpocapsae IJs, which were given 48 h to develop before 500 O. chongmingensis dauers were applied. After O. chongmingensis dauers were applied the plates were maintained another 48 h for nematodes to continue development before being monitored for growth and the appearance of adults. These experiments were repeated in triplicate. 2.6. Influence of O. chongmingensis on the reproduction and emergence of S. carpocapsae IJs in vivo To evaluate the effects of O. chongmingensis presence on the infection capabilities and reproduction of S. carpocapsae, infections of 3

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waxworms were performed using the standard filter paper method, described above (McMullen and Stock, 2014). Infections of waxworms were set up using the filter paper method with five G. mellonella per plate as described above (McMullen and Stock, 2014). Three doses were tested using a 1:1 ratio of inoculum containing 20 IJs/dauers of each species (S. carpocapsae IJs and O. chongmingensis dauers), 50 IJs/dauers of each species, or 100 IJs/dauers of each species. The nematodes were applied to plates of five waxworms, one dose per plate, six plates per dose. As a control, S. carpocapsae alone was applied in doses of 20 IJs, 50 IJs or 100IJs to plates of five waxworms each, six plates for each dose. Plates were incubated for 7 d at room temperature. On the 7th day, three of the five cadavers were evaluated for the presence of S. carpocapsae within the host and the other two cadavers were each placed on individual standard white traps (White, 1927) to evaluate IJ emergence as a measure of reproduction. White traps were incubated at room temperature for 5 d before being collected and counted. The entire experiment was performed in triplicate.

orthogonally by another bacterial species. The antagonism interaction is analyzed by the observation and measurement of the size of the inhibition zone, or the distance between the first bacteria and the second. Each of these experiments was replicated 3 times at different times. 2.10. Statistical analysis All data collected were analyzed and plotted with the Graphpad Prism 5 software package. Chemotaxis indices data from chemotaxis assays was analyzed using ordinary one-way ANOVA and Tukey’s multiple comparisons post-test. Participation data from chemotaxis assays was analyzed using ordinary two-way ANOVA with Tukey’s multiple comparisons test. Data regarding O. chongmingensis fecundity data were analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons test. Data regarding interspecific-competition effects on colonizing a G. mellonella host/cadaver, and IJ/dauer yield were performed using two-way ordinary ANOVA with Bonferroni multiple comparisons post-test. Data regarding antagonistic effects of symbiotic bacterial species were analyzed using ordinary one-way ANOVA with Tukey’s multiple comparisons post-test. For all graphs the mean is show with error bars representing standard error of the mean (SEM).

2.7. Influence of S. carpocapsae presence on the reproduction and emergence of O. chongmingensis dauers To evaluate the effect of S. carpocapsae presence on the reproduction of O. chongmingensis and its ability to establish itself within dead hosts, infections of waxworms were performed using the standard filter paper method described above (McMullen and Stock, 2014). To a plate of five freeze-killed G. mellonella, an equal number of O. chongmingensis dauers and S. carpocapsae IJs were applied. We tested two doses at a 1:1 ratio of each species: 20 nematodes (IJs and dauers as above) of each species and 100 nematodes of each species. As a control, 20 or 100 dauers were applied to a plate of five freeze-killed G. mellonella. All cadavers were incubated at room temperature for 7 d, after which each cadaver was placed onto a standard White trap. The emerging O. chongmingensis dauers and S. carpocapsae IJs were collected 4 d later and counted to evaluated reproductive output. For each condition and dose, six plates of the five G. mellonella were prepared and the entire set of experiment was performed in triplicate.

3. Results 3.1. O. chongmingensis has low virulence against waxworms To begin our investigation of O. chongmingensis as a potential EPN, we first performed virulence assays under a variety of different conditions (Fig. 1). We found that O. chongmingensis dauers had low virulence against waxworm larvae under standard infection conditions (Fig. 1A and B). In soil, O. chongmingensis dauers were no more virulent than water, and only slightly more virulent than water in the standard filter paper virulence assay (Kaya and Stock, 1997; McMullen and Stock, 2014; Woodring and Kaya, 1988), killing approximately 25% of waxworms within 5 d (Fig. 1A and B). Using assay conditions where the waxworms were in standing water, without filter paper, or in conditions using agar and sand we found that O. chongmingensis was much more virulent (Fig. 1C and D), but so were other non-EPN species such as C. elegans and P. pacificus. Under standard virulence assay conditions, where neither water nor free-living and necromenic nematodes were significantly pathogenic, we found that O. chongmingensis dauers had severely attenuated virulence. In contrast, S. carpocapsae IJs were highly virulent under all these conditions, killing 100% of exposed waxworm larvae within 48 h post-infection (Fig. 1).

