Construction of a cDNA library and preliminary analysis of the expressed sequence tags of the earthworm Eisenia fetida (Savigny, 1826).

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full‑length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5'End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high‑quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (P‑value <1x10‑8), of which 168 were annotated through Gene Ontology analysis. The STRING database was used to determine relationships among the 168 ESTs, identifying associated genes involved in protein‑protein interactions and gene expression regulation. Based on nucleic acid and protein sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a high‑quality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms.

Introduction

Earthworms are terrestrial annelids in the oligochaeta subclass, with a generally preferred habitat of damp and loose soil. They include ~3,000 species worldwide, with 229 species in China (1,2). In a wide variety of soil types, earthworms serve vital roles in converting large pieces of organic matter into rich humus to enrich soil fertility. The earthworms are the highest evolutionary species capable of regenerating an anterior portion containing the central nerve system, heart and clitellum (3). The anterior regeneration is a unique developmental process that requires cell proliferation, re-differentiation and sophisticated cell-cell communication. This process can serve as a useful model for investigating normal development and differentiation (4).

Over the past several years, cDNA library construction and analysis have become established as indispensable methods for functional genome analysis since they provide detailed information about the genomic mechanisms underlying the diverse processes of an organism (5). However, conventionally generated cDNA libraries contain a high percentage of 5′-truncated clones, limiting the utility of such libraries. The Switching Mechanism at 5′End of RNA Template (SMART) technique (6) amplifies and enriches the full-length mRNA, and thus generates cDNA libraries with a significantly improved ratio of full-length to partial cDNA sequences. In the present study, the SMART technique was adopted to construct a high quality library of full-length cDNAs representative of adult earthworms, namely of the earthworm Eisenia fetida (Savigny, 1826).

Unlike other model organisms, none of the oligochaete genomes have been sequenced to the best of our knowledge, and genomic research on earthworms lags behind that of other model species such as Mus musculus. In the absence of the full genome sequences, expressed sequence tags (ESTs) aid the rapid detection of expressed genes via sequence analysis, and are a significant resource for comparative and functional genomic studies (2). In addition, among the biological techniques for transcriptome analysis, the determination of ESTs is considered the simplest method for profiling the transcriptome, which is also particularly useful in the development of cDNA microarrays for systematic identification of differentially expressed genes (7). Analysis of ESTs is an effective method for rapidly analyzing gene expression, characterizing gene functions and discovering new genes that are important for specific developmental and physiological processes (8). The present study established 593 ESTs, representing 168 genes and 425 unknown tags, providing a gene expression profile of earthworm development. This collection of ESTs may provide a valuable basis for future research on the physiology of earthworms.

Materials and methods

Isolation of total RNA and mRNA

Eisenia fetida earthworms were purchased from Beijing Shuangqiao Farm (Beijing, China). Fully developed adult Eisenia fetida earthworms weighing 0.3–0.6 g (live weight) were selected for all experiments. All earthworm tissues were harvested and total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The integrity of total RNA was analyzed by electrophoresis using 1% agarose gels. Isolation of poly(A) mRNA from total RNA was carried out using an MN-NucleoTrap®mRNA kit (Machery-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer's protocol. Oligo(dT) beads suspension was applied to total RNA and incubated at 68°C for 5 min before eluting mRNA. Then ethidium bromide (EB) staining was applied and 1% agarose gel was used to visualize the result. The isolated mRNA was further vacuum concentrated using Concentrator plus™ (Eppendorf, Hamburg, Germany). The quantity and integrity of isolated mRNA were determined using a nanodrop spectrophotometer and agarose gel electrophoresis, respectively.

cDNA library construction

A total of ~8,048.4 ng mRNA was used for single-stranded cDNA synthesis. The purified mRNA was used as a template, Oligod(T)18 with XhoI cleavage site was used as the primer, and first strand cDNA was transcribed at 42°C using SuperScript™ II RnaseH-Reverse (Thermo Fisher Scientific, Inc.). Then the mRNA was digested using RNaseH, and the resultant mRNA fragments were used as further primers. The first cDNA chain was used as a template for double-stranded cDNA synthesis, using DNA Polymerase I (Takara Biotechnology Co., Ltd., Beijing, China). The ends of the double-stranded cDNA were ligated by T4 DNA polymerase and the ligation products were purified by phenol/chloroform/isoamyl alcohol to remove excess impurities such as protein. Subsequently, the double-stranded DNA fragments were ligated into EcoRI Adaptor using T4 DNA ligase at 4°C overnight. Then the double-stranded DNA fragments were phosphorylated with T4 Polynucleotide Kinase and digested with XhoI. Following XhoI digestion of the double-stranded cDNA, producing XhoI sticky ends, a QIAEXII Gel Extraction kit (Beijing BioDev-Tech, Beijing, China) was used to recycle 0.5–4 Kb fragments. The recycled cDNA was preserved at −20°C. Then the cDNA was ligated into the pBluescript II SK(+) XR vector (Promega Corporation, Madison, WI, USA) in a 3:1 molar ratio with T4 DNA ligase at 4°C overnight. To reduce the redundancy and avoid the underrepresentation of different transcript species, cDNA fragments with different fractionated sizes were balanced and subjected to library construction (9,10). Prior to transformation, mixing of all ligated products with microporous membranes was performed to remove salt ions. Subsequently the products were transformed into 5×107/ml DH10B competent cells (Thermo Fisher Scientific, Inc.), plated on agar plates (10 cm diameter) by pipetting the cells onto the middle of the plate and spreading, and monoclonal colonies were selected for PCR amplification. The inserted sequences in the plasmids were amplified by PCR using T3 primers (5′-ATTAACCCTCACTAAAGGGA-3′) and T7 primers (5′-TAATACGACTCACTATAGGG-3′). The total volume of PCR reaction mixture was 20 µl, containing 1 µl template, 10 µl 2XTaq MasterMix (CWBIO, Beijing, China), 1 µl T3 primers (10 pmol), 1 µl T7 primers (10 pmol) and 7 µl ddH2O (CWBIO). Cycling conditions were: 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 40 sec and 72°C for 60 sec, followed by 72°C for 5 min.

Bioinformatic analysis

cDNA clones were selected randomly from the cDNA library and the vector sequences were trimmed from the raw sequence data using Vecscreen tool (www.ncbi.nlm.nih.gov/tools/vecscreen/) from the National Center for Biotechnology Information. The sequence of each EST was also edited, mainly to remove ambiguous bases and poor-quality sequences (nucleotide sequences <100 bp). All edited sequences were assembled into groups using SeqMan software version 8.0 (DNASTAR, Madison, WI, USA). The processed cDNA sequences were used to perform a BLAST search in the GenBank database to compare all available ESTs and genes to date (11). The Basic Local Alignment Search Tool (BLAST; blast.ncbi.nlm.nih.gov/Blast.cgi) results with P-values <1×10−8 were generally regarded as a significant match (12,13). A large-scale Unigene assembly of the ESTs was initiated to identify and functionally annotate as many unique transcripts as possible. BLAST analysis against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and protein and nucleic acid databases was conducted for examination of biological functions. The ESTs homologous to known proteins were further annotated for Gene Ontology (GO; www.geneontology.org) terms and the GO analysis was carried out using WEB-based GEne SeT AnaLysis Toolkit (WebGestalt; www.webgestalt.org/option.php) (14,15).

