Macroinvertebrate detritivores directly and indirectly affect ecosystem processes and nutrient cycling, but the effects of detritivore interactions on ecosystem processes remain poorly understood. Furthermore, mounting evidence emphasizes the importance of understanding the effects and implications of environmental disturbance on interactions between soil organisms, including invasive species and climate change.
In this study, we asked: Is there evidence of competitive or complementary interactions between a native detritivore and two non-native detritivores? Additionally, can detritivore species-specific and interspecific interaction effects on soil ecosystem processes and microbial activity, be moderated by temperature?
To answer these questions, we performed a mesocosm experiment which included three detritivore species, a millipede native to North America (N. americanus) and two introduced earthworm species (L. rubellus and L. terrestris). We fully replicated a Simplex mixture design using these species under two temperature treatments, ambient and warmed 3.3°C.
We expected to observe species-specific and complementary effects of the study organisms due to differences in functional traits. Furthermore, we anticipated that temperature would alter species interactions, and warming would exert a disproportionately greater negative effect on surface dwelling millipedes. Overall, we expected invasive earthworm effects to overwhelm the effects of millipede presence.
An interaction between L. rubellus and N. americanus predicted an increase in litter mass lost and microbial biomass C, indicating a potential complementarity effect. N. americanus reduced both NAG and BG enzyme activity. L. terrestris reduced NAG and BG activity at warmed temperature, but increased activity at ambient temperature. An interaction effect between L. rubellus and L. terrestris also predicted a reduction in NAG activity. Earthworm biomass was significantly reduced over the duration of the experiment regardless of temperature treatment. L. terrestris significantly increased NH4+ leaching, and detritivores did not significantly affect carbon (CO2) efflux.
There was no evidence to indicate that interspecific interactions between these detritivores are moderated by temperature. However, these results indicate that N. americanus may exert some biotic resistance to invasion pressures by L. rubellus and L. terrestris. Future experiments may consider manipulations of food resources, additional trophic levels, and physical soil characteristics to parse out underlying mechanisms.
These data were collected with the purpose of better understanding interspecific interactions between native and non-native forest floor detritivores. Furthermore, these data were used to better understand the role temperature plays in moderating detritivore interactions and the effects of detritivores on ecosystem processes and microbial communities
Mesocosms were established on a covered porch at the University of Michigan Biological
Station (UMBS) in Pellston, MI, USA (45-35.5°N, 84-43°W). UMBS soils (used in this
experiment) are acidic (pH 4.8), and classified as mixed, frigid Entic Haplorthods of the Rubicon
series (92.9% sand, 6.5% silt, 0.6% clay). Local dominant tree species include bigtooth aspen
(Populus grandidentata), red maple (Acer rubrum), northern red oak (Quercus rubra), paper
birch (Betula papyrifera), Eastern white pine (Pinus strobus), and American beech (Fagus
grandifolia). Average (1979-2010) summer (June-August) temperature and precipitation at
UMBS is 17.8 °C and 219 mm respectively (Nave et al., 2011). Experimental Design A total of 40 mesocosms were constructed and subjected to one of two temperature
treatments, ambient or elevated 3.3°C above ambient (which corresponds to IPCC projections for
northern Michigan (IPCC, 2013)), resulting in 20 mesocosms per temperature treatment. Within
each temperature treatment, detritivore assemblages comprised of two invasive earthworm
species (L. rubellus and L. terrestris) and a native millipede (N. americanus) were introduced to
mesocosms following a Simplex design (Cornell, 2011) (Figure 1).
The Simplex design, which has been successfully implemented in other studies on
diversity-functioning relationships among soil detritivores (Andriuzzi et al., 2015; O’Hea et al.,
2010; Piotrowska et al., 2013; Sheehan et al., 2006; Sheehan et al., 2007; Sheehan et al., 2008),
allowed us to manipulate detritivore biomass on a continuous spectrum. Biomass was used as a
proxy for species richness and evenness, and explicitly manipulated on the assumption that, in
soil systems, biomass is intrinsically linked to an organism’s functional impact (Bradford et al.,
2002; Bílá et al., 2014; Turnbull et al., 2014; van Geffen et al., 2011). By controlling biomass as
a measure of evenness, the Simplex design further allowed us to probe into the strength of
species interactions (Petchey & Gaston, 2002; Kirwan et al., 2007).
