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The train millipede (Parafontaria laminata) mediates soil aggregation and N dynamics in a Japanese larch forest
Geoderma 159 (2010) 216–220
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Geoderma j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e o d e r m a
The train millipede (Parafontaria laminata) mediates soil aggregation and N dynamics in a Japanese larch forest Reiji Fujimaki 1, Yui Sato, Naoyuki Okai, Nobuhiro Kaneko ⁎ Graduate School of Environment and Information Sciences, Yokohama National University, 79-7, Tokiwadai, Hodogaya, Yokohama, Kanagawa 240-8501, Japan
a r t i c l e
i n f o
Article history: Received 15 December 2009 Received in revised form 8 May 2010 Accepted 15 July 2010 Available online 19 August 2010 Keywords: Larch forest soil N mineralization N2O emission Soil aggregation Train millipede (Parafontaria laminata)
a b s t r a c t The contributions of soil macroinvertebrates such as earthworms and termites to soil structure and nutrient cycling are well recognized, but few studies have examined the influence of geophagous millipedes on soil nutrients and structure. We conducted a soil incubation experiment to evaluate the effects of the train millipede (Parafontaria laminata), which is endemic to central Japan, on N mineralization, N2O emissions, and aggregation in soils. Larvae of P. laminata significantly increased development of soil aggregates N 2 mm during the 28-day incubation experiment. This soil aggregation was attributed to fecal pellets and molting chamber walls of P. laminata larvae. N mineralization, nitrification, and N2O–N emissions were also promoted by P. laminata, although these changes in N dynamics did not result in changes in the total amounts of C and N in the soil. N mineralization and N2O–N emissions were positively correlated with the amount of large (N 2 mm) soil aggregates. These correlations indicate that the biogenic structure produced by P. laminata significantly influences soil N dynamics, and suggest that the soil micro-environment associated with large aggregates promotes microbial processes related to N dynamics, especially N2O production. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The contribution of macroinvertebrates to soil processes is well recognized. Some groups of soil macroinvertebrates, including earthworms, termites, and ants, are known as ecosystem engineers, as they modify soil structure by creating cast aggregates, mounds, and subterranean galleries (Jones et al., 1994; Lavelle et al., 1997; Folgarait, 1998). These biogenic structures often persist for much longer than the lifetimes of the organisms that produced them. Such biogenic structures influence the activities of other organisms, and their function is referred to as “soil ecosystem engineering” (Lavelle et al., 1997; Lavelle, 2002). Biogenic structures also influence the dynamics of soil organic matter and nutrients. For example, earthworms can produce soil macroaggregates N1 mm in diameter (Ketterings et al., 1997) and up to 2 mm (Blanchart, 1992; Winsome and McColl, 1998) through their feeding and excreting activities. Soil aggregates derived from earthworm-worked soil contain organic materials from fresh residues of plant debris (Bossuyt et al., 2004; Fonte et al., 2007), and influence the structure and activity of the microbial community, resulting in alteration of the N transformation pattern (Schutter and Dick, 2002; Noguez et al., 2008). Bohlen and Edwards (1995) demonstrated that earthworms increased soil inorganic N from 1.26- to 4.00-fold in a ⁎ Corresponding author. Tel./fax: + 81 45 339 4379. E-mail address: [email protected] (N. Kaneko). 1 Present address: Faculty of Life and Environmental Science, Shimane University, 1060, Nishikawatsu, Matsue, Shimane 690-8504, Japan. 0016-7061/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.geoderma.2010.07.014
microcosm experiment. In addition, Borken et al. (2000) reported that the introduction of earthworms resulted in a 57% increase in soil N2O emissions. While there are more studies on earthworms than on other soil macroinvertebrates, it has been reported that millipedes also play an important role in the facilitation of nutrient and carbon dynamics in soils (Cárcamo et al., 2000; Toyota et al., 2006). Most millipedes are litter feeders (Hopkin and Read, 1992). Their ability to facilitate nutrient dynamics is attributed to litter comminution through their ingestive activity, which promotes nutrient release and utilization of litter by microbes (Cárcamo et al., 2000). However, there have been few studies on geophagous millipedes and their functions in modifying soil structure (but see Niijima, 1984; Toyota et al., 2006). We examined the effect of the