https://orcid.org/0000-0001-7398-1733
Figures
Abstract
Different biochars have diverse properties, with ambiguous effects on soil nematodes. This study investigated how aspen sawdust (ABC), bamboo powder (BBC), maize straw (MBC) and peanut-shell biochars (PBC) affected Caenorhabditis elegans via culture assays and RNA-seq analysis. The results showed that biochars derived from different agricultural materials varied significantly in physicochemical properties, and PBC produced more volatile organic compounds (VOCs) to attract C. elegans than ABC, BBC and MBC. Moreover, worms in ABC experienced the worst outcomes, while worms in PBC experienced milder impacts. Nematode body length decreased to 724.6 μm, 784.0 μm and 799.7 μm on average in ABC, BBC and MBC, respectively, compared to the control (1052 μm) and PBC treatments (960 μm). The brood size in ABC, MBC, BBC and PBC decreased 41.1%, 39.4%, 39.2% and 19.1% compared to the control, respectively. Furthermore, the molecular mechanisms of biochar-induced developmental effects on C. elegans were explored. Although several differentially expressed genes (DEGs) were different among the four biochars, worm phenotypic changes were mainly related to col genes (col-129; col-140; col-40; col-184), bli-6, sqt-3, perm-2/4, cdk-8, daf-16 and sod-1/2/5, which are associated with cuticle collagen synthesis, eggshell formation in postembryonic growth and rhythmic processes. Our study suggests that different properties of biochars could be crucial to soil nematodes, as well as the worms’ biochemical changes are important for the health in agriculture soil.
Citation: Chen Y, Wang X, Li J, Wang Z, Song T, Lai X, et al. (2023) The effects of different biochars on Caenorhabditis elegans and the underlying transcriptomic mechanisms. PLoS ONE 18(9): e0284348. https://doi.org/10.1371/journal.pone.0284348
Editor: Xiaoping Pan, East Carolina University, UNITED STATES
Received: January 21, 2022; Accepted: March 29, 2023; Published: September 22, 2023
Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This study was funded by National Science Foundation of China (grant number 41571292) awarded to GZ who provided the study design and data collection; Fundamental Cutting-edge Projects of Research Institutes (grant number 2021-jcqyrw-xwm) awarded to GZ who provided the financial support for materials in nematode culture; Qinghai Science and Technology Major Project (grant number 2019-NK-A11-01) awarded to JL who provided the financial support for the Transcriptome data analysis and preparation of the manuscript, and the Young Scientists Fund - National Science Foundation of China (grant number 42207048) awarded to JL who can support the publishing costs.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
With the rapid increase in the production of agricultural wastes in China, the recycling and utilization of these potential biomass resources have become crucial for alleviating environmental pollution and improving soil fertility [1]. In recent decades, biochar has been widely researched for its advantages in recycling agricultural wastes [2]. Studies claim that the application of biochars made from various agricultural wastes will not only enhance the recycling rate of wastes but also improve the properties and structure of soil [3]. Nevertheless, it is necessary to explore how biochar changes the soil biota since the application of biochar changes soil properties [4, 5].
Nematodes play a crucial role in soil biota systems, being dominant in abundance, having a diversity of life cycles, and occurring at various trophic levels [6]. Previous studies have reported the interactions between biochar and soil nematodes. The main results can be concluded as follows: i) biochar improves nutrient supply and habitat conditions, which would support more microbivorous nematodes and exert competitive pressure on other types of nematodes as well as change the community composition of soil nematodes [7, 8]. ii) Biochars improve soil porosity and root growth to inhibit the growth of some harmful phytophagous nematodes [9]. iii) In recent years, researchers have also stated that some substances in biochar can disturb the movement and growth of some nematodes [10, 11]. In general, those studies mainly focused on assessing the effect of biochar addition on soil nematode community abundance, structure and biodiversity or focused on certain plant parasitic nematodes. Few studies have tested the direct impacts of biochar on nematodes. Additionally, researchers further concluded that the properties of biochars vary widely due to the marked differences in the raw materials used [12, 13], so clarifying and comparing the soil nematode response to different biochars is essential to reveal the underlying mechanisms of biochar interactions with soil fauna.
Contemporary studies also demonstrated that the composition of different raw materials resulted in various yields and characteristics of biochars. A higher lignin content can result in a higher biochar yield; the percentage of lignocellulosic biomass (cellulose, hemicellulos, lignin) or minerals in biochar can result in different pH values, pore compositions or ash contents [14]. According to Lebrun [15], even biochars made from different layers of oak tree trunk (bark, sapwood, heartwood) have different properties. These differences between biochars caused different effects on the abundance, activity and feedback of soil nematodes. Total nematode abundance and faunal activity were different under wheat or corn biochar application [7, 16]. Biochars derived from rice husk and sawdust showed negligible toxic effects on C. elegans, while Acorus calamus (a wetland plant) biochar showed significant toxicity toward the worms [17]. Phenolic and polycyclic aromatic hydrocarbons (PAHs) [18] or persistent free radicals [11] from some biochars resulted in worm growth inhibition or avoidance, which implied that some biochars may have significant negative effects on the growth of nematodes. Based on these phenomena, the effects of different biochars on soil nematodes vary. However, few studies have investigated the response mechanisms of nematodes exposed to biochars produced by different raw materials.
Maize, aspen, bamboo and peanut are very important wood and agricultural resources in China [19, 20]. Moreover, these wastes or byproducts are difficult to degrade, recycle and manage. The direct use of these wastes may not only cause many problems in field production, such as unfavorable root fixation, more disease for the next crop, and lower seedling survival [21], but also result in high costs during recycling [22]. Therefore, the application of biochar derived from these wastes to arable land is a novel and convenient approach. One of the most highly debated points prior to biochar application might be how different biochars affect the development of soil nematodes.
C. elegans is a model organism in the fields of molecular biology, developmental biology and environmental toxicology because of its well-studied genetic background. Therefore, it is a perfect tool to investigate the effect of biochars on nematodes at the individual and molecular levels. Therefore, the aim of our study was 1) to compare the differences among the four biochars (peanut shell biochar, PBC; aspen sawdust biochar, ABC; bamboo powder biochar, BBC; maize straw biochar, MBC), including their physico-chemical properties, VOCs and PAHs. 2) Assessment of the body length, SOD activity, reproduction and lifespan of C. elegans exposed to different types of biochar was also performed. 3) Furthermore, RNA-seq was performed to obtain transcript sequences of the worms. Additionally, differentially expressed genes (DEGs) related to differential phenotypes revealed genome-wide transcript changes in C. elegans in response to biochar exposure.
