Ant-mediated effects on spruce litter decomposition, solution chemistry, and microbial activity

Ant-mediated effects on spruce litter decomposition,

solution chemistry, and microbial activity

Bernhard Stadlera,d,*, Andreas Schrammb,e, Karsten Kalbitzc

aDepartment of Animal Ecology, Bayreuth Institute for Terrestrial Ecosystem Research (BITOK),

University of Bayreuth, D-95440 Bayreuth, GermanybDepartment of Ecological Microbiology, Bayreuth Institute for Terrestrial Ecosystem Research (BITOK),

University of Bayreuth, D-95440 Bayreuth, GermanycDepartment of Soil Ecology, Bayreuth Institute for Terrestrial Ecosystem Research (BITOK),

University of Bayreuth, D-95440 Bayreuth, GermanydHarvard University, Harvard Forest, P.O. Box 68, Petersham, MA 01366, USA

eDepartment of Microbiology, Institute of Biological Sciences, University of Aarhus, Ny Munkegade,

Building 540, DK-8000 Aarhus C, Denmark

Received 18 January 2005; received in revised form 7 June 2005; accepted 11 June 2005

Available online 20 July 2005

Abstract

Forest management practices often generate clear-cut patches, which may be colonized by ants not present in the same densities in mature

forests. In addition to the associated changes in abiotic conditions ants can initiate processes, which do not occur in old-growth stands. Here,

we analyse the effects of ants and aphid honeydew on litter solution of Norway spruce, microbial enzyme activities, and needle

decomposition in a field and greenhouse experiment during summer 2003. In the field, low ant densities had relatively little effects on litter

solution 30 cm away from a tree trunk, but significantly increased organic carbon concentrations and decreased inorganic nitrogen

concentrations next to a trunk where ants tend to build their nests. In a greenhouse experiment, the addition of ants to lysimeters containing

spruce litter significantly increased dissolved organic carbon (DOC), dissolved organic nitrogen (DON), NH4–N, NO3–N and K

concentrations in litter solutions compared to the control treatment, while the simulation of aphid infestation (addition of honeydew)

significantly increased DOC as a direct result of honeydew leaching, and decreased inorganic N concentrations in leachates. The presence of

ants resulted in a changed composition of dissolved organic matter (DOM) with more aromatic and complex compounds, and microbial

enzyme activity was significantly higher in litter extracts from the ant treatment compared to the honeydew and control treatment. However,

mass loss, litter %C and %N were not affected by ants or honeydew. Our results suggest that ants have a distinct and immediate effect on

solution composition and microbial activity in the litter layer indicating accelerated litter decay whereas the effect of honeydew was

insignificant.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Ants; Decomposition; Formica polyctena; Honeydew; Litter solution chemistry; Microbial activity; Needle litter

1. Introduction

In Central European forests stand structure is now often

converted from monospecific into mixed species stands,

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.soilbio.2005.06.010

* Corresponding author. Address: Department of Animal Ecology,

Bayreuth Institute for Terrestrial Ecosystem Research (BITOK), University

of Bayreuth, D-95440 Bayreuth, Germany. Tel.: C49 921 555622; fax:

C49 921 555799.

E-mail address: [email protected] (B. Stadler).

thus providing new habitats for different ant species, which

they eventually colonise (Brian, 1952; Punttila et al., 1991).

For example, species like Formica rufa L. and F. lugubris

Zett. prefer early successional habitats while species like

F. aquilonia Yarrow tend to show a preference for later

successional stages, less fragmented habitats and a reduced

insulation (Punttila, 1996). Ants are an important com-

ponent in forest ecosystems both, with respect to abundance

(Holldobler and Wilson, 1990) and ecological function

(Wilson, 1987). For example, ants belonging to the genus

Formica have significant negative effects on the abundance

of phytophagous insects in the canopies of coniferous

Soil Biology & Biochemistry 38 (2006) 561–572

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B. Stadler et al. / Soil Biology & Biochemistry 38 (2006) 561–572562

and deciduous trees (Laine and Niemela, 1980; Wellenstein,

1980; Warrington and Whittaker, 1985a,b). In southern

Finland, workers of a single colony of the Formica-rufa

group were shown to collect 12 kg of arthropod prey (fresh

mass; mainly aphids), 1.8 kg of seeds, 36 kg of nest material

and 240 kg of honeydew (fresh mass) per year (Rosengreen

and Sundstrom, 1991). Searching intensity is a decreasing

function of nest distance (Sudd, 1983; Whittaker, 1991)

sometimes leading to ‘green islands’ around ant nests with

reduced herbivore pressure on adjacent trees (Laine and

Niemela, 1980; Wellenstein, 1980). Aphids, however, might

benefit from the presence of ants when their tending partners

collect honeydew and provide shelter against natural

enemies eventually leading to locally higher population

densities (Fowler and Macgarvin, 1985; Buckley, 1987).

Honeydew is a major source of energy in the canopy of

trees, which may reach concentrations of up to

1300 mg C lK1 in throughfall of Norway spruce during

periods of heavy infestation (Stadler and Michalzik, 1998a).

This energy is eventually flushed to the forest floor where it

may affect needle decay or is consumed by insects such as

ants. Because ants significantly affect the abundance and

distribution of different herbivores they may have a strong

imprint on the insect community in forested ecosystems

(Skinner and Whittaker, 1981; Fowler and Macgarvin,

1985). In addition, ant mounds appear to be more suitable

habitats for earthworms and micro-organisms leading to

higher densities due to higher temperatures and humidity

(Dauber and Wolters, 2000; Dauber et al., 2001).

