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What are the limits of the drilosphere? An incubation experiment using Metaphire posthuma
Pedobiologia 54S (2011) S113–S117
Contents lists available at SciVerse ScienceDirect
Pedobiologia - International Journal of Soil Biology journal homepage: www.elsevier.de/pedobi
9th International Symposium on Earthworm Ecology
What are the limits of the drilosphere? An incubation experiment using Metaphire posthuma Pascal Jouquet a,b,∗ , Gaëlle Huchet a,b , Nicolas Bottinelli c,d , Thuy Doan Thu a,b a
IRD – SFRI, Dong Ngac, Tu Liem, Hanoi, Viet Nam IRD, UMR 211 BIOEMCO, Centre IRD Bondy, 32 Avenue H. Varagnat, 93143 Bondy Cedex, France INRA, UMR 1069 Sol Agro et hydrosystème Spatialisation, F-35000 Rennes, France d Université Européenne de Bretagne, France b c
a r t i c l e
i n f o
Article history: Received 25 October 2010 Received in revised form 30 June 2011 Accepted 18 July 2011 Keywords: Earthworms Drilosphere Cast production Soil aggregate organization Near infrared reflectance spectroscopy (NIRS) Metaphire posthuma
a b s t r a c t The near infrared reflectance spectroscopy (NIRS) method was used in the present study to compare earthworm-made soil aggregates to aggregates found in the surrounding bulk soil. After initially assessing the daily cast production of Metaphire posthuma, boxes with soil incubated with M. posthuma and control soils were subjected to wetting in order to reorganize the soil structure. After two months of incubation, soil aggregates produced by earthworms (casts and burrows), soil aggregates that were appeared to be unaffected by earthworms (bulk soil without visible trace of earthworm bioturbation from the earthworm treatment) and soil aggregates that were entirely unaffected by earthworms (control – no earthworm – treatment) were sampled and their chemical signatures analyzed by NIRS. The production of belowground and surface casts reached 14.9 g soil g worm−1 d−1 and 1.4 g soil g worm−1 d−1 , respectively. Soil aggregates from the control soils had a significantly different NIRS signature from those sampled from boxes with earthworms. However, within the earthworm incubation boxes the NIRS signature was similar between cast and burrow aggregates and soil aggregates from the surrounding bulk soil. We conclude that the high cast production by M. posthuma and the regular reorganization of the soil structure by water flow in and through the soil lead to a relatively homogenous soil structure. Given these results, we question the relevance of considering the bulk soil that has no visible activity of earthworm activity as a control to determine the effect of earthworms on soil functioning. © 2011 Elsevier GmbH. All rights reserved.
Introduction Soil engineers are organisms that create biogenic structures with different physical and chemical properties compared to the surrounding soil (Lavelle et al. 1997; Jouquet et al. 2006, 2007b). Together these structures constitute the sphere of influence, or functional domain of soil engineers (i.e. the drilosphere, termitosphere and myrmecosphere, respectively, for earthworms, termites and ants, Lavelle 2002). Among soil engineers, earthworms are considered to be the most widespread and abundant in terrestrial ecosystems (Lavelle and Spain 2001) and their importance in the regulation of biogeochemical processes and ecosystem services have been largely demonstrated (Lavelle et al. 2006). Biogenic aggregates produced by earthworms are usually sampled in the field or in laboratory experiments and identified with regard to their spherical or rounded and smooth shapes (Bullock
∗ Corresponding author at: IRD, UMR 211 BIOEMCO, Centre IRD Bondy, 32 Avenue H. Varagnat, 93143 Bondy Cedex, France. E-mail address: [email protected] (P. Jouquet). 0031-4056/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2011.07.013
et al. 1985) or to their higher soil structural stability (reviewed in Edwards 2004). The effect of earthworms on soil properties is usually determined by comparing these earthworm-made soil aggregates and the surrounding soil environment that has no visible trace of earthworm activity, e.g. the physicogenic aggregates. Physicogenic aggregates are angular blocky macroaggregates that result from both abiotic (e.g. tillage processes, wetting and drying processes) and biotic (e.g. microbial activity associated with root exudation and degradation but not soil macrofauna activity) processes. This distinction between biogenic and physicogenic aggregates has been successfully used as an indicator in the study of the influence of earthworms on soil functioning (Decaëns et al. 2002; Hedde et al. 2005; Velasquez et al. 2007; Jouquet et al. 2010). However, as emphasized by Darwin (1881): “(. . .) castings are extremely liable to crumble during dry weather into small fragments, which are soon acted on by rain, and then sink down so as to be no longer distinguishable from the surrounding soil”. More recently, Jouquet et al. (2009) confirmed this observation and showed that raindrop impact led to the loss of the rounded shape of earthworm casts. Although these soil aggregates can be classified as physicogenic aggregates, studies have shown that they
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keep the specific chemical properties of biogenic aggregates. Consequently, the choice of control soil aggregates is questionable when only based on aggregate appearance. Another indicator is therefore required to determine true physicogenic or control soil aggregates, which can then be compared to earthworm biogenic aggregates. Near infrared reflectance spectroscopy (NIRS) has recently emerged as a promising technique for the assessment of soil quality (Velasquez et al. 2005; Cécillon et al. 2009). This method has also been used for the identification of soil aggregate origin and evolution (Hedde et al. 2005; Velasquez et al. 2007; Jouquet et al. 2009; Cécillon et al. 2010; Chapuis-Lardy et al. 2010; Jouquet et al. 2010). In the present study, NIRS was used to determine the value of comparing earthworm made soil aggregates (casts and burrows) to their surrounding soil environment (i.e. the control soil aggregates without visible influence of earthworms). The main hypothesis of this work was that the control soil has a NIRS signature different to that of the biogenic aggregates produced by earthworms. A laboratory experiment was carried out and the comparison was made between biogenic and physicogenic aggregates sampled in boxes where earthworms were added and physicogenic aggregates sampled in boxes without earthworms.