Results

Construction of cDNA library

Obtaining an adequate quantity of high quality mRNA initially is the key to yielding a sufficient quantity of first-strand full-length cDNA by reverse transcription. In the present study, total RNA was extracted from the tissues of earthworms. As shown in Fig. 1, 28s and 18s bands were clearly visible in the electrophoresis gel of total RNA, indicating that the total RNA was obtained. The optical density (OD)260/OD280 ratio for the total RNA was 2.04, well within the range of 1.8–2.1, indicating that the isolated total RNA was suitable for cDNA library construction.

Figure 1.

Result of the electrophoresis of total RNA. A total of 1,056 µg RNA was obtained from the tissues of earthworms. The optical density values were A260=0.880 and A280=0.430; A260/A280=2.04.

Once the double-stranded cDNA was synthesized as described in the Materials and methods, the present study determined the size distribution of the products. Diffuse strips between 0.5–4.0 kb could be detected by 1% agarose gel electrophoresis, which indicated that double-stranded cDNAs were successfully synthesized. A cDNA library of 4.12×105 clones was obtained and half of the bacteria were cultured for amplification, which produced a total of 1.4×1011 clones. Several colonies were selected, and the inserted sequences in the plasmids were amplified by PCR using T3 and T7 primers. The PCR products were detected using 1% agarose gel electrophoresis as clear bands. No nonspecific bands were identified and the recombinant rate was 97% (Fig. 2).

Figure 2.

Amplified inserts of cDNA clones from the constructed cDNA library. M, DNA marker; Lanes 1–32, insert cDNA (>500 bp) clones from phage plaques.

To investigate the quality of the full-length cDNA library, the lengths of the cDNA inserts were assessed. Sequence outputs were manually edited to remove vector and ambiguous sequences. Then, the sequence data of the cDNA clones obtained by random partial sequencing were searched in the NCBI GenBank using BLAST to identify similarities with sequences in the nucleic acid databases. An evaluation of cDNA insert size and its distribution revealed a low level of insert size bias in the final cDNA library. The majority of the cDNA inserts were larger than 500 bp.

EST analysis

Instead of the amplified library, the primary cDNA library was used to generate ESTs to reduce the redundancy of cDNA clones. Following the removal of the redundant sequences and low-quality sequences (<100 bp), 1,148 effective sequences (>100 bp) from the total cDNA sequences were obtained. As shown in Fig. 3, 53 ESTs were 100–300 bp, 261 ESTs were 300–500 bp, 828 ESTs were 500–700 bp and 6 ESTs were larger than 700 bp. Taken together, 1,148 ESTs were larger than 100 bp. Among them, the shortest sequence was 100 bp, the longest was 718 bp and the average length was ~452 bp. Following sequencing, a homology BLAST search and assembling of the data, 368 singletons and 225 contigs were obtained out of the 1,140 high-quality ESTs, as shown in Table I. Additionally, a total of 593 individual ESTs were analyzed and 168 ESTs annotated in GenBank with nematode homology (Table I).

Figure 3.

Different groups of ESTs, separated by length. ESTs, expressed sequence tags.

Table I.

Summary of ESTs obtained from the cDNA library of earthworms.

ESTs	Number
Total number of ESTs	1,256
Total length of ESTs (bp)	568,140
Average length of ESTs (bp)	452.34
Unique genes	593
Contigs	225
Singletons	368
Annotation	168

ESTs, expressed sequence tags.

GO annotation and bioinformatic analysis

The cDNA functions were classified using the GO database into the three main categories of molecular functions, cell components and biological processes.

GO annotation of genes associated with molecular functions indicated that among the 168 ESTs, 46% (77/168 ESTs) were associated with growth and metabolic pathways, with the distribution of the 77 ESTs shown in Fig. 4A. Out of the 77 ESTs, 21% (16 ESTs) were associated with ‘proteolytic enzymes’, 16% (12 ESTs) with ‘protein ligases’, 14% (11 ESTs) with ‘oxido-reductases’, 13% (10 ESTs) with ‘energy release’, 10% (8 ESTs) with ‘signal transduction and cell communication’, 5% (4 ESTs) with ‘transport’ and only 3% (2 ESTs) with ‘post-translational modification’, ‘protein turnover’ and ‘chaperones’ (Fig. 4B).

Figure 4.

Distribution of GO molecular functions. (A) The distribution of 77 expressed sequence tags with molecular functions. (B) The classification of identified genes based on the relevant molecular functions. GO, Gene Oncology.

Cellular components associated with the cDNAs included ‘myosin’, the ‘citrate lyase compound’, the ‘mitochondrial inner membrane translocase compound’, ‘microtubules’, the ‘mitochondrial inner membrane’ and ‘nucleosomes’. The proportions of cellular components are presented in Fig. 5. It can be observed that the proportion of ‘myosin’ among cellular components was the largest. Myosin, an actin-dependent molecular motor, is involved in a number of important functions in earthworms. In particular, the myosin network can drive movement and support different moving speeds of earthworms. This specific feature is closely related to the free moving ability of earthworms (16).

Figure 5.

Distribution of GO cellular components. GO, Gene Oncology.

Regarding biological processes, known genes were determined as those presenting significant matches to protein sequences with known functions in non-redundant nucleotide databases. According to these biological functions, the biological processes component was divided into different functions including ‘larval development’ (46%), ‘changes of cell morphogenesis’ (6%), ‘the process of cytokinesis’ (12%), ‘post-translational protein modification’ (15%), ‘stress response’ (5%), ‘cell redox homeostasis’ (2%), ‘protein polymerization’ (2%) and ‘protein catabolism’ (12%), as shown in Fig. 6. It was concluded from the above data that promoting growth was considered to be an important biological function of genes associated with biological processes.

Figure 6.

Classification of identified genes based on relevant biological processes.