2.8. Effects of phase variation of X. nematophila on O. chongmingensis nematodes Primary phase and secondary phase X. nematophila were cultivated in LB liquid culture overnight, and then streaked on 9-cm NBTA plates and incubated for 24 h, then 500 O. chongmingensis dauers were placed onto each inoculated NBTA plate. Bacterial colony color was checked and photographs were taken daily for total 8 d. IJs of O. chongmingensis were collected into 15-mL centrifuge tubes from NBTA plates after 12 d, rinsed 3 times with 15 mL autoclaved tap water, and centrifuged to remove suspended debris. Fifty rinsed IJs were then transferred into a sterilized 15-mL centrifuge tube and treated with 10 mL Hyamin for 30 min (Kaya and Stock, 1997) to remove fungal and bacterial contamination, then the IJS were rinsed 3 times with 15 mL autoclaved tap water and centrifuged to remove the Hyamin. The IJs were then resuspended in 50 µL of distilled water, before being transferred to a 1.5 mL sterilized centrifuge tube where they were homogenized with a plastic motorized pestle. The homogenized IJs were plated on NBTA plates and incubated for 2 d at 28 °C. The plates were then examined to determine the phase of the bacteria based on the color of the colonies. The experiment was repeated 3 times.

3.2. O. chongmingensis prefers infected hosts over dead hosts To gain insight into differences in behavioral preferences between O. chongmingensis and S. carpocapsae IJs, we performed two-choice chemotaxis assays. We found that although O. chongmingensis dauers are strongly attracted to uninfected waxworms compared to air, and these were their least preferred odor sources. In competitive assays, where they chose between two different odor sources, O. chongmingensis exhibited preference for either freeze-killed or S. carpocapsae-infected cadavers; however, when choosing between freeze-killed or alreadyinfected host, the preference appeared to be for the S. carpocapsae-infected host (Fig. 2A). Overall, O. chongmingensis dauers chemotax well, with > 50% of the population participating in chemotaxis behavior (Fig. 2B). In contrast to O. chongmingensis, participating S. carpocapsae IJs were repelled by both freeze-killed waxworms and waxworms infected with heterospecifics (Fig. 2C). Also, S. carpocapsae IJs participated poorly in chemotaxis assays, with < 40% of the population exhibiting chemotaxis behaviors, as has been previously observed (Baiocchi et al., 2017) (Fig. 2E and F). In competitive assays, where the IJs chose between two different odor sources, participating S.

2.9. Antagonism between S. nematodiphila and X. nematophila The cross-streak method was used to screen for antagonism between S. nematodiphila and X. nematophila as previously described (Lertcanawanichakul and Sawangnop, 2008; Madigan et al., 1997). Briefly, one bacterial strain is seeded by a single streak in the center of an LB plate. After incubating 2 d at 28 °C, the plate is streaked 4

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Fig. 1. Virulence of Oscheius chongmingensis compared to known EPNs and FLBNs (A) Assay on a plate filled with moistened sand to compare virulence of Oscheius chongmingensis dauers with EPN Steinernema carpocapsae IJs and tap water control treatments (B) Virulence assay on an infection plate using moistened filter paper as a pseudo-soil substrate (Kaya and Stock, 1997), to compare virulence of O. chongmingensis dauers, with EPN S. carpocapsae IJs (tap water control treatment). (C) Virulence assay on an infection plate set-up, but without filter paper; standing water in the dish weakens the insect hosts. Assay compared virulence of O. chongmingensis dauers with EPN S. carpocapsae IJs, as well as FLBNs Caenorhabditis elegans and Pristionchus pacificus (tap water control treatment). (D) Virulence assay on agar plate with sand layer on top of agar to facilitate nictation and jumping behavior. This assay conducted with O. chongmingensis, EPN S. carpocapsae and FLBNs C. elegans and P. pacificus.

