Mississippi Gruntworm (Diplocardia mississippiensis)

source: usda.gov

Abstract

Direct and indirect effects of earthworm feeding and activity can alter soil microbial and nutrient dynamics. Little is known about inØuence

of native North American earthworms on these dynamics. We investigated effects of native D. mississippiensis activity on total and soluble

C, N and microbial biomass pools in Spodosols from the Apalachicola National Forest in North Florida, USA. Soil native to D. mississip-

piensis was collected and reconstructed by horizon (A, E, B h) in laboratory chambers and incubated with earthworms at zero, low or high

density, respectively 0, three, or six individuals, for 28±31 days. Total C and N were measured by dry combustion methods. Soluble C and N

and microbial biomass were measured in 0.5 K 2SO 4 extracts from non-fumigated and fumigated soil samples. Substrate induced respiration

(SIR) was used with inhibitors to measure microbial respiration and shifts in bacterial and fungal components. Earthworm activity mixed soil

horizons, largely through burrowing and casting. Total C in A and Bh horizons averaged 16.7 mg C g21 soil and was signiÆcantly greater than

in the E (2.6 mg C g21 soil). Soil N concentration was highest in the A (0.73 mg g21 soil) and lowest in the E (0.11 mg N g21 soil). N

concentration was signiÆcantly increased over two fold in the E with high earthworm density. Microbial biomass C was greatest in the A and

E, averaging 8.6 mg g21 C, and decreased with earthworm activity. In all horizons, soluble C and N also increased with earthworm density.

Net N mineralization increased signiÆcantly with earthworm density and ranged from ±0.22 to 68 mg g21 N across all treatments and layers.

Changes in proportion of soluble to total soil nutrients indicated that greater turnover and mineralization occurred with earthworm activity.

SIR indicated earthworm activity induced microbial respiration in A and E horizons. Earthworm activity stimulated fungal and bacterial

respiration, respectively, by 13 and 30% in the A and by 123% and 450% in the E. Although bacterial respiration was stimulated more than

fungal there was no signiÆcant shift in B:F ratio, which ranged from 0.80 to 0.99. SIR did not provide evidence of any selective microbial

effects of earthworm activity. Direct and indirect effects on nutrient dynamics by earthworms were induced by casting activity and were

associated with passage of material through the gut (direct effect) and nutrient and microbial enrichment through the mixing of soil horizons

(indirect effect). This study established the importance of D. mississippiensis to nutrient availability in its native soil. q 2001 Elsevier

Science Ltd. All rights reserved1.

Introduction

Earthworm activity, namely comminution, burrowing

and casting in the soil, indirectly affects microorganisms

(Brown, 1995). Earthworm activity modiÆes soil structure,

moisture and aeration, redistributes carbon resources, and

aggregates soil with castings (Brown, 1995; Doube and

Brown, 1998). Direct effects of earthworms on microorgan-

isms are caused by feeding on and dispersing microbial

propagules (Brown, 1995). Devliegher and Verstraete

(1995) characterized indirect and direct effects with respect

to nutrient-enrichment (NEP) and gut-associated (GAP)

processes. GAP may encompass increased soluble organic

matter, microbial activity, and colony forming units of

microorganisms observed in ingested material during gut

passage (Parle, 1963a,b; Barois and Lavelle, 1986; Barois,

1992; Kristufek et al., 1992). Fischer et al. (1995) proposed

that dormant bacterial cells were activated during gut

passage. Bacterial activity can continue in fresh casts

(Parle, 1963a; Daniel and Anderson, 1992) and result in

local increases of total microbial activity in bulk soil.

Fungal colonization and succession of species occurs

in aging casts (Parle, 1963a; Tiwari and Mishra, 1993).

Earthworm feeding activity may inØuence microbial

dynamics. Some experiments report selective consumption

of fungi over bacteria (Cooke and Luxton, 1980; Brown,

1995; Doube and Brown, 1998). Selective assimilation or

stimulation of fungi and bacteria can inØuence long term

microbial community dynamics in bulk soil (Scheu and

Parkinson, 1994; McLean and Parkinson, 2000).

