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Article

Transcriptome Analysis of the Nematodes Caenorhabditis elegans and Litoditis marina in Different Food Environments

by
Peiqi Sun
1,2,3,4,
Xuwen Cao
1,2,3,4 and
Liusuo Zhang
1,2,3,*
1
CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
2
Laboratory of Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China
3
Center for Ocean Mega-Science, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China
4
University of Chinese Academy of Sciences, Beijing 100049, China
*
Author to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2022, 10(5), 580; https://doi.org/10.3390/jmse10050580
Submission received: 18 March 2022 / Revised: 19 April 2022 / Accepted: 21 April 2022 / Published: 25 April 2022
(This article belongs to the Special Issue Genetic Diversity in Marine Organisms: Assessment, Conservation and Sustainable Utilization)
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Abstract

:
Diets regulate animal development, reproduction, and lifespan. However, the underlying molecular mechanisms remain elusive. We previously showed that a chemically defined CeMM diet attenuates the development and promotes the longevity of C. elegans, but whether it impacts other nematodes is unknown. Here, we studied the effects of the CeMM diet on the development and longevity of the marine nematode Litoditis marina, which belongs to the same family as C. elegans. We further investigated genome-wide transcriptional responses to the CeMM and OP50 diets for both nematodes, respectively. We observed that the CeMM diet attenuated L. marina development but did not extend its lifespan. Through KEEG enrichment analysis, we found that many of the FOXO DAF-16 signaling and lysosome and xenobiotic metabolism related genes were significantly increased in C. elegans on the CeMM diet, which might contribute to the lifespan extension of C. elegans. Notably, we found that the expression of lysosome and xenobiotic metabolism pathway genes was significantly down-regulated in L. marina on CeMM, which might explain why the CeMM diet could not promote the lifespan of L. marina compared to bacterial feeding. Additionally, the down-regulation of several RNA transcription and protein generation and related processes genes in C. elegans on CeMM might not only be involved in extending longevity, but also contribute to attenuating the development of C. elegans on the CeMM diet, while the down-regulation of unsaturated fatty acids synthesis genes in L. marina might contribute to slow down its growth while on CeMM. This study provided important insights into how different diets regulate development and lifespan, and further genetic analysis of the candidate gene(s) of development and longevity will facilitate exploring the molecular mechanisms underlying how diets regulate animal physiology and health in the context of variable nutritional environments.

1. Introduction

Diets regulate animal development, reproduction, and lifespan [1]. Different dietary habits can affect the structure and metabolome of gut microbiota and may contribute to health or the pathogenesis of disorders such as atherosclerosis, coronary vascular disease, and inflammatory bowel disease both in humans and mice [2,3]. Diets modulate lifespan, consumption, and fat deposition in flies [4]. Dietary restriction (DR), which influences organelle function, gene expression, neural signaling, can extend the lifespan of every species that has been tested, including invertebrates and mammals [5].
Caenorhabditis elegans has been widely used to study the nutritional regulation of biological processes such as development and longevity [6,7,8,9,10]. It was known that C. elegans developed from L1 larva to adult in about 3 days by feeding on the bacteria E. coli OP50, the standard laboratory food, at 20 °C [11]. In C. elegans, distinctive food can affect its development, reproduction, and lifespan [12,13,14]. For instance, feeding on the bacteria Comamonas DA1877 led C. elegans to lay fewer eggs and exhibit a shorter lifespan compared to the E. coli OP50 diet [1]. The C. elegans maintenance medium (CeMM) diet was developed as an axenic, chemically defined food source, and is a suboptimal food source for wild-type C. elegans [15,16]. Our previous study showed that only about 10% of the N2 larvae had developed into adults after 8 days post-hatching when grown on CeMM, and their development was significantly attenuated compared to those that underwent bacterial feeding [17]. The advantages of the axenic chemically defined diet have made CeMM widely applied in the study of the effects of space flight and pharmaceutical drugs on the essential biological processes in C. elegans [10,15,17,18,19].
After five years of intensive efforts, our research group has developed Litoditis marina as a promising marine nematode model, which could be used as a marine satellite animal model to the terrestrial biomedical nematode model C. elegans [20,21,22,23]. Given that L. marina and C. elegans belong to the same family of Rhabditidae in the phylum Nematoda (Xie et al., 2020), this allows us to compare the physiological changes of marine and terrestrial relatives in response to changing nutritional environments. L. marina is widely distributed along coasts and estuaries around the world [24,25,26]. It has been reported that the overall embryonic cell lineage homology is 95.5% between L. marina and C. elegans, while the fate homology is 76.4%, of which differences lay in nerve, epidermis, and pharyngeal tissues [25]. Similar to C. elegans, the generation cycle of L. marina is short, and it takes 4–5 days for newly hatched L1 larvae to develop into adults at room temperature [22]. Xie et al. conducted an in-depth analysis on the association of developmental phenotypes and transcriptome characteristics of L. Marina in response to low and high salt stress [21]. Cao et al. performed transcriptome analysis in L. marina on the CeMM diet with stearic acid supplementation [27]. Therefore, L. marina is a potential marine nematode model to comparatively study the essential biological processes, such as development, reproduction, and longevity, between the marine and terrestrial nematode relatives in response to environmental changes in the context of global climate change.
In this report, we found that CeMM diets attenuated the development of L. marina compared to feeding on bacteria, which is in line with C. elegans. However, we observed that the lifespan of L. marina on the CeMM diet was comparable to that of bacterial feeding, in contrast to the previous report that the CeMM diet promotes the longevity of C. elegans. We further investigated their transcriptomic signatures via RNA sequencing (RNA-seq) in different food environments. The transcriptomic analysis of C. elegans on CeMM can deepen our understanding of how CeMM regulates development and longevity. Further, the transcriptome signature of C. elegans can serve as a base point to help compare the differences and similarities with L. marina, thus giving us more insight into marine and terrestrial organisms in response to changing nutritional environments. Further genetic analysis of the key candidate gene(s) will provide an understanding of the molecular mechanisms underlying how diets regulate animal physiology and health in the context of variable nutritional environments.

