Compositional changes and ecological characteristics of earthworm mucus under different electrical stimuli

Differences in mucus physicochemical factors and nutrient elements among electrical stimuli

Physical and chemical factors

Mucus contains electrolytes, such as potassium and multivalent calcium and magnesium ions, which participate in the osmoregulation of the earthworm body to maintain the metabolic balance of the organism^7,23. When earthworms are subjected to different stimuli, the mucus composition changes¹⁰. As shown in Fig. 1a, earthworms produced mucus with significant (P < 0.05) differences in pH and EC among the six different electrical stimuli. The mucus pH consistently exhibited weak alkalinity (7.50 < pH < 8.00) and reached a maximum value of 7.85 at 5 V 10 mA. The mucus pH also showed a tendency to decrease to different degrees as voltage and current increased, and the decrease was significant for most stimuli (P < 0.05, except for 6 V 20 mA, which was not significant), and a minimum value of 7.54 occurred at 5 V 30 mA. However, the EC value of mucus induced by different electrical stimuli varied more. EC significantly differed between all groups (P < 0.05), and the variation at 6 V was more obvious than that at 5 V. Additionally, both the maximum and minimum values appeared at 6 V, and the EC values of 6 V 10 mA and 6 V 20 mA were 1.5–2 times higher than those of other treatments (5 V 10 mA, 5 V 20 mA, 5 V 30 mA, 6 V 30 mA). Thus, different electrical stimuli induced clearly different effects in mucus EC.

External stimulation of cells changes the distribution of substances and potential of the surface of the cell membrane, which affects membrane permeability and alters the pH^24,25. As mucus is produced by the outermost columnar gland cells and somatic lumen cells in the earthworm epidermis¹⁰, both pH and EC changed when electrically stimulated at different intensities. When the intensity of electrical stimulation increased, the permeability of the cell membrane gradually increased, promoting the secretion of small intracellular organic acids, NH₄⁺, and other acids, resulting in a gradual decrease in pH^12,26. The change in membrane permeability also increased the release of inorganic salts, metabolic waste and minerals, and some minerals were also converted into soluble forms in the form of electrolytic ionization²⁷, changing the EC of the mucus such that it increased with voltage, resulting in a higher EC at 6 V. The EC of mucus abruptly decreased at 6 V 30 mA. At this point, the stimulation probably did not destroy the cell membrane integrity, and the earthworm secreted mucus while reducing material efflux in order to respond to the stress of stronger electrical stimulation and maintain body fluid homeostasis.

Nutritional elements

Mucus contains nutrients that are required for plant growth, such as N, P and K, so it is important to study changes in nutrient contents of mucus produced under different electrical stimuli¹⁷. As shown in Fig. 1b, TN and TK contents were lowest at 6 V 30 m A and 5 V 30 mA, respectively, significantly lower levels than those of the other treatment groups (Note: The other treatment groups for total nitrogen were 5 V 10 mA, 5 V 20 mA, 5 V 30 mA, 6 V 10 mA, 6 V 20 mA, and total potassium was 5 V 10 mA, 5 V 20 mA, 6 V 10 mA, 6 V 20 mA, 6 V 30 mA; P < 0.05), and there was no significant difference between the remaining five groups (Note: The other treatment groups for total nitrogen were 5 V 10 mA, 5 V 20 mA, 5 V 30 mA, 6 V 10 mA, 6 V 20 mA, and total potassium was 5 V 10 mA, 5 V 20 mA, 6 V 10 mA, 6 V 20 mA, 6 V 30 mA; P > 0.05), with TN and TK higher at 5 V and 6 V, respectively. The variation in TP among electrical stimuli was greater, and the electrical stimuli treatments can be ranked in the following descending order of TP content: 5 V 30 mA, 5 V 20 mA, 6 V 30 mA, 6 V 20 mA, 6 V 10 mA, 5 V 10 mA. Although the lowest TP content appeared at 5 V, the content of TP was generally higher at 5 V voltage.