carpocapsae IJs exhibited a preference opposite to that of O. chongmingensis dauers (Fig. 2A and E). In order, they preferred uninfected waxworms first, and then freeze-killed hosts (Fig. 2E). In agreement with previous observations, we did not observe attraction of S. carpocapsae IJs to waxworms infected with heterospecific nematodes. It has been previously noted that S. carpocapsae displays repulsion or lack of attraction in response to hosts infected with heterospecific species (Grewal et al., 1997), and even conspecific infections (Baiocchi et al., 2017; Ramos-Rodriguez et al., 2007). Furthermore, the presence of heterospecific species has been shown to reduce the ability of S. carpocapsae to act as a secondary invader to an already-colonized host (Glazer, 1997).

freeze-killed cadavers or either of these over uninfected G. mellonella hosts. We found that fitness, as measured by reproductive output (Fig. 3), was inversely related to behavioral preference (Fig. 2A). The behavioral assays (Fig. 2A) showed that, compared to uninfected host volatiles, there is a preference for either freeze-killed or S. carpocapsaeinfected cadaver odors, despite the fact that uninfected hosts yield the highest level of reproductive success (Fig. 3). Additionally, given the choice between freeze-killed and S. carpocapsae-infected hosts, there is a preference for the infected hosts (Fig. 2A) even though this type of host yields the lowest reproductive success (Fig. 3). We noted that, under standard assay conditions, the success of O. chongmingensis dauers infecting uninfected waxworms is quite low (Fig. 1A and B).

3.3. The fitness of O. chongmingensis is highest in uninfected hosts

3.4. O. chongmingensis outcompetes S. carpocapsae in vitro

We evaluated whether O. chongmingensis fitness tracked with behavioral host preference for S. carpocapsae-infected cadavers over

Because it appeared that there was low reproduction of O. chongmingensis in S. carpocapsae-infected waxworms, we tested whether there 5

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Fig. 2. Odor-competition assays and behavioral responses of Oscheius chongmingensis (Oc) and Steinernema carpocapsae (Sc). Odor competition included blank syringes (air), uninfected waxworms (UN), freeze-killed waxworms (FK), and Sc-infected waxworms (IF). Panels A-B: Competition assays assessing the preferences of O. chongmingensis dauers for various host types. (A) Chemotaxis indices (CI) of O. chongmingensis in response to odor-competition assays. Bars indicate level of attraction to the contents of the syringes (contents are listed either at +1 or −1 for each competition under the grey dotted line). (B) Data from the O. chongmingensis odor-competition chemotaxis assays (Panel A), indicating the proportion of the population that showed chemotaxis behavior (moved more than 1 cm directionally toward origin of odors). Panels C-D: Evaluation of host-seeking behavior exhibited by S. carpocapsae IJs (C) Chemotaxis indices indicating attraction or repulsion from each of the potential host types compared to air as a control. (D) Participation results from S. carpocapsae chemotaxis assays shown in panel C. Panels E-F: Competition assays assessing the preferences of S. carpocapsae IJs for various host types. (E) Chemotaxis indices (CI) of S. carpocapsae in response to odor-competition assays. Bars indicate level of attraction to the contents of the syringes within the competition (contents are listed either at + 1 or −1 for each competition under the grey dotted line). (F) Participation data resulting from the O. chongmingensis odor-competition chemotaxis assays (shown in panel E). All error bars represent SEM. Plots with same letter above error bar indicate no significant difference among them, while differing letters indicate a statistically significant difference. For participation statistical tests: *p < 0.05, ***p < 0.001.

on a lawn of secondary phase X. nematophila and a lawn of S. nematodiphila, whereas they grow slower on lawns of primary phase X. nematophila (Fig. 4A). However, S. carpocapsae IJs grew faster on lawns of primary phase X. nematophila, slower on secondary phase X. nematophila, and they were unable to grow on a lawn of S. nematodiphila

was antagonism between the two nematodes or between their respective symbionts and the nematodes. To do this, we assessed the growth of IJs of both O. chongmingensis and S. carpocapsae on primary and secondary phase X. nematophila, as well as on S. nematodiphila (Fig. 4). We found that O. chongmingensis dauers grew equally quickly 6

