- Research article
- Open Access
Comparative transcriptome sequencing of germline and somatic tissues of the Ascaris suum gonad
© Ma et al; licensee BioMed Central Ltd. 2011
- Received: 11 May 2011
- Accepted: 1 October 2011
- Published: 1 October 2011
Ascaris suum (large roundworm of pigs) is a parasitic nematode that causes substantial losses to the meat industry. This nematode is suitable for biochemical studies because, unlike C. elegans, homogeneous tissue samples can be obtained by dissection. It has large sperm, produced in great numbers that permit biochemical studies of sperm motility. Widespread study of A. suum would be facilitated by more comprehensive genome resources and, to this end, we have produced a gonad transcriptome of A. suum.
Two 454 pyrosequencing runs generated 572,982 and 588,651 reads for germline (TES) and somatic (VAS) tissues of the A. suum gonad, respectively. 86% of the high-quality (HQ) reads were assembled into 9,955 contigs and 69,791 HQ reads remained as singletons. 2.4 million bp of unique sequences were obtained with a coverage that reached 16.1-fold. 4,877 contigs and 14,339 singletons were annotated according to the C. elegans protein and the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases. Comparison of TES and VAS transcriptomes demonstrated that genes participating in DNA replication, RNA transcription and ubiquitin-proteasome pathways are expressed at significantly higher levels in TES tissues than in VAS tissues. Comparison of the A. suum TES transcriptome with the C. elegans microarray dataset identified 165 A. suum germline-enriched genes (83% are spermatogenesis-enriched). Many of these genes encode serine/threonine kinases and phosphatases (KPs) as well as tyrosine KPs. Immunoblot analysis further suggested a critical role of phosphorylation in both testis development and spermatogenesis. A total of 2,681 A. suum genes were identified to have associated RNAi phenotypes in C. elegans, the majority of which display embryonic lethality, slow growth, larval arrest or sterility.
Using deep sequencing technology, this study has produced a gonad transcriptome of A. suum. By comparison with C. elegans datasets, we identified sets of genes associated with spermatogenesis and gonad development in A. suum. The newly identified genes encoding KPs may help determine signaling pathways that operate during spermatogenesis. A large portion of A. suum gonadal genes have related RNAi phenotypes in C. elegans and, thus, might be RNAi targets for parasite control.
- Sperm Motility
- Parasite Control
- Sperm Extract
- RNAi Phenotype
- Germline Development
The genus Ascaris, also known as the "giant intestinal roundworms", contains the largest intestinal nematode species. Ascaris lumbricoides causes the commonest helminth infection of humans, whereas a closely related species, Ascaris suum, typically infects pigs and causes substantial financial losses to the meat industry. The A. suum female is capable of producing more than 200,000 eggs per day and these eggs can survive and remain infective after many years in soil . At present, there is no effective alternative to chemical control of intestinal parasites and resistance to anthelmintics has become an emerging problem . Greater knowledge of nematode biology is urgently needed to enable the development of new biotechnological tools (e.g., RNA interference) for parasite control.
To better understand the molecular and biochemical basis of nematode development, nematode EST projects have generated more than 250,000 ESTs from 30 species, including A. suum. Large-scale EST datasets have also been acquired by next-generation sequencing (NGS) technologies and the associated bioinformatic pipeline has been developed [4, 5]. This vast collection of ESTs combined with the extensive knowledge of Caenorhabditis elegans biology provides opportunities to elucidate functionally conserved mechanisms in nematode biology. Employment of NGS technologies has greatly accelerated the 959 Nematode Genomes project http://www.nematodes.org/nematodegenomes/index.php/Main_Page. Genome sequencing of A. suum somatic cells is ongoing http://www.sanger.ac.uk/resources/downloads/helminths/ascaris-suum.html, and a draft genome and transcriptome of A. suum is now available http://www.nematode.net/NN3_frontpage.cgi?navbar_selection=home&subnav_selection=asuum_ftp. To date, there have been 38 A. suum EST libraries with ~55,000 sequences available in the NEMBASE4 database http://www.nematodes.org/nembase4/ and these ESTs were obtained using conventional cDNA library sequencing technology.