Activity of native North American earthworms and their

direct or indirect interactions with the microbial community

are little studied. Previous Æeld results suggested geophagic

feeding behavior of native southeastern earthworms (Mega-

scolecidae: Diplocardia Garman, 1888) based on d13

C

signatures which indicated soil organic matter assimilation

by these earthworms (Hendrix et al., 1999). Although, soil

organic matter encompasses microbial d13

C signatures

selective feeding on microbial tissues could not be resolved.

The objective of the present study was to determine the

impact of the activity of Diplocardia mississippiensis

Smith, 1924 on microbial community dynamics by examin-

ing carbon and nitrogen turnover through soil organic matter

and microbial pools. We hypothesized that D. mississippien-

sis would have greater selective effects on fungal than on

bacterial dynamics. D. mississippiensis may ingest fungi,

which release oxalates, to derive calcium, a potentially

limiting nutrient in the Apalachicola National Forest

(ANF). Capability to ingest and metabolize oxalates binding

calcium (Khambata and Bhat, 1955; Cromack et al., 1977;

Graustein et al., 1977) has been suggested in Arctiostrotus

sp., another Nearctic megascolecid earthworm (Spiers et al.,

1986). Observing a direct impact of earthworms on

microbes is usually conÆned to short term laboratory feed-

ing experiments, therefore an attempt was made to deter-

mine selective effects of activity on bacterial:fungal (B:F)

ratios using substrate induced respiration (SIR) with inhibi-

tors (Anderson and Domsch, 1973). SIR was successfully

used to show changes in B:F ratios due to earthworms under

laboratory conditions (Scheu and Parkinson, 1994). Our

analyses were made through comparison of zero, low and

high earthworm densities.

2. Materials and methods

2.1. Site description

The system under study is the Apalachicola National

Forest (ANF) located in the panhandle region of northwest

Florida, USA. The ANF system is dominated by longleaf

pine (Pinus palustris) with a shrub±grassland understory

dominated by wire grass (Aristida stricta). More informa-

tion on the ecology of this habitat can be obtained from Vail

(1972); Callaham and Hendrix (1998); Hendrix et al.

(1999). The USDA forest service manages this area with a

prescribed burning program for the risk of forest Æres. Vail

(1972) recorded presence of three native species of Diplo-

cardia (Megascolecidae) in the ANF. Reynolds (1994);

Hendrix et al. (1999) conÆrmed the distribution of D. missis-

sippiensis, and D. Øoridana in the ANF area. D. komareki

may also occur but is rarely found (M.A. Callaham Jr.,

personal communication). Hendrix et al. (1994) reported

that population densities of Diplocardia spp. changed

seasonally and were reduced by earthworm harvesting in

burn areas for the Æshing bait industry. Hendrix et al.

(1994) recorded greatest fresh biomass of 6.0 ^ 1.0 g m22

attributed to a density of one±three earthworms m22

. Diplo-

cardia sp. in this system likely forage on soil microbial

biomass and decayed organic material (Vail, 1972; Hendrix

et al., 1994). The geophagous feeding behavior of both D.

mississippiensis and D. Øoridana was suggested by stable

isotopes (Hendrix et al., 1999) and gut content analysis.

Soils in the area of ANF used for this study are acidic

(pH à 4.3) and classiÆed as a sandy, siliceous, thermic

Aeric Alaquod. Three soil horizons were distinguished for

this study. The A horizon, from 0 to 17 cm depth, occurs as a

dark gray Æne sand. The E horizon at 17±29 cm depth, is a

light gray Æne sand with little organic materials. The third, a

B h horizon occurring at 29±70 cm depth is an organic

matter-rich very dark brown Æne sand. The depths of these

horizons are variable along a topographical sequence. The

soil nutrient and microbial status of each soil horizon

collected from the Æeld and after an initial week long condi-

tioning of reconstructed soil horizons at increased moisture

content are given in Tables 1 and 2.

2.2. Experimental design

Soils from the three horizons (A, E and B h) were retrieved

along with native Diplocardia sp. from the ANF in manage-

ment Compartment 47 in June 1998. Sieved (1 cm mesh)

soil from each horizon was reconstructed as a unit in 15

PVC cylinders (15 cm dia, 30 cm height). Soil was packed

at Æeld bulk density, 10 cm thick each for A and E horizons,

5 cm thick for B h, and wetted with deionized water to

increase moisture to 25% gravimetric weight. After

1 week of conditioning, three units were sampled for preli-

minary studies. Earthworms were then added into two-thirds

of the remaining units to generate three Ænal treatments with

four replicates for each treatment. The treatments were as

follows: zero density treatment (ZDT) with no earthworms;

low density treatment (LDT) with three earthworms; and

high density treatment (HDT) with six earthworms. Treat-

ments were incubated at constant moisture and temperature

(20 ^ 18C) in the laboratory. Three units, one replicate from

each treatment, were deconstructed on each day over days

28±31 of incubation and analyzed for microbial biomass

and substrate induced respiration.