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(Fig. 4B). We found evidence of interspecific competition between O. chongmingensis and S. carpocapsae IJs when nematodes were concurrently placed on primary phase X. nematophila; S. carpocapsae IJs were unable to develop to adulthood (Fig. 4C). This effect was abrogated when S. carpocapsae IJs were placed on plates with primary phase X. nematophila 2 d before adding O. chongmingensis dauers (Fig. 4D). Having determined that O. chongmingensis has lower reproductive fitness in S. carpocapsae-infected waxworms when S. carpocapsae is inoculated first (Fig. 3), and having also found that O. chongmingensis dauers had an antagonistic effect on S. carpocapsae IJ growth and development in vitro (Fig. 4C), we next wanted to determine if these effects would persist in an in vivo infection where IJs of the two nematodes were added at the same time. To do this, first we tested whether the presence of O. chongmingensis dauers would affect either the colonization of waxworm larvae by S. carpocapsae, or the emergence of S. carpocapsae from these larvae. We found no significant differences in either colonization or emergence of S. carpocapsae from waxworm larvae in the presence of O. chongmingensis dauers (Fig. 5A and B). We performed the reciprocal experiment, to determine whether the presence of S. carpocapsae IJs would affect either the colonization or

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Fig. 4. Effects of in vitro bacterial and nematode competition on growth and reproduction of Oscheius chongmingensis (Oc) and Steinernema carpocapsae (Sc). Panels AB. S. carpocapsae IJs and O. chongmingensis dauers were grown on lawns of bacteria: Either primary or secondary phase Xenorhabdus nematophila (Sc symbiont) or on Serratia nematodiphila (Oc symbiont) (A) Growth and reproduction of O. chongmingensis on various bacterial substrates (B) Growth and reproduction of S. carpocapsae on various bacterial substrates. Panels C-D: Using X. nematophila (Sc symbiont), both O. chongmingensis and S. carpocapsae were grown to evaluate competition. (C) Competition with O. chongmingensis and S. carpocapsae plated at the same time on primary phase of X. nematophila (D) Competition between O. chongmingensis and S. carpocapsae on bacterial lawn of primary phase X. nematophila; S. carpocapsae plated 2 days before O. chongmingensis dauers were introduced. 7

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Fig. 5. In vivo effects of interspecific competition between Oscheius chongmingensis (Oc) and Steinernema carpocapsae (Sc). Panels A-B: Evaluation of S. carpocapsae fecundity (IJ production) in live healthy waxworms; treatments of S. carpocapsae alone compared to interspecific species competition. (A) Competition effects on the ability of S. carpocapsae to kill G. mellonella hosts. (B) Competition effects on ability of S. carpocapsae to reproduce after colonizing hosts. Panels C-D: Evaluation of O. chongmingensis fecundity using freeze-killed hosts; treatments of O. chongmingensis alone compared to interspecific species competition. (C) Competition effects on ability of O. chongmingensis to grow on freeze-killed hosts. (D) Competition effects on ability of O. chongmingensis to reproduce in colonized hosts. All error bars represent SEM. No statistical differences were observed between single species or competition treatments in any case, using Two-Way ANOVA.

4. Discussion

emergence of O. chongmingensis from freeze-killed waxworm larvae. We found no significant differences in either colonization or emergence of O. chongmingensis from freeze-killed waxworm larvae in the presence of S. carpocapsae IJs (Fig. 5C and D).

O. chongminensis, formerly known as Heterorhabditidoides chongmingensis, has been previously characterized as an EPN with high pathogenicity towards G. mellonella larvae and Tenebrio molitor hosts (Liu et al., 2009; Zhang et al., 2008). However, there is a conflicting report of low virulence (35%) in waxworms, in which mortality occurred in 5–7 d in contrast to the 1–2 d that is typical of most EPNs infecting waxworms (Liu et al., 2012). We used a variety of assays to evaluate the ability of O. chongmingensis to cause pathogenicity in waxworm hosts, which are a commonly used EPN host and are used for the sampling and propagation of EPNs (Bedding and Akhurst, 1975; Orozco et al., 2014). We found that O. chongmingensis was not very efficient at killing waxworms as reported by Liu et al. (2012). Using standard conditions, with waxworms on a slightly moistened plate of filter paper (Kaya and Stock, 1997), O. chongmingensis killed 20–30% of the waxworms, whereas other EPN species, such as S. carpocapsae infect and kill 100% of exposed waxworm hosts within 2 d (Fig. 1A–D). Efficiency of O. chongmingensis infections improves dramatically if the infection conditions are altered such that the hosts are weakened (Fig. 1C), but these conditions favor host death even in the presence of free-living nematodes and control experiments. These findings are consistent with previous

