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Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas
Soil Biology & Biochemistry 37 (2005) 1043–1048 www.elsevier.com/locate/soilbio
Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas P. Moraa,*, E. Miambia, J.J. Jime´nezb, T. Decae¨nsc, C. Roulanda a
Laboratoire d’Ecologie des Sols Tropicaux UMR 137 Biosol, Universite´ Paris 12, 61, avenue du Ge´ne´ral de Gaulle, 94010 Cre´teil Cedex, France b Soil and Plant Nutrition Unit, CIAT, P.O. Box 6713, Cali, Columbia c Laboratoire d’Ecologie UPRES-EA 1293 UFR Sciences et Techniques Universite´ de Rouen F-76821 Mont Saint Aignan Cedex, France Received 29 March 2004; received in revised form 21 October 2004; accepted 27 October 2004
Abstract Soil-engineering organisms (earthworms, termites and ants) affect the soil and litter environment indirectly by the accumulation of their biogenic structures (casts, pellets, galleries, crop sheetings nests.). An enzymatic typology was conducted on six types of biogenic structures: casts produced by two earthworms (Andiodrilus sp. and Martiodrilus sp.), a nest built by a soil-feeding termite (Spinitermes sp.), crop galleries built by another soil-feeding termite (Ruptitermes sp.) and soil pellets produced by two species of leaf-cutting ant (Acromyrmex landolti and Atta laevigata) and an control soil from a natural Colombian savanna. A total of 10 enzymes (xylanase, amylase, cellulase, a-glucosidase, b-glucosidase, b-xylosidase, N-acteyl-glucosaminidase, alkaline and acid phosphatases and laccase) were selected to characterize the functional diversity of the biogenic structures. Our results showed that (i) Martiodrilus casts were characterized by a broad enzymatic profile that was different from that of the soil. (ii) A. laevigata pellets and termite structures had a profile broadly similar to the soil only with some enzymes (iii) Andiodrilus casts had an enzyme profile very similar to that of the soil. These results suggest that the functional diversity of these structures is related to differences between species and not to differences between taxonomic groups. For the first time, we evaluated differences in enzyme typology between biogenic structures collected on the same site but produced by different organisms. These differences suggested species dependent pathways for the decomposition of organic matter. q 2004 Elsevier Ltd. All rights reserved. Keywords: Polysaccharidases; Heterosidases; Phenol-oxidases; Phosphatases; Termites; Earthworms; Ants; Casts; Crop galleries
1. Introduction ‘Ecological engineers’, or ‘ecosystem engineers’, are organisms that produce physical structures through which they can modify the availability or accessibility of a resource for other organisms (Jones et al., 1994). Earthworms, termites and ants form the main groups recognized as soil engineers (Lavelle et al., 1997). These invertebrates are important for the soil and their action is believed to have a significant effect on functional processes in the soil. The biogenic structures (casts, sheetings, nests, burrows, crop galleries.) built by these organisms are believed to modify living conditions for other smaller and less mobile soil organisms (collembola, nematodes, protozoa, fungi, * Corresponding author. Fax: C33 1 45 17 15 05. E-mail address: [email protected] (P. Mora). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.10.019
bacteria), and hence to influence their abundance and diversity (Brown, 1995; Jones et al., 1997; Decae¨ns et al., 1999). These ecosystem engineers modify the soil structure and particle size as well as the microbial community and the dynamics of the chemical processes by building their biogenic structures (Lavelle et al., 1997; Blanchart et al., 1989; Blanchart et al., 1993; Tiunov and Scheu, 1999; Brauman, 2000; Tiunov and Scheu, 2000; Mora and Seuge´, 2003). Moreover, very little research has been carried out into the relationship between the diversity of the soil engineers and their biogenic structures and the functional diversity in the soil (Lavelle et al., 1997). Enzymes are the main mediators of soil biological processes (degradation of organic matter, mineralization and nutrient recycling) and so they provide an effective way of examining functional diversity in soils (Dick, 1994; Kandeler et al., 1996). Enzyme activities were measured to compare the functional
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aspects of different biogenic structures. The criteria for choosing the enzymes assayed were based on their importance in nutrient cycles and organic matter decomposition. Cellulase, xylanase and amylase were chosen because they hydrolyze major plant molecules transferred to soils. a-glucosidase, b-glucosidase and b-xylanase were chosen for their critical role in releasing low molecular weight sugars which are important as energy sources for microorganisms. Alkaline and acid phosphatase were included because of their role in releasing inorganic phosphorus in the P cycle. N-acetyl-glucosaminidase hydrolyzes N-acetyl-b-D-glucosamine residues from the non-reducing end of chitooligosaccharides in soils. This is one of the enzymes that play a major role in N mineralization in soils. Laccase is a polyphenol oxidase that oxidizes polyphenols, methoxy-substituted phenols, diamine and a wide range of other compounds (Thurston, 1994). This assay, therefore, provides a broad-spectrum indicator of soil biological activity. The aim of this work was (i) to assess whether these structures have different enzyme properties from those of the soil on which they are built (ii) to determine whether the functional diversity of these structures is related to the functional diversity of ecosystem engineers.