Materials and methods Study site and study model The study was carried out from May to June 2009 at the Soil and Fertilizer Research Institute (SFRI, Hanoi, Vietnam, 20◦ 57 N, 105◦ 29 E). The soil is a loamy soil (Clay: 13%, Silt: 50%, Sand: 37%) with low organic matter (24 g kg−1 ) sampled in the upper 10 cm from a garden. The garden is almost exclusively colonized by the earthworms Metaphire posthuma (Oligochaeta, Megascolecidae), which is a medium size endogeic geophagous earthworm (∼10 cm in length and 5 mm diameter on average at the adult stage). In northern Vietnam, M. posthuma produces approximately 5-fold its own weight per day (Bottinelli et al. 2010). Its casts range from 2 to 3 mm and are mainly found belowground (Bottinelli et al. 2010).
Soil incubation procedure Aggregates of <1 mm (462 g) were put in polyvinyl chloride boxes (9 cm wide × 12 cm long × 10 cm high) with a plastic pipe, closed by a waterproof silicone cap, running along the bottom of the box, above a 1 cm layer of sand and gravel covering the bottom of the boxes. Soil aggregates were compacted manually to 1.40 g cm−3 . Boxes were incubated at 30 ◦ C in the dark after remoistening (75% of the field capacity) and inoculated (W+), or not (W−), with one earthworm (∼0.5 g in average). Twice a week, the boxes were opened to allow air exchange. Every 15 days, 200 ml of distilled water was gently applied to the surface of the soil with a small watering can in order to dissolve earthworm biogenic structures (casts and burrows). Excess water leached through the plastic pipe in the bottom of the boxes. Soil aggregate sampling and NIRS fingerprint Soils were collected from the boxes incubated with earthworms and from the controls two months after incubation (n = 5). At this time, different categories of casts were established. Aboveand below-ground casts and burrows were considered as earthworm biogenic aggregates. Casts were identified according to their granular shapes, as described by Bottinelli et al. (2010). The soils without visible activity of earthworms were considered as physicogenic aggregates. A distinction was made between the physicogenic aggregates sampled on the soil surface to that sampled below the surface. In the treatment without earthworms, soils were considered true controls. They were also sampled above and below the surface of the soil. Soil aggregates were air-dried, crushed and passed through 250 m sieves. 5 g of each sample was scanned with a FOSS 5000 spectrophotometer (Foss NIRSystems, Silver Spring, MD, USA) in the 1100–2500 nm spectral range. The spectral data obtained were recorded as the logarithm of the inverse of reflectance [log(1/R)]. They were conducted using WinISI III-version 1.50e software (Foss NIRSystems, Infrasoft International). A 20-nm sampling interval was used for data analysis. Statistical analyses
Cast production Cast production was assessed following the procedure of Bottinelli et al. (2010). Approximately 50 kg of soil was sampled in the upper 10 cm of the soil, air-dried, ground-down and sieved to obtain 0.25–1 mm soil aggregates. Aggregates were put in polyvinyl chloride cylinders (20 cm in diameter and 10 cm high) and manually packed down to reach 1.4 g cm−3 . One earthworm was added per cylinder and no additional food was given during the experiment. Before inoculation, juvenile earthworms were placed on moist tissue paper for 24 h to void their guts and subsequently weighed (fresh weight). Individual earthworms weighed 0.5 g on average. A perforated plastic lid covered the top of the cylinders to allow air circulation. Cylinders were incubated at 30 ◦ C in the dark after remoistening (75% of the field capacity). Surface- and below-ground casts were sampled after 1, 5, 9, 12 and 15 days of incubation (15 cylinders in total, n = 3 for each date). Casts were easily distinguished from the bulk soil by their rounded shape and their size. They were hand-sorted cautiously with a spatula, airdried and weighed. After each incubation time, earthworms were removed, purged and their fresh weights determined as above. Earthworm development was assessed through the comparison of earthworm biomass at each sampling date minus the initial earthworm biomass.
Data were tested for homogeneity of variance using Levene’s test. Analysis of variance (ANOVA) was performed when variance homogeneity assumptions were met, and Mann–Whitney Wilcoxon’s test was used for non-parametric data. A Principal Component Analysis (PCA) was carried out to differentiate the samples based on their NIRS signature. Briefly, we used a matrix of 35 samples and 67 variables from NIRS wavelengths quantifying the adsorptions between 1100 and 2500 nm with a 20-nm sampling interval. All statistical calculations were carried out using R software. Differences among treatments were declared at the <0.05 probability level of significance.
