# Effects of Different Earthworms on Calcium Speciation and Base Cation Release in Terra Rossa Soil: A Case Study from South China

Jialong Wu, Chi Zhang, Menghao Zhang, Ting Deng, Mikael Motelica-Heino, Hesen Zhong, Christian Défarge, Yingmei Huang, Changchao Xu, Juntao Zhang

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Jialong Wu, Chi Zhang, Menghao Zhang, Ting Deng, Mikael Motelica-Heino, et al.. Effects of Different Earthworms on Calcium Speciation and Base Cation Release in Terra Rossa Soil: A Case Study from South China. Forests, 2025, 16 (2), pp.312. 10.3390/f16020312. insu-04991657

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# Effects of Different Earthworms on Calcium Speciation and Base Cation Release in Terra Rossa Soil: A Case Study from South China

Jialong Wu<sup>1,2,3</sup>, Chi Zhang<sup>4,5,6,7</sup>①, Menghao Zhang<sup>4,5,6,7</sup>, Ting Deng<sup>8</sup>, Mikael Motelica-Heino<sup>9</sup>, Hesen Zhong<sup>4,5,6,7</sup>, Christian Défarge<sup>9</sup>, Yingmei Huang<sup>1,2,3</sup>, Changchao Xu<sup>1,2,3</sup>① and Juntao Zhang<sup>1,2,3,*</sup>

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Academic Editor: Xiankai Lu

Received: 20 November 2024

Revised: 10 December 2024

Accepted: 28 December 2024

Published: 11 February 2025

**Citation:** Wu, J.; Zhang, C.; Zhang, M.; Deng, T.; Motelica-Heino, M.; Zhong, H.; Défarge, C.; Huang, Y.; Xu, C.; Zhang, J. Effects of Different Earthworms on Calcium Speciation and Base Cation Release in Terra Rossa Soil: A Case Study from South China. *Forests* **2025**, *16*, 312. https://doi.org/10.3390/f16020312

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**Abstract:** Soil calcium is a vital component in plant growth and soil health. Earthworm activities impact metal distribution and speciation a lot by changing soil pH. Nevertheless, little is known about how ecological earthworm species, particularly in Terra Rossa soil, affect soil Ca speciation distribution. This research examined the effects of the activities of four different earthworm species (epigeic species *Eisenia fetida* (noted as EF), endogeic species *Amynthas robustus* (noted as AR) and *Pontoscolex corethrurus* (noted as PC), anecic species *Amynthas aspergillum* (noted as AA)) on Ca speciations (water-soluble ($Ca_{WS}$), exchangeable ($Ca_{Ex}$), acid-soluble bound ($Ca_{Ac}$), organic-bound ($Ca_{Or}$), and residual ($Ca_{Re}$)), soil pH, the release contents of exchangeable cations (Ca, Mg, K, and Na), total calcium ($Ca_{Total}$) contents, total nitrogen (TN) contents, soil organic carbon (SOC) concentrations, cation-exchange capacity (CEC), and NIRS spectral characteristics in Terra Rossa soil for 40 days under lab conditions. In contrast to control soil, 108.3%, 158.3%, 91.7%, and 125.0% of $Ca_{WS}$ contents in casts and 116.6%, 108.3%, 58.3% and 91.6% of $Ca_{WS}$ in uningested soil increased significantly with the inoculation of EF, PC, AR, and AA, respectively. In addition, compared with control, for casts, the contents of exchangeable Ca, Mg, K, CEC, and available-K were significantly increased in the presence of EF, PC, AR, and AA, respectively. In the casts of EF, PC, AR, and AA, soil pH values declined by 0.72, 0.80, 0.45, and 0.60 units relative to control soil, while they decreased by 0.65, 0.84, 0.34, and 0.59 units in uningested soil. The soil inoculated with PC had higher soil pH values and $Ca_{WS}$ contents than those with the other three earthworm species. Principal component analysis revealed significant differences in soil pH, Ca speciation, NIR spectra, and exchangeable base cation release between casts and uningested soil in treatments with EF, PC, AR, and AA inoculation. These findings expand, for the first time, to the ecological functions of earthworm species, especially for PC, demonstrating a capacity to alter soil Ca

<footnote n="1">1 Guangzhou Institute of Forestry and Landscape Architecture, Guangzhou 510405, China; jialw07@163.com (J.W.)</footnote>

<footnote n="2">2 Guangdong Guangzhou National Urban Forestry Science and Technology Demonstration Park, Guangzhou 510405, China</footnote>

<footnote n="3">3 Guangzhou Collaborative Innovation Center on Science-Tech of Ecology and Landscape, Guangzhou 510405, China</footnote>

<footnote n="4">4 College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China</footnote>

<footnote n="5">5 Key Laboratory of Arable Land Conservation in South China, Ministry of Agriculture, Guangzhou 510642, China</footnote>

<footnote n="6">6 Key Laboratory of the Ministry of Land and Resources for Construction Land Transformation, Guangzhou 510640, China</footnote>

<footnote n="7">7 Guangdong Province Key Laboratory of Land Use and Consolidation, Guangzhou 510140, China</footnote>

<footnote n="8">8 Agricultural Environment and Cultivated Land Quality Protection Center of Guangdong Province, Guangzhou 510500, China</footnote>

<footnote n="9">9 Institut des Sciences de la Terre d'Orléans, UMR 7327 Université d'Orléans—CNRS—BRGM, 45071 Orléans, France; christian.defarge@univ-orleans.fr (C.D.)</footnote>

<footnote>* Correspondence: juntaozhang2024@163.com; Tel.: +86-(20)-3650-1075 or +86-137-9816-6827</footnote>
speciation, decrease soil pH, affect the exchangeable base cations' release, and participate in and regulate the geochemical circulation processes in limestone regions.

**Keywords:** earthworm; soil calcium speciation distribution; pH value; exchangeable base cations; Terra Rossa

# 1. Introduction

Calcium (Ca) is an essential element for plant productivity. Its migration and transformation have a significant impact on the formation of good soil structures, increasing SOC stocks and ecosystem functioning [1-4]. This is directly related to plant growth and the sustainability of ecosystems. Generally, in soils, Ca can be separated into several components, such as $Ca_{Ws}$ (water-soluble), $Ca_{Ex}$ (exchangeable), $Ca_{Ac}$ (acid-soluble), $Ca_{Or}$ (organically bound), and $Ca_{Re}$ (residual) [5]. Numerous elements, such as parent rocks, climate, acid rain, organic matter, biology, etc., affect the migration and change between these forms [6]. By using European Community Bureau of Reference (BCR) methodologies, Yang et al. [7] investigated the seasonal migration features and soil Ca speciation of calcareous soil in karst regions. Although calcareous or calcium-rich soil areas account for more than 30% of the land area, the role of soil fauna in the migration and transformation of Ca elements in calcareous soil remains unclear, and previous research has mainly focused on karst and Mediterranean areas [2,8]. Terra Rossa soil is a common calcareous soil found in subtropical regions, and its soil calcium has always been neglected. Currently, there is no information available about how soil fauna affects soil Ca speciation distribution and transformation in Terra Rossa soil.

