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DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY

Differential Effects of Earthworms on Nitrogen Cycling from Various Nitrogen-15-Labeled Substrates

^a MacArthur Agro-Ecology Research Center, 300 Buck Island Ranch Rd., Lake Placid, FL 33852 USA
^b Dep. of Entomology, Ohio State Univ., Columbus, OH 43210 USA
^c Environmental Sciences Graduate Program, Ohio State Univ., Columbus, OH 43210 USA

pbohlen{at}archbold-station.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Earthworms incorporate organic matter into soil but their influence on cycling of N from incorporated materials is not well understood. This study examined the role of earthworms in the turnover of N from different ¹⁵N-labeled inputs. We incubated intact soil cores with or without earthworms (Lumbricus terrestris) and with ¹⁵N-labeled KNO₃, rye litter (Secale cereale L.), or cow manure, added to the soil surface at a rate of 150 kg N ha^-1. The cores were destructively sampled after 2, 6, and 10 wk and assayed for total soil N, KCl-extractable N, microbial-biomass N (MBN), two size fractions (53–2000 µm and >2000 µm) of particulate organic matter (POM), and ¹⁵N enrichment of all N pools. Earthworms increased the incorporation of ¹⁵N into the soil in organically treated cores but had little effect on the distribution of ¹⁵N in the inorganically fertilized cores. The percentage of initial ¹⁵N that was incorporated into the soil after 10 wk was 71 and 45% in cores with earthworms and 34 and 25% in cores without earthworms, in the manure and rye treatments, respectively. Earthworms incorporated the manure more rapidly than the rye, as shown by temporal patterns of incorporation of total N and POM. Earthworms increased MBN in the manure treatment by about 20 to 30%, but slightly decreased MBN on one sample date (2 wk) in the rye treatment. Interactions between earthworms and organic materials of different quality influence both the rate at which the materials are incorporated into the soil and the subsequent mineralization of N from those materials.

Abbreviations: MBN, microbial-biomass nitrogen • POM, particulate organic matter

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
EARTHWORMS CONSUME AND INCORPORATE into soil large amounts of organic matter (MacKay and Kladivko, 1985; Parmelee et al., 1990) and play an important role in the carbon cycle and the mineralization of nutrients from organic matter in many ecosystems (Hendrix et al., 1987; Edwards et al., 1995). Despite their known importance to organic matter cycling, surprisingly little is known about the influence of earthworms on the rates of incorporation of specific types of organic material and the subsequent release of nutrients from that material. Furthermore, there is a lack of information on how earthworms influence N cycling in agroecosystems (Parmelee and Crossley, 1988; Blair et al., 1997), especially with respect to the incorporation and release of N from different kinds of organic amendments and residues common to agricultural systems.

Lumbricus terrestris is one of the most important species of earthworm in temperate agroecosystems because of its abundance and ability to incorporate large amount of surface litter into the soil surface (MacKay and Kladivko, 1985; Bohlen et al., 1997). Populations of L. terrestris have been estimated to incorporate 2000 kg ha^-1 of leaf litter in orchards in 5 mo (Raw, 1962) and are capable of consuming or incorporating the entire leaf fall of a deciduous forest annually (Nielson and Hole, 1964). There are few data for arable fields, but Bohlen et al. (1997) estimated that a population of L. terrestris in a corn (Zea mays L.) field had the potential to increase the disappearance of surface litter by 840 kg ha^-1 yr^-1. Furthermore, earthworms can significantly alter the C/N ratio of crop litter remaining on the soil surface, apparently by selectively consuming litter fractions with the highest N content (Bohlen et al., 1997). Ketterings et al. (1997) showed a similar result for coarse organic matter, which, in field plots with decreased earthworm populations, had a higher total N content than did organic matter in plots with unmodified or increased populations.

The incorporation of surface litter into the soil by earthworms would be expected to increase the mineralization of nutrients in the litter, but there are few data to show this. In laboratory experiments in which ¹⁵N labeled ryegrass (Lolium perenne L.) litter was placed on the soil surface of pots containing actively growing plants, L. terrestris was shown to increase the uptake of ¹⁵N by the plants and, thus, to facilitate the transfer of N from the litter to the plants (Binet and Trehen, 1992; Hameed et al., 1993).

