Soil Biology & Biochemistry 43 (2011) 351e358

Contents lists available at ScienceDirect

Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

The oribatid Scheloribates moestus (: ) alters litter chemistry and nutrient cycling during decomposition

Kyle Wickings*, A. Stuart Grandy

Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824, USA article info abstract

Article history: It is widely accepted that microarthropods influence decomposition dynamics but we know relatively Received 3 May 2010 little about their effects on litter chemistry, extracellular enzyme activities, and other finer-scale Received in revised form decomposition processes. Further, few studies have investigated the role of individual microarthropod 27 October 2010 species in litter decomposition. The oribatid mite Scheloribates moestus Banks (Acari: Oribatida) is Accepted 28 October 2010 abundant in many U.S. ecosystems. We examined the potential effects of S. moestus on litter decom- Available online 13 November 2010 position dynamics and chemical transformations, and whether these effects are influenced by variation in initial litter quality. We collected corn and oak litter from habitats with large populations of S. moestus Keywords: Oribatid and in microcosms with and without mites measured respiration rates, nitrogen availability, enzyme Litter decomposition activities, and molecular-scale changes in litter chemistry. Mites stimulated extracellular enzyme Extracellular enzymes activities, enhanced microbial respiration rates by 19% in corn litter and 17% in oak litter over 62 days, Nutrient cycling and increased water-extractable organic C and N. Mites decreased the relative abundance of poly- saccharides in decomposing corn litter but had no effect on oak litter chemistry, suggesting that the effects of S. moestus on litter chemistry are constrained by initial litter quality. We also compared the chemistry of mite feces to unprocessed corn litter and found that feces had a higher relative abundance of polysaccharides and phenols and a lower relative abundance of lignin. Our study establishes that S. moestus substantially changes litter chemistry during decomposition, but specific effects vary with initial litter quality. These chemical transformations, coupled with other observed changes in decom- position rates and nutrient cycling, indicate that S. moestus could play a key role in soil C cycling dynamics. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction communities will influence ecosystem function may require knowledge of individual species, their behavior, and their effect on The soil fauna consists of an array of species that collectively soil processes under different environmental conditions. have a profound influence on ecosystem processes, including One potentially important but largely unexplored way that soil decomposition and nutrient cycling (Wolters, 2000; Coleman, could affect decomposition dynamics is by altering litter 2008; Ekschmitt et al., 2008). Soil animals affect litter decomposi- chemistry during decomposition. As litter passes through the tion directly by ingesting and fragmenting litter and indirectly by digestive tract its chemical composition may be altered by altering the biomass and community structure of microbial enzymatic activity, organic matter mineralization and nutrient decomposers (Hanlon and Anderson, 1979; Cortet et al., 2003; absorption (Gasdorf and Goodnight, 1963; Ziechman, 1994; Coleman et al., 2004). However, faunal contributions to decompo- Guggenberger et al., 1996; Hopkins et al., 1998; Gillon and David, sition and nutrient cycling are variable and depend on the identity, 2001; Rawlins et al., 2006; Hedde et al., 2007; Filley et al., 2008; density and activity of the resident species, as well as on their Geib et al., 2008). These changes in organic matter chemistry may interactions with environmental conditions and substrate quality have implications for soil aggregation and sorption dynamics, as (Anderson et al., 1985; Bardgett and Chan, 1999; Lenoir et al., 2007; well as for other biogeochemical processes that influence long- Ayres et al., 2010). Thus, predicting how changes in soil faunal term C stabilization in soil (Golchin et al., 1994; Kleber et al., 2007). However, chemical transformations by soil animals have only been shown in a limited number of studies, have rarely used modern * Corresponding author. Tel.: þ1 517 355 0271x1247; fax: þ1 517 355 0270. spectroscopic chemical approaches, and have focused mostly on E-mail address: [email protected] (K. Wickings). macrofauna such as , millipedes and isopods (Teuben

