Project description:Over a billion people are infected by Ascaris spp. intestinal parasites. We investigated gene expression by various tissues of adult A. suum, and used that information to help resolve basic functions of these tissues, basic biology of parasitism and functional aspects of nematode genomes. The A. suum genome was sequenced and assembled to allow generation of microarray elements. Expression of over 40,000 60-mer elements was investigated in a variety of tissues from both male and female adult worms. Nearly 50 percent of the elements for which signal was detected exhibited differential expression among different tissues. The unique profile of transcripts identified for each tissue clarified functional distinctions among tissues, such as chitin binding in the ovary and peptidase activity in the intestines. Interestingly, hundreds of gender specific elements characterized female or male intestinal tissues, respectively. A. suum genes from the same family were frequently expressed differently among tissues. Transcript abundance for genes specific to A. suum, by comparison to Caenorhabditis elegans, varied to a greater extent among tissues than for genes conserved between A. suum and C. elegans. Analysis using C. elegans protein interaction data identified functional modules conserved between these two nematodes, resulting in identification of functional predictions of essential subnetworks of protein interactions and how these networks may vary among nematode tissues. A notable finding was very high module similarity between adult reproductive tissues and intestine. Our data identified gene and predicted protein determinants that distinguish functions of individual adult A. suum tissues as well those that likely serve all the tissues investigated in our study. The size of A. suum adults (30-40 cm compared to less than 1 cm for many nematodes) enabled this resolution and will facilitate experimental dissection of individual and interactive functions of these tissues in this and other parasitic nematodes.

Project description:Over a billion people are infected by Ascaris spp. intestinal parasites. We investigated gene expression by various tissues of adult A. suum, and used that information to help resolve basic functions of these tissues, basic biology of parasitism and functional aspects of nematode genomes. The A. suum genome was sequenced and assembled to allow generation of microarray elements. Expression of over 40,000 60-mer elements was investigated in a variety of tissues from both male and female adult worms. Nearly 50 percent of the elements for which signal was detected exhibited differential expression among different tissues. The unique profile of transcripts identified for each tissue clarified functional distinctions among tissues, such as chitin binding in the ovary and peptidase activity in the intestines. Interestingly, hundreds of gender specific elements characterized female or male intestinal tissues, respectively. A. suum genes from the same family were frequently expressed differently among tissues. Transcript abundance for genes specific to A. suum, by comparison to Caenorhabditis elegans, varied to a greater extent among tissues than for genes conserved between A. suum and C. elegans. Analysis using C. elegans protein interaction data identified functional modules conserved between these two nematodes, resulting in identification of functional predictions of essential subnetworks of protein interactions and how these networks may vary among nematode tissues. A notable finding was very high module similarity between adult reproductive tissues and intestine. Our data identified gene and predicted protein determinants that distinguish functions of individual adult A. suum tissues as well those that likely serve all the tissues investigated in our study. The size of A. suum adults (30-40 cm compared to less than 1 cm for many nematodes) enabled this resolution and will facilitate experimental dissection of individual and interactive functions of these tissues in this and other parasitic nematodes. 10 samples were prepared from the tissues of adult worms of both genders to be analyzed. Two to three replicates were done for each sample, swapping dyes and channel for the test and the control for each sample. The control consisted of pooled worm tissues from both genders with an equal amount being included from each sample. These samples and the control were hybridized against an Agilent 4x44k format custom chip spotted with 42,212 probes. This probeset was created from 38,768 predicted protein coding sequences from an Ascaris suum genome survey sequence (GSS) project, and also from 5,176 EST contigs that were not covered by this predicted GSS gene-set. These 43,944 putative gene sequences from Ascaris suum were filtered for cross-reaction to non-coding sequences using Agilent eArray, and our final set of 42,212 probes was determined.

Project description:BackgroundCaenorhabditis elegans has traditionally been used as a model for studying nematode biology, but its small size limits the ability for researchers to perform some experiments such as high-throughput tissue-specific gene expression studies. However, the dissection of individual tissues is possible in the parasitic nematode Ascaris suum due to its relatively large size. Here, we take advantage of the recent genome sequencing of Ascaris suum and the ability to physically dissect its separate tissues to produce a wide-scale tissue-specific nematode RNA-seq datasets, including data on three non-reproductive tissues (head, pharynx, and intestine) in both male and female worms, as well as four reproductive tissues (testis, seminal vesicle, ovary, and uterus). We obtained fundamental information about the biology of diverse cell types and potential interactions among tissues within this multicellular organism.Methodology/principal findingsOverexpression and functional enrichment analyses identified many putative biological functions enriched in each tissue studied, including functions which have not been previously studied in detail in nematodes. Putative tissue-specific transcriptional factors and corresponding binding motifs that regulate expression in each tissue were identified, including the intestine-enriched ELT-2 motif/transcription factor previously described in nematode intestines. Constitutively expressed and novel genes were also characterized, with the largest number of novel genes found to be overexpressed in the testis. Finally, a putative acetylcholine-mediated transcriptional network connecting biological activity in the head to the male reproductive system is described using co-expression networks, along with a similar ecdysone-mediated system in the female.Conclusions/significanceThe expression profiles, co-expression networks and co-expression regulation of the 10 tissues studied and the tissue-specific analysis presented here are a valuable resource for studying tissue-specific biological functions in nematodes.

Project description:BackgroundAscaris 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.ResultsTwo 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.ConclusionsUsing 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.

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Gene expression analysis distinguishes tissue-specific and gender-related functions among adult Ascaris suum tissues.

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Gene expression analysis distinguishes tissue-specific and gender-related functions among adult Ascaris suum tissues.

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