The assimilation and turnover of N in earthworm tissue and the influence of different food sources on those processes is also poorly understood. Many feeding trials have demonstrated that earthworms grow better on some food sources than on others (Edwards and Bohlen, 1996), but few studies have examined how different food sources affect N turnover in earthworms. Furthermore, the few published estimates of N turnover in earthworm tissue vary widely, even for a particular species of earthworm (Hameed et al., 1994a and 1994b; Curry et al., 1995).

Nearly all studies that have examined the influence of earthworms on the mineralization of N from surface litter have focussed on a single type of litter. To manage crop residues and nutrient inputs more effectively in agroecosystems it is essential that we understand the influence of earthworms on the incorporation of, and nutrient release from, a wide array of organic materials and fertilizers. Knowledge of the interaction of earthworms with different types of organic matter is essential for understanding how they influence the temporal patterns of mineralization of nutrients from different organic sources, and this understanding becomes important as practices that favor the build-up of earthworm populations (Edwards et al., 1995) and the conservation of crop residues on the soil surface (Hubbard and Jordan, 1996) are adopted more widely.

In order to increase our understanding of the interactions between earthworms and different nutrient inputs, we examined the incorporation and turnover of N from three different ¹⁵N-labeled nutrient inputs (KNO₃, rye, cow manure) in intact soil cores incubated in the laboratory with or without individuals of the earthworm, Lumbricus terrestris. We traced the movement of the added ¹⁵N into the soil and into different pools of N in the soil, including soil microbial biomass and POM. We examined the degree to which differences in the quality of different organic materials as food for earthworms influences the rate at which earthworms incorporate those materials into the soil, as well as the mineralization of N from those materials.

	Materials and methods

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Soil Core Preparation and Experimental Design
This experiment was performed with intact soil cores (10-cm-diam. PVC) taken to a depth of 20 cm from a field site in Wooster, Ohio. The site is being used to investigate the role of earthworms in conservation and loss of C and N in agroecosystems with different nutrient inputs (Bohlen et al., 1995). The cores were driven into the soil with a hammer, immediately removed, and returned to the laboratory on 27 January 1993. The soil was a Canfield silt loam (fine-loamy, mixed, mesic Aquic Fragiudalfs), which is a well drained silty soil with moderate slopes and a fragipan at 45 to 70 cm. Soils at the field site have a pH of 6.3 ± 0.4, cation exchange capacity of 10 ± 2 cmol kg^-1, and an average organic matter content of 3.7% ± 0.9 (Walkley-Black wet digestion).

Soil cores were frozen (-20°C) for 1 wk to destroy any earthworm cocoons. After thawing they were saturated and flushed four times with water and left to equilibrate at 250 g kg^-1 soil moisture and room temperature for 2 wk prior to adding the experimental treatments. An additional five cores were left unfrozen and otherwise treated identically to the frozen cores. These unfrozen controls were used to determine the effect of freezing, if any, on concentrations of soil inorganic N and microbial biomass (see below). The six experimental treatments were (i) KNO₃ with earthworms, (ii) KNO₃ without earthworms, (iii) rye litter with earthworms, (iv) rye litter without earthworms, (v) cow manure with earthworms, and (vi) cow manure without earthworms.

The soil cores were arranged in a 2 x 3 x 3 factorial randomized complete block design with five replicates, for a total of 90 cores. The first factor consisted of two earthworm treatments (with or without earthworms), the second factor consisted of three different nutrient treatments (KNO₃, rye litter, and manure), and the third factor was sampling time (2, 6, and 10 wk after adding earthworms).

Preparation of Nitrogen-15 Labeled Materials
The three nutrient treatments consisted of (i) ¹⁵N-labeled KNO₃ (10 atom % ¹⁵N, Isotech, Inc., Miamisburg, OH), (ii) rye litter (12.4 atom % ¹⁵N), and (iii) artificial cow manure (4.3 atom % ¹⁵N) made from the labeled rye litter. All nutrient amendments were added to the soil surface of the microcosms at a rate of 150 kg N ha^-1 (118 mg N core^-1) on 2 April 1993. The inorganic fertilizer was dissolved in deionized water and applied to the microcosms in solution. The rye litter was obtained from rye plants grown under field conditions with K¹⁵NO₃ (99 % atom ¹⁵N) fertilizer in a raised bed lined with plastic sheeting. Plants were fertilized three times during a 3-wk period when nutrient uptake was maximal. Whole rye plants were harvested before flowering and oven-dried (60°C). Roots were removed and shoots were coarsely chopped by hand before being added to the microcosms.

