DOI: 10.5061/dryad.x3ffbg7np
title: Fear of predators alters herbivore regulation of soil microbial community function

description: Fear of predation can affect important ecosystem processes by altering the
 prey traits' expression that, in turn, regulates the quantity and
 quality of nutritional inputs to soil. Here, we aimed to assist in
 bridging a knowledge gap in this cascading chain of events by exploring
 how risk of spider predation may affect grasshopper prey performances, and
 the activity of various microbial extracellular enzymes in the soil. Using
 a mesocosms field‐experiment, we found that grasshoppers threatened by
 spider predation ate less, grew slower, and had a higher body carbon to
 nitrogen ratio. Herbivory increased activity of all microbial
 extracellular enzymes examined, likely due to higher availability of root
 exudates. Predation risk had no effect on C‐acquiring enzymes but
 decreased activity of P‐acquiring enzymes. We found contrasting results
 regarding the effect of predation on the activity of
 N‐acetyl‐glucosaminidase and leucine arylamidase N‐acquiring enzymes,
 suggesting that predation risk may alter the composition of N‐inputs to
 soil. Our work highlighted the importance of soil microbial enzymatic
 activity as a way to predict how changes in the aboveground food‐web
 dynamics may alter key ecosystem processes like nutritional‐cycling.
description: Site description and
 experiment design We conducted our field
 experiment at Jilin Songnen Grassland Ecosystem National Observation and
 Research Station, Changling, China (44°45′ N, 123°45′ E). The climate in
 this site is semi‐arid continental and monsoon with average annual
 precipitation ranging from 280 to 400 mm. This system is dominated by the
 grass <em>L. chinensis</em>, and its main insect
 herbivore <em>E. unicolor</em> grasshopper. Wolf spiders are
 common predators of grasshoppers in this system.
 We tested how the risk of spider predation
 regulates grasshoppers' trophic function and subsequently soil
 microorganism enzymatic activity and stoichiometry, by setting a
 randomized block experiment with three‐trophic‐level treatments: (i)
 plants (control), (ii) plants and grasshoppers (herbivore), and (iii)
 plants, grasshoppers, and spiders (predation risk; Figure 1). We
 constructed 10 blocks of three 0.25‐m<sup>2</sup>‐basal area,
 0.9‐m‐high mesocosms covered by 0.8 mm × 1.2 mm mesh size screen. All
 mesocosms were constructed over naturally growing vegetation of similar
 composition, in which <em>L. chinensis</em> accounted for
 about 75% of the soil surface coverage. The blocks were at least 10 meters
 apart. Before stocking the mesocosms, we removed all naturally occurring
 invertebrates using a vacuum cleaner. Grasshopper density in this field
 site reaches a maximum of 20–30 per m<sup>2</sup> (Zhong et
 al., 2014). Thus, we stocked each
 mesocosm with eight third-instar grasshoppers and added one wolf spider to
 each predation mesocosm. We glued the spider chelicerae with quick‐drying
 glue to prevent them from preying upon grasshoppers (Hawlena &
 Schmitz, 2010). Previous work in our own
 study system, and elsewhere have shown that spiders with glued chelicerae
 can remain active for more than 2 months (Barton, 2010; Hawlena et
 al., 2012). Our experiment lasted from
 10 August to 20 September 2020. Grasshopper feeding
 frequency, survival rate, growth rate, and body C:N We conducted feeding measurements 7 days after
 stocking the mesocosms, to allow grasshoppers and spiders to settle within
 the mesocosms. We randomly selected a
 third‐instar <em>E. unicolor</em> from each mesocosm and
 painted a mark on its thorax to enable focal observations. The observer
 recorded the times of grasshopper feeding behavior (eating foliage without
 retracting the heat from the plants) in their most active period
 (07:00–09:00, 11:00–13:00, and 15:00–17:00), for a total of 6 h, and
 calculated total number of feeding time in 6 h as feeding frequency (Zhong
 et al., 2017). All grasshoppers were
 weighed at the beginning and end of the experiment, and the grasshopper
 growth‐rate was calculated as the difference between the final and initial
 wet weight divided by the number of days elapsed. Grasshopper survival
 rate was calculated as the final density divided by the initial density in
 each mesocosm. At the end of the experiment, we starved all surviving
 grasshoppers for 24 h to allow gut evacuation, and then freeze‐dried and
 homogenized their carcasses using MM400 ball mill (Retsch GmbH Rheinische
 Strabe 3642781). We used C:N:H elemental analyzer to quantify
 grasshoppers' body C and N content (Vario EL cube;
 Elementar). Soil sampling and analysis
 of soil enzymatic activity In each mesocosm, we
 collected five soil subsamples from 0 to 10 cm depth. The samples were
 thoroughly mixed to make a composite homogenous soil sample per mesocosm.
 We stored all samples at 4°C for 14 days and then analyzed them for
 microbial extracellular enzymatic activity. We measured the activity of
 one P-acquiring enzyme (acid phosphatase, PHO), two C-acquiring enzymes
 (β‐d‐Glucoside, BG; Cellobiohydrolase, CBH), and
 two N-acquiring enzymes (N‐Acetyl‐glucosaminidase, NAG; Leucine
 arylamidase, LAP), following the method described by DeForest (2009). In
 addition, the enzymatic stoichiometries of (BG + CBH):(NAG + LAP),
 (BG + CBH):PHO, and (NAG + LAP):PHO were calculated as the indicators for
 microbial C versus N, C versus P, and N versus P demand, respectively.
 Higher microbial C:N and C:P enzymatic ratios mean higher microbial C demand relative to N and P
 demand respectively. Higher microbial N:P enzymatic ratio represents higher microbial
 N demand relative to P demand (Luo et al., 2017; Waring et al., 2014).