In the present study, we applied 454 pyrosequencing technology to unravel the transcriptome of the male A. suum gonad, the organ for reproduction. A. suum males have a large gonad that can be readily isolated by dissection to provide large numbers of sperm that are suitable for biochemical and cell biological studies . The male A. suum gonad is composed of three distinct regions; the testis and seminal vesicle form germline tissue and the glandular vas deferens forms somatic tissue. Sperm are stored in the seminal vesicle. During copulation, the spherical, non-motile sperm are activated into bipolar, amoeboid spermatozoa by an unknown component secreted by the glandular vas deferens. The motility of amoeboid sperm is driven by the regulated assembly and disassembly of major sperm protein (MSP) cytoskeleton [7, 8]. The mechanism of sperm activation is poorly understood and the details of MSP-based sperm motility are yet to be determined, although several proteins (e.g., MPOP, MFPs and PP2A) that participate in the dynamics of the MSP cytoskeleton have been identified [9–12]. Despite the advantages of large gametes and the easy isolation of reproductive fluids from A. suum, there have been few studies focusing on sperm chromatin or on distinctions between germline and somatic tissues in A. suum. In addition, chromatin diminution in A. suum represents a fascinating exception to the general rule of the constancy of the genome. However, the complex mechanism of this phenomenon, involving DNA degradation and new telomere addition remain an enigma [13–18]. One of the barriers to answering the above questions is the lack of gene expression data for the reproductive tissues of A. suum.
To facilitate diverse studies concerning reproductive biology in A. suum, we acquired the transcriptomes of germline and somatic tissues of A. suum gonad using the RNA-seq approach. Comparison of these two tissues showed that the nucleic acid metabolic and proteasome-ubiquitin pathways are more active in the germline than in the soma. Further comparison with C. elegans microarray data identified 165 conserved germline-enriched genes in A. suum. We also categorized the RNAi phenotypes for A. suum gonadal genes, taking advantage of the C. elegans RNAi phenotype database. Therefore, these A. suum transcriptome data provide a valuable platform for both fundamental biological studies (e.g., MSP-based sperm motility and spermatogenesis studies) and for research concerning parasite control (e.g. use of RNAi).
454 sequencing and de novo assembly of A. suum gonad transcriptome
Summary of 454 sequencing and assembly
No. of reads
No. of base-pairs
Average read length (bp)
No. of singletons (> 50 bp)
Average length of singletons
No. of contigs
Average length of contigs (bp)
Total coverage (bp)
The current A. suum testis EST library in NEMBASE4 has collected 2,868 ESTs. These ESTs correspond to 595 homologous genes in C. elegans (BLAST cutoff E-value = 1e-5). In contrast, our A. suum gonad transcriptome corresponds to 4,207 homologous genes in C. elegans and 3,686 novel gonadal genes were identified (Additional file 1). This suggests our A. suum gonad transcriptome has a deeper coverage than the conventional EST library.
Functional assignments of A. suum 454 sequencing data
To annotate the A. suum 454 transcriptome data, we compared all unique sequences against the C. elegans protein database in WormBase, as well as against the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases using BLASTX (cutoff E-value = 1e-5). A total of 4,877 contigs (49%) and 14,339 singletons (20.5%) were annotated. A large portion of the 454 sequences have not been functionally defined. Some sequences can be annotated by increasing the E-value and others may represent A. suum specific genes. In summary, 9,822 unique sequences (corresponding to 5,683 gene models) were annotated in the TES dataset and 12,123 unique sequences (corresponding to 4,122 gene models) were annotated in the VAS dataset (Additional file 2). Although ~2,000 more sequences were assigned in the VAS dataset compared with the TES dataset, TES has ~1,500 more gene models than VAS suggesting that there are more diverse genes expressed in TES than in VAS tissues.
Tubulin genes (> 12,000 reads) are the most abundant transcripts in the A. suum gonad; the fibulin genes (> 10,000 reads), whose activity is essential for gonad and body morphology in C. elegans, are also highly abundant. The expression of genes encoding intermediate filament proteins, heat shock proteins, ribosomal proteins, aldehyde reductase and major sperm proteins were also enriched. It should be noted that among the 100 most highly enriched genes, over half have not been functionally characterized.