Results Earthworm biomass and cast production The evolution of earthworm biomass is shown in Fig. 1. Although significant differences were observed between the different sampling dates (ANOVA, F4,20 = 10.7, P = 0.002), our data did not show any temporal trend. The daily below-ground cast production decreased during the first 9 days of the experiment, reaching a threshold of 9.3 (SE: 2.8) g soil g worm−1 d−1 (Fig. 2 and Table 1). Conversely, the daily
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Fig. 1. Evolution of earthworm biomass (% initial soil weight) during the 15 days of the experiment (n = 3).
Fig. 3. Results of the principal components analysis showing the absorbance in the near infrared of soil aggregates sampled after 2 months of incubation. The circles are the barycenters of clusters of points for each aggregate class: control soil surface (S), control bulk soil (B), cast on the soil surface (Cs), cast in the bulk soil (Cb) and burrow (Bu). Circles in white were sampled in containers without earthworms and circles in grey were sampled in boxes with earthworms (n = 5).
NIRS signature of all the soil categories incubated with earthworms (Mann–Whitney Wilcoxon test, P < 0.05). Discussion Fig. 2. Evolution of the cast daily production (g soil g worm−1 d−1 ) of Metaphire posthuma in the soil (in white) or on the soil surface (in grey) during the 15 days of the experiment (n = 3).
above-ground cast production was very low and constant throughout the experiment at only 1.5 (SE: 0.3) g soil g worm−1 d−1 (Fig. 2). NIRS signature PCA performed using the NIRS data allowed soil aggregates to be differentiated mainly on the first axis (Fig. 3). Positive values on the first axis (62% of the total variability) of the PCA corresponded to the soil aggregates incubated without earthworms, whereas negative values described soil biogenic aggregates and the control soil sampled in the vicinity of soil biogenic aggregates. Univariate statistical analyses were subsequently performed on data obtained from the projection of the plots on the first axis of the PCA (Fig. 4). Although the NIRS signature of biogenic aggregates (casts and the soil covering the burrow) had the same signature as the surrounding bulk soil, we observed differences between casts sampled on the soil surface and the surrounding soil surface without visible trace of earthworm activity (Mann–Whitney Wilcoxon test, P < 0.05). However, the most striking result was the significant difference between the signatures of the soil incubated without earthworms (bulk soil and soil surface from the no earthworm treatment) and the
Earthworm biomass and cast production With the exception of Day 1 of the experiment, earthworm weight loss was very low, suggesting that earthworm activity was only slightly affected by the incubation experiment. The higher cast production at the beginning of the experiment is commonly observed in incubation experiments with earthworms and is likely to be the consequence of stress resulting from the earthworm manipulation and by the creation of a new habitat by the earthworms (Whalen et al. 2004; Bottinelli et al. 2010). Our cast production results were higher than those found by Bottinelli et al. (2010) who found a daily cast production of about 5 g soil−1 g worm−1 . This difference might be explained by the developmental stage of earthworms. The experiment carried out
Table 1 Two-way ANOVA table for the effect of the location (above vs. below-ground cast) and sampling date on the daily production of casts by Metaphire posthuma. d.f. Location (1) Date (2) 1×2
1,20 4,20 4,20
F-Values
P-Values
97.7 4.7 3.6
<0.001 0.007 0.021
Fig. 4. Projection of the plot on the first axis of the PCA for the bulk soil (B) and soil surface (S) sampled in boxes without earthworms (no earthworm treatment, in white) and sampled in boxes with earthworms (earthworm treatment, in grey), casts on the soil surface (Cs) or inside the soil (Cb) and soil covering the burrows (Bu). Histograms with the same letter are not different at P = 0.05 (n = 5).
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by Bottinelli et al. (2010) was made at the end of the rainy season, in September 2008 when the earthworms were adult. In our study, the experiment was done earlier in the season (May to June 2009) when the earthworms were juvenile and therefore probably required more energy for their development. This hypothesis is in agreement with Lavelle (1975) and Barois et al. (1999) who indicated that juveniles produce more casts than adults.
Conclusion In addition to confirming the relevance of the NIRS method to distinguish soil aggregates of different origins, this study suggests limitations in comparing biogenic structures to the surrounding bulk soil environment. Specifically, the present work shows that the only true control soils are non-drilosphere soils that have not been incubated with earthworms.