2. Materials and methods 2.1. Samples The biogenic structures were collected at the Carimagua (CIAT-CORPOICA) biological station in a savanna on oxisol where the vegetation was dominated by Trachypogon vestitus and Paspalum pectinatum (Decae¨ns et al., 2001). The morphological aspects of these biogenic structures were described by Decae¨ns et al. (2001). The biogenic structures collected belonged to two species of earthworms Andiodrilus sp. and Martiodrilus sp. (Jime´nez et al., 1998). Andiodrilus sp. is an endogeic earthworm that builds casts in the form of pellets whereas the biogenic structures of Martiodrilus sp., an anecic species, are cylindrical structures. The termite crop galleries collected on the site were built by Ruptitermes sp., a soil-feeding termite, and the nest fragments collected were built by Spinitermes sp. (another soil-feeding species). The biogenic structures of two leafcutting ants Acromyrmex landolti Forel and Atta laevigata Smith were small pellets forming a cone at the entrance to the nest or nearby. In the savanna, approximately 500 g of fresh biogenic structures and 3!100 g of control soil (0–10 cm depth) were collected in three plots in 2001 (July). For each biogenic structure and control soil, three composite samplings were taken (21 samples on the whole). Samples were mixed, and slightly air-dried to allow the seiving (2 mm mesh sieve) and stored at 4 8C for 2 weeks in sealed plastic bags before being analyzed.
2.2. Enzyme assays For each sample, the assays were carried out in triplicate to determine the reproducibility of the laboratory analyses. Enzyme measurements were made on samples reset to their water holding capacity and then incubated at 25 8C for 2 weeks to reactivate the microflora. Incubations were in plastic containers with a perforated plastic cover to restrict evaporation and yet permit gas exchange. Moisture levels were maintained gravimetrically every 2 days using deionized water. The acid and alkaline phosphatase activities were determined according to Tabatabai (1982): the enzymatic unit (U) is expressed as the quantity (mg) of p-nitrophenol (PNP) released minK1 under standard conditions (pH 6, or pH 9, 37 8C). The laccase activities were measured using the method described by Bourbonnais and Paice (1990): the enzymatic unit (U) is defined as the activity which oxidizes 1 mmole of ABTS minK1 (pH 5, 37 8C). The xylanase, amylase and cellulase activities were assayed using the quantity of reducing sugars that appeared in the sample in the presence of microcrystalline cellulose, xylan or starch (0.05 g of substrate per gram of sample). 0.1 g of soil was mixed with 400 ml of phosphate buffer pH 5 since the pH of the biogenic structures and the soil varied between 4.5–5.5 (Decae¨ns et al., 1998). After incubation at 37 8C for 1 h, the samples were then centrifuged at 14,000g for 5 min and 250 ml of the supernatant were used to determine the quantities of reducing sugars using the methods of Somogyi (1945); Nelson (1944). The enzymatic unit was defined as the quantity in mg of reducing sugars produced hK1. Samples incubated without substrate were used as controls. The b-xylosidase, b-glucosidase and a-glucosidase activities were measured using p-nitrophenyl-b-D-xylopyranoside, p-nitrophenyl-b-D-glucopyranoside and p-nitrophenyl-a-D-glucopyranoside as substrats: Soil (0.1 g) was incubated at 37 8C for 1 h in 0.4 ml of phosphate buffer (pH 5) with 3 mM of substrat. Samples incubated without substrate were used as controls. After incubation, the samples were then centrifuged at 14,000g for 5 min and 120 ml of the supernatant were used to determine the quantities of p-nitrophenyl released from the hydrolysis of the susbtrat. Three milliliters of 2% Na2CO3 solution was added to the supernatant and the absorbance was measured at 410 nm. The enzymatic activity (U) is expressed as the quantity in mg of p-nitrophenol released minK1. 2.3. Statistical analysis Principal component analysis (PCA) was performed by ADE-4 software (Thioulouse et al., 1997) using a matrix of 21 samples!10 variables (enzyme activities). A permutation test was used to test the significance of the groupings suggested by PCA.Values were considered as different when probability to reject hypothesis nil was lower than 0.05. The similarity dendrogram was produced using the same software. The averages were compared using ANOVA after
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0.7G0.7 (A) 1.5G2.4 (A) 2.6G1.0 (B) 15.1G1.2 (C) 0.8G0.6 (A) 2.1G1.3 (B) 2.0G1.1 (B)
verifying that the data were normal and checking that the variances were homogeneous. The post hoc test applied following ANOVA was the Tukey test (P!0.05).
3. Results
0.4G0.6 (B) 1.2G2.2 (B) 0.1G0.2 (A) 2.0G0.6 (D) 6.5G4.1 (E) 2.0G1.1 (C) 1.2G0.6 (C)
3.1. Enzyme activities
Enzyme activities are expressed as U gK1 dry soil. Within a column, values with different letters are significantly different (P!0.05) (G: standard deviation, nZ9).