2. Materials and Methods

2.1. Cultivation of Nematodes

The C. elegans wild-type Bristol N2 strain was obtained from the Caenorhabditis Genetics Center (CGC). At 20 °C, OP50 bacteria were used to culture the C. elegans strain on nematode growth medium (NGM) plates using standard methods [11]. For the CeMM part, the 1.7% agarose CeMM plate was used directly at 20 °C when conducting worm growth observation [10]. L. marina was grown at 20 °C on seawater NGM plates inoculated with E. coli OP50. 15‰ sea salt CeMM-agarose plates were prepared for the L. marina experiments.

2.2. Worm Synchronization

To obtain synchronized nematodes, the hermaphrodites of C. elegans with full eggs were bleached with a basic hypochlorite solution at room temperature until each adult worm was digested [28]. Synchronized L1s of C. elegans were obtained, followed by egg collection and hatching overnight on unseeded nematode growth medium NGM plates at 20 °C for about 12 h. To obtain synchronized nematodes of L. marina, a mass of L. marina, including male and female, were cultivated on a 9 cm medium. When there were many eggs on the plate, the eggs were collected into a 15 mL centrifuge tube at 1300× g for 1 min. After being washed twice with sterilized water, the eggs were treated with alkaline hypochlorite solution at room temperature, 60–90 s, 1300× g for 1 min. The egg liquid was incubated in sterilized seawater for 21 h. After incubation, the unhatched eggs and remnants were removed by 450 mesh filtrations with sterilized seawater after extraction. Then 500 mesh filter was used for a second filtration for L1 larvae filtrate to further remove remnants. After centrifugation for 1 min, the synchronized L1s were obtained.

2.3. CeMM Preparation

CeMM stock media (2×) was prepared using a predefined list of components, including vitamins, salt, amino acids, and nucleic acid substituents [17,18]. In brief, the above chemical solutions were prepared separately and then mixed. Next, L-reduced glutathione, dihydrocholine citrate, inositol, cytochrome C, glucose, β-sitosterol, streptomycin (200 mg/L), and kanamycin (40 mg/L) were added to the above mixture. The pH of the mixture was adjusted to 5.2 using a 10% (w/v) potassium hydroxide solution. The mixed media was filter-sterilized using a Corning 0.2 mm filter system, and the final stock media were stored at 4 °C. When using, 100 mL 2× CeMM final stock media were mixed with 100 mL 3.4% agarose, diluting the final CeMM content to 1× and agarose to 1.7%. Nystatin (200 mg nystatin/14 mL 95% ethanol) was subsequently added to the mixed solution. For 15‰ sea salt CeMM-agarose, 3 g sea salt were added when prepared 3.4% agarose. All operations were performed in a sterile environment. The mixed medium was divided into Petri dishes of 3.5 cm diameter, which contained 3 mL of CeMM-agarose. The CeMM-agarose plate was kept at 4 °C, away from light.

2.4. Growth and Lifespan Assay

For growth assay, 70 newly hatched L1 larvae of L. marina were transferred and cultured in each 3-cm-diameter seeded SW-NGM (10 µL OP50 per plate) and 15‰ sea salt CeMM plates. The worms were observed using a Stereo Microscope, and the adults were counted and picked out every day until 15 days. The adulthood rate of worms was calculated by counting the adult number every day and dividing by the total number. Females were age-assessed by the developmental stage of the vulva, while males were age-assessed based on the developmental stage of their proctodeum. Three replicates were performed for each assay. For lifespan assay, 40–60 L4 females were transferred to SW-NGM with OP50 and 15‰ sea salt CeMM-agarose plates, respectively. The number of live and dead worms was determined using a dissecting microscope every 48 h. Live worms were transferred to fresh 15‰ sea salt CeMM-agarose plates or SW-NGM with OP50. Worms were scored dead if no response was detected after prodding with a platinum wire. Dead worms on the wall of the plate were not counted. The number of live and dead worms was counted every day. The mean lifespan of worms was calculated by the total days of all the worms divided by the total number. The comparisons between the two groups were performed using the Log-rank (Mantel–Cox) test. A p value of <0.05 was considered statistically significant. All data were analyzed by GraphPad Prism 8 software.