train millipede (Parafontaria laminata), which is endemic to central Japan, on soil aggregation and N dynamics. The millipede has an 8-year life cycle with annual molting. The 1st to 7th-instar larvae inhabit soils, mostly at a depth of 0–10 cm in summer and autumn, and at lower depths in early spring (Niijima, 1984; Toyota and Kaneko 2004). Throughout central Japan, adult millipedes swarm the soil surface at 8-year intervals, often with extremely high densities and biomasses (Yoshida et al., 1985; Niijima and Shinohara, 1988). The larvae feed on mineral soils and associated old organic matter, whereas adults feed on a mixture of mineral soil and fresh leaf litter (Hashimoto et al., 2004). High densities of adult-stage train millipedes can reduce the accumulation of organic matter in surface soils (Niijima and Shinohara, 1988). In addition, their mixed feeding on litter and soil increases carbon sequestration in mineral soils through promoting the interactions of organic matter and mineral particles
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(Toyota et al., 2006). Together with the periodic, high-density swarming, the feeding habits of the train millipede can be expected to significantly affect ecosystem processes through changing the accumulation of soil organic matter and nutrient dynamics (Niijima, 1984; Hashimoto et al., 2004; Toyota et al., 2006). However, there is little information available about the effects of the train millipede on N dynamics and soil aggregation. The objective of this study was to determine the effects of the train millipede on soil aggregation and N dynamics in forest soil. We conducted an incubation experiment using forest soil and train millipede larvae, and evaluated their effects on soil aggregation, N mineralization, and N2O emissions. We hypothesized that millipede-worked soil would contain more large-sized soil aggregates, and would show increased N mineralization and N2O emissions. 2. Materials and methods 2.1. Feeding experiments with millipede larvae We conducted a soil incubation experiment with controlled densities of train millipedes (P. laminata). In July 2007, we collected 6th-instar train millipede larvae and surface soil (ca. 0–20 cm depth) from a Japanese larch (Larix kaempferi) forest. The train millipede had been found at high densities in this forest, which is located in the foothills of Mt. Tennyo in the Yatsugatake Mountains, central Japan (Tennyo site, 35°54′ N, 138°23′ E; 1400 m elevation). A detailed description of the site is given elsewhere (Toyota et al., 2006). Hashimoto et al. (2004) reported that the dry weight biomass of 7thinstar millipedes at the site was 15.7 g m− 2 in 1999 and of adult millipedes was 28.6 g m− 2 in 2000. These values are substantially higher than those generally reported for insect density in forest soils (e.g., Petersen and Luxton, 1982). Soils were classified as Andosols (FAO, ISSS, ISRIC, 1998). Freshly collected soils were passed through a 2-mm mesh sieve to remove gravel, plant debris, and large-sized aggregates, and then homogenized. Portions of approximately 88-g of fine-size (b2 mm) fresh soil, which was equivalent to 36 g of dry weight soil, were placed into 200-ml glass vials (5.1 cm in diameter and 10 cm in height), and soil moisture was adjusted to 60% field water holding capacity. The headspace volume in the vial was approximately 168 cm3. Train millipede larvae (6th-instar) were placed into the vials. Each vial contained 0, 2, 4, or 8 individuals, which approximated to 0, 6.4, 12.8, and 25.6 mg dry weight per vial, with five replicates of each density. These values were equivalent to densities of 0, 1000, 2000, and 4000 individuals m− 2 in field conditions. The observed field density of 6th-instar larvae around Mt. Yatsugatake ranged from 0 to 2156 m− 2 in 1999 (Toyota and Kaneko, 2004). Each vial was sealed with a polyethylene lid to prevent the soil from drying out, and to prevent escape of the millipedes. The polyethylene lid was equipped with a rubber septum for gas sampling. The soils and millipedes were incubated for 28 days in the dark at 18 °C. Three empty vials were prepared for use as blanks in the gas measurements. Every 7 days, the vials were opened for aeration and moisture adjustment. Some individuals died during the course of the experiment; the mean final densities were 0, 1.2, 3.4, and 7.4 individuals per vial (Table 1). Some of the survived individuals were molting at the end of the experiment.
Table 1 Mean number of survival, dead and molting individuals of P. laminata after 28 days of feeding. Introduced number
Survival
Dead
Molting
2 4 8
1.2 (0.45) 3.4 (0.89) 7.4 (0.89)
0.8 (0.45) 0.6 (0.89) 0.6 (0.89)
0.8 (0.45) 0.6 (0.89) 4.8 (1.78)
Values in parenthesis are standard deviations (n = 5).