Mucus is relatively rich in N, P, and K. Mucus N is mainly derived from substances such as NH₄⁺, urea, small molecule metabolites, amino acids, and proteins^26,28. In contrast, mucus P is derived from bacterial microorganisms and organophosphorus compounds⁷, while mucus K is mainly in cytoplasm and electrolytes^29,30. Under 5 V voltage stimulation, the earthworm body surface normally released mucus, which contains various nutrients required by plants. As the voltage increased to 6 V, the stimulation and pressure on the earthworm increased, inducing the release of more mucus, while the earthworm also released more potassium ions into the mucus in order to maintain the balance of sodium and potassium ions in its body fluid, resulting in a higher potassium ion content at 6 V³¹. While the cell membrane remained intact and active throughout the process, it was still selectively permeable to organic material and did not allow the excessive flow of organic material into the mucus³². Therefore, N and P from organic matter fractions were higher at 5 V, while inorganic salt ions K were higher at 6 V.

FTIR and amino acid differences of mucus among electrical stimuli

The FTIR spectra can reflect the chemical structure and functional groups of organic substances; thus, the infrared spectra of earthworm mucus produced under different voltage–current combinations show that the absorption peaks of all groups of mucus appeared at 3432 cm⁻¹, 2965–2873 cm⁻¹, 1647 cm⁻¹, 1575 cm⁻¹, 1408 cm⁻¹, 1315 cm⁻¹_, 1085–1045 cm⁻¹, and 769–540 cm⁻¹ (Fig. 2). Additionally, among treatments, the waveforms and crests were very similar, while the intensity of the crests changed significantly. Absorption peaks at 3432 cm⁻¹ correspond to carbohydrates and proteins, while those at 2965–2873 cm⁻¹ correspond to fatty acids mainly subjected to anti-symmetric stretching vibrations of C–H₃ and C–H. Peaks at 1647 cm⁻¹ correspond to C = O stretching binding, N–H bending and C-H variable angle vibrations of amide I bands and polysaccharide absorption peaks, while the peak appearing at 1575 cm⁻¹ is probably the amide II bands of C–N stretching and N–H bending. The peak at 1408 cm⁻¹ can be attributed to C–O asymmetric stretching, O–H bending vibrations, and symmetric stretching of COO– from carboxyl groups, while C–O stretching vibrations and O–H planar bending vibrations of ethers, phenols and esters would appear at 1315 cm⁻¹. The broad peak at 1085–1045 cm⁻¹ mainly corresponds to C–O–C and C–H–O functional groups of alcohols and polysaccharides and H–C–H stretching vibrations, and O–C–N bending, out-of-plane C = O bending of amide IV and out-of-plane N–H bending of amide V appear at 769–540 cm^{−17,16,33,34}. It is thus clear that earthworm mucus in this experiment consisted mainly of proteins, carbohydrates, fatty acids, and polysaccharides and contains small amounts of organic substances such as alcohols, phenols and esters. Thus, the absorption spectra of proteins and carbohydrates dominate substantially, which is also consistent with the results of Guhra et al.⁷.

Of the six bands in the spectral range of 400–4000 cm⁻¹, 3432 cm⁻¹, 1647 cm⁻¹, and 1575 cm⁻¹ were the three most dominant absorption bands, representing water-soluble proteins and amide I and II bands, respectively. Additionally, 9 of the 20 amino acids that constitute proteins showed strong absorbance corresponding to the amide I and II bands, so the amide I and II bands could also reflect the amino acid content^16,35. The absorbance of 5 V 10 mA, 6 V 10 mA, and 6 V 30 mA in this band was at a higher level in the Fig. 2, indicating high protein and amino acid content, followed by 5 V 20 mA in the middle, and finally 5 V 20 mA and 6 V 20 mA with the lowest absorption wavelengths. Thus, it was found that the protein and amino acid contents of the mucus produced under different electrical stimuli changed significantly, probably owing to the presence of defense proteins in the earthworm, which secrete amino acids with immune functions to respond to acute stress after different stimuli^36,37.