3.5. Antagonism between S. nematodiphila and X. nematophila After identifying that O. chongmingensis dauers can grow well on X. nematophila, the bacterial symbiont of S. carpocapsae, we sought to further investigate the competitive relationship between X. nematophila and S. nematodiphila (the symbiont carried by O. chongmingensis). Specifically, we evaluated antagonism between the two species of bacteria and found that primary phase X. nematophila had significant antagonistic activity toward S. nematodiphila (Fig. 6A). This was not true of secondary phase X. nematophila, which had little to no effect against S. nematodiphila growth (Fig. 6B). We also found that S. nematodiphila had significant antagonistic effects on the growth of both primary and secondary phase X. nematophila (Fig. 6C and D). In measuring the zone of inhibition created in the various pairings, we found that primary phase X. nematophila had the strongest antagonistic effect and created a significantly larger zone of inhibition than that created by S. nematodiphila. 8

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Fig. 6. Antagonism between X. nematophila (Xn) and Serratia nematodiphila (Sn). Panels A–D: Representative photos of antagonism effects between bacterial symbiotes of S. carpocapsae (Xn) and O. chongmingensis (Sn). (A) Antagonism of primary phase Xn to Sn. (B) Antagonism of secondary phase Xn to Sn. (C) Antagonism of Sn against primary phase Xn. (D) Antagonism of Xn against secondary phase Xn. (E) Quantification of the zone of inhibition lengths for graphical representation of antagonism effects between the species of bacteria.

heterospecific infections (Grewal et al., 1997) and that even resourcedepleted conspecific infections produce odors that are repulsive to IJs (Baiocchi et al., 2017). These previous findings support our results that for the participating IJs of S. carpocapsae, as demonstrated by the standard chemotaxis index (CI values) there is a preference for uninfected host odors over air (Fig. 2C and E), and IJs are repelled by both freeze-killed and O. chongmingensis-infected cadavers. Taken together, O. chongmingensis dauers have very different odor preferences than would be expected from an EPN like S. carpocapsae, exhibiting a preference for odors that are produced by dead hosts over living ones. Given the disparity between S. carpocapsae and O. chongmingensis host preferences, it was conceivable that there might be a reproductive advantage to infecting a host that was previously infected or had died. However, our findings did not support this expectation. O. chongmingensis reproduction was highest in uninfected cadavers, and lowest in S. carpocapsae-infected cadavers. The reasons for this negative correlation between fecundity and virulence are not yet known. An uninfected host would provide more and possibly higher quality resources to the invading nematodes, thus allowing for a higher number of progeny than a dead or already infected host. However, access to the nutrients and resources contained within a potential host is contingent on the nematode’s ability to infect the host, overcome or evade the immune system, and kill the host. EPNs host-killing utilizes entomopathogenic bacteria (Chaston and Goodrich-Blair, 2010; Dillman et al., 2012a) and, in some species, nematode-derived toxins or immune modulators (Balasubramanian et al., 2010; Hao et al., 2010; Lu et al., 2017). It may be that, because O. chongmingensis is not very effective at infecting and killing naïve hosts, a dead or infected host is preferable to a naïve host, and the likelihood of reproductive success in much higher in these environments. Thus, the ability to detect, seek out and survive within an already-dead or infected host, as a scavenger, may provide O. chongmingensis with opportunities for reproduction where there would otherwise be none.

reports that Oscheius spp. is generally not adept at infecting and killing insect hosts (Campos-Herrera et al., 2015; Liu et al., 2012). It is possible that when O. chongmingensis was isolated it was more efficient at killing and colonizing uninfected hosts and has subsequently lost virulence due to lab culturing (Bilgrami et al., 2006; Chaston et al., 2011), or that certain microbes necessary for virulence were lost after extensive lab culturing. A similar loss of virulence has occurred in other species of Oscheius; O. onrici was originally characterized as an EPN (Torrini et al., 2015) but subsequent poor pathogenicity observed in other populations of this species suggests that this species should be recharacterized as a scavenger rather than an EPN (Blanco-Pérez et al., 2019; CamposHerrera et al., 2015). The evolution of insect-parasitism by nematodes is thought to have resulted from a sequence of host-nematode associations (Dillman et al., 2012a; Poinar, 1983; Sudhaus, 2008), therefore, it is possible that O. chongmingensis is a species that is in a transitional stage of evolution between necromeny and entomopathogeny. It also is possible that inbreeding or a reduction of selection pressure for maintaining virulence could result in decreased pathogenicity over time. However, our results suggest that O. chongmingensis is a facultatively-parasitic scavenger rather than an EPN (Fig. 1). O. chongmingensis behaves like a scavenger; the trends in attraction, repulsion, and participation are all consistent with that of a facultatively-parasitic scavenger. O. chongmingensis dauers prefer odors associated with S. carpocapsae-infected cadavers over freeze-killed cadavers or uninfected hosts (Fig. 2A and B). O. chongmingensis is proficient at chemotaxing (we observed a relatively high percentage of participation in chemotaxis behavior), and host-seeking behavior complements the chemotaxis data in a way that reveals additional odor preference (Fig. 2A and B). For example, we observed a higher percentage of chemotaxis when freeze-killed and S. carpocapsae-infected cadavers were used as an odor source than when naïve hosts were used (Fig. 2A and B). Previous research reports that EPNs are often repelled by 9

