A White Paper on Nematode Comparative Genomics

In response to the new opportunities for genome sequencing and comparative genomics, the Society of Nematology (SON) formed a committee to develop a white paper in support of the broad scientific needs associated with this phylum and interests of SON members. Although genome sequencing is expensive, the data generated are unique in biological systems in that genomes have the potential to be complete (every base of the genome can be accounted for), accurate (the data are digital and not subject to stochastic variation), and permanent (once obtained, the genome of a species does not need to be experimentally re-sampled). The availability of complete, accurate, and permanent genome sequences from diverse nematode species will underpin future studies into the biology and evolution of this phylum and the ecological associations (particularly parasitic) nematodes have with other organisms. We anticipate that upwards of 100 nematode genomes will be solved to varying levels of completion in the coming decade and suggest biological and practical considerations to guide the selection of the most informative taxa for sequencing.     

The use of Caenorhabditis elegans in parasitic nematode research

There is increasing interest in the use of the free-living nematode Caenorhabditis elegans as a tool for parasitic nematode research and there are now a number of compelling examples of its successful application. C. elegans has the potential to become a standard tool for molecular helminthology researchers, just as yeast is routinely used by molecular biologists to study vertebrate biology. However, in order to exploit C. elegans in a meaningful manner, we need a detailed understanding of the extent to which different aspects of C. elegans biology have been conserved with particular groups of parasitic nematodes. This review first considers the current state of knowledge regarding the conservation of genome organisation across the nematode phylum and then discusses some recent evolutionary development studies in free-living nematodes. The aim is to provide some important concepts that are relevant to the extrapolation of information from C. elegans to parasitic nematodes and also to the interpretation of experiments that use C. elegans as a surrogate expression system. In general, examples have been specifically chosen because they highlight the importance of careful experimentation and interpretation of data. Consequently, the focus is on the differences that have been found between nematode species rather than the similarities. Finally, there is a detailed discussion of the current status of C. elegans as a heterologous expression system to study parasite gene function and regulation using successful examples from the literature.     

The mitochondrial genome of Strongyloides stercoralis (Nematoda) - idiosyncratic gene order and evolutionary implications

The complete mitochondrial genome sequence of the parasitic nematode Strongyloides stercoralis was determined, and its organisation and structure compared with other nematodes for which complete mitochondrial sequence data were available. The mitochondrial genome of S. stercoralis is 13,758 bp in size and contains 36 genes (all transcribed in the clockwise direction) but lacks the atp8 gene. This genome has a high T content (55.9%) and a low C content (8.3%). Corresponding to this T content, there are 16 (poly-T) tracts of >/=12 Ts distributed across the genome. In protein-coding genes, the T bias is greatest (76.4%) at the third codon position compared with the first and second codon positions. Also, the C content is higher at the first (9.3%) and second (13.4%) codon positions than at the third (2%) position. These nucleotide biases have a significant effect on predicted codon usage patterns and, hence, on amino acid compositions of the mitochondrial proteins. Interestingly, six of the 12 protein-coding genes are predicted to employ a unique initiation codon (TTT), which has not yet been reported for any other animal mitochondrial genome. The secondary structures predicted for the 22 transfer RNA (trn) genes and the two ribosomal RNA (rrn) genes are similar to those of other nematodes. In contrast, the gene arrangement in the mitochondrial genome of S. stercoralis is different from all other nematodes studied to date, revealing only a limited number of shared gene boundaries (atp6-nad2 and cox2-rrnL). Evolutionary analyses of mitochondrial nucleotide and amino acid sequence data sets for S. stercoralis and seven other nematodes demonstrate that the mitochondrial genome provides a rich source of phylogenetically informative characters. In conclusion, the S. stercoralis mitochondrial genome, with its unique gene order and characteristics, should provide a resource for comparative mitochondrial genomics and systematics studies of parasitic nematodes.     

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.     

Analysis of a 43-kDa glycoprotein from the intracellular parasitic nematode Trichinella spiralis.