KEGG pathway annotation

KEGG is a collection of online databases describing pathways associated with biochemical, genomic and enzymatic processes. Furthermore, it provides annotations of biochemical pathways for the species in which the genome has been sequenced (17). In this analysis, proteins are not viewed as individual gene products but are organized into pathways and networks according to their biological function(s). In the present study, from the data in Table II, 15 of the 168 ESTs were revealed to be involved in metabolism. Notably, 4 of the 15 ESTs (27%) were involved in the ubiquitin-mediated proteolysis pathway, which was the most represented. The glutathione metabolism (3 ESTs) and arachidonic acid metabolism (2 ESTs) pathways were the second and third most represented pathways, respectively. Additionally, chondroitin sulfate biosynthesis, heparan sulfate biosynthesis, riboflavin metabolism, selenoamino acid metabolism, γ-hexachlorocyclohexane degradation, and the fructose and mannose metabolism pathways were also represented.

Table II.

Pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes database.

Pathway	Count	P-value	Q-value	Protein	Input symbol
Ubiquitin-mediated proteolysis	4	1.12×10−5	1.73×10−4	ubc-14;ubc-13; ubc-18;let-70	Y87G2A.9;Y54G2A.31; R01H2.6;M7.1
Glutathione metabolism	3	2.55×10−5	2.63×10−4	W07G4.4;F26E4.12; C11E4.1	W07G4.4;F26E4.12; C11E4.1
Arachidonic acid metabolism	2	2.01×10−4	1.25×10−3	F26E4.12; C11E4.1	F26E4.12; C11E4.1
Chondroitin sulfate biosynthesis	1	6.54×10−3	2.03×10−2	sqv-8	ZK1307.5
Heparan sulfate biosynthesis	1	1.14×10−2	2.75×10−2	sqv-8	ZK1307.5
Riboflavin metabolism	1	1.14×10−2	2.75×10−2	F02E9.7	F02E9.7
Selenoamino acid metabolism	1	3.39×10−2	4.62×10−2	seld-1	Y45F10A.4
γ-hexachlorocyclohexane degradation	1	4.02×10−2	4.62×10−2	F02E9.7	F02E9.7
Fructose and mannose metabolism	1	4.18×10−2	4.62×10−2	R04B5.5	R04B5.5

Mutual relationship between 287 genes

Relationships among the 168 ESTs were analyzed using the STRING database, and a functional association network was determined with 287 nodes, as shown in Table III. Each node corresponds to a gene and each (weighted) edge represents the evidence of a functional association between the gene pair. Predicted potential regulators are presented in Fig. 7. It can be seen from the STRING results that the most associated nodes (blue) included the 19 genes: Ribosomal protein L (rpl)-1, ribosomal protein S (rps)-0, rpl-4, rpl-5, rps-13, rps-2, acidic ribosomal protein (rla)-1, translocon-associated protein-4, transcription factor BTF3 homolog, rpl-7A, iff-1, rps-17, elongation factor 1α3, polyadenylate-binding protein-1, rps-28, translationally-controlled tumor protein homolog-1, rpl-18, rps-4 and adenosine triphosphate synthase subunit β mitochondrial precursor-2. Of note, the genes rpl-4, rps-13, rpl-5, rpl-1, rps-0, rla-1 and rpl-7A served critical roles in overall gene expression networks.

Table III.

Relationships between the 287 associated genes, including several important parameters.