Metabolic pathway mapping
Comparative analysis of TES and VAS datasets
To highlight the functions of differentially expressed genes between TES and VAS, the contigs having 10-fold higher levels of expression were searched against the STRING database http://string-db.org/ to identify the functional associations of these genes. The result demonstrated that, compared with the somatic VAS tissues, the germline TES tissues has a more complex gene/protein interaction network (Additional file 5). In the germline, the genes encoding proteins involved in DNA replication and RNA transcription are highly enriched; the germline also expresses a large number of genes participating in the proteasome and ubiquitin-mediated proteolysis pathways. These data underpin the nature of the germline, which functions through cell cycle progression and differentiation. It also shows the necessity of the proteasome in germline development. In addition, as expected, the expression of genes encoding MSPs and sperm specific proteins was highly enriched in the germline.
To validate the gene expression changes observed between TES and VAS tissues, we selected 18 genes (Daf-21, Cul-1, Skr-1, Ubc-7, Rbx-1, Rpn-1, Pas-4, Pbs-2, Let-70, Eel-1, Kin-19, Sel-12, Paa-1 Gsk-3, Cdc-42, Smo-1, Exos-7 and Pri-1) having significantly higher levels of expression in TES than in VAS for semi-quantitative RT-PCR analysis. These genes are involved in processes including, protein processing, ubiquitin-proteasome pathways, Wnt signaling, cell division and nucleic acid metabolism. The results (Additional file 6) showed that the expression in the majority of these genes is either down-regulated or absent in VAS as compared with TES.
Comparison with C. elegans microarray and RNAi screening datasets
Germline development in C. elegans has been extensively studied http://www.wormbook.org/toc_germline.html. A. suum and C. elegans belongs to Clade III and V, respectively, in the phylum Nematoda, and it is estimated that these two clades have an evolutionary divergence of 350 million years . To identify the conserved genes regulating gonad development, we compared the A. suum gonad transcriptome with two C. elegans datasets acquired by microarray and genome-wide RNAi analyses.
Using microarray technology, Reinke et al. identified 1,092 and 340 genes that have enriched expression in C. elegans adult male germline and soma, respectively (14 of these genes are pseudogenes or are no longer available in WormBase) . BLAST analysis showed that 532 (49.1%) of the C. elegans germline-enriched genes and 139 (32.8%) of the soma-enriched genes have homologues in A. suum TES and VAS tissues, respectively (Additional file 7). The corresponding A. suum germline-enriched genes include 259 contigs and 37 singletons and the expression profiling of the 259 contigs is illustrated in Figure 3B. 165 genes of these contigs have over 10-fold higher levels of expression in TES than in VAS, and thus might represent conserved genes controlling germline development in different nematode species (Additional file 8). Among them, 137 genes (83%) are spermatogenesis-enriched and the rest are involved in other aspects of germline development (e.g., mitotic proliferation). Substantial numbers of serine/threonine kinases and phosphatases (KPs), as well as tyrosine KPs were identified, suggesting pivotal roles of phosphorylation during spermatogenesis. It should be noted that the genes encoding KPs are over-represented among the sperm-enriched genes in C. elegans.
Categorization of RNAi phenotypes of A. suum gonadal genes
Nematodes are one of the most diverse phyla and they make up approximately 80% of all individual animals on earth [3, 26]. As the most prevalent nematode parasite in pigs, A. suum causes massive losses to the swine industry worldwide. The 959 Nematode Genomes Project has included this species for whole-genome sequencing and a draft genome and transcriptome of A. suum has just become available. In this study, we adopted 454 sequencing technology to determine the transcriptome of the A. suum gonad so as to facilitate further studies of this organism.