The artificial manure was prepared from a subsample of the ¹⁵N-labeled rye shoots using an in vitro rumen fermentation technique (Goering and Van Soest, 1970; Craig et al., 1984). Six hundred grams of finely ground rye litter were incubated anaerobically for 48 h with a buffered solution of rumen extract collected from a cannulated dairy cow. The digested litter was centrifuged at low speed, after which the supernatant was decanted and the remaining manure air-dried. Two subsamples each of oven-dried (60°C) rye shoots and artificial manure were taken for analysis of total C and N content and ¹⁵N enrichment.

Addition of Earthworms
Earthworms were collected from the field site using a formalin extraction technique (Raw, 1959). Collected worms were placed in containers filled with field soil, returned to the lab, and stored at room temperature until used. On 31 March 1993, the worms were sorted and identified, and three small, immature individuals of L. terrestris were added to the microcosms designated to receive worms (equivalent to 382 worms m^-2). The total fresh weight of worms added to each microcosm was recorded. The average initial oven-dry mass of worms added was .

Core Incubation
Cores were arranged in five replicate blocks of the six treatments and were incubated for 2, 6, or 10 wk under a natural light regime at 18 ± 2°C. Soil moisture was kept constant (200 g kg^-1) by regularly adding water to the soil surface with a hand-held sprayer to a constant weight. Pieces of fiberglass mesh (2 mm) were secured around the top and bottom of each core to prevent the escape of the added worms.

Sampling Procedure and Sample Analyses
At 2, 6, and 10 wk after the addition of earthworms, 30 soil cores consisting of five cores each of the six experimental treatments were destructively sampled. Prior to sampling the soil, any rye litter or manure remaining on the soil surface was collected. The soil was then removed from the cores in two layers; the upper 5 cm of soil was collected and analyzed separately from the lower 5- to 20-cm layer. The soil was mixed thoroughly by hand and a subsample was sieved through a 2-mm mesh. Sieved soil (12 g) was extracted in 25 mL of 0.5 M K₂SO₄ and extractable NH⁺₄ and NO^-₃ analyzed on a QuikChem AE Autoanalyzer (Lachat Instruments, Mequon, WI). Microbial-biomass N of sieved soil was determined using a chloroform fumigation–extraction technique (Brookes et al., 1985). Subsamples of 12 g were fumigated with chloroform for 5 d and then extracted with 0.5 M K₂SO₄. Extracts were digested with an alkaline persulfate oxidation technique (Cabrera and Beare, 1993) and analyzed for NO^-₃ on a QuikChem AE Autoanalyzer. Unsieved soil was oven-dried (60°C), ground to pass through a 250-µm screen, and analyzed for total C and N using a Carlo Erba NA 1500 CHN analyzer (Carlo Erba Instruments, Milan, Italy).

Two different size fractions (53–2000 µm and >2000 µm) of POM were determined on unsieved, oven-dried (60°C) soil samples using a wet sieving technique (Cambardella and Elliot, 1992). Only the upper 5 cm of soil in each core was analyzed for these fractions. Four 30-g subsamples of oven-dried soil were shaken overnight in 125-mL Erlenmeyer flasks with 60 mL of 0.55 metaphosphate solution to disperse clay particles. The samples were then poured through a stack of two sieves with mesh sizes of 2000 and 53 µm and the material retained on the sieves was collected and oven-dried (60°C). The organic matter content of the POM fractions retained on the sieves was determined by burning the samples at 450°C for 4 h, and total C and N contents were determined on a Carlo Erba NA 1500 CHN analyzer.

The ¹⁵N enrichment of the samples was determined by continuous flow isotope ratio mass spectrometry (Europa Tracer Mass, Europa Scientific, Franklin, OH) after combustion of the samples and separation of N gas on a Carlo Erba NA 1500 CHN analyzer. The atom % ¹⁵N enrichment of total soil N was determined in both soil layers, but enrichment of the two POM fractions, soil inorganic N, and MBN was determined only in the 0- to 5-cm layer of soil. Dissolved samples (inorganic N, MBN) were prepared using the micro-diffusion technique of Brookes at al. (1989) as modified by Sorenson and Jensen (1991). The ¹⁵N enrichments of the samples were corrected by using a blank calculated by comparing a diffused and non-diffused isotope standard (Kelly et al., 1991). The method used was identical to that described in Stark and Hart (1996) except that 5 µL of a solution of ¹⁵NH₄NO₃ (10 g N L^-1 at 5 APE) rather than (¹⁵NH₄)SO₄ were added to diffused and non-diffused blanks. The ¹⁵N enrichment of all diffused samples was determined on a Europa 20/20 continuous flow isotope ratio mass spectrometer (Europa Scientific, Franklin, OH) with helium as the carrier gas. Samples were combusted and N gas separated on a Europa Scientific Roboprep automated CN analyzer hooked directly to the mass spectrometer.

