1. Introduction
In an ecosystem, various species play different roles and have different functions [1]. Some species are known as controllers because they exert a significant influence on the ecosystem’s structure and dynamics [2]. Top–down controllers regulate the population size and behaviour of their prey, which reduces competition and predatory pressure on lower trophic levels [3,4]. The role of controllers in an ecosystem is to regulate the population dynamics, community structure, energy flow, and nutrient cycling [5,6]. Earthworms are significant soil organisms that are recognised as soil engineers and act as controllers in the soil ecosystem [7,8].
Earthworms are the main large soil animals and an important component of many terrestrial ecosystems, and they strongly affect soil ecosystems [9]. For example, earthworms are able to influence the soil nitrogen cycle and promote the mineralisation of fresh organic matter (FOM) and soil organic matter (SOM) in tropical soils [10,11]. In addition to affecting the soil ecosystem through burrowing, defecation, and soil ingestion, earthworms can also affect soil properties through their mucus. The mucus secreted by earthworms promotes the rate of mineralisation and humification of organic matter, increases the soil’s organic carbon content, strongly regulates microbial activity and community structure, and even influences soil nematodes [12,13,14,15].
Within the soil fauna, nematodes are an extremely rich and diverse group and an important component of the detritus food web, widely distributed in various types of ecosystems and participating in and influencing many ecological processes [16]. According to their feeding habits, soil nematodes are classified into four groups: bacterivores, herbivores, fungivores and omnivores/predators [17]. They control the soil microbe community and influence nutrient cycling [18,19,20].
Previous studies have shown that earthworm mucus is a good lubricant and an important immune substance that influences other soil organisms [21,22,23]. In our past studies, earthworm mucus was found to alter the feeding habits of soil nematodes by inhibiting the growth of some free-living soil nematodes and reducing their feeding rates [15]. Moreover, we identified the active substance in this earthworm mucus that interferes with nematodes as an earthworm cyclic peptide [24]. This earthworm cyclic peptide damages nematode DNA, forcing the nematode to divert some of the energy allocated for reproduction to DNA repair [24]. This has caused nematodes that previously preferred to feed on high-quality bacteria (which maintain higher reproductive rates and shorter lifespans, supporting a reproduction-dominant mode of energy allocation) to switch to feeding on more nonpreferred bacteria (which maintain low reproductive rates, repair DNA, and complete lifespans), helping the nematodes shift to a survival-dominant mode of energy allocation [24]. This food selection driven by earthworm cyclic peptides may result in changes in the structure and function of soil-feeding microfood webs. However, it is unclear how this active substance (cyclic peptide) affects soil nematodes, microbes, and the soil environment under natural conditions. We hypothesised that earthworm cyclic peptides affect soil nematodes and modulates soil microbe community structure, thereby affecting soil physicochemical properties. This research was conducted to understand the effect of earthworm cyclic peptides, such as nematodes and microorganisms, on soil biological communities and soil physicochemical properties.
2. Materials and Methods
2.1. Experimental Design and Sampling
The cyclic peptide used in this study is cyclo (LLLIII), synthesised by Zhejiang Ontores Biotechnology Co. Ltd. (Hangzhou, China). It will be referred to as earthworm cyclic peptide. Its cLogP value is 9.146.
The experiment used alluvial soil from the Fengqiu State Key Agro-Ecological Experimental Station, Fengqiu County, Henan Province, China, formed by the impact of the Yellow River. The soil underwent a 2 mm mesh sieving process to eliminate any plant tissues, roots, rocks, or other debris. The moisture content was adjusted to 21.93%. To prepare a suspension, the cyclic peptide (0 mg and 0.02 mg) was dissolved in 10 mL of methanol and ultrasonicated. The suspension was mixed with 10 g of fresh soil and dried thoroughly in a fume hood. Ten grams of air-dried soil were added to 190 g of fresh soil and mixed in 300 mL glass culture flasks. The final concentrations of cyclic peptide in the soil were 0 parts per million (CK) and 0.1 parts per million (w/w, 0.1 ppm). Eleven groups of cultivation bottles were set up for each treatment. The bottles were cultured at 25 °C in darkness and replenished with sterile distilled water every two days. Destructive sampling was conducted on day 5 (five glass culture flasks) and day 21 (six glass culture flasks).