0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.10.023 352 K. Wickings, A.S. Grandy / Soil Biology & Biochemistry 43 (2011) 351e358 and Verhoef, 1992; Wolters, 2000; Rawlins et al., 2006; Filley et al., retention 11 mm) to remove most particulate organic matter while 2008; Geib et al., 2008). retaining bacteria or fungal spores. After soaking, arenas were Oribatid mites are among the most abundant soil mesofauna, allowed to drain for 5 h before incubating in the dark at 22 C for 5 d ranging from 25,000 to 500,000 individuals m 2 in terrestrial to allow microbial activity to increase. At the end of the 5-d incu- ecosystems (Coleman et al., 2004). Studies have shown that the bation period, all arenas were placed into glass jars. In an effort to presence of oribatid mites can increase litter mass loss, stimulate maintain adequate litter moisture contents, we placed 10 mL microbial respiration rates and decrease fungal biomass (Siepel and of water in the bottom of each jar. The litter arenas were Maaskamp, 1994; Kaneko, 1998; Hansen, 1999; Heneghan et al., elevated w5 mm above the water using polypropylene stands. 1999). However, little is known about their effects on other aspects Twenty adult individuals of S. moestus were added to each of 60 of decomposition including the alteration of microbial extracellular arenas (n ¼ 30 corn and 30 oak) with the remaining 60 arenas enzymes and transformations of litter chemistry. Further, predicting receiving no mites. Mite numbers were chosen to be consistent the effects of oribatids on decomposition dynamics requires a better with densities of S. moestus measured in field sites at KBS (Wickings understanding of the ways in which individual species interact with et al., Unpublished results). All microcosms were covered with litter quality (Hansen, 1999; Gergócs and Hufnagel, 2009). plastic wrap to retain moisture but still allow for some gas Our recent experiments show that the oribatid mite Schelor- exchange. Microcosms were kept at approximately 21 Cona12h ibates moestus Banks () is found in many Michigan lightedark cycle for the duration of the experiment (62 d). ecosystems at densities approaching 200 individuals g 1 dry litter (Wickings, Unpublished results). Both adults and juveniles may 2.3. Microcosm analysis contribute significantly to decomposition through feeding on litter and microbes as well as through the production of feces. Given its At 6, 13, 22, 34, 48 and 62 d of incubation, 20 microcosms were population size and potential activity, S. moestus may play a key role destructively harvested for the measurement of enzyme activity, in regulating decomposition in cultivated and native ecosystems. inorganic N, and dissolved organic carbon (DOC) and nitrogen Our overall objective was to evaluate the potential effects of S. (DON). Numbers of live mites were recorded at each harvest date at moestus on corn and oak litter decomposition dynamics and, more 400 magnification. specifically, to determine whether S. moestus changes C and N cycling rates, enzyme activities, and chemical transformations in 2.3.1. Litter C min and composition decomposing litter. At 3-d intervals, the plastic covering was removed from 20 litter arenas, which were then transferred to dry 120 mL jars and capped 2. Methods with lids fitted with rubber septa to quantify microbial respiration rate. The arenas used for CO2 measurements were randomly 2.1. Litter and mite collection selected at the beginning of the experiment. These same arenas were used for all CO2 measurements and were subsequently the Red oak (Quercus rubra L.) and corn (Zea mays L.) leaf litter were last to be destructively harvested. CO2 concentration of the jar collected from the Michigan State University W.K. Kellogg Biolog- headspace was measured using an LI-820 infrared CO2 analyzer ical Station Long-Term Ecological Research Site (KBS, LTER) in (Licor Biosciences, Lincoln, NE) at 30-m intervals over the course of October 2008. Micro-invertebrates were extracted from litter using 1 h (0, 30 and 60 m) and used to determine production rate 1 1 Berlese funnels (Bioquip, Rancho Dominguez, CA) over a 3 d period (mg CO2eCg litter d ). into liter-sized plastic boxes lined with a moistened plaster-char- Chemical composition of litter from time zero and after 62 d of coal mixture (10:1 by weight). S. moestus was identified from decomposition was determined using pyrolysis gas chromatogra- samples and compared with type specimens from the Museum of phyemass spectroscopy (py-gc/ms) using previously described Comparative Zoology, Harvard University, Cambridge, MA. methods (Grandy et al., 2009; Wickings et al., 2010). Briefly, Approximately 4000 S. moestus adults were collected in total samples were pulse-pyrolyzed in quartz tubes on a Pyroprobe 5150 (approximately 3000 individuals from corn, 1000 individuals from (CDS Analytical Inc., Oxford, PA) using a pyrolysis temperature of oak) and all individuals were transferred to separate live boxes to 600 C. Pyrolysis products were transferred onto a gas chromato- avoid by other . Approximately 2 g of either graph (Trace GC Ultra, Thermo Scientific, West Palm Beach, FL) corn or oak leaf litter, moistened with deionized water, were placed where they were further separated upon passage through a heated, in each box to provide food and habitat for S. moestus. After micro- fused silica capillary column (60 m 0.25 mm i.d.). GC oven invertebrates were extracted, corn and oak litter were removed temperature was increased from 40 to 270 C at a rate of 5 Cm 1 from Berlese funnels and dried completely at 60 C. All litter was with a final temperature ramp to 310 Cat30Cm 1. Finally, cut and sieved to fragments ranging 2e5 mm after drying and compounds were transferred to a mass spectrometer (Polaris Q, further defaunated using two successive freezeeheat cycles (80 Thermo Scientific, West Palm Beach, FL) via a 270 C heated transfer and þ80 C) similar to those used by Bardgett et al. (1998). line. The abundance of all ions was scaled relative to the largest peak. Peaks were compared to reference spectra using the Auto- 2.2. Microcosm setup mated Mass Spectral Deconvolution and Identification program (AMDIS V 2.65) and the National Institute of Standards and Tech- Microcosms consisted of a 70 mL polypropylene bottle (litter nology library (NIST). arena), from which the bottom was removed and replaced with In order to better understand the direct effects of S. moestus on 38 mm stainless steel mesh, contained within a 120 mL glass jar. litter chemistry we also analyzed the chemical composition of its Before being placed in jars, 120 litter arenas received 1 g of feces. Two additional S. moestus live boxes, containing approxi- defaunated corn or oak litter (n ¼ 60 corn and 60 oak arenas). Once mately 1000 adult mites each, were also maintained for the dura- litter was arranged in an arena, it was soaked in a litter homogenate tion of the study. Four nylon filter disks were placed in each live box for 2 h to inoculate the microcosms with microbes. Homogenates and each disk received approximately 1 g of litter (one box con- were created by agitating 500 g of field-moist corn or oak litter in tained corn and one contained oak). Live boxes were kept at 4 L of deionized water for approximately 15 m. Suspensions were approximately 21 C and deionized water was added to the plaster then filtered through a paper filter (Whatman, grade 1, particle base of each box as needed to maintain adequate moisture. At K. Wickings, A.S. Grandy / Soil Biology & Biochemistry 43 (2011) 351e358 353