2.5. RNA-Seq Library Preparation and Analysis

To acquire synchronized C. elegans newly hatched L1 larvae, the adults with full eggs were bleached using standard hypochlorite to obtain embryos. Larvae were hatched and underwent growth arrest on unseeded plates [28]. The synchronized L1 larvae of N2 were transferred to E. coli OP50 and CeMM plates, respectively, and treated for 2.5 h. Similarly, the synchronized L1s of L. marina were transferred to 90 mm Sea-Water (SW) plates full of E. coli and 15‰ sea salt CeMM-agarose plates for feeding for 2.5 h. Three replicates (around 60,000 worms per replicate) were performed for both N2 and L. marina. The worms were washed with M9 three times to remove remnants of the medium. The samples were then transferred to 1.5 mL tubes, and the excess supernatants were removed via centrifugation (1300× g, 1 min). The supernatant liquid was removed as soon as possible and the samples were frozen using liquid nitrogen for 5 min and stored at −80 °C. The samples were swiftly ground in the liquid nitrogen and then RNA was extracted with the Trizol method. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. First-strand cDNA was synthesized with fragmented mRNA as a template and random hexamers as primers, followed by second-strand synthesis with the addition of a PCR buffer, dNTPs, RNase H, and DNA polymerase I. The purification of cDNA was processed with the AMPure XP system (Beckman Coulter, Beverly, CA, USA). After reverse transcription, cDNA fragments of preferentially 370~420 bp in length were selected and purified to prepare for the library (VAHTS mRNA-seq V3 Library Prep Kit for Illumina®, Nanjing, China). After the library was constructed, Qubit3.0 Fluorometer was used for initial quantification. Nanodrop 2000 was used to detect concentration and Agilent 2100 BioAnalyzer was then used to detect the insert size of the library, and then Qsep-400 system (Bioptic) was used to ensure library quality, followed by a qualified library inspection. Illumina sequencing was performed by illumina NovaSeq 6000, and 150 bp paired-end reads were generated. Reference genome and gene model annotation files were downloaded from Ensembl (Caenorhabditis elegans (WBcel235)). The used reference genome of L. marina was the genome generated in our laboratory [22]. Paired-end reads were processed with fastp for adapters and low-quality reads (Q10) removal and SOAP for rRNA and mRNA filtering. Clean reads were mapped to the reference genome with Hisat2 [29] and quantified by StringTie in expression estimation mode(-eB) [30]. KEGG function was assigned by best hit (with an E value cutoff of 1 × 10−5) to the KEGG database (http://www.genome.jp/kegg/ accessed on 27 July 2020) by using KOBAS 3.0 [31], and then FPKM was calculated based on the length of the gene and the reads count mapped for each gene [32]. Differential expression analysis of two groups (three biological replicates per condition) was performed using the DESeq2 R package (1.20.0) [33]. Benjamini and Hochberg’s approach was applied to the false discovery rate. |Log2Fold change| > 1 and an adjusted p-value (padj) < 0.01 were used as the threshold for significantly differential expressions. Log2FC in Figures was further calculated by the ratio of the FPKM of treatment groups (CeMM) to the average FPKM of control groups (OP50). The KEGG pathway enrichment analysis of differentially expressed genes (DEGs) was performed by the clusterProfiler R package [34].

2.6. Real-Time Quantitative PCR (qPCR)

Some of the key genes of our interest were randomly selected for qPCR validation, including cytochrome P450 pathway related genes; glutathione S-transferase gene gst-4 in C. elegans; glutathione S-transferase gene EVM0002486/gst-8 and UDP-glucuronosyl transferase EVM0005671/ugt-19 in L. marina; peroxisomal β-oxidation pathway related gene acyl-coenzyme A oxidase acox-3 in C. elegans; daf-16 target gene superoxide dismutase sod-3 in C. elegans; ribosome biogenesis pathway gene eukaryotic initiation factor eif-6 in C. elegans; protein processing in endoplasmic pathway gene heat shock protein hsp-1 in C. elegans; retinol metabolism pathway gene dehydrogenases short-chain EVM0002754/dhs-2 in L. marina; and fatty acid elongation gene EVM0013887/elo-3 in L. marina.
The total RNA used for quantitative PCR was from RNA-seq experiments, and standard cDNA synthesis was performed using a cDNA synthesis kit (Toyobo, Osaka, Japan, FSQ-301). We performed reverse transcription with 500 ng RNA per sample. PCR amplification, using SYBR Green (Toyobo, Osaka, Japan, QPK-201) via ABI QuantStudio 6 Flex system, was performed in 96-well microtiter plates. qPCR program was as follows: 95 °C, 60 s; then 40 cycles of 95 °C, 15 s, 60 °C, 15 s; 72 °C, 45 s; dissolution curve was detected by increasing from 1.6 °C/s to 95 °C for 15 s, decreasing from 1.6 °C/s to 60 °C for 1 min, and, finally, increasing from 0.05 °C/s to 95 °C for 15 s. cdc-42 for C. elegans and EVM0013809/cdc-42 for L. marina were used as the internal reference genes, respectively [21,35]. Gene names and primers used for real-time PCR are shown in Supplementary File S1. Quantitative PCRs (qPCRs) were performed in triplicate. The fold change was calculated by the average ratio of three biological replicates for each treatment group (CeMM) to the control group (OP50) using the delta-delta Ct method. The correlation analysis was computed by Pearson correlation analysis using GraphPad Prism 8 software.

3. Results

3.1. CeMM Diet Attenuates the Development of L. marina but Does Not Extend Its Longevity

To ask whether the CeMM diet affects the development of L. marina, we transferred newly hatched L1 larvae into 15‰ sea salt CeMM and found that only about 8% of them developed into adulthood after 6 days post-hatching when grown on CeMM (Figure 1A). On the contrary, nearly 95% of the newly hatched L1 larvae of L. marina developed into adulthood after 5 days at 20 °C (Figure 1B). Under CeMM feeding conditions, a previous report showed that the mean life span of C. elegans was about 58 days, and the maximum was approximately 90 days. In contrast, under bacterial OP50 feeding conditions, the mean life span of C. elegans was 18–20 days [36], while the maximum was approximately 25 days [37]. To ask whether the CeMM diet promotes the lifespan of L. marina compared to bacterial feeding, we transferred L4 female larvae to 15‰ SW-CeMM plates. We observed that L. marina could live as long as 28 days, with an average lifespan of about 16 days on CeMM (Figure 1C). Similarly, in OP50 feeding environment, L. marina could live as long as 26 days, with an average lifespan of about 16 days (Figure 1C). Our data suggested that the CeMM diet attenuated the development of L. marina but did not promote its longevity compared to bacterial feeding.

3.2. RNA-Seq Analysis in C. elegans and L. marina under CeMM Food Environment

To investigate the genome-wide transcriptional responses of C. elegans and L. marina in CeMM and OP50 dietary conditions, we used RNA-seq analysis to describe their transcriptomic signatures. At least 5.64 Gb of clean data were generated for each sample with a minimum of 93.16% of clean data that achieved a quality score of Q30. Reads which were sequenced from each library are listed in Supplementary Table S1. The mapping ratio ranged from 70.05% to 71.25% for L. marina and from 97.44% to 97.75% for C. elegans. In order to avoid genetic changes in the growth of the nematode itself, the newly hatched L1s of both C. elegans and L. marina were treated for 2.5 h on CeMM and OP50 plates, respectively. We used OP50 as the control group and CeMM as the treatment group, and a |Log2Fold change| > 1 and a padj < 0.01 were applied as the differential gene-screening threshold. A total of 2571 DEGs for C. elegans and 678 DEGs for L. marina were identified in our analysis (Figure 2). The details of significantly up- and down-regulated DEGs of C. elegans and L. marina are listed in Supplementary File S2.