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After the feeding experiment, the vials were sealed for 4 h and the headspace N2O concentration was analyzed. A 1.5-ml sample of the headspace gas was taken from the rubber septum of each incubation vial using a gas-tight syringe. N2O concentrations were determined using a gas chromatograph equipped with an electron capture detector and 2 m of Porapak Q (60/80 mesh, GC-14B; Shimadzu, Kyoto, Japan). The temperatures of the injector, column, and detector were 80, 60, and 340 °C, respectively. The carrier gas (N2) was supplied at a rate of 10 ml min− 1, and both standard and atmospheric gases were measured before and after sample gas measurements were taken. The N2O emissions in each vial were calculated by multiplying the headspace volume by the difference in N2O concentrations between the sample gas and the atmosphere. Before and after the incubation experiment, soil samples were dried at 40 °C and the C and N concentrations were determined by combustion (NC-95A; Sumika Chemical Analysis Service, Osaka, Japan). In addition, before and after the 28-day incubation, inorganic N was extracted from 3 g soil samples using 30 ml 2 M KCl. The − inorganic N (NH+ 4 –N and NO3 –N) concentration was determined using a continuous flow analyzer (Futura, Alliance Instruments, Frépillon, France). The nitrogen mineralization rate was determined by calculating the difference between the inorganic N concentrations in the soils before and after the 28-day incubation. After the experiment, the soils were again sieved through a 2-mm mesh to separate the fine- (b2 mm) and large- (N2 mm) size fractions. Then, the soil fractions were dried at 40 °C and weighed to evaluate the aggregation. 2.2. Statistical analyses A linear regression was performed to evaluate the relationship between the millipede density and soil aggregation (N2 mm size fraction). The effect of the millipedes on N mineralization, nitrification, and CO2 and N2O emissions in the incubation experiment was determined using a one-way analysis of variance (ANOVA) after checking the data for normality and homoscedasticity by Shapiro– Wilk and Bartlett tests, respectively. Data from the N mineralization was log-transformed prior to the ANOVA to improve the homoscedasticity. When a significant effect (P b 0.05) was detected by the ANOVA, a pair-wise t-test with Holm's adjustment was performed as a post hoc test. A Spearman's rank correlation coefficient was calculated to evaluate the relationship between soil aggregation and N mineralization or N2O emissions. 3. Results Millipede larvae promoted soil aggregation, i.e., increased the proportion of the large-size fraction (N2 mm), during the 28-day experiment (Fig. 1). The amount of the large-size aggregates was positively correlated with the number of introduced larvae (y = 1.72x + 3.65, P b 0.001), although the difference between the four larvae and eight larvae treatments was not significant (paired t-test with Holm's adjustment, P = 0.35). The millipede larvae also significantly stimulated soil N dynamics (Fig. 2; F = 21.0, P b 0.001 for N mineralization; F = 34.6, P b 0.001 for nitrification; and F = 7.6, P b 0.05 for N2O emissions). Compared with control soils, soils containing larvae showed increased N mineralization and nitrification (paired t-test with Holm's adjustment, P b 0.05), especially those with a high density of larvae. The millipedes had a greater effect on N mineralization than on nitrification, and the proportions of nitrification in N mineralization decreased with the addition of millipedes. Soil samples containing four or eight larvae showed higher N2 O emissions and greater amounts of large aggregates, while the effects of two larvae were not significant. Both the N mineralization and N2O emissions were positively correlated with the amount of soil in the N2-mm size fraction (Fig. 3;
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Fig. 1. Relationship between numbers of introduced P. laminata and amount of large soil aggregates (N2 mm size fraction) after the 28-day incubation experiment. Line shows linear regression (y = 1.72x + 3.65, P b 0.001).
Spearman's rank correlation test, rs = 0.74, P b 0.01 and rs = 0.52, P b 0.05 for N mineralization and N2O emissions, respectively). The soil C and N concentrations and C:N ratio did not differ among the treatments (Table 2). 4. Discussion 4.1. Aggregate formation The larvae of P. laminata are geophagous (Hashimoto et al., 2004; Toyota et al., 2006), which is uncommon among millipedes (Hopkin and Read, 1992). Geophagous soil macrofauna such as earthworms facilitate soil aggregation into large-size fractions, mainly as casts (Blanchart, 1992; Ketterings et al., 1997; Bossuyt et al., 2004; Fonte et al., 2007; Loranger-Merciris et al., 2008). While individual fecal pellets
Fig. 3. Relationships between soil aggregate (N2 mm size) and N mineralization (A) and between soil aggregates and N2O–N emissions (B) in the soil incubation experiment using 6th larvae of P. laminata.
of P. laminata are smaller than 2 mm, the pellets can gather fine particles of soil or other pellets (Fujimaki, R., personal observation; Niijima, 1984), presumably because polysaccharides in the feces bind to neighboring soil particles. These pellet aggregates likely account for the increased soil aggregation observed in the incubation experiment. Our observation that the millipede activity resulted in greater amounts of N2 mm aggregates in this experiment is consistent with results of previous studies on soil-feeding earthworms (Blanchart, 1992; Ketterings et al., 1997; Winsome and McColl, 1998; Bossuyt et al., 2004; Loranger-Merciris et al., 2008). In addition to the effect of fecal pellets, the molting habits of the millipede may have contributed to the large-size aggregates (Table 1). The larvae of P. laminata molt annually in molting chambers made from their feces (Niijima, 1984; Niijima and Shinohara, 1988). Unlike fecal pellets of other diplopods that mainly feed on fresh organic matter, the feces of train millipedes contain a high proportion of mineral soils resulting from their geophagous feeding habit (Niijima, 1984; Toyota et al., 2006). This results in molting chamber walls with Table 2 Mean concentrations of the soil C and N and mean C:N ratios after the feeding experiment.
Fig. 2. N mineralization (white) and nitrification (grey) during the experiment (A) and N2O–N emissions at the end of the experiment (B). Error bars indicate standard deviations (n = 5). Letters indicate significant differences among the millipede numbers.
Number of millipedes
C [mg kg− 1]
N [mg kg− 1]
C:N
0 2 4 8
224.8 221.9 213.5 227.6
13.5 13.3 13.1 13.2
17 16 16 17
(1.5) (2.3) (11.4) (2.3)
Values in parenthesis are standard deviations.
