Introduction

In Amazonia, few studies on soil fauna have indicated earthworms as having an important biomass (Lavelle and Pashanasi 1989; Römbke and Verhaagh 1992; Barros 1999; Barros et al. 2001, 2002), or as playing a fundamental role on the litter decomposition process (Luizão 1985; Luizão and Schubart 1987). The geographical distribution of earthworms is mainly limited by macroclimate, but litter quality is a factor that determines their local distribution and activity (González et al. 1996). In both the soil and the litter layer, earthworms can increase by up to five times the number of edaphic microorganisms (Atlavinyte and Lugauskas 1971; Guerra and Asakawa 1981). Thus, interactions between earthworms and microorganisms can change microbial composition and activity, making earthworms important in the regulation of decomposition processes and nutrient recycling (Parle 1963; Fragoso et al. 1997; Lavelle et al. 1997; Wardle and Lavelle 1997; Lavelle 2000). For instance, earthworms can influence N mineralization, either directly, through N release by their metabolic products (faeces, urine, mucus) and dead tissues, or indirectly, through changes caused in soil physical properties, fragmentation of organic material, and through their interactions with other elements of the soil biota (Lee 1985).

Despite being very well known in the temperate zones, limited information is available to date for the tropics on the role of earthworms in the N cycle, partly disclosed only in the last decade. Pashanasi et al. (1992) have shown that the introduction of Pontoscolex corethrurus increased significantly the soil microbial biomass-N (bio-N) and mineral-N availability in experiments with seedlings of fruit species in Peru. Other studies in the tropics showed that earthworm populations are highly sensitive to soil management practices (Lavelle and Pashanasi 1989; Decaens et al. 1994, 1999; Fragoso et al. 1997; Barros et al. 2002). A better understanding of earthworm biology and ecology, including relationships with the litter layer and biochemical soil characteristics, is an essential step towards the adoption of ecologically sound management practices and maintaining the nutrient recycling processes (Jiménez et al. 1998; Decaens et al. 1999).

This study aimed to determine the effects of introducing two earthworm species, common in the region, Rhinodrilus contortus and Pontoscolex corethrurus, on the availability of soil mineral N (NH4 + and NO3 −), on the soil microbial biomass-N (bio-N), and on litter decomposition rates of leaf species differing in nutritional quality, in mesocosms containing soils from second-growth vegetation.

Material and methods

Two sizes of mesocosms were used for the different earthworm species: mesocosms measuring 20×20×20 cm for the smaller earthworm species P. corethrurus, and 30×30×40 cm mesocosms for the larger R. contortus earthworms. Both mesocosms were filled with a Xathic Ferralsol soil (Oxisol) collected from 8-year-old second-growth forest (Araujo 2000), located at the EMBRAPA/CPAA Experimental Station, 30 km along the AM-010 Road, in Manaus, Amazonas, Brazil (geographic coordinates: 2°51′S and 59°52′W). This soil had a high content of clay (60%), extreme acidity (pH=4), low concentrations of mineral N (1.6 mg g−1), cation exchange capacity of 0.5 cmol kg−1, and base saturation of 33%. Before filling the mesocosms, the soil samples were air-dried and sieved through a 6-mm mesh in order to remove larger organic matter fragments. The earthworms were collected from plots of primary forest and second growth (“capoeira”) at the EMBRAPA station. In the treatments with earthworms, two immature individuals of R. contortus were added to each large mesocosm, and four individuals of P. corethrurus (three adults and one immature) to each smaller mesocosm. In all the mesocosms belonging to the two size classes (small or large), the biomass of inoculated earthworms by mesocosm was similar at the beginning of the experiment (9.14 g of R. contortus and 2.17 g of P. corethrurus).