2.2. Soil Nematodes and Physicochemical Analysis
The soil nematodes were isolated using Baermann’s shallow tray method [25]. In brief, 50 g of fresh soil was placed on a shallow tray lined with nematode filter paper and submerged in water. The nematodes were then isolated at 25 °C for 48 h and collected by passing them through a 500-mesh sieve. The nematodes were then transferred from the sieve to a 60 mm Petri dish. The nematodes were counted using a stereomicroscope and changed to 100 g of dry soil based on the soil water content, and the number was converted. The nematodes were prepared as microsections. Specifically, nematodes were carefully aspirated under a stereomicroscope using a 100 μL pipette, and then all nematodes were transferred to a microscope slide (controlling the final volume of liquid on the slide to within 200 μL). The cover slips were covered, and the gap between the cover slips and the microscope slides was carefully closed with transparent nail polish. The prepared microsections were placed in a refrigerator at 4 °C and microscopically analysed and classified to genus level within 2 weeks.
Soil respiration was measured by means of a gas chromatograph [26]. Samples were preincubated in the dark at 25 °C for 24 h. In a well-ventilated area, 10 g of dry soil equivalent were weighed and placed into 300 mL respiration vials. Vials were then cultured in darkness at 25 °C for three hours. A 10 mL gas sample was collected and analysed using gas chromatography (Agilent, 7890A, Santa Clara, CA, USA).
Soil nitrate nitrogen (NO3-N) and ammoniacal nitrogen (NH4-N) were extracted with 2 M KCl and analysed determined using a flow analyser [27]. The pH of the soil was measured using a glass electrode with a ratio of soil to water of 1:2.5 [28]. The soil’s available potassium (AK) was determined using a flame photometer [28]. The soil’s available phosphorus (AP) was determined using the molybdenum-antimony anti-coloration method [28].
2.3. DNA Extraction and PCR Amplification
Five grams of fresh soil samples were weighed and immediately frozen at −80 °C. The frozen samples were then sent to Beijing Allwegene Technology Co., Ltd. for DNA extraction and sequencing according to their standard sequencing Illumina MiSeq process. The PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) was used to extract soil DNA according to the manufacturer’s instructions. Genomic DNA purity and quality were assessed on a 0.8% agarose gel, and DNA concentration was quantified using NanoDrop (Thermo Scientific, Waltham, MA, USA). The primers used for amplifying the highly variable V3-4 region of the bacterial 16S rRNA gene were 357F (5-GTACTCCTACGGGGAGGCAGCA-3) and 806R (5-GTGGACTACHVGGGTWTCTAAT-3). The primers used for amplifying the fungal ITS region were ITS1F (5-GGAAGTAAAAGTCGTAACAAGG-3) and ITS2R (5-ATCCTCCGCTTATTGATATGC-3). Illumina Analysis Pipeline Version 2.6 was used for image analysis, base calling, and error estimation after the run.
2.4. Sequencing Data Analysis
The genetic sequences were analysed using the standard OTU (operational Taxonomic units) analysis process on the BMKCloud (
2.5. Statistics and Analysis
Graphs were created using GraphPad Prism 8.0.2. SPSS® 23 was used for the Mann–Whitney U test and t-test. Redundancy analysis (RDA) was used to investigate the correlations among microbes and nematodes, as well as those among microbes and physicochemical indicators. The analysis was performed using Wekemo Bioincloud (
3. Results
3.1. Soil Physicochemical Properties
The physicochemical indices of the samples were measured, and it was found that the earthworm cyclic peptide significantly reduced soil basal respiration on day 5 and soil nitrate nitrogen content on day 21 only (Table 1, p < 0.05, Mann–Whitney U Test).
3.2. Soil Nematodes
At 5 days, the earthworm cyclic peptide significantly (Figure 1, p < 0.05, t-test) reduced the number of fungivorous nematodes. At 21 days, the earthworm cyclic peptide significantly reduced the total number of soil nematodes (Figure 1, p < 0.05, t-test).
3.3. Soil Microbial Community
The sample’s number of OTUs was calculated, along with the ACE, Chao1, Simpson, and Shannon index. Bacterial alpha diversity did not show any significant differences between the treatments at either sampling time point. However, for the fungal community, the earthworm cyclic peptide significantly increased the ACE index on day 5 and the number of OTUs, ACE, and Chao1 indices on day 21 (Figure 2A, p < 0.05, t-test).
PCoA and Adonis analysis results showed significantly different soil bacterial community structures between CK and 0.1 ppm on day 5 according to Bray–Curtis distance (Figure 2B, p < 0.05). For the fungal community, significant differences were observed between CK and 0.1 ppm on days 5 and 21 according to the Bray–Curtis distance (Figure 2B, p < 0.05).
