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Entomopathogenic nematodes increase predation success by inducing cadaver volatiles that attract healthy herbivores

  1. Xi Zhang
  2. Ricardo AR Machado
  3. Cong Van Doan
  4. Carla CM Arce
  5. Lingfei Hu
  6. Christelle AM Robert  Is a corresponding author
  1. University of Bern, Switzerland
  2. University of Neuchatel, Switzerland
Research Article
Cite this article as: eLife 2019;8:e46668 doi: 10.7554/eLife.46668
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Root herbivore recruitment dynamics of plant-herbivore-natural enemy complexes reveal that herbivore cadavers infected by entomopathogenic nematodes attract healthy herbivores.

(A) Proportions (mean ± SEM) of western corn rootworm (WCR) choosing between healthy maize plants and maize plants infected with conspecifics and entomopathogenic nematodes (EPNs) in belowground olfactometers. WCR choice was measured 0 hr, 24 hr, 48 hr and 96 hr after infection (n = 45). (B) Root removal (mean ± SEM) by WCR larvae 0 hr, 24 hr, 48 hr and 96 hr after infection (n = 5–8). (C) (E)-β-caryophyllene (EBC) production (mean ± SEM) of maize roots 0 hr, 24 hr, 48 hr and 96 hr after infection (n = 3–5). (D) WCR infection by EPNs (mean ± SEM) 0 hr, 24 hr, 48 hr and 96 hr after infection (n = 8). (E) Proportions (mean ± SEM) of WCR larvae choosing healthy plants or plant+WCR+EPN complexes (n = 20), healthy plants or WCR-infested plants (n = 20), healthy plants or plant+WCR+EPN complexes (n = 20), caged uninfected or EPN-infected WCR cadaver (n = 33). Larval preference was assessed in belowground olfactometers 96 hr post infection. (F) Proportion of WCR larvae orienting towards uninfected and EPN-infected WCR cadavers in the presence of maize root pieces in petri dish assays (n = 10–15) (G-H) Number and duration of visits (mean ± SEM) of WCR larvae exposed to uninfected and EPN-infected WCR cadavers in the presence of maize root pieces (n = 6). Stars indicate significant differences based on analysis of variance (*: p<0.05, **: p<0.01, ***: p<0.001). Raw data are available in Figure 1—source data 1.

https://doi.org/10.7554/eLife.46668.002
Figure 1—figure supplement 1
Attraction of the western corn rootworm to nematode-infected cadavers requires plant background odors.

(A) Proportions (mean ± SEM) of WCR choosing between uninfected cadavers and EPN-infected cadavers in petri dish assays without maize roots (n = 20). (B) Proportions (mean ± SEM) of WCR choosing between maize roots previously exposed to uninfected or EPN-infected cadavers in petri dish assays (n = 20). Attraction of the western corn rootworm to nematode-infected cadavers requires plant background odors.

https://doi.org/10.7554/eLife.46668.003
Figure 2 with 1 supplement
The presence of EPN-infected cadavers increases herbivore recruitment, nematode predation success and offspring production.

(A) Visual representation of experimental setup. Entomopathogenic nematodes (EPNs) were applied on both sides of the arenas, in presence of a healthy or infected WCR cadavers. Eight western corn rootworm (WCR) larvae were then released in the middle and recollected after five days (n = 9). (B) Proportions (Mean ± SEM) of WCR larvae recovered from each side (n = 9). (C) WCR infection rates (Mean ± SEM) on each side (n = 9). (D) Number (Mean ± SEM) of emerging EPN juveniles per infected WCR larva (nControl = 4, ninfected = 9). (E) Total number of EPNs (Mean ± SEM) emerging from the WCR larvae on each side (n = 9). Stars indicate significant differences based on analysis of variance (**: p<0.01, ***: p<0.001). Ns: non-significant. Raw data are available in Figure 2—source data 1.

https://doi.org/10.7554/eLife.46668.005
Figure 2—figure supplement 1
Picture of the adapted white-traps used in the study.