In temperate forests ants often build mounds at the base

of trees or tree trunks because of more suitable micro-

climatic conditions there, especially in early spring

(Gosswald, 1938). Searching activities in their territory

and mound building is associated with frequent needle

turnover and resource input, which can be expected to affect

needle decomposition and subsequently the export of

nutrients from the litter layer with litter solution.

Needle decomposition is a complex process involving

interactions between chemical properties of litter, herbi-

vores, soil fauna; micro-organisms and the physical

environment (Swift et al., 1979; Chapman et al., 2003) as

well as the external input of nutrients and energy, which

varies with stand age, stand structure, tree species and

faeces of the associated insect communities (Hollinger,

1986; Stadler and Michalzik, 1998b; Stadler et al., 2001a;

Prescott, 2002). During litter decomposition its surface will

increase, tissue becomes permeable and thus is more prone

to leaching of organic matter and mineral nutrients. Fresh

litter may release highly soluble and readily degradable low

molecular weight organic compounds (Norden and Berg,

1990) and the relative enrichment of lignin-derived

compounds during litter decomposition (Norden and Berg,

1990; Baldock et al., 1992) should result in release of more

aromatic organic matter.

The first, and probably rate-limiting, step in the

degradation of complex organic matter like litter is

the hydrolysis of polymers by enzymes released by

micro-organisms (e.g. Burns, 1983; Schimel and Wein-

traub, 2003). In spruce needle litter, a major polymer

(together with hemicellulose and lignin) is cellulose (e.g.

Sjoberg et al., 2004); it is hydrolysed by the combined

action of the cellulase system, i.e. endo-(1,4)-b-glucanases

and exo-(1,4)-b-glucanases split cellulose into cellobiose

which is subsequently split into two glucose molecules by

b-glucosidases (Walker and Wilson, 1991). An ant-

mediated increase in extracellular cellulase activity might

therefore suggest accelerated litter decomposition. Another

important polymer is chitin, which might also be related to

the activity of ants. Major sources of chitin in soil are

fungal cell walls and the exoskeletons of arthropods (Ueno

et al., 1991); the latter are abundantly collected by ants.

Chitin is hydrolysed by chitinases (chitobiases) into N,N 0-

diacetylchitobiose which is further split by hexosamin-

idases into two N-acetylglucosamine molecules. Chitin

may play an important role as source of carbon and

nitrogen in soils (Sinsabaugh and Moorhead, 1995). We

hypothesize that chitinase and hexosaminidase activities

will increase in ant-affected litter, supplying increased

amounts of dissolved organic carbon (DOC) and dissolved

organic nitrogen (DON) to the microbial community.

Proteins are further important yet short-lived sources of C

and N in soils which are tapped by a range of different

proteases (Bach and Munch, 2000). Ants may amend litter

with proteins (thereby stimulating proteolytic activity) by

fragmentation of fresh needle litter and resource

(prey) input.

To understand the chain of mechanisms relating trophic

aspects between ants, aphid honeydew and micro-organ-

isms, and their combined effects on needle decomposition

and local ecosystem processes in forest habitats, a multi-

disciplinary approach comprising zoological, microbiologi-

cal and ecosystem ecological aspects is required. The core

hypothesis is that ants are the driving force, which

significantly affects microbial activity and ultimately needle

decomposition and nutrient export from the litter layer. We

emphasise that we did not study mature ant mounds, but the

effects of ants on litter in their foraging area during an early

period of colonisation of a forest stand, which was

converted from a spruce dominated to mixed species stand

including ash, oak and beech.

Here, we follow such a cross disciplinary approach by

studying the effects of ants and honeydew on litter solution

chemistry of Norway spruce and subsequent effects on

needle breakdown and microbial enzyme activities com-

bining a field and a greenhouse experiment. In particular, we

performed (1) a field experiment to study the effects of

F polydena on litter solution. Given that there is a

measurable effect of ants we (2) performed a greenhouse

experiment to separate the relative magnitude of the effects

of ants and nutrient input (honeydew) on litter leachates,

microbial activity and needle decomposition.

B. Stadler et al. / Soil Biology & Biochemistry 38 (2006) 561–572 563

2. Materials and methods

2.1. Site and organisms

The field studies were performed in close proximity to

the main investigation plots of the Bayreuth Institute for

Terrestrial Ecosystem Research (BITOK) in the Fichtelge-

birge situated in northern Bavaria/Germany (508, 09 0N, 118

52 0E). The site was originally stocked with Norway spruce

150 years old, which had been harvested in 1999.

Subsequently, the site was planted with saplings of beech

(65%), fir (30%), and ash (5%). By now, the dominant tree

species is Norway spruce (Picea abies (L.) Karst.) with seed

grown saplings ranging in height from 10 to 80 cm and

densities O300 individuals mK2 in some patches. Mean

annual precipitation is 1100 mm and mean annual tempera-

ture is 5 8C.

At high altitude sites ants usually build their nests close

to tree trunks because these sites provide suitable

microenvironmental conditions e.g. higher spring tempera-

tures (Eichhorn, 1964). At our study site F. polyctena was

the dominant ant species, which is usually found in

fragmented sites, forest edges or sites, which underwent a

substantial change, such as a clear-cut or wind throw. At

these clear-cut sites estimated nest density might be more

than 10–15 nests haK1. However, this density might

subsequently drop to 1–3 nests haK1 during later periods

of succession (Rosengreen and Sundstrom, 1991). Due to

the logging and relatively recent colonization of this site by

ants no mounds were yet visible.

