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

Abstract

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.

Soldier‐biased gene expression in a subterranean termite implies functional specialization of the defensive caste

“…This transcriptome is assembled from whole body tissue mRNAs sampled from three different North American populations. As such, it provides a reasonable estimate of gene composition and expression for this species that compliments prior genomic studies of Reticulitermes (Dedeine et al, 2015;Hayashi et al, 2013;Mitaka et al, 2016;Raychoudhury et al, 2013;Scharf et al, 2003;Scharf et al, 2005;Scharf et al, 2017;Sen et al, 2013;Steller et al, 2010; and other termites (Huang, Sun, Zhou, & Lei, 2012;Ishikawa et al, 2010;Korb et al, 2015;Koshikawa et al, 2005;Poulsen et al, 2014;Terrapon et al, 2014;Wu et al, 2016). Established indicators of transcriptome quality such as N50 value and average gene length generated for the present assembly (Table 3) are consistent with benchmark expectations for non-model organisms (Francis et al, 2013).…”

“…For the transcriptome as a whole, the proportion of unknown but presumably functional genes is ∼20% (Table 4). This proportion is lower than previous R. flavipes EST-sequencing (Steller et al, 2010;Tartar et al, 2009), and is comparable to other termite RNA-seq studies (Dedeine et al, 2015;Hayashi et al, 2013;Wu et al, 2016). The set of 93 casteinformative genes presented in Figure 1 are well characterized by homology from other organisms in the SwissProt database: only n = 29 genes (31%) did not have a strict homolog (Table S2).…”

High juvenile hormone titre and abdominal activation of JH signalling may induce reproduction of termite neotenics

“…Partial cDNA sequences of Met , Kr‐h1 and Br‐C homologues ( RsMet , RsKr‐h1 and RsBr‐C ) were obtained from normalized cDNA libraries of R. speratus (Hayashi et al ., ). We designed gene‐specific primers [ RsMet for rapid amplification of cDNA ends (RACE) PCR, RsKr‐h1 and RsBr‐C for subcloning; Fig.…”

“…The amino acid sequence is written under the cDNA sequence. Shaded areas show the partial cDNA sequence obtained from normalized cDNA libraries (Hayashi et al, 2013). The boxed area and solid underline show the sequences of the Per-Arnt-Sim (PAS) and basic-helix-loop-helix (bHLH) domains, respectively.…”

Omic research in termites: an overview and a roadmap

“…Results also suggested that DNA methylation in R. flavipes is targeted to genes with ubiquitous (rather than differential) expression among castes and morphs. A third study examined host transcriptomes of three termite species that included two lower ( Hodotermopsis sjostedti , R. speratus ) and one higher termite ( Nasutitermes takasagoensis ; Hayashi et al, 2013 ). Pyrosequencing was done in combination with 69 caste and phenotypic libraries from the three termite species.…”