Changes in diversity, protein content, and amino acid composition of earthworms from a paddy soil under different long-term fertilizations in the Tai Lake Region, China

ACTA ECOLOGICA SINICA Volume 26, Issue 6, June 2006 Online English edition of the Chinese language journal Cite this article as: Acta Ecologica Sinica, 2006, 26(6), 1667−1674.

RESEARCH PAPER

Changes in diversity, protein content, and amino acid composition of earthworms from a paddy soil under different long-term fertilizations in the Tai Lake Region, China Xiang Changguo1, 2, Zhang Pingjiu1, Pan Genxing1, *, Qiu Duosheng3, Chu Qiuhua3 1 Institute of Resources, Ecosystem and Environment of Agriculture, Nanjing Agricultural University, Nanjing 210095, China 2 College of Junketing Sciences, Jishou University, Zhangjiajie 217000, China 3 Bureau of Ecological Agriculture of Wujiang Municipality, Suzhou 222300, China

Abstract: Influence of the agricultural management practices on soil quality and the ecosystem functioning has been an increasing concern in soil science and ecology with sustainable agriculture. This study deals with the changes of soil earthworm communityfrom a paddy soil under different long-term fertilizations. The soil earthworms were collected and counted from different fertilizer treated plots in the field after the rape harvest in May 2004, and their taxonomic groups were determined under a binocular stereoscope at the laboratory. The body of the earthworm (Metaphire californica) was crushed by a cell crusher to collect protein, and the protein molecules with different sizes were analyzed by electrophoresis. Furthermore, the Metaphire californica collected was hydrolyzed and the aliquots were subject to an amino acid auto-analyzer. The results showed that totally seven species of earthworms were recognized in the paddy field with the number varying with different fertilization treatments. The structure of earthworm communities was dramatically affected by the fertilization practice. Under chemical fertilization only, both the number of earthworm species and the quantity of individuals were significantly smaller than those under other treatments, or even than those under no fertilization. Furthermore, there was an obvious decrease in the total amino acid and the contents of most individual amino acids of Metaphire californica under chemical fertilization only, compared with those under the combined fertilization of chemical and organic fertilizers. Although chemical fertilizers in combination with rice straw return increased earthworm amino acid content, long-term pig manure application tended to increase earthworm protein content. As a molecular footprint, long-term chemical fertilization caused a reduction in the content of protein with MW less than 25 kd, but a significant increase in that of protein with molecule size around 33 kd. Our study demonstrated that different fertilizations affected not only earthworm population but also diversity and richness in the paddy soil after 16 years of treatment, and that long-term chemical fertilization may impact the soil animal community and, thus, influence the paddy ecosystem functioning for yield stability. This study implicated that not only the community structure but also the amino acid metabolism for life functioning of earthworms in cropland soils may pose significant responses to the agricultural management practices. Key Words: long-term trial; soil earthworm; amino acid and protein; paddy soil; fertilization

1

Introduction

Soil fauna, the main consumers and decomposers of the soil ecosystem, have a significant role in soil quality[1]. As early as 1988, Lal recognized the impact of soil fauna on the soil fertility and functioning of a tropical ecosystem[2]. Recently, in-

creasing interests on soil fauna have been focused on the changes of the community structure associated with ecosystem degradation and restoration[3]. The abundance or activity of soil macrofauna has been considered as an indicator of the biological health of soils[4]. Earthworms being a dominant group of macrofauna in soils of a relatively large size and a

Received date: 2005-04-08; Accepted date 2005-10-27. *Corresponding author. E-mail: [email protected] Copyright © 2006, Ecological Society of China. Published by Elsevier BV. All rights reserved.