2. Materials and methods
2.1. Biochar preparation and characterization
Four different biochars were produced by grinding maize straw (M), aspen sawdust (A), bamboo powder (B) and peanut shells (P) through a 0.85 mm sieve. These raw materials were pyrolyzed at 500°C without oxygen (N2) for 120 min in a muffle furnace (HBYQ 2200) and then cooled to room temperature and passed through a 1.5 mm sieve. The lignocellulosic biomass of the materials was tested with an ANKOM A2000i fiber analyzer through Soest [23] detergent analysis and calculated by the difference between neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin and crude fiber.
The porosity and specific surface area, pH value and relative atomic ratio of these biochars were determined by SEM TM-1000 (Hitachi, Japan), an MP511 lab pH meter (San-xin, China) and XPS Escalab 250Xi (Thermo Scientific, USA). The ash content was measured by the China National Academy of Nanotechnology & Engineering with a thermal decomposition furnace following GB/T17664-1999. The dissolved biochar was obtained by washing 15g each biochar in 500 mL sddH2O and passed through a 0.65 μm filter. And the dissolved organic carbon of biochar was detected by total organic carbon analyzer (TOC-VCPH, Shimadzu, Japan).
We also examined the volatile organic compounds (VOCs) and water-extracted polycyclic aromatic hydrocarbons (PAHs) of ABC, BBC, MBC and PBC. The composition of VOCs was determined by the solid phase microextraction method (SPME, 75 μm Carboxen/PDMS, Supelco, Bellefonte, PA, USA) and then characterized by pyrolysis-gas chromatography/mass spectrometry (GC-MS, 7890-5975C, Agilent Inc., USA). Details of the parameters of VOC measurement were reported in Li, Chen [24]. The water-extracted PAH16 in biochar was detected according to Rogovska, Laird [25]. An Agilent 7890GC-7000C triple quadrupole MS (MDL: 1 μg/kg) was used to analyze the samples. Standard substances purchased from Sigma-Aldrich (USA) and Sangon Biotech (China) were used to generate external standard curves for quantifying samples.
2.2. Nematode culture and physiological and biochemical indexes
Wild-type N2 (Bristol) nematodes were obtained from NanKai University, and the worms were grown on nematode growth medium (NGM). The Escherichia coli OP50 strain was prepared as the food source for the nematodes. The strain was cultured in LB liquid medium for a day before it was used to feed the worms. We applied OP50 at a concentration of 10x to the NGM. All worms were synchronized by 1x lysis buffer and transferred to M9 buffer to grow on NGM in the presence of different biochars. The preparations of media and buffers were made according to the protocol which was originated from Brenner [26] and modified by He [27]. Ten milligrams of biochar was placed in the center of each petri dish (6.5 cm) with the help of an Oxford cup (d = 0.5 cm). A 1 mL buffer of age-synchronized eggs (~500) of the wild type C. elegans was placed on the biochar surface in each petri dish.
Body length was not measured until the C. elegans reached the day 1 adult stage (56 h). The media were washed off of the worms with 2 mL M9 buffer. Then, the nematodes were killed with gentle heat, and 1 mL of worms was drained randomly, and their body length was recorded [28]. Nematodes were observed under an Olympus SZX16 microscope, and nematode morphology and body lengths were measured by cellSens Dimension software.
Reproduction assays were carried out after morphological observations. One L4-stage worm was transferred to each petri dish (3.5 cm) containing NGM and 50 μL OP50 in LB, totaling 25 worms for each treatment. Nematodes grew at 25°C for 72 h, after which the number of worms at all stages except eggs was recorded.
L4 stage worms were also used in the SOD assay according to a previous reference [29]. We first collected the worm samples and ensured that there was 1 mL, 2 mL, and 4 mL of ice-cold PBS buffer in the tubes containing nematodes. Then, glass grinders were utilized to homogenize the nematodes and obtain the protein on ice (4°C). Then, the tubes were centrifuged at 4°C at 5,000 xg for 5 min, and any supernatants were discarded from each sample. The diluted samples were used with the SOD Assay Kit (Sigma) to determine the activity of superoxide dismutase in C. elegans by Multiskan Spectrum (Thermo Scientific, 450 nm).
According to Mishra [30], lifespan was evaluated by the rate of worms developed into the L4 larval stage. After 40 hours transfer from the synchronized eggs, the worms would be reaching the L3/L4 molt stage, and the number of L4 worms could be counted and recorded during the 44–55 h. Every hour afterward, the number of worms was recorded until they all reached the L4 stage.
In chemotaxis assays, 1 mL buffer of the wild type C. elegans are placed in the center of the 9 cm NGM petri dish with the peanutshell biochar on one side and each other biochars (MBA/ABC/BBC) on the other side (Madžarić, Kos [31], Baiocchi, Dillman [32]). Worms in regions with a radius of 1.5 cm around each Oxford cup were counted at 2 h, 4 h, 6 h, 12 h, 24 h, 36 h and 48 h. During the test, five replicates (Petri dishes) were conducted. The details and experimental setup of the biochar chemotaxis test protocol are shown in S1 Fig.
2.3. Transcriptome sequencing
The duration of biochar exposure prior to RNA-sequencing is about 56 hours. Once the age-synchronized worms grew to young adult worms, they were harvested and stored at -80°C after quick freezing by liquid nitrogen for the RNA-sequencing. Total RNA of young adult worms was extracted by TRIzol reagent (Sangong, Shanghai). Then, the RNA was purified by silicon membrane. RNase-free water was used to dissolved RNA. RNA sequencing was carried out by Sangong Biotech. Briefly, RNA was quantified and qualified by a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and an RNA Nano 6000 Assay Kit with the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
A total amount of 3 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext ® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample [28]. Then, 3 μl USER Enzyme (NEB, USA) was used for size selection and adaptor ligation of cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. To analyze the sequences, HISAT2, RSeQC, Qualimap and BEDTools software was utilized. GO and KEGG enrichment analysis were performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at p-value≤0.05 compared with the whole-transcriptome background. GO functional enrichment and KEGG pathway analysis were carried out by Gene Ontology database (http://www.geneontology.org/), R (ClusterProfiler) and KAAS (https://www.genome.jp/tools/kaas/) [33].