The LEfSe analysis of the bacterial community showed that 48 and 88 taxonomic clades were enriched in CK and 0.1 ppm, respectively, on day 5 (Figure 2C). The enriched clades were mainly from bacterial phyla Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Elusimicrobia, Firmicutes, Gemmatimonadetes, Nitrospirae, Proteobacteria, and Planctomycetes. Similarly, the fungal community analysis identified 53 and 36 taxonomic clades that were enriched in CK and 0.1 ppm, respectively, on day 5 (Figure 2C). The fungi are primarily distributed among the phyla Ascomycota, Basidiomycota, and Zygomycota. On day 21, the LEfSe analysis of the bacterial community revealed that 105 and 64 taxonomic clades were enriched in CK and 0.1 ppm, respectively. These were mainly from the bacterial phyla Acidobacteria, Actinobacteria, Bacteroidetes, Nitrospirae, Proteobacteria, and Verrucomicrobia. The analysis of the fungal community revealed 38 and 67 taxonomic clades that were enriched in CK and 0.1 ppm, respectively. They were mainly distributed in fungal phyla Ascomycota, Basidiomycota, and Zygomycota.
3.4. Relationships between Soil Nematodes and Soil Factors with Microbial Communities
The RDA results revealed a significant correlation between fungivores, predatory and omnivorous nematodes, total nematodes, and fungal communities on day 5 (Figure 3A). On day 21, the bacterial community showed a significant relation to the bacterivorous, herbivores, predatory and omnivorous, and total nematodes, while the fungivores and total nematodes correlated significantly with the fungal community (Figure 3A).
RDA analyses were conducted on environmental factors and microbial communities. The RDA results revealed a significant correlation between NO3-N, basal respiration, and bacterial community structure on day 5 (Figure 3B). On day 21, the fungal community was significant in correlating with pH, NO3-N, and AK (Figure 3B).
3.5. PLS-PM Analysis
PLS-PM was used to establish potential regulatory pathways through which the earthworm cyclic peptide regulates soil biology and the environment (Figure 4). The results showed the nematodes influenced the bacterial community structure, accounting for 18.7% and 8.2% of the variation on days 5 and 21, respectively (Figure 4). Moreover, the nematodes influenced the fungal community, accounting for 27.3% and 23.5% of the variation on days 5 and 21, respectively. On day 5, the soil physicochemical response to the bacterial and fungal community had path coefficients of −0.013 and 0.025, respectively. Meanwhile, the response of the bacterial and fungal communities to nematodes had path coefficients of 0.433 and −0.523, respectively. Moreover, soil nematodes responded to the earthworm cyclic peptide with a path coefficient of −0.613. On day 21, the soil physicochemical response to bacterial and fungal community structures had path coefficients of −0.219 and 0.563, respectively. Meanwhile, the bacterial and fungal community structures’ response to nematodes was stronger, with path coefficients of 0.287 and −0.484, respectively. Soil nematodes also responded to the earthworm cyclic peptide with a path coefficient of −0.611.
4. Discussion
This research has revealed that earthworms, in addition to their predatory role, can mediate tri-trophic interactions among nematodes, microbes, and environmental factors by secreting an active substance called earthworm cyclic peptide.
Earthworm cyclic peptides reduce soil nematode populations and alter nematode community structure. On day 5, the fungivores were significantly reduced by the earthworm cyclic peptide. Additionally, the total number of nematodes was reduced, although this reduction was not statistically significant (Figure 1). On day 21, the earthworm cyclic peptide significantly reduced the total number of soil nematodes. The study found that the earthworm cyclic peptide reduced the mean number of bacterivores, herbivores, and fungivores, but the differences were not statistically significant. This may be due to experimental error. Our frontline research has found that earthworm cyclic peptides induce a trade-off between bacterial nematode reproduction and survival, reducing the ability of bacterial nematodes to reproduce [24]. However, this has not been confirmed for other types of nematodes. The target of cyclic peptides acting on nematodes is the destruction of nematode DNA, which causes a modification of the nematode’s survival strategy [24]. The physiology of other nematodes is largely similar to that of bacterivores [31,32,33,34,35]. It is possible that cyclic peptides can also affect other types of nematodes. In fact, our unpublished research also suggests that earthworm cyclic peptides can have a similar effect on plant-parasitic nematodes.