A total of 0.4 billion bp were obtained by 454 sequencing, which were assembled into 25 million bp, which is equivalent to the C. elegans exome (26 million bp). 86% of the high-quality reads were assembled into longer contigs, suggesting that these sequencing data had a high coverage. We annotated half of the contigs and 20% of the singletons; a large fraction of sequences have not been functionally assigned. The unannotated 454 ESTs may contain precursor non-coding RNAs (e.g., pre-miRNA, pre-snoRNAs) as well as the polyadenylated ncRNA classes; for example, over 13% and 26% of full-length cDNAs in mice and human, respectively, are proposed to be polyadenylated mRNA-like ncRNAs [27–29]. The germline (TES) encompasses more gene models than the soma (VAS). Metabolic pathway mapping analysis also showed that TES and VAS datasets have distinct groups of genes involved in their respective metabolic processes.
To investigate germline-soma distinctions, we compared the digital transcriptomes of TES and VAS, and identified numerous TES-specific pathways, including DNA replication and proteasome and ubiquitin-mediated proteolysis pathways. These pathways might be required to regulate germline proliferation and differentiation. The proteasome has been documented to regulate the balance between cell proliferation and meiotic entry in C. elegans. The genes encoding MSPs, sperm specific proteins and pyruvate dehydrogenase were the most highly expressed genes in the A. suum germline. MSP comprises 10-15% of the total proteins in nematode sperm  and sperm motility is driven by the regulated assembly and disassembly of MSP [7, 8, 32, 33]. Hence, the high levels of expression of MSP genes were expected. With regard to pyruvate dehydrogenase, we speculate it might promote the tricarboxylic acid (TCA) cycle to supply energy for germ cell development.
We have a particular interest in A. suum spermatogenesis. Based on the C. elegans microarray data, 165 A. suum genes (83% are spermatogenesis-enriched) were identified as conserved genes controlling germline development. The most abundant proteins involved in A. suum spermatogenesis consist of sperm specific proteins, PDZ domain proteins, tyrosine kinases and phosphatases (KPs), and serine/threonine KPs. Identification of the genes encoding KPs in this analysis underpins the essential role of phosphorylation in the regulation of spermatogenesis. As the most common posttranslational modification, phosphorylation has been established to link to sperm function in a variety of species. In mammalians, the processes regulated by phosphorylation include capacitation, hyperactivated motility, zona pellucida binding, acrosome reaction and sperm-oocyte binding and fusion [34–36]. In C. elegans, the genes encoding KPs are over-represented among the sperm-enriched genes . Clues to the phosphorylation signaling pathway that controls MSP-based cell motility were also documented in A. suum. A phosphorylated membrane protein (named MPOP) recruits a soluble casein kinase 1 (named MPAK) to the inner leaflet of the plasma membrane to initiate sperm motility ; MPAK, in turn, phosphorylates a second cytosolic protein (named MFP2) to accelerate MSP assembly [10, 11]. A putative PP2A homologue was shown to trigger the retraction of MSP cytoskeleton . The newly identified KPs in this study may aid in determining the signaling pathways that operate during spermatogenesis in A. suum. We provide evidence that a ~45 KD protein in sperm is associated with strong tyrosine phosphorylation (pY). Because tyrosine phosphorylation and dephosphorylation act as a molecular switch to regulate MSP assembly , we propose this ~45 KD protein may be involved in MSP-based sperm motility. Immunolabeling of pY on the leading edge of spermatozoa has also been observed (Zhao Y. and Miao L., unpublished observations), which reinforces the notion that phosphorylation plays a role in A. suum spermatogenesis/spermiogenesis.
Lastly, C. elegans genes that are homologous to genes represented in the A. suum gonad transcriptome were examined for associated RNAi phenotypes. Although the genes involved in spermatogenesis are possibly insensitive to RNAi , a variety of RNAi phenotypes, mostly embryonic lethality, arrested growth or sterility, were retrieved from the C. elegans database. This RNAi phenotypic categorization is in line with the functional classification of the gonad developmental genes, the majority of which control reproduction, embryo development and growth. Due to the growing concern of anthelminth resistance, RNAi provides a new means to combat parasitic nematodes. RNAi has been successfully used to knock down target genes in a few parasitic nematode species, including B. malayi, H. glycines, G. pallida, O. volvulus, T. colubriformis and notably, A. suum. Recently, serine/threonine phosphatases have been recommended as targets for new nematicidal drugs . Therefore, we anticipate that these A. suum gonad transcriptome sequencing data will provide opportunities to use RNAi as a novel anti-parasite agent for parasite control.