NIRS signature
Acknowledgments
Recent studies have emphasized the suitability of the NIRS method for the assessment of soil quality (see Cécillon et al. 2009 for a review). In our study, this method was used to determine the optical signature of soil biogenic (e.g. casts and burrows) and physicogenic aggregates (e.g. the control aggregates not apparently influenced by earthworms). This study emphasized the limitation of comparing earthworm casts to their surrounding soil in laboratory experiments and questions the relevance of using this surrounding soil as a control soil in laboratory experiment and also perhaps in the field. The drilosphere has been described as the part of the soil influenced by earthworm movement, secretions and castings (Lavelle 2002). This functional domain is traditionally limited to well identifiable structures: the cast aggregates, the soil covering the burrows, the middens and the earthworm biomass in itself. NIRS analysis shows that the bulk soil, which does not present any visible activity of earthworms, might also be influenced by earthworms and therefore also might be included in the drilosphere. Three main hypotheses can explain the modification of the signature of the bulk soil surrounding the casts and burrows produced by earthworms. First, earthworms might influence the chemical properties of the soil underneath their burrows by promoting the transfer of mineral or organic molecules (Decaëns et al. 1999; Amador et al. 2006; Jouquet et al. 2007a; Don et al. 2008). It can therefore be suggested that the activity of M. posthuma under a constrained environment fosters the leaching of nutrients such as ammonium, nitrate and dissolved organic carbon, and therefore leads to a homogenization of the soil chemical characteristics. Another explanation relates to the lifetime of earthworm biogenic structures. In the same study field, Bottinelli et al. (2010) showed that casts produced by M. posthuma have lower soil organic matter content and are less resistant to water impact than the surrounding soil aggregates. As a consequence, its casts collapse rapidly after significant rainfall events, leading to a reduction of soil porosity. In our experiment, excess water was applied every 2 weeks in order to dissolve non-stable soil aggregates. Therefore, it is likely that some casts and burrows collapsed when water was applied to the soil surface and were then mixed with the soil matrix, leading to a homogenization of the soil properties and to lack of differences as detected with the NIRS. Finally, considering an average cast production of 10 g soil g worm−1 d−1 , and applying the assumption that earthworms do not feed on their own fresh or degraded casts (Lavelle, personal communication), we can assume that 300 g of casts was produced during the 2 months of the experiment. This data therefore suggests that around 65% of the soil initially put in the container had been consumed by earthworms. Consequently, it is likely that earthworm activity led to a homogenization of the bulk soil and that it was not possible to distinguish the NIRS signature of casts from that of the surrounding soil aggregates. These three hypotheses are reinforced by the higher variability of the NIRS data of the control soil incubated without earthworms compared to the physicogenic aggregates and biogenic aggregates sampled in the boxes with earthworms (standard error: 4.0 and 0.36, respectively, for bulk soil from boxes without earthworms and bio- and physicogenic aggregates sampled from boxes with earthworms).
We would like to thank Patrick Lavelle for constructive discussions. This project was supported financially by CNRS/INSU (VERAGREGAT project under the framework of the EC2CO program), IRD (unit research UMR-211-BIOEMCO) and CNRS (unit research UMR-7618-BIOEMCO) French institutes. References Amador, J.A., Gorres, J.H., Savin, M.C., 2006. Effects of Lumbricus terrestris L. on nitrogen dynamics beyond the burrow. Appl. Soil Ecol. 33, 61–66. Barois, I., Lavelle, P., Brossard, M., Tondoh, J., Martinez, M.d.l.A., Rossi, J.P., Senapati, B.K., Angeles, A., Fragoso, C., Jimenez, J.J., Decaëns, T., Lattaud, C., Kanyonyo, J., Blanchart, E., Chapuis, L., Brown, G., Moreno, A., 1999. Ecology of earthworm species with large environmental tolerance and/or extended distributions. In: Lavelle, P., Brussaard, L., Hendrix, P. (Eds.), Earthworm Management in Tropical Agroecosystems. CABI Publishing, Wallingford, UK, pp. 57–86. Bottinelli, N., Henry-des-Tureaux, T., Hallaire, V., Benard, Y., Mathieu, J., Duc Tran, T., Jouquet, P., 2010. How earthworms accelerate soil porosity under watering events. Geoderma 156, 43–47. Bullock, P., Federoff, N., Jongerius, A., Stoops, G., Tursina, T., 1985. Handbook for Soil Thin Section Description. Waine Research Publications, Albrighton, UK. Cécillon, L., Barthès, B.G., Gomez, C., Ertlen, D., Genot, V., Hedde, M., Stevens, A., Brun, J.J., 2009. Assessment and monitoring of soil quality using near-infrared reflectance spectroscopy (NIRS). Eur. J. Soil Sci. 60, 770–784. Cécillon, L., de Mello, N.A., De Danieli, S., Brun, J.J., 2010. Soil macroaggregate dynamics in a mountain spatial climate gradient. Biogeochemistry 97, 31–43. Chapuis-Lardy, L., Brauman, A., Bernard, L., Pablo, A.L., Toucet, J., Mano, M.J., Weber, L., Brunet, D., Razafimbelo, T., Chotte, J.L., Blanchart, E., 2010. Effect of the endogeic earthworm Pontoscolex corethrurus on the microbial structure and activity related to CO2 and N2 O fluxes from a tropical soil (Madagascar). Appl. Soil Ecol. 45, 201–208. Darwin, C., 1881. The Formation of Vegetable Mould through the Action of Worms with some Observations on their Habits. John Murray, London. Decaëns, T., Rangel, A.F., Asakawa, N., Thomas, R.J., 1999. Carbon and nitrogen dynamics in ageing earthworm casts in grasslands of the eastern plains of Colombia. Biol. Fertil. Soils 30, 20–28. Decaëns, T., Asakawa, N., Galvis, J.H., Thomas, R.J., Amézquita, E., 2002. Surface activity of soil ecosystem engineers and soil structure in contrasted land use systems of Colombia. Eur. J. Soil Biol. 38, 267–271. Don, A., Steinberg, B., Schöning, I., Pritsch, K., Joschko, M., Gleixner, G., Schulze, E.D., 2008. Organic carbon sequestration in earthworm burrows. Soil Biol. Biochem. 