1.4G1.8 (C) 1.7G3.2 (C) 0.4G0.5 (B) 3.9G0.6 (C) 9.8G4.7 (D) 0.2G0.4 (B) 0.0G0.0 (A) 0.0G0.0 (A) 0.0G0.0 (A) 0.0G0.0 (A) 0.9G0.3 (C) 0.2G0.2 (B) 0.3G0.4 (C) 0.0G0.0 (A) 1.6G0.7 (D) 1.3G1.3 (D) 0.1G0.1 (AB) 1.7G1.5 (D) 0.3G0.3 (C) 0.1G0.2 (C) 0.0G0.0 (A) 422.2G203.7 (D) 342.7G201.3 (C) 852.0G451.0 (E) 251.9G156.2 (C) 2.2G4.1 (A) 650.8G247.3 (E) 56.9G31.8 (B) Ruptitermes sp. Spinitermes sp. Andiodrilus sp. Martiodrilus sp. Atta laevigata Atta landolti Soil
861.8G347.1 (B) 2585.3 G904.3 (E) 1354.7G365.4 (C) 1544.2G199.5 (D) 275.5G147.0 (A) 1367.9G303.0 (C) 901.4G147.0 (B)
4.1G3.2 (C) 26.8G29.3 (C) 32.5G24.5 (D) 109.3G52.1 (E) 1.2G1.9 (B) 3.5G4.3 (C) 0.0G0.0 (C)
2.6G1.4 (A) 8.2G2.5 (C) 2.1G0.6 (A) 3.2G1.7 (B) 2.0G0.7 (A) 3.2G1.7 (B) 2.5G1.5 (A)
0.7G0.6 (B) 0.3G0.9 (B) 0.3G0.2 (B) 2.7G1.0 (C) 0.3G0.1(B) 0.4G0.1 (B) 0.0G0.0 (A)
LAC ACP ALP NAG b-Glu a-Glu b-Xyl CEL AMY XYL
Table 1 Enzyme activities of the biogenic structures and the soil. XYL, Xylanase; AMY, Amylase; CEL, Cellulase, b-Xyl, b-Xylosidase; a-Glu, a-Glucosidase; b-Glu, b-Glucosidase; NAG, N-ace´tyl-glucosaminidase, ALP, Alkaline phosphatase; ACP, Acid phosphatase; LAC, Laccase
P. Mora et al. / Soil Biology & Biochemistry 37 (2005) 1043–1048
3.1.1. Polysaccharidases All the samples tested showed xylanase activity to various degrees (Table 1). The Spinitermes nest fragment, and to a lesser extent the casts and the A. landolti pellets had high xylanase activities. No significant differences in xylanase activities were observed between Ruptitermes crop galleries and the soil and between Andiodrilus and A. Landolti structures. A. laevigata pellets had the lowest activity. Andiodrilus casts and A. landolti pellets showed the highest amylase activity (Table 1). In addition, Martiodrilus casts and both termites structures showed enzyme activities which were 4 to 6–7.5 times higher than those in the soil, respectively. No significant differences were observed between Spinitermes sp. and Martiodrilus sp. The lowest amylase activity was observed in A. laevigata pellets. The Martiodrilus casts and to a lesser degree Andiodrilus structures exhibited the highest cellulolytic activities in comparison with those obtained for the other biogenic structures (Table 1). No significant differences were observed between both termite structures, A. landolti pellets and soil. 3.1.2. Heterosidases The Spinitermes sp. nest fragments had the highest b-xylosidase activity while the activities of the Ruptitermes Andiodrilus and A. laevigata structures were the lowest and similar to those in the soil (Table 1). Martiodrilus and A. landolti structures had intermediate activities. With activities higher than 1 U gK1 dry soil, the termite and Martiodrilus structures showed the most significant a-glucosidase activities. In the other biogenic structures, the activities were low (!0.5 U gK1 dry soil) and no activity was measured in the soil (Table 1). The highest b-glucosidase activities were found in the Martiodrilus casts with values higher than 2 U gK1 dry soil. In the other structures the values were close to zero (Table 1). The activities measured for PNP-N-acetyl-glucosamine were relatively low and only found in the Martiodrilus and A. laevigata structures (Table 1). 3.1.3. Phosphatases The acid and alkaline phosphatases activities in A. laevigata pellets were significantly higher than those of the other samples (Table 1). The phosphatase activities measured for the Martiodrilus casts were lower but still very significant.
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In the other samples, the acid phosphatase activities were lower than the activity measured in the soil (Table 1). The soil differed from the other samples in that no alkaline phosphatase activity was detected. 3.1.4. Laccases Martiodrilus had the highest laccase activity, higher than 15 U gK1 dry soil (Table 1). The Andiodrilus and A. landolti structures had activities comparable with that measured for the soil (2 U gK1 dry soil). The lowest laccase activities were found in the Ruptitermes, Spinitermes and A. landolti structures. 3.2. Multivariate analysis A principal component analysis (PCA) was carried out (Fig. 1 A). Axes 1 and 2, respectively, explained 38.3 and 23.3% of the total variance. The significance test carried out on 10,000 permutations showed that the PCA was highly significant (P!0.00001). The samples with high cellulase, b and a-glucosidase, N-acetyl-glucosaminidase, and laccase activities were separated to those with low activities along axis 1. The samples with high b-xylosidase, xylanase,
Fig. 1. Results of the principal components analysis (PCA). (A) Correlation circle. (B) Ordination of the samples in the plane defined by axes 1 and 2 of the PCA.
amylase activities were opposed to those with high phosphatase activities along axis 2 (Fig. 1 A). The position of the samples along the first two PCA axes (Fig. 1 B) showed that the Martiodrilus casts were very clearly different from the other samples. Martiodrilus casts were characterized by a large number of enzymes with activities that were high in comparison with the activities measured in the other samples. A. laevigata pellets were also clearly different from the other samples, mainly because of their phosphatase activities. Despite their differences, the other samples were fairly close to the soil. 3.3. Dendrogram analysis The similarity dendrogram (Fig. 2) shows the hierarchy of the differences observed between the samples. The Martiodrilus casts were very clearly different from all the other structures. In these structures, the enzyme profile showed significant differences in the activities. The A. laevigata pellets also differed from the other structures but, unlike the Martiodrilus casts, the activities were low and only the phosphatase activities were significant. In these structures, there was a significant modification of the enzyme profile in comparison with the soil. The third section separated the fragments of Spinitermes nests from the other samples. This is the result of the relatively high b-xylosidase and xylanase activities. To a lesser extent, the Ruptitermes crop galleries also formed a cluster different from that of the soil. Despite some differences, the Andiodrilus and A. landolti structures were not really distinct from the soil.