Earthworms are an essential component of the soil fauna [9] and are crucial to the movement and changes in the chemical elements in soil, such as aluminum (Al), zinc (Zn), copper (Cu), cadmium (Cd), lead (Pb), and so forth [10–13]. Wu JL et al. [10] demonstrated that the gut processes of earthworm *Pontoscolex corethrurus* (noted as PC), *Amynthas robustus* (noted as AR), and *Amynthas aspergillum* (noted as AA) significantly increased exchangeable Ca, Mg, and K content of lateritic red soil. García-Montero LG et al. [14] evaluated the effect of *Prosello-drilus* sp. casts on calcium carbonate contents and soil pH in black truffle burns, and their findings demonstrated that, compared to control soils, earthworm casts dramatically increased soil pH value by 0.2 units and total calcium carbonate by 66%. Nevertheless, information about how these earthworm species' ecosystems impact soil Ca speciation in calcareous soils is lacking. It was previously reported that *Eisenia fetida* (noted as EF), endogeic species PC, and AR and anecics species AA are common earthworm species in south China [10,15]. However, there are no data on how theses ecological earthworm species affect soil Ca speciation in subtropical calcareous soil.

In the present study, we aim to investigate the effects of four earthworm species, including EF, PC, AR, and AA, on soil Ca fractions and exchangeable base cation release, and evaluate the impacts of earthworm presence and activity on Ca speciation and transformation in tropical soils. We hypothesize that (1) earthworm activity can affect the distribution of soil Ca speciation and the release of base cations, and that this is related to the types of earthworm activity (ingestion and mucus secretion). (2) The distribution of soil Ca speciation and the release of base cations are species-dependent.
## 2. Materials and Methods

### 2.1. Preparation of Soil and Biology

Natural uncontaminated Terra Rossa soil was collected from the surface horizon (0–10 cm) of a subtropical evergreen broad-leaved forest in Yingde, Qingyuan city, northern Guangdong province, China (24°18′21″ N, 113°22′50″ E). After air drying, it was sieved at a 3 mm mesh. The soil's basic properties are shown in Table 1.

<table><caption>**Table 1.** Main physico-chemical characteristics of soil (mean ± S.E).</caption><thead><tr><th rowspan="2">pH</th><th rowspan="2">SOC</th><th rowspan="2">Total N</th><th rowspan="2">C:N Ratio</th><th rowspan="2">Clay (%)</th><th colspan="2">Cation-Exchange Capacity (CEC)</th><th colspan="2">Exchangeable K</th><th colspan="2">Exchangeable Na</th><th colspan="2">Exchangeable Ca</th><th colspan="2">Exchangeable Mg</th><th rowspan="2">Total Ca</th><th rowspan="2">Total P</th><th rowspan="2">Total K</th><th rowspan="2">Hydrolyzable N</th><th rowspan="2">Available P</th><th rowspan="2">Available K</th></tr><tr><th>cmol·kg<sup>-1</sup></th><th>mg·kg<sup>-1</sup></th><th>g·kg<sup>-1</sup></th><th>mg·kg<sup>-1</sup></th><th>g·kg<sup>-1</sup></th><th>mg·kg<sup>-1</sup></th><th>g·kg<sup>-1</sup></th><th>mg·kg<sup>-1</sup></th><th>g·kg<sup>-1</sup></th><th>mg·kg<sup>-1</sup></th></tr></thead><tbody><tr><td>(H<sub>2</sub>O)</td><td>g·kg<sup>-1</sup></td><td>g·kg<sup>-1</sup></td><td></td><td></td><td>13.28</td><td>94.53</td><td>38.56</td><td>1.68</td><td>156.31</td><td>2.40</td><td>0.43</td><td>22.02</td><td>184.34</td><td>1.86</td><td>97.73</td><td></td><td></td><td></td><td></td></tr><tr><td>6.65 ± 0.03</td><td>21.94 ± 0.26</td><td>2.92 ± 0.03</td><td>7.52 ± 0.14</td><td>32.1 ± 2.08</td><td>0.18 ± 0.18</td><td>± 3.30</td><td>± 4.24</td><td>± 0.02</td><td>± 6.43</td><td>± 0.08</td><td>± 0.01</td><td>± 2.74</td><td>± 9.30</td><td>± 0.16</td><td>± 2.25</td><td></td><td></td><td></td><td></td></tr></tbody></table>

Apart from the fact that *Eisenia fetida* (noted as EF) was purchased from a farm in Qingyuan city, the same location's three earthworm species, *Pontoscolex corethrurus* (noted as PC), *Amynthas robustus* (noted as AR), and *Amynthas aspergillum* (noted as AA) were hand-sorted in the field where soil was collected. The candidates for the laboratory testing were mature earthworms with a developed clitellum. Reference was made to previous studies to determine the amount of earthworm inoculum in the soil [10,12]. The weights of EF were 1.59 ± 0.05 g (average fresh weight, 1.39 to 1.75 g ind⁻¹), and 29 individuals were introduced to 0.5 kg of soil. Then, 0.5 kg of soil was supplemented with 41 PC individuals, with their fresh weights averaging 0.29 ± 0.01 g (in the range of 0.27 to 0.31 g ind⁻¹). We gave four worms 0.5 kg of soil, and the fresh weight range of AR was 2.10 to 3.17 g, with an average of 2.91 ± 0.09 g ind⁻¹. Six AA individuals were introduced to 0.5 kg of soil, with an average fresh weight of 2.34 ± 0.07 g (range from 1.10 to 4.07 g ind⁻¹).

### 2.2. Experimental Design

We designed five treatments using 1.5 L plastic plots of 9 cm by 12 cm by 13 cm, and placed 0.5 kg of soil inside.

1. CS: no earthworms;

2. ES: 12.0 g of EF was added;

3. PS: 12.0 g of PC was added;

4. RS: 12.5 g of AR was added;

5. AS: 12.1 g of AA was added.

Every treatment, consisting of five replicates, was incubated in the lab for forty days at a constant temperature condition of 25 °C. Every two days, the moisture was adjusted using a weighing method. In order to avoid affecting the qualities of the soil, throughout the entire culture period, no organic materials were added. Surface castings were collected from the incubation period. Following an incubation period of 40 days, Zhang C et al. [12] methodically collected both internal and surface casts. The total casts in this study comprised a mixture of the surface and internal casts, which were obtained following a drying process in an air-circulating atmosphere. Weighing the uningested soil and casts allowed us to measure the overall amount of cast production [16].