node1	node2	node1_string_id	node2_string_id	node1_external_id	node2_external_id	coexpression	combined_score
rpl-5	atp-2	502,561	496,479	F54C9.5.1	C34E10.6.3	0.866	0.871
rpl-18	snr-2	510,406	508,903	Y45F10D.12.2	W08E3.1	0.426	0.468
trap-4	rps-2	511,330	497,379	Y56A3A.21.2	C49H3.11.1	0.243	0.919
sod-2	daf-21	498,735	497,248	F10D11.1.1	C47E8.5.1	0	0.572
rla-2	rps-4	511,588	510,280	Y62E10A.1.1	Y43B11AR.4.2	0.981	0.998
rpl-1	drs-1	511,889	493,869	Y71F9AL.13a.1	B0464.1.1	0.492	0.523
skr-1	sdhd-1	501,798	500,588	F46A9.5.3	F33A8.5.2	0	0.587
prdx-6	sod-2	509,858	498,735	Y38C1AA.11	F10D11.1.1	0.233	0.726
act-4	rps-0	505,013	493,798	M03F4.2a	B0393.1.1	0.426	0.425
daf-21	rps-13	497,248	495,308	C47E8.5.1	C16A3.9.1	0.402	0.474
rpl-1	rpl-4	511,889	493,528	Y71F9AL.13a.1	B0041.4.1	0.8	0.999
cyc-1	atp-2	497,719	496,479	C54G4.8.1	C34E10.6.3	0.849	0.873
rla-1	act-4	509,817	505,013	Y37E3.7.2	M03F4.2a	0.538	0.538
rpl-7A	rps-17	509,604	506,860	Y24D9A.4a	T08B2.10.1	0.872	0.923
rps-28	rpl-5	510,228	502,561	Y41D4B.5.2	F54C9.5.1	0.77	0.887
rpl-18	cyc-1	510,406	497,719	Y45F10D.12.2	C54G4.8.1	0.387	0.411
tpi-1	rps-0	509,457	493,798	Y17G7B.7.2	B0393.1.1	0.477	0.49
daf-21	drs-1	497,248	493,869	C47E8.5.1	B0464.1.1	0.281	0.543
lsm-3	rla-2	511,591	511,588	Y62E10A.12.2	Y62E10A.1.1	0	0.408
rpl-1	rps-4	511,889	510,280	Y71F9AL.13a.1	Y43B11AR.4.2	0.819	0.989
tpi-1	atp-2	509,457	496,479	Y17G7B.7.2	C34E10.6.3	0.448	0.877
lev-11	unc-60	509,147	496,717	Y105E8B.1d	C38C3.5b.1	0	0.431
rpl-5	icd-1	502,561	497,806	F54C9.5.1	C56C10.8.1	0.864	0.87
rla-2	pab-1	511,588	509,182	Y62E10A.1.1	Y106G6H.2a.4	0.102	0.904
unc-54	mlc-3	498,853	498,666	F11C3.3.1	F09F7.2a.2	0.452	0.452
alp-1	cyc-1	507,123	497,719	T11B7.4d	C54G4.8.1	0	0.409
mca-3	cmd-1	511,739	507,723	Y67D8C.10b	T21H3.3.1	0	0.581
cyc-1	rps-2	497,719	497,379	C54G4.8.1	C49H3.11.1	0.579	0.579
rpl-1	daf-21	511,889	497,248	Y71F9AL.13a.1	C47E8.5.1	0.251	0.415
rla-2	iff-1	511,588	506,602	Y62E10A.1.1	T05G5.10	0.436	0.521
rpl-1	rla-1	511,889	509,817	Y71F9AL.13a.1	Y37E3.7.2	0.794	0.997
tpi-1	vha-4	509,457	506,234	Y17G7B.7.2	T01H3.1.1	0.483	0.483
vha-4	atp-2	506,234	496,479	T01H3.1.1	C34E10.6.3	0.304	0.724
tpi-1	sod-2	509,457	498,735	Y17G7B.7.2	F10D11.1.1	0.213	0.84
rps-4	act-4	510,280	505,013	Y43B11AR.4.2	M03F4.2a	0.429	0.469
rpl-18	rps-2	510,406	497,379	Y45F10D.12.2	C49H3.11.1	0.999	0.999
rpl-18	rps-13	510,406	495,308	Y45F10D.12.2	C16A3.9.1	0.999	0.999
rps-17	tct-1	506,860	499,979	T08B2.10.1	F25H2.11.2	0.874	0.884
nmy-1	mlc-4	502,225	497,828	F52B10.1	C56G7.1.2	0	0.698
drs-1	rpl-4	493,869	493,528	B0464.1.1	B0041.4.1	0.581	0.74
rpl-5	rps-0	502,561	493,798	F54C9.5.1	B0393.1.1	0.999	0.999
sdhd-1	cey-1	500,588	500,586	F33A8.5.2	F33A8.3.2	0.224	0.624
rpl-18	rps-28	510,406	510,228	Y45F10D.12.2	Y41D4B.5.2	0.78	0.928
rps-28	rps-13	510,228	495,308	Y41D4B.5.2	C16A3.9.1	0.794	0.979
pab-1	rpl-5	509,182	502,561	Y106G6H.2a.4	F54C9.5.1	0.762	0.978
rps-4	icd-1	510,280	497,806	Y43B11AR.4.2	C56C10.8.1	0.826	0.833
eft-3	atp-2	505,324	496,479	R03G5.1a.2	C34E10.6.3	0.478	0.602
iff-1	rps-0	506,602	493,798	T05G5.10	B0393.1.1	0.604	0.623
rpl-18	pab-1	510,406	509,182	Y45F10D.12.2	Y106G6H.2a.4	0.522	0.95
iff-1	rpl-4	506,602	493,528	T05G5.10	B0041.4.1	0.603	0.656
rla-2	rpl-18	511,588	510,406	Y62E10A.1.1	Y45F10D.12.2	0.967	0.997
crt-1	rpl-5	509,852	502,561	Y38A10A.5.1	F54C9.5.1	0.452	0.465
rps-28	tpi-1	510,228	509,457	Y41D4B.5.2	Y17G7B.7.2	0.278	0.451
rps-4	iff-1	510,280	506,602	Y43B11AR.4.2	T05G5.10	0.64	0.888
icd-1	rps-13	497,806	495,308	C56C10.8.1	C16A3.9.1	0.856	0.856
trap-4	rla-1	511,330	509,817	Y56A3A.21.2	Y37E3.7.2	0.176	0.923
nmy-1	unc-54	502,225	498,853	F52B10.1	F11C3.3.1	0.102	0.565
trap-4	rps-13	511,330	495,308	Y56A3A.21.2	C16A3.9.1	0.148	0.942
iff-1	rps-13	506,602	495,308	T05G5.10	C16A3.9.1	0.614	0.614
maoc-1	ftn-2	498,218	497,902	E04F6.3	D1037.3.4	0.997	0.996
rpl-18	eft-3	510,406	505,324	Y45F10D.12.2	R03G5.1a.2	0.428	0.938
exos-2	cpf-1	512,052	500,247	Y73B6BL.3	F28C6.3	0.403	0.403
rla-2	rla-1	511,588	509,817	Y62E10A.1.1	Y37E3.7.2	0.999	0.999
rla-2	eft-3	511,588	505,324	Y62E10A.1.1	R03G5.1a.2	0.104	0.91
iff-1	rps-2	506,602	497,379	T05G5.10	C49H3.11.1	0.696	0.903
rpl-7A	rpl-4	509,604	493,528	Y24D9A.4a	B0041.4.1	0.955	0.996
tpi-1	daf-21	509,457	497,248	Y17G7B.7.2	C47E8.5.1	0.313	0.404
crt-1	rps-0	509,852	493,798	Y38A10A.5.1	B0393.1.1	0.402	0.462
rpl-7A	iff-1	509,604	506,602	Y24D9A.4a	T05G5.10	0.477	0.536
pab-1	rps-17	509,182	506,860	Y106G6H.2a.4	T08B2.10.1	0.217	0.936