Fig. 2. Dendrogram of similarity calculated on the basis of PCA values. MA, Martiodrilus sp.; Alea, A. laevigata; S, Spinitermes sp.; Alan, A. landolti; AN, Andiodrilus sp.; So, Soil; R, Ruptitermes sp.
P. Mora et al. / Soil Biology & Biochemistry 37 (2005) 1043–1048
4. Discussion 4.1. Earthworm structures Martiodrilus casts showed a different enzyme typology from the other biogenic structures and soil samples. The high enzyme activities measured in these structures suggest that the organic matter was strongly degraded here. On the other hand, the Andiodrilus casts had an enzyme profile close to that of the soil. These differences can be explained by their feeding behavior. The effects of earthworms on soil processes differ between ecological categories and species (Bouche´, 1977). Epigeic species mainly ingest litter and humus and do not mix organic and inorganic matter extensively (McLean and Parkinson, 1998). In contrast, endogeic earthworms live in the mineral soil layers and mainly ingest mineral soil matter. Andiodrilus sp. is an endogeic polyhumic earthworm while Martiodrilus sp. is an anecic earthworm (Jime´nez et al., 1998). Analysis of the intestinal contents of this earthworm showed that Martiodrilus sp. can ingest roots (Mariani et al., 2001). The high proportions of plant fragments in the digestive tract is consistent with the presence in the casts of the broad range of enzymes involved in the degradation of plant matter. Conversely Andiodrilus sp. was characterized by low enzyme activities, for enzymes that are involved in hydrolysis of plant polysaccharides. These activities can be compared with those found in the digestive tracts of three other endogeic earthworms Pontoscolex corethrurus, Polypheretima elongata and Milsonia anomala (Lattaud et al., 1998). This paper showed that endogeic earthworms had polysaccharidase activities which were rather low compared with other invertebrates such as fungus-growing termites and xylophagous termites. Furthermore, the high laccase (a polyphenol oxydase) activity suggested that Andiodrilus sp. ingested organic matter primarily comprising residues such as phenolic compounds. Finally, it seems that the enzyme profile of each earthworm species reflected their feeding behavior: one preferentially ingesting plant matter, the other consuming organic matter mainly comprised of polyhumic substances. The contrasting effects of these two functional groups of earthworms on the incorporation of organic matter into the mineral soil led to the differences between the enzyme activity profiles. 4.2. Termite structures The enzyme typology of the termite biogenic structures was close to that of the soil but there were some significant differences: cellulase, a and b-glucosidase, and alkaline phosphatase were found in these structures but not in the soil samples. Therefore, these termites modified the enzyme profile of the soil by building these structures. Such modifications have been observed in fungus growing termite sheetings (Seuge´, unpublished thesis). Moreover, physical and chemical differences in the walls of termite mounds
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and crop sheetings by comparison with the top soil samples, have been observed by several authors (Brauman, 2000; Fall et al., 2001; Mora and Seuge´, 2003). The similarity analysis (dendrogram) regroups Spinitermes nest fragments in a cluster distinct from that of Ruptitermes crop galleries. There may be two reasons for the differences observed between these two biogenic structures. Firstly, the soil used by the two species is not the same. It is well established that termites use different strategies depending on whether the structure to be built is permanent (a nest) or temporary (crop sheetings and crop galleries) (Jouquet et al., 2002). In this study the orange color of the Ruptitermes galleries indicated that the soil used for construction was a deep soil (Decae¨ns et al., 1999) In contrast Spinitermes sp used the top soil to build their nest. Secondly, the organic characteristics of the soil might have an impact on soil enzyme activities. It is well known that the enzyme activities are greatly affected by organic matter content of the soil (Ladd and Buttler, 1972; Dalal, 1975; Speir et al., 1980; Tabatabai, 1977). Chemical analyses showed that the concentration of organic matter was higher in the Spinitermes sp. nest fragments than in the Ruptitermes sp. crop galleries (Decae¨ns et al., 2001). The differences between the soil layers used and the method of construction could explain the differences in enzyme activity observed between these two biogenic structures. 4.3. Ant structures The enzyme typology of A. landolti pellets was close to that of the soil. According to Decae¨ns et al. (2001) few significant differences were observed when comparing physical (aggregate size and stability, bulk density) and chemical (C, N, P contents, pH, Al saturation) properties of these biogenic structures and the soil. These results suggested that these structures were the result of a simple mechanical movement of the soil when digging the subterranean galleries. A. laevigata structures were characterized by low enzyme activities with the exception of the acid and alkaline phosphatase activities. Phosphatases in soils are derived mainly from the microbial population and have been suggested as a satisfactory indicator of microbial activity (Frankenberger and Dick, 1983; Dick and Tabatabai, 1993). However, the exact origin, quantity and quality of the organic substrates and/ or the stimulation of microbial communities associated with the high levels of alkaline and acid phosphatase activities observed in this study cannot be determined from this study.
5. Conclusion This enzyme typology made it possible to demonstrate the functional diversity of the biogenic structures produced on the soil surface. This study shows three
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groups of structure: structures characterized by a broad enzyme profile (Martiodrilus casts), structures with enzyme profiles similar to the soil (Andiodrilus casts and A. landolti pellets) and those which differ from the soil by the presence of specific enzymes (termite structures and A. laevigata pellets). The production of a diversity of biogenic structures by ecosystem engineers may result in efficient regulation of soil processes through specific enzyme profiles. However, it will be necessary to describe other structures and to assay other enzymes to confirm the role that these biogenic structures play in nutrient cycles in tropical soils.