Ants collect honeydew from aphids on Norway spruce

with Cinara pilicornis (Hartig) and C. pruinosa (Harig) as

the most abundant species at our study site (Stadler and

Muller, 1996), especially on young spruce and spruce

saplings. The abundance of the aphids was not surveyed at

the managed site but ants were frequently observed to

collect honeydew from colonies on spruce saplings close to

their previously established nests at the base of trunks.

2.2. Collection of litter solution (field)

From late April to September 2003, needle litter solution

was collected by putting two zero tension lysimeters

(diameter 10 cm) beneath the litter layer, one close to the

trunk (2 cm) and the other at a distance of 30 cm from the

trunk of a harvested tree. Two relatively small lysimeters

were chosen rather than a single big one to capture the local

variability in litter solution better. Treatments were either

trunks with ant nests (mounds not visible) or trunks without

ants. Each treatment and distance class was replicated five

times. Every two weeks litter solutions were collected and

transferred to the laboratory to measure organic and

inorganic compound concentrations. All solution samplers

were replaced with clean ones during each sampling

interval.

2.3. Manipulation experiment (greenhouse)

In early May 2003, fifteen lysimeters (diameter 17 cm)

mere placed in a temperature controlled greenhouse

(21G3 8C) and each was filled with 115 g (fresh mass)

spruce needles, which were collected at the site in the

Fichtelgebirge one week before the experiment started.

The lysimeters were equipped with a ceramic plate (pore

size 1 mm) for collection of the litter solution. Prior to

the first use, each ceramic plate was cleaned (NaOH,

HCl, ultra-pure water) and conditioned with an aqueous

extract obtained from litter sampled at the same site.

Equilibration was continued until a constant UV

absorbance at 254 nm in the filtrate from the

suction plate.

Three different treatments were set up each comprising

five lysimeters (Fig. 1). Treatments were: addition of (a) ants

(ant treatment), (b) honeydew (honeydew treatment), (c)

water (control). All lysimeters were then flushed with 250 ml

H2Odeionized. The next day to five of the lysimeters (ant

treatment) 100 workers of F. polyctena were added by

transferring them individually with a pair offine tweezers. To

prevent them from escaping the lysimeters were sealed at the

top with insect glue. Those individuals, which were

entangled in the glue were substituted the next day. Only

workers were added to the lysimeters because we did not

want to create functional nests during the experimental

period but study the effects of ant activity (needle turnover)

on soil solution and needle decomposition close to their nest

(similar to the field experiment). Each ant colony was given

access to a nearby spruce sapling planted in a five litre pot

filled with compost soil. A bridge made out of wooden sticks

was connected to the main shoot of the spruce sapling to

facilitate access. All spruce saplings were infested with

C. pilicornis from which the ants could collect honeydew or

use the aphids as food. We could not reliably estimate the

quantity of honeydew or the number of aphids, which were

carried to the nests but we frequently observed workers doing

so. Ants were prevented from leaving the trees by applying a

ring of insect glue at the base of the trunk. In this way, each

combination of lysimeter and spruce sapling was a separate

experimental unit. In addition, ants had access to water

provided in a shallow glass vial plugged with a Cotton ball.

One week after the ants had settled we added to each

lysimeter of the ant and control treatment 50 ml H2Odeionized

and 50 ml honeydew solution to the honeydew treatment to

maintain a relative constant humidity. Each week the

lysimeters were weighed to control water loss. Honeydew

was collected from C. pilicornis on Norway spruce the

previous year and a filtered solution was stored in a freezer

until the beginning of the manipulation experiment.

Honeydew consists almost exclusively of different sugars

and we applied the honeydew solution with a concentration

of 100 mg DOC lK1. Concentrations of other compounds

were (mg lK1): Ntotal: 0.240, NH4–N: 0.038, NO3–N: not

detectable, K: 2.03. These concentrations are equivalent to

115 g needles (FM)(=98.9 g DM)200 ml +50 ml H20

115 g needles (FM) 200 ml +50 ml honey dew

Ants Honeydew Control

Parameters measured: Litter solution,filtered: DOC, DON, NH4, NO3, K,UV, fluorescence spectra

Litter: Dry mass, C, N, enzyme activities

115 g needles (FM) 200 ml+50 ml H20 100 workers ofFormica polyctena

Treatments:

17 cm

20 cm

Leachates were collected eight times during a three month experimentalperiod in 2003

Fig. 1. Conceptual sketch of the experimental design to test the effects of ants and honeydew on litter solution chemistry, humification and microbial enzyme

activities. Samples of litter solution were collected fortnightly when 200 ml H2Odeionized or honeydew were added. Every second week 50 ml of the respective

solutions were added to maintain moisture. The temperature in the greenhouse ranged between 18–24 8C and the experiment lasted 18 weeks. FMZfresh mass;

DMZdry mass.


low infestation rates of aphids on Norway spruce at our

experimental site and are mirrored in throughfall concen-

trations (Stadler et al., 2001a). One week later another

200 ml of solution (honeydew or water) was added to each

lysimeter of the respective treatments with a fine sprayer

and the leaching solution was immediately collected from

the lysimeters by applying a negative pressure of 35 kPa.