The purpose of the Simplex design is to assess mixture performances of species
combinations including: single species monocultures, ‘centroids’ where each species contributes
equally to total detritivore biomass, and intermediary combinations of biomass (Figure 1). Single
species monocultures and centroids were duplicated to account for losses due to mortality, and
three detritivore-free mesocosms served as controls under each temperature treatment.
Replicating the other species combination treatments was not necessary because they encompass
a continuous range of species co-occurrences as opposed to factorial combinations, which allows
the data to be analyzed using multiple regression. The Simplex design is advantageous to
diversity-functioning studies as it makes possible the ability to differentiate (a) species identity
effects from the effects of interspecific interactions as well as (b) interaction effects from
biomass effects on ecosystem processes (Kirwan et al., 2007; Kirwan et al., 2009).
Each mesocosm containing detritivores received approximately 6 grams of detritivore
biomass (which is comparable to the higher end of total detritivore biomass densities observed at
this site, (Crumsey et al., 2014)). Total biomass was relatively consistent across mesocosms with
small discrepancies between ideal values dictated by the Simplex design, and actual values (±
1.8% on average, Range: 0.808 g). Discrepancies in biomass by species for each combination
were also relatively small (± 4.5% on average). The number of individuals in each bucket was
also kept as even as possible (± 1 N. americanus individual, ± 1 L. terrestris individual, ± 1 to 2
L. rubellus individuals) across treatments to reduce variation associated with the number of intra-
and interspecific interactions.
Mesocosm Construction and Maintenance Mesocosms were constructed using 5-gallon plastic buckets (height 14.5”, diameter at top 11.9”, diameter at bottom 10.33”). A circular hole 6 cm in diameter was cut into the center of the bottom of each bucket and covered with 2 mm mesh to allow for adequate drainage and to
prevent organisms from escaping. Funnels were then attached underneath each hole and fixed
with removable plastic bottles that were used to collect leachate (Figure 2). The buckets were
placed underneath a porch overhang and suspended 3 feet off the ground between wooden planks
Soil was collected during the second week of May 2016. Litter and surface detritus were
raked away before soil was removed in layers by shovel. Collected soil was separated into B, E,
and mixed O/A horizons and placed into separate plastic bins by layer for 24 hours. Each layer
was then sieved through ¼ inch wire, which removed large rocks and macroinvertebrates, but
maintained much of the naturally occurring soil aggregates in each layer. Each layer was
homogenized separately using the cone and quarter method (Gerlach et al., 2002). This process
involves: piling soil onto a plastic tarp forming a cone shape, raking quartered sections of the
pile towards four opposing directions, and shoveling the distributed soil around to other quarters
to evenly disperse the soil before reforming the original cone. The cone and quarter method was
applied to each layer 3 times, and each layer was visually inspected for residual
macroinvertebrates before being added to the mesocosms. Four liters of B horizon were added
first to each bucket, followed by four liters of E horizon and finally seven liters of O/A horizon.
These proportions deviate slightly from field proportions. More organic O/A horizon was
included to support detritivore and microbial populations in the mesocosms. The mesocosms
were then allowed stabilize for four weeks prior to start of the experiment. During this time, 200
mL of distilled water was added to each mesocosms weekly to prevent drying and death of
Circles (25.4 cm diameter) were cut out of the accompanying bucket lids so that only the
lip remained; this allowed for ease of removal and replacement when measuring respiration.
Mesh screening (1 mm) was fitted to cover the frame to allow for opening air exchange, as well
as to prevent organisms from entering and leaving the mesocosms.
Mixed diversity litter composed of six species (see Study System for species descriptions)
from a forest stand burned in 1954 was collected, dried, and stored in trash bags in a climate
controlled laboratory prior to the start of the experiment. Litter bags, composed of 10 g dried,
leaf litter representative of dominant local hardwood species and 4 g of quaking aspen wood
chips, were constructed using fish netting (hole width approximately 12.7 mm). This material
was used as it facilitated large detritivore movement through the litter bags.
Litter bags were randomly assigned to buckets, and mesocosms were randomly assigned
temperature and detritivore treatments. Heat bulbs (Phillips® 250W 120V) were suspended
above the surface of mesocosms to raise air temperature 3.3 °C (± 0.25°C) above ambient at the
surface of the soil in the elevated temperature treatments (Figure 4). A single heat lamp was used
to warm 4 mesocosms, so mesocosms were clustered into groups of 4 for a total of 10 groups of
4, with 5 groups under each temperature treatment (Figure 3). These groups were treated as
blocks in the statistical analyses.
