The ascarids, Toxocara canis and Toxascaris leonina, are probably the most common gastrointestinal helminths encountered in dogs. These ascarids have similar aspects; their genera are included in the Family Ascarididae and their morphological features are difficult to distinguish from one another by naked eyes of a non-professional person [1]. However, they also exhibit some different biological features. Prenatal transmission assures that virtually all puppies are born infected with T. canis [1,2]. This transplacental transmission is observed in the life cycle of other Toxocara sp.; it is probably the major route of infection for kittens with T. cati. T. canis can cause a serious disease in puppies; however, T. leonina is generally less pathogenic because they have no prenatal transmission ability. In a young host, T. canis can migrate to several internal organs (intestine-lung-trachea-intestine) during their juvenile stage for maturation into adult worms; however, T. leonina larvae can mature to be adults in the intestinal cavity without tissue migration [1]. Finally, T. canis can grow into an adult only in canid hosts; however, T. leonina can grow to be an adult in both canid and felid hosts [1].

In order to characterize the biology, pathogenesis, and differential diagnosis of the 2 genera, many genetic variation studies have been reported [3-6]. Most studies dealt with genetic and serologic characteristics for the diagnosis of infection with 2 parasites. The parasites have similar antigenic characteristics, and T. canis-infected host serum can be reacted with T. leonina antigens [3]. However, sequences of cox2 in mitochondrial DNA and ITS-2 region of ribosomal DNA of Toxocara and Toxascaris differed significantly from one another [4,7]. However, no study comparing functional gene annotation profiles between T. canis and T. leonina has been reported.

Functional genomic strategies, such as the expressed sequence tag (EST), are a powerful tool for identification of large numbers of genes [8]. EST analysis is not only one of the more effective methods for gene discovery, gene expression profiling, and functional genome studies, but also one of the more efficient methods for identification of differential genes [8]. EST analyses of T. canis and T. leonina have been reported, respectively [8-10]. An efficient functional annotation of DNA or protein sequences is a major requirement for successful application of these approaches as functional information on gene products is often the key for interpretation of experimental results [11]. Recently, a new functional annotation program, Blast2GO according to Gene Ontology (GO) criteria has been introduced; it is a powerful tool for comparison of functional gene expression profiles between 2 genetically similar organisms [12-14]. The GO project provides ontology of defined terms representing properties of gene products. The ontology covers 3 domains; cellular component (parts of a cell or its extracellular environment), molecular function (elemental activities of a gene product at the molecular level, such as binding or catalysis), and biological process (operations or sets of molecular events with a defined beginning and end, pertinent to functioning of integrated living units: cells, tissues, organs, and organisms) (http://www.geneontology.org).

The aim of this study was to understand parasite-specific functional gene expression profiles of ascarids, and difference of gene expression profiles between the 2 ascarids. We analyzed gene expression profiles of female adults of T. canis and T. leonina using CLC Genomics Workbench and the results were compared with those of a free-living nematode species, Caenorhabditis elegans. Conducts of functional genomic studies give to us understanding of parasitism, development, metabolism, and reproduction of these parasites.

Functional annotation of sequence data is a key requirement for successful generation of functional genomics in biological researches. In this study, we evaluated functional annotation of ESTs of T. leonina, T. canis, and C. elegans using the CLC Genomics Workbench program, in order to find some genetic difference for interpretation of biological differences between 2 ascarids and a free-living nematode.

Instead of our expectation, overall, our results showed that most functional gene annotations of 2 ascarids were quite similar to one another. We were not surprised by this result because they are included in the same Family Ascarididae. In particular, comparative analysis of biological process level 2 GO showed similar profiles between the 2 ascarids. Most ascarids, including T. canis, require internal organ migration periods for growth to the adult. However, T. leonina can grow to the adult without any migration period [1]. T. leonina can grow to be adults without any larval migration; this fact suggests several hypotheses. One is that molting and development of T. leonina can occur easily, compared with T. canis; in other words, T. leonina might have more abundant proteins related to molting and development. The other is that T. leonina does not require certain molecules that may be released from host tissues (lung, airway tract, blood, etc.) and may be critical for molting and development of other ascarids. In the molecular function category, structural molecule activity proteins showed greater expression in T. leonina than in T. canis. Structural molecule activity category, including motor proteins (actin, myosins, and dyneins), are important to molting and development as well as to movement [18]. Ubiquitin, heat-shock protein 70, α-actin, and β-actin are overexpressed in the abdominal muscle of marine shrimp Litopenaeus vannamei during the molting cycle [18]. This result suggested that perhaps T. leonina have more moving activity and ability to molt easily compared with T. canis. We cautiously suggested that these abilities articulated that molting and development of T. leonina could occur without internal organ migration during their life cycle. However, our analyzed data were obtained from adult female worms, and the data were not sufficiently complete to explain the hypothesis.

However, we found some difference between free-living nematodes and parasites. Membrane and membrane part categories were composited with intrinsic to, extrinsic to, and integral to membrane proteins. These proteins were more enriched in free-living nematodes than in parasites (Fig. 1). This result suggested that membrane complex might have been uncomplicated during evolutionary periods of parasites. In molecular function categories, we found a significantly different proportion of subcategories between parasites and free-living nematodes. In particular, the electron carrier activity category showed higher expression in C. elegans than in the other 2 parasites. This category included hydrolase activity, transferase activity, and oxidoreductase activity related proteins, which were closely related to the metabolic system. These results suggested that free-living nematodes have a more complex metabolic system than parasitic nematodes. Most digestive, metabolite systems of parasites might have degenerated, compared with those of their ancestors during evolutionary periods [1]. The most highly expressed transcripts of C. elegans were classified as single-organism process category in Biological Process level 2 GO terms, but a few transcripts of parasites were classified in this category. However, some subcategories in the single organism process category were also classified in the cellular process category. In order to identify the transcripts in this category, we have to classify the transcripts according to the subcategory level in the single-organism process and cellular process category.

In this study, although no significant difference was observed between T. leonina and T. canis female adults to explain differences in their life cycle, we observed difference of transcript expression between parasites and free-living nematodes. Thus, it might be helpful to understand evolutionary genetic changes of parasites. Analysis of detailed functional individual gene study is needed in order to understand parasite-specific transcript expression patterns.