The L1 larvae of the parasitic nematode Trichinella spiralis invade skeletal muscle and initiate a process that has been interpreted to represent skeletal muscle dedifferentiation. In this process, the infected region of the muscle cell is converted into a unique structure, called the Nurse cell. The nematode T. spiralis can survive for tens of years within the cytoplasm of the Nurse cell and secretes proteins into the cytoplasm that are believed to play a role in mediating the Nurse cell formation or maintenance. We have cloned a cDNA encoding the T. spiralis-derived, 43-kDa secreted protein. Structural analysis of the predicted 344-amino acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the main body of the protein. Antibodies raised against the 43-kDa recombinant protein were used in immunocytolocalizations of T. spiralis-infected skeletal muscle sections. These antibodies strongly stained the Nurse cell nuclei and the nematode itself. Specific, though slightly weaker staining also occurred in the Nurse cell cytoplasm. In Western blots, the antibodies react with the 43-kDa protein but also detected at least two other T. spiralis-secreted proteins. DNA hybridizations reveal at least one additional 43-kDa-related sequence encoded in the T. spiralis genome. We conclude that either the 43-kDa protein and/or a closely related 43-kDa family member is secreted into the muscle and translocates to the muscle-derived nuclei. This model may provide insights into the mechanisms involved in Nurse cell formation.     

Evolutionary and functional genomics of the Archaea

In the past two years, archaeal genomics has achieved several breakthroughs. On the evolutionary front the most exciting development was the sequencing and analysis of the genome of Nanoarchaeum equitans, a tiny parasitic organism that has only approximately 540 genes. The genome of Nanoarchaeum shows signs of extreme rearrangement including the virtual absence of conserved operons and the presence of several split genes. Nanoarchaeum is distantly related to other archaea, and it has been proposed to represent a deep archaeal branch that is distinct from Euryarchaeota and Crenarchaeota. This would imply that many features of its gene repertoire and genome organization might be ancestral. However, additional genome analysis has provided a more conservative suggestion - that Nanoarchaeum is a highly derived euryarchaeon. Also there have been substantial developments in functional genomics, including the discovery of the elusive aminoacyl-tRNA synthetase that is involved in both the biosynthesis of cysteine and its incorporation into proteins in methanogens, and the first experimental validation of the predicted archaeal exosome.     

Biological consequences of ancient gene acquisition and duplication in the large genome of Candidatus Solibacter usitatus Ellin6076

Members of the bacterial phylum Acidobacteria are widespread in soils and sediments worldwide, and are abundant in many soils. Acidobacteria are challenging to culture in vitro, and many basic features of their biology and functional roles in the soil have not been determined. Candidatus Solibacter usitatus strain Ellin6076 has a 9.9 Mb genome that is approximately 2-5 times as large as the other sequenced Acidobacteria genomes. Bacterial genome sizes typically range from 0.5 to 10 Mb and are influenced by gene duplication, horizontal gene transfer, gene loss and other evolutionary processes. Our comparative genome analyses indicate that the Ellin6076 large genome has arisen by horizontal gene transfer via ancient bacteriophage and/or plasmid-mediated transduction, and widespread small-scale gene duplications, resulting in an increased number of paralogs. Low amino acid sequence identities among functional group members, and lack of conserved gene order and orientation in regions containing similar groups of paralogs, suggest that most of the paralogs are not the result of recent duplication events. The genome sizes of additional cultured Acidobacteria strains were estimated using pulsed-field gel electrophoresis to determine the prevalence of the large genome trait within the phylum. Members of subdivision 3 had larger genomes than those of subdivision 1, but none were as large as the Ellin6076 genome. The large genome of Ellin6076 may not be typical of the phylum, and encodes traits that could provide a selective metabolic, defensive and regulatory advantage in the soil environment.     

Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders

The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda.     

Plant parasitic nematode proteins and the host parasite interaction.