Total and extractable soil C and N were determined on

each layer in every incubation cylinder and are expressed

per gram oven dry soil weight (1058C). Total soil C and N

were determined by dry combustion on a Carlo Erba CN

analyzer. Soluble C and N were extracted with 0.5 M K2SO 4

at 3:1 solution to soil mass ratio. Soluble organic C (C sol)

Table 1

Total soil N, inorganic N (NH4

1-N 1 NO 3

2-N), microbial biomass N

(MBN), and net mineralized N, of initial and incubated soils by layer (A,

E, and Bh ) and by earthworm density treatment (ZDT à zero, LDT à three

earthworms, and HDT à six earthworms). (Data are given as means (s.e.);

for Initial n à 2, for earthworm density treatments n à 4)

Layer

A E B h

Total N (mg g21 soil)

Initial 0.71 (0.02) 0.06 (0.00) 0.48 (0.00)

ZDT 0.74 (0.01) Aa a 0.07 (0.003) Ba 0.49 (0.01) Ca

LDT 0.71 (0.03) Aa 0.12 (0.003) Bb 0.50 (0.01) Ca

HDT 0.73 (0.03) Aa 0.14 (0.003) Bc 0.53 (0.02) Ca

Inorganic N (mg g21 N)

Initial 3.2 (0.2) 11.0 (0.3) 2.7 (0.1)

ZDT 4.4 (0.3) Aa 10.5 (0.5) Ba 2.5 (0.5) Ca

LDT 21.2 (2.9) Ab 34.4 (5.3) Bb 11.2 (3) Cb

HDT 33.4 (3.5) Ac 72.6 (20.5) Bc 42.7 (11.6) Cc

MBN (mg g21 N)

Initial 29.7 (14.6) 37.5 (17.6) 11.3 (2.0)

ZDT 21.7 (1) Aa 20.5 (3.9) Aa 10.2 (1) Ba

LDT 12.4 (2.6) Ab 10.7 (4) Ab 13.7 (1.4) Aa

HDT 18.2 (1.7) Aab 7.2 (5) Bb 14.0 (3.8) ABa

Net mineralized N (mg g21 N)

ZDT 1.35 (0.32) Aa 0.14 (0.44) Aa 20.22 (0.42) Ba

LDT 17.99 (2.82) Ab 28.04 (5.26) Ab 9.13 (2.98) Bb

HDT 30.29 (3.88) Ac 67.63 (20.74) Ac 40.28 (23.28) Bc

a SigniÆcant differences within each pool, by multiple comparison (Fish-

er's LSD test, P , 0.05), among layers (within treatment row) are indicated

by capital letters and among treatments (within layer column) are indicated

by lower case letters.

Table 2

Total soil C, soluble organic C, and microbial biomass C (MBC), of initial

and incubated soils by layer (A, E, Bh ) and by earthworm density treatment

(ZDT à zero, LDT à three earthworms, and HDT à six earthworms).

(Data are given as means (s.e.); for Initial n à 2, for earthworm density

treatments n à 4)

Layer

A E Bh

Total C (mg g21 soil)

Initial 16.5 (0.5) 1.6 (0.1) 16.1 (0)

ZDT 17.5 (0.3) Aa a 1.71 (0.5) Ba 16.2 (0.5) Aa

LDT 16.6 (0.7) Aa 2.72 (0.14) Ba 16.5 (0.6) Aa

HDT 17.1 (0.7) Aa 3.35 (0.08) Ba 16.4 (0.6) Aa

Soluble organic C (mg g21 C)

Initial 1.8 (0.1) 9.1 (0.7) 4.1 (0.6)