40, 1803–1812. Edwards, C.A., 2004. Earthworm Ecology, 2nd ed. CRC Press. Hedde, M., Lavelle, P., Joffre, R., Jimenez, J.J., Decäens, T., 2005. Specific functional signature in soil macro-invertebrate biostructures. Funct. Ecol. 19, 785–793. Jouquet, P., Dauber, J., Lagerlof, J., Lavelle, P., Lepage, M., 2006. Soil invertebrates as ecosystem engineers: intended and accidental effects on soil and feedback loops. Appl. Soil Ecol. 32, 153–164. Jouquet, P., Bernard-Reversat, F., Bottinelli, N., Orange, D., Rouland, C., Tran Duc, T., Podwojewski, P., 2007a. Influence of land use change and earthworm activity on carbon and nitrogen dynamics in a steepland ecosystem in Northern Vietnam. Biol. Fertil. Soils 44, 69–77. Jouquet, P., Mathieu, J., Choosaï, C., Barot, S., 2007b. Soil Engineers as ecosystem het˜ S.I. (Ed.), Ecology Research Progress. Nova Science erogeneity drivers. In: Munoz, Publishing. Jouquet, P., Zangerlé, A., Rumpel, C., Brunet, D., Bottinelli, N., Tran Duc, T., 2009. Relevance of the biogenic and physicogenic classification. A comparison of approaches to discriminate the origin of soil aggregates. Eur. J. Soil Sci. 60, 1117–1125. Jouquet, P., Henry-des-Tureaux, T., Doan Thu, T., Tran Duc, T., Orange, D., 2010. Utilization of near infrared reflectance spectroscopy (NIRS) to quantify the impact of earthworms on soil and carbon erosion in steep slope ecosystem. A study case in Northern Vietnam. Catena 81, 113–116. Lavelle, P., 1975. Consommation annuelle de terre par une population naturelle de vers de terre (Millsonia anomala Omodeo, Acanthodrilidae-Oligochetes) dans la savane de Lamto (Côte d’Ivoire). Rev. Ecol. Biol. Sol 12, 11–24. Lavelle, P., Bignell, D., Lepage, M., 1997. Soil function in a changing world: the role of invertebrate ecosystem engineers. Eur. J. Soil Biol. 33, 159–193.
P. Jouquet et al. / Pedobiologia 54S (2011) S113–S117 Lavelle, P., Spain, A.V., 2001. Soil Ecology. Kluwer Academic Publishers, Dordrecht, the Netherlands, 654 pp. Lavelle, P., 2002. Functional domains in soils. Ecol. Res. 17, 441–450. Lavelle, P., Decaëns, T., Aubert, M., Barot, S., Blouin, M., Bureau, F., Margerie, P., Mora, P., Rossi, J.-P., 2006. Soil invertebrates and ecosystem services. In: European Journal of Soil Biology ICSZ – Soil Animals and Ecosystems Services, Proceedings of the XIVth International Colloquium on Soil Biology, vol. 42, pp. 3–15. Velasquez, E., Lavelle, P., Barrios, E., Joffre, R., Reversat, F., 2005. Evaluating soil quality in tropical agroecosystems of Colombia using NIRS. Soil Biol. Biochem. 37, 889–898.
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Contents lists available at SciVerse ScienceDirect
Pedobiologia - International Journal of Soil Biology journal homepage: www.elsevier.de/pedobi
9th International Symposium on Earthworm Ecology
What are the limits of the drilosphere? An incubation experiment using Metaphire posthuma Pascal Jouquet a,b,∗ , Gaëlle Huchet a,b , Nicolas Bottinelli c,d , Thuy Doan Thu a,b a
IRD – SFRI, Dong Ngac, Tu Liem, Hanoi, Viet Nam IRD, UMR 211 BIOEMCO, Centre IRD Bondy, 32 Avenue H. Varagnat, 93143 Bondy Cedex, France INRA, UMR 1069 Sol Agro et hydrosystème Spatialisation, F-35000 Rennes, France d Université Européenne de Bretagne, France b c
a r t i c l e
i n f o
Article history: Received 25 October 2010 Received in revised form 30 June 2011 Accepted 18 July 2011 Keywords: Earthworms Drilosphere Cast production Soil aggregate organization Near infrared reflectance spectroscopy (NIRS) Metaphire posthuma
a b s t r a c t The near infrared reflectance spectroscopy (NIRS) method was used in the present study to compare earthworm-made soil aggregates to aggregates found in the surrounding bulk soil. After initially assessing the daily cast production of Metaphire posthuma, boxes with soil incubated with M. posthuma and control soils were subjected to wetting in order to reorganize the soil structure. After two months of incubation, soil aggregates produced by earthworms (casts and burrows), soil aggregates that were appeared to be unaffected by earthworms (bulk soil without visible trace of earthworm bioturbation from the earthworm treatment) and soil aggregates that were entirely unaffected by earthworms (control – no earthworm – treatment) were sampled and their chemical signatures analyzed by NIRS. The production of belowground and surface casts reached 14.9 g soil g worm−1 d−1 and 1.4 g soil g worm−1 d−1 , respectively. Soil aggregates from the control soils had a significantly different NIRS signature from those sampled from boxes with earthworms. However, within the earthworm incubation boxes the NIRS signature was similar between cast and burrow aggregates and soil aggregates from the surrounding bulk soil. We conclude that the high cast production by M. posthuma and the regular reorganization of the soil structure by water flow in and through the soil lead to a relatively homogenous soil structure. Given these results, we question the relevance of considering the bulk soil that has no visible activity of earthworm activity as a control to determine the effect of earthworms on soil functioning. © 2011 Elsevier GmbH. All rights reserved.