### 2.3. Earthworm Responses

At the start of the culture phase, after 20 days, and at its conclusion, earthworms that were still alive were gathered, numbered, and weighed. The ingestion rate (I, represented
in grams of air-dried soil consumed per gram of fresh weight of earthworm) could be calculated using the following formula:

$$I = \frac{(C/d)}{(W_0 + W_{40})/2} \times 100\% \qquad (1)$$

where *d* is the culture time in days, *W*₀ and *W*₄₀ are the earthworm fresh weight at the start and end of the whole culture period, and the total cast production, expressed in grams of air-dried weight (internal + surface cast), is C [10,12,17].

## 2.4. Soil Analyses

A water suspension (1: 2.5 (w/v) soil/distilled water ratio) was used to measure the pH of the soil [18]. The saturation method of CH₃COONH₄ (pH 7.0, 1 mol·L⁻¹) was used to calculate the CEC. The potassium dichromate titrimetric method was used to determine SOC content. Soil total calcium content was assessed using the heated digestion of HClO₄-H₂SO₄ and aqua regia perchloric acid. This was followed by measurements using Atomic Absorption Spectrometry (AAS). Soil Ca speciation was determined by an optimized European Community Bureau of Reference (BCR) sequential extraction. To review this, refer to the method of Yang H et al. [7] and Hu LN et al. [19]. Soil Ca speciation was classified as follows: water-soluble (CaWS), exchangeable (CaEx), acid-soluble (CaAc), organically bound (CaOr), and residual (CaRe). These are shown in Table 2. All Ca concentrations in the extracts were also determined by AAS (HITACHI ZA3300, Hitachi Ltd., Tokyo, Japan). Following a previously published protocol [20], exchangeable Ca, Mg, K, and Na in soil were extracted using ammonium acetate (pH 7.0, 1 mol·L⁻¹) and then determined by AAS as well. We use dry weight bases for all following data expressions.

<table><caption>**Table 2.** Method for soil calcium speciation.</caption><thead><tr><th>Step</th><th>Abbreviation</th><th>Ca Speciation</th><th>Chemical Reagents and Analytical Conditions</th></tr></thead><tbody><tr><td>1</td><td>CaWS</td><td>Water-soluble</td><td>Redistilled H<sub>2</sub>O (w/v = 1/10) for 2 h, centrifugation for 30 min at 3000 rpm</td></tr><tr><td>2</td><td>CaEx</td><td>Exchangeable</td><td>CH<sub>3</sub>COONH<sub>4</sub> (1 mol·L<sup>-1</sup>, w/v = 1.5/10) for 2 h, centrifugation for 30 min at 3000 rpm</td></tr><tr><td>3</td><td>CaAc</td><td>Acid-soluble</td><td>HCl (3 mol·L<sup>-1</sup>, w/v = 1.5/10) for 2 h, centrifugation for 30 min at 3000 rpm</td></tr><tr><td>4</td><td>CaOr</td><td>Organic-bound</td><td>Na<sub>2</sub>SO<sub>4</sub> (0.5 mol·L<sup>-1</sup>, w/v = 3/10) for 2 h, centrifugation for 30 min at 3000 rpm</td></tr><tr><td>-</td><td>CaRe<sup>#</sup></td><td>Residual</td><td>Ca<sub>Total</sub> - &Sigma;Ca</td></tr><tr><td>-</td><td>CaTotal</td><td>Total Ca</td><td>Decomposed by a heated combination of HF, HNO<sub>3</sub>, HClO<sub>4</sub> and H<sub>2</sub>O<sub>2</sub>.</td></tr></tbody></table>

For NIRS analysis, we manually crushed and sieved 20 g of each sample (less than 2 mm). An NIR FOSS DS2500 spectrometer (FOSS Ltd., Seattle, WA, USA) with a 1100–2440 nm spectral range was used to scan them. Every sample was placed inside a quartz glass-covered cell and covered, and the spectrometer was used to measure the reflected light. At 2 nm sampling intervals, spectra showed the reflectance of reemitted light as a function of wavelength. Between 1120 and 2480 nm, the relative reflectance in wavelengths was measured at regular intervals of 20 nm. The following formula was used to convert reflectance (R) into absorbance (A): A = log (1/R). The ISI software version 1.50e was used to analyze the data first, and then the data were converted into second derivatives [21]. The permutation test was employed to compare the groups. Casts of earthworms, uningested soil, and control soil were the nine treatments × four replicates × three that made up the data matrix that was analyzed in this case. The absorbance values for 70 wavelengths were the variables.

<footnote># most soils include multilayer silicates that occlude calcium, making it impossible to remove with the chemical reagents mentioned above; CaRe = CaTotal - &Sigma;Ca, &Sigma;Ca = CaWS + CaEx + CaAc + CaOr [5].</footnote>
Among other uses, the current technique known as laser diffraction analysis (LDA) can be used to detect the texture of soil. The soil sample's particle size distribution is measured using LDA [22]. A dispersion unit, a measuring cell, and an optical unit make up the Malvern Instruments laser analyzer LS-909 (Zhuhai OMEC Instrument Co., Ltd., Zhuhai, China). It is a wet dispersion device filled with distilled water. The range of measurements was between 0.01 and 2100 µm. The LDA samples were sieved with a mesh size of 2 mm. About 20 g was measured from this quantity; then, quartering was performed. The identical, chemical-free procedures were used to prepare each sample. For practical reasons, samples were dried at different temperatures (some at room temperature, others at 105 °C), but all were completely dry when the measurement was taken. Dry soil is best sieved through a sieve with small pores.

## 2.5. *Statistics Analysis*

R version 3.0.2 (ADE 4 library) and SPSS statistics 23 (SPSS Inc., Chicago, IL, USA) were the software programs used to analyze all of the data. One-way ANOVA was used to examine the variations in means. This was evaluated using Duncan's test. Statistical significance was defined as a *p*-value of less than 0.05. The results were presented as mean ± standard error. Multivariate analyses, including principal component analysis (noted as PCA) [23,24] and correlation analysis, were performed using ADE4 package and Mantel test in R, respectively.