Surviving earthworms were carefully removed from the soil cores during sampling. Worms were placed in deionized water for 48 h to allow them to completely void their guts, and their fresh weight was recorded before they were oven-dried (60°C). Earthworm growth rates were calculated as the change in mass between sampling periods divided by the average of the beginning and final weights for that period. Oven-dried worms were finely ground with a mortar and pestle and their atom % ¹⁵N enrichment was determined using the same method described above for total soil and POM fractions.

Statistical Analysis
Differences among treatments were determined using a general linear model with earthworms, nutrient treatments, and time as the main effects. Samples from different dates from the two soil layers were analyzed separately. When there were significant interactions between earthworms and the different nutrient treatments, nutrient treatments were analyzed separately for significant earthworm effects. Differences among means were determined using a Tukey's HSD mean separation tests with a significance level.

	Results and discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Total Soil Nitrogen
Earthworms greatly increased the proportion of total ¹⁵N recovered in cores treated with organic inputs but had little effect in cores treated with inorganic fertilizer (Fig. 1 and 2 , Table 1) . Earthworms incorporated the manure more rapidly and to a greater degree than they incorporated the rye. The percentage of initially added N that was incorporated into the soil after 10 wk was 26% without worms and 43% with worms in microcosms treated with rye, and 34% without worms and 71% with worms in microcosms treated with manure, assuming that there was no isotopic fractionation of N during incorporation. Although we did not examine the amount of ¹⁵N remaining in the residues removed from the soil surface at each sample date, we assume that most of the original added N that is not accounted for in the soil remained on the soil surface. In microcosms treated with KNO₃, nearly all of the initially added ¹⁵N was recovered in the soil after 10 wk, and there was no difference in the percentage of initial ¹⁵N recovered in cores with (94.6%) or without (93.1%) earthworms. The lack of an earthworm effect on total soil ¹⁵N in the inorganic treatment is not surprising because the N was added in a soluble form that percolated readily into the soil.

View larger version (27K): [in this window] [in a new window]   Fig. 1 The percentage of initially added excess 15N that was incorporated into the 0–5 cm soil layer in cores treated with 15N-labeled KNO3 (upper panel), rye (middle panel) or manure (lower panel), and incubated with (shaded bars) or without (open bars) earthworms. Asterisks indicate significant differences (P < 0.05) between cores with or without worms. Error bars are standard errors

View larger version (30K): [in this window] [in a new window]   Fig. 2 The percentage of initially added excess 15N that was incorporated into the 5–20 cm soil layer in cores treated with 15N-labeled KNO3 (upper panel), rye (middle panel) or manure (lower panel), and incubated with (shaded bars) or without (open bars) earthworms . Asterisks and error bars are as for Fig. 1

View this table: [in this window] [in a new window]   Table 1 ANOVA table showing F values and levels of significance for the effects of main treatments and interactions on the excess atom% 15N of total soil N, microbial-biomass N (MBN), inorganic soil N and the 53–2000 µm and >2000 µm particu- late organic matter (POM) fractions. Data used in the analysis were expressed as a percentage of the initial 15N added to the microcosms and were arcsine-square-root-transformed prior to analysis

Earthworms increased the incorporation of ¹⁵N into the deeper soil layer (5–20 cm) in the manure treatment but did not in the other two nutrient treatments. After 2 wk most of the ¹⁵N label was restricted to the upper soil layer in all nutrient treatments, but enrichment of the deeper layer increased over time, especially in the inorganic treatment where downward movement of the added nitrate was facilitated by the small amounts of water added to maintain constant moisture. Over 85% of the added inorganic fertilizer N was in the lower soil layer at the end of the experiment. In the organic treatments the amount of initial ¹⁵N remaining in the deeper soil layer after 10 wk ranged from 12.5% for the rye-treated cores with no earthworms to 27.8% in the manure-treated cores with earthworms.