2e3 d intervals, mite feces were collected from the surface of each filtrates were stored at 20 C until further analysis. Bioavailable filter disk using a modified vacuum apparatus consisting of a 70 mL nitrate and ammonium were quantified in litter filtrates using photo- quartz tube filled half way with glass wool and attached to metric procedures (Sinsabaugh et al., 2000; Doane and Horwath, a vacuum pump. All feces were stored at 80 C prior to analysis. 2003). Total organic C and N were also measured on filtrates Oven-dried (60 C) and pulverized samples of mite feces were using a Shimadzu TOC-TN combustion analyzer (Shimadzu Scien- analyzed using py-gc/ms following the procedures outlined above. tific Instruments, Columbia, MD) and leaf litter C and N values were Analyzing fecal material chemistry allowed us to compare chem- determined using an elemental analyzer (Costech ECS 4010, Cost- istries among the following: 1) initial undecomposed litter; 2) litter ech Analytical Technologies, Inc., Valencia, CA). Total organic N was plus microbes after decomposition for 62 d; 3) litter plus microbes determined by subtracting nitrate and ammonium from total N. plus mites after 62 d of decomposition (mites were removed from litter prior to chemical analysis); and 4) mite feces after feeding on 2.4. Statistical analysis litter. Juvenile S. moestus were present in live boxes after approxi- mately one month and we were unable to separate their feces from Microbial respiration data were analyzed using mixed model that of the adults although their numbers were low relative to analysis for repeated measures in SAS (PROC MIXED) with mite adults. We attempted to collect mite feces from oak for three presence/absence and time as fixed effects and block as a random months but were unable to obtain enough material for analysis. effect. Extracellular enzymes were analyzed using two-way anal- ysis of variance in SAS (ANOVA) with mite presence/absence and 2.3.2. Extracellular enzyme activity time as treatment factors. Nitrate, ammonium, total organic The activity of seven extracellular enzymes was analyzed using nitrogen, total organic carbon and molecular chemical data were all procedures outlined in Saiya-Cork et al. (2002) and Grandy et al. analyzed using one-way analysis of variance in SAS (ANOVA). Litter (2007). Briefly, the activities of N-acetyl-b-D-glucosaminidase types were analyzed independently for both one- and two-way (chitinase), b-glucosidase (cellulase), b-D-1,4-cellobiosidase (cellu- analyses. lase), tyrosine amino peptidase and acid phosphatase were assessed in black, 96-well microplates using substrates containing 3. Results the synthetic fluorescing molecules methylumbelliferone (MUB) and methylcoumarin (MC). The activity of urease was measured 3.1. Mite numbers and observations of feeding activity using colorimetric methods outlined in Sinsabaugh et al. (2000) and modified for analysis in 96-well microplates. Finally, the The number of mites per microcosm dropped from 20 to a mean activity of the oxidative enzyme phenol oxidase was measured of 15 in corn and 13 in oak within one week of the start of the in clear 96-well microplates using L-3,4-dihydroxyphenylalanine experiment. This number was maintained throughout the experi- (L-DOPA). Litter suspensions were prepared using 0.25 g subsam- ment in corn microcosms, however, the number of live mites in oak ples from each microcosm in a kitchen blender with 50 mM sodium litter dropped to a mean of 4.5 individuals g 1 of litter from 48 to acetate buffer at the initial pH (6.5) of the litters used for the 62 d of incubation. Juvenile mites were observed in both corn and experiment. After incubation at 14 C (approximately 6 h for all oak microcosms beginning on day 34 and were present in low hydrolases and 24 h for oxidases), enzyme activity was determined numbers for the remainder of the incubation. Daily observations using a flourometer (355 nm excitation and 460 nm emission for all confirmed that both adults and juveniles fed throughout the fluorescent substrates) and spectrophotometer (phenol oxidase experiment and the majority of individuals examined contained 450 nm, and urease 620 nm) (Thermo Fisher Flouroskan and multiple fecal boluses. Additionally, slide mounts of fecal material Multiskan, Thermo Scientific, Hudson, NH). All data are presented during the course of the experiment showed that boluses contained as potential activity (mmol of substrate g 1 of dry litter h 1). both plant and microbial material. We additionally examined the potential enzymatic activity of S. moestus collected from corn litter during our live Berlese 3.2. Litter C and N dynamics extractions to better understand chemical transformations of litter resulting from gut passage. Prior to conducting the enzyme assay, Corn and oak litter differed qualitatively at the start of the 100 mites were washed to remove associated external microor- experiment. Although corn litter had a higher proportion of lignin ganisms following a modification of the procedure outlined in than oak (38 vs. 14% of sample composition) it also had higher Siepel (1990). Briefly, mites were soaked for 10 min in 3% H2O2, proportions of nitrogen bearing compounds (13 vs. 5%) and poly- followed by 10 m in a 1:5 w/v solution of MnO2 and deionized saccharides (20 vs. 9%). Additionally, oak litter had a higher water. Finally, all mites were rinsed in deionized water for 10 m. proportion of lipids (50 vs. 11%) and a higher C/N ratio than corn. Twenty adult mites were crushed in 200 mL of sodium acetate CO2 flux was higher in corn than in oak microcosms for the dura- buffer solution using a glass pestle. Mite slurries were diluted to tion of the incubation (Fig. 1). Mites increased cumulative CO2 20 mL with additional buffer solution. All enzymes were assayed production by 19% in corn (F1, 168 ¼ 23.00, P < 0.0001) and 17% in following the same 96-well plate methods described for litter. oak (F1, 168 ¼ 51.72, P < 0.0001). The C/N ratio of oak increased Fluorometric assays, including N-acetyl-b-D-glucosaminidase, b- through time regardless of whether or not S. moestus was present. glucosidase, tyrosine amino peptidase and acid phosphatase, were In contrast, corn C/N ratios only increased during decomposition in incubated for approximately 6 h and photometric assays were the absence of mites (Table 1). incubated for 18 (urease) and 24 h (phenol oxidase). Five replicate Polysaccharide relative abundance increased in corn over 62 d of samples (each containing 20 randomly selected adult mites) were decomposition in the absence of mites (F2, 6 ¼ 11.01, P < 0.01) (Fig. 2 run for all gut enzymes measured. top). This increase was due primarily to the compound 2,3-dihydro- benzofuran and was not observed when mites were present 2.3.3. Aqueous C and N (Appendix Table 1). Oak litter was dominated by lipids which At 13, 34 and 62 d of incubation, 0.5 g subsamples of litter from comprised 40e50% of all identified compounds. Of the lipids the destructively harvested microcosms were shaken with 50 mL of present, 60e90% were long-chain (>C20) compounds. Unlike corn, deionized water for 1 h on a bench-top shaker (200 rpm). All the chemistry of oak litter was not affected by the presence of mites samples were then passed through a Whatman GF/A filter and (Fig. 2 bottom). 354 K. Wickings, A.S. Grandy / Soil Biology & Biochemistry 43 (2011) 351e358

oxidase activity was also marginally suppressed by the presence of Cumulative CO2 25 mites during sampling week 1 but was greater in microcosms with corn oak ¼ S. moestus than without mites during the following week (F5, 48 2.50,

dry litter) dry w/ 20 S. moestus < -1 w/o P 0.05). Urease, cellobiosidase and tyrosine amino peptidase g