This review focuses on the proteins and secretions of sedentary plant parasitic nematodes potentially important for plant-nematode interactions. These nematodes are well equipped for parasitism of plants. Having acquired the ability to manipulate fundamental aspects of plant biology, they are able to hijack host-cell development to make their feeding site. They feed exclusively from feeding sites as they complete their life cycle, satisfying their nutritional demands for development and reproduction. Biochemical and genomic approaches have been used successfully to identify a number of nematode parasitism genes. So far, 65 204 expressed sequence tags (ESTs) have been generated for six Meloidogyne species and sequencing projects, currently in progress, will underpin genomic comparisons of Meloidogyne spp. with sequences of other pathogens and generate genechip microarrays to undertake profiling studies of up- and down-regulated genes during the infection process. RNA interference provides a molecular genetic tool to study gene function in parasitism. These methods should provide new data to help our understanding of how parasitic nematodes infect their hosts, leading to the identification of novel pathogenicity genes.     

Bioinformatic analysis of P granule-related proteins: insights into germ granule evolution in nematodes

Germ cells in many animals possess a specialized cytoplasm in the form of granules that contain RNA and protein complexes essential for the function and preservation of the germline. The mechanism for the formation of these granules is still poorly understood; however, the lack of conservation in their components across different species suggests evolutionary convergence in the assembly process. Germ granules are assumed to be present in all nematodes with a preformed germline. However, few studies have clearly identified these structures in species other than Caenorhabditis elegans and even less have carried functional analysis to provide a broader panorama of the granules composition in the phylum. We adopted a bioinformatics approach to investigate the extension of conservation in nematodes of some known C. elegans germ granule components, as a proxy to understand germ granules evolution in this phylum. Unexpectedly, we found that, in nematodes, the DEAD box RNA helicase Vasa, a conserved protein among different phyla, shows a complex history of clade-specific duplications and sequence divergence. Our analyses suggest that, in nematodes, Vasa’s function might be shared among proteins like LAF-1, VBH-1, and GLH-1/-2/-3 and GLH-4. Key components of P granules assembly in C. elegans, like the PGL protein family, are only preserved in Caenorhabditis species. Our analysis suggests that germ granules assembly may not be conserved in nematodes. Studies on these species could bring insight into the basic components required for this pathway.     

A Lover and a Fighter: The Genome Sequence of an Entomopathogenic Nematode Heterorhabditis bacteriophora

Heterorhabditis bacteriophora are entomopathogenic nematodes that have evolved a mutualism with Photorhabdus luminescens bacteria to function as highly virulent insect pathogens. The nematode provides a safe harbor for intestinal symbionts in soil and delivers the symbiotic bacteria into the insect blood. The symbiont provides virulence and toxins, metabolites essential for nematode reproduction, and antibiotic preservation of the insect cadaver. Approximately half of the 21,250 putative protein coding genes identified in the 77 Mbp high quality draft H. bacteriophora genome sequence were novel proteins of unknown function lacking homologs in Caenorhabditis elegans or any other sequenced organisms. Similarly, 317 of the 603 predicted secreted proteins are novel with unknown function in addition to 19 putative peptidases, 9 peptidase inhibitors and 7 C-type lectins that may function in interactions with insect hosts or bacterial symbionts. The 134 proteins contained mariner transposase domains, of which there are none in C. elegans, suggesting an invasion and expansion of mariner transposons in H. bacteriophora. Fewer Kyoto Encyclopedia of Genes and Genomes Orthologies in almost all metabolic categories were detected in the genome compared with 9 other sequenced nematode genomes, which may reflect dependence on the symbiont or insect host for these functions. The H. bacteriophora genome sequence will greatly facilitate genetics, genomics and evolutionary studies to gain fundamental knowledge of nematode parasitism and mutualism. It also elevates the utility of H. bacteriophora as a bridge species between vertebrate parasitic nematodes and the C. elegans model.     