ZDT 1.9 (0.1) Aa 8.8 (0.3) Ba 4.0 (0.2) Ca

LDT 2.4 (0.2) Aa 8.2 (0.3) Ba 4.3 (0.2) Ca

HDT 3.1 (0.1) Ab 9.9 (0.2) Bb 4.7 (0.2) Cb

MBC (mg g21 C)

Initial 10.3 (1.09) 12.0 (0.22) 4.1 (0.13)

ZDT 8.9 (0.2) Aa 10.4 (0.8) Ba 3.3 (0.1) Ca

LDT 8.5 (0.3) Ab 7.6 (0.5) Ab 3.6 (0.1) Bb

HDT 8.5 (0.6) Ab 7.6 (0.5) Ab 3.5 (0.3) Bb

a SigniÆcant differences within each pool, by multiple comparison (Fish-

er's LSD test, P , 0.05), among layers (within treatment row) are indicated

by capital letters and among treatments (within layer column) are indicated

by lower case letters.

was determined on a Shimadzu total organic carbon (TOC)

analyzer. Total inorganic N (NH 4

1-N 1 NO 3

2-N) was

measured on an Alpkem nitrogen autoanalyzer. Extractable

C and N are expressed in mg g21 of total soil C or N as

appropriate. Net N mineralization was calculated as the

difference in inorganic N in mg between incubated and

initial values expressed g21 of N after incubation.

Microbial biomass was determined by chloroform fumi-

gation direct extraction (Brookes et al., 1985; Vance et al.,

1987). Microbial biomass C (MBC) was measured by TOC

and microbial biomass N (MBN) was determined following

persulfate digestion (Cabrera and Beare, 1993). Microbial

biomass results are reported as 0.5 K 2SO4 -extractable C or N

of fumigated minus non-fumigated soil expressed in mg g21

of total C or total N, as appropriate.

Microbial activity was determined by substrate induced

respiration (SIR) on only A and E horizons using strepto-

mycin and cycloheximide as inhibitors, respectively, of

prokaryotic and eukaryotic activity (Anderson and Domsch,

1973). Basal respiration was optimized at 30% gravimetric

weight, equivalent to 65±70% of the water holding capacity

of the soil layers. Glucose and inhibitor concentrations were

optimized by preliminary studies on conditioned Æeld soil.

Glucose, streptomycin and cycloheximide, were applied at

concentrations of 1, 8, and 10 mg g21 soil dry wt, respec-

tively. Inhibitors were initially applied dry 16 h prior to

glucose addition (Johnson et al., 1996). Glucose was applied

in solution, increasing soil moisture to 30% gravimetric

weight. Minimal supplementary moisture was added to the

A horizon soil to aid distribution of inhibitors. Respiration

was determined simultaneously on basal, glucose and

glucose-plus-inhibitor treatments, after 3 h incubation.

Respiration was measured as CO 2 on an infrared gas analy-

zer (IRGA) using a continuous Øow apparatus modiÆed

from Cheng and Coleman (1989); Cheng and Virginia

(1993). Respiration as CO 2-C in mg g21 soil dry wt h21

was calculated from averaged Øow rate and IRGA reading

measured over two minutes. Bacterial respiration was calcu-

lated as respiration induced by glucose addition alone (GIR)

minus respiration that resulted with addition of glucose plus

streptomycin. Fungal respiration was calculated as GIR

minus respiration that resulted with addition of glucose

plus cycloheximide.

Data were analyzed with Sigma Stat software (Jandel

ScientiÆc). Main effects of earthworm density treatment

and soil layer were analyzed by two-way ANOVA. Non-

normal data were log or square root transformed for analy-

sis. Fisher's LSD test was used as a post-hoc test to analyze

multiple comparisons among treatments and layers. Two-

way comparisons with a signiÆcant interaction occurring

between treatment and layer were analyzed within treatment

or layer with Fisher's LSD test. SigniÆcance of all tests was

set at P , 0.05.

3. Results

During the incubation period earthworms moved soil from

the A horizon layer into the E horizon layer. Burrowing activ-

ity maintained open macropores in the A layer and casting

activity Ælled macropores in the E layer. The appearance of

the HDT exhibited more earthworm activity than LDT. The

ZDT appeared unchanged over the time of the incubation.