XIANG Changguo et al. / Acta Ecologica Sinica, 2006, 26(6): 1667–1674

significant displacement of colonization, changes in the community structure and the diversity of earthworms have been frequently studied for developing bioindicators[5,6] of soil health associated with environmental pollution and ecosystem degradation under impacts of pesticides, heavy metals, and soil － degradation[7 9]. There has been increasing attention paid to the changes in the physiological and biological property of earthworms under environmental stresses. Guo et al. investigated the effect of heavy metal pollution on Pheretime Califonica’s stomach—intestinal mucosa[10] and Xue et al[11]. reported a study on the toxicity of a dimethoate pesticide on earthworms via the respiratory activity. Some recent works have shown the significance in developing bioindicators of the changes in contents and composition of bioactive components such as amino acids and special proteins from earthworms living in the soil body. A study by Hua et al[12]. showed a response of earthworm amino acid composition to spiked rare earth in soils, and the study by Nadeau et al[13]. demonstrated an induction of Hsp-70 in earthworm blood when exposed to organic pollutants and spiked heavy metals (Pb, Cd, Cu, and Hg) in soils, suggesting a possible biomarker for monitoring soil pollution. However, little knowledge is available on the effect of different fertilizations on earthworm populations and on the biomarker of living components. Rice paddies are considered as a unique anthropogenic soil type formed under long-time seasonally submerged hydroagric managements for rice production in China. The changes in the soil quality and the potential effects on environmental feedbacks under intensified farming, targeted for a high yield, have been much concerned with the sustainable development of agriculture[14,15]. In particular, the problem of soil quality decline and the environmental emissions from the paddy soils in the Tai Lake region, China, has raised a great deal of public concern with the over production of rice using increasingly large amounts of chemical fertilizers[16]. The changes in community structure and biodiversity, and the bioactive components of earthworms in relation to soil quality and ecosystem functioning of the paddy soils have not been understood yet, although the changes in the soil microbial community have been studied as a key factor of soil quality[17,18]. In the present study, earthworm community, protein, and amino acid content were analyzed with a focus on the effect of long-term fertilizations on the health of earthworms and a possible link to soil health by taking an example of rice paddy under a long-term

fertilization trial from the Tai Lake region, China. The authors aim at a better awareness of changes in soil quality and biodiversity of paddy soils and the possible link to soil functioning under different farm managements.

2

Materials and methods

2.1 Soil and fertilizer treatments The studied paddy soil (Ferric-accumulic Stagnic Anthrosols[19], which is located in Jinjiaba Township, Wujiang Municipality, Jiangsu Province, China (N: 31°05′900″; E: 120°46′924″), has been cultivated continuously under ricerape rotation under different fertilizer application treatments since 1987. The local climate was humid subtropical with mean annual temperature and precipitation of 22℃ and 1100 mm, respectively. Fertilizer treatments were as follows: no fertilizer application (NF); chemical fertilizer only (CF); chemical fertilizer plus rice straw return (CSF), and chemical fertilizer plus pig manure (CMF). The treatments were performed in a randomized block design and conducted in triplicates. The basic properties of topsoil in 1987 are listed in Table 1 and those under different treatments in 2002 are shown in Table 2. Table 1 Basic properties of the studied soil, sampled and tested in 1987 Depth

SOC

Total N

pH

Clay content

CEC

(cm)

(g/kg)

(g/kg)

(H2O)

(< 2μm, g/kg)

(cmol/kg)

0–5

16.40

1.72

5.60

249.30

20.20

5 – 15

16.00

1.68

6.00

279.70

20.90

2.2 Earthworm collection and identification Field collection of earthworms was conducted in May 2004 when rape was just harvested without rice. In a single plot, three sampling areas of 1 m × 1 m were randomly chosen and earthworms collected and counted from the topsoil at a depth of 0–20 cm. Species identification was done following the procedure described in “Atlas of Fauna of China” by Chen[20]. 2.3 Protein extraction and amino acid determination 2.3.1 Protein extraction and electrophoresis Protein extraction from the body of the earthworms and electrophoresis were performed following the Manual of Practical Molecular Biology Methods[21]. All tests were done under a constant room temperature of 25℃.

Table 2 Fertilizer treatments and basic properties of the treated plots, sampled and measured in 2002[18] Fertilization rate (kgFW/(hm2·a))

Treated plot

Straw

NF

0

Manure

N

0

0

P2O5

Straw

pH

Organic C

Total N

Total P

(H2O)

(g/kg)

(g/kg)

(g/kg)