Nematodes are often thought to determine the structure of soil microbial communities, as many studies to date clearly demonstrate this relationship [36,37]. Nematodes prey on fungi, reducing their abundance and diversity [38,39]. Earthworm cyclic peptides might also affect feeding by fungivores, leading to changes in the alpha diversity of the fungal community. RDA showed that soil nematode communities were correlated with microbial communities at both sampling times (Figure 3A). The correlation was stronger on day 21. This may be due to the fact that cyclic peptides take a longer time to act on nematodes to affect the microbial community structure. Many of the changing “biomarkers” in the microbial community have been shown to be regulated by soil nematodes (Figure 2C). For instance, we found that the addition of cyclic peptide reduced Bacteroidetes abundance on days 5 and 21, possibly because cyclic peptides inhibit nematodes, which can enhance Bacteroidetes abundance [40]. The reduced abundance of some microorganisms belonging to the phylum Nitrospirae in the cyclic peptide treatment may be due to the fact that nematodes can increase the abundance of this phylum; however, the cyclic peptide inhibited soil nematodes [41]. Earthworm cyclic peptide treatment also increased the abundance of OTUs belonging to Basidiomycota, probably because these fungi are food for fungivores [42], and the cyclic peptide reduced the number of fungivores.
Bacteria and fungi play a crucial role as decomposers in ecosystems. Soil microorganisms can influence the physical and chemical properties of soil [43]. They promote the natural mineralising of the organic substances in the soil and thus the nutrient cycle [43,44]. RDA showed a significant correlation between NO3-N, basal respiration, and bacterial community structure on day 5 (Figure 3B). Soil basal respiration is a measure of soil microbial activity [45]. A decrease in soil basal respiration means a decrease in microbial activity. This may have been due to the suppression of soil nematodes, which were able to stimulate the increase in soil basal respiration [46]. On day 21, pH, NO3-N, and AK were significantly correlated with fungal community structure. Fungi are also involved in the soil nitrogen cycle, affecting nitrate nitrogen levels [47,48]. Similarly, fungi can be involved in the process of cycling potassium from the soil [49].
Earthworm cyclic peptides can influence soil physicochemical properties by modulating microbial community structure via their effects on soil nematodes. PLS-PM also corroborates this idea (Figure 4). On days 5 and 21, the pathway coefficients of earthworm cyclic peptides influencing nematodes were negative and had similar values. This implies that the nematode inhibitory effect of earthworm cyclic peptide can occur rapidly and persist for a while. The effects of soil nematodes were generally positive for soil bacteria and negative for fungi, with the intensity of the effects slightly diminishing over time (Figure 4). It was found that the impact of bacteria and fungi on soil physicochemical factors increased over time. This may be because top–down regulation of ecosystems takes some time [50]. The models did not include the factor of variation in nematode feeding preference because it was not available to measure the factor of each nematode feeding preference in the soil. However, even so, our modelling shows the existence of a regulatory pathway by which earthworm cyclic peptides are able to influence the soil microbial community by modulating nematode community structure, which in turn alters soil environmental factors.
5. Conclusions
In conclusion, the study found that earthworm cyclic peptide, an active substance in earthworm mucus that modulates nematodes, can regulate the soil environment by influencing the structure of soil bacterial and fungal communities through the soil nematode. The earthworm cyclic peptide mediates tri-trophic interactions between earthworms, nematodes, microbes, and environmental factors. The report presents new insights into the interactions and feedbacks among soil biota in dynamic soil food webs. It also highlights the regulatory role of earthworms in soil ecosystems through their secretion of mucus, which modifies the structure of the soil nematode community, nutrient cycling, and microbial community composition. These findings offer new perspectives on soil ecology.
Conceptualisation, F.Y. and J.S.; methodology, F.Y.; software, Q.D.; validation, C.Z.; formal analysis, Y.Y.; investigation, C.J.; resources, H.X.; data curation, Y.Q.; writing—original draft preparation, F.Y.; writing—review and editing, J.S. and H.X.; visualisation, C.J.; supervision, Y.Q.; project administration, F.Y.; funding acquisition, F.Y. All authors have read and agreed to the published version of the manuscript.
The original data presented in this study will be made available by the authors without undue reservation, and the corresponding author can be contacted directly for further inquiries.
We would like to thank Huixin Li from the College of Resources and Environment Sciences, Nanjing Agricultural University, for his support in experimental design.
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 1. Effects of earthworm cyclic peptide (LLLIII) on free-living soil nematodes. B: bacterivores; H: herbivores; F: fungivores; O and C: omnivores/predators; TN: total number of nematodes in 100 g of soil; CK: the concentrations of cyclic peptide in the soil were 0 parts per million (w/w); 0.1 ppm: the concentrations of earthworm cyclic peptide in the soil were 0.1 parts per million (w/w). The samples were incubated in the dark at 25 °C for 5 days. The samples were incubated in the dark at 25 °C for 21 days. Error bars are standard errors and * indicates a t-test, p < 0.05.
