The flatworm planarian Dugesia japonica inhabits fresh water in East Asian areas and is the most common planarian in Japan . Planarians are members of the phylum Platyhelminthes, a group of organisms that are thought to have acquired a central nervous system (CNS) with a simple brain structure early during evolution [2–4]. The constitution of the planarian CNS and neural network has been visualized in detail by whole mount in situ hybridization and immunofluorescence studies, and fluorescent dye tracing experiments [5–8]: the CNS is composed of a brain (anterior cephalic ganglion) in the head region and a pair of ventral nerve cords extending anterior to posterior along the ventral side of the body, with the brain and ventral nerve cords being morphologically distinguishable structures. Neurons from a variety of sensory organs, such as eyes and auricles, project to different portions of the main lobes [6, 8, 9]. These interneurons are involved in information processing of external signals and work to regulate behavior and learning/memory [10–12]. The planarian brain acts as an information center of the nervous system, and shows fundamental evolutionarily conserved features of the animal brain not only morphologically but also functionally . In addition, planarians are attracting attention as new model organism for regeneration, including brain regeneration, research. Interestingly, planarians can regenerate their well-organized brains from any portion of their bodies utilizing adult somatic pluripotent stem cells (neoblasts) [13–15]. The RNA interference technique can be applied in planarians to identify gene functions , and single-cell level gene profiling is also possible based on a combination of fluorescence activated cell sorting (FACS) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) [17, 18].
In recent years, resources for comparative transcriptome analysis among members of the phylum Platyhelminthes have accumulated rapidly. The planarian Schmidtea mediterranea and blood-fluke Schistosoma mansoni genome sequences have been analyzed [19, 20], and transcriptome resources and analyses have been reported [21–23]. Unigene Build #4 for planarian S. mediterranea, which is based on the Sanger sequencing method, contains 10,173 clusters from 69,699 EST sequences, which were obtained from juvenile and adult libraries [24, 25]. Some studies with massive numbers of sequencing reads produced from next generation sequencing technologies, include the Illumina HiSeq, Roche 454 and Life Technologies SOLiD, have been reported [26–28]. However, there is no genomic resource for D. japonica, and only limited transcriptome information is available for this species. Despite the large evolutionary distance between these two planarians , they share not just morphological similarity, but also genes, CNS features and regeneration ability .
The cDNA libraries of the schistosome S. mansoni cover its various life stages: egg, miracidium, sporocyst, cercaria, larva and adult, with a total of 152,704 sequences and 10,061 clusters (Unigene Build #19) . Schistosomes are triploblastic animals and members of Platyhelminthes, like planarians, with which they share not only body shape but also basic organismal functions. Specifically, they have bilateral symmetry, a functional brain and peripheral nerves, ventral suckers, digestive and excretory organs; and lack a cardiovascular system . Moreover, many genes and their amino acid sequences are well conserved between schistosomes and planarians. However, whereas planarians are free-living flatworms that prey on other organisms, schistosomes, which are major agents of the disease schistosomiasis and parasitize multiple hosts and organs, change their morphology to adapt to their living environments . The life cycles of these two genuses are thus in sharp contrast, requiring brain functions and metabolic processes that are quite different.
To establish a database of genetic information for planarian transcriptome studies, we performed a large-scale EST project for the planarian D. japonica using head cDNA libraries. We adopted Sanger sequencing in order to decrease the sequence gaps, frame-shift errors, and the misassembly that can occur due to splice variants and to the short reads produced by next generation sequencing. These factors are important for the identification of long consensus sequences between conserved proteins. We compared the percentage of amino acid substitutions between D. japonica and its sister species S. mediterranea using the homologue proteins to identify genes whose mutability enables accommodation to different environmental conditions. For this analysis, we developed a method to extract gene groups that have different rates of evolution in close species that have very-well-conserved proteins.
We have already published a partial analysis of D. japonica transcriptome , and have identified several genes that are specifically expressed in the CNS [7, 9, 34]. However, those studies were insufficient for the exhaustive comparative analyses between planarians and members of the same family or the same phylum necessary for clarifying the composition and evolution of the CNS. As compared with model organisms, the gene information of Platyhelminthes is very limited. For these reasons we used Gene Ontology , which is based on information across many species, including vertebrates and non-vertebrates, and serves as a common platform to compare and annotate non-model organisms.
In this study, we focused on the CNS-development genes, which should give information about the evolutionary position of Platyhelminthes. To examine the genomic evolution and the presence of gene expression, we compared the D. japonica unigenes with not only S. mansoni unigenes but also the predicted protein information from the genome sequence. The traces we thereby found on the genome suggested the possibility that these genes were derived from the common ancestor of these two genuses, and the divergent gene expression between these genuses provided information about their adaptation to their specific habitats.