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studied in more detail. The interactions between different bacterial symbionts may influence the competition between different nematodes. During infections by EPNs, the bacterial symbiont is thought to facilitate pathogenicity in the host, prevent putrification of the cadaver, and provide a suitable environment for the EPNs to grow and reproduce (Gaugler and Kaya, 1990). Many bacterial species, including Xenorhabdus spp., secrete antimicrobial compounds that help prevent opportunistic soil microbes and the innate bacterial communities from the digestive tract of the host insect from utilizing the resources of the insect cadaver. We found that primary phase X. nematophila was highly antagonistic towards S. nematodiphila, while secondary phase X. nematophila was not able to effectively to impede S. nematodiphila growth (Fig. 6). These results support previous reports that primary phase X. nematophila produces antimicrobial compounds (Akhurst, 1982) and sequesters the resources for use by the EPN. We also found that S. nematodiphila was capable of impeding X. nematophila growth and, moreover, it was able to impede the growth of both primary and secondary phases of X. nematophila with similar effectiveness (Fig. 6C–E). In addition to the antagonism between symbiotic bacteria, our results indicated that the presence of O. chongmingensis might be able to influence the phase of X. nematophila bacterial lawns in vitro (Supplementary Fig. S3). As previously noted, under more natural conditions, the phase I bacteria dominates within the insect cadaver, whereas phase II of X. nematophila has only been observed under laboratory conditions in vitro, in conjunction with subculturing of the bacteria, in this study as well as in previous work (Campos-Herrera et al., 2009). Although phase I may typically dominate, our study has shown evidence that when phase I X. nematophila is exposed to O. chongmingensis, there is a shift from primary to secondary phase. We have found that nematodes can influence the phase of bacterial symbiont of other nematodes, but the underlying mechanism remains to be studied. This occurrence, however, may be important for O. chongmingensis survival, allowing the nematode to garner a competitive advantage– creating a more suitable food source for itself while potentially limiting ability of its competitor to develop. It is important to note, however, that these results are different from the in vivo results from this study and previous findings (Blanco-Pérez et al., 2017, 2019), where competition between EPNs and FLBNs (in this case, S. carpocapsae and O. chongmingensis, respectively) did not appear to be detrimental to the EPN colonization of, or proliferation within, the host. This may be due to community dynamics, and other microbes that may affect competition, not evaluated in this study, that remain to be elucidated. Regardless, the ability of O. chongmingensis to modulate the phase of X. nematophila may be key to its ability to compete with EPNs or other nematodes and further suggests that O. chongmingensis is a facultatively-parasitic scavenger.