Enlarge this image.Figure 2. Effects of earthworm cyclic peptide (LLLIII) on the structure of soil bacterial and fungal communities. (A): Effects of earthworm cyclic peptide on the alpha diversity of bacterial and fungal communities; error bars are standard errors, and * indicates a t-test with p < 0.05. (B): PCoA of bacterial and fungal communities (principal coordinates PCo1 and PCo2) with Bray–Curtis distances (permutational multivariate analysis of variance (PERMANOVA) by Adonis). (C): Discriminant taxa significantly retrieved by LEfSe analysis for rhizosphere bacterial and fungal communities. The cladogram illustrates the taxonomic representation of statistically significant differences between bacterial and fungal communities. The figure illustrates taxonomic levels from phylum to species, with each circle representing a taxon at that level and the diameter of the circle indicating its relative abundance. Species with no significant differences are coloured in yellow, while those with significant differences are highlighted in Biomarker according to their grouping. Red nodes indicate microbial taxa that play an important role in the CK, while blue-green nodes indicate those that play an important role in the 0.1 ppm. CK: the concentrations of cyclic peptide in the soil were 0 parts per million (w/w); 0.1 ppm: the concentrations of earthworm cyclic peptide in the soil were 0.1 parts per million (w/w). The samples were incubated in the dark at 25 °C for 5 days. The samples were incubated in the dark at 25 °C for 21 days.
Enlarge this image.Figure 3. Ordination plots of the results from the RDA (redundancy analysis) to determine relationships between microbial and nematodes and between microbial and the soil environment. (A): RDA was performed between microbial and nematodes, bacterivores (B), herbivores (H), fungivores (F), and omnivores/predators (O and P). TN: total number of nematodes in 100 g of soil. (B): RDA was performed between microbial and soil environmental variability, ammonium nitrogen (NH4-N), nitrate nitrogen (NO3-N), available phosphorus (P), available potassium (K), potential of hydrogen (pH), and soil basal respiration (BR). CK: the concentrations of cyclic peptide in the soil were 0 parts per million (w/w); 0.1 ppm: the concentrations of cyclic peptide in the soil were 0.1 parts per million (w/w). The samples were incubated in the dark at 25 °C for 5 days. The samples were incubated in the dark at 25 °C for 21 days.
Enlarge this image.Figure 4. Direct and indirect effects of earthworm cyclic peptide on the relationship between soil nematodes, soil microbial community structure, and soil environmental variability. Red and blue-green arrows indicate positive significant and negative significant relationships, respectively. R2 denotes the proportion of variance explained. The samples were incubated in the dark at 25 °C for 5 days. The samples were incubated in the dark at 25 °C for 21 days.
Soil physical and chemical indicators.
| Time | Treatment | pH | NH4-N | NO3-N | AK | AP | BR |
|---|---|---|---|---|---|---|---|
| 5 days | CK | 7.9 ± 0.03 | 4.84 ± 0.14 | 38.71 ± 0.94 | 284.78 ± 2.51 | 12.63 ± 0.40 | 2.34 ± 0.08 |
| 0.1 ppm | 7.84 ± 0.02 | 4.86 ± 0.22 | 38.85 ± 0.44 | 284.53 ± 1.95 | 11.91 ± 0.20 | 1.90 ± 0.18 * | |
| 21 days | CK | 7.83 ± 0.04 | 2.46 ± 0.15 | 59.32 ± 1.58 | 239.59 ± 2.43 | 15.20 ± 0.38 | 2.19 ± 0.13 |
| 0.1 ppm | 7.77 ± 0.01 | 2.75 ± 0.25 | 51.88 ± 1.58 * | 240.80 ± 1.87 | 15.68 ± 0.23 | 2.06 ± 0.26 |
CK: The concentrations of cyclic peptide in the soil were 0 parts per million (w/w); 0.1 ppm. The concentrations of earthworm cyclic peptide in the soil were 0.1 parts per million (w/w); pH: potential of hydrogen; NH4-N: ammonium nitrogen; NO3-N: nitrate nitrogen; AK: available potassium; AP: available phosphorus; BR: basis respiration. The samples were incubated in the dark at 25 °C for 5 days. The samples were incubated in the dark at 25 °C for 21 days. Data are presented as means plus or minus standard errors.; * means p < 0.05 after Mann–Whitney U Test.
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