2.4. Quantitative RT-PCR
According to the physiological and biochemical variation of C. elegans in this study, eight target genes (col-184, col-140, col-129, lys-7, ilys-5, cdk-8, sod-1, perm-4) were found from wormbase and previous studies [34–37]. Then they were validated by quantitative real-time PCR (qPCR). EasyScriptct® One-Step gDNA Removal and cDNA Synthesis Super Mix were used to synthesize cDNA. A Thermal Cycler® was used for quantitative PCR performed with 2×Trans Start® Top/Tip Green qPCR Super Mix. Three independent biological replicates were applied for each gene, and the expression levels of each gene were normalized on the basis of the reference gene pmp-3 [38]. The relative expression ratio of target genes was calculated using the ΔΔCt method.
2.5. Statistical analysis
The statistics were analyzed by SPSS 22 using t-tests if variances were unequal. Moroever, we compared the differences among different groups through univariate analysis (ANOVA). If the data did not pass the variance homogeneity test, Games-Howell tests were applied in one-way ANOVA. The mean values and standard errors of the mean are presented below and were calculated using SPSS 22.
Chemotaxis indexes (CIs) were used to evaluate the results for the worms in biochar chemotaxis experiment and were calculated as follows:
Chemotaxis index (CI) = (worm count in PBC-treated group) − (worm count in MBC/PBC/ABC-treated group)/(worm count in PBC-treated groups) + (worm count in MBC/PBC/ABC-treated groups).
A CI value of 0.5 to 1.0 indicated that the majority of worms were in/around the PBC-treated group, while a CI of -1.0 to -0.5 indicated that most worms were in/around the other biochar (MBC/ABC/BBC-treated) groups. Additionally, a CI value near zero indicated that there was no difference between the number of worms in both treatments.
3. Results
3.1. Properties of different biochars
The lignocellulosic biomass of raw materials and physicochemical properties of biochars are described in Tables 1 and 2. The four raw materials differed in the percentages of hemicellulose, lignin and fiber, with the lowest NDF and ADF for maize straw and the highest for aspen sawdust. All biochars were typically alkaline, with pH values ranging from 8.76 to 9.92. Moreover, the value of DOC in PBC, namely 29.46%, was the highest, followed by ABC, BBC, and MBC of 7.66 mg/L, 5.50 mg/L, and 7.43 mg/L, respectively. The biochars also possessed a range of 11.45–19.36 m2/g specific surface area, 0.89–4.77 nm porosity, 3.0%-16.2% ash, 83.05%-96.34% fixed carbon, 5.50–29.46 mg/L dissolved organic carbon and 0.66%-1.43% volatiles.
Most of the PAH contents of the biochars could be attributed to Pyr, which accounted for 49.0–59.7%. Finally, we observed that the VOC profiles from the four biochars varied greatly (Table 3). The most positively identified volatile compounds, seven, were in PBC compared to the three compounds identified in ABC and MBC, as well as only one compound identified in BBC (S2 Fig). Additionally, the results also suggested that the VOCs were almost the same among ABC, MBC and BBC, with similar silicon compounds and retention times.
3.2. Developmental observation of C. elegans under different biochars
The brood size, body length, SOD activity and the lifespan (L4 reaching time) were evaluated in the four biochar (ABC, BBC, MBC and PBC)-treated groups in comparison with the water control group. The results showed that exposure to these biochars significantly decreased the body length of C. elegans. The rates of decrease were 31.12%, 25.48%, 23.99% and 10.16% for ABC, BBC, MBC and PBC exposure, respectively (Fig 1a). Among the treatments, worms in ABC had the shortest average body length, with values of 724.6±26.7 μm, which was significantly shorter than that of worms in PBC (945.2±24.3 μm) and the control (1052.1±10.3 μm). Worms in the BBC and MBC treatments were similar in body length, with mean values of 799.7±15.8 μm and 784.0±20.4 μm, which were also significantly shorter than the control value.
Different lowercase letters indicated significant differences between treatments (Tukey’s test, P < 0.05); the stars indicated significant differences between peanut-treated group and each other biochar (ABC/BBC/MBC-treated) group.
The brood size refers to the number of hatched worms during the observation period among treatments. The brood size was significantly decreased following biochar exposure, and the reduction reached 39.44%, 39.21%, 41.07% and 19.10% in the MBC-, BBC-, ABC- and PBC- exposed groups, respectively, compared to the control group (Fig 1b).
The results also demonstrated that the SOD density in the biochar-treated group was significantly lower than that in the control group (Fig 1c). The SOD assay results showed that the control group had the highest SOD activity, which was 196.5±3.86 U/mL. The MBC treatment resulted in the lowest SOD activity (123.9±3.89 U/mL), which was nearly 1/3 of the control level. ABC, BBC and PBC decreased the SOD activity to 154.2±6.49, 145.0±3.95 and 165.81±3.82 U/mL, respectively.
According to Fig 1d, the C. elegans exposed to peanut shell biochar had a shorter lifespan than those exposed to other biochars or the control group. Almost all worms took 51 hours to developed into young adult in PBC treatment. However, most worms took 53–55 hours from synchronized eggs in the other biochars (MBC, BBC, ABC) treatments and the control to reach adulthood.
Moreover, C. elegans shows chemotaxis to PBC compared to other biochars (Fig 2). The variation of CI between PBC and each other biochars were 0.66 ~ 0.93 during the cultivation period, which indicated that more nematodes moved around PBC than ABC/MBC/BBC after 48 h of treatment. However, the statistically significance of chemotaxis indices were not significant between PBC-treated and ABC/BBC/MBC-treated group.
3.3. RNA sequencing analysis of C. elegans in different biochars
To determine the mechanisms of the depressive response, transcriptome sequencing and analysis were conducted on C. elegans exposed to ABC, MBC, BBC and PBC for 56 h. To identified differential expression among various groups of C. elegans, false discovery rate (FDR-) adjusted p-values <0.05 and absolute fold change (>1.0) among treatments were regarded as thresholds. Compared to the control, 9778 (8929 up- and 849 downregulated genes), 9936 (8982 up- and 954 downregulated genes), 9567 (8648 up- and 919 downregulated genes) and 1649 genes (1394 up- and 255 downregulated genes) were differentially expressed following ABC, BBC, MBC and PBC treatments, respectively (Fig 3a).