The white trap consisted of a 1.5 mL Eppendorf lid (Sarstedt AG and Co., Germany) placed upside down in a solo cup. The lid was covered with 2.5 cm diameter filter paper. Tap water was then added on the filter paper and around the lid. One insect larva was placed onto the filter paper and emerging EPNs could reach the water.

https://doi.org/10.7554/eLife.46668.006
Figure 3 with 2 supplements
Western corn rootworm larvae that are infected by entomopathogenic nematodes release distinct volatile bouquets.

(A) Venn diagram showing the numbers of overlapping and non-overlapping GC-MS headspace features of uninfected (white) and EPN-infected (brown) western corn rootworm (WCR) cadavers (n = 5). (B) Principal component analysis (PCA) of volatile emissions of uninfected (white) and EPN-infected (brown) WCR cadavers (n = 5). (C) Representative GC-MS volatile profile of uninfected (white) and EPN-infected WCR larvae (brown). 1, 3, 5, 6, 8: unknown; 2: aceptophenone; 4: 1-hexanol; 7: benzene; 9: 5-cyclohexadien-1-one; 10: butylated hydroxytoluene (BHT); 11: 2,6-bis (1,1-dimethylethyl)−4-(1-oxopropyl) phenol; Cont.: contamination. cps: count per second. (D) Volatile peak areas (mean ± SEM) of uninfected (white) and EPN-infected WCR larvae (brown) (n = 5). cps: count per second. L: identification based on libraries. S: identification based on pure standards. Stars indicate significant differences based on analyis of variance (*: p<0.05, **: p<0.01, ***: p<0.001). Raw data are available in Figure 3—source data 1.

https://doi.org/10.7554/eLife.46668.008
Figure 3—figure supplement 1
Infection by entomopathogenic nematodes does not alter CO2 emissions from western corn rootworm cadavers.

CO2 content (mean peak area ± SEM) in an empty vial (Blank, n = 8), in a vial containing five uninfected western corn rootworm (WCR) cadavers (n = 12) or five WCR cadavers infected by entomopathogenic nematodes (EPNs; n = 12). CO2 emissions were recorded by GC-FID over 20 min. cps: counts per second. Different letters indicate significant differences between treatments based on one-way ANOVA, p<0.05.

https://doi.org/10.7554/eLife.46668.009
Figure 3—figure supplement 2
Butylated hydroxytoluene emission is specific to western corn rootworm infection by entomopathogenic nematodes.

(A) Butylated hydroxytoluene (BHT) emissions (mean ± SEM) of western corn rootworm (WCR) cadavers infected by entomopathogenic nematodes (EPNs) at 0, 1, 3 and 5 days after infection (n = 4–5). Different letters indicate significant differences between treatments based on one-way ANOVA, p<0.05. (B) BHT emissions (mean ± SEM) of uninfected WCR cadavers (n = 8), EPN-infected WCR cadavers (n = 5), ground uninfected WCR (n = 3), EPNs (n = 3), EPN symbiontic bacteria (n = 3), bacteria-injected WCR (n = 5) and bacterial medium (LB)-injected WCR (n = 4). n.d.: not detected. Stars indicate significant differences based on two-tailed Student’s test between uninfected control WCR and the different treatments (***: p<0.001).

https://doi.org/10.7554/eLife.46668.010
Figure 4 with 2 supplements
Butylated hydroxytoluene attracts herbivores and increases infection by entomopathogenic nematodes.