To further explain the mechanism of protein amino acid changes in mucus under different electrical stimuli, proteins in mucus were hydrolyzed, and the changes in the total content of 17 amino acids in mucus were analyzed¹². As shown in Table 1, glutamic acid, alanine, aspartic acid, proline, and leucine were the most dominant components of the observed 17 amino acids (44.6–47.4%), with glutamic acid being the most abundant at 13.2–14.3%, while cystine accounted for only 0.3–0.4% of all amino acids and was the least abundant in the mucus. Under the six electrical stimuli, the vast majority of amino acids reached the highest and lowest levels in the mucus at 5 V 10 mA and 5 V 20 mA, respectively, with total concentrations of 4.3 and 3.6 mg/g, while the amino acid content in the 6 V treatment groups changed more moderately, without any large fluctuations at 5 V. The stimuli can be ranked in descending order of their amino acid content as follows: 5 V 10 mA, 6 V 30 mA, 6 V 10 mA, 6 V 20 mA, 5 V 30 mA, 5 V 20 mA. Among all amino acids, except alanine, cystine, and arginine, the amino acid content of 5 V 10 mA mucus was higher than the amino acid content of other treatment groups. Among them, the content of glycine, phenylalanine, threonine, proline, and tyrosine at 5 V 20 mA decreased at a higher rate compared to 5 V 10 mA, by 32.4%, 28.4%, 28.0%, 26.4% and 26.1%, respectively. In comparison, alanine and cystine reached a maximum at 5 V 30 mA, with an elevation of 13.8% and 16.0% compared to 5 V 10 mA, and arginine at 6 V 30 mA reached a maximum value, which was 8.4% higher than 5 V 10 mA.

Among the nine amino acids that correspond to the amide I and II bands, this mucus contains seven, namely glutamic acid, aspartic acid, lysine, phenylalanine, tyrosine, arginine, and histidine¹⁶, accounting for 47.3% to 48.8% of the total amino acid content, which is also consistent with Fig. 2. Glutamic acid is the most abundant excitatory neurotransmitter in the central nervous system and can stabilize the central nervous system³⁸, and aspartic acid performs important roles related to nervous system development and hormone regulation³⁹. Additionally, lysine has a stress-protective function against external stimuli⁴⁰. Accordingly, the content of these three amino acids is high in the mucus. Earthworm mucus also exhibited changes in amino acid fractions under stimulation. For example, exposure of earthworms to Cu produced histidine⁴¹, which had a detoxification mechanism, and exposure to pesticides causes significant increases in isoleucine, alanine, and glutamate⁴², which can also be used as indicators in earthworm exposure tests⁴³. Thus, when earthworms are electrically stimulated, they protect themselves by changing the humoral environment through stress responses. For example, aspartic acid has a protective effect on the myocardium, and electrical stimulation can change its secretion to maintain the electrolyte balance of the animal’s myocardium and thus myocardial function³⁹. Additionally, glutamate affects the central nervous system of earthworms under electrical stimulation, and its secretion was indeed changed. Earthworms also produce defense proteins to protect themselves from damage, and there are also 16 amino acids present in the five earthworm defense proteins identified by Roch et al.⁴⁴; these defense proteins are also present in the mucus to protect the earthworm. It is thus clear that earthworms produce different amino acids to regulate homeostasis in the body when subjected to different electrical stimuli, allowing the earthworm to protect itself from damage in a cost-effective manner. Among the six voltage–current combinations examined, 5 V 10 mA stimulated the earthworms the least and induced the most abundant levels of amino acids.

Correlation between levels of physicochemical factors, nutritional elements, and amino acids in mucus under different electrical stimuli

The physiochemical factors, nutrient elements, and amino acids in mucusre crucial for plant growth, organic matter mineralization, soil remediation and sludge compost stabilization and are among the current criteria for evaluating the performance of mucus. Therefore, it is important to conduct correlation analysis to explore the intrinsic connection between different voltage currents^5,7,12,16. As shown in Fig. 3, there were significant negative correlations of pH with TP (P < 0.05), EC with Ala (P < 0.01), TP with Leu and Pro (P < 0.05), and pH with Met (P < 0.05) at various voltage and current combinations, while the correlations between levels of amino acids varied more closely. The contents of the 10 amino acids Asp, Thr, Ser, Gly, Val, Ile, Leu, Phe, Lys, and Pro all showed significant positive correlations with each other (P ≤ 0.05, except Ile, Ser, Leu, Phe, Lys), and levels of Glu, Tyr, and His also showed significant positive correlations with each other (P ≤ 0.05). However, levels of Ala, Cys, Met, and Arg were not significantly correlated with each other or with those of other amino acids (P > 0.05). Indeed, positive correlations between levels of amino acids are likely to indicate common origins⁴⁵. Based on the data in Table 1, it was further found that Asp, Thr, Ser, Gly, Val, Ile, Leu, Phe, Lys, and Pro showed the same trend, and Glu, Tyr, and His showed almost the same trend. Thus, Asp, Thr, Ser, Gly, Val, Ile, Leu, Phe, Lys, and Pro appear to have the same origin in the production of mucus at various voltage–current combinations, and Glu, Tyr, and His similarly originated from the same source conditions.

