DOI: 10.5061/dryad.ngf1vhhsk
title: Bioturbation by endogeic earthworms facilitates entomopathogenic nematode movement toward herbivore-damaged maize roots

description: Entomopathogenic nematodes (EPNs) have been extensively studied as
 potential biological control agents against root-feeding crop pests. Maize
 roots under rootworm attack have been shown to release volatile organic
 compounds, such as (E)-β-caryophyllene (Eβc) that guide EPNs toward the
 damaging larvae. As yet, it is unknown how belowground ecosystems
 engineers, such as earthworms, affect the biological control capacity of
 EPNs by altering the root Eβc-mediated tritrophic interactions. We here
 asked whether and how, the presence of endogeic earthworms affects the
 ability of EPNs to find root-feeding larvae of the
 beetle Diabrotica balteata. First, we performed a field mesocosm
 experiment with two diverse cropping systems, and revealed that the
 presence of earthworms increased the EPN infection potential of larvae
 near maize roots. Subsequently, using climate-controlled,
 olfactometer-based bioassays, we confirmed that EPNs response to Eβc alone
 (released from dispensers) was two-fold higher in earthworm-worked soil
 than in earthworm-free soil. Together our results indicate that endogeic
 earthworms, through burrowing and casting activities, not only change soil
 properties in a way that improves soil fertility but may also enhance the
 biocontrol potential of EPNs against root feeding pests. For an
 ecologically-sound pest reduction in crop fields, we advocate agricultural
 practices that favour earthworm community structure and diversity.
description: <em>Mesocosm
 experiment</em> - To study the effect of earthworms’ bioturbation
 activities on EPNs recruitment by maize roots, we performed an outdoor
 mesocosm experiment at the Botanical Garden of Neuchâtel, Switzerland. The
 mesocosms consisted of 50 × 50 × 30 cm wooden raised garden beds layered
 with a 1 mm diameter plastic mesh to prevent earthworm escape (Figure S1
 in Supplementary material). Each mesocosm was filled with approximately 60
 L of the A-layer of an Anthrosol (organo-mineral horizon) enriched with
 10% compost and 10% sand. The initial soil was sieved once at 2 cm, and
 subsequently hand-sieved twice to remove all potential indigenous
 earthworms present. Before adding compost and sand, natural soil samples
 were collected, homogenized, dried at 40 °C for 48 hours, sieved at 2 mm,
 and ground using agate mortars for subsequent physicochemical analyses.
 Specifically, we measured the particle size distribution (modified
 Robinson pipette method), the organic matter content through loss on
 ignition by weighing before and after burning 10 gr of soil at 450°C for 2
 hours, carbon to nitrogen ratio (CN) using an elemental analyser
 (FLASH2000, Thermo Fisher Scientific, Waltham, Massachusetts, United
 States); the pH in 1:2.5 soil to water ratio; the cation exchange capacity
 (CEC) following the cobaltihexamine chloride method; and the total
 phosphorous (using the Kjeldahl digestion method)
 <sup>1</sup>. The initial A-layer of the Anthrosol so was thus
 characterized as a silty-loamy soil (23% sand, 65% silt, 11% clay), with
 7.06% organic matter content, 2.95% organic carbon, with a CN of 11.36,
 and with 19 ppm of total phosphorus content. The soil pH was 7.65 and the
 CEC was 5.0 cmolc/kg. Four experimental treatments were
 tested (Figure S1): monocultures with and without the endogeic earthworm
 species <em>Allolobophora icterica</em>; and polycultures with
 and without the same earthworms. To reduce risks of interspecific
 variation in earthworms, we used a commercial strain of <em>A.
 icterica</em> supplied by the Ecotoxicology Department of National
 Institute for Agricultural Research (INRA Versailles, France).
 The simulated monoculture
 consisted of three maize plants (<em>Zea mays </em>var.