I-Buttons (DS1925, Maxim Integrated, San Jose, CA) were used to monitor temperature
changes over the duration of the experiment. I-Buttons were placed in each mesocosm (40 total)
at the soil surface at the edge of the bucket furthest from the heat lamps and bucket cluster
Mesocosm moisture content was monitored using gravimetric methods. The mass of each
bucket with soil at field moisture capacity was measured at the start of the experiment and was
adjusted weekly using DO water as needed. Volumetric water content of each mesocosm was
only recorded at the conclusion of the experiment in order to avoid altering detritivore burrowing
and efflux measurements. Detritivore Assemblages Detritivores were hand collected from forests surrounding UMBS during late May 2016.
L. rubellus and L. terrestris were retrieved using an electroshock extraction method (Crumsey et
al., 2014). Due to difficulties finding mature L. terrestris individuals in late Spring, individuals
purchased from bait shops were used as supplements. To avoid biases towards using purchased
versus collected L. terrestris individuals in mesocosms, all L. terrestris individuals were pooled
in single terrarium and selected randomly when assigned to mesocosms. All detritivores were
housed in the climate controlled Lakeside Laboratory (21°C) at UMBS and separated into
terrariums by species for two weeks prior to the start of the study. Each terrarium contained soils
and litter representative of UMBS forests.
Before being introduced to mesocosms, earthworms were identified to species, and only
individuals that appeared healthy were used. Due to difficulties in collecting adult L. terrestris
with low enough biomass requirements for some Simplex proportions dictated by the design,
juveniles were used as necessary. To avoid the possibility of confusing L. rubellus juveniles with
L. terrestris, only juveniles that were observed leaving burrows in habitat types primarily
dominated by L. terrestris during electroshocking were used in the study on the assumption that
these burrows were early indications of anecic burrowing behavior. At the conclusion of the
study, the matured juveniles were identified and confirmed as L. terrestris in all cases.
The earthworms and millipedes were placed in containers with moist paper towels and
allowed to clear their guts for 24 hours prior to being introduced to the mesocosms. The mass of
each organism was recorded before being placed on the mesocosm surface soil and allowed to
Temperature treatments began one week after detritivore treatments to allow for adequate
acclimation. Casting, molting, and soil surface mortality were observed and recorded throughout
the duration of the experiment.
Carbon Efflux was measured weekly using a LI-6400 (LICOR, Lincoln, NE).
Bottles of collected leachate were analyzed at the end of the mesocosm stabilization period, at four weeks, and at mesocosm harvest, for a total of three separate collection dates. The day before each collection date, each mesocosm received 500 mL of DO water and bottles were
collected 24 hours later. Each sample was acidified with 6N TMG HCl and filtered prior to analysis (Fisherbrand™ Glass Fiber Circles, Whatman GF/F). Ammonium (NH4+) concentration was analyzed using the automated phenate method (Bran + Leubbe® AA3). Nitrate (NO3-)
concentration was determined using IC (ThermoFisher™/Dionex™ Integrion HPIC™); column set AS-11 HC (4 micron), isocratic separation using 30 mM KOH as eluent. Total leachate NH4+ and NO3- are reported as µg/L and mg/L respectively. Mesocosm Harvest The experiment ran for a total of 8 weeks and mesocosms were harvested randomly over
a three-day period.
On the first day of harvest, litter bags were removed from the surface of the soil and
visually inspected to detect if any of the study organisms were inside the bags. The litter bags
were then sealed inside plastic bags to ensure no litter was lost prior to drying. Two soils cores at
a depth of 15 cm were taken from the soil surface; one core was extracted from directly beneath
the litter bag, and another core was taken randomly around the circumference of the surface soil.
When detritivore burrows were present at the soil surface, the second core was targeted on those
areas. The reasoning behind this comes from evidence that suggests the chemical and physical
properties of detritivore burrows and casts are species-specific and affect microbial community
functioning as well as soil ecosystem dynamics (Jégou et al., 2001). Millipede molts were not
collected, but recorded, due to similar evidence which suggests that arthropod molts affect
microbial community functioning (Cabib, 1987). Soil samples were placed in large plastic bags,
homogenized for 5 minutes, and stored in a 4°C refrigerator.