(0.3) (0.2) (0.9) (0.2)
(0.4) (0.3) (0.3) (0.3)
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a tightly bound structure. The chamber wall can reach several millimeters in thickness (Fujimaki, R., personal observation). Although the chamber structure would likely decay within several years (Yamaguchi, K., personal comm.), pieces of the chamber wall could last for a longer period (Niijima, 1984). We confirmed that the activity of P. laminata can increase soil aggregation under laboratory conditions in this short-term (28-day) experiment. The characteristics of their fecal pellets and molting chamber walls also suggest that they would contribute to maintaining the aggregate structure in field soils for longer periods. 4.2. N dynamics in fresh millipede-worked soil Our feeding experiment confirmed that the geophagous larvae of P. laminata increased N mineralization (Fig. 2), which is consistent with previous reports on the effects of soil-dwelling earthworms on soil (Scheu, 1987; Bohlen and Edwards, 1995; Subler et al., 1998; Decaëns et al., 1999) and litter-feeding millipedes (Cárcamo et al., 2000). Millipede larvae had greater effects on N mineralization than on nitrification. Mineralization of N was 2.8–4.7-fold greater in soil containing millipedes than in control soil, whereas the increase in nitrification in millipede-worked soil was less than 1.9-fold. It is possible that the extent of nitrification was offset by N2O production. However, N2O–N emission at the end of the experiment occurred at the centesimal order of nitrification (Fig. 1). Generally, N2 O production is far too low to offset nitrification in well-drained soils (e.g. Davidson et al., 2000). Thus, we assumed that the consumption of NO− 3 –N by N2O production was minor. The increase in N mineralization can be attributed to millipede excretions. Many studies have reported that fresh excrement from soil animals contains high concentrations of ammonium (e.g., Teuben and Verhoef, 1992; Decaëns et al., 1999; Cárcamo et al., 2000). The high content of NH+ 4 –N can induce nitrification (Decaëns et al., 1999), and N2O is emitted as a by-product of nitrification (Davidson et al., 2000). The correlation between aggregation and N mineralization also demonstrates the effect of millipede activity on soil N mineralization. As discussed above, the feces of the millipede larvae probably induced the increase in soil aggregation in these feeding experiments. Thus, the relationship between soil aggregation and N mineralization would reflect the effect of excretion on soil N mineralization. Soil containing millipedes showed 1.8- to 4.0-fold greater N2O emissions than control soil. Although many studies have examined the effects of earthworms on soil N2O emissions (e.g., Matthies et al., 1999; Borken et al., 2000; Drake and Horn, 2007; Rizhiya et al., 2007; Speratti and Whalen, 2008), to our knowledge, this is the first study on the effects of soil macroarthropods on N2O emissions in forest soil. N2O can be emitted as a by-product of nitrification and denitrification, and denitrification also depends on NO− 3 –N availability (Davidson et al., 2000). The higher nitrification conditions of the millipede-worked soil would also likely promote increased N2O emissions in our feeding experiment, although we did not evaluate the denitrification process. In addition, N2O emissions in the millipede-worked soil might be directly associated with soil aggregation. Højberg et al. (1994) showed that soil aggregates contain interior anaerobic microsites, in which N2O concentrations are higher than at the surface of the aggregates, presumably due to the favorable conditions for denitrifying bacterial activity. In addition, we observed a positive correlation between soil aggregation and N2O emissions in our study. This suggests that together with the higher inorganic N conditions, soil microbial activity related to N2O production was mediated by soil aggregation. While millipedes significantly influenced the dynamics of inorganic N, they did not affect the total C and N concentrations and the C:N ratio in the soils (Table 2). We concluded that total C and N in mineral soil were not significantly affected by feeding activity, at least in this 28-day experiment. This result is not consistent with those of Cárcamo et al.
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(2000), who reported that the litter-feeding millipede Harpaphe haydeniana enhanced litter decomposition and nutrient release in a 14-day feeding experiment. This may be partly due to the characteristics of the soil organic matter in the Andic soils used in our experiment, and partly due to the feed-preferences of these millipedes. In the present study, the larvae of P. laminata fed on Andic soil, which contains a substantial proportion of soil organic matter bound to clay particles. These organic matters are recalcitrant against degradation. Such characteristics may result in a low efficiency of digestion of the soil organic matter by P. laminata larvae, resulting in minor or undetectable effects on total C and N concentrations in the soil. The difference between our results and those of Cárcamo et al. (2000) suggests that feeding habits are an important factor in the effects of soil macrofauna on nutrient dynamics in soil ecosystems.
Acknowledgments The authors thank Dr. Muneoki Yoh and Dr. Keisuke Koba at Tokyo University of Agriculture and Technology for providing facilities to carry out the gas analysis. We thank Dr. Tatsuyuki Seino and the staff at the Yatsugatake Forest, Tsukuba University, for their help with soil sampling. Dr. Kazuya Nishina, Dr. Minori Hashimoto, Dr. Lucero Mariani, Mr. Tatsuya Kawaguchi, Mr. Hiroto Asanuma, and Ms. Kumiko Yamaguchi (Yokohama National University) provided valuable comments on our earlier manuscript. This study was supported by the Global Environment Research Fund of the Ministry of the Environment, Japan (F-073).