tpi-1	rps-17	509,457	506,860	Y17G7B.7.2	T08B2.10.1	0.341	0.412
act-4	rpl-4	505,013	493,528	M03F4.2a	B0041.4.1	0.471	0.572
crt-1	rps-2	509,852	497,379	Y38A10A.5.1	C49H3.11.1	0.507	0.523
rpl-7A	tct-1	509,604	499,979	Y24D9A.4a	F25H2.11.2	0.793	0.794
exos-2	lsm-3	512,052	511,591	Y73B6BL.3	Y62E10A.12.2	0.164	0.449
rla-1	icd-1	509,817	497,806	Y37E3.7.2	C56C10.8.1	0.835	0.835
crt-1	cmd-1	509,852	507,723	Y38A10A.5.1	T21H3.3.1	0.116	0.609
pab-1	atp-2	509,182	496,479	Y106G6H.2a.4	C34E10.6.3	0.432	0.432
inf-1	rpl-5	503,072	502,561	F57B9.6a.3	F54C9.5.1	0.409	0.409
trap-4	rps-0	511,330	493,798	Y56A3A.21.2	B0393.1.1	0.174	0.911
rpl-18	atp-2	510,406	496,479	Y45F10D.12.2	C34E10.6.3	0.476	0.659
rps-2	drs-1	497,379	493,869	C49H3.11.1	B0464.1.1	0.664	0.663
rpl-5	drs-1	502,561	493,869	F54C9.5.1	B0464.1.1	0.332	0.403
rpl-5	daf-21	502,561	497,248	F54C9.5.1	C47E8.5.1	0.559	0.676
rpl-18	rpl-5	510,406	502,561	Y45F10D.12.2	F54C9.5.1	0.999	0.999
trap-4	rpl-5	511,330	502,561	Y56A3A.21.2	F54C9.5.1	0.273	0.949
snr-2	rps-0	508,903	493,798	W08E3.1	B0393.1.1	0.35	0.414
unc-54	daf-21	498,853	497,248	F11C3.3.1	C47E8.5.1	0	0.674
rpl-18	rps-0	510,406	493,798	Y45F10D.12.2	B0393.1.1	0.999	0.999
rpl-7A	icd-1	509,604	497,806	Y24D9A.4a	C56C10.8.1	0.737	0.756
tpi-1	rps-2	509,457	497,379	Y17G7B.7.2	C49H3.11.1	0.484	0.521
qua-1	nas-4	506,538	494,442	T05C12.10	C05D11.6	0.42	0.419
try-1	cpn-1	513,205	501,580	ZK546.15	F43G9.9.1	0.408	0.408
rla-1	iff-1	509,817	506,602	Y37E3.7.2	T05G5.10	0.551	0.691
rpl-18	iff-1	510,406	506,602	Y45F10D.12.2	T05G5.10	0.564	0.577
pab-1	eft-3	509,182	505,324	Y106G6H.2a.4	R03G5.1a.2	0.398	0.708
abcf-2	rps-2	508,270	497,379	T27E9.7.1	C49H3.11.1	0.268	0.452
tct-1	rps-0	499,979	493,798	F25H2.11.2	B0393.1.1	0.987	0.987
alp-1	daf-21	507,123	497,248	T11B7.4d	C47E8.5.1	0	0.998
daf-21	rps-0	497,248	493,798	C47E8.5.1	B0393.1.1	0.44	0.445
lev-11	act-4	509,147	505,013	Y105E8B.1d	M03F4.2a	0.244	0.441
daf-21	atp-2	497,248	496,479	C47E8.5.1	C34E10.6.3	0.415	0.745
rla-1	tct-1	509,817	499,979	Y37E3.7.2	F25H2.11.2	0.998	0.999
rps-17	iff-1	506,860	506,602	T08B2.10.1	T05G5.10	0.569	0.678
rps-28	rla-1	510,228	509,817	Y41D4B.5.2	Y37E3.7.2	0.785	0.963
try-1	cyp-31A2	513,205	503,484	ZK546.15	H02I12.8	0.543	0.543
rpl-1	rpl-5	511,889	502,561	Y71F9AL.13a.1	F54C9.5.1	0.799	0.995
rps-28	rps-0	510,228	493,798	Y41D4B.5.2	B0393.1.1	0.76	0.966
act-4	unc-54	505,013	498,853	M03F4.2a	F11C3.3.1	0.168	0.474
rpl-5	tct-1	502,561	499,979	F54C9.5.1	F25H2.11.2	0.996	0.996
rla-1	rps-13	509817	495,308	Y37E3.7.2	C16A3.9.1	0.999	0.999
rpl-1	rps-17	511,889	5068,60	Y71F9AL.13a.1	T08B2.10.1	0.799	0.993
rpl-1	rla-2	511,889	511,588	Y71F9AL.13a.1	Y62E10A.1.1	0.772	0.996
cey-1	cpf-1	500,586	500,247	F33A8.3.2	F28C6.3	0	0.904
pab-1	rps-13	509,182	495,308	Y106G6H.2a.4	C16A3.9.1	0.327	0.928
sqv-8	ret-1	512,932	508,785	ZK1307.5	W06A7.3f	0	0.579
rps-28	rpl-7A	510,228	509,604	Y41D4B.5.2	Y24D9A.4a	0.752	0.838
icd-1	rps-0	497,806	493,798	C56C10.8.1	B0393.1.1	0.875	0.88
rla-1	tpi-1	509,817	509,457	Y37E3.7.2	Y17G7B.7.2	0.501	0.501
icd-1	rps-2	497,806	497,379	C56C10.8.1	C49H3.11.1	0.913	0.913
sod-2	atp-2	498,735	496,479	F10D11.1.1	C34E10.6.3	0.271	0.595
hum-5	arx-6	506,256	496,567	T02C12.1	C35D10.16	0	0.421
rla-1	eft-3	509,817	505,324	Y37E3.7.2	R03G5.1a.2	0.189	0.92
iff-1	rpl-5	506,602	502,561	T05G5.10	F54C9.5.1	0.58	0.61
atp-2	rpl-4	496,479	493,528	C34E10.6.3	B0041.4.1	0.828	0.835
rpl-5	rps-13	502,561	495,308	F54C9.5.1	C16A3.9.1	0.999	0.999
unc-87	unc-60	498,495	496,717	F08B6.4a	C38C3.5b.1	0	0.407
mlc-3	unc-87	498,666	498,495	F09F7.2a.2	F08B6.4a	0.516	0.516
gpd-3	atp-2	504,646	496,479	K10B3.7.2	C34E10.6.3	0.214	0.523
rpl-5	rps-2	502,561	497,379	F54C9.5.1	C49H3.11.1	0.999	0.999
tct-1	icd-1	499,979	497,806	F25H2.11.2	C56C10.8.1	0.746	0.751
rpl-1	rps-2	511,889	497,379	Y71F9AL.13a.1	C49H3.11.1	0.798	0.995
pab-1	icd-1	509,182	497,806	Y106G6H.2a.4	C56C10.8.1	0.458	0.458
rps-4	rps-13	510,280	495,308	Y43B11AR.4.2	C16A3.9.1	0.999	0.999
rps-4	rpl-7A	510,280	509,604	Y43B11AR.4.2	Y24D9A.4a	0.949	0.989
pab-1	rpl-4	509,182	493,528	Y106G6H.2a.4	B0041.4.1	0.69	0.966
rps-17	rps-0	506,860	493,798	T08B2.10.1	B0393.1.1	0.999	0.999
rps-17	icd-1	506,860	497,806	T08B2.10.1	C56C10.8.1	0.856	0.861
rpl-7A	drs-1	509,604	493,869	Y24D9A.4a	B0464.1.1	0.451	0.519
rpl-18	rla-1	510,406	509,817	Y45F10D.12.2	Y37E3.7.2	0.996	0.999
rpl-1	rpl-18	511,889	510,406	Y71F9AL.13a.1	Y45F10D.12.2	0.832	0.993
daf-21	unc-60	497,248	496,717	C47E8.5.1	C38C3.5b.1	0	0.911
sqv-8	cyp-31A2	512,932	503,484	ZK1307.5	H02I12.8	0.411	0.411