The surface of the mesocosms was covered with freshly fallen leaf litter, also collected at the EMBRAPA station, air-dried, and stored in a dry place. Leaves were cut into 2×2-cm pieces, in order to speed their decomposition in the short period of the experiment. Four treatments were applied, adding leaves of distinct nutritional quality, as defined by their C:N ratios: Hevea brasiliensis Muell. Arg.; Carapa guianensis Aubl.; Vismia sp.; and a mixture of the three species, with equal weights. (H. brasiliensis and C. guianensis have been used by EMBRAPA to form rows of valuable tree species, growing together with the natural regeneration, after deforesting abandoned and sick rubber-tree plantations). In this study, the H. brasiliensis leaves (C:N=27) with a low C:N ratio were considered to be of high nutritional quality, while Vismia sp. leaves (C:N=42) with a high C:N ratio were considered to be of low nutritional quality, and the C. guianensis leaves (C:N=32) and mixed litter (C:N=34) were considered to be of intermediate nutritional quality (Beck et al. 1998). The tops of the mesocosms were covered by a 1-mm nylon mesh to prevent the earthworms from leaving the boxes. Each of the treatments with earthworms had four replicates; the treatments without earthworms had three replicates, making up a total of 56 mesocosms, besides the controls.

The experiment was carried out from August to November 1999 in a protected (walls in nylon mesh) glasshouse located at INPA-V8 secondary campus, in the city of Manaus (3°88′S; 60°1′O). Soil moisture of the 0- to 10-cm layer was kept between 30 and 42%. The water matrix potential in the upper 10-cm soil layer of mesocosms corresponded to pF=2–2.5. Air temperature and relative humidity in the glasshouse, during the experiment, ranged from 24–31 °C and 38–61%, respectively.

After 97 days, the mesocosms were emptied and all earthworms found were retrieved. All individuals were counted and their fresh weight determined using a precision balance. Estimates of the proportion of faecal residues present on the soil surface in the mesocosms were obtained by counting the number of faecal pellets present on the surface of each mesocosm at the end of the experiment. The relative proportion (percentage) of faecal material produced by each earthworm species was then calculated, giving an estimate of the activity of the earthworms (Guerra 1988).

In each mesocosm, composite soil surface samples (0–5 cm) were taken using a fine corer (2 cm diameter), and soil analyses were made on fresh soil, immediately after collection. The concentrations of NO3 − and NH4 + were determined after extraction with 1 M KCl (20 g of soil and 150 ml of KCl) (Maynard and Kalra 1993). Measurements were taken using a flow injection system (SFA-2, Burkard Scientific). Bio-N was measured using the fumigation-extraction method (Brookes et al. 1985). Decomposition rates of the litter added to mesocosms were estimated, at the end of the experiment, by the weight loss of the leaves in relation to the initial dry weight.

Multiple analyses of variance (MANOVA) were used to assess the differences between the concentrations of mineral-N and bio-N with and without earthworms, as well as for each of the added earthworm species, and for each litter species. For all analyses, the statistical package SYSTAT 6.0 for Windows (Wilkinson 1996) was used.

Results

Density and biomass of earthworms

Earthworm density and biomass decreased in all treatments at the end of the experiment for the species R. contortus (Table 1). The greatest loss of earthworm biomass occurred in the mesocosms receiving the addition of Carapa guianensis leaves (0.5 g per mesocosm); the least loss of earthworm biomass was found in the treatment with addition of Hevea brasiliensis leaves (3.5 g per mesocosm).

Table 1 Mean number of individuals and mean initial and final biomass of earthworms in the mesocosms (n=4). Litter species: H Hevea brasiliensis; C Carapa guianensis; M mixed litter; V Vismia sp
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In the mesocosms inoculated with the species P. corethrurus, earthworm densities increased and immature individuals were abundant, besides the presence of some cocoons. For this species, the total number of individuals in the mesocosms showed no significant differences between the different leaf types in the litter addition treatments (10.2–11.4 ind. per mesocosm). At the end of the experiment, the earthworm biomass (2.4–3.8 g per mesocosm) was greater than the initial (2.1 g per mesocosm), in all treatments (Table 1).

Earthworm activity

The presence of faecal material on the surface of the mesocosms allowed an estimate of earthworm activity, which was greater in the species P. corethrurus than in R. contortus (Fig. 1). Both species were more active under the treatment with addition of H. brasiliensis, while smaller activities were found under the treatments with additions of Vismia and the mixture of leaf species. The species P. corethrurus had similar activity in the two latter treatments. The lowest activity was found with the addition of C. guianensis leaves, for both earthworm species.