(A) Proportions (mean ± SEM) of western corn rootworm (WCR) larvae orienting towards BHT or H2O (n = 20), uninfected WCR cadavers or cadavers infected by entomopathogenic nematodes (EPNs, n = 10), uninfected WCR cadavers covered with BHT or H2O (n = 15), EPN-infected WCR cadavers covered with BHT or H2O (n = 15). (B) WCR infection rate (Mean ± SEM) after exposure to BHT or H2O (n = 10). (C) Proportions (Mean ± SEM) of EPNs orienting towards Diabrotica balteata exudates complemented with BHT or H2O, uninfected WCR cadavers or cadavers infected by EPNs, uninfected WCR cadavers covered with BHT or H2O, EPN-infected WCR cadavers covered with BHT or H2O (n = 20). (D) WCR infection by EPNs (Mean ± SEM) after pre-incubation of EPNs with BHT or H2O for 24 hr (n = 15). Stars indicate significant differences based on analysis of variance (*: p<0.05, **: p<0.01, ***: p<0.001). All treatment solutions contained 0.01% ethanol. Raw data are available in Figure 4—source data 1.

https://doi.org/10.7554/eLife.46668.012
Figure 4—figure supplement 1
Rinsing WCR larvae after exposure to BHT reduces BHT emissions to traces.

(A) Representative chromatograms of WCR headspace of EPN-infected larvae and BHT-exposed larvae 24 hr after rinsing with 100% ethanol and tap water. Cps: counts per second. (B) EPN choice between control vs. BHT-exposed and rinsed WCR larvae after rinsing.

https://doi.org/10.7554/eLife.46668.013
Figure 4—figure supplement 2
Butylated hydroxytoluene attracts entomopathogenic nematodes and increases their predation success in the soil.

(A) Visual representation of experimental setup. Each arena contained two pairs of plants separated by 15 cm. BHT or water (H2O) was added to the soil of one of the plant pairs. All treatment solutions contained 0.01% ethanol. EPNs were added to the soil of the second plant pair. After two days, 150 mg soil was collected from the BHT and H2O treated plants and placed in cups containing three Galleria mellonella larvae for infection tests. (B) Proportion of EPN-infected G. mellonella larvae (mean ± SEM) exposed to soil collected from water (H2O) and BHT sides of the different arenas (n = 12). Star indicate significant differences based on two-tailed Student’s test (*: p<0.05).

https://doi.org/10.7554/eLife.46668.014
Butylated hydroxytoluene increases herbivore recruitment, predation success and reproduction of entomopathogenic nematodes in the soil.

(A) Visual representation of experimental setup. Entomopathogenic nematodes (EPNs) were applied on both sides of the arenas, and each side was either watered with butylated hydroxytoluene (BHT) or water (H2O). Eight western corn rootworm (WCR) larvae were then released in the middle and recollected after five days (n = 12). (B) Proportions (Mean ± SEM) of WCR larvae recovered from each side (n = 12). (C) WCR infection rates (Mean ± SEM) on each side (n = 12). (D) Number (Mean ± SEM) of EPN juveniles emerging per WCR larva (nControl = 6, ninfected = 12). (E) Total number of EPNs (Mean ± SEM) emerging from the WCR larvae on each side (n = 12). Stars indicate significant differences based on analysis of variance (**: p<0.01, ***: p<0.001). Raw data are available in Figure 5—source data 1.

https://doi.org/10.7554/eLife.46668.016
Figure 6 with 1 supplement
Attraction to cadavers infected by entomopathogenic nematodes is widespread.

(A) Proportions (mean ± SEM) of larvae orienting towards conspecific cadavers that were uninfected or infected by the entomopathogenic nematode (EPNs) H. bacteriophora. A total of seven species were tested: Diabrotica virgifera (WCR, n = 10), D. balteata (n = 19), Tenebrio molitor (n = 16), Drosophila melanogaster (n = 14), Spodoptera littoralis (n = 18), Plutella xylostella (n = 16) and Galleria mellonella (n = 10). (B) Proportions (mean ± SEM) of WCR larvae to their conspecific cadavers that were uninfected or infected by other four species of EPNs. These four EPN species were H. bacteriophora (n = 12), H. beicherriana (n = 15), H. georgiana (n = 13) and S. feltiae (n = 15). (C) Proportions (mean ± SEM) of WCR larvae for insect cadavers of different species that were uninfected or infected by H. bacteriophora. The four species were D. balteata (n = 15), Tenebrio molitor (n = 15), Spodoptera littoralis (n = 15), and Galleria mellonella (n = 15). (D) Proportions (mean ± SEM) of larvae orienting towards H. bacteriophora uninfected or infected WCR larvae. Replicate numbers were the same as figure C. Stars indicate significant differences based on analysis of variance (*: p<0.05, **: p<0.01, ***: p<0.001). Raw data are available in Figure 6—source data 1.

https://doi.org/10.7554/eLife.46668.018
Figure 6—figure supplement 1
Attraction of insects to EPN-infected cadavers is widespread.