The number above each column indicated the differentially expressed genes of C. elegans.
A hierarchical clustering of correlation heat-map demonstrated the expressed gene data of ABC/MBC/BBC were grouped together, while the PBC/Control cluster were grouped together (Fig 3b). Moreover, according to correlation analyze and principal component analysis (PCA), it was confirmed that worms in the ABC, BBC and MBC treatments were very different from those in the PBC and control (Fig 4, S3 Fig). The first, second and third principal components explained 65.6%, 15.62% and 12.33% of the variation, respectively.
3.4. Functional analysis of DEGs in biochar-exposed C. elegans
The identified DEGs under the biochar treatments were further annotated with GO analysis and classified into biological process (BP), cellular component (CC), and molecular function (MF) categories. Every DEGs were annotated into different sub-categories belonging to the following three GO categories in biochar treatments (Fig 5).
Dark/light colour of columns and numbers in the figure and right axis represent the differentially expressed genes and total genes.
According to Fig 5, in BP sub-categories, the majority DEGs were related to organic substance metabolic process, metabolic process, cellular metabolic process and etc. Cellular metabolic process and translation were different among treatments. Among the CC sub-categories, the majority of GO terms were grouped into cell, intracellular, nuclear, endoplasmic reticulum, etc. Protein complex, ribonucleoprotein complex and pseudopodium were differentially expressed among biochars during environmental information processing. In MF sub-categories, genes for binding activity, catalytic activity and protein binding were in the top three terms, in which structural molecule activity and structural constituent of cuticle were more in ABC, BBC and MBC than the PBC groups.
Moreover, scatter plots displayed the GO enrichment among the different biochar groups (S4 Fig). The rich factors of ABC-, BBC- and MBC-treated groups were 0.5~0.8, which were higher than the value of PBC-treated group (0.1~0.5). And the GO enrichment analysis demonstrated that the cell and cellular progress was the largest cluster, followed by cell, cell part, intracellular and etc.
Hierarchical clustering analyses were performed on the heat-map of 20 most-expressed genes in the four biochar treatment groups (Fig 6). Among the genes analyzed, there was a significant separation of the ABC-, BBC- and MBC-treated groups from the control and PBC-treated groups.
Different colors represent the Z values of the DEGs abundance after standardization.
In addition, based on KEGG pathway mapping, the identified DEGs were classified into the following five KEGG functional categories (cellular processes, environmental information processing, genetic information processing, metabolism and organismal systems). A summary of the DEGs are demonstrated in Fig 7. According to further KEGG analysis, the top sub functional categories were N-Glycan biosynthesis, MAPK signaling pathway, TNF signaling pathway, ribosome biogenesis in eukaryotes and glycerophospholipid metabolism in ABC, BBC and MBC. Results showed that worms in the PBC treatment showed maximum alterations in proteasome, pyrimidine metabolism, RNA degradation, ribosome and apoptosis compared to the control. However, these progressed were less important in ABC, BBC and MBC groups, indicating the nematodes in other biochars varied in different ways.
Dark/light colour of columns and numbers in the figure and right axis represent the differentially expressed genes and total genes.
3.5. Analysis of target genes related to the differential phenotypes of C. elegans
Five extracellular structure component-encoding genes (col-184, col-140, col-129, lys-7, ilys-5), one life span-related gene (cdk-8), one oxidative stress-related gene (sod-1) and one eggshell vitelline layer-related gene (perm-4) were confirmed through quantitative real-time PCR. The permeable eggshell-encoding gene perm-4 was significantly downregulated under biochar treatment, whereas extracellular structure component-encoding genes such as the col-gene family (col-129, col-140, col-184) and lys-7 were differentially regulated compared to those in the control group. Life span-related genes (cdk-8) were expressed slightly up-regulated under biochar treatment compared with nematodes in the control, while the expression of oxidative stress-related genes (sod-1) was down-regulated (Fig 8).
4. Discussion
4.1. Development of C. elegans exposed to different biochars
Biochars were obtained from a variety of agricultural and forest sources so that the physicochemical components, PAHs, and VOC profiles would be correspondingly different. This study showed that the four raw materials differed in lignocellulosic biomass, and the biochars varied in ash content, pH, porosity, etc. (Table 2), similar to the differences in physico-chemical properties reported in Ronsse, van Hecke [39] and Kan, Strezov [40].
The total PAH16 content of the four biochars was less than 0.1 mg/kg, which was much lower than the European (3 mg/kg, Communities S.O.O., 2011) [41] and American (6 mg/kg, Khair et al., 2000) standards [42]. The result was similar to those of De la Rosa, Sánchez-Martín [43] and Hale, Lehmann [44], who observed significantly lower PAHs in crop residue biochars developed under low oxygen and 450–600°C pyrolysis than in biochars derived from animal manure.
As previous studies mentioned that mobile organic compounds were produced during the pyrolysis process and identified widely in biochars [45, 46], our research confirmed that both the contents and composition of VOCs were significantly varied among biochars made from different biomasses. Moreover, worms in our study showed a preference for biochar derived from peanut shells over those derived from aspen, bamboo and maize straw (Fig 2). As Baiocchi, Dillman [32] and Busch, Kammann [47] noted that nematodes can detect specific mobile compounds by using their sensory cues, the fact that the highest number of VOCs was identified from PBC might play an important role in worm attraction.
Given the exposure of nematodes on the surface of biochars, our study demonstrated that biochar shortened the life span of C. elegans and decreased body length and brood size, which was different from the results obtained with biochar applied to farmland soils. Zhang, Li [7] found no significant difference in free-living nematode abundance among 2.4–48 t/ha biochar treatments after a 7-month application. Pressler, Foster [48] also demonstrated that one year of biochar amendment did not contribute to the negative effects of soil nematodes in a crop field. Thus, without soil buffer capacity, biochar-exposed treatment may result in environmental stress of the growth of C. elegans.