Fig. 1
figure 1

Relative proportion (%) of amount of faecal material produced by each earthworm species in the mesocosms. Values represent means ± 1 standard error (n=4). H Hevea brasiliensis; C Carapa guianensis; M mixed litter; V Vismia sp

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Nitrate concentrations

The upper soil layer in the mesocosms with R. contortus showed NO3 − concentrations higher than soil with P. corethrurus (Table 2). The presence of R. contortus had a significant effect on soil NO3 − concentrations (ANOVA; F= 20.4; p<0.01). Treatments with addition of H. brasiliensis and Vismia sp. showed the greatest NO3 − concentrations (13 µg N g−1 for both), followed by the mixture of three leaf species (10.2 µg N g−1) and C. guianensis (6.2 µg N g−1). However, no significant differences (p>0.05) were found among the four litter addition treatments. Mesocosms with P. corethrurus presented NO3 − concentrations significantly higher (ANOVA; F=17.8; p<0.01) than mesocosms without earthworms (Table 2). The concentrations ranged from 4.5 µg N g−1 under C. guianensis to 5.6 µg N g−1 under H. brasiliensis, but the differences between the four litter addition treatments were not significant (p>0.05). Nitrate concentrations did not vary significantly in the control treatments.

Table 2 Soil NO3 −, NH4 + and microbial biomass-N (bio-N) concentrations (µg N/g) in the experiment with (n=4) and without (n=3) inoculation of R. contortus and P. corethrurus under different litter addition treatments, 97 days after inoculation. Values are means ± standard deviation Different letters a and b in the lines indicate significant differences between treatments with and without earthworm inoculation (NO3 −: p<0.01), (NH4 +: p<0.05). Bio-N showed no significant differences between treatments with and without both species of earthworms
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Ammonium concentrations

Ammonium concentrations in the mesocosms with R. contortus showed a similar pattern as for NO3 −; thus, addition of C. guianensis leaves presented the smallest value (1.7 µg N g−1), and the greatest was found in the treatment with H. brasiliensis leaves (4.7 µg N g−1) (Table 2). However, such differences between litter addition treatments were not significant (p>0.05). Mesocosms without R. contortus also showed no significant differences among litter addition treatments; soil NH4 + concentrations ranged from 1.2–2.3 µg N g−1. Nonetheless, the species R. contortus had a general positive and significant effect on soil NH4 + concentrations (F=9.9; p<0.01), when compared to the control. Soil NH4 + concentrations in the mesocosms inoculated with P. corethrurus ranged from 1.5 µg N g−1 (H. brasiliensis) to 1.9 µg N g−1 (Vismia). Mesocosms without P. corethrurus had concentrations ranging from 0.7 (Vismia) to 1.4 µg N g−1 (H. brasiliensis), but these differences were not significant (p>0.05).

Soil microbial biomass-nitrogen (bio-N)

The greatest values for bio-N were found in the mesocosms with R. contortus (Table 2), especially with addition of H. brasiliensis (6 µg N g−1) and Vismia (1.6 µg N g−1) leaves. The controls (with no inoculation of R. contortus) presented very low values of bio-N, ranging from 0.03–0.1 µg N g−1. Bio-N values in the mesocosms with P. corethrurus were greater than in the mesocosms without earthworms (Table 2). The greatest value of bio-N was recorded under the treatment with H. brasiliensis (0.4 µg N g−1), while the smallest was found under the mixed litter treatment (0.1 µg N g−1). The mesocosms without P. corethrurus showed bio-N values between 0.05 and 0.1 µg N g−1. Though consistently greater than the control (without earthworm inoculation), the bio-N values recorded in the treatments with earthworms were not significantly (p>0.05) different, neither in the treatment with R. contortus nor with P. corethrurus, presenting a very high data variability among mesocosms.

Leaf litter weight loss

Disregarding the earthworm treatment, the greatest weight losses were always found on the H. brasiliensis litter, followed by the mixed litter and the C. guianensis; the slowest weight loss occurred in the Vismia litter (Table 3). However, the weight loss rates of the mixed litter and C. guianensis were not significantly different (Tukey, p>0.05). Neither the species R. contortus nor the species P. corethrurus had significant effect on leaf litter decomposition (p>0.05 for both species) during the 97-day-long experiment.