# 3. Results

## 3.1. *Reaction of Earthworms to Soil*

Table 3 displayed the biomass, weight loss rate, and earthworm survival percentage for each treatment. When the experiment was over, *Amynthas aspergillum* (noted as AA) had the highest survival percentage (97.5%); the survival percentage of *Eisenia fetida* (noted as EF), *Pontoscolex corethrurus* (noted as PC) and *Amynthas robustus* (noted as AR) were, however, significantly lower than those seen at the beginning of the experiment (*p* < 0.05). PC had the highest biomass (10.1 g) and lowest weight loss rate, although the biomass was significantly lower than at the beginning of the experiment (*p* < 0.05). Significantly, the surface cast production levels of PC (71.9 g·kg⁻¹) and AR (69.6 g·kg⁻¹) were higher than those of earthworm AA (46.1 g·kg⁻¹) and EF (5.0 g·kg⁻¹) (*p* < 0.05, Figure 1a). Significantly, the four earthworms' intercast productions levels were, notably ranked, as PC > AA ≈ AR > EF, with the average values of 509.6, 86.8, 84.0 and 50.1 g·kg⁻¹, respectively (Figure 1b). Inter-cast production levels of EF, PC, AA and AR were 9.0, 6.1, 0.9 and 0.2 folds, which were significantly higher than those of the surface (*p* < 0.05), respectively. In general, the total cast production was 10.55, 3.78 and 4.38 folds higher, respectively, for PC and AR, AA and EF (*p* < 0.05, Figure 1c). The soil ingestion rate was 7.3, 3.1 and 2.8 folds higher, respectively, for PC than AR, AA, and EF (*p* < 0.05, Figure 1d).

<table><caption>**Table 3.** Variations in survival and growth of earthworms in treatments.</caption><thead><tr><th rowspan="3">Earthworms</th><th colspan="6">Incubation Days</th></tr><tr><th colspan="2">0 d</th><th colspan="2">20 d</th><th colspan="2">40 d</th></tr><tr><th>Survival Percentage (%)</th><th>Biomass (g)</th><th>Survival Percentage (%)</th><th>Biomass (g)</th><th>Survival Percentage (%)</th><th>Biomass (g)</th><th>Weight Loss Rate (%)</th></tr></thead><tbody><tr><td>EF</td><td>100 ± 0.00 a</td><td>12.0 ± 0.01 a</td><td>75.2 ± 4.52 b</td><td>7.64 ± 0.33 b</td><td>65.5 ± 5.24 b</td><td>4.68 ± 0.35 c</td><td>61.04 ± 2.92 A</td></tr><tr><td>PC</td><td>100 ± 0.00 a</td><td>12.0 ± 0.02 b</td><td>94.3 ± 3.26 ab</td><td>13.0 ± 0.47 a</td><td>88.2 ± 2.21 b</td><td>10.1 ± 0.35 c</td><td>15.87 ± 2.89 B</td></tr><tr><td>AR</td><td>100 ± 0.00 a</td><td>12.2 ± 0.04 a</td><td>100 ± 0.00 a</td><td>14.49 ± 0.42 a</td><td>56.0 ± 10.42 b</td><td>7.06 ± 1.68 b</td><td>53.85 ± 9.38 A</td></tr><tr><td>AA</td><td>100 ± 0.00 a</td><td>12.0 ± 0.02 a</td><td>100 ± 0.00 a</td><td>10.5 ± 1.11 a</td><td>97.5 ± 2.50 a</td><td>7.55 ± 0.75 b</td><td>37.12 ± 6.28 AB</td></tr></tbody></table>

EF: *E. fetida*; PC: *P. corethrurus*; AR: *A. robustus*; AA: *A. aspergillum*. Different lowercase letters indicated that there were significant differences in the survival percentage and biomass of earthworms at different cultivation days. Different capital letters indicate that there were significant differences in the weight loss rates of four earthworms at the end of the cultivation period (*p* < 0.05).
<figure><img src="image_3.png" /><figcaption>**Figure 1.** ES: soil + *E. fetida*; PS: soil + *P. corethrurus*; RS: soil + *A. robustus*; AS: soil + *A. aspergillum*. (a) Surface casts production; (b) internal casts production; (c) total casts production; (d) ingestion rate. n = 5. Cast production and ingestion rate for different earthworms. Different lowercase letters indicate that there were significant differences in the cast production and ingestion rates of four earthworms (n = 5, *p* < 0.05).</figcaption></figure>

## 3.2. Differences in Soil pH Between Treatments

The cast and uningested soil pH values were considerably reduced (*p* < 0.05, Figure 2) upon the addition of the four species of earthworms. In contrast to the control treatment, EF, PC, AR and AA decreased pH values by 0.72, 0.80, 0.45 and 0.60 units, respectively, in casts (Figure 2). The uningested soil pH values were 0.65, 0.84, 0.34 and 0.59 lower, respectively, in uningested soil with four earthworms compared to the control soil. Moreover, casts of EF and AR had significantly lower pH values than the corresponding uningested soil, respectively, in casts with PC than in uningested soil (*p* < 0.01, Figure 2).

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Soil pH values in soils affected by different earthworms. Different capital letters indicated that there were significant differences in the cast group. Different lowercase letters indicated that there were significant differences in the uningested soil group (n = 5, *p* < 0.05). (**) *p* < 0.01 relate to the significant differences between casts and the uningested soil group of the *T* test, respectively.</figcaption></figure>

## 3.3. Variation in Particle Size and Distribution in Treatments

The particle distribution in casts and the uningested soil of the four species earthworm varied notably among treatments (Refer to Figure S1a,b). Compared to control soil, EF and PC
increased the peak value of particle distribution by 4.69 and 1.44 units, respectively, in casts. For EF, PC, AR and AA earthworms, it was also noted that the particle size (1200 nm, 1518 nm, 1200 nm and 1200 nm, respectively) corresponded to the peak value of particle distribution in casts, which was higher than that in uningested soil (all 469 nm) and control soil (750 nm).

### 3.4. Variation in NIRS Spectra in Casts and Uningested Soils

Samples of casts and uningested soil were significantly different according to the PCA of 70 variables defining the NIRS spectra. However, all treatments were confused along axis 1 (Figure 3). Factor 1 (Ax1: 48.6%) represents the casts that were separated clearly from uningested soils according to the species of earthworm. Samples from treatments with the casts of EF had high reflectance at wavelengths, such as 1220, 1300–1340, 1400, 1480, 1700, 1800–1820, 1880, 1900–1920, 2140–2200, 2400–2480, and 2260–2340, whereas treatments with the uningested soil of all earthworms had high reflectance values at wavelengths of 1100, 1420, 1460, 1940–1980, 2000–2020, 2200, and 2360 on the Ax2.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** PCA analysis of NIRS spectra in casts and uningested soils. (a) Projection of NIR spectra in soils; (b) sore plots in soils. n = 4.</figcaption></figure>