2 activities were not affected by mites in oak litter. 15 3.5. Enzymes e enzymatic potential of S. moestus (mg CO

2 10 Chitinase was the most active enzyme detected within S. 5 moestus averaging 62.7 mmol h 1 g 1 dry mite biomass followed by cellulase, phosphatase and peptidase (44.8, 17 and 0.13 mmol 0 h 1 g 1 dry mite biomass, respectively). Urease and phenol oxidase

Cumulative CO 0 6 12 18 24 30 36 42 48 54 60 activities were not detected in S. moestus. days of incubation 3.6. Aqueous C and N in microcosms Fig. 1. Cumulative CO2 production from litter over the 62 d incubation. Data represent means SE. The presence of mites led to a significant increase in available nitrate, ammonium and both dissolved organic C and N (Tables 2 3.3. Fecal material chemistry and 3). The effects of mites on ammonium concentrations peaked in corn litter at 34 d (4.9 fold increase) and in oak litter at 13 d Mites that were fed corn litter generated feces that were (12.26 fold increase). In contrast, ammonium concentrations were chemically different from the original litter (Fig. 2 top). While the greatest in corn microcosms without mites at 13 d. Mites increased relative abundance of lignin was lower in feces than litter, that of nitrate concentrations in corn litter at 34 d (35.19 times more than phenol was higher (Fig. 2 top). In addition, polysaccharide relative in litter without mites), as well as dissolved organic C and N (1.3 abundance was significantly higher in feces than in both the initial and 4.2 times more, respectively) (Table 3). litter and in litter decomposed for 62 d with microbes and mites. Ratios of lignin to nitrogen, and lignin to polysaccharides, were also 4. Discussion lower in feces than in corn litter by approximately 88 and 87% respectively. Further, the relative abundance of five compounds The soil fauna strongly influences ecosystem processes but the including one polysaccharide, a phenol, a compound of unknown effects of individual species on C and N cycling and microbial origin and two lignin derivatives differed between feces and litter activity during decomposition remain contentious (Ayres et al., (Appendix Table 2). We planned to also include feces from oak-fed 2010). Further, the effects of soil animals on changes in litter mites in our analyses but were unable to gather an amount suffi- chemistry, extracellular enzyme activities and other key ecosystem cient for analysis. processes during decomposition are poorly understood but could have a substantial, long-term effect on soil C cycling dynamics. We 3.4. Enzymes e extracellular enzymes within litter found that the widely distributed and often abundant oribatid mite S. moestus: 1) increased the mineralization of C and N; 2) acceler- The presence of mites altered the activity of both hydrolytic and ated extracellular enzyme activities; 3) altered the molecular oxidative extracellular enzymes. In corn microcosms, the activities chemistry of decomposing litter; and 4) interacted with variation in initial litter chemistry, indicating that the overall effect of S. moestus of urease (32 d, F5, 48 ¼ 2.88, P < 0.05), glucosidase (48 d, F5, on ecosystem processes depends upon substrate quality. 48 ¼ 2.49, P < 0.05) and acid phosphatase (48 d F4, 40 ¼ 7.32, P < 0.05) were greater in the presence than absence of mites (Fig. 3). The presence of mites also resulted in a 30% increase in the 4.1. C and N cycling and enzyme activities activity of phenol oxidase averaged across sample dates (F1, Some microarthropods have been shown to stimulate C 48 ¼ 8.20, P < 0.01) (Fig. 3). The most marked increase occurred in week 3, when oxidase activity was approximately 2.5 times higher mineralization (Kaneda and Kaneko, 2008) but studies focusing on in microcosms with than without mites. Chitinase, cellobiosidase oribatids are rare (c.f. Kaneko 1998). We observed that C mineral- and tyrosine amino peptidase activities in corn were not affected by ization in the presence of S. moestus increased by 19% in corn and 17% in oak litter. Previous estimates suggest that the entire meso- mites. In oak litter, the presence of mites stimulated glucosidase (F5, faunal community directly accounts for approximately 5% of the 48 ¼ 3.48, P < 0.01) and chitinase (F5, 48 ¼ 3.64, P < 0.01) activity at 48 and 62 d but suppressed chitinase, glucosidase and phosphatase CO2 released from soil during decomposition and mathematical activity during the initial week of the experiment (Fig. 4). Phenol models predict that oribatids should have little impact on microbial C mineralization (Berg et al., 2001). Although our microcosms do not simulate field conditions with fluctuating environmental Table 1 conditions and substrate availability, our results indicate that some Carbon (C) and nitrogen (N) content (mg g 1 dry mass) of corn and oak litter at time zero and after 62 d of incubation with and without mites (P<0.05). species of oribatid mites can strongly enhance mineralization rates (Siepel and Maaskamp, 1994). We also show that S. moestus 62 days increases concentrations of both nitrate and ammonium in Time zero w/ S. moestus w/o S. moestus decomposing litter. The increases in inorganic N that we observed Corn N (mg) 14.33a 11.29b 10.43b were temporally variable but generally similar to or greater than C (mg) 312.39 323.38 327.14 those found in studies using collembolans or other soil animals C:N 21.80a 29.10ab 31.56b (Ineson et al., 1982; Anderson et al., 1985; Bardgett and Chan, 1999). Oak N (mg) 7.48a 6.73b 6.85b The observed changes in C and N cycling are likely due to the direct C (mg) 352.68 343.15 349.33 effects of S. moestus on organic matter mineralization as well as to C:N 47.15a 50.97b 51.04b enhanced microbial enzyme activities and decomposition rates K. Wickings, A.S. Grandy / Soil Biology & Biochemistry 43 (2011) 351e358 355