 Delprim, UFA Delley Semences et Plantes, Delley-Portalban, Switzerland)
 per mesocosm, whereas one squash plant (<em>Curcubita
 pepo</em>, var. Rondini, Sativa Rheinau AG, Switzerland)<em>
 </em>and two bean plants (<em>Phaseolus vulgaris</em>,
 var. Neckargold, Sativa Rheinau AG) were growing with three maize plants
 in the simulated polycultures (Figure S1). In total we built 10 mesocosms
 per treatment (N = 40 mesocosms). All plants were sown early July and
 grown until mid-October 2018 before the onset of the experiment (Figure
 S2). Seven weeks after sowing, half of the mesocosms were inoculated with
 15 earthworms each. The earthworms were standardized to 6 g of total fresh
 weight biomass per mesocosm. In mid-October, the roots of one
 maize plant per mesocosm were mechanically damaged with a cork borer
 (punched three times near root area) and watered with 25 ml of a solution
 containing 500 μg (2.4 µmoles) of jasmonic acid (JA; (±)-Jasmonic acid,
 CAS Number: 77026-92-7, Sigma, St Louis, IL, USA) per plant to induce
 emission of volatile defence compounds <sup>2</sup>. JA is a
 growth phytohormone also called the “wound hormone” as it plays a central
 role in plant defence and has been shown to induce the release of Eβc in
 herbivore-attacked maize plants <sup>3</sup>. Mechanical
 damage was preferred over direct herbivory on roots to ensure damage
 reproducibility and to standardize the production of defence volatile
 compounds. Two days later, four <em>Galleria mellonella</em>
 (Lepidoptera: Pyralidae) larvae per mesocosm were placed in the soil as
 sentinel hosts to quantify EPN infection success. Specifically, in each
 mesocosm, the first two <em>G. mellonella </em>larvae were
 buried 5 cm deep in the soil and 5 cm away from the stem of a root-broken
 maize plant (damaged roots), while the second pair of larvae was placed in
 the same conditions, but close to the roots of an undamaged plant (control
 roots). Because late-instars <em>G. mellonella </em>larvae are
 immobile and highly susceptible to EPN infection, they have been
 extensively used for monitoring EPNs’ presence in soil
 <sup>4,5</sup>, as was done here. One day after <em>G.
 mellonella </em>addition, a solution of less than two-week old 3000
 infective juveniles <em>H. megidis </em>EPNs was inoculated at
 the centre of all mesocosms. The used <em>H. megidis </em>EPNs
 (Nematoda: Heterorhabditidae) were supplied by Andermatt Biocontrol AG,
 Switzerland, and reared on late-instar <em>G. mellonella</em>
 larvae in accordance with an <em>in vivo</em> rearing protocol
 described step by step <sup>6</sup>. Five days after EPN
 inoculation, all <em>G. mellonella</em> larvae were collected.
 Dead larvae were directly transferred into White traps<strong>
 </strong>to confirm infestation by EPNs, while living larvae were
 kept in soil-filled 5×6×4 cm plastic boxes for measuring potential EPN
 infection. Next, we collected plant traits related to biomass
 accumulation, including: total aboveground biomass, total vegetative
 height, and fitness, as the total biomass of all corncobs on each plant.
 Finally, a fraction of the soil was sampled in each mesocosm for
 fertility-related analyses; which included CEC and CN measures.
 <em>Olfactometer-based
 bioassays</em> To dissect the interactive effect
 of root herbivory and earthworm presence near the roots of maize plants on
 EPN recruitment, a first (<em>four-arm</em>) olfactometer
 bioassay was conducted in controlled conditions of temperature, light and
 humidity (22±2 °C day / 16±2 °C night, 55% RH, daytime 08:00am – 06:00pm,
 230 μmol/m2.s). The belowground olfactometer device (Figure 2A), modified
 from Rasmann et al. <sup>7</sup>, consisted of a central glass
 chamber filled with white sand (Spielsand classic, Hamann Mercatus GmbH,
 Germany) extending in side arms connected to terminal glass pots (10 cm
 high, 15 cm diameter). Pots were filled with 1.2 L of soil (1/4 sand, and
 3/4 standard potting soil; Ricoter, Aarberg, Switzerland, 10% relative
 humidity) as a standard for growing maize in the non-soil substrate when
 tested in olfactometers <sup>8</sup>. Three <em>A.