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Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas P. Moraa,*, E. Miambia, J.J. Jime´nezb, T. Decae¨nsc, C. Roulanda a
Laboratoire d’Ecologie des Sols Tropicaux UMR 137 Biosol, Universite´ Paris 12, 61, avenue du Ge´ne´ral de Gaulle, 94010 Cre´teil Cedex, France b Soil and Plant Nutrition Unit, CIAT, P.O. Box 6713, Cali, Columbia c Laboratoire d’Ecologie UPRES-EA 1293 UFR Sciences et Techniques Universite´ de Rouen F-76821 Mont Saint Aignan Cedex, France Received 29 March 2004; received in revised form 21 October 2004; accepted 27 October 2004
Abstract Soil-engineering organisms (earthworms, termites and ants) affect the soil and litter environment indirectly by the accumulation of their biogenic structures (casts, pellets, galleries, crop sheetings nests.). An enzymatic typology was conducted on six types of biogenic structures: casts produced by two earthworms (Andiodrilus sp. and Martiodrilus sp.), a nest built by a soil-feeding termite (Spinitermes sp.), crop galleries built by another soil-feeding termite (Ruptitermes sp.) and soil pellets produced by two species of leaf-cutting ant (Acromyrmex landolti and Atta laevigata) and an control soil from a natural Colombian savanna. A total of 10 enzymes (xylanase, amylase, cellulase, a-glucosidase, b-glucosidase, b-xylosidase, N-acteyl-glucosaminidase, alkaline and acid phosphatases and laccase) were selected to characterize the functional diversity of the biogenic structures. Our results showed that (i) Martiodrilus casts were characterized by a broad enzymatic profile that was different from that of the soil. (ii) A. laevigata pellets and termite structures had a profile broadly similar to the soil only with some enzymes (iii) Andiodrilus casts had an enzyme profile very similar to that of the soil. These results suggest that the functional diversity of these structures is related to differences between species and not to differences between taxonomic groups. For the first time, we evaluated differences in enzyme typology between biogenic structures collected on the same site but produced by different organisms. These differences suggested species dependent pathways for the decomposition of organic matter. q 2004 Elsevier Ltd. All rights reserved. Keywords: Polysaccharidases; Heterosidases; Phenol-oxidases; Phosphatases; Termites; Earthworms; Ants; Casts; Crop galleries
1. Introduction ‘Ecological engineers’, or ‘ecosystem engineers’, are organisms that produce physical structures through which they can modify the availability or accessibility of a resource for other organisms (Jones et al., 1994). Earthworms, termites and ants form the main groups recognized as soil engineers (Lavelle et al., 1997). These invertebrates are important for the soil and their action is believed to have a significant effect on functional processes in the soil. The biogenic structures (casts, sheetings, nests, burrows, crop galleries.) built by these organisms are believed to modify living conditions for other smaller and less mobile soil organisms (collembola, nematodes, protozoa, fungi, * Corresponding author. Fax: C33 1 45 17 15 05. E-mail address: [email protected] (P. Mora). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.10.019
bacteria), and hence to influence their abundance and diversity (Brown, 1995; Jones et al., 1997; Decae¨ns et al., 1999). These ecosystem engineers modify the soil structure and particle size as well as the microbial community and the dynamics of the chemical processes by building their biogenic structures (Lavelle et al., 1997; Blanchart et al., 1989; Blanchart et al., 1993; Tiunov and Scheu, 1999; Brauman, 2000; Tiunov and Scheu, 2000; Mora and Seuge´, 2003). Moreover, very little research has been carried out into the relationship between the diversity of the soil engineers and their biogenic structures and the functional diversity in the soil (Lavelle et al., 1997). Enzymes are the main mediators of soil biological processes (degradation of organic matter, mineralization and nutrient recycling) and so they provide an effective way of examining functional diversity in soils (Dick, 1994; Kandeler et al., 1996). Enzyme activities were measured to compare the functional
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aspects of different biogenic structures. The criteria for choosing the enzymes assayed were based on their importance in nutrient cycles and organic matter decomposition. Cellulase, xylanase and amylase were chosen because they hydrolyze major plant molecules transferred to soils. a-glucosidase, b-glucosidase and b-xylanase were chosen for their critical role in releasing low molecular weight sugars which are important as energy sources for microorganisms. Alkaline and acid phosphatase were included because of their role in releasing inorganic phosphorus in the P cycle. N-acetyl-glucosaminidase hydrolyzes N-acetyl-b-D-glucosamine residues from the non-reducing end of chitooligosaccharides in soils. This is one of the enzymes that play a major role in N mineralization in soils. Laccase is a polyphenol oxidase that oxidizes polyphenols, methoxy-substituted phenols, diamine and a wide range of other compounds (Thurston, 1994). This assay, therefore, provides a broad-spectrum indicator of soil biological activity. The aim of this work was (i) to assess whether these structures have different enzyme properties from those of the soil on which they are built (ii) to determine whether the functional diversity of these structures is related to the functional diversity of ecosystem engineers.