Moreover, there is an obvious difference between PBC-exposed nematodes and ABC/BBC/MBC-exposed worms. PBC-exposed C. elegans had milder decreases in body length, brood size and SOD activity relative to other biochar-exposed worms. Dissolved organic matter (DOM) may be an important factor to alleviate adverse impact on C. elegans. Hoss [49] implied that DOM could potentially be an additional carbon source for bacterial cells and serving as food for nematodes. Lieke [50] found fulvic acid played an essential role in the stability of environmental persistent free radicals (EPFRs) and may protect aquatic organism species from the harms of persistent free radicals. The dissolved organic carbon of PBC was 29.46 mg/L, which was approximately 4–5 times than that of other biochars in Table 2. Thus, higher DOM of PBC was assumed to alleviate the harmful environmental impact on C. elegans. Nonetheless, further investigation is needed to clarify the effect of DOM derived from biochar on nematodes.
4.2. Transcriptome analysis of C. elegans exposed to different biochars
Based on the phenotypic variation of C. elegans under biochar treatment, RNA-seq analysis can reveal the mechanism of transcription. According to gene ontology (GO) enrichment analysis, biochar mainly impacts genes involved in sensory processing, cuticle development, reproductive processes and immune system processes in C. elegans (Table 4). It was also found that several DEGs were obviously different among the four kinds of biochars (Table 5).
Although C. elegans seemed not to sense and/or be attracted to the ABC/BBC/MBC, the odr-4 and odr-10 genes upregulated significantly in ABC/BBC/MBC treatment. These genes were important to encode a tail-anchored transmembrane protein of ODR-10, which mediates C. elegans chemotaxis to volatile odors [63]. It seemed that the worms in ABC/BBC/MBC treatment attempt to strengthen their olfactory receptor, even if there is very few volatile odors detected from ABC/BBC/MBC. The regulation of odor sensory processing of nematode is complex, therefore, further confirmation of the adaptive process is needed.
Cuticle collagen synthesis is a complex process throughout life, and it has been shown that a multigene family of approximately 154 members could encode collagen-like proteins [34]. The most important genes were involved in the modification of the worm exoskeleton belong to the col, vit and rpl gene families [64]. Specific target genes varied among biochars. For instance, col-40/81/129 was associated with PBC amendment, while col-20/124/106, col-20/124/140 and col-119/122/140 were related to the MBC, ABC and BBC treatments, respectively.
Biochar significantly impacts the reproductive process, resulting in a smaller brood size. Perm-2 and perm-4 were reported as key genes for eggshell formation [35, 64] and were the top downregulated genes under all biochar treatments in this study (Table 4). Recent studies stated that perm-2 and perm-4 encode the vitelline layer to protect the embryonic soluble factors from leaking to extracellular matrix [58]. The process is as follows: proteins PERM-2 and PERM-4 hinge to each other and then stabilize on CBD-1 to form the vitelline layer, and inhibiting any of them makes the adhesive component of the eggshell surface absent, which might prevent the connection alternatively between uterine secretions and the outer eggshell layer. Therefore, losing this protective barrier would cause not only soluble factor leakage but also embryonic death [65]. As a result, the down-regulating perm-2/4 genes could be associated with the brood size inhibition in biochar treatment.
Other genes, such as members of the sqt, bli, and dpy gene families encoding major protein components of the cuticle [51, 52, 56]. Furthermore, Frankie found that the mutants of these target genes are significantly shorter and fatter than the wild type [56]. Thereby, variations of sqt-3, bli-6 and dpy-17 gene expression would be associated with shorter body length in biochar-treated groups [66, 67].
Additionally, the downregulation of sip-1 and upregulation of irg-7 were also detected in the ABC, BBC and MBC treatments (Table 5) and are associated with embryo development [61, 62] and carbohydrate binding activity [60, 68], indicating more inhibition of brood size in the other three biochars than in PBC.
Genes involved in rhythmic processes were also detected via transcriptome analysis. The cdk-8 gene was upregulated in all biochar treatments (Table 4). According to Steimel, Suh [36], CDK-8 is required in neurons for correct navigation of commissures and interneuron axons in the ventral cord. Further research demonstrated that the cdk-8 mutant had commissure navigation defects [61, 69], suggesting that C. elegans had to upregulate cdk-8 to repress a specific pathway to ensure proper commissure navigation [36]. Moreover, the daf-16, sip-1 and icl-1 genes are also associated with determinate worm lifespan by encoding DAF-16, which is a necessary component of promotor orthologs (FOXO1, FOXO3, and FOXO4) [53, 70]. In this study, the downregulation of the sip-1 gene was found in the ABC, BBC and MBC treatments (Table 5), while the downregulation of icl-1 was found in the PBC treatment only, indicating that different biochars regulated different genes to shorten the nematode lifespan.
The C. elegans genome encodes catalase (CTL) and superoxide dismutase (SOD) to eliminate mitochondrial and cytoplasmic ROS. According to previous reports, sod-1 encodes cytosolic Cu/Zn SOD, sod-2/5 encodes mitochondrial (Mn-SOD) isoforms [37, 53], and ctl-1/2 encodes an unusual cytosolic isoform or a typical peroxisomal catalase [37]. Corroborated by Schaar, Dues [71], this study demonstrated that the SOD activity, brood size and lifespan of C. elegans were significantly changed in the ABC, BBC and MBC treatments in comparison with those in PBC and the control (Table 5), which confirmed that the interaction among ctl-1 and sod-2 may cause a lower brood size and an abnormal lifespan under oxidative stress.
In summary, C. elegans was significantly underdeveloped in body growth, reproduction and life span under different biochar treatments, and these changes were associated with key gene variation under biochar exposure. Genes in the col, dpy, and bli families were differentially expressed, affecting coding of the major proteins of the cuticle and disrupting the development of the exoskeleton; genes in the perm, sip and irg families were associated with eggshell formation during embryonic development. Other genes, such as members of the daf, sip, icl, lys, ctl and sod families, were involved in life-span regulation and immune systems. Our study explored the effects of different biochars on C. elegans and the related mechanisms, providing insights for the use of biochar in agricultural management.
5. Conclusion
Biochar made from different agricultural wastes varied in physicochemical properties, total PAH16 contents and VOCs. We tested the phenotypic variation, selective behavior and transcriptome mechanisms of C. elegans in response to four different biochars. This study noted that most worms were located near the biochar made from peanut shell after 56 h of exposure, indicating that PBC could produce more VOCs to attract C. elegans than ABC, BBC and MBC. Furthermore, RNA-seq analysis demonstrated that ABC, BBC MBC and PBC downregulated 849, 862, 919 and 255 C. elegans genes and upregulated 8929, 8236, 8648 and 1394 genes, respectively, in comparison to the control. The main target genes included those related to collagen (bli-6, sqt-3, dpy-17 and col gene families), reproductive processes (perm-2/4, sip-1 and irg-7), rhythmic processes (daf-16, sip-1, icl-1 and cdk-8) and immune systems (sod-1/2/5, ctl-1/2 and lys-7). Variations in those genes might have contributed to a significantly shorter body length and life span as well as a smaller brood size. Overall, our study clarified the response of C. elegans to different biochars and revealed the underlying mechanisms of biochar interactions with nematodes.