Table 3 Dry weight loss (%) of litter in the mesocosms with (n=4) and without inoculation (n=3) of R. contortus and P. corethrurus in the litter addition treatments, 97 days after earthworm inoculation. Values are means ± standard deviation. (No significant differences were found between treatments with and without earthworms)
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Discussion

The decrease in both density and biomass of R. contortus in the larger mesocosms indicates that this species was severely affected by the environmental conditions developed within the mesocosms during the experiment, causing the earthworms to die or to flee. Still very little is known on the biology and ecology of R. contortus; information on the appropriate conditions in captivity is unavailable. They appear to be very mobile animals which do not tolerate disturbances in the soil. Alternatively, their low densities may be a result of insufficient amount of the specific resources they need to survive (Lavelle 2000, personal communication). Apparently, the earthworms did not die or leave the mesocosms in the first weeks of the experiment, considering that faeces were found on the surface and bottom of the mesocosms, as well as distributed in all soil layers, in addition to the galleries within the soil profile. This confirms R. contortus as an endogeic and geophagous species (i.e., one that lives and feeds in soil).

On the other hand, the observed increase in P. corethrurus densities within the mesocosms indicates that mature individuals of this species are reproducing during the experiment, which means they were not severely affected by the treatment stress. P. corethrurus was able to maintain or even increase its biomass within the mesocosms during the whole experiment. This species presents a large geographical distribution and high adaptability to diverse environments (Lavelle et al. 1987; Barros 1999). Very likely because of such adaptability, P. corethrurus showed higher activity within the mesocosms (as estimated by the presence of faeces on the soil surface) than R. contortus.

In spite of this activity, none of the earthworm species had a significant effect on litter decomposition, independent of its nutritional quality. Both earthworm species are geophagous, i.e., soil feeders; thus, they are not directly feeding on litter. However, their possible indirect effect on the decomposition rate of different litter species should not be dismissed; that may occur through the enhancement of the microbial activity, by their faeces and N release. In fact, earthworms were more active in the mesocosms covered with Hevea brasiliensis leaves, a treatment in which were also found the highest values of bio-N and NO3 −. Possibly, the fast decay of the Hevea leaves produced soluble compounds which stimulated microbial growth and nitrification activity (Atlavinyte and Lugauskas 1971; Guerra and Asakawa 1981; Barois et al. 1987; Bohlen and Edwards 1995). However, in principle, such nitrification activity could only be carried out by fungi, in view of the strong soil acidity of the substrate used here (pH=4). Under these conditions, heterotrophic nitrification by fungi prevails over bacterial nitrification, which only converts NH4 + into NO3 − when oxygen is present, allowing the chemioautotrophic process to take place (Brookes et al. 1985; Maynard and Kalra 1993). Apparently, all fungi are able to utilize some organic N sources for their nitrification activities (Jennings 1989). The possible stimulation of nitrification and nitrifying organisms remains an open and important issue for future research.

The highest weight loss of Hevea leaves at the end of the experiment was likely due to its better nutritional quality and relatively low C/N ratio (C/N=27), allowing a faster decomposition rate (Palm and Sanchez 1990). Among all treatments, the lowest earthworm activity in the mesocosms was found in the treatment with C. guianensis leaves, possibly because of its low nutritional quality (C/N=32) and the thickness and hardness of the leaves.

In spite of the decrease in density and biomass of R. contortus, observed at the end of the experiment, the apparent death of earthworms did not produce an increase of soil mineral N, nor of bio-N, in the mesocosms. Even in the mesocosms from which the earthworms disappeared or died (thus, being decomposed ‘in situ’), the concentrations of mineral N and bio-N were not significantly higher than in the mesocosms where the earthworms remained alive to the end of the experiment.

Most of the mineral N produced during the experiment was found as NO3 −. The presence of R. contortus led to a near-doubling of N-NO3 − concentrations and increased the bio-N by four- to 14-fold in the treatments with addition of Vismia leaves. Increases in the mineral and microbial N concentrations in soil as a result of earthworm presence and activity have been reported in other studies in the tropics (Lee 1985; Barois et al. 1987; Pashanasi et al. 1992). The positive influence of earthworms on N mineralization may have been a result of either the N release in their metabolic products (faeces, urine, mucus) and dead tissues, or the changes caused in soil physical properties, and fragmentation of organic material (Lee 1985). The highest concentrations of N-NH4 + were found under Hevea leaves, the fastest decomposing leaf species; however, in all treatments, there were losses of NH4 + in relation to the control. Probably higher N mineralization happened due to the decomposition process (highly controlled by fungi), and this stimulated the transformation of N-NH4 + into N-NO3 − in the presence of R. contortus. Higher losses of N-NH4 + have also been recorded in temperate locations; for instance, Anderson et al. (1985) found substantial increases in N-NH4 + losses in microcosms where animals were introduced.