act-4	rps-13	505,013	495,308	M03F4.2a	C16A3.9.1	0.53	0.53
tct-1	rps-13	499,979	495,308	F25H2.11.2	C16A3.9.1	0.996	0.996
crt-1	eft-3	509,852	505,324	Y38A10A.5.1	R03G5.1a.2	0.573	0.573
rps-2	daf-21	497,379	497,248	C49H3.11.1	C47E8.5.1	0.658	0.721
let-70	cyp-31A2	505,189	503,484	M7.1	H02I12.8	0	0.522
rps-13	drs-1	495,308	493,869	C16A3.9.1	B0464.1.1	0.528	0.528
crt-1	pdi-3	509,852	503,539	Y38A10A.5.1	H06O01.1.3	0.726	0.739
rpl-18	rpl-7A	510,406	509,604	Y45F10D.12.2	Y24D9A.4a	0.925	0.984
rps-13	rps-0	495,308	493,798	C16A3.9.1	B0393.1.1	0.999	0.999
rps-4	rpl-4	510,280	493,528	Y43B11AR.4.2	B0041.4.1	0.999	0.999
rps-4	rps-2	510,280	497,379	Y43B11AR.4.2	C49H3.11.1	0.999	0.999
rla-1	rpl-7A	509,817	509,604	Y37E3.7.2	Y24D9A.4a	0.872	0.912
rps-28	rps-17	510,228	506,860	Y41D4B.5.2	T08B2.10.1	0.805	0.989
rpl-18	tct-1	510,406	499,979	Y45F10D.12.2	F25H2.11.2	0.963	0.966
rla-2	rps-28	511,588	510,228	Y62E10A.1.1	Y41D4B.5.2	0.799	0.879
eft-3	rpl-4	505,324	493,528	R03G5.1a.2	B0041.4.1	0.713	0.97
tpi-1	rpl-4	509,457	493,528	Y17G7B.7.2	B0041.4.1	0.489	0.498
tpi-1	gpd-3	509,457	504,646	Y17G7B.7.2	K10B3.7.2	0.608	0.998
unc-54	unc-60	498,853	496,717	F11C3.3.1	C38C3.5b.1	0	0.587
tpi-1	cey-1	509,457	500,586	Y17G7B.7.2	F33A8.3.2	0	0.403
hum-5	unc-54	506,256	498,853	T02C12.1	F11C3.3.1	0	0.54
rps-4	pab-1	510,280	509,182	Y43B11AR.4.2	Y106G6H.2a.4	0.344	0.955
rla-2	icd-1	511,588	497,806	Y62E10A.1.1	C56C10.8.1	0.809	0.81
rpl-7A	rpl-5	509,604	502,561	Y24D9A.4a	F54C9.5.1	0.934	0.994
rps-17	eft-3	506,860	505,324	T08B2.10.1	R03G5.1a.2	0.187	0.916
rpl-18	rpl-4	510,406	493,528	Y45F10D.12.2	B0041.4.1	0.999	0.999
rla-2	daf-21	511,588	497,248	Y62E10A.1.1	C47E8.5.1	0.38	0.548
rps-4	tct-1	510,280	499,979	Y43B11AR.4.2	F25H2.11.2	0.941	0.946
rps-4	rps-28	510,280	510,228	Y43B11AR.4.2	Y41D4B.5.2	0.76	0.971
rla-2	rpl-5	511,588	502,561	Y62E10A.1.1	F54C9.5.1	0.967	0.999
rps-4	rpl-5	510,280	502,561	Y43B11AR.4.2	F54C9.5.1	0.999	0.999
rla-1	pab-1	509,817	509,182	Y37E3.7.2	Y106G6H.2a.4	0.29	0.929
rla-2	rps-2	511,588	497,379	Y62E10A.1.1	C49H3.11.1	0.991	0.999
iff-1	tct-1	506,602	499,979	T05G5.10	F25H2.11.2	0.483	0.483
rla-1	rps-2	509,817	497,379	Y37E3.7.2	C49H3.11.1	0.997	0.999
rpl-1	pab-1	511,889	509,182	Y71F9AL.13a.1	Y106G6H.2a.4	0.237	0.918
rps-28	rps-2	510,228	497,379	Y41D4B.5.2	C49H3.11.1	0.732	0.981
rpl-5	rpl-4	502,561	493,528	F54C9.5.1	B0041.4.1	0.999	0.999
rps-4	rps-17	510,280	506,860	Y43B11AR.4.2	T08B2.10.1	0.995	0.999
rpl-18	rps-17	510,406	506,860	Y45F10D.12.2	T08B2.10.1	0.956	0.996
rpl-1	rpl-7A	511,889	509,604	Y71F9AL.13a.1	Y24D9A.4a	0.796	0.96
inf-1	rps-0	503,072	493,798	F57B9.6a.3	B0393.1.1	0.414	0.433
alp-1	unc-87	507,123	498,495	T11B7.4d	F08B6.4a	0.413	0.421
rla-1	rpl-4	509,817	493,528	Y37E3.7.2	B0041.4.1	0.997	0.999
rps-2	rps-13	497,379	495,308	C49H3.11.1	C16A3.9.1	0.998	0.999
pdi-3	rps-2	503,539	497,379	H06O01.1.3	C49H3.11.1	0.457	0.457
cpl-1	cpf-1	506,335	500,247	T03E6.7.2	F28C6.3	0	0.408
gyg-1	cey-1	502,889	500,586	F56B6.4a	F33A8.3.2	0	0.55
hum-5	nmy-1	506,256	502,225	T02C12.1	F52B10.1	0	0.544
gpd-3	sod-2	504,646	498,735	K10B3.7.2	F10D11.1.1	0	0.588
rps-4	tpi-1	510,280	509,457	Y43B11AR.4.2	Y17G7B.7.2	0.444	0.444
rla-2	trap-4	511,588	511,330	Y62E10A.1.1	Y56A3A.21.2	0.076	0.916
rps-28	rpl-4	510,228	493,528	Y41D4B.5.2	B0041.4.1	0.756	0.847
drs-1	rps-0	493,869	493,798	B0464.1.1	B0393.1.1	0.464	0.494
sdhd-1	cyc-1	500,588	497,719	F33A8.5.2	C54G4.8.1	0.382	0.54
rpl-1	icd-1	511,889	497,806	Y71F9AL.13a.1	C56C10.8.1	0.777	0.787
rps-17	rps-13	506,860	495,308	T08B2.10.1	C16A3.9.1	0.997	0.999
rla-1	rps-0	509,817	493,798	Y37E3.7.2	B0393.1.1	0.999	0.999
daf-21	rpl-4	497,248	493,528	C47E8.5.1	B0041.4.1	0.59	0.634
tpi-1	cyc-1	509,457	497,719	Y17G7B.7.2	C54G4.8.1	0.442	0.444
trap-4	rpl-4	511,330	493,528	Y56A3A.21.2	B0041.4.1	0.308	0.951
rpl-1	rps-28	511,889	510,228	Y71F9AL.13a.1	Y41D4B.5.2	0.793	0.939
rps-28	tct-1	510,228	499,979	Y41D4B.5.2	F25H2.11.2	0.683	0.683
eft-3	rpl-5	505,324	502,561	R03G5.1a.2	F54C9.5.1	0.701	0.974
sod-2	cyc-1	498,735	497,719	F10D11.1.1	C54G4.8.1	0.456	0.604
pdi-3	phy-2	503,539	500,798	H06O01.1.3	F35G2.4.1	0	0.4
rpl-18	daf-21	510,406	497,248	Y45F10D.12.2	C47E8.5.1	0.387	0.401
rpl-1	tct-1	511,889	499,979	Y71F9AL.13a.1	F25H2.11.2	0.751	0.757
rpl-1	iff-1	511,889	506,602	Y71F9AL.13a.1	T05G5.10	0.612	0.624
rpl-1	tpi-1	511,889	509,457	Y71F9AL.13a.1	Y17G7B.7.2	0.396	0.614
rps-0	rpl-4	493,798	493,528	B0393.1.1	B0041.4.1	0.999	0.999
eft-3	rps-13	505,324	495,308	R03G5.1a.2	C16A3.9.1	0.362	0.956
