Construction and Characterization of Normalized cDNA Libraries by 454 Pyrosequencing and Estimation of DNA Methylation Levels in Three Distantly Related Termite Species

In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST) libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti , Reticulitermessperatus and Nasutitermestakasagoensis . We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3) and methyl-CpG binding domain (mbd) were performed against the EST libraries. All four of these genes were found in the H . sjostedti library, while all except dnmt3 were found in R . speratus and N . takasagoensis . The ratio of the observed to the expected CpG content (CpG O/E), which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1), and the distributions of CpG O/E were bimodal, suggesting the presence of DNA methylation.

[1]  N. Galtier,et al.  Comparative Analysis of Transcriptomes from Secondary Reproductives of Three Reticulitermes Termite Species , 2015, PloS one.

[2]  D. Fonseca,et al.  The ubiquity and ancestry of insect doublesex , 2015, Scientific Reports.

[3]  D. Emlen,et al.  Soldier morphogenesis in the damp-wood termite is regulated by the insulin signaling pathway. , 2013, Journal of experimental zoology. Part B, Molecular and developmental evolution.

[4]  M. Goodisman,et al.  Evidence of a conserved functional role for DNA methylation in termites , 2013, Insect molecular biology.

[5]  T. Miura,et al.  Exploration and characterization of genes involved in the synthesis of diterpene defence secretion in nasute termite soldiers , 2012, Insect molecular biology.

[6]  Xuguo Zhou,et al.  Characterization of Head Transcriptome and Analysis of Gene Expression Involved in Caste Differentiation and Aggression in Odontotermes formosanus (Shiraki) , 2012, PloS one.

[7]  Evgeny M. Zdobnov,et al.  OrthoDB: a hierarchical catalog of animal, fungal and bacterial orthologs , 2012, Nucleic Acids Res..

[8]  K. Matsuura.  Multifunctional Queen Pheromone and Maintenance of Reproductive Harmony in Termite Colonies , 2012, Journal of Chemical Ecology.

[9]  T. Miura,et al.  Expression and function of a limb‐patterning gene Distal‐less in the soldier‐specific morphogenesis in the nasute termite Nasutitermes takasagoensis , 2012, Evolution & development.

[10]  N. Lo,et al.  DNA methylation in the termite Coptotermes lacteus , 2012, Insectes Sociaux.

[11]  W. Nierman,et al.  Carbohydrate‐active enzymes revealed in Coptotermes formosanus (Isoptera: Rhinotermitidae) transcriptome , 2012, Insect molecular biology.

[12]  C. McGregor,et al.  Transcriptome profiling of female alates and egg-laying queens of the Formosan subterranean termite. , 2012, Comparative biochemistry and physiology. Part D, Genomics & proteomics.

[13]  S. Roberts,et al.  Is There a Relationship between DNA Methylation and Phenotypic Plasticity in Invertebrates? , 2011, Front. Physio..

[14]  M. Goodisman,et al.  DNA methylation in insects: on the brink of the epigenomic era , 2011, Insect molecular biology.

[15]  K. Maekawa,et al.  Effects of juvenile hormone III on morphogenetic changes during a molt from each nymphal instar in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae) , 2010 .

[16]  Doina Caragea,et al.  Comparative analysis of expressed sequence tags from three castes and two life stages of the termite Reticulitermes flavipes , 2010, BMC Genomics.

[17]  D. Zilberman,et al.  Genome-Wide Evolutionary Analysis of Eukaryotic DNA Methylation , 2010, Science.

[18]  G. K. Davis,et al.  Genome Sequence of the Pea Aphid Acyrthosiphon pisum , 2010, PLoS biology.

[19]  M. Goodisman,et al.  Computational approaches for understanding the evolution of DNA methylation in animals , 2009, Epigenetics.

[20]  T. Miura,et al.  Presoldier Induction by a Juvenile Hormone Analog in the Nasute Termite Nasutitermes takasagoensis (Isoptera: Termitidae) , 2009, Zoological science.

[21]  A. Moczek,et al.  The basis of bee‐ing different: the role of gene silencing in plasticity , 2008, Evolution & development.

[22]  Albert Jeltsch,et al.  Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism. , 2008, RNA.

[23]  A. Bird,et al.  DNA methylation landscapes: provocative insights from epigenomics , 2008, Nature Reviews Genetics.

[24]  K. Maekawa,et al.  Effect on mandibular length of juvenile hormones and regulation of soldier differentiation in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae) , 2008 .

[25]  R. Kucharski,et al.  Nutritional Control of Reproductive Status in Honeybees via DNA Methylation , 2008, Science.

[26]  A. Vogler,et al.  A comprehensive phylogenetic analysis of termites (Isoptera) illuminates key aspects of their evolutionary biology. , 2007, Molecular phylogenetics and evolution.

[27]  Keith Bradnam,et al.  CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes , 2007, Bioinform..

[28]  Adrian Bird,et al.  CpG methylation is targeted to transcription units in an invertebrate genome. , 2007, Genome research.

[29]  Gang Liu,et al.  Automatic clustering of orthologs and inparalogs shared by multiple proteomes , 2006, ISMB.

[30]  Lin Fang,et al.  WEGO: a web tool for plotting GO annotations , 2006, Nucleic Acids Res..

[31]  Robin B. Gasser,et al.  A hitchhiker's guide to expressed sequence tag (EST) analysis , 2006, Briefings Bioinform..

[32]  A. Bird,et al.  Genomic DNA methylation: the mark and its mediators. , 2006, Trends in biochemical sciences.