Fig. 2. Chemical composition of corn (top) and oak (bottom) litter at time zero, after 62 d incubation with and without mites, and after passage through the mite gut. Fecal matter from mites feeding on oak litter was not available. In corn, mite feces had a significantly lower relative abundance of lignin and higher relative abundance of phenols than the other treatments. Polysaccharides also differed (feces > litter; feces > litter þ microbes þ mites; litter þ microbes > litter þ microbes þ mites). All differences noted were significant (P < 0.05) and were determined by one-way analysis of variance. Oak chemical composition did not differ among treatments. Abbreviations: nbear e nitrogen bearing compounds; polysac e polysaccharides; unknown e compounds of unknown origin.

when animals are present (Petersen and Luxton, 1982; Kandeler et al., 1999; Ayres et al., 2010). Indeed, we found that extracellular enzyme activity was 30 urease 15 glucosidase generally higher in litter with mites than without, ranging from 1.3 25 -w/ S. moestus 10 1.2 chitinase glucosidase 20 -w/o S. moestus 1.2 15 1 1 )

-1 5 0.8 0.8 g 10 -1

) 0.6 0.6

5 -1

g 0.4 0.4 0 -w/ S. moestus 0 -1 0.2 -w/o S. moestus 0.2 8 10 0 0 phosphatase phenol oxidase 8 6 2.5 2.5

Activity (µmol h phosphatase phenol oxidase 6 4 2 2 4 Activity (µmol h 1.5 1.5 2 2 1 1 0 0 0.5 0.5 0 15 30 45 60 0 15 30 45 60 0 0 days 0 15 30 45 60 0 15 30 45 60

Fig. 3. Potential extracellular enzyme activity in corn microcosms (mean SE). Phenol days oxidase activity was significantly greater in the presence of S. moestus when averaged across all sample dates and there were significant mite by time interactions for chi- Fig. 4. Potential extracellular enzyme activity in oak microcosms (mean SE). tinase, glucosidase and phosphatase (P < 0.05). Data represent means SE. Significant mite by time interactions were observed for all enzymes (P < 0.05). 356 K. Wickings, A.S. Grandy / Soil Biology & Biochemistry 43 (2011) 351e358

Table 2 Ammonium and nitrate from litter filtrates collected at 13, 34 and 62 d of incubation (nd - not determined, na - not applicable) .

-1 - 1 NH4 -N (mgg dry litter) NO3 -N (mgg dry litter) Corn Oak Corn Oak

13 34 62 13 34 62 13 34 62 13 34 62 w/ S. moestus 3.78 120.00 70.48 10.91 3.52 4.76 nd 7.39 5.16 nd nd nd w/o S. moestus 8.75 24.54 93.49 0.89 2.98 4.00 nd 0.21 6.09 nd nd nd

ANOVA F-test P-value 0.027 0.034 0.636 0.017 0.432 0.417 na 0.001 0.685 na na na

to 2.4 times higher in corn litter and 1.2 to 5 times higher in oak transformations within the mite gut, although we cannot rule out litter. The increases in extracellular enzyme activity may have the possibility that the continuing activity of microbes and extra- resulted from litter fragmentation, which can increase microbial cellular enzymes in feces during the time between defecation and access to previously protected plant polysaccharides (Moorhead sample collection also played a role. Our results contrast with two and Sinsabaugh, 2006; Saqib and Whitney, 2006; Herman et al., past reports using different oribatid species, which showed higher 2008). Microbial activity and extracellular enzyme activities may lignin concentrations in feces than in unprocessed litter (Gasdorf have also been stimulated by microbial grazing as well as the and Goodnight, 1963; Ziechman, 1994). Since we found no addition of labile mite feces with relatively low ratios of lignin:ni- evidence for oxidative enzyme activity in the mite gut, the low trogen and lignin:polysaccharide (Jenkinson et al., 1985; Schimel percentage of lignin in feces likely resulted from selective feeding and Weintraub, 2003). on labile microbial and plant compounds (Hubert et al., 2000). While selective feeding likely explains differences in lignin relative abundance between fecal material and unprocessed plant 4.2. Litter chemistry litter, differences in other chemical constituents may have been due to enzymatic activity within the S. moestus gut. We found that S. Determining the effects of soil animals on C and N cycling also moestus had high activities of chitinase (27 times higher than plant requires a more complete understanding of how they influence litter), cellulase (8 times higher) and phosphatase (5 times higher), litter chemistry during decomposition, as variations in chemistry indicating that along with other microarthropods it possesses can affect nutrient cycling and long-term soil organic matter a variety of digestive enzymes capable of catabolizing diverse dynamics (Rubino et al., 2009; Kleber and Johnson, 2010). Recent organic compounds (Siepel and de Ruiter-Dijkman, 1993; Berg studies have shown that ecosystem N enrichment (Neff et al., 2002; et al., 2004; Norton and Behan-Pelletier, 2009). Our study did not Gallo et al., 2005; Grandy et al., 2008) and agricultural conversion determine the origin of this enzymatic activity but microbial gut (Grandy et al., 2009; Wickings et al., 2010) can alter organic matter symbionts within the oribatid gut likely play a key role in gut chemistry. Wickings et al. (2010) argued that differences in litter enzyme production and litter processing (Stefaniak and Seniczak, chemistry during decomposition may arise from variations in 1976; Seniczak and Stefaniak, 1978; Ayres et al., 2010). Whether decomposer communities. However, these studies were not able to these enzymes are produced by the animal itself or by gut-inhab- isolate the effects of decomposer communities from other abiotic or iting microbes, the soil animal gut is thus a hotspot of enzymatic biogeochemical processes that influence soil C dynamics in the activity, as well as of chemical transformations. field. Our study firmly establishes that a single oribatid species can influence changes in litter chemistry during decomposition. We compared the chemistry of decomposing litter with and without 4.3. Litter quality mites and found that S. moestus decreased the relative abundance of polysaccharides in corn litter by 7% during decomposition. This Changes in litter chemistry due to the presence of S. moestus difference is likely due in part to the indirect effects of S. moestus on were limited to corn litter. We attribute this to the initial differ- microbial activity. S. moestus stimulated microbial extracellular ences in chemical structure of corn and oak litter and its effects on enzyme activity in litter, and changes in enzyme activity may alter microbial activity. Before decomposition, oak litter had a greater the trajectory of chemical changes in decomposing organic matter relative abundance of long-chain lipids, which are found in unde- (Grandy et al., 2008, 2009; Wickings et al., 2010). composed plant material (Spaccini et al., 2009) and likely origi- We also found that S. moestus feces, which contain both plant nated in the thick, waxy cuticle of oak leaves. While lignin may and microbial material, are relatively depleted in lignin, enriched in break down relatively rapidly (Rasse et al., 2006; Denef et al., 2009), phenols and possess lower ratios of lignin:polysaccharide and lig- long-chain lipids can be very recalcitrant and selectively preserved nin:nitrogen than unprocessed . These measured differ- during decomposition (Lorenz et al., 2007; Grandy et al., 2008; ences between litter and feces chemistry primarily reflect Denef et al., 2009). Indeed, C and N mineralization rates and