0

0

6.13

16.54

1.65

0.24

CF

0

0

28.5

3.0

0

5.93

16.85

1.87

0.37

CMF

0

1120

28.5

3.0

0

5.74

17.75

1.91

0.72

CSF

300

0

28.5

3.0

300

5.88

16.79

1.87

0.37

XIANG Changguo et al. / Acta Ecologica Sinica, 2006, 26(6): 1667–1674

Sample pretreatment: Ten mature earthworms of Metaphire californica randomly selected from each plot, were washed, cut, crushed, and mixed. About 2 ml of the crushed fragments were placed in a U tube with sand-sized quartz grains inside. An ultrasonic cell crusher (FP120HY-230) was used to crush earthworm cells at a vibration speed of 6.5 times/s for 20 s for two cycles. The homogenate was centrifuged under 4℃ at 12000 r/min for 15 min. A supernatant of 0.08 ml with equal volume of buffer solution (pH 6.8) was incubated in a metal bath at 100℃ for 5 min for denaturing. Electrophoresis was carried out in glycine solution (volume ratio of glycine to sample=5:1) at 200 V/15mA for 1 h with an electrophoretor (EC250-90, Shanghai, China). The preparation of gels was performed as follows: Preparation of 12% separating gel: water, 1.6 ml; 30% ACR (acrylamide), 2.0ml; 1.5mol/L Tris-HCl (pH 8.8), 1.3 ml; 10% SDS (Sodium dodecyl sulfate or sodium lauryl sulfate), 0.5 ml; TEMED (N,N,N,N'-tetramethylethlenediamine), 0.002 ml. Preparation of 5% stacking gel: water, 0.68 ml; 30% ADR (Adriamycin), 0.17 ml; 1 mol/L Tris-HCl (pH 6.8), 0.13 ml. Mixed and stored. Staining and destaining procedure: the gels were incubated in a staining solution (Coomassie Blue) for 2 h, and then incubated in a destaining solution [TBST(10 mmol/L TRIS, 150 mmol/L NaCl (pH 8.0), and 0.05％ Tween-20)] for 30 min. 2.3.2 Amino acid determination Amino acids were determined following the method used by Ding et al[22]. Ten mature earthworms of Metaphire californica selected randomly from each fertilizer plot were washed, dried at 65℃ for 6 h, weighed, and dried for 3 h more to constant weight. Samples were parched again after grinding. Then 40 mg of dried earthworms were put into a hydrolysis tube in 10 ml HCl (6 mol/L). The tubes were vacuumized and then sealed under alcohol spurt flame. The contents were allowed for acid hydrolysis at 110℃ for 24 h, transferred to a centrifuge tube and then centrifuged at 10000 r/min for 15 min. The supernatant was diluted to 200 ml and the amino acids in the aliquot were determined with an amino acid automatic analyzer(Hitachi 835-50). Temperature of the column was set at 53℃. The testing column was 150 mm in length and 2.6 mm in diameter, and kept under 53℃ constantly during determination. A sulfonated polystyrene resin (＃2619) was used for separating the amino acids. Flow rate of No.1 pump was 0.255 ml/min and pressure at 80–120 kg/cm2, while those of No.2 was 0.3 ml/min and 15–30 kg/cm2, respectively. A reference sample from FLUKA company, USA, was inserted in each patch of the determination for the quality control.

3

Results and analysis

3.1 Earthworm population composition and diversity 3.1.1 Earthworm population composition The composition of the earthworm communities under dif-

ferent fertilizer treatments is shown in Fig. 1. The earthworms recognized, belonged to Megascolecidae, Moniligastridae and Lumbricidae in order, to Amynthas, Microscolex, Metaphire, Eisenia, Drawida, and Allolobophora in genera, and to Amynthas hupeiensis, Microscolex wuxiensis, Metaphire californica, Metaphire guillelmi, Drawida japonica, Allolobophora caliginosa, and Eisenia foetida in species. All the seven species could be found under CFM and CFS, while only three species under CF. Drawida japonica, a general species with feeding pattern of decomposed organic matter, could not been found under NF and CF. Moreover, the rare species of Amynthas hupeiensis, Microscolex wuxiensis and/or Allolobophora caliginosa disappeared under CF. The earthworm population density under CFS and CFM was 17 individuals/m2 and 16.9 individuals/m2, respectively, compared with 13.3 individuals/m2 under NF and only 3 individuals/m2 under CF. Although earthworm population density was small under NF, both earthworm populations reduced and some sensitive species vanished under CF.

Fig. 1 Earthworm population and species under different fertilizations

3.1.2 Earthworm diversity index and species richness Soil fauna diversity and species richness indicate good variability of soil fauna at the community level. The soil fauna abundance index d and Shannon diversity index H can be calculated by the following equations: (1) d = ( s − 1) / ln N Where s is the gross number of the community and N is the gross number of the individuals. s

H = −∑ Pi ln Pi

(2)

i =1

Where Pi = Ni/N, N is the gross quantity of individuals and Ni is the number of the individuals of the i community. Earthworm diversity and species richness are shown in Fig. 2. As shown by the Shannon index, the diversity of earthworms turned to be slightly higher under CFS and CFM than that under NF. However, the lowest diversity was observed under CF. A similar trend occurred for variation of the richness indicator with the treatments. No fertilization affected the diver-