Introduction Soil engineers are organisms that create biogenic structures with different physical and chemical properties compared to the surrounding soil (Lavelle et al. 1997; Jouquet et al. 2006, 2007b). Together these structures constitute the sphere of influence, or functional domain of soil engineers (i.e. the drilosphere, termitosphere and myrmecosphere, respectively, for earthworms, termites and ants, Lavelle 2002). Among soil engineers, earthworms are considered to be the most widespread and abundant in terrestrial ecosystems (Lavelle and Spain 2001) and their importance in the regulation of biogeochemical processes and ecosystem services have been largely demonstrated (Lavelle et al. 2006). Biogenic aggregates produced by earthworms are usually sampled in the field or in laboratory experiments and identified with regard to their spherical or rounded and smooth shapes (Bullock
∗ Corresponding author at: IRD, UMR 211 BIOEMCO, Centre IRD Bondy, 32 Avenue H. Varagnat, 93143 Bondy Cedex, France. E-mail address: [email protected] (P. Jouquet). 0031-4056/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2011.07.013
et al. 1985) or to their higher soil structural stability (reviewed in Edwards 2004). The effect of earthworms on soil properties is usually determined by comparing these earthworm-made soil aggregates and the surrounding soil environment that has no visible trace of earthworm activity, e.g. the physicogenic aggregates. Physicogenic aggregates are angular blocky macroaggregates that result from both abiotic (e.g. tillage processes, wetting and drying processes) and biotic (e.g. microbial activity associated with root exudation and degradation but not soil macrofauna activity) processes. This distinction between biogenic and physicogenic aggregates has been successfully used as an indicator in the study of the influence of earthworms on soil functioning (Decaëns et al. 2002; Hedde et al. 2005; Velasquez et al. 2007; Jouquet et al. 2010). However, as emphasized by Darwin (1881): “(. . .) castings are extremely liable to crumble during dry weather into small fragments, which are soon acted on by rain, and then sink down so as to be no longer distinguishable from the surrounding soil”. More recently, Jouquet et al. (2009) confirmed this observation and showed that raindrop impact led to the loss of the rounded shape of earthworm casts. Although these soil aggregates can be classified as physicogenic aggregates, studies have shown that they
S114
P. Jouquet et al. / Pedobiologia 54S (2011) S113–S117
keep the specific chemical properties of biogenic aggregates. Consequently, the choice of control soil aggregates is questionable when only based on aggregate appearance. Another indicator is therefore required to determine true physicogenic or control soil aggregates, which can then be compared to earthworm biogenic aggregates. Near infrared reflectance spectroscopy (NIRS) has recently emerged as a promising technique for the assessment of soil quality (Velasquez et al. 2005; Cécillon et al. 2009). This method has also been used for the identification of soil aggregate origin and evolution (Hedde et al. 2005; Velasquez et al. 2007; Jouquet et al. 2009; Cécillon et al. 2010; Chapuis-Lardy et al. 2010; Jouquet et al. 2010). In the present study, NIRS was used to determine the value of comparing earthworm made soil aggregates (casts and burrows) to their surrounding soil environment (i.e. the control soil aggregates without visible influence of earthworms). The main hypothesis of this work was that the control soil has a NIRS signature different to that of the biogenic aggregates produced by earthworms. A laboratory experiment was carried out and the comparison was made between biogenic and physicogenic aggregates sampled in boxes where earthworms were added and physicogenic aggregates sampled in boxes without earthworms.
Materials and methods Study site and study model The study was carried out from May to June 2009 at the Soil and Fertilizer Research Institute (SFRI, Hanoi, Vietnam, 20◦ 57 N, 105◦ 29 E). The soil is a loamy soil (Clay: 13%, Silt: 50%, Sand: 37%) with low organic matter (24 g kg−1 ) sampled in the upper 10 cm from a garden. The garden is almost exclusively colonized by the earthworms Metaphire posthuma (Oligochaeta, Megascolecidae), which is a medium size endogeic geophagous earthworm (∼10 cm in length and 5 mm diameter on average at the adult stage). In northern Vietnam, M. posthuma produces approximately 5-fold its own weight per day (Bottinelli et al. 2010). Its casts range from 2 to 3 mm and are mainly found belowground (Bottinelli et al. 2010).