Table 3 Dissolved organic carbon and total organic nitrogen from litter filtrates collected at 13, 34 and 62 d of incubation.

DOC (mg g 1 dry litter) DON (mg g 1 dry litter)

Corn Oak Corn Oak

13 34 62 13 34 62 13 34 62 13 34 62 w/ S. moestus 11.36 30.68 17.33 5.51 9.82 8.54 0.70 2.40 1.30 0.08 0.23 0.20 w/o S. moestus 8.97 16.42 19.74 5.58 10.57 10.18 0.57 1.16 1.44 0.09 0.22 0.23

ANOVA F-test P-values 0.453 0.000 0.686 0.981 0.179 0.226 0.353 0.005 0.591 0.549 0.853 0.373 K. Wickings, A.S. Grandy / Soil Biology & Biochemistry 43 (2011) 351e358 357 enzyme activities, all indicators of microbial activity, were lower in Cortet, J., Joffre, R., Elmholt, S., Krogh, P.H., 2003. Increasing species and trophic oak litter. This provides additional evidence that oak litter was diversity of mesofauna affects fungal biomass, mesofauna community structure and organic matter decomposition processes. Biology and Fertility of Soils 37, more recalcitrant than corn litter, but also points to the probability 302e312. that the effects of decomposer communities on litter chemical Denef, K., Plante, A.F., Six, J., 2009. Characterization of soil organic matter. In: transformations may depend upon the quality of the substrate Kutsch, W.L., Bahn, M., Heinemeyer, A. (Eds.), Soil Carbon Dynamics: an Integrated Methodology. Cambridge University Press, Cambridge UK, pp. being decomposed (Kampichler and Bruckner, 2009; Wickings 91e126. et al., 2010). Doane, T.A., Horwath, W.A., 2003. Spectrophotometric determination of nitrate with a single reagent. Analytical Letters 36, 2713e2722. Ekschmitt, K., Liu, M., Vetter, S., Fox, O., Wolters, V., 2008. Strategies used by soil 4.4. Summary biota to overcome soil organic matter stability e why is dead organic matter left over in the soil? Geoderma 128, 167e176. Soil animals are broadly known to have a range of effects on Filley, T.R., McCormick, M.K., Crow, S.E., Szlaveck, K., Whigham, D.F., Johnston, C.T., fi van den Heuvel, R.N., 2008. Comparison of the chemical alteration trajectory of decomposition dynamics. However, at a ner-scale there remain Liriodendron tulipifera L. leaf litter among forests with different many key uncertainties, including the effects of individual species abundance. Journal of Geophysical Research 113, 1e14. on decomposition processes, and the degree to which animals can Gallo, M.E., Lauber, C.L., Cabaniss, S.E., Waldrop, M.P., Sinsabaugh, R.L., Zak, D.R., 2005. Soil organic matter and litter chemistry response to experimental N alter the chemistry of litter during decomposition (Ayres et al., deposition in northern temperate deciduous forest ecosystems. Global Change 2010). We have demonstrated that the presence of a single orib- Biology 11, 1514e1521. atid mite species can enhance C mineralization rates by almost 20%, Gasdorf, E.C., Goodnight, C.J., 1963. Studies on the ecology of soil . Ecology e stimulate hydrolytic and oxidative enzyme activities and change 44, 261 268. Geib, S.M., Filley, T.R., Hatcher, P.G., Hoover, K., Carlson, J.E., Jiminez-Gasco, M., litter chemistry during decomposition. No other studies have come Nakagawa-Izumi, A., Sleighter, R.L., Tien, M., 2008. Lignin degradation in wood- to our attention that show molecular-scale differences in the feeding insects. Proceedings of the National Academy of Sciences 105, e chemistry of oribatid feces compared to litter, or of litter decom- 12932 12937. Gergócs, V., Hufnagel, L., 2009. Application of oribatid mites as indicators (review). posing with and without mites. These changes in litter chemistry Applied Ecology and Environmental Research 7, 79e98. during decomposition varied with initial substrate quality and Gillon, D., David, J.F., 2001. The use of near infrared reflectance spectroscopy to could have long-term implications for soil C cycling. Future studies study chemical changes in the leaf litter consumed by saprophagous inverte- brates. Soil Biology & Biochemistry 33, 2159e2161. comparing different oribatid mite species, different groups of Golchin, A., Oades, J.M., Skjemstad, J.O., Clarke, P., 1994. Study of free and occluded microarthropods and different trophic levels under more realistic particulate organic-matter in soils by solid-state C-13 CP/MAS NMR-spectros- conditions will help elucidate the effects of individual species and copy and scanning electron-microscopy. Australian Journal of Soil Research 32, 285e309. functional groups on decomposition. Grandy, A.S., Sinsabaugh, R., Neff, J., Stursova, M., Zak, D., 2008. Nitrogen deposition effects on soil organic matter chemistry are linked to variation in enzymes, e Acknowledgements ecosystems and size fractions. Biogeochemistry 91, 37 49. Grandy, A.S., Neff, J.C., Weintraub, M.N., 2007. Carbon structure and enzyme activities in alpine and forest ecosystems. Soil Biology & Biochemistry 39, The authors thank Roy Norton, SUNY Environmental Science 2701e2711. and Forestry, Syracuse, NY for assistance in identification of