Particulate Organic Matter
Most of the increase due to earthworms in the ¹⁵N content of the soil in cores treated with rye litter and cow manure was attributable to their effect on the incorporation of POM. Earthworms had a significant effect on the amount of ¹⁵N in the 53- to 2000-µm and >2000-µm size fractions of POM, but not on the amount of ¹⁵N in the inorganic soil N pool or microbial biomass of the upper soil layer (Table 1). The temporal patterns of incorporation of the ¹⁵N-labeled, 53- to 2000-µm POM fraction reflect those of total soil ¹⁵N in the organically treated cores. In the manure-treated cores, earthworms greatly increased the amount of ¹⁵N in >2000-µm POM after 2 wk (Fig. 3) and increased the amount of ¹⁵N in the 53- to 2000-µm size fraction throughout the experiment (Fig. 4) . The amount of ¹⁵N in the >2000-µm size fraction in cores with earthworms declined rapidly after 2 wk probably because earthworms processed the coarser size fraction into finer fragments. Such processing of coarse organic matter into finer fractions was demonstrated in another pot study in which Lumbricus rubellus greatly reduced the amount of maize residue recovered in the >2000-µm size class (MacKay and Kladivko, 1985). In our study, after 10 wk, nearly 20% of the initial manure N was in the 53- to 2000-µm size fraction of POM in the upper 5 cm of soil with earthworms, whereas only 8% was in this size fraction after 10 wk in cores without earthworms (assuming there was no isotopic fractionation of N in POM). Nearly half of the total ¹⁵N in the upper 5 cm of soil was in the fine POM fraction in manure-treated cores with earthworms, but only one third of total ¹⁵N in the soil was in this fraction in manure-treated cores without worms.

View larger version (23K): [in this window] [in a new window]   Fig. 3 The percentage of initially added excess 15N that was recovered in the >2000-µm size fraction of POM in the 0–5 cm soil layer of cores treated with 15N-labeled KNO3 (upper panel), rye (middle panel) or manure (lower panel), and incubated with or without worms . Asterisks and error bars are as for Fig. 1

View larger version (20K): [in this window] [in a new window]   Fig. 4 The percentage of initially added excess 15N that was recovered in the 53–2000 µm size fraction of POM in the 0–5 cm soil layer of cores treated with 15N-labeled KNO3 (upper panel), rye (middle panel) or manure (lower panel), and incubated with or without worms . Asterisks and error bars are as for Fig. 1

The temporal patterns of incorporation of ¹⁵N-labeled POM provide insight into the feeding activities of the earthworms and explain their effect on total soil N. The rapid incorporation of POM by earthworms in the manure treatment indicates that worms in this treatment fed vigorously on the manure from the outset of the experiment and pulled fine fragments into the soil profile. By contrast, worms in the rye-treated cores were slow to incorporate POM from the labeled rye. In part, the slower incorporation of rye litter compared with the manure may have been related to the coarseness of the rye particles, which would have been difficult for the small worms to grasp with their small mouthparts. Larger individuals of L. terrestris, which can be over 10 to 20 times as large as the individuals used in this experiment, may have been able to incorporate larger particles and may have incorporated the rye at a greater rate and to a greater depth than did these smaller individuals. Throughout the course of the experiment, the rye litter softened and the decrease in tensile strength along with the increasing size of the worms probably enabled the worms to feed more actively on the rye litter and shred the larger fragments into finer particles, which they could then pull into the soil.

In addition to the physical differences between rye and manure, chemical differences between these two substrates may have affected feeding rates of the worms and incorporation of the substrates into the soil. The manure had a lower C/N ratio than the rye and contained large amounts of microbial and dissolved organic components from the rumen contents, which is likely to have stimulated feeding activity and growth. The rye litter probably became more palatable to the worms after it had been colonized by microorganisms. Weathering of raw litter and its colonization by microorganisms increases the palatability of many types of litter to earthworms (Edwards and Fletcher, 1988). For example, the acceptability of apple leaves to earthworms was increased when bacterial cells were added to the leaves (Wright, 1972). Large differences in litter quality can cause even greater differences in the rate of incorporation of litter than we observed in our study. When offered leaf litter from five different tree species in a laboratory soil-column experiment, individuals of L. terrestris began feeding immediately on rapidly decomposing, higher quality leaf litter, such as that of maple (Acer platanoides L.), but did not feed on the lower quality litter of beech (Fagus spp.) and oak (Quercus spp.) until it had weathered for several months (Zicsi, 1983).