### 3.5. Ca Speciation Variation and Treatment Distribution

In both the uningested soil and the casts, the Ca speciation determined by chemical extractions showed considerable variation between treatments (Table 4). In contrast to the control, 110% (0.25 g·kg⁻¹), 115% (0.31 g·kg⁻¹), 96.0% (0.23 g·kg⁻¹), and 129% (0.27 g·kg⁻¹) increases were observed in Ca<sub>Ws</sub> contents in the casts of ES, PS, RS and AS, respectively, and 119% (0.26 g·kg⁻¹), 109% (0.25 g·kg⁻¹), 59.7% (0.19 g·kg⁻¹) and 96.0% (0.23 g·kg⁻¹) increases were found in uningested soil as well. The Ca<sub>Ws</sub> contents in casts of PC, AR, and AA were significantly higher than the content of uningested soil (*p* < 0.05). Ca<sub>Ex</sub> speciations in casts and uningested soil varied notably when earthworms were present. In contrast to control soil (1.38 g·kg⁻¹), the contents of Ca<sub>Ex</sub> in uningested soil were significantly decreased by 8.35%, 10.9%, 7.75%, and 9.36% due to the activities of EF, PC, AR and AA, respectively. We saw 15.8% (1.16 g·kg⁻¹), 15.4% (1.17 g·kg⁻¹), 14.7% (1.18 g·kg⁻¹), and 11.6% (1.22 g·kg⁻¹) decreases in uningested soil as well. Only the Ca<sub>Ex</sub> content in casts with AR was significantly higher than that of uningested soil (*p* < 0.05). In comparison with control, earthworm addition decreased Ca<sub>Ac</sub> contents in both casts and uningested soil (*p* < 0.05, Table 4).
<table><caption>**Table 4.** Variation in Ca speciation and contents in soils.</caption><thead><tr><th rowspan="3">Treatments</th><th rowspan="3"></th><th colspan="5">Ca Speciations</th></tr><tr><th rowspan="2">Ca<sub>Ws</sub></th><th rowspan="2">Ca<sub>Ex</sub></th><th colspan="3">(Ca g·kg<sup>-1</sup>)</th></tr><tr><th>Ca<sub>Ac</sub></th><th>Ca<sub>Or</sub></th><th>Ca<sub>Re</sub></th></tr></thead><tbody><tr><td rowspan="5">Casts</td><td>ES</td><td>0.25 ± 0.04 BC</td><td>1.27 ± 0.05 B ns</td><td>0.18 ± 0.02 B ns</td><td>0.04 ± 0.00 A ns</td><td>0.67 ± 0.04 A</td></tr><tr><td>PS</td><td>0.31 ± 0.01 A *</td><td>1.23 ± 0.10 B ns</td><td>0.15 ± 0.02 D ns</td><td>0.03 ± 0.00 A</td><td>0.68 ± 0.08 A</td></tr><tr><td>RS</td><td>0.23 ± 0.01 C **</td><td>1.27 ± 0.05 B *</td><td>0.16 ± 0.01 BC</td><td>0.04 ± 0.00 A</td><td>0.70 ± 0.05 A</td></tr><tr><td>AS</td><td>0.27 ± 0.04 AB **</td><td>1.25 ± 0.08 B</td><td>0.17 ± 0.02 BC</td><td>0.04 ± 0.00 A</td><td>0.67 ± 0.08 A</td></tr><tr><td>ES</td><td>0.26 ± 0.02 a ns</td><td>1.16 ± 0.06 b</td><td>0.16 ± 0.02 bc</td><td>0.03 ± 0.00 c</td><td>0.78 ± 0.06 a *</td></tr><tr><td rowspan="3">Uningested soil</td><td>PS</td><td>0.25 ± 0.03 a</td><td>1.17 ± 0.03 b</td><td>0.15 ± 0.02 d</td><td>0.07 ± 0.00 b ***</td><td>0.76 ± 0.03 ab ns</td></tr><tr><td>RS</td><td>0.19 ± 0.01 c</td><td>1.18 ± 0.02 b</td><td>0.18 ± 0.03 b ns</td><td>0.07 ± 0.00 a ***</td><td>0.78 ± 0.03 a *</td></tr><tr><td>AS</td><td>0.23 ± 0.03 b</td><td>1.22 ± 0.04 b</td><td>0.17 ± 0.01 bc ns</td><td>0.07 ± 0.00 ab ***</td><td>0.71 ± 0.05 b ns</td></tr><tr><td>Control</td><td>CS</td><td>0.12 ± 0.00 Dd</td><td>1.38 ± 0.05 Aa ns</td><td>0.25 ± 0.01 Aa ns</td><td>0.04 ± 0.00 Ac</td><td>0.62 ± 0.05 Ac</td></tr></tbody></table>

The content of total Ca in soil is 2.40 ± 0.08 g·kg⁻¹. Different capital letters indicated that there were significant differences among Ca contents in the cast group. Different lowercase letters indicated that there were significant differences among Ca contents in uningested soil group (*p* < 0.05, n = 5). (***) *p* < 0.001, (**) *p* < 0.01, (*) *p* < 0.05, and (ns) *p* ≥ 0.05 relate to the significant differences between casts and the uningested soil group in a T test, respectively.

Ca<sub>Or</sub> contents in uningested soil affected by PC, AR and AA were significantly higher than those of CS (*p* < 0.05, Table 4). Comparing casts, the uningested soil of PC, AR, and AA had significantly higher Ca<sub>Or</sub> levels (*p* < 0.001). Compared to CS, the Ca<sub>Re</sub> contents in uningested soil were generally increased (*p* < 0.05). It was also noted that Ca<sub>Re</sub> contents of EF, AR, and uningested soil were higher than the content in casts, significantly (Table 4).

The average percentage contents of five Ca speciations varied notably among treatments (Figure 4). Ca<sub>Ws</sub> contents in casts and uningested soil were 7.95–12.7%. This level was higher than that of the control (4.98%), especially for EF and PC. This indicated that the activities of four earthworms could increase the water-soluble Ca contents. In comparison with control soil, there were 0.25–0.41-fold decreases in Ca<sub>Ac</sub> contents in four casts and uningested soil. The uningested soil of PC, AR and AA had the highest proportions of Ca<sub>Or</sub> contents (2.73–2.99%) compared to casts (1.44–1.50%) and control (1.50%).

<figure><img src="image_6.png" /><figcaption>**Figure 4.** The percentage of five speciations of Ca in casts and the uningested soil of four earthworm species (n = 5). CS: control soil; ESC: cast of EF; PSC: cast of PC; RSC: cast of AR; ASC: cast of AA; ESU: uningested soil of EF; PSU: uningested soil of PC; RSU: uningested soil of AR; ASU: uningested soil of AA.</figcaption></figure>
### 3.6. General Impacts on Uningested Soils and Casts' Ca Speciations

The linkage between the Ca speciation of casts and uningested soil was explained using PCA (Figure 5). The variation in Ca speciations among the wide range of factors examined was discernible from the soil Ca speciations in casts and the uningested soils of four earthworms (Figure 5a,b). Along axis 1, which accounts for 54.4% of the total inertia, casts were easily distinguished from those involving uningested soils. Casts of all earthworms were linked to greater $Ca_{WS}$ levels when compared to the corresponding uningested soil: four earthworms were helpful in promoting the water solubility of soil Ca and reducing $Ca_{EX}$ and $Ca_{Ac}$. Ax2 (27.1%) refers to the differences in earthworms in terms of $Ca_{WS}$ and $Ca_{Or}$, which showed that the digestion of earthworms was helpful in increasing the Ca content in soil solutions. The correlation analysis results demonstrated a strong relationship between $Ca_{WS}$, $Ca_{EX}$, and pH in casts ($r = -0.964$ **, 0.962 **) and $Ca_{WS}$ and $Ca_{Ac}$ in uningested soil ($r = -0.958$ *, 0.966 **), respectively.