On the second harvest day, half of the mesocosms were destructively sampled to remove
surviving organisms. The remaining half of mesocosms were harvested on the third day. The
remaining organisms were then placed in containers with moist paper towels for 24 hours to
clear their guts after being extracted from the mesocosms. After 24 hours, the final biomass of
each organism was recorded to determine change in biomass. Enzyme Assays Immediately following collection, soils were placed in sealed plastic bags and stored at
4°C for six days. Soils were then placed into large coolers with ice and transported to the
University of Toledo for enzyme analysis. Hydrolytic (β-1,4-glucosidase (BG) and N
acetylglucosaminidase (NAG)) and oxidative (phenol oxidase (POX) and peroxidase (PER))
microbial enzyme activity were measured using fluorimetric methods six days after soil
collection following the methods outlined in (Saiya-Cork et al., 2002). The oxidative enzyme
levels were found to be below detection limits, and are therefore not reported here.
Enzyme activity is typically used as a proxy for microbial nutrient demand (Schimel &
Weintraub, 2003). The main function of BG is to hydrolyze cellobiose (a dissacharide derivative
of cellulose) to release glucose for microbial C-acquisition (Sinsabaugh, 2005). NAG exhibits an
analogous function, but acts on chitin, hydrolyzing chitobiose (a nitrogenous glucosamine dimer)
to monomers for microbial N-acquisition (Sinsabaugh et al., 2008). Enzyme activity is reported
as nmol per hour per gram dry soil.
Fluorometrically labelled enzyme substrates were prepared one day prior to assays using
4-methylumbelliferone (MUF) to make 4-MUF-β-D-glucopyranoside and 4-MUF-N-acetyl-β-D
glucosaminide for BG and NAG respectively. The average soil pH of 10 randomly sampled
mesocosms (~ 5.3) was used as the reference point for preparing the modified universal buffer
(MUB) pH. Approximately 1 g (± 0.1 g) of each sample was added to 125 mL plastic bottles and
slurried with ~125 mL of MUB. Each sample then was poured into a wide mouth plastic
container and was continuously stirred. While stirring, 200 μL aliquots of each sample was
pipetted into 96-well microplates for a total of 16 replicates per sample per assay. Plates were
allowed to incubate for 5 hours at 20°C prior to reading. Following the incubation period,
fluorescence was measured for each well using a BioTek Synergy HT microplate reader (BioTek
Instruments Inc., Winooski VT, USA). Microbial Biomass C and N Extractable microbial C (DOC) and N (DON) were determined using the chloroform
fumigation-extraction technique outlined in Brookes et al. (1985) and modified by Scott-Denton
et al. (2006). Approximately 5 g (± 0.1 g) of each soil sample was added to separate centrifuge
tubes and extracted with 25 mL 0.5 M K2SO4. Two sets of centrifuge tubes per sample as well as
blanks were prepared to account for fumigated and non-fumigated samples. Prepared centrifuge
tubes were placed horizontally on a shaker table and allowed to shake for 1 hour. One set of
centrifuge tubes was placed under a fume hood with 2 mL ethanol-free chloroform where it was
allowed to fumigate for 24 hours. Additionally, both sets of extracts were filtered using
Whatman #1 filter paper and diluted at a 1:10 ratio prior to TOC analysis. The diluted extracts
were analyzed using a Shimadzu TOC-Vcpn total organic carbon analyzer with a total N module
(Shimadzu Scientific Instruments Inc., Columbia, MD, USA). Microbial biomass carbon and
nitrogen were determined by subtracting DOC and TN in the unfumigated samples from the
DOC and TN in the fumigated samples. Organic C and N (DOC and DON) are reported as µg
per gram dry soil.
Following removal from the mesocosms, litter bags were placed in a drying oven at 70°C
for 24 hours. Soil that had adhered to the bags was carefully removed before the mass of the
litter was determined.
Automated Phenate (Bran + Leubbe® AA3)
IC (ThermoFisher™/Dionex™ Integrion HPIC™); column set AS-11 HC (4 micron), isocratic separation using 30 mM KOH as eluent
BioTek Synergy HT microplate reader (BioTek Instruments Inc., Winooski VT, USA)
Shimadzu TOC-Vcpn total organic carbon analyzer with a total N module (Shimadzu Scientific Instruments Inc., Columbia, MD, USA)
I-Buttons (DS1925, Maxim Integrated, San Jose, CA)
Heat bulbs (Phillips® 250W 120V)
LI-6400 (LICOR, Lincoln, NE)