Collection of A. suum gonad samples
A. suum males were collected from the intestines of infected hogs at Zhongrui Pork Processors (Liucun, Beijing, China) and were stored in worm buffer (phosphate buffered saline containing 100 mM NaHCO3, pH 7.0, 37°C). A. suum gonads were dissected into two parts: (1) testis and seminal vesicle; (2) glandular vas deferens. Dissected samples were immediately frozen in liquid nitrogen prior to storage at -80°C.
cDNA synthesis and 454 pyrosequencing
Total RNAs from A. suum testis and vas deferens were prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) before removal of trace genomic DNA using DNAse I (Promega, Madison, WI, USA). Poly A+ RNA was purified using an Oligotex Direct mRNA Kit (Qiagen, Hilden, Germany) followed by first and second-strand cDNA synthesis using the Universal RiboClone cDNA Synthesis System (Promega) with a modification as follows. We designed a poly(T) adaptor with a Bsg I site flanking the poly(T) sequence (5'-CGTGTGCAGT(20)VN-3') for cDNA synthesis. The purified second-strand cDNAs were digested by Bsg I and recovered with a QIAquick PCR Purification Kit (Qiagen). Both double-strand cDNA samples were subjected to 454 pyrosequencing using the GS FLX Titanium Kit.
454 sequencing data analyses
High-quality sequences (> 99.5% accuracy on single base reads) were filtered to remove short ESTs (< 50 bp) before assembly using Newbler (version 2.3). For assembly, the quality score threshold was set to 40. All unique sequences containing both contigs and singletons were compared against the C. elegans confirmed protein database (derived from Wormbase) as well as the KEGG protein databases for all organisms http://www.genome.jp/kegg/download/ by BLASTX; the BLAST cutoff E-value was set at < 1e-5. The A. suum gonad developmental genes were functionally assigned by GO Slimer http://amigo.geneontology.org/cgi-bin/amigo/slimmer?session_id. The A. suum genes were mapped onto metabolic pathways according to C. elegans pathways http://www.genome.jp/kegg/download/. Total read numbers of TES and VAS datasets were normalized to equal levels, and the relative gene abundance was defined by log10 of the normalized read number. Heat-maps were generated using R software (version 2.12.0).
Reverse Transcription PCR
Total RNAs from A. suum testis and vas deferens were isolated before being reverse transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). The cDNA products were diluted 10-fold for use as RT-PCR templates. PCR was performed using High Fidelity PCR SuperMix (TransGen, Beijing, China) under the following conditions: 94°C for 3 min; 30 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 1 min; 72°C for 8 min. The genes Act-1 and eIF-4A were used as controls.
Preparation of TES and VAS protein extracts and of sperm extracts (S100)
Fresh A. suum TES and VAS tissues were disrupted in HKB buffer (50 mM Hepes, 65 mM KCl, 10 mM NaHCO3, pH 7.1) in a homogenizer. An equal volume of lysis buffer (100 mM Hepes, 300 mM NaCl, 2% Triton-100) was added and samples were then placed on ice for 30 min before centrifugation for 30 min at 20,000 rpm. Supernatants were then collected for Western blot analysis. To prepare sperm extracts (S100), frozen sperm were thawed on ice for 1 hr and centrifuged for 10 min at 13,000 rpm. Supernatant was centrifuged at 100,000 × g for 1 hr at 4°C. Supernatant (S100) was further analyzed by Western blotting.
SDS-PAGE and Western blotting
SDS-PAGE and Western blotting were performed as previously described . Western blots were probed with anti-phosphotyrosine primary antibody (Millipore, Billerica, MA, USA) at 0.2 μg/mL, followed by peroxidase-conjugated secondary antibody, and developed with enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA).
We thank Dr. Yanmei Zhao for suggestions on total protein extraction from TES and VAS tissues and we thank Fugang Chen for dissections of TES and VAS tissues. This research was supported by grants 2012CB94502, 31171337 and 30971648 (to LM) and 81130069 (to SC) from the Chinese government. LM is supported by the Chinese Academy of Sciences 100-talents program.
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