3.3. Shared Transcriptomic Signatures of L. marina and C. elegans under CeMM Food Conditions

We found that the expression of several pathways, such as cytochrome P450 (Figure 3A and Figure S1A), glutathione metabolism (Figure 3B and Figure S1B), retinol metabolism (Figure 3C and Figure S1C), carbon metabolism (Figure 3D and Figure S1D), glyoxylate and dicarboxylate metabolism (Figure 3E and Figure S1E), arachidonic acid metabolism (Figure 3F and Figure S1F), tryptophan metabolism (Figure 3G and Figure S1G), and fatty acid degradation (Figure 3H and Figure S1H) pathway related genes, were significantly increased in both L. marina and C. elegans on the CeMM medium (Figure 2B). These shared features indicate that these genes may play essential roles in attenuating the development of both L. marina and C. elegans in CeMM food environments.

3.4. Different Transcriptomic Signatures of C. elegans under CeMM Conditions

Based on the KEGG analysis, we found that the longevity-regulating pathway genes were significantly up-regulated in C. elegans on CeMM, including seven daf-16 target genes (mtl-1, mtl-2, ctl-1, ctl-2, ctl-3, lgg-1, and sod-3) (Figure 4A). In addition, we found that the forkhead box (FOXO) signaling pathway genes were also significantly up-regulated in C. elegans on CeMM (Figure 4B), which included nine potential daf-16 target genes. In addition to the five daf-16 target genes shared in the longevity regulation pathway, four additional genes were mfb-1, dct-1, aakg-4, and pck-1. Furthermore, we found that lysosome- (Figure 4C) and peroxisome- (Figure 4D) related genes were significantly up-regulated in C. elegans under CeMM food conditions compared to bacterial feeding. Our data suggest that daf-16 target genes and lysosome- and peroxisome-related genes might play essential roles in promoting the longevity of C. elegans when feeding on the CeMM diet.
We found that ribosome biogenesis, ribosome, RNA transcription, protein generation, processing, and transport-related genes were significantly down-regulated in C. elegans on CeMM (Figure 5). The expression level of ribosome biogenesis genes such as eif-6, fib-1, mac-1, nxt-1, and ran-1 (Figure 5A); ribosome machinery-associated genes such as rpl-, rps-, mrpl-, and mrps- (Figure 5B); and RNA polymerase genes such as rpc-1, rpb-6, rpoa-12, rpoa-2, and rpb-12 (Figure 5C) were significantly down-regulated in C. elegans on CeMM. Ribosomes are macromolecular machines that support biological protein synthesis [38], and RNA polymerase transcribes DNA into RNA such as mRNA, tRNA, and rRNA, among others [39]. The down-regulation of these transcription- and translation-related genes would negatively affect RNA and protein generation and functions. Similarly, spliceosome-related genes (eftu-2, emb-4, isy-1, mog-2, and snr-1, Figure 5D), RNA transport-related genes (such as eif-3. B~eif-3. I, hoe-1, and ife-2, Figure 5E), aminoacyl-tRNA biosynthesis-related genes (such as ears-1, fars-1, fars-3, nars-1, pars-1, tars-1, and wars-1, Figure 5F), and protein processing in endoplasm genes (dad-1, hsp-90, dnj-12, hsp-1, and hsp-17, Figure 5G), as well as protein export associated genes (such as emo-1, hsp-3, oxa-1, spcs-2, and sec-61, Figure 5H), were significantly down-regulated in response to the CeMM diet in C. elegans. Our data suggest that the CeMM diet might attenuate the development and promote a lifespan extension of C. elegans through the down-regulation of several RNA transcription and protein generation and related processes.

3.5. Different Transcriptomic Signatures of L. marina under CeMM Conditions

In L. marina, 678 DEGs were observed under CeMM food conditions compared with bacterial OP50 feeding (Figure 2). We found that drug and cytochrome P450 pathway genes such as ugt-19, ugt-65, ugt-22, and ugt-23 were significantly down-regulated in L. marina on CeMM (Figure 6A). In contrast to C. elegans, we found that ‘lysosome’-related genes such as slc-17.9, ptr-22, lcn-3.3, and crn-6 were significantly down-regulated on CeMM (Figure 6B).
In addition, we found that the biosynthesis of unsaturated fatty acid-related genes such as elo-3 and acox-1.5 was also down-regulated (Figure 6C). A previous study showed that mutants with severe unsaturated fatty acid deficiencies display growth and neurological defects in C. elegans [40]. Our data suggest that a CeMM diet might attenuate the development of L. marina through the down-regulation of unsaturated fatty acid synthesis genes.

3.6. Quantitative Real-Time PCR Validation

We conducted qPCR to verify the expression of the genes of interest identified from our RNA-seq results (Figure 7, Supplementary File S3). Consistent trends were demonstrated, as seen in Figure 7B. The expression of the glutathione S-Transferase gene gst-4, acyl-Coenzyme A oxidase gene acox-3, and superoxide dismutase gene sod-3 of C. elegans, and glutathione S-Transferase gene EVM0002486/gst-8, and dehydrogenases short chain gene EVM0002754/dhs-2 in L. marina were significantly up-regulated when feeding on CeMM compared with OP50 feeding, while the expression of the eukaryotic initiation factor gene eif-6 and heat shock protein gene hsp-1 of C. elegans, and UDP-Glucuronoxylan transferase EVM0005671/ugt-19 and fatty acid elongation EVM0013887/elo-3 in L. marina were significantly down-regulated under the CeMM diet.