Sche- Grandy, A.S., Strickland, M.S., Lauber, C.L., Bradford, M.A., Fierer, N., 2009. The influence of microbial communities, management, and soil texture on soil loribates moestus to species and for advice on the maintenance of organic matter chemistry. Geoderma 150, 278e286. mite populations in the laboratory. We also thank David Weed and Guggenberger, G., Thomas, R.J., Zech, W., 1996. Soil organic matter within earth- Steve Hamilton at the Kellogg Biological Station LTER, Hickory worm casts of an anecic-endogeic tropical pasture community. Colombia Applied Soil Ecology 3, 263e274. Corners, MI for their assistance in the measurement of dissolved Hanlon, R.D.G., Anderson, J.M., 1979. The effects of Collembola grazing on microbial organic carbon and nitrogen from litter filtrates. Roy Norton and activity in decomposing leaf litter. Oecologia 38, 93e99. Deborah Neher provided insightful comments on earlier drafts of Hansen, R.A., 1999. Red oak litter promotes a microarthropod functional group that e this manuscript. accelerates its decomposition. Plant and Soil 209, 37 45. Hedde, M., Bureau, F., Akpa-Vinceslas, M., Aubert, M., Decaens, T., 2007. Beech leaf degradation in laboratory experiments: effects of eight detritivorous inverte- Appendix. Supplementary data brate species. Applied Soil Ecology 35, 291e301. Heneghan, L., Coleman, D.C., Zou, X., Crossley, D.A., Haines, B.L., 1999. Soil micro- contributions to decomposition dynamics: tropical-temperate Supplementary data related to this article can be found online at comparisons of a single substrate. Ecology 80, 1873e1882. doi:10.1016/j.soilbio.2010.10.023. Herman, J., Moorhead, D., Berg, B., 2008. The relationship between rates of lignin and cellulose decay in aboveground forest litter. Soil Biology & Biochemistry 40, 2620e2626. References Hopkins, D.W., Chudek, J.A., Bigness, D.E., Frouz, J., Webster, E.A., Lawson, T., 1998. Application of 13C NMR to investigate the transformations and biodegradation Anderson, J.M., Leonard, M.A., Ineson, P., Huish, S.A., 1985. Faunal biomass: a key of organic materials by wood- and soil-feeding termites, and a coprophagous component of a general model of nitrogen mineralization. Soil Biology & litter-dwelling dipteran . Biodegradation 9, 423e431. Biochemistry 17, 735e737. Hubert, J., Kubatova, A., Sarova, J., 2000. Feeding of Scheloribates laevigatus (Acari: Ayres, E., Wall, D.H., Bardgett, R.D., 2010. Trophic interactions and their implications Oribatida) on different stadia of decomposing grass litter (Holcus lanatus). for soil carbon fluxes. In: Kutsch, W.L., Bahn, M., Heinemeyer, A. (Eds.), Soil Pedobiologia 44, 627e639. Carbon Dynamics: an Integrated Methodology. Cambridge University Press, Ineson, P., Leonard, M.A., Anderson, J.M., 1982. Effect of collembolan grazing upon Cambridge UK, pp. 187e206. nitrogen and cation leaching from decomposing leaf litter. Soil Biology and Bardgett, R.D., Chan, K.F., 1999. Experimental evidence that soil fauna enhance Biochemistry 14, 601e605. nutrient mineralization and plant nutrient uptake in montane grassland Jenkinson, D.S., Fox, R.H., Rayner, J.H., 1985. Interactions between fertilizer nitrogen ecosystems. Soil Biology & Biochemistry 31, 1007e1014. and soil-nitrogen e the so-called priming effect. Journal of Soil Science 36, Bardgett, R.D., Keiller, S., Cook, R., Gilburn, A.S., 1998. Dynamic interactions between 425e444. soil animals and microorganisms in upland grassland soils amended with sheep Kampichler, C., Bruckner, A., 2009. The role of microarthropods in terrestrial dung: a microcosm experiment. Soil Biology & Biochemistry 30, 531e539. decomposition: a meta-analysis of 40 years of litterbag studies. Biological Berg, M.P., de Ruiter, P., Didden, W., Janssen, M., Schouten, T., Verhoef, H., 2001. Reviews 84, 375e389. Community food web, decomposition and nitrogen mineralisation in a strati- Kaneda, S., Kaneko, N., 2008. Collembolans feeding on soil affect carbon and fied Scots pine forest soil. Oikos 94, 130e142. nitrogen mineralization by their influence on microbial and nematode activi- Berg, M.P., Stoffer, M., van den Heuvel, H.H., 2004. Feeding guilds of Collembola ties. Biology and Fertility of Soils 44, 435e442. based on digestive enzymes. Pedobiologia 48, 589e601. Kandeler, E., Kampichler, C., Joergensen, R.G., Mölter, K., 1999. Effects of mesofauna Coleman, D.C., 2008. From peds to paradoxes: linkages between soil biota and their in a spruce forest on soil microbial communities and N cycling in field meso- influences on ecological processes. Soil Biology & Biochemistry 40, 271e289. cosms. Soil Biology and Biochemistry 31, 1783e1792. Coleman, D.C., Crossley Jr., D.A., Hendrix, P.F., 2004. Fundamentals of Soil Ecology, Kaneko, N., 1998. Do mites and Collembola affect pine litter fungal biomass and second ed. Elsevier Academic Press, Burlington, MA. microbial respiration? Applied Soil Ecology 9, 209e213. 358 K. Wickings, A.S. Grandy / Soil Biology & Biochemistry 43 (2011) 351e358