[33]  Xiaoyu Zhang,et al.  Methylation of tRNAAsp by the DNA Methyltransferase Homolog Dnmt2 , 2006, Science.

[34]  Gregory Butler,et al.  OrfPredictor: predicting protein-coding regions in EST-derived sequences , 2005, Nucleic Acids Res..

[35]  O. Kitade,et al.  Patterns of neotenic differentiation in a subterranean termite, Reticulitermes speratus (Isoptera: Rhinotermitidae) , 2004 .

[36]  T. Miura,et al.  Comparative studies on alate wing formation in two related species of rotten-wood termites: Hodotermopsis sjostedti and Zootermopsis nevadensis (Isoptera, Termopsidae) , 2004, Insectes Sociaux.

[37]  J. Kojima,et al.  Parthenogenetic reproduction in neotenics of the subterranean termite Reticulitermes speratus (Isoptera: Rhinotermitidae) , 2003 .

[38]  S. Rudd.  Expressed sequence tags: alternative or complement to whole genome sequences? , 2003, Trends in plant science.

[39]  K. Robertson.  DNA methylation and chromatin – unraveling the tangled web , 2002, Oncogene.

[40]  Christian E. V. Storm,et al.  Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. , 2001, Journal of molecular biology.

[41]  Rolf Apweiler,et al.  InterProScan - an integration platform for the signature-recognition methods in InterPro , 2001, Bioinform..

[42]  T. Miura,et al.  Caste developmental system of the Japanese damp-wood termite Hodotermopsis japonica (Isoptera: Termopsidae) , 2000, Ecological Research.

[43]  C. V. Jongeneel,et al.  Searching the Expressed Sequence Tag (EST) Databases: Panning for Genes , 2000, Briefings Bioinform..

[44]  E. Li,et al.  Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases , 1998, Nature Genetics.

[45]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[46]  Tadao Matsumoto,et al.  Juvenile Hormone Analogue,S-31183,Causes a High Level Induction of Presoldier Differentiation in the Japanese Damp-Wood Termite , 1993 .

[47]  Y. Takematsu.  Biometrical Study on the Development of the Castes in Reticulitermes speratus (Isoptera, Rhinotermitidae) , 1992 .

[48]  D. Schorderet,et al.  Analysis of CpG suppression in methylated and nonmethylated species. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[49]  H. Noda,et al.  Small-Scale Rearing of a Subterranean Termite, Reticulitermes speratus(Isoptera:Rhinotermitidae) , 1991 .

[50]  Diana E. Wheeler,et al.  Juvenile Hormone and the Physiological Basis of Insect Polymorphisms , 1982, The Quarterly Review of Biology.

[51]  A. Bird.  DNA methylation and the frequency of CpG in animal DNA. , 1980, Nucleic acids research.

[52]  E. Wilson,et al.  Caste and ecology in the social insects. , 1979, Monographs in population biology.

[53]  J. Ott,et al.  Detection of rare major genes in lipid levels , 1979, Human Genetics.

[54]  Philip J. Farabaugh,et al.  Molecular basis of base substitution hotspots in Escherichia coli , 1978, Nature.

[55]  C. Brooke Worth,et al.  The Insect Societies , 1973 .

[56]  E. Wilson.  Chemical Communication in the Social Insects. , 1965, Science.

[57]  Ļ. Cleveland,et al.  Symbiosis between Termites and Their Intestinal Protozoa. , 1923, Proceedings of the National Academy of Sciences of the United States of America.

[58]  Yves Roisin,et al.  Biology of Termites: A Modern Synthesis , 2011 .

[59]  T. Miura,et al.  Molecular Basis Underlying Caste Differentiation in Termites , 2010 .

[60]  M. Ohkuma,et al.  Diversity, Structure, and Evolution of the Termite Gut Microbial Community , 2010 .

[61]  Hiromi Tanaka,et al.  RNA interference in the termite Reticulitermes speratus: silencing of the hexamerin gene using a single 21 nucleotide small interfering RNA-promoted differentiation of nymph to nymphoid. , 2010 .

[62]  M. Soares,et al.  Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\ normalization and subtraction of cDNA libraries. , 2009, Methods in molecular biology.

[63]  A. S. Shcheglov,et al.  Normalization of cDNA Libraries , 2007 .

[64]  A. Buzdin,et al.  Nucleic Acids Hybridization Modern Applications , 2007 .

[65]  Qunfeng Dong,et al.  Comparative EST analyses in plant systems. , 2005, Methods in enzymology.

[66]  T. Miura,et al.  Developmental pathways and plasticity of neuter castes in Nasutitermes takasagoensis (Isoptera: Termitidae) , 2004 .

[67]  Yves Roisin,et al.  Diversity and evolution of caste patterns , 2000 .

[68]  T. Yoshimura,et al.  Symbiotic Associations with Protists , 2000 .

[69]  C. Bandi,et al.  Characterizing the Ancestors: Paedomorphosis and Termite Evolution , 2000 .

[70]  安部 琢哉,et al.  Termites: Evolution, Sociality, Symbioses, Ecology , 2000, Springer Netherlands.

[71]  P. Eggleton,et al.  Taxonomy and Phylogeny of Termites , 2000 .

[72]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[73]  C. Noirot,et al.  Caste differentiation in Isoptera: basic features, role of pheromones , 1991 .

[74]  V B WIGGLESWORTH,et al.  INSECT HORMONES. , 1965, Endeavour.