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Geoderma j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e o d e r m a
The train millipede (Parafontaria laminata) mediates soil aggregation and N dynamics in a Japanese larch forest Reiji Fujimaki 1, Yui Sato, Naoyuki Okai, Nobuhiro Kaneko ⁎ Graduate School of Environment and Information Sciences, Yokohama National University, 79-7, Tokiwadai, Hodogaya, Yokohama, Kanagawa 240-8501, Japan
a r t i c l e
i n f o
Article history: Received 15 December 2009 Received in revised form 8 May 2010 Accepted 15 July 2010 Available online 19 August 2010 Keywords: Larch forest soil N mineralization N2O emission Soil aggregation Train millipede (Parafontaria laminata)
a b s t r a c t The contributions of soil macroinvertebrates such as earthworms and termites to soil structure and nutrient cycling are well recognized, but few studies have examined the influence of geophagous millipedes on soil nutrients and structure. We conducted a soil incubation experiment to evaluate the effects of the train millipede (Parafontaria laminata), which is endemic to central Japan, on N mineralization, N2O emissions, and aggregation in soils. Larvae of P. laminata significantly increased development of soil aggregates N 2 mm during the 28-day incubation experiment. This soil aggregation was attributed to fecal pellets and molting chamber walls of P. laminata larvae. N mineralization, nitrification, and N2O–N emissions were also promoted by P. laminata, although these changes in N dynamics did not result in changes in the total amounts of C and N in the soil. N mineralization and N2O–N emissions were positively correlated with the amount of large (N 2 mm) soil aggregates. These correlations indicate that the biogenic structure produced by P. laminata significantly influences soil N dynamics, and suggest that the soil micro-environment associated with large aggregates promotes microbial processes related to N dynamics, especially N2O production. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The contribution of macroinvertebrates to soil processes is well recognized. Some groups of soil macroinvertebrates, including earthworms, termites, and ants, are known as ecosystem engineers, as they modify soil structure by creating cast aggregates, mounds, and subterranean galleries (Jones et al., 1994; Lavelle et al., 1997; Folgarait, 1998). These biogenic structures often persist for much longer than the lifetimes of the organisms that produced them. Such biogenic structures influence the activities of other organisms, and their function is referred to as “soil ecosystem engineering” (Lavelle et al., 1997; Lavelle, 2002). Biogenic structures also influence the dynamics of soil organic matter and nutrients. For example, earthworms can produce soil macroaggregates N1 mm in diameter (Ketterings et al., 1997) and up to 2 mm (Blanchart, 1992; Winsome and McColl, 1998) through their feeding and excreting activities. Soil aggregates derived from earthworm-worked soil contain organic materials from fresh residues of plant debris (Bossuyt et al., 2004; Fonte et al., 2007), and influence the structure and activity of the microbial community, resulting in alteration of the N transformation pattern (Schutter and Dick, 2002; Noguez et al., 2008). Bohlen and Edwards (1995) demonstrated that earthworms increased soil inorganic N from 1.26- to 4.00-fold in a ⁎ Corresponding author. Tel./fax: + 81 45 339 4379. E-mail address: [email protected] (N. Kaneko). 1 Present address: Faculty of Life and Environmental Science, Shimane University, 1060, Nishikawatsu, Matsue, Shimane 690-8504, Japan. 0016-7061/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.geoderma.2010.07.014
microcosm experiment. In addition, Borken et al. (2000) reported that the introduction of earthworms resulted in a 57% increase in soil N2O emissions. While there are more studies on earthworms than on other soil macroinvertebrates, it has been reported that millipedes also play an important role in the facilitation of nutrient and carbon dynamics in soils (Cárcamo et al., 2000; Toyota et al., 2006). Most millipedes are litter feeders (Hopkin and Read, 1992). Their ability to facilitate nutrient dynamics is attributed to litter comminution through their ingestive activity, which promotes nutrient release and utilization of litter by microbes (Cárcamo et al., 2000). However, there have been few studies on geophagous millipedes and their functions in modifying soil structure (but see Niijima, 1984; Toyota et al., 2006). We examined the effect of the train millipede (Parafontaria laminata), which is endemic to central Japan, on soil aggregation and