Differences in contents of trace elements in mucus among electrical stimuli

As shown in Fig. 4, the following nine trace metal elements were identified in the mucus produced under different electrical stimuli in the following descending order of content: Al, Mg, Fe, Cu, Cr, Zn, Mn, Ni, and Pb. Among these trace elements, their contents were 652.6–1159.8 μg/g, 392.4–697.8 μg/g, 36.4–64.7 μg/g, 29.7–47.9 μg/g, 12.7–22.7 μg/g, 8.8–17.3 μg/g, 4.7–9.3 μg/g, 1.3–2.9 μg/g, and 0.14–0.25 μg/g respectively. The Al and Mg content is much higher than the other metal elements. In the mucus produced by earthworms under different electrical stimuli, the lowest content of all metallic elements occurred at 5 V 10 mA, except for Cu at 6 V 20 mA. Among them, the contents of Al, Mg, Fe, Cr, Zn, Mn, Ni, and Pb were significantly decreased at 5 V 10 mA compared to other treatment groups by 22.6–77.7%, 22.5–77.8%, 17.3–77.8%, 32.3–79.0%, 46.9–96.7%, 46.1–97.1%, 78.1–114.4%, and 36.4–79.4% (Note: The other treatment groups were 5 V 20 mA, 5 V 30 mA, 6 V 10 mA, 6 V 20 mA, 6 V 30 mA, respectively, P < 0.05). Moreover, the Cu content at 6 V 30 mA was significantly lower than the other treatment groups, 25.5%-61.2% (Note: The other treatment groups were 5 V 10 mA, 5 V 20 mA, 5 V 30 mA, 6 V 10 mA, 6 V 30 mA, respectively, P < 0.05). The analysis of the trends of the nine metal elements revealed that differences in Al, Mg, Fe, Cu, Zn, and Mn contents among the different voltage–current combinations were almost the same, and their contents could all be ranked in the same descending order according to treatment: 5 V 20 mA, 6 V 30 mA, 5 V 30 mA, 6 V 10 mA, 6 V 20 mA, 5 V 10 mA (except Cu at 5 V 10 mA and Zn at 6 V 20 mA). There were significant differences in trace metal levels between these treatments (P < 0.05), while Cr, Ni, and Pb showed similar trends in their change, but non-significant differences (P < 0.05) between multiple electrical stimuli treatments and thus less variability in metal element level.

Earthworms, as ecological engineers in soil ecosystems, can bioaccumulate and utilize different metal elements, and thus, their body metal element content also depends on their environment⁴⁶. For example, Song et al.⁴⁷ found that the Cu, Zn, and Pb contents of earthworms living in a mixed substrate of pig manure and mushroom residues increased significantly, while Zhang et al.⁴⁸ found that earthworms living in soil with high levels of Cu, Cd, Pb, and Zn contamination also had elevated metal contents in their bodies. In contrast, earthworms in the present experiment lived in a mixed substrate of fermented cow dung and mud, and as the earthworms grew and reproduced, the metal elements in the substrate continuously accumulated in their bodies, thus affecting the metal element content in the mucus. The contents of Al and Mg were higher, probably owing to the substrate, and possibly because Al and Mg have important biological roles⁴⁹. After earthworms received different electrical stimuli, the content of each metal element in the mucus produced changed to some extent, probably because the transport mechanisms of metal elements inside and outside the cell changed, such as the membrane potential of the cell membrane surface, the osmotic pressure inside and outside cells, and transport proteins themselves⁴⁹. At the same time cells also have sensors that regulate the storage of metal ions in order to differentiate them, and thus, the regulation of metal ions changed when the electrical stimulation changed⁵⁰, which altered the release of metal ions as well.