This fortnightly sampling procedure with applying 200 ml

water and honeydew and 50 ml between sampling dates was

continued for 18 weeks.

2.4. Chemical analyses of litter solutions

After transfer to the laboratory all leachate samples were

immediately filtered (0.45 mm, cellulose-acetate) and kept at

2 8C until the next day for the chemical analyses. Dissolved

organic carbon (DOC) and total nitrogen were determined

by high temperature oxidation (High-TOC, Elementar,

Hanau Germany). Ammonium-N (NH4–N) and nitrate-N

(NO3–N) was determined by ion chromatography (DX-500,

Dionex Corp., Sunnyvale, CA). Dissolved organic nitrogen

(DON) was calculated from the following relationship:

DONZNtotalK(NH4–NCNO3–N). Potassium was

analyzed by ICP-atomic emission spectrometry (Integra

XMP GBC).

2.5. Chemical analyses of solid litter samples

We analyzed the water content and total C and N in the

litter before and after the greenhouse experiment. Aliquots

sampled from the initial litter and from each lysimeter after

the experiment were oven-dried (80 8C) for 24 h to

determine the moisture content. The dried samples were

then ground and analyzed for total C and N contents on a CN

elemental analyzer (CHN–O–Rapid, Foss Heraeus,

Germany).

2.6. Exoenzyme activities

The potential activities of selected enzymes in litter

extracts were analyzed by a microplate assay using

fluorigenic substrates as described by Marx et al. (2001).

Enzymes tested and their respective fluorigenic substrates

(obtained from Sigma-Aldrich, Germany) are listed in

Table 1. Two grams of litter from each lysimeter (geenhouse

experiment) was collected at the end of the experiment,

mixed with 200 ml sterile, distilled water, and pre-incubated

for 30 min at room temperature. Subsequently, the slurry

was shaken horizontally for 20 min (230 rev minK1, 25 8C),

sonicated for 1 min (output energy, 50 J sK1), then allowed

to settle for 2 min, and the supernatant (Zlitter extract) was

sampled. Triplicate incubations were set up for each litter

extract in black 96-well microplates (Greiner Bio-One,

Solingen, Germany) with 50 ml extract, 50 ml buffer (0.05 M

Trizma buffer, pH 7.8 for protease assay, 0.1 M MES buffer,

pH 6.1 for all others; Sigma-Aldrich) and 100 ml substrate

(final concentration, 500 mM) per well. Plates were

incubated at 26 8C directly in a computerized fuorimeter

plate reader (Mithras LB940, Berthold, Germany). Plates

were allowed to equilibrate for 5 min, then were shaken for

5 s, and fluorescence in all wells was recorded every minute

(readout time, 0.3 s wellK1) for 35 min; plates were shaken

for 5 s between measurement cycles. Controls were

performed without substrate for each extract, and with

Table 1

Fluorigenic substrates used to test for exoenzyme activity in litter extracts

Enzymes tested Fluorigenic substrate Short name

Cellulase

(1,4-b-Glucanase)

4-Methylumbelliferyl-b-D-

cellobioside

MUF-Cel

b-Glucosidase 4-Methylumbelliferyl-b-D-

glucopyranoside

MUF-Glu

Chitobiase 4-Methylumbelliferyl-b-D-

N,N 0-diacetylchitobioside

MUF-2-

NAG

Hexosaminidase 4-Methylumbelliferyl-N-

acetyl-b-D-glucosaminide

MUF-NAG

Leucine protease L-Leucine-7-amido-4-

methylcoumarin

Leu-AMC

Arginine protease L-Arginine-7-amido-4-

methylcoumarin

Arg-AMC

Serine protease Ala-Ala-Phe-7-amido-4-

methylcoumarin

Ser-AMC


sterile water instead of litter extract for each

substrate. Standard curves of the fluorescent enzyme

products (4-methylumbelliferone [MUF] and 7-amino-4-

methylcoumarin [AMC], respectively, 0–70 pmol wellK1)

were recorded in buffer plus litter extract for each treatment.

Since activities of cellulose, chitobiase, and serine

proteases were too low for reliable quantification in the

dilute extracts, these enzymes were assayed a second time in

more concentrated litter extracts, i.e. 5 g litter per 50 ml of

sterile water; other conditions were identical. Only a subset

of samples was tested.

A third series of measurements was used to estimate

kinetic parameters of the three enzymes with highest

potential activities (b-glucosidase, hexosaminidase, and

leucine protease); only one extract per treatment (ants,

honeydew, control) was used. Again, extracts were prepared

with 5 g litter in 50 ml of sterile water, and incubations were

set up in triplicate with increasing concentrations of

fluorigenic substrate (2, 8, 20, 50, 75, 100, 200, 300, 500,

750 mM); other parameters were identical to the ones

described above.

Enzyme activities were calculated as the increase of

fluorescence over time from the linear part of the curve

(usually between 8 and 25 min), and transformed into nmol

MUF or AMC g litter (dry weighty)K1 minK1 according to

the extract-specific standard curve and the initial dilution

factor. From the kinetic experiment, substrate affinities (Km)

and maximum reaction rates (Vmax) of the enzymes were

estimated by fitting the experimental data to a non-linear

regression assuming Michaelis-Menten kinetics, using the

curve-fitting tools in SigmaPlot (Jandel Scientific,

Germany).