XIANG Changguo et al. / Acta Ecologica Sinica, 2006, 26(6): 1667–1674

Table 3 Composition (%) of amino acids of Metaphire californica

Fig. 2 Earthworm diversity and species richness

sity but only the richness of earthworms, while chemical fertilization alone reduced both the diversity and species richness of the earthworm community. 3.2 Amino acid content of Metaphire californica Seventeen species of amino acids were obtained from the earthworm body of Metaphire californica by acid hydrolysis, and their composition is listed in Table 3. The total amount of amino acids from the different plots decreased in order of CFS(26.68%) ＞ NF(24.65%) ＞ CFM(23.89%) ＞CF(22.47%) although the variation of a single acid with the treatments was not consistent. However, the contents of most amino acids were higher in the earthworms from the plots CFS, NF, and CFM than those from CF, independent of the soil N content. 3.3 Content of protein and the molecular size of Metaphire californica Metaphire californica, is a common species in paddy soils, was dominant in the total abundance of earthworms from the treated plots. The molecular size of proteins from Metaphire californica ranged from 7 kd to 175 kd (Fig. 3). Obviously, the fertilizer treatments affected the content of proteins in different molecular sizes of the Metaphire californica bodies. Compared with the earthworms under NF and CF, the earthworms under CFS and CFM are abundant in proteins in molecular sizes ranging from 7 kd to 25 kd. As for the proteins in sizes ranging from 25 kd to 33 kd, the NF plot was basically of lower molecular size and the CF plot was of larger size of 33 kd, while the CFS and CFM plots of an even distribution of protein molecular sizes. As shown in Fig. 3, the earthworms from CFS and CFM exhibited two bright protein bands in molecular size between 33 kd and 83 kd, which disappeared under CF. This implied an effect of different fertilization treatments on the protein composition of earthworm Metaphire californica. Long-term chemical fertilization increased the protein amount to 33 kd in size, while conjunct fertilization of inorganic fertilizers with organic fertilizers tended to enhance the proteins in size between 25 kd and 33 kd.

Amino acid

CF

CFM

NF

Plenylalanine

1.05

1.12

1.14

CFS 1.23

Alanine

1.35

1.35

1.40

1.59

Methionine

1.18

1.34

1.52

1.33

Proline

0.89

0.90

0.85

1.04

Glycine

1.21

1.25

1.35

1.47

Glutamic acid

2.99

3.18

3.24

3.75

Cystine

0.86

0.77

0.88

0.78

Arginine

1.43

1.60

1.69

1.85

Lysine

1.52

1.62

1.67

1.84

Tyrosine

0.88

1.00

1.04

0.92

Leucine

1.71

1.83

1.89

2.16

Serine

1.06

1.12

1.16

1.27

Threoine

1.03

1.10

1.12

1.21 2.68

Aspartic acid

2.21

2.37

2.41

Valine

1.36

1.42

1.49

1.56

Isoleucine

1.19

1.36

1.39

1.44

Histidine Total amount

0.56

0.57

0.41

0.56

22.47

23.89

24.65

26.68

Fig. 3 Metaphire californica protein profile

4

Discussions

4.1 Earthworm population and paddy productivity It has been well known that fertilization practices could result in changes of soil fauna population composition. In this study, not only abundance but also species of earthworms were lower under chemical fertilization only than those under no fertilization. However, conjunct fertilization of inorganic with organic fertilizers increased the abundance of earthworms without significant effect on the species of earthworms.

XIANG Changguo et al. / Acta Ecologica Sinica, 2006, 26(6): 1667–1674

In a previous study, although no significant difference in microbial biomass C was found between earthworms under no fertilization treatment and under chemical fertilization treatment, gene diversity was higher under no fertilization than under chemical fertilization. Of course, gene diversity was highest under conjunct fertilization[18]. Therefore, soil macrofauna seemed insensitive to fertilization compared with microbes. In general, the change of earthworm abundance and population under different fertilization practices was attributed to the variation of soil nutrient pools. Qiao et al. suggested that the rapid rise of inorganic nutrients under sole application of chemical fertilizers was not beneficial for earthworm living and competition in the low production of the agro-ecosystem of North China[23]. Abundance of earthworms was reduced because of limited access to N nutrient input compared with the microbial organisms under acid deposition. Earthworm species and diversity could be further constrained by decrease of organic matter due to declined vegetation under acid deposition[25]. The deterioration of physical and chemical properties of soils under long-term chemical fertilization could be the main reason for the decline of earthworm population in cropland soils[8]. In the present study, without any fertilization treatment, plots sustained higher abundance of earthworms than those under fertilization treatments, indicating that there