Minimal activity of earthworms was noted in the Bh layer of

both LDT and HDT. Casting material recognized in the soil

and collected from earthworms after incubation indicated

preferential ingestion of A layer material. All earthworms in

the LDT survived, however, one replicate of HDT experienced

the loss of three worms. Initial earthworm weight was

2.16 ^ 0.20 g per individual. LDT averaged a gain of 0.08 g

and HDT averaged a loss of 0.16 g per worm. Outlying values

due to earthworm mortality were determined by analysis of

residuals and dropped from statistical tests.

Total soil N concentration was lowest in the E horizon

initially and, after incubation, increased with increasing

earthworm density, up to two-fold in the HDT (Table 1).

Final total N was signiÆcantly different among all layers,

however, was signiÆcantly different among all treatments

within the E layer only (df à 4; F à 34.9; P , 0.0001;

LSD). An increase in total N in the E occurred with an

increase in inorganic N and decrease in MBN. Inorganic

N was signiÆcantly greatest in the E (F à 20.797 (log)

P , 0.0001) and increased with earthworm density

(F à 74.135 (log), P , 0.0001, Table 1). SigniÆcant effects

on MBN were due to an interaction between treatments and

layers (df à 4, F à 2.9; P à 0.0407). Mean MBN was high-

est in the A layer in the ZDT and lowest in the E layer in the

HDT; MBN was signiÆcantly reduced in the A layer by

LDT and in the E layer by both LDT and HDT (LSD,

P , 0.001; Table 1). A shift in N balance from the MBN

pool to the inorganic pool was apparent in the A layer where

there was no change in total N. Net mineralization of N

increased signiÆcantly with earthworm density in all layers

(F à 116.834 (log), P , 0.001) and had lowest mean value

across treatments in the Bh layer (F à 6.369 (log),

P à 0.005; Table 1). Although, net mineralization occurred

in all soil layers and treatments, N was immobilized in the

ZDT B h layer only.

Total soil C was lowest initially and remained signiÆcantly

reduced across treatments in the E layer (F à 1037.11;

P , 0.0001; Table 2). Although treatments had no signiÆcant

effect across layers, total C in the E layer increased nearly two-

fold from the ZDT to HDT. A signiÆcant one-way effect of

earthworm density on C content in the E layer was obscured by

the greater content and variance of total C in the A and Bh

layers (F à 60.1, P , 0.0001). Both Csol and MBC were high-

est initially in the E layer (Table 2). After incubation Csol was

signiÆcantly different among the layers but remained greatest

in the E (F à 358.00, P , 0.0001). Across layers mean Csol

was signiÆcantly greater in HDT than in LDT or ZDT (5.9, 5.0,

and 4.9 mg g21 C, respectively; F à 10.22; P à 0.0005,

LSD). After incubation, MBC was less than initial quantities

in all layers (Table 1). Final MBC was signiÆcantly lower in

the Bh layer and was signiÆcantly different in the ZDT from

LDT and HDT across all layers (df à 4, F à 4.22, P à 0.0088,

LSD). MBC in ZDT was highest in A and E layers, but lowest

in the Bh layer (Table 1). Earthworm density treatments had no

apparent effect on total C in any layer, however, in A and E

layers, C balance was shifted from MBC to Csol pools with

increasing earthworm density.

As with microbial biomass, basal soil respiration was signif-

icantly greater in A than in E layer soil (0.82 ^ 0.02 and

0.24 ^ 0.04 mg CO2-C g21 h21

, respectively; F à 139.40,

P , 0.001). There were no signiÆcant differences in GIR

between layers or treatments, however respiration was stimu-

lated by 228±237% in the A layer and by 80±137% in the E

layer. Respiration was signiÆcantly greater in the A layer for

both bacteria (F à 326.497, P , 0.001) and fungi

(F à 512.577, P , 0.001). Stimulation of both bacterial and

fungal respiration by LDT and HDT compared to ZDT was

most pronounced in the E layer (Fig. 1). Bacterial respiration

was signiÆcantly stimulated in HDT and LDT over ZDT on

average by 30% in the A layer and by 450% in the E layer

(F à 8.714, P à 0.002). Fungal respiration in LDT and HDT

was signiÆcantly stimulated over ZDT on average by 13% in

the A layer and by 123% in the E layer (F à 5.814 (sqrt),

P , 0.012). Fungal respiration (1.50 ^ 0.06 mg CO2-

C g21 h21

) was signiÆcantly greater than bacterial

(1.32 ^ 0.06 mg CO2-C g21 h21

) across treatments in the A

layer (F à 7.34, P à 0.014). The B:F ratio ranged from 0.80

to 0.93 in the A layer, from 0.80 to 0.99 in the E; B:F was

higher in LDT and HDT, but the effects were not signiÆcant.