2.7. Spectroscopic properties of dissolved organic matter

In the litter leachates of the greenhouse experiment the

specific UV absorbance at 280 nm (UVIKON 930, BIO-

TEK Instruments) was measured to estimate the aromaticity

of dissolved organic matter (Chin et al., 1994; McKnight

et al., 1997). Furthermore, we recorded synchronous and

emission fluorescence spectra followed by calculation of

two humification indices (SFM 25, BIO-TEK Instruments;

Zsolnay et al., 1999; Kalbitz and Geyer, 2001) as an

expression of the complexity and condensation of the

molecules. These indices are also correlated to the aromatic

content of organic matter (Kalbitz et al., 2003). The

humification index deduced from emission spectra

(HIXem) is defined as the area in the upper quarter

(S 435–480 nm) of the usable emission spectra divided by

the area in the lower usable quarter (S 300–345 nm). We

calculated the humification index deduced from synchro-

nous spectra (HIXsyn) by dividing the intensity at bands or

shoulders of a longer by a shorter wavelength (460 nm/

345 nm). We used two fluorescence methods because the

resolution of synchronous spectra is better than emission

spectra but emission spectra are less susceptible to changes

in pH, concentration or ionic strength (Yang and Zhang,

1995; Zsolnay et al., 1999). For the UV and fluorescence

measurements the concentration of dissolved organic C was

adjusted to 10 mg lK1 to ensure a comparability of all

samples.

2.8. Data analysis

Differences in concentrations of major compounds in

the litter solution were analyzed using Generalized

Linear Models (GLM) ANOVA with distance from the

trunk and presence/absence of ants as fixed factors. Data

from all sampling dates were pooled to produce an

overall mean value for each lysimeter. This approach was

preferred to a Repeated measures ANOVA because at

this stage we were not interested in seasonal patterns of

the influential variables (distance, ants). The response

variables were log(10) transformed to meet assumptions

of variance homogeneity (Sokal and Rohlf, 1995), but

untransformed data are presented in the figures to

facilitate viewing and interpretation. To test the ant

effect for a particular distance class a post-hoc t-test was

applied. For the greenhouse experiment the same

compounds as well as enzyme activities and kinetic

parameters were analyzed using one-way ANOVAs with

ants, honeydew and control as treatment factors.

Bonferroni correction was applied in the post-hoc tests.

Because no interaction effects were present we do not

show a table on the statistical details but include the

information of the post-hoc tests in the graphs.

3. Results

3.1. Field experiment

The effect of ants on litter solution chemistry was only

apparent close to the trunk, that is in close proximity to an


ants’ nest. For example, in the presence of ants average

DOC concentrations increased significantly in leachates in

close proximity to the trunk and was significantly lower at a

distance of 30 cm from the trunk (Fig. 2a), while DON

concentrations showed no significant distance and ant

effects (Fig. 2b, Table 2). Average concentrations of

NH4–N and NO3–N significantly decreased when ants

were present close to the trunk (Fig. 2c, d) with an overall

significant distance effect for NH4–N (Table 2). Nitrate

nitrogen concentrations tended to increase at a distance of

30 cm from the trunk when ants were present leading to

significant distance!ant interactions (Table 2). The DOC/

DON ratio tended to be higher in the presence of ants but the

ant effect was not significantly different (values not shown)

(Fig. 2e). Potassium showed a highly significant distance

dependent effect with lower mean concentrations further

DO

C (

mg

l-1)

NH

4-N

(m

g l-1

)

Distance from trunk

30 cm2 c m

DO

C/D

ON

80

70

60

50

40

2.6

2.2

1.8

1.4

1.0

40

35

30

25

20

b

a

aa

b

aa

a

a

a

a

a

(a)

(c) (d

(b

(e) (f

Fig. 2. Concentrations of DOC, DON, NO3–N, NH4–N, K and DOC/DON ratios in

a tree trunk or at a distance of 30 cm. Litter was either influenced by the activity o

with different letters indicate significant differences in the concentrations between

absent; solid linesZants present. MeansG1 SE.

away from the trunk, while the ant effect was insignificant

(Fig. 2f, Table 2).

3.2. Manipulation experiment in the greenhouse

3.2.1. Leachate and needle chemistry

Simulating low aphid infestation rates, the addition of

honeydew with throughfall resulted in significantly higher

DOC concentrations in the litter solution compared to the

ant and control treatments (Fig. 3a). In addition, ants also

significantly increased the carbon concentrations in the litter

solution compared to the control treatment. Dissolved

organic nitrogen was highest in the ant treatment but did

not differ significantly between the honeydew and the

control treatment (Fig. 3b). Inorganic nitrogen concen-

trations followed the same pattern. In the honeydew

DO

N (

mg

l-1)

NO

3-N

(m

g l-1

)

Distance from trunk

30 cm2 cm

K (

1mg

l-1)

2.6

2.4

2.2

2.0

1.8

1.6

1.4

1.6

1.4

1.2

1.0

0.8

0.6

0.4

18

16

14

12

10

8

6

4

a

a

a

a

ba

a

a

a

a a

a

)

)

)

leachates of Norway spruce needle litter collected in the field either close to

f ants (F. polyctena) or remained unaffected by ants. Data points separated

two treatments (P!0.05, t-test); n.s.Znot significant. Dashed linesZants

Table 2

GLM ANOVA performed on litter solution containing different compounds collected during May to September in 2003 at a managed spruce site in the