were no constraints of organic matter input on earthworm abundance. However, conjunct application of inorganic fertilizers with organic fertilizers enhanced earthworm abundance possibly because of increased organic input by way of straw or pig manure. Amendments to the earthworm diversity index were observed to be significantly correlated with the increment of soil organic matter and the increment of total nitrogen (Fig. 4). Therefore, a change in the quality of the soil organic matter under different fertilization treatments could be the key factor affecting the competing and living of earthworms. The present study also indicates that paddy soils receiving combined fertilization of both inorganic and organic fertilizers sustains a high soil quality. 4.2 Earthworm diversity and crop productivity The primary production and the stability plays the key role in agroecosystem health. The yearly variation of rice yield of the different plots is shown in Fig. 5. Clearly, plots with conjunct application of inorganic and organic fertilizers sustained not only high grain yield but also the lowest yield variability, showing a high and stable crop productivity coincident with high earthworm diversity as shown by the good correlation of earthworm diversity index and grain yield variation coefficient (Fig. 6). Thus, an earthworm community of higher diversity facilitated a higher and more stable production of rice of the paddy. Mäder et al. stated that soil fauna diversity has been

Fig. 4 Correlation of earthworm diversity with soil organic C (a) and soil total N (b) of the studied plots

Fig. 5 Grain yield variability of treated plots for the last six years

Fig. 6 Correlation of earthworm diversity to

(▲, NF; ◇, CF; □, CFS; ■, CMS)

grain yield variability

XIANG Changguo et al. / Acta Ecologica Sinica, 2006, 26(6): 1667–1674

shown as a definite indicator of the stability of agroecosystem under organic farming[26]. In a previous study with the same experiment plots, good correlation between soil microbial gene diversity and stability of rice production was also found[18]. The results obtained from this study could support that, as an important component of soil biota, earthworms could have played an indispensable role in mediating some biophysical processes for the primary production in this agrosystem of paddy. Therefore, the paddy soils receiving combined fertilization of both inorganic and organic fertilizers could sustain and promote high soil biodiversity, which in turn was crucial for sustaining a healthy agroecosystem. 4.3 Earthworm protein and amino acid contents as indicators of paddy agroecosystem. Increasing studies have been focused on effect of soil contamination on the health of the soil ecosystem using soil vertebrate as a bioindicator[27,28]. Protein Hsp from earthworms or other organisms induced under pollution stress was explored as a biomarker of soil ecosystem, especially a particular species, Hsp-70, was proposed for soil and water environment[13,29]. In the present study, Hsp-70 could not been detected in earthworms from any of the treated plots. The failure of Hsp-70 protein to respond to fertilization could be accounted for its sensitivity to the short-term spike of metal pollutants. The paddy has been treated by different fertilizations for more than a decade and the earthworms should be accustomed to a certain fertilization plot. From Fig. 3 and Table 3, a remarkable decrease in content of a single amino acid of earthworms and a sharp increase of proteins with a size of 33 kd was observed in earthworms from the plot with long-term chemical fertilization; whereas, there was no correlation of the amino acid content of earthworms with soil N content of the different plots. It seemed that the amino acid composition was not preferably affected by soil N, but by the metabolism of the soil N in its body in response to soil fertilization practice. The real mechanism of the variation of amino acid and protein synthesis behind different fertilizations deserves further study for developing biochemical indicators for paddy health.

5

Conclusions

(1) Totally four orders, five genera, and seven species of earthworms were identified in a paddy soil under different fertilization treatments in Tai Lake region, China. Although the number of earthworm individuals increased, the species declined under a long-term of no fertilization. Both number of individuals and number of species were observed to decrease under long-term chemical fertilization compared with those under conjunct application of inorganic and organic fertilizers; (2) Both the abundance and the diversity of earthworm communities were affected by different long-term fertilizations. Although only the abundance declined under no fertilization,

the diversity and abundance of earthworms decreased under chemical fertilization compared with those under conjunct application of organic and inorganic fertilizers. Furthermore, the earthworm diversity was found to correlate with the rice productivity of the paddy. (3) The protein and amino acid composition of earthworms also seemed to change in response to different fertilizations. Although the content of total amino acids of earthworms decreased, content of protein decreased in size of <25 kd and increased in size of around 33 kd under chemical fertilization compared with that under the conjunct fertilization. However, why the N metabolism and protein synthesis changed under chemical fertilization is still unclear.

Acknowledgement This project was partially funded by the National Natural Science Foundation of China (No. 40231016).

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