Volatile bouquets of entomopathogenic nematode infected cadavers are species-specific. Differentially regulated volatiles (p<0.10) released by EPN-infected cadavers compared to uninfected cadavers of the different insect species (square root transformed data mean ± SEM, n = 10–19). Red lines indicate volatiles that are emitted in higher amounts by EPN-infected cadavers than by uninfected cadavers. Green lines indicate volatiles that are emitted in lower amounts by EPN-infected cadavers than by uninfected cadavers.

https://doi.org/10.7554/eLife.46668.019

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditionnal
information
Biological sample (Zea mays L.)Maize; Zea maysDelley semences et plantes SA, SwizerlandAkku
Biological sample (Diabrotica virgifera virgifera LeConte)Western corn rootworm; WCRUSDA-ARS-NCARL, Brookings, SD, USA
Biological sample (Diabrotica balteata LeConte)Diabrotica balteata; D. balteataSyngenta Crop Protection AG, Stein, Switzerland
Biological sample (Plutella xylostella)Plutella xylostella; P. xylostellaSyngenta Crop Protection AG, Stein, Switzerland
Biological sample (Drosophila melanogaster)Drosophila melanogaster; D. melanogasterUniversity of Bern (Bern, Switerland)
Biological sample (Spodoptera littoralis)Spodoptera littoralis; S. littoralisUniversity of Neuchâtel, Neuchâtel, Switzerland
Biological sample (Galleria melonella)Galleria melonella; G. melonellaFischereibedarf Wenger, Bern, Swizerlandhttps://www.fischen-wenger.ch/
Biological sample (Tenebrio molitor)Tenebrio molitor; T. molitorFischereibedarf Wenger, Bern, Swizerland
Biological sample (Heterorhabditis bacteriophora)Heterorhabditis bacteriophora; H. bacteriophoraAndermatt Biocontrol, Grossdietwil, SwizerlandMeginem (643C)https://www.biocontrol.ch/de_bc/meginem-pro
Biological sample (Steinernema feltiae)Steinernema feltiae; S. feltiaeAndermatt Biocontrol, Grossdietwil, SwizerlandTraunem (1008C)https://www.biocontrol.ch/de_bc/traunem
Biological sample (H. beicherriana)H. beicherrianaHebei Academy of Agricultural and Forestry Science/IPM center of Hebei Province, ChinaMa et al., 2013
Biological sample (H. georgiana)H. georgianaOwn collection from the USA
Chemical compound, drugButylated hydroxytoluene; BHTSigma AldrichW218405https://www.sigmaaldrich.com/catalog/product/aldrich/w218405?lang=de&region=CH
Chemical compound, drugEthanolSigma Aldrich51976https://www.sigmaaldrich.com/catalog/product/sial/51976?lang=de&region=CH
Software, algorithmR 3.2.2R Foundation for Statistical Computing, Vienna, Austriahttps://www.r-project.org/
Software, algorithmProgenesis QIinformatics package from Waters, MA, USAhttps://www.waters.com/waters/de_CH/Progenesis-QI/nav.htm?cid=134790652&locale=de_CH
Software, algorithmNIST search Mass Spectral Library, NIST 2.2National Institute of Standards and Technology Standard Reference Data Program Gaithersburg, USA
OtherGC-MSAgilent Technologies (Schweiz) AG, Basel, Switzerland
OtherSPME fiberSupelco, USA100 μm polydimethylsiloxane coating

Data availability

Source data files have been provided for all main figures.

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