The species P. corethrurus also increased mineral-N and bio-N contents within the mesocosms; however, such increases were lower than those produced by R. contortus. Probably, this is a result of the highest biomass of R. contortus, a species that can eat and process much more soil than P. corethrurus, thus causing a higher N turnover.

Other studies have shown partially similar results to those found here. For instance, Bohlen and Edwards (1995) and Blair et al. (1997) found that earthworms increased the concentrations of soil N-NO3 −, but the bio-N concentrations decreased with an increase in earthworm populations. In contrast to the above-mentioned authors, an increase of the microbial activity, as an indirect effect of earthworms, has been reported by several authors (Scheu 1987; Lavelle 1988; Barros 1999; Decaens et al. 1999). Apparently, earthworms have a variable influence on the microbial biomass and activities, inducing decreases in bio-N and/or in N transformations in some experiments, and increases in others.

The effect of the species P. corethrurus on mineral-N and microbial biomass has been studied in the tropics by Barois et al. (1987), who found in a laboratory experiment using pasture soil 2.7 times more N-NH4 + in the earthworms’ faecal material than in the original soil; N-NO3 − concentrations also increased, but the differences were not significant. Pashanasi et al. (1992), in pots with soils from second-growth areas, planted with seedlings of three tropical fruit species, found positive correlations between the concentrations of N-NH4 + and earthworm biomass, while nitrification was generally low, and the concentrations of N-NO3 − were smaller than those of N-NH4 +; in all treatments, there was an increase of bio-N. In the present study, however, concentrations of N-NH4 + were always smaller than those of N-NO3 −, suggesting the existence of mechanisms other than autotrophic nitrification occurring in the presence of earthworms. In temperate agrosystems, Blair et al. (1997) also found a significant increase of N-NO3 − concentration when earthworm population was also increased, despite recording only a modest increase in N-NH4 +, and a low bio-N. In a temperate deciduous woodland soil, a 50% increase in the net mineralization was recorded due to the presence of soil animals, which significantly disrupt the time course of microbial process and enhance the turnover of microbial populations, with release of N-NO3 − from the dead microbial bodies (Anderson et al. 1985).

In the Amazon, few published works report the effects of earthworms on the soil microflora and nutrients. Guerra and Asakawa (1981) showed that the presence of P. corethrurus, in pots, increased the microbial population over time, while Barros (1999) found a positive correlation between the bio-C and earthworms biomass. In the present study, the presence of both earthworm species increased the soil bio-N, due to the edible substrates (carbon) made available to the microorganisms by the earthworms, by the secretion of cutaneous and intestinal mucus (Barois et al. 1987; Blair et al. 1997). Within the earthworm intestines, the mucus is mixed with the ingested soil, or deposited along the soil galleries (Barois et al. 1987). Thus, earthworm faeces have more organic matter, in a partly digested form which can be easily assimilated by the microorganisms (Shaw and Pawluk 1986; Blanchart et al. 1993; Decaens et al. 1999; Barros et al. 2001).

The effect of earthworms on microbial biomass seems to depend upon many factors, and the experimental design must be taken into account before making generalizations regarding effects of earthworms on soil biogeochemistry. Additionally, attention should be paid to the specific role of fungi in the nitrification process in such acidic soils. Future research should determine both heterotrophic and autotrophic nitrification and should address the question of whether the nitrification and nitrifiers are or are not stimulated by organic compounds, by directly monitoring changes in nitrifiers.

Despite the limitations of the present study, including its short duration, the positive role of earthworms in the N cycle of tropical soils is confirmed, thus being similar to that of earthworms from temperate zones (where most of the cited studies were made). The importance of earthworms for organic-N mineralization and for the control of microbial populations, which makes the mineral-N available in soil, is now demonstrated. Both NH4 + and NO3 − are easily taken up by plant roots, favoring considerably plant growth; thus, earthworms can stimulate the transfer of nutrients from microbial biomass to the plants.