N dynamics. The millipede has an 8-year life cycle with annual molting. The 1st to 7th-instar larvae inhabit soils, mostly at a depth of 0–10 cm in summer and autumn, and at lower depths in early spring (Niijima, 1984; Toyota and Kaneko 2004). Throughout central Japan, adult millipedes swarm the soil surface at 8-year intervals, often with extremely high densities and biomasses (Yoshida et al., 1985; Niijima and Shinohara, 1988). The larvae feed on mineral soils and associated old organic matter, whereas adults feed on a mixture of mineral soil and fresh leaf litter (Hashimoto et al., 2004). High densities of adult-stage train millipedes can reduce the accumulation of organic matter in surface soils (Niijima and Shinohara, 1988). In addition, their mixed feeding on litter and soil increases carbon sequestration in mineral soils through promoting the interactions of organic matter and mineral particles
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(Toyota et al., 2006). Together with the periodic, high-density swarming, the feeding habits of the train millipede can be expected to significantly affect ecosystem processes through changing the accumulation of soil organic matter and nutrient dynamics (Niijima, 1984; Hashimoto et al., 2004; Toyota et al., 2006). However, there is little information available about the effects of the train millipede on N dynamics and soil aggregation. The objective of this study was to determine the effects of the train millipede on soil aggregation and N dynamics in forest soil. We conducted an incubation experiment using forest soil and train millipede larvae, and evaluated their effects on soil aggregation, N mineralization, and N2O emissions. We hypothesized that millipede-worked soil would contain more large-sized soil aggregates, and would show increased N mineralization and N2O emissions. 2. Materials and methods 2.1. Feeding experiments with millipede larvae We conducted a soil incubation experiment with controlled densities of train millipedes (P. laminata). In July 2007, we collected 6th-instar train millipede larvae and surface soil (ca. 0–20 cm depth) from a Japanese larch (Larix kaempferi) forest. The train millipede had been found at high densities in this forest, which is located in the foothills of Mt. Tennyo in the Yatsugatake Mountains, central Japan (Tennyo site, 35°54′ N, 138°23′ E; 1400 m elevation). A detailed description of the site is given elsewhere (Toyota et al., 2006). Hashimoto et al. (2004) reported that the dry weight biomass of 7thinstar millipedes at the site was 15.7 g m− 2 in 1999 and of adult millipedes was 28.6 g m− 2 in 2000. These values are substantially higher than those generally reported for insect density in forest soils (e.g., Petersen and Luxton, 1982). Soils were classified as Andosols (FAO, ISSS, ISRIC, 1998). Freshly collected soils were passed through a 2-mm mesh sieve to remove gravel, plant debris, and large-sized aggregates, and then homogenized. Portions of approximately 88-g of fine-size (b2 mm) fresh soil, which was equivalent to 36 g of dry weight soil, were placed into 200-ml glass vials (5.1 cm in diameter and 10 cm in height), and soil moisture was adjusted to 60% field water holding capacity. The headspace volume in the vial was approximately 168 cm3. Train millipede larvae (6th-instar) were placed into the vials. Each vial contained 0, 2, 4, or 8 individuals, which approximated to 0, 6.4, 12.8, and 25.6 mg dry weight per vial, with five replicates of each density. These values were equivalent to densities of 0, 1000, 2000, and 4000 individuals m− 2 in field conditions. The observed field density of 6th-instar larvae around Mt. Yatsugatake ranged from 0 to 2156 m− 2 in 1999 (Toyota and Kaneko, 2004). Each vial was sealed with a polyethylene lid to prevent the soil from drying out, and to prevent escape of the millipedes. The polyethylene lid was equipped with a rubber septum for gas sampling. The soils and millipedes were incubated for 28 days in the dark at 18 °C. Three empty vials were prepared for use as blanks in the gas measurements. Every 7 days, the vials were opened for aeration and moisture adjustment. Some individuals died during the course of the experiment; the mean final densities were 0, 1.2, 3.4, and 7.4 individuals per vial (Table 1). Some of the survived individuals were molting at the end of the experiment.
Table 1 Mean number of survival, dead and molting individuals of P. laminata after 28 days of feeding. Introduced number
Survival
Dead
Molting
2 4 8
1.2 (0.45) 3.4 (0.89) 7.4 (0.89)
0.8 (0.45) 0.6 (0.89) 0.6 (0.89)
0.8 (0.45) 0.6 (0.89) 4.8 (1.78)
Values in parenthesis are standard deviations (n = 5).