4. Discussion

C. elegans develop from L1 larvae to adults in about 3 days when fed OP50 at 20 °C [11], whereas we previously showed that about 10% of the larvae developed into adults after 8 days post-hatching when fed CeMM [17]. A previous report showed that the mean life span of C. elegans was about 16 days when fed on OP50 in 20 °C, with a maximum life span of days [10], while under CeMM conditions, the mean life span of C. elegans was about 30 days, and the maximum was 93 days [10]. In line with these data, we previously found that the mean life span of C. elegans was about 58 days, with the maximum life span of approximately 90 days [17]. The above-mentioned reports suggest that the CeMM diet attenuates C. elegans development and extends its lifespan compared with that of bacterial feeding [10,17]; however, the physiological mechanisms underlying how the CeMM diet attenuates the development and promotes the longevity extension of C. elegans are not completely understood, and whether the CeMM diet regulates the growth and lifespan of other nematodes is unknown. We recently showed that approximately 91% of the newly hatched L1 larvae of L. marina developed into adulthood after 5 days at 20 °C, and the mean life span of L. marina is about 16 days [21,41], which is similar to our observations in this study (Figure 1B,C). In the current report, we observed that the CeMM diet attenuated the development of the marine nematode L. marina, though it did not extend the nematode’s lifespan (Figure 1). Previous microarray analysis identified 48 significantly altered genes in C. elegans on CeMM compared to that of bacterial feeding [10]. We compared these 48 genes to our DEG data and found that 12 of them were significantly enriched in C. elegans on CeMM, 6 of them were expressed in the same trend, and the expression of another 6 genes was in the opposite direction (Supplementary File S4). Among genes changing in the same direction, two potential daf-16 target genes, including mtl-1 and mtl-2, were significantly increased, while gale-1, zhit-3, K05C4.5, and dnj-10 were significantly reduced on the CeMM diet in our data. Notably, we found that more daf-16 target genes are up-regulated in CeMM food conditions in C. elegans (Figure 4A), suggesting that we might observe a more complete picture of the physiological change in C. elegans on the CeMM diet compared to the previous microarray analysis [10,17], which we will discuss in detail in the next part.

4.1. CeMM Diet Promotes the Longevity of C. elegans but Not L. marina

FOXO transcription factor DAF-16 and its target genes play essential roles in aging [42,43,44,45,46,47]. Through KEEG enrichment analysis, we found that 11 potential daf-16 target genes, such as mtl-1, mtl-2, ctl-1, ctl-2, ctl-3, lgg-1, sod-3, mfb-1, dct-1, aakg-4, and pck-1, were significantly up-regulated in C. elegans on CeMM compared to that of bacterial feeding (Figure 4A,B), whereas only three known daf-16 downstream targets being upregulated on CeMM were identified in previous microarray analysis [10,17]. The RNAi of the metallothionein mtl-1, the cytosolic and peroxisomal catalases ctl-1 and ctl-2, and the mitochondrial superoxide dismutase sod-3 impaired the lifespan-extending properties of active daf-16 [43], and the RNAi of lgg-1 suppresses the increased lifespan induced by LIPL-4 overexpression [48]. Further, we found another 30 daf-16 direct targets through comparing our significantly changed DEG genes to previously reported potential daf-16 direct targets [42,43,44,45,46,47]. Among them, 22 genes were significantly increased, and 8 were significantly down-regulated in C. elegans on CeMM (Supplementary File S5). Of note, some of the daf-16 targets, such as sams-1 and ubh-4, were anti-longevity genes, and the RNAi knock-down mrpl-12, which encodes a mitochondrial ribosomal protein, sams-1, which encodes S-adenosyl methionine synthetase, and ubh-4, which encodes ubiquitin C-terminal hydrolase, could extend the lifespan of C. elegans, respectively [49,50,51,52]. The expression of mrpl-12, sams-1, and ubh-4 was significantly repressed in C. elegans on CeMM in the current study. Additionally, it was reported that decreased insulin/IGF-1-like signaling (IIS) promotes C. elegans lifespan extension by increasing the stress response (class I) and repressing other (class II) genes in a daf-16-dependent manner (Tepper et al., 2013). We compared the top 50 class I and class II genes in Tepper’s paper with our significantly changed DEGs in C. elegans on CeMM; among these 100 top genes, 30 class I genes were up-regulated, and 10 class II genes were down-regulated (Supplementary File S6). All of these data suggested that the CeMM diet might extend the longevity of C. elegans on CeMM via FOXO DAF-16 signaling. Notably, the longevity and FOXO pathway-related genes were not enriched in L. marina on the CeMM diet, which might explain why CeMM could not promote the longevity of the marine nematode L. marina.
Lysosome has been reported as a ‘signaling hub’ that integrates metabolic inputs, organelle interactions, and the control of lifespan in C. elegans [53,54,55,56,57]. The lysosomal acid lipase (lipl) has been found to increase autophagy, which extends longevity in C. elegans [58,59], and the overexpression of LIPL-4 significantly increases the lifespan of C. elegans [60,61]. In this report, three lipl genes, lipl-2, lipl-4, and lipl-7, were significantly up-regulated in C. elegans on CeMM (Figure 4C). Peroxisomes support key metabolic platforms for the oxidation of various fatty acids, regulate redox conditions, and play crucial roles in the biosynthesis of essential lipid molecules and longevity [62,63]. For instance, dietary restriction and AMPK-increased lifespan requires coordination between mitochondria and peroxisomes [63]. The peroxisomal catalase gene ctl-2, which function as an antioxidant enzyme that protects C. elegans cells from reactive oxygen species, contributes to the extended lifespan of C. elegans [64,65,66]. Of note, ctl-2 is a daf-16 target gene. Our data suggested that the CeMM diet might promote the longevity of C. elegans on CeMM via lysosome- and peroxisome-related functions. Of note, the expression of lysosome-related genes was significantly down-regulated in L. marina on the CeMM diet (Figure 6B), which might be part of the reason why CeMM could not promote the longevity of the marine nematode L. marina.
A previous report showed that the increased levels of bile acids in long-lived mice up-regulate xenobiotic detoxification genes [67]. The expression of cytochrome P450 genes is significantly increased in a long-lived daf-2 mutant of C. elegans [68]. Consistent with this, we found that drug and cytochrome P450 xenobiotic detoxification pathway genes were significantly up-regulated in C. elegans on CeMM (Figure 3A). Our data suggest that the CeMM diet might extend the longevity of C. elegans on CeMM via the up-regulation of the xenobiotic detoxification pathway. Notably, drug and cytochrome P450 xenobiotic detoxification pathway-related genes were significantly down-regulated in L. marina on the CeMM diet (Figure 2B). Although the genes of one P450 pathway, including genes such as gst-7 and gst-8, were significantly up-regulated in L. marina on the CeMM diet (Supplementary Figure S1A), we supposed that these genes might not be involved in longevity regulation.
The reduction of protein synthesis prolongs the lifespan of C. elegans [69,70,71,72,73]. The general decreases in protein synthesis can reduce the cellular load of erroneously synthesized polypeptides and the production of toxic metabolic derivatives in C. elegans [70,74]. We found that RNA transcription and protein generation-related genes, including ribosome biogenesis, ribosome, RNA transcription, splicing, and protein processing in transport associated genes, were significantly down-regulated when C. elegans fed on CeMM (Figure 5), indicating that the CeMM diet might extend the longevity of C. elegans via decreased protein synthesis-related processes. Notably, the above transcription- and protein synthesis-related pathway genes were not enriched in L. marina on the CeMM diet, which might also contribute to the reason that CeMM could not extend the lifespan of the marine nematode L. marina.
Taken together, the CeMM diet slowed down the growth and extended the lifespan of C. elegans, while only attenuating the development of L. marina but not promoting its longevity. We proposed that increased expression of FOXO DAF-16 signaling, lysosome, xenobiotic metabolism-related genes, and decreased expression of protein synthesis-related genes might play important roles in extending the lifespan of C. elegans on the CeMM diet. Given that DEGs were identified from L1 larvae comparisons in this report, it will be interesting to verify their functions in other developmental stages in further studies. A previous report showed that metformin extends lifespan when adult worms are exposed to the drug, while treating the larvae with metformin does not extend the longevity of C. elegans [75]. However, it was reported that early-life, low-dose oxidant exposure could extend the lifespan of the fly [76]. Maternal protein restriction during rat pregnancy has also been identified to shorten the lifespan of offspring [77,78]. Thus, it is important to systematically explore the mechanisms underlying the developmental stage-dependent nutritional regulation of aging in future studies. Given that dietary restriction retards the aging of a wide range of species [5], it will be interesting to ask whether the CeMM diet represents a type of DR in C. elegans and how L. marina in different developmental stages responds to DR conditions in future studies.