 icterica </em>earthworms were inoculated in two terminal pots
 (Figure 2A). Simultaneously, one maize seedling was sown in each of the
 four terminal pots, and left to grow for 20 days (two-leaf to three-leaf
 stage). After 20 days of growth, which is considered as a minimum period
 for significant bioturbation of the soils <sup>9</sup>, three
 second instars of the banded cucumber beetle <em>Diabrotica
 balteata</em> (Coleoptera: Chrysomelidae) were added to two opposite
 glass pots containing the plants for the herbivore treatment setup (Figure
 3A). We used <em>D. balteata </em>instead of <em>D. v.
 virgifera </em>because of quarantine restrictions impeding the use
 of this species in the climate chambers in Switzerland. <em>D.
 balteata</em> is a generalist beetle that can feed on maize roots,
 and has been previously shown to induce maize plants to produce Eβc and
 attract EPNs <sup>10</sup>. Therefore, while slight
 differences might exist in terms of defence induction between the two
 <em>Diabrotica </em>species, they should be negligible and the
 results generalizable across Diabroticine beetles. Eggs of <em>D.
 balteata </em>were supplied by Syngenta Crop Protection (Stein,
 Switzerland) and larvae reared on a corn-based diet. Overall, the
 treatments followed a two-by-two factorial design experiment with the
 presence or absence of earthworms and root herbivores in each four-arm
 olfactometer (Figure 2A). After three days, a solution of 2000
 <em>H. megidis</em> infective juveniles was inoculated 5 mm
 below the sand surface in the middle of the central arena. After an
 additional 24 hours, EPNs were retrieved from each side arm using the
 Baermann decantation funnel method. Next, roots were harvested, carefully
 washed and flash frozen in liquid nitrogen. Roots were ground in liquid
 nitrogen and Eβc production from each root system was measured using
 solid-phase microextraction (SPME) coupled to gas chromatography-mass
 spectrometry (GCMS) as described in Rasmann et al.
 <sup>7</sup>. Finally, for each plant, we scored root and
 shoot fresh biomass and plant height, measured as the longest leaf length
 from the ground. The same experiment consisting of 5 olfactometers each
 time was repeated four times for a total of N = 20 replicates.
 A second
 (<em>two-arm</em>) olfactometer bioassay was performed in
 order to explore whether earthworm-emitted exudates via the epidermal
 mucus or faeces would directly interfere with the production of Eβc from
 maize roots, and the subsequent EPN movement. For this, maize seeds were
 sown in olfactometer glass pots (10 cm high, 5 cm diameter) with
 commercial substrate in similar conditions as described above (see Figure
 S3). Half of the plants were watered with tap water (control) while the
 other plants were watered with mucus solution. The mucus solution was
 obtained by caging 10 earthworms (adults and juveniles) in two 1mm plastic
 mesh sieves in contact with each other’s open edge. The cage was submerged
 in 3 mm of tap water and left in darkness at room temperature (25 °C) for
 one hour, allowing earthworms to move into water and rub their skin
 against the sieve. The mucus of 20 earthworms (two cages) was collected to
 water 10 plants (equivalent of two earthworms per plant). The mucus
 solution (200 mL) was freshly prepared an hour before direct application
 onto the plants. All plants received the same volume of liquid at the same
 interval in order to keep substrate between plants as homogenously moist
 as possible. After 20 days, three second-instar <em>D.</em>
 <em>balteata</em> larvae were placed in every pot (controls
 and treatments) and left to feed on maize roots for three days. Connection
 with the olfactometer system was made 1 day before EPN inoculation, after
 which, a solution of 2000 infective juvenile EPNs was inoculated in the
 olfactometer central arena. After 24 hours, EPNs were extracted from the
 two side arms of each olfactometer with the Baermann funnel method and
 counted under the microscope. Finally, root biomass and Eβc emissions were
 recorded as described above. The experiment was replicated 10
 times. A third
 (<em>two-arm</em>) olfactometer bioassay was performed to the
 test whether earthworm bioturbation activity in bulk soil affects nematode
 mobility alone, independently of earthworms being in contact with the
 maize root system. For this, soil-filled side arms and central arena and
 sand-filled terminal pots (10 cm high, 5 cm diameter) were assembled into
 a two-arm olfactometer (Figure 4A). The same natural soil that was used
 for the mesocosm experiment at the Botanical Garden was used and sieved to
 2 mm to ensure effective bioturbation and burrowing by earthworms. Ten
 soil samples were randomly taken from the soil stock, put on filter paper,
 emerged in water and left for decantation for 48 hours to test the
 presence of any indigenous nematodes. None were observed and the soil was
 consequently not sterilized. Based on the study of Chiriboga et al.