Supporting information
S1 Fig. Experimental setup of the chemotaxis assay.
PBC was placed in A, and each other biochar (MBC/ABC/BBC) was places in B, respectively. C point refers the center of the petri dish. Gray color indicated the scoring region for counting C. elegans.
https://doi.org/10.1371/journal.pone.0284348.s001
(TIF)
S2 Fig. Chromatograms of biochars derived from peanut shell, aspen, bamboo and maize straw, corresponding to PBC, ABC, BBC and MBC, respectively.
The numbers refer to the volatile organic compounds in Table 3.
https://doi.org/10.1371/journal.pone.0284348.s002
(TIF)
S3 Fig. Correlation of C. elegans expressed genes between biochar treatments and the control.
https://doi.org/10.1371/journal.pone.0284348.s003
(TIF)
S4 Fig. Major gene ontology (GO) terms for C. elegans exposed to different biochar, respectively.
Rich factor was the ratio of significant to annotated DEGs.
https://doi.org/10.1371/journal.pone.0284348.s004
(TIF)
S1 Table. Lists of differentially expressed genes of C. elegans for each biochar.
https://doi.org/10.1371/journal.pone.0284348.s005
(XLSX)
S2 Table. All identified GO categories of C. elegans for each biochar.
https://doi.org/10.1371/journal.pone.0284348.s006
(XLSX)
S3 Table. All identified KEGG categories of C. elegans for each biochar.
https://doi.org/10.1371/journal.pone.0284348.s007
(XLSX)
References
- 1. Wang B, Dong F, Chen M, Zhu J, Tan J, Fu X, et al. Advances in recycling and utilization of agricultural wastes in China: based on environmental risk, crucial pathways, influencing factors, policy mechanism. Procedia Environmental Sciences.2016;31:12–17.
- 2. Beesley L, Moreno-Jimenez E, Gomez-Eyles JL, Harris E, Robinson B, Sizmur T. A review of biochars’ potential role in the remediation, revegetation and restoration of contaminated soils. Environmental Pollution. 2011; 159(12): 3269–3282. pmid:21855187
- 3. Oliveira MND, Santos PA, Silva G, Guenther C, Mary C, De AB, et al. Biochar dosage and granulomaetry influencing soil density and water retention. International Journal of Agriculture Sciences. 2018;10(4):5153–5157.
- 4. Lehmann J, Rillig MC, Thies J, Masiello CA, Hockaday WC, Crowley D. Biochar effects on soil biota—A review. Soil Biology and Biochemistry. 2011;43(9):1812–1836. doi:
- 5. Zhu X, Chen B, Zhu L, Xing B. Effects and mechanisms of biochar-microbe interactions in soil improvement and pollution remediation: A review. Environmental Pollution. 2017; 227: 98–115. pmid:28458251
- 6. Darby B, Neher D, Belnap J. Soil nematode communities are ecologically more mature beneath late- than early- successional stage biological soil crusts. Applied Soil Ecology. 2007; 35(1): 203–212.
- 7. Zhang XK, Li Q, Liang WJ, Zhang M, Bao XL, Xie ZB. Soil nematode response to biochar addition in a Chinese wheat field. Pedosphere. 2013; 23(1): 98–103.
- 8. Rahman L, Whitelaw-Weckert MA, Orchard B. Impact of organic soil amendments, including poultry-litter biochar, on nematodes in a Riverina, New South Wales, vineyard. Soil Research. 2014;52(6): 604–619.
- 9. Huang WK, Ji HL, Gheysen G, Debode J, Kyndt T. Biochar-amended potting medium reduces the susceptibility of rice to root-knot nematode infections. BMC Plant Biol. 2015; 15: 267. pmid:26537003
- 10. Cao Y, Gao Y, Qi Y, Li J. Biochar-enhanced composts reduce the potential leaching of nutrients and heavy metals and suppress plant-parasitic nematodes in excessively fertilized cucumber soils. Environ Sci Pollut Res Int. 2018; 25(8): 7589–7599. pmid:29282668
- 11. Lieke T, Zhang X, Steinberg CEW, Pan B. Overlooked Risks of Biochars: Persistent Free Radicals trigger Neurotoxicity in Caenorhabditis elegans. Environ Sci Technol. 2018; 52(14): 7981–7987. pmid:29916700
- 12. Brassard P, Godbout S, Raghavan V. Pyrolysis in auger reactors for biochar and bio-oil production: A review. Biosystems Engineering. 2017; 161: 80–92.
- 13. McBeath AV, Smernik RJ, Krull ES, Lehmann J. The influence of feedstock and production temperature on biochar carbon chemistry: A solid-state 13C NMR study. Biomass and Bioenergy. 2014; 60: 121–129.
- 14. Dorez G, Ferry L, Sonnier R, Taguet A, Lopez-Cuesta JM. Effect of cellulose, hemicellulose and lignin contents on pyrolysis and combustion of natural fibers. Journal of Analytical and Applied Pyrolysis. 2014;107:323–331.
- 15. Lebrun M, Miard F, Hattab-Hambli N, Scippa GS, Bourgerie S, Morabito D. Effect of different tissue biochar amendments on As and Pb stabilization and phytoavailability in a contaminated mine technosol. Science of The Total Environment. 2019:135657. pmid:31784149
- 16. Domene X, Mattana S, Hanley K, Enders A, Lehmann J. Medium-term effects of corn biochar addition on soil biota activities and functions in a temperate soil cropped to corn. Soil Biology and Biochemistry. 2014;72:152–62.
- 17. Wang YY, Jing XR, Li LL, Liu WJ, Tong ZH, Jiang H. Biotoxicity evaluations of three typical biochars using a simulated system of fast pyrolytic biochar extracts on organisms of three kingdoms. Acs Sustainable Chemistry & Engineering. 2017;5(1): 481–488.