2. Materials and methods 2.1. Samples The biogenic structures were collected at the Carimagua (CIAT-CORPOICA) biological station in a savanna on oxisol where the vegetation was dominated by Trachypogon vestitus and Paspalum pectinatum (Decae¨ns et al., 2001). The morphological aspects of these biogenic structures were described by Decae¨ns et al. (2001). The biogenic structures collected belonged to two species of earthworms Andiodrilus sp. and Martiodrilus sp. (Jime´nez et al., 1998). Andiodrilus sp. is an endogeic earthworm that builds casts in the form of pellets whereas the biogenic structures of Martiodrilus sp., an anecic species, are cylindrical structures. The termite crop galleries collected on the site were built by Ruptitermes sp., a soil-feeding termite, and the nest fragments collected were built by Spinitermes sp. (another soil-feeding species). The biogenic structures of two leafcutting ants Acromyrmex landolti Forel and Atta laevigata Smith were small pellets forming a cone at the entrance to the nest or nearby. In the savanna, approximately 500 g of fresh biogenic structures and 3!100 g of control soil (0–10 cm depth) were collected in three plots in 2001 (July). For each biogenic structure and control soil, three composite samplings were taken (21 samples on the whole). Samples were mixed, and slightly air-dried to allow the seiving (2 mm mesh sieve) and stored at 4 8C for 2 weeks in sealed plastic bags before being analyzed.
2.2. Enzyme assays For each sample, the assays were carried out in triplicate to determine the reproducibility of the laboratory analyses. Enzyme measurements were made on samples reset to their water holding capacity and then incubated at 25 8C for 2 weeks to reactivate the microflora. Incubations were in plastic containers with a perforated plastic cover to restrict evaporation and yet permit gas exchange. Moisture levels were maintained gravimetrically every 2 days using deionized water. The acid and alkaline phosphatase activities were determined according to Tabatabai (1982): the enzymatic unit (U) is expressed as the quantity (mg) of p-nitrophenol (PNP) released minK1 under standard conditions (pH 6, or pH 9, 37 8C). The laccase activities were measured using the method described by Bourbonnais and Paice (1990): the enzymatic unit (U) is defined as the activity which oxidizes 1 mmole of ABTS minK1 (pH 5, 37 8C). The xylanase, amylase and cellulase activities were assayed using the quantity of reducing sugars that appeared in the sample in the presence of microcrystalline cellulose, xylan or starch (0.05 g of substrate per gram of sample). 0.1 g of soil was mixed with 400 ml of phosphate buffer pH 5 since the pH of the biogenic structures and the soil varied between 4.5–5.5 (Decae¨ns et al., 1998). After incubation at 37 8C for 1 h, the samples were then centrifuged at 14,000g for 5 min and 250 ml of the supernatant were used to determine the quantities of reducing sugars using the methods of Somogyi (1945); Nelson (1944). The enzymatic unit was defined as the quantity in mg of reducing sugars produced hK1. Samples incubated without substrate were used as controls. The b-xylosidase, b-glucosidase and a-glucosidase activities were measured using p-nitrophenyl-b-D-xylopyranoside, p-nitrophenyl-b-D-glucopyranoside and p-nitrophenyl-a-D-glucopyranoside as substrats: Soil (0.1 g) was incubated at 37 8C for 1 h in 0.4 ml of phosphate buffer (pH 5) with 3 mM of substrat. Samples incubated without substrate were used as controls. After incubation, the samples were then centrifuged at 14,000g for 5 min and 120 ml of the supernatant were used to determine the quantities of p-nitrophenyl released from the hydrolysis of the susbtrat. Three milliliters of 2% Na2CO3 solution was added to the supernatant and the absorbance was measured at 410 nm. The enzymatic activity (U) is expressed as the quantity in mg of p-nitrophenol released minK1. 2.3. Statistical analysis Principal component analysis (PCA) was performed by ADE-4 software (Thioulouse et al., 1997) using a matrix of 21 samples!10 variables (enzyme activities). A permutation test was used to test the significance of the groupings suggested by PCA.Values were considered as different when probability to reject hypothesis nil was lower than 0.05. The similarity dendrogram was produced using the same software. The averages were compared using ANOVA after
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0.7G0.7 (A) 1.5G2.4 (A) 2.6G1.0 (B) 15.1G1.2 (C) 0.8G0.6 (A) 2.1G1.3 (B) 2.0G1.1 (B)
verifying that the data were normal and checking that the variances were homogeneous. The post hoc test applied following ANOVA was the Tukey test (P!0.05).
3. Results
0.4G0.6 (B) 1.2G2.2 (B) 0.1G0.2 (A) 2.0G0.6 (D) 6.5G4.1 (E) 2.0G1.1 (C) 1.2G0.6 (C)
3.1. Enzyme activities
Enzyme activities are expressed as U gK1 dry soil. Within a column, values with different letters are significantly different (P!0.05) (G: standard deviation, nZ9).