4.2. CeMM Diet Attenuates the Development of Both C. elegans and L. marina

Given that the CeMM diet attenuates the development of both C. elegans and L. marina, the shared pathway in L. marina and C. elegans may play an essential role in attenuating their development in CeMM environments (Figure 3 and Figure S1). Of note, although there were several KEGG-enriched pathways shared between C. elegans and L. marina, the differentially expressed genes in each pathway are not the same, and only a few DEGs such as dhs-2, dhs-19, and alh-4 were shared between C. elegans and L. marina. Further studies are required to delineate which genes play major roles.
We found that a series of RNA and protein processing-related genes were significantly down-regulated in C. elegans under the CeMM diet (Figure 5). The breakdown of RNA assembly and transport can have catastrophic consequences, such as impaired RNA localization and distal protein synthesis that correlate with significant functional impairments such as neuronal growth defect [79,80]. The RNAi of eif-6, which encodes the ortholog of vertebrate anti-association factor eIF6, slows down the growth of C. elegans [81]. The RNAi of some structural constituents of ribosomes genes such as rps-12 and rps-26 results in larval developmental delay or arrest [82]. Similarly, the RNAi of rpc-1, which contributes to RNA polymerase III activity, and hel-1, which is predicted to enable RNA binding activity and RNA helicase activity, lead to slow growth [83,84]. The initiation factors are significant for protein transcription and regulation, of which down-regulation could decrease the translation for most mRNAs under starvation or stress conditions [85]. Methionyl-tRNA synthetase (MRS) down-regulation results in DNA damage and, further, leads to functional defects [86]. The RNAi of protein processing in the endoplasmic-related gene hsp-1 could result in larval developmental delay or arrest phenotypes [82]. RNAi knocking down oxa-1, which is involved in mitochondrial cytochrome C oxidase assembly and protein insertion into the mitochondrial inner membrane from the matrix, exhibits increased longevity and a reduced developmental rate [87]. Given that development requires building blocks such as proteins to support cell growth and differentiation, our data suggest that the CeMM diet might attenuate the development of C. elegans on CeMM via the down-regulation of protein synthesis-related genes.
Unlike C. elegans, RNA and protein synthesis processing-related genes were not enriched in L. marina on the CeMM diet. Of note, we found that the synthesis of unsaturated fatty acids pathway genes was significantly decreased in L. marina on the CeMM diet (Figure 6C). Unsaturated fatty acids have been reported to participate in numerous biological processes, such as inflammation and immune response [88], neuro-reception [89], and reproduction [90]. The mutants, fat-2, fat-6, and fat-7, which have defected to synthesize 18- and 20-carbon unsaturation fatty acid, grow slowly compared to their wild-types [40,91,92]. The depletion of elo-1 and elo-2 results in decreased PUFA, slowed growth, and disrupted reproduction [93]. The RNAi treatment of elo-3 in both the ire-1 and atf-6 mutants’ backgrounds exhibited synthetic growth defects in C. elegans [94]. In L. marina, we found that the expression of elo-3 was significantly repressed under the CeMM diet. The data indicated that the CeMM diet might attenuate the development of L. marina on CeMM via the down-regulation of unsaturated fatty acid synthesis genes.
Collectively, down-regulated protein synthesis-related genes may not only contribute to longevity extension, but also lead to growth delay in C. elegans on CeMM, while reduced unsaturated fatty acids pathway genes may be the reason for developmental attenuation in the marine nematode L. marina on CeMM compared with bacterial feeding. Different species may have both common and distinct requirements for nutrients, which in turn affect the animal biological processes such as development and lifespan. Consistent with this, CeMM was derived from C. briggsae Maintenance Medium (CbMM) [16]. It was reported that there are some different nutritional requirements between C. briggsae and C. elegans [95,96], for instance, the multiple absences of nonessential amino acids attenuate the reproduction of C. briggsae while having no noticeable influence on C. elegans [96]. A previous report showed that an E. coli endogenous noncoding RNA, OxyS, impacts the physiology of C. elegans but not C. briggsae [97]. CeMM medium might not be an optimal food source condition for the development of both C. elegans and L. marina, as CeMM slows the development of both nematodes. Given that CeMM is a chemically defined synthetic food, it will be interesting to ask which components and their amounts are essential for the optimal development of C. elegans and the marine nematode, L. marina, respectively. Our previous report suggested that the tmc-1 mutant has the ability to better utilize the CeMM media [17], and it is interesting to explore if the L. marina homolog tmc-1 gene has a conserved developmental regulation role in relaxing the stress response to an unfamiliar food in future studies.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jmse10050580/s1, Supplementary Figures: Figure S1: Shared genes in L. marina and C. elegans under CeMM conditions. Supplementary Table S1: Sequencing data statistics. Supplementary File S1: The information of qPCR primers. Supplementary File S2: Details of DEGs. Supplementary File S3: The result of quantitative real-time PCR validation. Supplementary File S4: The comparison with microarray analysis. Supplementary File S5: The list of daf-16 targets genes. Supplementary File S6: The comparation with class I and class II genes in Tepper’s paper.