Soil incubation procedure Aggregates of <1 mm (462 g) were put in polyvinyl chloride boxes (9 cm wide × 12 cm long × 10 cm high) with a plastic pipe, closed by a waterproof silicone cap, running along the bottom of the box, above a 1 cm layer of sand and gravel covering the bottom of the boxes. Soil aggregates were compacted manually to 1.40 g cm−3 . Boxes were incubated at 30 ◦ C in the dark after remoistening (75% of the field capacity) and inoculated (W+), or not (W−), with one earthworm (∼0.5 g in average). Twice a week, the boxes were opened to allow air exchange. Every 15 days, 200 ml of distilled water was gently applied to the surface of the soil with a small watering can in order to dissolve earthworm biogenic structures (casts and burrows). Excess water leached through the plastic pipe in the bottom of the boxes. Soil aggregate sampling and NIRS fingerprint Soils were collected from the boxes incubated with earthworms and from the controls two months after incubation (n = 5). At this time, different categories of casts were established. Aboveand below-ground casts and burrows were considered as earthworm biogenic aggregates. Casts were identified according to their granular shapes, as described by Bottinelli et al. (2010). The soils without visible activity of earthworms were considered as physicogenic aggregates. A distinction was made between the physicogenic aggregates sampled on the soil surface to that sampled below the surface. In the treatment without earthworms, soils were considered true controls. They were also sampled above and below the surface of the soil. Soil aggregates were air-dried, crushed and passed through 250 m sieves. 5 g of each sample was scanned with a FOSS 5000 spectrophotometer (Foss NIRSystems, Silver Spring, MD, USA) in the 1100–2500 nm spectral range. The spectral data obtained were recorded as the logarithm of the inverse of reflectance [log(1/R)]. They were conducted using WinISI III-version 1.50e software (Foss NIRSystems, Infrasoft International). A 20-nm sampling interval was used for data analysis. Statistical analyses
Cast production Cast production was assessed following the procedure of Bottinelli et al. (2010). Approximately 50 kg of soil was sampled in the upper 10 cm of the soil, air-dried, ground-down and sieved to obtain 0.25–1 mm soil aggregates. Aggregates were put in polyvinyl chloride cylinders (20 cm in diameter and 10 cm high) and manually packed down to reach 1.4 g cm−3 . One earthworm was added per cylinder and no additional food was given during the experiment. Before inoculation, juvenile earthworms were placed on moist tissue paper for 24 h to void their guts and subsequently weighed (fresh weight). Individual earthworms weighed 0.5 g on average. A perforated plastic lid covered the top of the cylinders to allow air circulation. Cylinders were incubated at 30 ◦ C in the dark after remoistening (75% of the field capacity). Surface- and below-ground casts were sampled after 1, 5, 9, 12 and 15 days of incubation (15 cylinders in total, n = 3 for each date). Casts were easily distinguished from the bulk soil by their rounded shape and their size. They were hand-sorted cautiously with a spatula, airdried and weighed. After each incubation time, earthworms were removed, purged and their fresh weights determined as above. Earthworm development was assessed through the comparison of earthworm biomass at each sampling date minus the initial earthworm biomass.
Data were tested for homogeneity of variance using Levene’s test. Analysis of variance (ANOVA) was performed when variance homogeneity assumptions were met, and Mann–Whitney Wilcoxon’s test was used for non-parametric data. A Principal Component Analysis (PCA) was carried out to differentiate the samples based on their NIRS signature. Briefly, we used a matrix of 35 samples and 67 variables from NIRS wavelengths quantifying the adsorptions between 1100 and 2500 nm with a 20-nm sampling interval. All statistical calculations were carried out using R software. Differences among treatments were declared at the <0.05 probability level of significance.
Results Earthworm biomass and cast production The evolution of earthworm biomass is shown in Fig. 1. Although significant differences were observed between the different sampling dates (ANOVA, F4,20 = 10.7, P = 0.002), our data did not show any temporal trend. The daily below-ground cast production decreased during the first 9 days of the experiment, reaching a threshold of 9.3 (SE: 2.8) g soil g worm−1 d−1 (Fig. 2 and Table 1). Conversely, the daily
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Fig. 1. Evolution of earthworm biomass (% initial soil weight) during the 15 days of the experiment (n = 3).
Fig. 3. Results of the principal components analysis showing the absorbance in the near infrared of soil aggregates sampled after 2 months of incubation. The circles are the barycenters of clusters of points for each aggregate class: control soil surface (S), control bulk soil (B), cast on the soil surface (Cs), cast in the bulk soil (Cb) and burrow (Bu). Circles in white were sampled in containers without earthworms and circles in grey were sampled in boxes with earthworms (n = 5).
NIRS signature of all the soil categories incubated with earthworms (Mann–Whitney Wilcoxon test, P < 0.05). Discussion Fig. 2. Evolution of the cast daily production (g soil g worm−1 d−1 ) of Metaphire posthuma in the soil (in white) or on the soil surface (in grey) during the 15 days of the experiment (n = 3).