Similar to many EPNs, which each associate with one or more bacterial species (Akhurst, 1983; Lewis and Clarke, 2012), O. chongmingensis associates with S. nematodiphila as its bacterial symbiont. We found that O. chongmingensis development on S. nematodiphila is quite fast, with IJs developing into adults within approximately 2 days, and adult population growth expanding in 4–5 days (after initial inoculation with IJs). This indicates that the development time between egg and adult is also quite rapid. Although development time frames for O. chongmingensis have not been previously described in detail, another Oscheius species, O. andrassyi, develops from egg to adult in approximately 3–4 days, similar to our findings with O. chongmingensis. Population growth on bacterial lawns is much slower for S. carpocapsae. On primary phase X. nematophila, IJs develop into adults within approximately 2–3 days, but expansion of the population occurs between 6 and 9 days. On secondary phase X. nematophila, S. carpocapsae can develop, but the development appears to be delayed by about 24 h. Under normal circumstances however, S. carpocapsae would not encounter or utilize secondary phase X. nematophila as a food source, since it is the primary phase that dominates and is the phase typically isolated from S. carpocapsae infected cadavers (Campos-Herrera et al., 2009). S. carpocapsae does not appear to be able to grow or develop on S. nematodiphila. This supports previous findings that Steinernema spp. growth on non-symbiotic bacteria can greatly delay development timing (Ehlers et al., 1990). Our results suggest that O. chongmingensis development overall outpaces S. carpocapsae development, even on primary phase X. nematophila. Furthermore, the ability of O. chongmingensis to survive on multiple bacterial species and bacteria in different phases may allow it to exploit a wider variety of environments, a trait that would be advantageous to a scavenger. Campos-Herrera et al. (2015) recently demonstrated that Steinernema spp. and Oscheius spp. overlap in range and may compete for resources, and that in such situations Oscheius spp. (O. onirici and O. tipulae) outcompeted the Steinernema species. However, there are indications that co-infection with Steinernema spp. and Oscheius spp. does not always result in an apparent negative impact on Steinernema spp. fecundity (Blanco-Pérez et al., 2017; Blanco-Pérez et al., 2019). In addition to our finding that the development of O. chongmingensis outpaces S. carpocapsae population growth in vitro, even on the bacterial symbiont of S. carpocapsae, we found that the presence of O. chongmingensis had antagonistic effects on S. carpocapsae development in vitro (Fig. 4C and D). We found that S. carpocapsae did not develop in the presence of O. chongmingensis if the two species were plated at the same time. However, when S. carpocapsae was given 48 h to develop before O. chongmingensis was added, S. carpocapsae was able to compete and grow normally (Fig. 4D). Although S. carpocapsae can successfully develop in the presence of O. chongmingensis if given an advanced start, the O. chongmingensis population is capable of catching up to and matching S. carpocapsae’s development. We also note that although O. chongmingensis appears to develop well on X. nematophila under in vitro conditions, our in vivo infections did not corroborate this finding (Fig. 5A–D). We found that in vivo there were no discernible negative impacts on S. carpocapsae development or fecundity (Fig. 5A and B), nor did the presence of S. carpocapsae yield a statistically-significant negative impact on O. chongmingensis colonization of or emergence from freeze-killed cadavers (Fig. 5C and D). These results, despite a difference in species used, are similar to results of previous studies (BlancoPérez et al., 2017; Blanco-Pérez et al., 2019), where competition between FLBNs and EPNs was not observed to not have significant deleterious effects on EPN reproduction. The disparate results observed between the in vitro (Fig. 4C and D) and in vivo (Fig. 5A–D) experiments may be due to a variety of factors. The environments provided by a host and by a Petri dish are very different. Surface area, humidity, oxygen exposure, microbial communities, and host behavior and immunity, are all among the potential confounding factors that could impact the fitness of nematodes. The dynamics of how interspecies interactions or competition for resources works within the insect host remains to be

5. Conclusions Nematodes appear to have evolved to occupy every ecological niche imaginable. Entomopathogenic nematodes are members of a specialized guild of insect-parasitic nematodes and the number of described species in this guild is increasing. Some of the recently described EPNs are new species of heterorhabditids and steinernematids, but rhabditid nematodes also are being characterized as EPNs. In order for the epithet “entomopathogenic” to continue to be meaningful, it should only be applied to nematode species that possess the hallmark characteristics of nematode pathogens, namely causing rapid death (less than 5 d postinfection) and using a mutualistic association with bacteria to help facilitate their pathogenesis (Dillman et al., 2012a). Here, we evaluated the nematode Oscheius chongmingensis, examining the behavior and virulence of the nematode as well as certain features of its bacterial association. We found insufficient evidence to support its characterization as an EPN and instead have presented compelling data that suggest this nematode is a scavenger, competing with EPNs and other organisms for resource-rich insect cadavers in the soil. Additional 10

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studies of virulence from recently isolated strains may provide further insight, and more in-depth symbiont heritability studies would be helpful. We agree with the previous assessment that nematode-bacterium partnerships that do not explicitly demonstrate the hallmark characteristics of EPNs are interesting and may represent developing or nascent partnerships. The study their biology may increase our understanding of the tremendous specialization exhibited by EPNs (Dillman et al., 2012a).

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