Author Contributions

P.S. and L.Z. conceived and designed the experiments. P.S. carried out most of the experiments, analyzed the data, and wrote the manuscript. X.C. was involved in sampling and data analysis. L.Z. edited the manuscript and supervised the project. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Key R and D Program of China (No. 2018YFD0901301); “Talents from overseas Program, IOCAS” of the Chinese Academy of Sciences; “Qingdao Innovation Leadership Program” (Grant 16-8-3-19-zhc); and the Key deployment project of the Centre for Ocean Mega-Research of Science, Chinese Academy of Sciences (COMS2019Q16).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated from this study have been deposited in NCBI and the BioProject ID: PRJNA801608.

Acknowledgments

We are grateful to all members of the LZ laboratory for their helpful discussions.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. CeMM diet attenuates the development of L. marina but does not promote its longevity. (A) For 15‰ sea salt CeMM developmental analysis, 70 newly hatched L1s were transferred onto each indicated 3 cm dimeter agar plates of 15‰ sea salt CeMM (three trials, n = 70 worms in each trial). All data shown here are mean ± SEM (standard error of mean). (B) For OP50 developmental analysis, 70 newly hatched L1s were transferred onto each indicated 3 cm dimeter agar SW-NGM with E. coli OP50 (three trials, n = 70 worms in each trial). All data shown here are mean ± SEM. (C) For lifespan assay (one trial, n = 96, and 115 for OP50 and CeMM feeding, respectively), L4 females were transferred to each assay plate and incubated at 20 °C. The number of live and dead worms was counted every day. Live worms were assigned to 3 cm dimeter agar plates (15‰ sea salt CeMM plates and SW-NGM plates with fresh OP50) every two days.
Figure 1. CeMM diet attenuates the development of L. marina but does not promote its longevity. (A) For 15‰ sea salt CeMM developmental analysis, 70 newly hatched L1s were transferred onto each indicated 3 cm dimeter agar plates of 15‰ sea salt CeMM (three trials, n = 70 worms in each trial). All data shown here are mean ± SEM (standard error of mean). (B) For OP50 developmental analysis, 70 newly hatched L1s were transferred onto each indicated 3 cm dimeter agar SW-NGM with E. coli OP50 (three trials, n = 70 worms in each trial). All data shown here are mean ± SEM. (C) For lifespan assay (one trial, n = 96, and 115 for OP50 and CeMM feeding, respectively), L4 females were transferred to each assay plate and incubated at 20 °C. The number of live and dead worms was counted every day. Live worms were assigned to 3 cm dimeter agar plates (15‰ sea salt CeMM plates and SW-NGM plates with fresh OP50) every two days.
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Figure 2. The transcriptomic signature of C. elegans and L. marina. (A) Experimental design of this study and the resulting transcriptomic signature of worms. Differentially expressed genes (DEGs, |Log2Fold change| > 1; DESeq2 padj < 0.01) were determined for each condition. (B) KEGG enrichment analysis for DEGs. ‘HQ’ and ‘N2′ represent L. marina and C. elegans, respectively. ‘Up-regulation’ indicates increased gene expression in the CeMM diet compared to the OP50 diet. ‘Down-regulation’ indicates decreased gene expression in the CeMM diet compared to the OP50 diet. The color change from red to green represents the significance of the enrichment. We focused on the pathway of blue font enrichment. The Rich Factor was defined as the ratio between the number of differentially expressed genes and the number of all genes in the KEGG pathway.
Figure 2. The transcriptomic signature of C. elegans and L. marina. (A) Experimental design of this study and the resulting transcriptomic signature of worms. Differentially expressed genes (DEGs, |Log2Fold change| > 1; DESeq2 padj < 0.01) were determined for each condition. (B) KEGG enrichment analysis for DEGs. ‘HQ’ and ‘N2′ represent L. marina and C. elegans, respectively. ‘Up-regulation’ indicates increased gene expression in the CeMM diet compared to the OP50 diet. ‘Down-regulation’ indicates decreased gene expression in the CeMM diet compared to the OP50 diet. The color change from red to green represents the significance of the enrichment. We focused on the pathway of blue font enrichment. The Rich Factor was defined as the ratio between the number of differentially expressed genes and the number of all genes in the KEGG pathway.
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Figure 3. The expression of shared pathway related genes in C. elegans on CeMM. (A) Cytochrome P450-related genes in C. elegans. (B) Glutathione metabolism-related genes in C. elegans. (C) Retinol metabolism genes in C. elegans. (D) Carbon metabolism genes in C. elegans. (E) Glyoxylate and dicarboxylate metabolism-related genes in C. elegans. (F) Arachidonic acid metabolism-related genes in C. elegans. (G) Tryptophan metabolism-related genes in C. elegans. (H) Fatty acid degradation-related genes in C. elegans. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