<figure><img src="image_7.png" /><figcaption>**Figure 5.** PCA analysis of five Ca speciations in soils: (a) projection of variables; (b) sore plots in soils. n = 5.</figcaption></figure>

### 3.7. General Impacts of Four Earthworms on Soil Ca Speciation, pH, CEC, Exchangeable Base Cations, Soil C, N, and C/N Ratio

The main patterns of soil Ca speciations, exchangeable K, Na, Ca, Mg contents, soil C, N, and C/N ratio in casts, and uningested soils were identified by PCA (Figure 6a,b). The axis 1 (33.5% of total inertia) isolated earthworm casts and uningested soils according to their respective pH, $Ca_{EX}$, $Ca_{Ac}$, $Ca_{WS}$, Ex-Na, Ex-K, Ex-Ca, Ex-Mg, and available K values, especially compared to control (Figure 6). The uningested soils of four earthworms were clearly separated from their casts along axis 2, which explained 18.9% of the total inertia. This is because all earthworms are capable of increasing the contents of their casts relative to uningested soils, including $Ca_{WS}$, Ex-Na, Ex-K, Ex-Ca, Ex-Mg, available K, available P, SOC, and the C/N ratio ($p < 0.001$, Figure 6). Additionally, a strong association was seen between the pH in casts ($r = -0.965$ ** and $r = 0.960$ **, Figure 7) and the concentrations of $Ca_{WS}$ and $Ca_{EX}$. Unlike casts, $Ca_{WS}$ and $Ca_{Ac}$ contents had a correlation with pH values in uningested soil ($r = -0.958$ * and 0.965 **), respectively.
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Projection of soil Ca speciation, exchangeable K, Na, Ca, Mg contents, etc., in casts and uningested soils by PCA analysis. (a) Projection of variables; (b) sore plots in soils. n = 5.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 7.** Correlation analysis between soil Ca speciation and exchangeable K, Na, Ca, Mg contents, etc., in casts and uningested soil. n = 5. (***) *p* < 0.001, (**) *p* < 0.01, (*) *p* < 0.05 relate to the significant differences between corresponding soil indicators in a T test, respectively.</figcaption></figure>
# 4. Discussion

## 4.1. Earthworm Growth

It is known that *Pontoscolex corethrurus* (noted as PC), *Amynthas robustus* (noted as AR), and *Amynthas aspergillum* (noted as AA) are common earthworm in Guangdong province, South China [10], except for *Eisenia fetida* (noted as EF), which is not a native species. As expected, in this study, high weight loss rate and low biomass were observed for EF (Table 3). A low soil pH had a significant inhibitory effects on the survival of EF, beginning with 3.0. Some previous studies demonstrated that the survival rates of PC, AR and AA in acidic soil (pH 4.25) were 79.1%, 97.1%, and 85.8% [10], respectively. The field observation results showed that for typical alkaline greenbelt soil (filled with construction waste), three earthworms PC, AR, and AA were found in greenbelt soil with a pH of 6.77. Even the presence of AR earthworm was found in urban garden soil at pH 8.60. In earthworm inoculation experiments, both PC and AA were able to survive in green soil with a pH of 8.38 (unpublished results). In our laboratory culture experiments, the survival rates of PC, AR, and AA in acidic soil (pH 6.65) were 88.2%, 56.0% and 97.5%, respectively, and their extensive presence in south China could be explained by this adaptive advantage.

## 4.2. Impacts of Earthworm on Soil Physical Characteristics

In general, earthworms have been shown to improve soil structure [9]. It is believed that aggregation, a highly dynamic feature of soils, significantly affects the water-holding capacity, carbon sequestration, and nutrient retention [25,26]. Several researchers have emphasized the great contribution of earthworms to the formation of stable microaggregates [27], especially for macro-aggregate. In a tropical forest, earthworms can completely break down the surface soil (0–20 cm) in 50 years [28]. Compared to other earthworms, endogeic species, particularly PC, have a greater total cast output in our laboratory culture tests (Figure 1c). The soil ingestion rates of PC in Terra Rossa soil were higher than those of lateritic red soil in subtropical areas [10]. The fact that PC produced more castings than AR, EF, and AA suggests that it had a greater influence on soil characteristics, which was beneficial for soil quality. However, it is worth noting that PC is also considered an invasive species in most parts of the world, including southern China [15]. Therefore, we should objectively recognize the positive effects of PC earthworms on soil, and it is essential to conduct a thorough ecological assessment before applying them in agriculture and ecological restoration. Nonetheless, the cast production of earthworm only represented the ability of earthworms to ingest, and could not reflect the texture, particle size, and distribution of the soil they ingested. An alternative to traditional sedimentation techniques for figuring out particle size distribution is laser diffraction analysis. Bittelli M et al. [29] even suggested that the standards for particle size analysis be changed from sedimentation to LDA methods. Currently, the majority of research studies that use the LDA method concentrate on analyzing the particle sizes of different soils or comparison with measurement methods such as settlement method [22,29], and there are no published studies on the size and distribution of earthworm casts' particles in soil using the LDA method. In this study, LDA result analysis indicated that two earthworms, EF and PC, increased the peak value of particle distribution in casts, although the ingestion rate of EF was less one-tenth than that of PC (Figure S1). These effects show that the ingestion of earthworms is crucial for the formation of aggregates, but there was no direct correlation between the particle size distribution of earthworm' casts and the ingestion rate in this study. Ca is a common mineral found in soil that is essential to the development of soil aggregates [30]. Totsche KU et al. [31] found that Ca²⁺ functions as a bridging agent, promoting the formation of stable microaggregates by compounding inorganic clay particles and organic matter through the cation bridging of multivalent cations. In summary, we must ask how the combined
function of earthworms and Ca affects the formation and structural characteristics of soil aggregates? Further research is needed.

Many studies have focused on soil aggregates [10–12] and the NIRS spectral characteristics of earthworms [21]. Significant discrepancies in the NIRS spectral fingerprints of the aggregates suggest that they may differ in terms of origins [26], compositions, structures, and typical functional groups [21]. In this study, we discovered that certain particular wavelengths have increased reflectivity (Figure 3). We compared earthworm casts in soils with varying litter concentrations to wavelengths defined by Zhang C et al. [21] as particular to them. In a study conducted by Velasquez E et al. [26] on the soils of Nicaragua, there were several frequent wavelength values (1340, 1400–1460, 1700–1720, 1900–1920, 2100–2160, and 2240–2280) that were generally related to “good-quality” soils. In this study, the analysis of NIRS spectra and particle distribution in casts and uningested soils indicated that the ingestion of earthworms had a positive impact on the formation of good physical structure in the soil. NIRS spectra and particle distribution in casts were closely associated with earthworm species (Figure 3 and S1).