4. Discussion

The major effect of D. mississippiensis activity was the

obvious physical disruption of the reconstructed soil layers.

Cast material in the E layer corresponded with removal of A

layer material. Evidently, the near two fold increases in total

Fig. 1. Bacterial and fungal respiration of A and E layer soil by earthworm

density treatment (ZDT à zero, LDT à three earthworms, and HDT à six

earthworms). Bars show one standard error. See text for calculation of

respiration measurements. Different letters within a component of respira-

tion indicate signiÆcant differences among earthworm density treatment

means across layers (Fisher's LSD) at P , 0.05. For HDT means in the

A layer n à 3, for all other means n à 4.

C and N in the E layer resulted from casting activity. In

comparable soils, total C and N in casts of D. longa were

signiÆcantly greater than in bulk soil (Hendrix and Calla-

ham, 1994). The redistribution of A layer material by earth-

worms provided a source of soil organic matter and

microbial biomass for the E layer. Total C and N pools

within A and B h layers were unchanged across treatments,

however shifts in proportion of extractable and microbial

pools in LDT and HDT compared to ZDT indicated C and N

were mobilized in all soil layers. Nutrient mobilization in

the E layer was clearly due to both organic matter enrich-

ment and effects associated with passage of this material

through the earthworm gut. In the E layer, the distinction

between NEP and GAP, made through experiments of

Devliegher and Verstraete (1995) was not directly tested,

however, the function of both processes was recognized in

this study.

Earthworm activity had signiÆcant effects on N

dynamics. D. mississippiensis activity contributed to net N

mineralization in all soil layers (Table 1). In the E layer,

much more of the N deposited in casts was introduced in

mineral rather than microbial forms. Midwestern species of

Diplocardia studied by James (1991) released great

amounts of inorganic N through cast production. Based on

organic matter enrichment we estimated that D. mississip-

piensis could have released between 76 and 173 mg inor-

ganic N per gram of cast. These values are close to those of

James (1991) who found that around 40±170 mg of total

NH 4

1-N and NO 3

2-N could be added to the soil per gram

of cast. In the A layer, an increase in inorganic N (miner-

alized N) in the LDT, with a signiÆcant decrease in MBN

and no change in total N indicated N was mobilized in part

from microbial to mineral pools. Decreased MBN asso-

ciated with increased total N in the E layer could be attrib-

uted to organic matter enrichment from the A horizon.

Decreased microbial N was typical in other earthworm incu-

bations (Bohlen and Edwards, 1995; Willems et al., 1996;

Hendrix et al., 1998). Decreased microbial N was attributed

to residue incorporation and increased turnover of organic

matter N through the microbial biomass with earthworm

activity (Hendrix et al., 1998). However, Brown et al.

(1995) found that Lumbricus rubellus contributed to N

mobilization from residue to soil but with no effect on

microbial or mineral N forms.

Mobilization of C detected as changes in Csol and MBC

pools occurred with earthworm activity. Decreased MBC

indicated less immobilization of C within the microbial

biomass with earthworm activity. Stimulation of bacterial

and fungal respiration with earthworm activity suggested

that mobilization of C was associated with microbial turn-

over, however, net C mineralization, as a fractional change

in total C, was immeasurable. Soil respiration was substan-

tially increased in the presence of earthworms in a 16 day

incubation in accordance with increased microorganism

(protozoan) density (Binet et al., 1998). However, increased

microbial activity was not associated with microbial

biomass changes in a 225 day incubation with Octolasian

lacteum (Scheu, 1993). Bohlen and Edwards (1995) found

that organic matter amendments of green and manure ferti-

lizers to plots with high densities of earthworms produced

more CO 2 respiration than plots without amendments. As

with N mineralization, nutrient enrichment may also have

lead to increased C turnover in the E layer.