Kleber, M., Johnson, M.G., 2010. Advances in understanding the molecular structure Saiya-Cork, K.R., Sinsabaugh, R.L., Zak, D.R., 2002. The effects of long term nitrogen of soil organic matter: implications for interactions in the environment. deposition on extracellular enzyme activity in an Acer saccharum forest soil. Advances in Agronomy 106, 77e142. Soil Biology & Biochemistry 34, 1309e1315. Kleber, M., Sollins, P., Sutton, R., 2007. A conceptual model of organo-mineral Saqib, A.A.N., Whitney, P.J., 2006. Role of fragmentation activity in cellulose interactions in soils: self-assembly of organic molecular fragments into zonal hydrolysis. International Biodeterioration and Biodegradation 58, 180e185. structures on mineral surfaces. Biogeochemistry 85, 9e24. Schimel, J.P., Weintraub, M.N., 2003. The implications of exoenzyme activity on Lenoir, L., Persson, T., Bengtsson, J., Wallander, H., Wiren, A., 2007. Bottom-up microbial carbon and nitrogen limitation in soil: a theoretical model. Soil and top-down control in forest soil microcosms? Effects of soil fauna on Biology & Biochemistry 35, 1e15. fungal biomass and C/N mineralization. Biology and Fertility of Soils 43, Seniczak, S., Stefaniak, O., 1978. The microflora of the alimentary canal of Oppia 281e294. nitens (Acarina, Oribatei). Pedobiologia 18, 110e119. Lorenz, K., Lal, R., Preston, C.M., Nierop, K.G.J., 2007. Strengthening the soil organic Siepel, H., 1990. Niche relationships between two panphytophagous soil mites, carbon pool by increasing contributions from recalcitrant aliphatic bio (macro) Nothrus silvestris Nicolet (Acari, Oribatida, ) and Platynothrus peltifer molecules. Geoderma 142, 1e10. (Koch) (Acari, Oribatida, )*. Biology and Fertility of Soils 9, 139e144. Moorhead, D.L., Sinsabaugh, R.L., 2006. A theoretical model of litter decay and Siepel, H., de Ruiter-Dijkman, E.M., 1993. Feeding guilds of oribatid mites based on microbial interaction. Ecological Monographs 76, 151e174. their carbohydrase activities. Soil Biology & Biochemistry 25, 1491e1497. Neff, J.C., Townsend, A.R., Gleixner, G., Lehman, S.J., Turnbull, J., Bowman, W.D., Siepel, M., Maaskamp, F., 1994. Mites of different feeding guilds affect decomposi- 2002. Variable effects of nitrogen additions on the stability and turnover of soil tion of organic matter. Soil Biology & Biochemistry 26, 1389e1394. carbon. Nature 419, 915e917. Sinsabaugh, R.L., Reynolds, H., Long, T.M., 2000. Rapid assay for amidohydrolase (urease) Norton, R.A., Behan-Pelletier, V.M., 2009. Oribatida. In: Krantz, G.W., Walter, D.E. activity in environmental samples. Soil Biology & Biochemistry 32, 2095e2097. (Eds.), A Manual of Acarology. Texas Tech Univ. Press, Lubbock, TX, pp. 430e564. Spaccini, R., Sannino, D., Piccolo, A., Fagnano, M., 2009. Molecular changes in organic Petersen, H., Luxton, M., 1982. A Comparative analysis of soil fauna populations and matter of a compost-amended soil. European Journal of Soil Science 60, 287e296. their role in decomposition processes. Oikos 39, 287e388. Stefaniak, O., Seniczak, S., 1976. The microflora of the alimentary canal of Achipteria Rasse, D.P., Dignac, M.F., Bahri, H., Rumpel, C., Mariotti, A., Chenu, C., 2006. Lignin coleoptrata (Acarina, Oribatei). Pedobiologia 16, 185e194. turnover in an agricultural field: from plant residues to soil-protected fractions. Teuben, A., Verhoef, H.A., 1992. Direct contribution by soil arthropods to nutrient European Journal of Soil Science 57, 530e538. availability through body and fecal nutrient content. Biology and Fertility of Rawlins, A.J., Bull, I.D., Poirier, N., Ineson, P., Evershed, R.P., 2006. The biochemical Soils 14, 71e75. transformation of oak (Quercus robur) leaf litter consumed by the pill millipede Wickings, K., Grandy, A.S., Reed, S.C., Cleveland, C.C., 2010. Management intensity (Glomeris marginata). Soil Biology & Biochemistry 38, 1063e1076. alters decomposition via biological pathways. Biogeochemistry. doi:10.1007/ Rubino, M., Lubritto, C., D’Onofrio, A., Terrasi, F., Kramer, C., Gleixner, G., s10533-010-9510-x. Cotrufo, M.F., 2009. Isotopic evidences for microbiologically mediated and Wolters, V., 2000. Invertebrate control of soil organic matter stability. Biology and direct C input to soil compounds from three different leaf litters during their Fertility of Soils 31, 1e19. decomposition. Environmental Chemistry Letters 7, 85e95. Ziechman, W., 1994. Humic Substances. Wissenschaftsverlag, Mannheim.