1.4G1.8 (C) 1.7G3.2 (C) 0.4G0.5 (B) 3.9G0.6 (C) 9.8G4.7 (D) 0.2G0.4 (B) 0.0G0.0 (A) 0.0G0.0 (A) 0.0G0.0 (A) 0.0G0.0 (A) 0.9G0.3 (C) 0.2G0.2 (B) 0.3G0.4 (C) 0.0G0.0 (A) 1.6G0.7 (D) 1.3G1.3 (D) 0.1G0.1 (AB) 1.7G1.5 (D) 0.3G0.3 (C) 0.1G0.2 (C) 0.0G0.0 (A) 422.2G203.7 (D) 342.7G201.3 (C) 852.0G451.0 (E) 251.9G156.2 (C) 2.2G4.1 (A) 650.8G247.3 (E) 56.9G31.8 (B) Ruptitermes sp. Spinitermes sp. Andiodrilus sp. Martiodrilus sp. Atta laevigata Atta landolti Soil
861.8G347.1 (B) 2585.3 G904.3 (E) 1354.7G365.4 (C) 1544.2G199.5 (D) 275.5G147.0 (A) 1367.9G303.0 (C) 901.4G147.0 (B)
4.1G3.2 (C) 26.8G29.3 (C) 32.5G24.5 (D) 109.3G52.1 (E) 1.2G1.9 (B) 3.5G4.3 (C) 0.0G0.0 (C)
2.6G1.4 (A) 8.2G2.5 (C) 2.1G0.6 (A) 3.2G1.7 (B) 2.0G0.7 (A) 3.2G1.7 (B) 2.5G1.5 (A)
0.7G0.6 (B) 0.3G0.9 (B) 0.3G0.2 (B) 2.7G1.0 (C) 0.3G0.1(B) 0.4G0.1 (B) 0.0G0.0 (A)
LAC ACP ALP NAG b-Glu a-Glu b-Xyl CEL AMY XYL
Table 1 Enzyme activities of the biogenic structures and the soil. XYL, Xylanase; AMY, Amylase; CEL, Cellulase, b-Xyl, b-Xylosidase; a-Glu, a-Glucosidase; b-Glu, b-Glucosidase; NAG, N-ace´tyl-glucosaminidase, ALP, Alkaline phosphatase; ACP, Acid phosphatase; LAC, Laccase
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3.1.1. Polysaccharidases All the samples tested showed xylanase activity to various degrees (Table 1). The Spinitermes nest fragment, and to a lesser extent the casts and the A. landolti pellets had high xylanase activities. No significant differences in xylanase activities were observed between Ruptitermes crop galleries and the soil and between Andiodrilus and A. Landolti structures. A. laevigata pellets had the lowest activity. Andiodrilus casts and A. landolti pellets showed the highest amylase activity (Table 1). In addition, Martiodrilus casts and both termites structures showed enzyme activities which were 4 to 6–7.5 times higher than those in the soil, respectively. No significant differences were observed between Spinitermes sp. and Martiodrilus sp. The lowest amylase activity was observed in A. laevigata pellets. The Martiodrilus casts and to a lesser degree Andiodrilus structures exhibited the highest cellulolytic activities in comparison with those obtained for the other biogenic structures (Table 1). No significant differences were observed between both termite structures, A. landolti pellets and soil. 3.1.2. Heterosidases The Spinitermes sp. nest fragments had the highest b-xylosidase activity while the activities of the Ruptitermes Andiodrilus and A. laevigata structures were the lowest and similar to those in the soil (Table 1). Martiodrilus and A. landolti structures had intermediate activities. With activities higher than 1 U gK1 dry soil, the termite and Martiodrilus structures showed the most significant a-glucosidase activities. In the other biogenic structures, the activities were low (!0.5 U gK1 dry soil) and no activity was measured in the soil (Table 1). The highest b-glucosidase activities were found in the Martiodrilus casts with values higher than 2 U gK1 dry soil. In the other structures the values were close to zero (Table 1). The activities measured for PNP-N-acetyl-glucosamine were relatively low and only found in the Martiodrilus and A. laevigata structures (Table 1). 3.1.3. Phosphatases The acid and alkaline phosphatases activities in A. laevigata pellets were significantly higher than those of the other samples (Table 1). The phosphatase activities measured for the Martiodrilus casts were lower but still very significant.
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In the other samples, the acid phosphatase activities were lower than the activity measured in the soil (Table 1). The soil differed from the other samples in that no alkaline phosphatase activity was detected. 3.1.4. Laccases Martiodrilus had the highest laccase activity, higher than 15 U gK1 dry soil (Table 1). The Andiodrilus and A. landolti structures had activities comparable with that measured for the soil (2 U gK1 dry soil). The lowest laccase activities were found in the Ruptitermes, Spinitermes and A. landolti structures. 3.2. Multivariate analysis A principal component analysis (PCA) was carried out (Fig. 1 A). Axes 1 and 2, respectively, explained 38.3 and 23.3% of the total variance. The significance test carried out on 10,000 permutations showed that the PCA was highly significant (P!0.00001). The samples with high cellulase, b and a-glucosidase, N-acetyl-glucosaminidase, and laccase activities were separated to those with low activities along axis 1. The samples with high b-xylosidase, xylanase,
Fig. 1. Results of the principal components analysis (PCA). (A) Correlation circle. (B) Ordination of the samples in the plane defined by axes 1 and 2 of the PCA.
amylase activities were opposed to those with high phosphatase activities along axis 2 (Fig. 1 A). The position of the samples along the first two PCA axes (Fig. 1 B) showed that the Martiodrilus casts were very clearly different from the other samples. Martiodrilus casts were characterized by a large number of enzymes with activities that were high in comparison with the activities measured in the other samples. A. laevigata pellets were also clearly different from the other samples, mainly because of their phosphatase activities. Despite their differences, the other samples were fairly close to the soil. 3.3. Dendrogram analysis The similarity dendrogram (Fig. 2) shows the hierarchy of the differences observed between the samples. The Martiodrilus casts were very clearly different from all the other structures. In these structures, the enzyme profile showed significant differences in the activities. The A. laevigata pellets also differed from the other structures but, unlike the Martiodrilus casts, the activities were low and only the phosphatase activities were significant. In these structures, there was a significant modification of the enzyme profile in comparison with the soil. The third section separated the fragments of Spinitermes nests from the other samples. This is the result of the relatively high b-xylosidase and xylanase activities. To a lesser extent, the Ruptitermes crop galleries also formed a cluster different from that of the soil. Despite some differences, the Andiodrilus and A. landolti structures were not really distinct from the soil.