Figure 3. The expression of shared pathway related genes in C. elegans on CeMM. (A) Cytochrome P450-related genes in C. elegans. (B) Glutathione metabolism-related genes in C. elegans. (C) Retinol metabolism genes in C. elegans. (D) Carbon metabolism genes in C. elegans. (E) Glyoxylate and dicarboxylate metabolism-related genes in C. elegans. (F) Arachidonic acid metabolism-related genes in C. elegans. (G) Tryptophan metabolism-related genes in C. elegans. (H) Fatty acid degradation-related genes in C. elegans. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
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Figure 4. The expression of up-regulated pathway related genes of C. elegans on CeMM. (A) Longevity-regulating pathway-related genes in C. elegans. (B) FOXO signaling pathway-related genes in C. elegans. (C) Lysosome-related genes in C. elegans. (D) Peroxisome-related genes in C. elegans. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
Figure 4. The expression of up-regulated pathway related genes of C. elegans on CeMM. (A) Longevity-regulating pathway-related genes in C. elegans. (B) FOXO signaling pathway-related genes in C. elegans. (C) Lysosome-related genes in C. elegans. (D) Peroxisome-related genes in C. elegans. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
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Figure 5. The expression of down-regulated pathway related genes of C. elegans on CeMM. (A) Ribosome biogenesis in eukaryotes-related genes in C. elegans. (B) Ribosome genes in C. elegans. (C) RNA polymerase genes in C. elegans. (D) Spliceosome-related genes in C. elegans. (E) RNA transport-related genes in C. elegans. (F) Aminoacyl-tRNA biosynthesis-related genes in C. elegans. (G) Protein processing in endoplasmic-related genes in C. elegans. (H) Protein export-related genes in C. elegans. The fold changes indicate the ratio of the treatment group (CeMM) to the control group (OP50). Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
Figure 5. The expression of down-regulated pathway related genes of C. elegans on CeMM. (A) Ribosome biogenesis in eukaryotes-related genes in C. elegans. (B) Ribosome genes in C. elegans. (C) RNA polymerase genes in C. elegans. (D) Spliceosome-related genes in C. elegans. (E) RNA transport-related genes in C. elegans. (F) Aminoacyl-tRNA biosynthesis-related genes in C. elegans. (G) Protein processing in endoplasmic-related genes in C. elegans. (H) Protein export-related genes in C. elegans. The fold changes indicate the ratio of the treatment group (CeMM) to the control group (OP50). Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
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Figure 6. The expression of down-regulated pathway related genes of L. marina on CeMM. (A) Drug metabolism-cytochrome P450 pathway-related genes in L. marina. (B) Lysosome-related genes in L. marina. (C) Biosynthesis of unsaturated fatty acid-related genes in L. marina. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
Figure 6. The expression of down-regulated pathway related genes of L. marina on CeMM. (A) Drug metabolism-cytochrome P450 pathway-related genes in L. marina. (B) Lysosome-related genes in L. marina. (C) Biosynthesis of unsaturated fatty acid-related genes in L. marina. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM.
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Figure 7. Validation of the RNA-seq results using qPCR. (A) qPCR analysis of the genes of interest identified from the RNA-seq results. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM. (B) Correlation analysis of the results of RNA-seq and qPCR for the genes of interest. Each dot represents a gene, and detailed information is shown in Supplementary File S3. Pearson R = 0.9501, with a p-value of <0.0001.
Figure 7. Validation of the RNA-seq results using qPCR. (A) qPCR analysis of the genes of interest identified from the RNA-seq results. Log2FC: Log2(Fold Change). All data shown here are mean ± SEM. (B) Correlation analysis of the results of RNA-seq and qPCR for the genes of interest. Each dot represents a gene, and detailed information is shown in Supplementary File S3. Pearson R = 0.9501, with a p-value of <0.0001.
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Sun, P.; Cao, X.; Zhang, L. Transcriptome Analysis of the Nematodes Caenorhabditis elegans and Litoditis marina in Different Food Environments. J. Mar. Sci. Eng. 2022, 10, 580. https://doi.org/10.3390/jmse10050580

AMA Style

Sun P, Cao X, Zhang L. Transcriptome Analysis of the Nematodes Caenorhabditis elegans and Litoditis marina in Different Food Environments. Journal of Marine Science and Engineering. 2022; 10(5):580. https://doi.org/10.3390/jmse10050580

Chicago/Turabian Style

Sun, Peiqi, Xuwen Cao, and Liusuo Zhang. 2022. "Transcriptome Analysis of the Nematodes Caenorhabditis elegans and Litoditis marina in Different Food Environments" Journal of Marine Science and Engineering 10, no. 5: 580. https://doi.org/10.3390/jmse10050580

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