The Mitochondrial Genome of Xiphinema americanum sensu stricto (Nematoda: Enoplea): Considerable Economization in the Length and Structural Features of Encoded Genes

The complete sequence of the mitochondrial genome of the plant parasitic nematode Xiphinema americanum sensu stricto has been determined. At 12626bp it is the smallest metazoan mitochondrial genome reported to date. Genes are transcribed from both strands. Genes coding for 12 proteins, 2 rRNAs and 17 putative tRNAs (with the tRNA-C, I, N, S1, S2 missing) are predicted from the sequence. The arrangement of genes within the X. americanum mitochondrial genome is unique and includes gene overlaps. Comparisons with the mtDNA of other nematodes show that the small size of the X. americanum mtDNA is due to a combination of factors. The two mitochondrial rRNA genes are considerably smaller than those of other nematodes, with most of the protein encoding and tRNA genes also slightly smaller. In addition, five tRNAs genes are absent, lengthy noncoding regions are not present in the mtDNA, and several gene overlaps are present. [Reviewing Editor: Dr. Yues van de Peer] F. Lamberti: Deceased, 2004     

Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown Clades

Inference of evolutionary relationships between nematodes is severely hampered by their conserved morphology, the high frequency of homoplasy, and the scarcity of phylum-wide molecular data. To study the origin of nematode radiation and to unravel the phylogenetic relationships between distantly related species, 339 nearly full-length small-subunit rDNA sequences were analyzed from a diverse range of nematodes. Bayesian inference revealed a backbone comprising 12 consecutive dichotomies that subdivided the phylum Nematoda into 12 clades. The most basal clade is dominated by the subclass Enoplia, and members of the order Triplonchida occupy positions most close to the common ancestor of the nematodes. Crown Clades 8-12, a group formerly indicated as "Secernentea" that includes Caenorhabditis elegans and virtually all major plant and animal parasites, show significantly higher nucleotide substitution rates than the more basal Clades 1-7. Accelerated substitution rates are associated with parasitic lifestyles (Clades 8 and 12) or short generation times (Clades 9-11). The relatively high substitution rates in the distal clades resulted in numerous autapomorphies that allow in most cases DNA barcode-based species identification. Teratocephalus, a genus comprising terrestrial bacterivores, was shown to be most close to the starting point of Secernentean radiation. Notably, fungal feeding nematodes were exclusively found basal to or as sister taxon next to the 3 groups of plant parasitic nematodes, namely, Trichodoridae, Longidoridae, and Tylenchomorpha. The exclusive common presence of fungivorous and plant parasitic nematodes supports a long-standing hypothesis that states that plant parasitic nematodes arose from fungivorous ancestors.     

Identification of MicroRNAs in Meloidogyne incognita Using Deep Sequencing

MicroRNAs play important regulatory roles in eukaryotic lineages. In this paper, we employed deep sequencing technology to sequence and identify microRNAs in M. incognita genome, which is one of the important plant parasitic nematodes. We identified 102 M. incognita microRNA genes, which can be grouped into 71 nonredundant miRNAs based on mature sequences. Among the 71 miRANs, 27 are known miRNAs and 44 are novel miRNAs. We identified seven miRNA clusters in M. incognita genome. Four of the seven clusters, miR-100/let-7, miR-71-1/miR-2a-1, miR-71-2/miR-2a-2 and miR-279/miR-2b are conserved in other species. We validated the expressions of 5 M. incognita microRNAs, including 3 known microRNAs (miR-71, miR-100b and let-7) and 2 novel microRNAs (NOVEL-1 and NOVEL-2), using RT-PCR. We can detect all 5 microRNAs. The expression levels of four microRNAs obtained using RT-PCR were consistent with those obtained by high-throughput sequencing except for those of let-7. We also examined how M. incognita miRNAs are conserved in four other nematodes species: C. elegans, A. suum, B. malayi and P. pacificus. We found that four microRNAs, miR-100, miR-92, miR-279 and miR-137, exist only in genomes of parasitic nematodes, but do not exist in the genomes of the free living nematode C. elegans. Our research created a unique resource for the research of plant parasitic nematodes. The candidate microRNAs could help elucidate the genomic structure, gene regulation, evolutionary processes, and developmental features of plant parasitic nematodes and nematode-plant interaction.