In calcareous soil from south China, the largest decrease in pH values was effected by PC, confirming that it has strong ability to regulate the pH in Terra Rossa soil. According to García-Montero LG et al. [14], some earthworm species can release calcium carbonate granules and have the ability to regulate the soil acid-base equilibrium. Soil pH determines the rate at which earthworms secrete calcium carbonate, and the amount of calcium carbonate particles secreted increases with the increase in soil pH [32,33]. Lambkin DC et al. [34] found that the soil pH determines the rate at which earthworms secrete calcium carbonate, and the amount of calcium carbonate particles secreted increases with the increase in soil pH. The *Amynthas* earthworm (AR and AA) had a weaker ability to reduce soil pH than EF and PC in our study, which was potentially related to the absence of calcium glands [35].

### 4.3. Effects of Earthworm on Ca Speciation and Base Cation Release

Researching Ca speciation provides more information than analyzing the overall content since it relates to the substance and the chemical forms that are present [17]. Understanding the distribution of calcium in calcareous soils can aid in understanding the mechanisms governing the regeneration and cycle of calcium in karst areas. It can also be used to assess bioavailability and gain insight into the biogeochemical characteristics and ecological environmental changes taking place in the areas under investigation [7]. Usually, the calcium percentage in soil is categorized according to binding. Yuan KN [36] classified the calcium in soil into four groups: exchangeable Ca, organic Ca, mineral Ca, and water-soluble Ca. Mineral Ca is the main soluble speciation that weathers into Ca²⁺; CaEx is the main speciation in the entire soil system with good availability to plants; CaOr is primarily found in organisms; CaWs is the bioavailable speciation in soil solution that plants can use directly. Iron and manganese oxides are coupled with calcium carbonate and sulfate to form the majority of the CaAc found in soil. Consequently, CaAc and CaRe can be seen as potentially reactive Ca pools in soil. The higher CaWs contents in the presence of earthworms validated the effects of earthworms on raising the calcium availability of soil in subtropical environments. Earthworms altered the Ca speciations in the soils and moved Ca from an acid-soluble speciation to water-soluble speciation in our study. This was determined based on PCA analysis (Figure 5). The earthworm ingestion of soil organic materials and pH changes could be the cause of this phenomena [1]. The favorable effect of decreasing soil pH on the rise of soil water-soluble Ca was corroborated by the significant negative association between pH and CaWs seen in this study (Figure 6). Moreover, the digestion ability of the earthworms (PC, AR and AA) to release water-soluble
calcium was stronger than that of earthworm mucus (Table 4). Shabtai IA et al. [1] found that earthworm casts had significantly increased SOC values, and Ca could promote the maintenance of SOC by regulating the physico-chemical interactions between organic matter and soil minerals. It is worth noting that in this study, for PC, AR and AA, the higher CaOr level in soil not ingested by earthworms than that in casts may indicate that Ca organic complexation from the skin mucus is the main pathway instead of the gut (Table 4). Furthermore, species dependence may exist for Ca speciations impacted by earthworms. Since AR is an anecic species, it may generate a unique mucus that causes uningested soil to have a higher CaEx content than casts (Table 4). Furthermore, the inclination of various ecological earthworm species for feeding on soil organic matter varies [28]. Given their significant impact on Ca speciations, more research should be conducted on the physiological traits of earthworms, such as their calciferous glands, intestinal secretions (including gut functional microorganisms), and skin mucus.

Numerous studies have shown that earthworms enhance soil fertility by promoting the release of soil base ions [10,28]. Wu JL et al. [10] observed that for earthworms, PC, AR, and AA, their casts had higher contents of exchangeable Ca, Mg, K, TN, and SOC than that in uningested soil. PC, AR, and AA, respectively, significantly enhanced the exchangeable Ca contents in casts (78.7%, 37.7%, and 40.1%) and the exchangeable Mg contents in casts (12.3%, 24.7%, and 26.8%) when compared to control soil. Wen SH et al. [25] investigated the effects of earthworms on soil nutrient losses associated with surface runoff and sediment during rainy seasons. Findings showed that earthworms could increase the content of NO3⁻-N and OP, but decreased SOC, TN, TP, and NH4⁺-N contents in soil. This study found that the exchangeable K contents in the PC, AR, and AA castings were considerably higher than those of uningested soil (p < 0.05, Table 5). When the PC casts were compared to those of the uningested soil, they showed noticeably increased exchangeable Na and Mg levels, significantly better CEC, SOC, and TN levels, and an improved C:N ratio. Greater contents of hydrolyzable N and available P were found in EF casts compared to the uningested soil (Table 5).

As Wang AQ et al. [37] reported, there is a well-established, highly substantial positive association between pH and exchangeable Ca and Mg. One possible outcome of earthworm casts’ exchangeable base cation discharge in acidic soil is a rise in soil pH [38]. However, the release of calcium carbonate from earthworm calciferous glands may be an important influencing factor in soil pH increase [15]. These glands may play significant roles in the biological regulation of the acid-base balance [39]. However, our study found completely different results (Figure 6), which indicated that the decrease in soil pH effected by earthworm activity (ingestion, burrowing, secretion, etc.) is more conducive to the release of base ions. Above all, it remained unclear from this experiment whether the pH decrease in soil caused by earthworms was due to the disturbance in the balance of the dissolution of carbonate in the soil or something else entirely. To be frank, a single study is insufficient to explain these effects.