above-ground cast production was very low and constant throughout the experiment at only 1.5 (SE: 0.3) g soil g worm−1 d−1 (Fig. 2). NIRS signature PCA performed using the NIRS data allowed soil aggregates to be differentiated mainly on the first axis (Fig. 3). Positive values on the first axis (62% of the total variability) of the PCA corresponded to the soil aggregates incubated without earthworms, whereas negative values described soil biogenic aggregates and the control soil sampled in the vicinity of soil biogenic aggregates. Univariate statistical analyses were subsequently performed on data obtained from the projection of the plots on the first axis of the PCA (Fig. 4). Although the NIRS signature of biogenic aggregates (casts and the soil covering the burrow) had the same signature as the surrounding bulk soil, we observed differences between casts sampled on the soil surface and the surrounding soil surface without visible trace of earthworm activity (Mann–Whitney Wilcoxon test, P < 0.05). However, the most striking result was the significant difference between the signatures of the soil incubated without earthworms (bulk soil and soil surface from the no earthworm treatment) and the
Earthworm biomass and cast production With the exception of Day 1 of the experiment, earthworm weight loss was very low, suggesting that earthworm activity was only slightly affected by the incubation experiment. The higher cast production at the beginning of the experiment is commonly observed in incubation experiments with earthworms and is likely to be the consequence of stress resulting from the earthworm manipulation and by the creation of a new habitat by the earthworms (Whalen et al. 2004; Bottinelli et al. 2010). Our cast production results were higher than those found by Bottinelli et al. (2010) who found a daily cast production of about 5 g soil−1 g worm−1 . This difference might be explained by the developmental stage of earthworms. The experiment carried out
Table 1 Two-way ANOVA table for the effect of the location (above vs. below-ground cast) and sampling date on the daily production of casts by Metaphire posthuma. d.f. Location (1) Date (2) 1×2
1,20 4,20 4,20
F-Values
P-Values
97.7 4.7 3.6
<0.001 0.007 0.021
Fig. 4. Projection of the plot on the first axis of the PCA for the bulk soil (B) and soil surface (S) sampled in boxes without earthworms (no earthworm treatment, in white) and sampled in boxes with earthworms (earthworm treatment, in grey), casts on the soil surface (Cs) or inside the soil (Cb) and soil covering the burrows (Bu). Histograms with the same letter are not different at P = 0.05 (n = 5).
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by Bottinelli et al. (2010) was made at the end of the rainy season, in September 2008 when the earthworms were adult. In our study, the experiment was done earlier in the season (May to June 2009) when the earthworms were juvenile and therefore probably required more energy for their development. This hypothesis is in agreement with Lavelle (1975) and Barois et al. (1999) who indicated that juveniles produce more casts than adults.
Conclusion In addition to confirming the relevance of the NIRS method to distinguish soil aggregates of different origins, this study suggests limitations in comparing biogenic structures to the surrounding bulk soil environment. Specifically, the present work shows that the only true control soils are non-drilosphere soils that have not been incubated with earthworms.
NIRS signature
Acknowledgments
Recent studies have emphasized the suitability of the NIRS method for the assessment of soil quality (see Cécillon et al. 2009 for a review). In our study, this method was used to determine the optical signature of soil biogenic (e.g. casts and burrows) and physicogenic aggregates (e.g. the control aggregates not apparently influenced by earthworms). This study emphasized the limitation of comparing earthworm casts to their surrounding soil in laboratory experiments and questions the relevance of using this surrounding soil as a control soil in laboratory experiment and also perhaps in the field. The drilosphere has been described as the part of the soil influenced by earthworm movement, secretions and castings (Lavelle 2002). This functional domain is traditionally limited to well identifiable structures: the cast aggregates, the soil covering the burrows, the middens and the earthworm biomass in itself. NIRS analysis shows that the bulk soil, which does not present any visible activity of earthworms, might also be influenced by earthworms and therefore also might be included in the drilosphere. Three main hypotheses can explain the modification of the signature of the bulk soil surrounding the casts and burrows produced by earthworms. First, earthworms might influence the chemical properties of the soil underneath their burrows by promoting the transfer of mineral or organic molecules (Decaëns et al. 1999; Amador et al. 2006; Jouquet et al. 2007a; Don et al. 2008). It can therefore be suggested that the activity of M. posthuma under a constrained environment fosters the leaching of nutrients such as ammonium, nitrate and dissolved organic carbon, and therefore leads to a homogenization of the soil chemical characteristics. Another explanation relates to the lifetime of earthworm biogenic structures. In the same study field, Bottinelli et al. (2010) showed that casts produced by M. posthuma have lower soil organic matter content and are less resistant to water impact than the surrounding soil aggregates. As a consequence, its casts collapse rapidly after significant rainfall events, leading to a reduction of soil porosity. In our experiment, excess water was applied every 2 weeks in order to dissolve non-stable soil aggregates. Therefore, it is likely that some casts and burrows collapsed when water was applied to the soil surface and were then mixed with the soil matrix, leading to a homogenization of the soil properties and to lack of differences as detected with the NIRS. Finally, considering an average cast production of 10 g soil g worm−1 d−1 , and applying the assumption that earthworms do not feed on their own fresh or degraded casts (Lavelle, personal communication), we can assume that 300 g of casts was produced during the 2 months of the experiment. This data therefore suggests that around 65% of the soil initially put in the container had been consumed by earthworms. Consequently, it is likely that earthworm activity led to a homogenization of the bulk soil and that it was not possible to distinguish the NIRS signature of casts from that of the surrounding soil aggregates. These three hypotheses are reinforced by the higher variability of the NIRS data of the control soil incubated without earthworms compared to the physicogenic aggregates and biogenic aggregates sampled in the boxes with earthworms (standard error: 4.0 and 0.36, respectively, for bulk soil from boxes without earthworms and bio- and physicogenic aggregates sampled from boxes with earthworms).
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