Fichtelgebirge/Germany

Source df DOC DON NH4–N NO3–N K

F P F P F P F P F P

Distance (D) 1,19 5.923 0.027 1.322 0.267 11.881 0.003 1.661 0.216 21.124 !0.001

Ants (A) 1,19 4.912 0.042 0.133 0.721 4.034 0.062 0.240 0.631 0.622 0.442

D!A interactions 1,19 1.139 0.302 1.010 0.330 1.810 0.197 5.490 0.032 1.194 0.666

Values in bold indicate significant effects. Tests were performed on log-transformed values.


treatment virtually no ammonium-N and nitrate-N was

measurable, while the presence of ants significantly

increased inorganic nitrogen concentrations in the litter

solution (Fig. 3c, d). DOC/DON ratio in litter solution was

significantly higher when honeydew was added but did not

differ between the ant and control treatment (Fig. 3e).

DO

C (

mg

l-1)

140

120

100

80

60

40

20

a

b

c

NH

4-N

(m

g l-1

)

2.5

2.0

1.5

1.0

0.5

0.0

a

b

c

Ants

DO

C/D

ON

120

100

80

60

40

20

b

a

a

(a)

(e) (

(c) (

(

ControlHoneydew

Fig. 3. Concentrations of DOC, DON, NO3–N, NH4–N, K and DOC/DON ratios in

Needle litter was affected either by 100 workers of F. polyctena or honeydew, simu

with different letters indicate significant differences in the concentrations betwee

The concentrations of potassium were highest in the litter

solution of the ant treatment followed by the honeydew

addition treatment and the control treatment (Fig. 3f).

The average mass loss during the 18 weeks incubation

period was 9% (0–26%) with no significant differences

between the treatments (FZ0303, dfZ2, PZ0.727). We

DO

N (

mg

l-1)

3.5

3.0

2.5

2.0

1.5

1.0

0.5

a

b

b

NO

3-N

(m

g l-1

)

0.3

0.2

0.1

0.0

a

b

b

K (

mg

l-1)

18

16

14

12

10

8

a

b

c

f)

d)

b)

Ants ControlHoneydew

leachates of Norway spruce litter collected during a greenhouse experiment.

lating a moderate aphid infestation on Norway spruce. Data points separated

n treatments (P!0.05, one-way ANOVA). MeansG1 SE.

Table 3

Substrate affinities (Km) and maximal reaction rates (Vmax) of selected

enzymes in litter extracts from the greenhouse experiment

b-Glucosidase Km [mM MUF-Glu] Vmax [nmol MUF g

litterK1 minK1]

Ants 44.9G2.4 (a) 44.2G0.5 (a)

Honeydew 35.2G0.5 (b) 27.5G0.3 (b)

Control 35.0G1.1 (b) 30.2G0.3 (c)

Hexosaminidase Km [mM MUF-NAG] Vmax [nmol MUF g

littefK1 minK1]

Ants 47.6G1.3 (a) 18.9G0.1 (a)

Honeydew 43.6G3.0 (b) 9.4G0.3 (b)

Control 41.2G1.4 (b) 9.0G0.1 (b)

Leucine protease Km [mM Leu-AMC] Vmax [nmol AMC g

littefK1 minK1]

Ants 36.5G1.3 (a) 9.1G0.1 (a)

Honeydew 38.5G3.7 (a) 6.1G0.1 (b)

Control 66.8G4.2 (b) 2.4G0.0 (c)

Different letters indicate significant differences in the kinetic

parameters between treatments (P!0.05, one-way ANOVA). Means of

triplicates G1 SD.

0

2

cellu

lase

gluco

sidas

e

chito

biase

hexo

samini

dase

leucin

e pro

tease

argini

ne pr

oteas

e

4

6

8

10

12

serin

e pro

tease

nmol

MU

F or

AM

Cgl

itter

[dr

y w

eigh

t]-1

min

-1

Ants

Honeydew

Control

a

a

a

a

bb

b

a,b

b

a,bb b b

a

ab

ab

b b

b

Fig. 4. Potential activity (with 500 mM fluorigenic substrate added) of

selected enzymes in litter extract from the greenhouse experiment. Data are

from dilute extracts (2 g litter in 200 ml water; nZ15) except for cellulase,

chitobiase, and serine protease that had to be recorded in more concentrated

litter extracts (5 g in 50 ml water, nZ3) due to their low activities. Data

points separated with different letters indicate significant differences in

enzyme activities between treatments (P!0.05, one-way ANOVA).

MeansG1 SD.


did not find changes in the C and N content of the

needles between the treatments and during the incubation

in the greenhouse (C: 51.1%, N: 1.43%, C/N ratio: 35.7).

3.2.2. Exoenzyme activities

For most enzymes, potential activities in the ant

treatments were significantly higher than in the honeydew

and control treatments (Fig. 4). In contrast, the addition of

honeydew did not result in significantly higher enzyme

activities compared to the control. The kinetic tests

revealed, in agreement with the potential activities, higher

maximum reaction rates (Vmax values) of b-glucosidase,

hexosaminidase, and leucine protease in the ant treatment

compared to the control (Table 3). In the honeydew

treatment Vmax of b-glucosidase was slightly but signifi-

cantly decreased whereas Vmax of leucine protease was

more than 2-fold increased compared to the control.