Above all, these results extend to the ecological roles of earthworm species, particularly *P. corethrurus*, which can change the Ca speciation of the soil, lower the pH of the soil, influence the release of exchangeable base cations, and take part in and control the geochemical circulation processes in the limestone region. This study highlights earthworms’ possible contributions to soil fertility and nutrient cycling while offering fresh perspectives on their ecological roles in subtropical calcareous soils.
<table><caption>Table 5. Variations in series nutrients in casts and uningested soil.</caption><thead><tr><th>Treatments</th><th></th><th>Ex-K<br>mg·kg<sup>-1</sup></th><th>Ex-Na<br>mg·kg<sup>-1</sup></th><th>Ex-Ca<br>g·kg<sup>-1</sup></th><th>Ex-Mg<br>mg·kg<sup>-1</sup></th><th>CEC<br>cmol·kg<sup>-1</sup></th><th>SOC<br>g·kg<sup>-1</sup></th><th>Total N<br>g·kg<sup>-1</sup></th><th>C: N ratio<br>-</th><th>Hydrolyzable N<br>mg·kg<sup>-1</sup></th><th>Available P<br>mg·kg<sup>-1</sup></th><th>Available K<br>mg·kg<sup>-1</sup></th></tr></thead><tbody><tr><td rowspan="5">Casts</td><td>ES</td><td>121 ± 6 A</td><td>56.3 ± 1.5 A ns</td><td>1.79 ± 0.02 B ns</td><td>178 ± 5 A ns</td><td>13.8 ± 0.3 B **</td><td>24.3 ± 0.5 Aa **</td><td>3.22 ± 0.01 Aa *</td><td>7.54 ± 0.04 A **</td><td>197 ± 5 B *</td><td>2.46 ± 0.13 A *</td><td>124 ± 6 A ns</td></tr><tr><td>PS</td><td>120 ± 3 A *</td><td>61.5 ± 5.0 A *</td><td>1.92 ± 0.03 A ns</td><td>187 ± 1 A **</td><td>29.9 ± 2.4 A</td><td>21.9 ± 0.6 B</td><td>3.01 ± 0.02 B</td><td>7.31 ± 0.12 A ns</td><td>184 ± 8 A ns</td><td>1.59 ± 0.10 D</td><td>113 ± 1 B ns</td></tr><tr><td>RS</td><td>130 ± 5 A *</td><td>57.3 ± 6.9 A ns</td><td>1.86 ± 0.04 AB ns</td><td>178 ± 3 A ns</td><td>29.3 ± 1.5 A</td><td>21.8 ± 1.4 B ns</td><td>2.92 ± 0.01 C ns</td><td>7.48 ± 0.19 A ns</td><td>216 ± 16 B ns</td><td>2.02 ± 0.33 B ns</td><td>121 ± 10 AB *</td></tr><tr><td>AS</td><td>129 ± 4 A *</td><td>58.8 ± 3.4 A ns</td><td>1.86 ± 0.03 AB ns</td><td>189 ± 2 A **</td><td>26.6 ± 1.7 A</td><td>22.4 ± 0.5 B ns</td><td>3.02 ± 0.04 B ns</td><td>7.42 ± 0.07 A ns</td><td>196 ± 9 B ns</td><td>1.72 ± 0.10 CD</td><td>115 ± 8 B *</td></tr><tr><td>ES</td><td>133 ± 3 a *</td><td>38.0 ± 2.7 a</td><td>1.76 ± 0.01 a</td><td>173 ± 1 a</td><td>19.2 ± 0.3 d</td><td>28.2 ± 0.8 a</td><td>3.09 ± 0.03 a</td><td>6.97 ± 0.07 b</td><td>186 ± 6 b</td><td>1.83 ± 0.29 a</td><td>120 ± 4 a</td></tr><tr><td rowspan="3">Uningested<br>soil</td><td>PS</td><td>121 ± 2 b</td><td>56.0 ± 4.2 ab</td><td>1.81 ± 0.05 a</td><td>170 ± 3 a</td><td>33.0 ± 1.3 b ns</td><td>21.6 ± 0.8 a</td><td>3.12 ± 0.03 a ns</td><td>7.11 ± 0.10 b</td><td>187 ± 4 b</td><td>1.67 ± 0.19 a ns</td><td>112 ± 2 b</td></tr><tr><td>RS</td><td>125 ± 4 ab</td><td>46.1 ± 4.9 a</td><td>1.76 ± 0.06 a</td><td>172 ± 4 a</td><td>35.3 ± 1.4 a ns</td><td>22.2 ± 0.6 a ns</td><td>3.05 ± 0.04 a *</td><td>6.97 ± 0.15 b</td><td>216 ± 18 a</td><td>1.95 ± 0.29 a</td><td>115 ± 6 ab</td></tr><tr><td>AS</td><td>122 ± 4 b</td><td>55.3 ± 2.6 a</td><td>1.74 ± 0.05 a</td><td>168 ± 3 ab</td><td>29.2 ± 0.7 c ns</td><td>21.3 ± 0.7 a</td><td>3.08 ± 0.01 a ns</td><td>7.14 ± 0.09 b</td><td>191 ± 7 b</td><td>1.83 ± 0.14 a ns</td><td>109 ± 7 b</td></tr><tr><td>Control</td><td>CS</td><td>94.5 ± 3.3 Bc</td><td>53.3 ± 1.8 Bb</td><td>1.68 ± 0.02 Ca</td><td>156 ± 6 Bb</td><td>13.3 ± 0.2 Be</td><td>22.0 ± 0.6 Ba</td><td>2.92 ± 0.03 Cb</td><td>7.52 ± 0.14 Aa</td><td>184 ± 9 Ab</td><td>1.86 ± 0.16 BCa</td><td>97.7 ± 2.3 Cc</td></tr></tbody></table>

Different capital letters correspond to the significant differences in the cast group. Different lowercase letters correspond to the significant differences in uningested soil group (p < 0.05, n = 5). (**) p < 0.01, (*) p < 0.05, and (ns) p ≥ 0.05 relate to the significant differences between casts and the uningested soil group of T test, respectively.
**5. Conclusions**

Our research showed that in a south China Terra Rossa soil, earthworm *Eisenia fetida* (noted as EF), *Pontoscolex corethrurus* (noted as PC), *Amynthas robustus* (noted as AR), and *Amynthas aspergillum* (noted as AA), have different effects on Ca speciations, soil pH and base cation release. Firstly, four earthworms, especially PC, significantly increased soil water-soluble Ca content. Subsequently, the mucus, intestinal secretions, and calcium gland regulation of earthworms may be the cause of the pH decrease in soil. The primary route for the release of exchangeable Ca, Mg, K contents and CEC may be through the intestine. Finally, all four earthworms altered Ca speciations, especially PC, which increased water-soluble Ca contents in cast by 158.3%. Advancing the knowledge about the effects of earthworms on Ca speciations is key to providing useful information for sustainable agricultural production, ecological protection, and restoration management.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16020312/s1. Figure S1: The particle distribution in casts (a) and uningested soil (b) of four earthworm species (n = 5).

**Author Contributions:** Data curation, J.W., M.Z., M.M.-H., T.D., and H.Z.; formal analysis, C.Z. and J.Z.; funding acquisition, C.Z., C.X., and J.Z.; writing—original draft preparation, J.W.; writing—review and editing, C.Z., Y.H., M.M.-H., C.D., and J.Z.; supervision, J.W., C.Z., M.Z. and J.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by budget project of Guangzhou forestry and landscape Bureau department (202307130200), the Social Development Project of Guangzhou Municipal Science and Technology Bureau (202206010058), National Natural Science Foundation of China (41201305), Guangdong provincial Natural Science Foundation (2021A1515011543, 2023A1515012572).

**Data Availability Statement:** Data are contained within the article and Supplementary Materials.

**Conflicts of Interest:** The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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