 <sup>11</sup> on diffusion of Eβc in different soil textures,
 soil and sand moisture were set respectively at 20% and 10% to maximise
 diffusion of volatiles. On the first day, three <em>A. icterica
 </em>earthworms were added to one half of olfactometer, and none in
 the other half. Earthworm bioturbation was restricted to half of the
 central arena by a 0.5 mm-mesh screen dividing it and by an anti-EPN mesh
 screen at the end of the side arms. Earthworms were left to work the soil
 for 4 days in climatic chamber (18 ± 2 °C, continuous darkness). Four days
 were considered enough time for three earthworms to properly burrow 0.5 L
 of soil. On day 5, the terminal pots were connected to olfactometer
 central system, and custom-made dispensers containing
 CO<sub>2</sub> generating material (300 mg and sodium
 hydrogencarbonate and citric acid 3:1) and synthetic Eβc (300 μl,
 β-Caryophyllene, CAS Number 87-44-5, Sigma, St Louis, IL, USA) were
 prepared as described in Turlings et al. <sup>3</sup>, and
 inserted into the terminal pots filled with sand to ensure that EPN
 attraction was equally stimulated by both sides of the olfactometer
 (Figure 4A). Five hours after inserting the dispensers, a suspension of
 2000 <em>H. megidis </em>infective juveniles was inoculated in
 the central arena and left for 24 hours, after which EPN presence in each
 arm was retrieved using Baermann funnels. The experiment was replicated
 eight times. <em>Statistical
 analysis</em> – All statistical analyses were performed on R
 <sup>12</sup>. <em>Mesocosm outdoor
 experiment</em> – We scored the probability of infection by dividing
 the number of larvae infected by EPNs around each plant by two. We then
 assessed the full interactive effect of culture type (two levels),
 earthworms (two levels), and root induction (two levels) on the
 probability of infection with generalized linear model analysis (GLM) with
 quasi-binomial distribution. We next performed the same GLM, but by
 comparing the interactive effect of earthworms and root induction, by
 splitting the data into monoculture and polyculture systems. Probabilities
 of infection scores were visualized using the library
 <em>popbio</em> <sup>13</sup>. The interactive
 effect of culture type and earthworms on CEC, CN, plant biomass and corn
 earcobs biomass was assessed using two-ways ANOVAs, followed by TukeyHSD
 post-hoc tests. For plant traits, we included mesocosm as a blocking
 effect in the model. <em>Four-arm olfactometer
 bioassay</em> - Analysis of variation in nematode recruitment across
 earthworms by root herbivory treatments was performed using generalized
 linear model analysis (GLM) with quasi-Poisson distribution to take data
 overdispersion into consideration, and by including the experiment date as
 a blocking factor in the model. Differences among treatments were assessed
 using analyses of deviance and F statistics. The analysis of the effect of
 herbivores, earthworms and their interactions on log+1-transformed Eβc
 emissions were performed using two-way ANOVA, and by including experiment
 as blocking factor, and root biomass as covariate in the model.
 Differences among treatments were assessed using Tukey HSD tests. The
 effect of root herbivory and earthworms on plant biomass accumulation was
 assessed on the first principal component analysis (PCA) axis that
 included plant height, root and shoot fresh biomass (Figure
 S4).  <em>Mucus
 experiment</em> - Analysis of variation in nematode recruitment
 across treatments was performed using GLM with quasi-Poisson distribution.