act-4	unc-60	505,013	496,717	M03F4.2a	C38C3.5b.1	0	0.863
crt-1	pab-1	509,852	509,182	Y38A10A.5.1	Y106G6H.2a.4	0.425	0.453
tct-1	atp-2	499,979	496,479	F25H2.11.2	C34E10.6.3	0.575	0.583
rps-13	rpl-4	495,308	493,528	C16A3.9.1	B0041.4.1	0.998	0.999
rps-2	rpl-4	497,379	493,528	C49H3.11.1	B0041.4.1	0.999	0.999
rla-2	rps-17	511,588	506,860	Y62E10A.1.1	T08B2.10.1	0.997	0.999
rla-1	rps-17	509,817	506,860	Y37E3.7.2	T08B2.10.1	0.966	0.997
rps-17	rps-2	506,860	497,379	T08B2.10.1	C49H3.11.1	0.969	0.999
cyc-1	eat-6	497,719	493,780	C54G4.8.1	B0365.3.2	0.295	0.425
trap-4	rpl-18	511,330	510,406	Y56A3A.21.2	Y45F10D.12.2	0.37	0.956
tpi-1	rps-13	509,457	495,308	Y17G7B.7.2	C16A3.9.1	0.51	0.556
rla-2	rps-0	511,588	493,798	Y62E10A.1.1	B0393.1.1	0.994	0.999
icd-1	drs-1	497,806	493,869	C56C10.8.1	B0464.1.1	0.403	0.509
sod-2	ftn-2	498,735	497,902	F10D11.1.1	D1037.3.4	0	0.494
pab-1	cey-1	509,182	500,586	Y106G6H.2a.4	F33A8.3.2	0.317	0.447
pab-1	rps-0	509,182	493,798	Y106G6H.2a.4	B0393.1.1	0.546	0.958
rla-1	rpl-5	509,817	502,561	Y37E3.7.2	F54C9.5.1	0.996	0.999
rps-17	rpl-4	506,860	493,528	T08B2.10.1	B0041.4.1	0.995	0.999
lsm-3	rpl-4	511,591	493,528	Y62E10A.12.2	B0041.4.1	0.126	0.622
tct-1	rpl-4	499,979	493,528	F25H2.11.2	B0041.4.1	0.919	0.926
icd-1	daf-21	497,806	497,248	C56C10.8.1	C47E8.5.1	0.439	0.478
rpl-1	rps-0	511,889	493,798	Y71F9AL.13a.1	B0393.1.1	0.808	0.992
rpl-1	trap-4	511,889	511,330	Y71F9AL.13a.1	Y56A3A.21.2	0.258	0.92
rps-4	eft-3	510,280	505,324	Y43B11AR.4.2	R03G5.1a.2	0.402	0.939
rps-4	rla-1	510,280	509,817	Y43B11AR.4.2	Y37E3.7.2	0.997	0.999
atp-2	rps-0	496,479	493,798	C34E10.6.3	B0393.1.1	0.432	0.763
rpl-7A	rps-2	509,604	497,379	Y24D9A.4a	C49H3.11.1	0.914	0.978
rla-2	rpl-7A	511,588	509,604	Y62E10A.1.1	Y24D9A.4a	0.936	0.958
rps-28	icd-1	510,228	497,806	Y41D4B.5.2	C56C10.8.1	0.772	0.772
rpl-18	icd-1	510,406	497,806	Y45F10D.12.2	C56C10.8.1	0.857	0.863
inf-1	rps-2	503,072	497,379	F57B9.6a.3	C49H3.11.1	0.395	0.417
tpi-1	tct-1	509,457	499,979	Y17G7B.7.2	F25H2.11.2	0.428	0.476
rps-17	rpl-5	506,860	502,561	T08B2.10.1	F54C9.5.1	0.972	0.998
snr-2	cpf-1	508,903	500,247	W08E3.1	F28C6.3	0.245	0.919
pab-1	rps-2	509,182	497,379	Y106G6H.2a.4	C49H3.11.1	0.704	0.968
skr-1	pas-2	501,798	497,931	F46A9.5.3	D1054.2.1	0	0.899
rla-2	act-4	511,588	505,013	Y62E10A.1.1	M03F4.2a	0.508	0.508
icd-1	rpl-4	497,806	493,528	C56C10.8.1	B0041.4.1	0.826	0.858
eft-3	rps-2	505,324	497,379	R03G5.1a.2	C49H3.11.1	0.66	0.971
rpl-1	rps-13	511,889	495,308	Y71F9AL.13a.1	C16A3.9.1	0.8	0.997
rps-2	atp-2	497,379	496,479	C49H3.11.1	C34E10.6.3	0.943	0.947
unc-54	unc-87	498,853	498,495	F11C3.3.1	F08B6.4a	0.566	0.776
tct-1	rps-2	499,979	497,379	F25H2.11.2	C49H3.11.1	0.999	0.999
trap-4	rps-4	511,330	510,280	Y56A3A.21.2	Y43B11AR.4.2	0.245	0.921
rps-28	iff-1	510,228	506,602	Y41D4B.5.2	T05G5.10	0.432	0.635
rla-1	daf-21	509,817	497,248	Y37E3.7.2	C47E8.5.1	0.394	0.543
rps-4	crt-1	510,280	509,852	Y43B11AR.4.2	Y38A10A.5.1	0.491	0.5
rpl-18	crt-1	510,406	509,852	Y45F10D.12.2	Y38A10A.5.1	0.595	0.597
rps-2	rps-0	497,379	493,798	C49H3.11.1	B0393.1.1	0.999	0.999
lev-11	unc-87	509,147	498,495	Y105E8B.1d	F08B6.4a	0.272	0.454
trap-4	rps-17	511,330	506,860	Y56A3A.21.2	T08B2.10.1	0.308	0.929
tct-1	daf-21	499,979	497,248	F25H2.11.2	C47E8.5.1	0.648	0.713
crt-1	rpl-4	509,852	493,528	Y38A10A.5.1	B0041.4.1	0.472	0.604
rpl-18	rps-4	510,406	510,280	Y45F10D.12.2	Y43B11AR.4.2	0.999	0.999
eft-3	rps-0	505,324	493,798	R03G5.1a.2	B0393.1.1	0.523	0.959
rpl-1	eft-3	511,889	505,324	Y71F9AL.13a.1	R03G5.1a.2	0.348	0.948
iff-1	icd-1	506,602	497,806	T05G5.10	C56C10.8.1	0.47	0.47
rpl-7A	rps-13	509,604	495,308	Y24D9A.4a	C16A3.9.1	0.944	0.979
eft-3	ftn-2	505,324	497,902	R03G5.1a.2	D1037.3.4	0	0.999
rps-4	drs-1	510,280	493,869	Y43B11AR.4.2	B0464.1.1	0.422	0.434
snr-2	cey-1	508,903	500,586	W08E3.1	F33A8.3.2	0	0.899
rpl-1	atp-2	511,889	496,479	Y71F9AL.13a.1	C34E10.6.3	0.144	0.403
rla-2	rpl-4	511,588	493,528	Y62E10A.1.1	B0041.4.1	0.995	0.999
act-2	act-4	506,426	505,013	T04C12.5	M03F4.2a	0	0.481
cyn-7	daf-21	512,151	497,248	Y75B12B.2.2	C47E8.5.1	0.359	0.408
rla-2	tct-1	511,588	499,979	Y62E10A.1.1	F25H2.11.2	0.96	0.963
rps-4	rps-0	510,280	493,798	Y43B11AR.4.2	B0393.1.1	0.999	0.999
rpl-18	drs-1	510,406	493,869	Y45F10D.12.2	B0464.1.1	0.408	0.474
atp-2	eat-6	496,479	493,780	C34E10.6.3	B0365.3.2	0.666	0.707
rpl-7A	rps-0	509,604	493,798	Y24D9A.4a	B0393.1.1	0.942	0.947
lsm-3	snr-2	511,591	508,903	Y62E10A.12.2	W08E3.1	0.323	0.625
rla-2	rps-13	511,588	495,308	Y62E10A.1.1	C16A3.9.1	0.996	0.999