- 18. Zhang K, Mao J, Chen B. Reconsideration of heterostructures of biochars: Morphology, particle size, elemental composition, reactivity and toxicity. Environmental Pollution. 2019; 254: 113017. pmid:31415977
- 19. He K, Zhang J, Zeng Y, Zhang L. Households’ willingness to accept compensation for agricultural waste recycling: taking biogas production from livestock manure waste in Hubei, P. R. China as an example. Journal of Cleaner Production. 2016;131: 410–420.
- 20. Zhang Q, Wang J, Wang G, Shamsi IH, Wang X. Site-specific nutrient management in a bamboo forest in Southeast China. Agriculture, Ecosystems & Environment. 2014;197:264–270.
- 21. Suthar RG, Wang C, Nunes MC, Chen J, Sargent SA, Bucklin RA, et al. Bamboo Biochar Pyrolyzed at Low Temperature Improves Tomato Plant Growth and Fruit Quality. Agriculture. 2018;8,153: 1–13.
- 22. Peiris C, Gunatilake SR, Wewalwela JJ, Vithanage M. Biochar for Sustainable Agriculture. In: Ok YS, Tsang DCW, Bolan N, Novak JM, editors. Biochar from Biomass and Waste. 2019; Chapter 11: 211–224.
- 23. Soest P J Vax, Use of Detergents in the Analysis of Fibrous Feeds. II. A Rapid Method for the Determination of Fiber and Lignin. Journal of Association of Official Agricultural Chemists. 1963;45(5): 829–835.
- 24. Li J, Chen Y, Zhang G, Ruan W, Shan S, Lai X, et al. Integration of behavioural tests and transcriptome sequencing of C. elegans reveals how the nematode responds to peanut shell biochar amendment. Science of The Total Environment. 2020;707: 136024. pmid:31972909
- 25. Rogovska N, Laird D, Cruse RM, Trabue S, Heaton E. Germination tests for assessing biochar quality. J Environ Qual. 2012;41(4): 1014–1022. pmid:22751043
- 26. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94. pmid:4366476
- 27. He F. Common Worm Media and Buffers. Bio-protocol 2011;1(7): e55.
- 28. Zhou D, Yang J, Li H, Cui C, Yu Y, Liu Y, et al. The chronic toxicity of bisphenol A to Caenorhabditis elegans after long-term exposure at environmentally relevant concentrations. Chemosphere. 2016;154: 546–551. pmid:27085314
- 29. Zhang J, Chen R, Yu Z, Xue L. Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans. Bio-protocol. 2017;7(16): e2505. pmid:34541169
- 30. Mishra N, Mallick S, Negi VD. Salmonella Typhimurium infection causes defects and fastening of Caenorhabditis elegans developmental stages. Microbes Infect. 2022;24(3):104894. pmid:34756991
- 31. Madžarić S, Kos M, Drobne D, Hočevar M, Jemec Kokalj A. Integration of behavioral tests and biochemical biomarkers of terrestrial isopod Porcellio scaber (Isopoda, Crustacea) is a promising methodology for testing environmental safety of chars. Environmental Pollution. 2018;234:804–11. pmid:29247943
- 32. Baiocchi T, Dillman AR. Chemotaxis and Jumping Assays in Nematodes. Bio-protocol. 2015; 5(18):e1587.
- 33. Gui T, Yao C, Jia B, Shen K. Identification and analysis of genes associated with epithelial ovarian cancer by integrated bioinformatics methods. PLoS One. 2021;16(6): e0253136. pmid:34143800
- 34. Johnstone IL. Cuticle collagen genes: expression in Caenorhabditis elegans. Trends in Genetics. 2000;16(1):21–27. pmid:10637627
- 35. Carvalho A, Olson SK, Gutierrez E, Zhang K, Noble LB, Zanin E, et al. Acute drug treatment in the early C. elegans embryo. PloS one. 2011;6(9): e24656–e. pmid:21935434
- 36. Steimel A, Suh J, Hussainkhel A, Deheshi S, Grants JM, Zapf R, et al. The C. elegans CDK8 Mediator module regulates axon guidance decisions in the ventral nerve cord and during dorsal axon navigation. Developmental Biology. 2013;377(2): 385–398. pmid:23458898
- 37. Vanfleteren JR, Braeckman BP. Mechanisms of life span determination in Caenorhabditis elegans. Neurobiology of Aging. 1999;20(5):487–502. pmid:10638522
- 38. Zhang Y, Chen D, Smith MA, Zhang B, Pan X. Selection of reliable reference genes in Caenorhabditis elegans for analysis of nanotoxicity. PLoS One. 2012;7(3): e31849. pmid:22438870
- 39. Ronsse F, van Hecke S, Dickinson D, Prins W. Production and characterization of slow pyrolysis biochar: influence of feedstock type and pyrolysis conditions. Global Change Biology Bioenergy. 2013;5(2): 104–115.
- 40. Kan T, Strezov V, Evans TJ. ChemInform Abstract: Lignocellulosic Biomass Pyrolysis: A Review of product properties and effects of pyrolysis parameters. ChemInform. 2016;47(28).
- 41.
Communities S O O. Sustainable development in the European Union: Monitoring report of the EU sustainable development strategy[M]. Publications Office of the European Union, 2011.
- 42.
Khair M, Lemaire J, Fischer S. Achieving heavy-duty diesel NOx/PM levels below the EPA 2002 standards an integrated solution[C]. SAE 2000 World Congress, 2000.
- 43. De la Rosa JM, Sánchez-Martín ÁM, Campos P, Miller AZ. Effect of pyrolysis conditions on the total contents of polycyclic aromatic hydrocarbons in biochars produced from organic residues: Assessment of their hazard potential. Science of The Total Environment. 2019;667: 578–585. pmid:30833256
- 44. Hale SE, Lehmann J, Rutherford D, Zimmerman AR, Bachmann RT, Shitumbanuma V, et al. Quantifying the Total and Bioavailable Polycyclic Aromatic Hydrocarbons and Dioxins in Biochars. Environmental Science & Technology. 2012;46(5):2830–2838. pmid:22321025
- 45. McDonald JD, Zielinska B, Fujita EM, Sagebiel JC, Chow JC, Watson JG. Fine Particle and Gaseous Emission Rates from Residential Wood Combustion. Environmental Science & Technology. 2000;34(11): 2080–2091.