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After the feeding experiment, the vials were sealed for 4 h and the headspace N2O concentration was analyzed. A 1.5-ml sample of the headspace gas was taken from the rubber septum of each incubation vial using a gas-tight syringe. N2O concentrations were determined using a gas chromatograph equipped with an electron capture detector and 2 m of Porapak Q (60/80 mesh, GC-14B; Shimadzu, Kyoto, Japan). The temperatures of the injector, column, and detector were 80, 60, and 340 °C, respectively. The carrier gas (N2) was supplied at a rate of 10 ml min− 1, and both standard and atmospheric gases were measured before and after sample gas measurements were taken. The N2O emissions in each vial were calculated by multiplying the headspace volume by the difference in N2O concentrations between the sample gas and the atmosphere. Before and after the incubation experiment, soil samples were dried at 40 °C and the C and N concentrations were determined by combustion (NC-95A; Sumika Chemical Analysis Service, Osaka, Japan). In addition, before and after the 28-day incubation, inorganic N was extracted from 3 g soil samples using 30 ml 2 M KCl. The − inorganic N (NH+ 4 –N and NO3 –N) concentration was determined using a continuous flow analyzer (Futura, Alliance Instruments, Frépillon, France). The nitrogen mineralization rate was determined by calculating the difference between the inorganic N concentrations in the soils before and after the 28-day incubation. After the experiment, the soils were again sieved through a 2-mm mesh to separate the fine- (b2 mm) and large- (N2 mm) size fractions. Then, the soil fractions were dried at 40 °C and weighed to evaluate the aggregation. 2.2. Statistical analyses A linear regression was performed to evaluate the relationship between the millipede density and soil aggregation (N2 mm size fraction). The effect of the millipedes on N mineralization, nitrification, and CO2 and N2O emissions in the incubation experiment was determined using a one-way analysis of variance (ANOVA) after checking the data for normality and homoscedasticity by Shapiro– Wilk and Bartlett tests, respectively. Data from the N mineralization was log-transformed prior to the ANOVA to improve the homoscedasticity. When a significant effect (P b 0.05) was detected by the ANOVA, a pair-wise t-test with Holm's adjustment was performed as a post hoc test. A Spearman's rank correlation coefficient was calculated to evaluate the relationship between soil aggregation and N mineralization or N2O emissions. 3. Results Millipede larvae promoted soil aggregation, i.e., increased the proportion of the large-size fraction (N2 mm), during the 28-day experiment (Fig. 1). The amount of the large-size aggregates was positively correlated with the number of introduced larvae (y = 1.72x + 3.65, P b 0.001), although the difference between the four larvae and eight larvae treatments was not significant (paired t-test with Holm's adjustment, P = 0.35). The millipede larvae also significantly stimulated soil N dynamics (Fig. 2; F = 21.0, P b 0.001 for N mineralization; F = 34.6, P b 0.001 for nitrification; and F = 7.6, P b 0.05 for N2O emissions). Compared with control soils, soils containing larvae showed increased N mineralization and nitrification (paired t-test with Holm's adjustment, P b 0.05), especially those with a high density of larvae. The millipedes had a greater effect on N mineralization than on nitrification, and the proportions of nitrification in N mineralization decreased with the addition of millipedes. Soil samples containing four or eight larvae showed higher N2 O emissions and greater amounts of large aggregates, while the effects of two larvae were not significant. Both the N mineralization and N2O emissions were positively correlated with the amount of soil in the N2-mm size fraction (Fig. 3;
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Fig. 1. Relationship between numbers of introduced P. laminata and amount of large soil aggregates (N2 mm size fraction) after the 28-day incubation experiment. Line shows linear regression (y = 1.72x + 3.65, P b 0.001).
Spearman's rank correlation test, rs = 0.74, P b 0.01 and rs = 0.52, P b 0.05 for N mineralization and N2O emissions, respectively). The soil C and N concentrations and C:N ratio did not differ among the treatments (Table 2). 4. Discussion 4.1. Aggregate formation The larvae of P. laminata are geophagous (Hashimoto et al., 2004; Toyota et al., 2006), which is uncommon among millipedes (Hopkin and Read, 1992). Geophagous soil macrofauna such as earthworms facilitate soil aggregation into large-size fractions, mainly as casts (Blanchart, 1992; Ketterings et al., 1997; Bossuyt et al., 2004; Fonte et al., 2007; Loranger-Merciris et al., 2008). While individual fecal pellets
Fig. 3. Relationships between soil aggregate (N2 mm size) and N mineralization (A) and between soil aggregates and N2O–N emissions (B) in the soil incubation experiment using 6th larvae of P. laminata.
of P. laminata are smaller than 2 mm, the pellets can gather fine particles of soil or other pellets (Fujimaki, R., personal observation; Niijima, 1984), presumably because polysaccharides in the feces bind to neighboring soil particles. These pellet aggregates likely account for the increased soil aggregation observed in the incubation experiment. Our observation that the millipede activity resulted in greater amounts of N2 mm aggregates in this experiment is consistent with results of previous studies on soil-feeding earthworms (Blanchart, 1992; Ketterings et al., 1997; Winsome and McColl, 1998; Bossuyt et al., 2004; Loranger-Merciris et al., 2008). In addition to the effect of fecal pellets, the molting habits of the millipede may have contributed to the large-size aggregates (Table 1). The larvae of P. laminata molt annually in molting chambers made from their feces (Niijima, 1984; Niijima and Shinohara, 1988). Unlike fecal pellets of other diplopods that mainly feed on fresh organic matter, the feces of train millipedes contain a high proportion of mineral soils resulting from their geophagous feeding habit (Niijima, 1984; Toyota et al., 2006). This results in molting chamber walls with Table 2 Mean concentrations of the soil C and N and mean C:N ratios after the feeding experiment.
Fig. 2. N mineralization (white) and nitrification (grey) during the experiment (A) and N2O–N emissions at the end of the experiment (B). Error bars indicate standard deviations (n = 5). Letters indicate significant differences among the millipede numbers.
Number of millipedes
C [mg kg− 1]
N [mg kg− 1]
C:N
0 2 4 8
224.8 221.9 213.5 227.6
13.5 13.3 13.1 13.2
17 16 16 17
(1.5) (2.3) (11.4) (2.3)
Values in parenthesis are standard deviations.