Principal component analysis of each factor under different voltage and current combinations

To further clarify the effects of different voltage and current combinations on the physicochemical factors, nutrients, amino acids, and trace elements in the mucus produced, principal component analysis (PCA) was conducted (Fig. 5). Based on the PCA, a cumulative variance contribution of 77.57% for the two principal components was observed, with the first and second principal components (PC1 and PC 2 respectively) accounting for 63.8% and 13.8% of the variance. As shown in Fig. 5, 5 V 10 mA and 6 V 30 mA had significant positive effects on each factor in the PC1 and PC2 directions, respectively, and 5 V 20 mA and 6 V 20 mA had significant negative effects on each factor in the PC1 and PC2 directions, respectively; thus, it is clear that both 5 V 10 mA and 6 V 30 mA and both 5 V 20 mA and 6 V 20 mA are the main electrical stimulation groups affecting high and low levels of various factors inn mucus fractions, respectively. In addition, the factor loading of the two main components of each factor was determined to further clarify the contribution of each factor to the two main components, revealing that metal elements and most amino acids had the most significant negative and positive effects on PC1, respectively. Similarly, the factors pH, EC, TK, TP, and TK and the factors Glu, Ala, Tyr, His, and Arg had the most significant negative and positive effects on PC2. Figure 5 also shows three aggregations of mucus fractions produced by different electrical stimuli. Thus, pH, EC, TK, TK, and Met were more densely aggregated into one cluster, trace elements and TP were aggregated into another cluster, and the majority of amino acids were aggregated into the third cluster. This shows that the mucus extracted after stimulation with 5 V 10 mA was rich in amino acids and low in metal elements, the mucus extracted after stimulation with 5 V 20 mA was rich in metal elements and low in amino acids, while the mucus extracted after stimulation with 6 V 20 mA was richer in physicochemical factors and nutrient elements, with higher average levels of amino acids and metal elements.

Evaluation of appropriate applications for mucus generation under different electrical stimuli

From the above analysis, it is clear that the physicochemical factors, nutrient elements, amino acids, and trace elements in earthworm mucus induced by six different voltage–current combinations significantly differed; therefore, the object of study for earthworm mucus application may change with the composition. As shown in Table 2, which is in part based on previous studies on earthworm mucus, it is clear that mucus that can have an important ecological impact on soil, compost, and plants should have the following specific characteristics: neutral pH, low EC, and exogenous amino acid protease content to promote the germination of plant seeds, high amino acid protein content to promote plant growth as a liquid fertilizer, the ability to adsorb clay minerals and complex to heavy metals. High contents of nutrients can not only promote plant growth, but also promote soil mineralization and humification, while also passivating heavy metals. The micronutrient content of mucus obtained is in the plant-available range, which can better replenish the micronutrients required by plants and has a bridging effect on the organic matter-mineral combination in the substrate.

Based on the present study, we can tentatively conclude and propose that the mucus produced under different electrical stimuli can be applied to different study subjects. Stimuli of 5 V 10 mA, 6 V 10 mA, and 6 V 20 mA can generate mucus to be used for sludge and straw composting tests, soil remediation and amendment tests, and the mineralization and humification of organic residues owing to its high amino acid content, high nutrient content, and low trace element content. Under the same conditions, 5 V 10 mA mucus with the highest amino acid content and the lowest trace elements was selected as the experimental group. The mucus induced by 5 V 30 mA stimulation had the most neutral pH, lower potassium ion content and EC, higher levels of amino acids, and moderate levels of trace elements, indicating it can be used for seed germination tests. The mucus induced by 6 V 30 mA had high contents of nutrients, amino acids, and trace elements essential for plants, making it particularly suitable for plant growth and development tests. Unlike mucus stimulated by 5 V 20 mA, which contains lower levels of amino acids but was richest in nutrients and trace elements, it can be used for fertilizing plants deprived of trace elements and for improving soil agglomerate structure tests.

Source: Ecology - nature.com