Microbial Biomass Nitrogen
Earthworms significantly increased MBN throughout the experiment in the manure treatment but not in the other nutrient treatments (Fig. 5 , Table 2) . The increase in microbial biomass in the manure treatment was probably stimulated by the enhanced incorporation of manure into the soil by earthworms (Fig. 3 and 4). In cores treated with rye, earthworms did not increase microbial biomass at any time during the experiment and actually slightly decreased MBN on the first sample date (2 wk). It might be expected that earthworms would have stimulated microbial biomass in the rye treatment after 10 wk because they would have greatly increased the incorporation of fine particulate rye by that time (Fig. 4). The absence of an earthworm effect on microbial biomass after 10 wk in the rye treatment suggests that the POM derived from the rye was not as adequate an energy or nutrient source to support microbial growth as was that derived from cow manure. Cow manure contains readily mineralizable substrates that stimulate microbial growth, whereas rye contains more recalcitrant compounds that require a greater investment of energy to break down.

View larger version (19K): [in this window] [in a new window]   Fig. 5 Microbial biomass N in 0–5 cm soil layer of cores treated with 15N-labeled KNO3 (upper panel), rye (middle panel) or manure (lower panel), and incubated with or without worms. Asterisks and error bars are as for Fig. 1

View this table: [in this window] [in a new window]   Table 2 ANOVA table showing F values and levels of significance for the effects of main treatments and interactions on total N, atom% 15N enrichment, and total 15N in soil inorganic and microbial biomass N pools in the upper 5 cm of soil

View larger version (19K): [in this window] [in a new window]   Fig. 6 The percentage of initial excess 15N recovered in microbial-biomass N in the 0–5 cm layer of soil cores treated with 15N-labeled KNO3 (upper panel), rye (middle panel) or manure (lower panel), and incubated with or without worms. Asterisks and error bars are as for Fig. 1

Inorganic Nitrogen
Earthworms had less of an effect on inorganic N pools than they did on organic N pools. They had a statistically significant effect on soil inorganic N concentrations in the upper 5 cm of soil but did not affect the ¹⁵N-enrichment of the inorganic N pool (Table 2). Earthworms had no effect on inorganic N concentrations in the manure treatment and slightly decreased inorganic N concentrations on the first two sample dates in the KNO₃ treatment, and on the second sample date in the rye treatment (Fig. 7) . After 10 wk, the slight effects of earthworms on inorganic N had disappeared. Although earthworms have often been observed to increase inorganic N concentrations in soil (Lunt and Jacobson, 1944; Parle, 1963; Lavelle and Martin, 1992), in our experiment the large concentrations of inorganic N, which even after 10 wk were 15- to 25-µg g^-1 soil in the organically fertilized cores and 34-µg N g^-1 in inorganically fertilized cores, diminished the likelihood of observing an effect of earthworms on inorganic N concentrations. Furthermore, in the organically fertilized treatments, earthworms increased the incorporation of organic matter into the soil, which could have acted to immobilize any inorganic N mineralized by earthworm activity.

View larger version (20K): [in this window] [in a new window]   Fig. 7 The concentration of inorganic N in soil extracts from the upper 5 cm of soil cores treated with 15N-labeled KNO3 (upper panel), rye (middle panel) or manure (lower panel), and incubated with or without worms. Asterisks and error bars are as for Fig. 1

View larger version (19K): [in this window] [in a new window]   Fig. 8 Earthworm growth during the course of the experiment in soil cores treated with manure (·····), rye litter (– – – –) or potassium nitrate (•—•). Error bars are standard errors

View larger version (16K): [in this window] [in a new window]   Fig. 9 Accumulation of 15N in earthworm tissue in cores treated with 15N-labeled rye (•—•), manure (– – – –), or potassium nitrate (·····). Error bars are standard errors

	ACKNOWLEDGMENTS

 
We thank Dr. J.L. Firkins for providing laboratory facilities and invaluable assistance and advice for preparing the artificial ¹⁵N-labeled manure, D. Harris of the University of Michigan Stable Isotope Lab, and J. Fuller of the University of Georgia for timely and accurate ¹⁵N analysis by mass spectrophotometry. This research was supported by a grant from the National Science Foundation.

Received for publication July 8, 1998.

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