Differences of absolute values and significance levels

between potential activity assays (Fig. 4) and kinetic

experiments (Table 3) might be due to the different

extracts used but may also indicate that these enzymes

were not saturated at a substrate concentration of 500 mM.

It is, however, important to note that the trends of Vmax

and potential activities are in all cases identical. Km

values of b-glucosidase and hexosaminidase were slightly

higher in the ant treatment (indicating a lower substrate

affinity) but virtually unchanged in the honeydew

treatment compared to the control. In contrast, a clear

decrease of the Km values for leucine protease was

estimated for both ant and honeydew treatments,

indicating the occurrence of proteases with higher

substrate affinity in both treatments.

3.2.3. Spectroscopic properties

The addition of ants resulted in an increased aromaticity

and complexity of the organic molecules sampled in the

litter leachates. This is indicated by the largest values for all

measured spectroscopic properties (Fig. 5a, b), albeit not

always significantly different from the other treatments. The

effect was significant using humification indices deduced

from synchronous fluorescence spectra (Fig. 5a). Further-

more, application of honeydew resulted in significantly

smaller specific UV absorbance (Fig. 5a) compared to the

other treatments reflecting a distinct portion of the applied

honeydew in the litter leachate.

4. Discussion

Litter decomposition is a complex process dependent on

temperature, moisture, soil properties or the litter quality

itself. In addition, soil dwelling organisms and micro-

organisms with their specific enzymatic properties strongly

affect decomposition through litter fragmentation and

ultimate litter breakdown (Lavelle et al., 1997). Ants, in

particular, are known to modify the physical and chemical

properties of soil and litter especially in their nest mounds

(Pokarzhevskij, 1981; Petal and Kusinska, 1994; Frouz

et al., 1997; Nkem et al., 2000; Frouz et al., 2003). For

example, in Central Finland spruce forests F. aquilonia is

significantly increasing soil organic matter content in the

nest and it is suggested that most energy passing through a

mound is channeled via the bacteria based food web

(Laakso and Setala, 1997; Laakso and Setala, 1998).

However, most studies focus on nest cores and it is less

well understood how the activity of ants outside their nests

spec

ific

UV

abs

orba

nce

at 2

80 n

mL

10

mg

C-1

cm-1

hum

ific

atio

n in

dex

(syn

chro

nous

mod

e)

0.0

0.1

0.2

0.3

0.4

0.0

0.1

0.2

0.3

0.4

Ants Honeydew Control

A 280HIXsyn

a a

b

A

B B

hum

ific

atio

n in

dex

(em

issi

on m

ode)

0

1

2

3

4

Ants Honey dew Control

HIXema

aa

(a)

(b)

Fig. 5. (a) Specific UV absorbance at 280 nm, humification indices deduced from synchronous (HIXsyn) and (b) emission (HIXem) fluorescence spectra in

leachates of Norway spruce litter collected during a greenhouse experiment. Needle litter was affected either by 100 workers of F. polyctena or honeydew,

simulating a moderate aphid infestation on Norway spruce. Data points separated with different letters indicate significant differences in the concentrations

between treatments (P!0.05, one-way ANOVA). MeansG1 SE.


affects litter properties, litter solutions or trophic relation-

ships with micro-organisms especially in managed forests

where ants established recently and where aphid honeydew

is abundantly available on small tree saplings.

4.1. Field experiment

The activity of ants through collecting honeydew and

rearranging needle litter is manifested in higher DOC

concentrations in needle litter solution compared to

unaffected litter (Figs. 2a, 3a). But as long as the size of

ant colonies is small this effect only stretches a very short

distance from the site of a newly founded nest. During the

early period of nest establishment ants had no effect on

DON concentrations while inorganic N concentrations

decreased in the presence of ants (Fig. 2c, d). It is likely

that the collected honeydew fuels the growth of micro-

organisms on the needles, which immobilize inorganic N.

4.2. Greenhouse experiment

When the size of ant mounds increases with more workers

per unit area, like in the greenhouse experiment, organic and

inorganic compounds significantly increased in concen-

trations compared to the control treatment (Fig. 3b–d). This

indicates that in addition to collecting honeydew the

gathering of prey such as aphids, the excreta of ants (Hillery

and Fell, 2000), and needle rearrangement provide patches of

high nutrient turnover, which might eventually stimulate

plant growth and reproduction (Rissing, 1986; McGinley

et al., 1994; Wagner, 1997). The addition of honeydew with.

throughfall, which mirrors the condition beneath an aphid

infested tree, immobilizes inorganic N in the litter layer. The

controlling mechanisms appear to be similar to those in the

canopy where aphid infestation stimulates microbial growth

and eventually reduce concentrations of inorganic N in

throughfall (Stadler et al., 1998).

Potassium is considered to be a relatively mobile element

(Seastedt and Crossley, 1984; Stadler et al., 2001b) and

is thus expected to indicate biological activity. Ants

and honeydew addition both lead to an increase in K

concentrations in litter solution (Figs. 2f, 3f) with the largest

effect in the ant treatment. Therefore, we assume that ants

caused an increased microbial activity in the litter horizon

whereas larger K concentrations in the honeydew treatment

should be the result of added K by honeydew.