Change in microbial biomass C and N supported a possi-

ble change in the microbial composition of A and E hori-

zons. Decreased MBC and MBN possibly resulted from

greater bacterial activity. Saetre (1998) found decreased

microbial biomass and associated N with a decrease in

soil fungi and increase in actinomycetes. Casting activity

may have introduced more bacteria, which would result

from stimulation of microbial growth and activity inside

the earthworm gut (Parle, 1963a,b; Barois and Lavelle,

1986; Barois, 1992; Kristufek et al., 1992; Fischer et al.,

1995). However, both bacterial and fungal respiration

were stimulated in treatments with earthworms. Fungal

respiration was greater than bacterial respiration reØecting

the fungal dominance of microbial biomass typical in pine

habitats. Calcium, a proposed limiting nutrient in the ANF

system, is bound by fungal oxalates (Cromack et al., 1979;

Fox and Comerford, 1992; Delucia et al., 1997). To obtain

required calcium, Diplocardia spp. may be capable of

ingesting fungal biomass, however, there was no evidence

that fungal activity was affected by direct feeding of earth-

worms. The SIR technique (Anderson and Domsch, 1973)

was used previously to show increased B:F ratios with activ-

ity of O. lacteum and Dendrobaena octaedra (Lumbricidae)

under laboratory conditions (Scheu and Parkinson, 1994).

Although our results indicated a shift towards bacteria, as

B:F ratio increased with earthworm density, SIR was inade-

quate for measuring statistically signiÆcant differences. As a

residual of gut passage, nutrient enrichment and increased

microorganism biomass or activity, including viable propa-

gules of fungi, were found previously in earthworm cast-

ings, including those produced by Diplocardia spp. (Parle,

1963a; Shaw and Pawluk, 1986; James, 1991; Reddell and

Spain, 1991; Daniel and Anderson, 1992; Tiwari and

Mishra, 1993; Hendrix and Callaham, 1994). Although,

bacterial respiration was more greatly stimulated than

fungal respiration by earthworm activity in A and E layers,

there was no indication of strong selective changes due to

GAP. Stimulation and maintenance of greater fungal

respiration in earthworm treatments could have been caused

by fungal succession in aging cast material (Parle, 1963a;

Tiwari and Mishra, 1993; Tiunov and Scheu, 2000a,b).

We proposed that D. mississippiensis, like other well-

studied species, would stimulate microbial biomass and

activity and exhibit selective effects on microbial compo-

nents. This paper showed that earthworm activity, primarily

casting of A layer material, increased C and N content of the

E layer over two fold. Reduced nutrient immobilization by

the microbial biomass was indicated by reduced biomass in

proportion to total C and N pools with earthworm activity. A

strong relationship between D. mississippiensis and N turn-

over was apparent with shifts in balance of mineral and

microbial N pools. Increased availability of mineralized N

with earthworm activity, established by this study, concei-

vably induced the increased native plant biomass and N

content previously observed with activity of D. mississip-

piensis (Callaham and Hendrix, 1998). This study estab-

lished that D. mississippiensis functions comparably to

lumbricid species, in contributing to N availability (Robin-

son et al., 1992; Scheu, 1993; Willems et al., 1996). The

effects of D. mississippiensis were likely due to the mixing

of A horizon soil, a source of greater microbial biomass and

soil organic matter, into the E horizon soil. SIR with inhi-

bitors indicated that earthworm activity caused signiÆcant

stimulation of microbial respiration in the A and E layers.

Similar to experiments of Fischer et al. (1995); Wolter and

Scheu (1999) there was no evidence that earthworms fed on

or selectively stimulated any speciÆc microbial component.

This study established an important role for D. mississip-

piensis in nutrient availability in its native soil. Although

average Æeld densities of D. mississippiensis are relatively

low (ca. 3 m22

), their highly clumped distributions suggest

potential for signiÆcant localized effects on nutrient

dynamics, for example, in plant rhizospheres. Reduced

densities of D. mississippiensis caused by harvesting

(Hendrix et al. 1994) may impact forest regeneration in

burn management areas.

Acknowledgements

This study was supported by a grant from the National

Science Foundation to The University of Georgia Research

Foundation, Inc. We gratefully acknowledge collaborative

efforts of Fran James, FSU, and the US Forest Service,

Apalachicola Ranger District. Thanks also to Tom Maddox,

Todd Ackermann and the Soil Analysis Lab, Institute of

Ecology, University of Georgia. We appreciate the helpful

comments of two anonymous reviewers.