Fig. 2. Dendrogram of similarity calculated on the basis of PCA values. MA, Martiodrilus sp.; Alea, A. laevigata; S, Spinitermes sp.; Alan, A. landolti; AN, Andiodrilus sp.; So, Soil; R, Ruptitermes sp.
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4. Discussion 4.1. Earthworm structures Martiodrilus casts showed a different enzyme typology from the other biogenic structures and soil samples. The high enzyme activities measured in these structures suggest that the organic matter was strongly degraded here. On the other hand, the Andiodrilus casts had an enzyme profile close to that of the soil. These differences can be explained by their feeding behavior. The effects of earthworms on soil processes differ between ecological categories and species (Bouche´, 1977). Epigeic species mainly ingest litter and humus and do not mix organic and inorganic matter extensively (McLean and Parkinson, 1998). In contrast, endogeic earthworms live in the mineral soil layers and mainly ingest mineral soil matter. Andiodrilus sp. is an endogeic polyhumic earthworm while Martiodrilus sp. is an anecic earthworm (Jime´nez et al., 1998). Analysis of the intestinal contents of this earthworm showed that Martiodrilus sp. can ingest roots (Mariani et al., 2001). The high proportions of plant fragments in the digestive tract is consistent with the presence in the casts of the broad range of enzymes involved in the degradation of plant matter. Conversely Andiodrilus sp. was characterized by low enzyme activities, for enzymes that are involved in hydrolysis of plant polysaccharides. These activities can be compared with those found in the digestive tracts of three other endogeic earthworms Pontoscolex corethrurus, Polypheretima elongata and Milsonia anomala (Lattaud et al., 1998). This paper showed that endogeic earthworms had polysaccharidase activities which were rather low compared with other invertebrates such as fungus-growing termites and xylophagous termites. Furthermore, the high laccase (a polyphenol oxydase) activity suggested that Andiodrilus sp. ingested organic matter primarily comprising residues such as phenolic compounds. Finally, it seems that the enzyme profile of each earthworm species reflected their feeding behavior: one preferentially ingesting plant matter, the other consuming organic matter mainly comprised of polyhumic substances. The contrasting effects of these two functional groups of earthworms on the incorporation of organic matter into the mineral soil led to the differences between the enzyme activity profiles. 4.2. Termite structures The enzyme typology of the termite biogenic structures was close to that of the soil but there were some significant differences: cellulase, a and b-glucosidase, and alkaline phosphatase were found in these structures but not in the soil samples. Therefore, these termites modified the enzyme profile of the soil by building these structures. Such modifications have been observed in fungus growing termite sheetings (Seuge´, unpublished thesis). Moreover, physical and chemical differences in the walls of termite mounds
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and crop sheetings by comparison with the top soil samples, have been observed by several authors (Brauman, 2000; Fall et al., 2001; Mora and Seuge´, 2003). The similarity analysis (dendrogram) regroups Spinitermes nest fragments in a cluster distinct from that of Ruptitermes crop galleries. There may be two reasons for the differences observed between these two biogenic structures. Firstly, the soil used by the two species is not the same. It is well established that termites use different strategies depending on whether the structure to be built is permanent (a nest) or temporary (crop sheetings and crop galleries) (Jouquet et al., 2002). In this study the orange color of the Ruptitermes galleries indicated that the soil used for construction was a deep soil (Decae¨ns et al., 1999) In contrast Spinitermes sp used the top soil to build their nest. Secondly, the organic characteristics of the soil might have an impact on soil enzyme activities. It is well known that the enzyme activities are greatly affected by organic matter content of the soil (Ladd and Buttler, 1972; Dalal, 1975; Speir et al., 1980; Tabatabai, 1977). Chemical analyses showed that the concentration of organic matter was higher in the Spinitermes sp. nest fragments than in the Ruptitermes sp. crop galleries (Decae¨ns et al., 2001). The differences between the soil layers used and the method of construction could explain the differences in enzyme activity observed between these two biogenic structures. 4.3. Ant structures The enzyme typology of A. landolti pellets was close to that of the soil. According to Decae¨ns et al. (2001) few significant differences were observed when comparing physical (aggregate size and stability, bulk density) and chemical (C, N, P contents, pH, Al saturation) properties of these biogenic structures and the soil. These results suggested that these structures were the result of a simple mechanical movement of the soil when digging the subterranean galleries. A. laevigata structures were characterized by low enzyme activities with the exception of the acid and alkaline phosphatase activities. Phosphatases in soils are derived mainly from the microbial population and have been suggested as a satisfactory indicator of microbial activity (Frankenberger and Dick, 1983; Dick and Tabatabai, 1993). However, the exact origin, quantity and quality of the organic substrates and/ or the stimulation of microbial communities associated with the high levels of alkaline and acid phosphatase activities observed in this study cannot be determined from this study.
5. Conclusion This enzyme typology made it possible to demonstrate the functional diversity of the biogenic structures produced on the soil surface. This study shows three
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groups of structure: structures characterized by a broad enzyme profile (Martiodrilus casts), structures with enzyme profiles similar to the soil (Andiodrilus casts and A. landolti pellets) and those which differ from the soil by the presence of specific enzymes (termite structures and A. laevigata pellets). The production of a diversity of biogenic structures by ecosystem engineers may result in efficient regulation of soil processes through specific enzyme profiles. However, it will be necessary to describe other structures and to assay other enzymes to confirm the role that these biogenic structures play in nutrient cycles in tropical soils.
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