(0.3) (0.2) (0.9) (0.2)
(0.4) (0.3) (0.3) (0.3)
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a tightly bound structure. The chamber wall can reach several millimeters in thickness (Fujimaki, R., personal observation). Although the chamber structure would likely decay within several years (Yamaguchi, K., personal comm.), pieces of the chamber wall could last for a longer period (Niijima, 1984). We confirmed that the activity of P. laminata can increase soil aggregation under laboratory conditions in this short-term (28-day) experiment. The characteristics of their fecal pellets and molting chamber walls also suggest that they would contribute to maintaining the aggregate structure in field soils for longer periods. 4.2. N dynamics in fresh millipede-worked soil Our feeding experiment confirmed that the geophagous larvae of P. laminata increased N mineralization (Fig. 2), which is consistent with previous reports on the effects of soil-dwelling earthworms on soil (Scheu, 1987; Bohlen and Edwards, 1995; Subler et al., 1998; Decaëns et al., 1999) and litter-feeding millipedes (Cárcamo et al., 2000). Millipede larvae had greater effects on N mineralization than on nitrification. Mineralization of N was 2.8–4.7-fold greater in soil containing millipedes than in control soil, whereas the increase in nitrification in millipede-worked soil was less than 1.9-fold. It is possible that the extent of nitrification was offset by N2O production. However, N2O–N emission at the end of the experiment occurred at the centesimal order of nitrification (Fig. 1). Generally, N2 O production is far too low to offset nitrification in well-drained soils (e.g. Davidson et al., 2000). Thus, we assumed that the consumption of NO− 3 –N by N2O production was minor. The increase in N mineralization can be attributed to millipede excretions. Many studies have reported that fresh excrement from soil animals contains high concentrations of ammonium (e.g., Teuben and Verhoef, 1992; Decaëns et al., 1999; Cárcamo et al., 2000). The high content of NH+ 4 –N can induce nitrification (Decaëns et al., 1999), and N2O is emitted as a by-product of nitrification (Davidson et al., 2000). The correlation between aggregation and N mineralization also demonstrates the effect of millipede activity on soil N mineralization. As discussed above, the feces of the millipede larvae probably induced the increase in soil aggregation in these feeding experiments. Thus, the relationship between soil aggregation and N mineralization would reflect the effect of excretion on soil N mineralization. Soil containing millipedes showed 1.8- to 4.0-fold greater N2O emissions than control soil. Although many studies have examined the effects of earthworms on soil N2O emissions (e.g., Matthies et al., 1999; Borken et al., 2000; Drake and Horn, 2007; Rizhiya et al., 2007; Speratti and Whalen, 2008), to our knowledge, this is the first study on the effects of soil macroarthropods on N2O emissions in forest soil. N2O can be emitted as a by-product of nitrification and denitrification, and denitrification also depends on NO− 3 –N availability (Davidson et al., 2000). The higher nitrification conditions of the millipede-worked soil would also likely promote increased N2O emissions in our feeding experiment, although we did not evaluate the denitrification process. In addition, N2O emissions in the millipede-worked soil might be directly associated with soil aggregation. Højberg et al. (1994) showed that soil aggregates contain interior anaerobic microsites, in which N2O concentrations are higher than at the surface of the aggregates, presumably due to the favorable conditions for denitrifying bacterial activity. In addition, we observed a positive correlation between soil aggregation and N2O emissions in our study. This suggests that together with the higher inorganic N conditions, soil microbial activity related to N2O production was mediated by soil aggregation. While millipedes significantly influenced the dynamics of inorganic N, they did not affect the total C and N concentrations and the C:N ratio in the soils (Table 2). We concluded that total C and N in mineral soil were not significantly affected by feeding activity, at least in this 28-day experiment. This result is not consistent with those of Cárcamo et al.
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(2000), who reported that the litter-feeding millipede Harpaphe haydeniana enhanced litter decomposition and nutrient release in a 14-day feeding experiment. This may be partly due to the characteristics of the soil organic matter in the Andic soils used in our experiment, and partly due to the feed-preferences of these millipedes. In the present study, the larvae of P. laminata fed on Andic soil, which contains a substantial proportion of soil organic matter bound to clay particles. These organic matters are recalcitrant against degradation. Such characteristics may result in a low efficiency of digestion of the soil organic matter by P. laminata larvae, resulting in minor or undetectable effects on total C and N concentrations in the soil. The difference between our results and those of Cárcamo et al. (2000) suggests that feeding habits are an important factor in the effects of soil macrofauna on nutrient dynamics in soil ecosystems.
Acknowledgments The authors thank Dr. Muneoki Yoh and Dr. Keisuke Koba at Tokyo University of Agriculture and Technology for providing facilities to carry out the gas analysis. We thank Dr. Tatsuyuki Seino and the staff at the Yatsugatake Forest, Tsukuba University, for their help with soil sampling. Dr. Kazuya Nishina, Dr. Minori Hashimoto, Dr. Lucero Mariani, Mr. Tatsuya Kawaguchi, Mr. Hiroto Asanuma, and Ms. Kumiko Yamaguchi (Yokohama National University) provided valuable comments on our earlier manuscript. This study was supported by the Global Environment Research Fund of the Ministry of the Environment, Japan (F-073).
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