Figure 7.

Connectivity between the predicted regulators and the clusters in the STRING network: Experimentally-derived interactions (green), co-expression (navy blue), and co-occurrences (red) in the genomes are shown. Colored circles represent input genes.

Discussion

The present study reported on the construction of a high-quality cDNA library from tissues of the earthworm Eisenia fetida (Savigny, 1826), following preliminary analysis of ESTs, putative functional analysis of the ESTs and the gene expression pattern associated with the physiological functions of this organism. cDNA libraries are widely used to identify genes and splice variants and are considered to be a physical resource for the construction of full-length clones (18,19). In the present study, a cDNA library was utilized to provide a molecular resource for the analysis of genes involved in the specific biology of earthworms in terms of their development, survival, pathogenicity and virulence. There are two main factors to consider when assessing the quality of a cDNA library: Representation and cDNA lengths. According to Clarke-Carbon's formula (20), a cDNA library should contain at least 1.7×105 independent clones to ensure that 99% of low-abundance mRNAs will be represented in the library (21). Furthermore, the average length of the inserted cDNAs should be no less than 1.0 kb to ensure the integrity of cDNAs, indicating that in the present study the fragment sizes were effective for ensuring full-length cDNAs in the cDNA library. Since selection bias could favor the smaller cDNAs, the present study used fewer PCR cycles to minimize such bias as previously suggested (10). In addition, up to 25 PCR amplification cycles were used to generate an adequate amount of cDNA for cloning.

The generation of ESTs is an effective and unique approach in molecular studies as it allows for the analysis and measurement of gene expression, as well as simultaneous discovery of new genes. As each EST represents a copy of the functional part of a genome, the study of ESTs is believed to be a more effective way to discover functional genes (22). Furthermore, analysis of the expression of a large number of genes combined with the knowledge of their functions enables insight into the overall situation in terms of biological processes (23), for the current purposes in earthworms. In the present study, ~91% of the ESTs generated were sequences with known or putative functions, while the remainder represented unknown proteins or sequences with no similarities to those in the databases. Although close to 600 ESTs were reported, this is actually far from what could be considered as a ‘complete’ transcriptome (which usually includes between 15,000–20,000 to >100,000 ESTs). Therefore, the present characterization of this seemingly partial transcriptome may far from reflect the full transcriptomic profile of tissues in earthworms.

A comparison of the classification of ESTs with a C. elegans cDNA library based on their putative functions was conducted. Based on identification of clusters via GO analysis, 168 ESTs were matched to C. elegans genes by BLASTx. It is well known that earthworms serve significant roles in organic matter decomposition and mineral cycling, and thus are considered to be important contributors to soil fertility and humification processes (24). In the present study, hydrolytic enzyme activity, conjugating protein ligase activity, oxidation reduction and energy release activity of metabolic enzymes accounted for a large proportion of the molecular functions component. These molecular functions are considered to be a key part of the physiological functions of earthworms, which allow earthworms to survive in different soil environments.

Cell components, as part of GO annotation, are mainly categorized based on subcellular location (including cell cytoplasm, mitochondria, lysosomes, nucleus, microtubules, plasma membrane and myosin), which is highly important for the study of protein functions (25). The results of subcellular localization analysis can provide significant clues to aid the understanding of protein functions. In the present study, several cell components were determined through GO analysis of the 168 ESTs (annotated genes). It was evident that these cell components had strong associations with the regulation of gene expression during the biological development of earthworms, enabling the regeneration of the anterior portion, alterations in movement ability and tissue differentiation.

Due to the temporal specificity of gene expression and interactions with other gene products, the specific pathway undertaken, sequence of gene expression and expression pattern may ultimately change the effects of multiple pathways in earthworms (2). Therefore, the cDNA library and transcription profile of genes representative of fully developed adult earthworms may differ markedly to those of juvenile earthworms. In the present study, gene expression profiles representative of adult earthworm development were generated; however, gene expression profiling of juvenile earthworms was not performed, nor were analyses of the expected differential gene expression between juveniles and adults. In the present study, 168 individual ESTs of earthworms were analyzed in terms of KEGG pathway annotation, which identified 9 corresponding categories. Among them, glutathione metabolism is involved in antioxidant defense systems in Eisenia Andrei, and the associated enzymes are mainly identified in cytosolic fractions (26). Chondroitin sulfate and heparan sulfate biosynthesis are involved in biosynthesis pathways, which have important effects on growth and regeneration. In addition, heparan sulfate is also considered to be a type of anticoagulant, which may also be a function of arachidonic acid (27). Therefore, understanding the molecular function of earthworms may provide some basis for the treatment of thrombotic disorders.

Information on functional annotation and relevant biological interactions associated with a particular gene is available from many online resources. The gene network comprises a collection of genes that cooperate with each other to control the main biological processes. The STRING database suggested a functional context for earthworm lumbrokinase with unknown specificity. A previous study revealed that lumbrokinase and dilong administration can efficiently reduce the incidence of cardiac disease among nonsmokers exposed to second-hand smoke (28). In this regard, the discovery of genes and protein interactions in earthworms has provided a basis for further investigation into human diseases.