Besides increasing DOC and DON concentrations and

decreasing DOC/DON ratios ants resulted in increased

aromaticity and complexity of organic matter in the litter

leachates. All of these changes in organic matter properties

of the leachates are indications of increased microbial

processing of needles resulting in an increased contribution

of lignin-degradation products to dissolved organic matter

(Kalbitz et al., 2004). Furthermore, the stronger response of

DON than DOC to the activity of ants might also imply an

increased input of excreta into leachate solutions because

these compounds are rich in nitrogen. The enhanced

microbial processing of needles is supported by the higher

activities of cellulase and b-glucosidase, while the increased

activities of chitobiase, hexosaminidase, and possibly also

protease suggest increased microbial processing of

resources collected by ants (Fig. 4). Since, the analyzed

enzymes transform particulate or colloidal substrates into

DOM, and the degradation of chitin and proteins ultimately

results in the release of ammonia, which in turn is nitrified to

nitrate, these increased enzyme activities correspond well to

the increased DOC, DON, and inorganic N concentrations

in the ant treatments (Fig. 3a–d).

In addition to increasing potential enzyme activities, the

presence of ants also significantly altered the kinetic

properties of the three enzymes investigated (Table 3). In

an environmental sample, the maximum reaction rate (Vmax)

of an enzyme is a function of the fundamental kinetic

constant of the enzyme and its concentration (Roberts,

1977). The observed increased Vmax can therefore be either

the result of increased enzyme production (per cell or due to

growth of the bacterial population) or of the production of

different isoenzymes (per cell or due to a shift of the

microbial community structure). Similar changes in

substrate affinities (Km) strongly suggest a change in


isoenzyme expression and thus presumably in microbial

community structure.

The effect of the ants during a three month experimental

period was too small to being detectable in the needles, e.g.

via accelerated mass loss or decreasing C/N ratios of the

needle litter. Nevertheless, our findings support the idea that

dissolved organic matter is a link and bottleneck between

biomass, soil and hydrosphere and can therefore be used as a

sensitive indicator of shifts in ecological processes

(Zsolnay, 2003).

Increased DOC concentrations after honeydew appli-

cation were largely the result of the leached honeydew (sum

of DOC in the control and the 100 mg C lK1 in the

honeydew) which suggests that energy may be transferred

to deeper soil layers. This fact was also supported by the

significantly lower specific UV absorbance of honeydew

leachates compared to litter leachates. Furthermore,

honeydew addition did not change DON concentration,

because DON concentrations in the honeydew were very

low (0.2 mg lK1). The activity of exoenzymes in the

honeydew treatment was nearly unchanged compared to

the control (Fig. 4). However, we noted a trend for a slightly

reduced activity of cellulose degrading enzymes (not

significant for the potential activities (Fig. 4) but significant

for the kinetics of b-glucosidase (Table 3)). Inhibition by

glucose of fungal cellulases and some b-glucosidase has

been reported previously while bacterial enzymes seem to

be unaffected (e.g. Boschker and Cappenberg, 1994).

Therefore, our results suggest that the addition of honeydew

inhibits the fungal fraction of cellulose degrading enzymes

which seems, given the minor effect on the enzyme

activities, rather small in our samples. Furthermore, we

observed a trend for higher protease activities (again not

significant for the potential activities (Fig. 4) but significant

for the kinetics of leucine protease (Table 3)). Proteases, and

specifically leucine peptidase are often produced by

microorganisms under N-starvation (Chrost, 1991), a

situation most likely induced by the addition of honeydew,

and reflected in the depletion of inorganic N (Fig. 3c, d).

The significantly higher substrate affinity of leucine

protease (Table 3) can also be seen as a response to the

apparent N limitation, allowing for the utilization of even

minute amounts of proteins. As discussed for the ant

treatment, the change in kinetic parameters, most notably

Km, of leucine protease, suggests a change in isoenzyme

expression and possibly structure of the proteolytic

microbial community. Despite these effects, honeydew

addition did not affect the production of dissolved organic

matter from the litter horizon, nor did it stimulate the

production of microbial exoenzymes involved in litter

degradation.

4.3. Conclusions

From the results of the field and greenhouse experiments

we conclude that ants strongly affect litter leachate

composition and microbial activity. Increased concen-

trations of DOC and DON, a changed composition towards

more aromatic and complex compounds derived from

lignin, and higher activities of exoenzymes suggest

accelerated litter decay. We propose that this effect is

caused by ants through a combination of direct physical

effects on needle litter and indirect trophic effects with

aphids and micro-organisms. In contrast to ants, honeydew

resulted in immobilization of inorganic N but had little

short-term effects on litter decomposition or microbial

exoenzyme activities. Even though the duration of our

experiments was too short to detect direct effects on litter C,

N concentrations the amounts and composition of dissolved

organic matter and the activity of exoenzymes appear to be

good indicators for changes in the turnover of organic

matter caused by increased activity of soil fauna during the

ant-colonisation process of managed forested ecosystems.

Acknowledgements

We are grateful to Petra Dietrich and Kerstin Lateier who

helped with the chemical analyses. Uwe Hell provided the

experimental set-up for the greenhouse manipulation

experiment. We thank Ralf Mertel for his help during the

enzyme assays, and Berthold Technologies (Bad Wildbach,

Germany) for providing a test instrument for the measure-

ments. Aaron Ellison, Jan Frouz, Egbert Matzner and two

anonymous reviewers provided helpful suggestions for

improving an earlier draft of this paper. This work was

financially supported by the Bundesministeriums fur

Bildung, Wissenschaft, Forschung und Technologie

(BMBF).

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Ant-mediated effects on spruce litter decomposition, solution chemistry, and microbial activity