 Analysis of the effect of earthworm mucus on log+1-transformed Eβc
 emissions and root biomass were performed using one-way ANOVAs.
 <em>Bioturbation and
 synthetic </em>Eβc<em> olfactometer bioassay </em>- The
 analysis of variation in nematode recruitment across earthworm
 presence/absence treatment was performed using GLM with quasi-Poisson
 distribution, and followed by analysis of deviance. 1         
 Allen, S. E., Grimshaw, H. M., Parkinson, J. A. & Quarmby, C.
 <em>Chemical analysis of ecological materials</em>. 
 (Blackwell Scientific Publications., 1974). 2          van
 Dam, N. M., Witjes, L. & Svatos, A. Interactions between
 aboveground and belowground induction of glucosinolates in two wild
 <em>Brassica</em> species. <em>New Phytol.</em>
 <strong>161</strong>, 801-810,
 doi:10.1111/j.1469-8137.2004.00984.x (2004). 3         
 Turlings, T., Hiltpold, I. & Rasmann, S. The importance of
 root-produced volatiles as foraging cues for entomopathogenic nematodes.
 <em>Plant Soil</em> <strong>358</strong>, 51-60,
 doi:10.1007/s11104-012-1295-3 (2012). 4         
 Woodring, J. L. & Kaya, H. K. <em>Steinernematid and
 Heterorhabditid Nematodes: a Handbook of Techniques.</em>, 
 (Arkansas Agricultural Experiement Station, 1988). 5         
 Rasmann, S., Erwin, A. C., Halitschke, R. & Agrawal, A. A. Direct
 and indirect root defences of milkweed (<em>Asclepias
 syriaca</em>): trophic cascades, trade-offs and novel methods for
 studying subterranean herbivory. <em>J. Ecol.</em>
 <strong>99</strong>, 16-25 (2011). 6         
 McMullen, J. G. & Stock, S. P. In vivo and in vitro rearing of
 entomopathogenic nematodes (Steinernematidae and Heterorhabditidae).
 <em>Journal of Visualized Experiments</em>, doi:10.3791/52096
 (2014). 7         
 Rasmann, S.<em> et al.</em> Recruitment of entomopathogenic
 nematodes by insect-damaged maize roots. <em>Nature</em>
 <strong>434</strong>, 732-737, doi:10.1038/Nature03451
 (2005). 8         
 Degenhardt, J.<em> et al.</em> Restoring a maize root signal
 that attracts insect-killing nematodes to control a major pest.
 <em>Proceedings of the National Academy of Sciences of the United
 States of America</em> <strong>106</strong>,
 13213-13218, doi:10.1073/pnas.0906365106 (2009). 9          Le
 Couteulx, A., Wolf, C., Hallaire, V. & Pérès, G. Burrowing and
 casting activities of three endogeic earthworm species affected by organic
 matter location. <em>Pedobiologia</em>
 <strong>58</strong>, 97-103, doi:10.1016/j.pedobi.2015.04.004
 (2015). 10       
 Rasmann, S. & Turlings, T. C. J. First insights into specificity
 of belowground tritrophic interactions. <em>Oikos</em>
 <strong>117</strong>, 362-369,
 doi:doi:10.1111/j.2007.0030-1299.16204.x (2008). 11       
 Chiriboga, M. X., Campos-Herrera, R., Jaffuel, G., Röder, G. &
 Turlings, T. C. J. Diffusion of the maize root signal
 (<em>E</em>)-β-caryophyllene in soils of different textures
 and the effects on the migration of the entomopathogenic nematode
 <em>Heterorhabditis megidis</em>.
 <em>Rhizosphere</em> <strong>3</strong>, 53-59,
 doi:10.1016/j.rhisph.2016.12.006 (2017). 12        R
 Development Core Team. R: A language and environment for statistical
 computing.  (2019). &lt;http://www.R-project.org/.&gt;.
 13       
 Stubben, C. & Milligan, B. Estimating and analyzing demographic
 models using the popbio package in r. <em>2007</em>
 <strong>22</strong>, 23, doi:10.18637/jss.v022.i11
 (2007).