- 46. Spokas KA, Novak JM, Stewart CE, Cantrell KB, Uchimiya M, DuSaire MG, et al. Qualitative analysis of volatile organic compounds on biochar. Chemosphere. 2011;85(5):869–882. pmid:21788060
- 47. Busch D, Kammann C, Grunhage L, Muller C. Simple biotoxicity tests for evaluation of carbonaceous soil additives: establishment and reproducibility of four test procedures. Journal of Environmental Quality. 2012;41(4):1023–1032. pmid:22751044
- 48. Pressler Y., Foster E.J., Moore J.C. and Cotrufo M.F. (2017), Coupled biochar amendment and limited irrigation strategies do not affect a degraded soil food web in a maize agroecosystem, compared to the native grassland. GCB Bioenergy, 9: 1344–1355.
- 49. Hoss S, Bergtold M, Haitzer M, Traunspurger W, Steinberg CEW. Refractory dissolved organic matter can influence the reproduction of Caenorhabditis elegans (Nematoda). Freshwater Biology, 2001,46(1): 1–10.
- 50. Lieke T, Steinberg CEW, Pan B, Perminova IV, Meinelt T, Knopf K, et al. Phenol-rich fulvic acid as a water additive enhances growth, reduces stress, and stimulates the immune system of fish in aquaculture. Sci. Rep. 2021. 11, 174. pmid:33420170
- 51. Lang AE, Lundquist EA. The collagens DPY-17 and SQT-3 direct anterior-posterior migration of the Q neuroblasts in C. elegans. Journal of developmental biology. 2021; 9(7): 1–15. pmid:33669899
- 52. Novelli J, Page AP, Hodgkin J. The C terminus of collagen SQT-3 has complex and essential functions in nematode collagen assembly. Genetics. 2006;172(4): 2253–2267. pmid:16452136
- 53. Curtis C, Landis GN, Folk D, Wehr NB, Hoe N, Waskar M, et al. Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes. Genome Biol. 2007;8(12): R262. pmid:18067683
- 54. Johnston AD, Ebert PR. The Redox system in C. elegans, a phylogenetic approach. Journal of Toxicology 2012: 546915. pmid:22899914
- 55. Levy AD, Kramer JM. Identification, sequence and expression patterns of the Caenorhabditis elegans col-36 and col-40 collagen-encoding genes. Gene. 1993;137(2): 281–285. pmid:8299960
- 56. Engel J, Furthmayr H, Odermatt E., Mark HVD, Aumailley M, Fleischmajer R, et al. Structure and macromolecular organization of the type VI collagen. Biology, Chemistry, and Pathology of Collagen. 1985; 460(1): 25–37.
- 57. Schulenburg H., Boehnisch C. Diversification and adaptive sequence evolution of caenorhabditis lysozymes (Nematoda: Rhabditidae). BMC Evol Biol. 2008;8:114. pmid:18423043
- 58. Gonzalez DP, Lamb HV, Partida D, Wilson ZT, Harrison MC, Prieto JA, et al. CBD-1 organizes two independent complexes required for eggshell vitelline layer formation and egg activation in C. elegans. Developmental Biology. 2018; 442(2): 288–300. pmid:30120927
- 59. Zhang Y, Zhao C, Zhang H, Liu R, Wang S, Pu Y, et al, Integrating transcriptomics and behavior tests reveals how the C. elegans responds to copper induced aging, Ecotoxicology and Environmental Safety, 2021; 222: 112494. pmid:34265532
- 60. Yunger E, Safra M, Levi-Ferber M, Haviv-Chesner A, Henis-Korenblit S. Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7. PLoS Genet. 2017; 13(2): e1006577. pmid:28196094
- 61. Lee S S. DAF-16 Target Genes That Control C. elegans Life-Span and Metabolism. Science.2003;300(5619): 644–647. pmid:12690206
- 62. Murphy C., McCarroll S., Bargmann C. Fraser A, Kamath R, Ahringer J, et al. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature. 2003;424: 277–283. pmid:12845331
- 63. Chen C, Itakura E, Weber KP, Hegde RS, de Bono M. An ER complex of ODR-4 and ODR-8/Ufm1 specific protease 2 promotes GPCR maturation by a Ufm1-independent mechanism. PLoS Genet. 2014;10(3): e1004082. pmid:24603482
- 64. Boxem M, Maliga Z, Klitgord N, Li N, Lemmens I, Mana M, et al. A protein domain-based interactome network for C. elegans early embryogenesis. Cell. 2008;134(3): 534–545. pmid:18692475
- 65. Ko Frankie C.F, Chow King L, A mutation at the start codon defines the differential requirement of dpy-11 in Caenorhabditis elegans body hypodermis and male tail. Biochemical and Biophysical Research Communications. 2003;309(1): 201–208. pmid:12943683
- 66. Kramer J, Johnson J, Edgar R, Basch C, Robebli S. The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis, Cell. 1988;55(4): 555–565. pmid:3180220
- 67. Linder B, Jin Z, Freedman JH, Rubin CS. Molecular characterization of a novel, developmentally regulated small embryonic chaperone from Caenorhabditis elegans. The Journal of biological chemistry. 1996; 271(47): 30158–30166.
- 68. Sun YY, Huang XM, Wang YH, Shi ZH, Liao YY, Cai P. Lipidomic alteration and stress-defense mechanism of soil nematode Caenorhabditis elegans in response to extremely low-frequency electromagnetic field exposure. Ecotoxicology and Environmental Safety. 2019;170: 611–619. pmid:30579161
- 69. Knuesel MT, Meyer KD, Donner AJ, Espinosa JM, Taatjes DJ. The human CDK8 subcomplex is a histone kinase that requires Med12 for activity and can function independently of mediator. Molecular and Cellular Biology. 2009; 29(3): 650–661. pmid:19047373
- 70. Schulenburg H, Hoeppner MP, Weiner J, Bornberg-Bauer E. Specificity of the innate immune system and diversity of C-type lectin domain (CTLD) proteins in the nematode Caenorhabditis elegans. Immunobiology. 2008; 213(3–4): 237–250. pmid:18406370
- 71. Schaar CE, Dues DJ, Spielbauer KK, Machiela E, Cooper JF, Senchuk M, et al. Mitochondrial and Cytoplasmic ROS Have Opposing Effects on Lifespan. PLoS Genet. 2015;11(2): e1004972. pmid:25671321