Insect Polyphenism Research Articles

Histone Deacetylase 3 Is Involved in Maintaining Queen Hallmarks of a Termite.

The role of epigenetics in regulating caste polyphenism in social insects has been debated. Here, we tested the importance of histone de/acetylation processes for the maintenance of queen hallmarks like a high fecundity and a long lifespan. To this end, we performed RNA interference experiments against histone deacetylase 3 (HDAC3) in the termite Cryptotermes secundus. Fat body transcriptomes and chemical communication profiles revealed that silencing of HDAC3 leads to signals indicative of queen hallmarks. This includes fostering of queen signalling, defence against ageing and a reduction of life-shortening IIS (insulin/insulin-like growth factor signalling) and endocrine JH (juvenile hormone) signalling via Kr-h1 (Krüppel-homologue 1). These observed patterns were similar to those of a protein-enriched diet, which might imply that histone acetylation conveys nutritional effects. Strikingly, in contrast to solitary insects, reduced endocrine JH signalling had no negative effect on fecundity-related vitellogenesis in the fat bodies. This suggests an uncoupling of longevity pathways from fecundity in fat bodies, which can help explain queens' extraordinary lifespans combined with high fecundity.

Sex- and caste-specific developmental responses to juvenile hormone in an ant with maternal caste determination.

Juvenile hormone is considered to be a master regulator of polyphenism in social insects. In the ant Cardiocondyla obscurior, whether a female egg develops into a queen or a worker is determined maternally and caste-specific differentiation occurs in embryos, so that queens and workers can be distinguished in a non-invasive manner from late embryogenesis onwards. This ant also exhibits two male morphs - winged and wingless males. Here, we used topical treatment with juvenile hormone III and its synthetic analogue methoprene, a method that influences caste determination and differentiation in some ant species, to investigate whether hormone manipulation affects the development and growth of male, queen- and worker-destined embryos and larvae. We found no effect of hormone treatment on female caste ratios or body sizes in any of the treated stages, even though individuals reacted to heightened hormone availability with increased expression of krüppel-homolog 1, a conserved JH first-response gene. In contrast, hormone treatment resulted in the emergence of significantly larger males, although male morph fate was not affected. These results show that in C. obscurior, maternal caste determination leads to irreversible and highly canalized caste-specific development and growth.

MiR-2765 Modulates the Seasonal Polyphenism in Cacopsylla chinensis by Targeting a Novel Cold Rreceptor CcTRPC3.

Polyphenism is a beneficial way in organisms to better cope with changing circumstances and is a hot topic in entomology, evolutionary biology, and ecology. Until now, this phenomenon has been proven to be season-, density-, and diet-dependent; however, there are very few reports on temperature regulation. Cacopsylla chinensis showed seasonal polyphenism, namely as summer- and winter-form, with obvious diversity in phenotypic characteristics in response to seasonal variation. Previous studies have found that low temperature in autumn is an extremely important element in inducing summer-form change to winter-form, but the underlying regulatory mechanism is still a mystery. Herein, we provided the initial evidence that the third instar of the summer-form is the critical period for developing to the winter-form, and 10 °C induces this transition by affecting the total pigment, chitin level, and thickness of the cuticle. Second, CcTPRC3 was proven to function as a novel cold receptor to control this seasonal polyphenism. Moreover, miR-2765 was found to mediate seasonal polyphenism by inhibiting CcTRPC3 expression. Last, we found that cuticle binding proteins CcCPR4 and CcCPR9 function as the downstream signals of CcTRPC3 to regulate the seasonal polyphenism in C. chinensis. In conclusion, our results displayed a novel signal pathway of miR-2765 and CcTRPC3 for the regulation of seasonal polyphenism in C. chinensis. These findings provide insights into the comprehensive analysis of insect polyphenism and are useful in developing potential strategies to block the phase transition for the pest control of C. chinensis.

FoxO and rotund form a binding complex governing wing polyphenism in planthoppers

Wing polyphenism is found in a variety of insects and offers an attractive model system for studying the evolutionary significance of dispersal. The Forkhead box O (FoxO) transcription factor (TF) acts as a wing-morph switch that directs wing buds developing into long-winged (LW) or short-winged morphs in wing-dimorphic planthoppers, yet the regulatory mechanism of the FoxO module remains elusive. Here, we identified the zinc finger TF rotund as a potential wing-morph regulator via transcriptomic analysis and phenotypic screening in the brown plathopper, Nilaparvata lugens. RNA interference-mediated knockdown of rotund antagonized the LW development derived from in the context of FoxO depletion or the activation of the insulin/insulin-like growth factor signaling cascade, reversing long wings into intermediate wings. Invitro binding assays indicated that rotund physically binds to FoxO to form the FoxO combinatorial code. These findings broaden our understanding of the complexity of transcriptional regulation governing wing polyphenism in insects.

Comparison of gene expression profiles among caste differentiations in the termite Reticulitermes speratus

Termite castes express specialized phenotypes for their own tasks and are a good example of insect polyphenism. To understand the comprehensive gene expression profiles during caste differentiation, RNA-seq analysis based on the genome data was performed during the worker, presoldier, and nymphoid molts in Reticulitermes speratus. In this species, artificial induction methods for each molt have already been established, and the time scale has been clarified. Three different periods (before the gut purge (GP), during the GP, and after the molt) were discriminated in each molt, and two body parts (head and other body regions) were separately sampled. The results revealed that many differentially expressed genes (head: 2884, body: 2579) were identified in each molt. Based on the independent real-time quantitative PCR analysis, we confirmed the different expression patterns of seven out of eight genes in the presoldier molt. Based on the GO and KEGG enrichment analyses, the expressions of genes related to juvenile hormone titer changes (e.g., JH acid methyltransferase), nutrition status (e.g., Acyl-CoA Delta desaturase), and cell proliferation (e.g., insulin receptor), were shown to specifically fluctuate in each molt. These differences may have a crucial impact on caste differentiation. These data are important resources for future termite sociogenomics.

Photoperiod controls wing polyphenism in a water strider independently of insulin receptor signalling.

Insect wing polyphenism has evolved as an adaptation to changing environments and a growing body of research suggests that the nutrient-sensing insulin receptor signalling pathway is a hot spot for the evolution of polyphenisms, as it provides a direct link between growth and available nutrients in the environment. However, little is known about the potential role of insulin receptor signalling in polyphenisms which are controlled by seasonal variation in photoperiod. Here, we demonstrate that wing length polyphenism in the water strider Gerris buenoi is determined by photoperiod and nymphal density, but is not directly affected by nutrient availability. Exposure to a long-day photoperiod is highly inducive of the short-winged morph whereas high nymphal densities moderately promote the development of long wings. Using RNA interference we demonstrate that, unlike in several other species where wing polyphenism is controlled by nutrition, there is no detectable role of insulin receptor signalling in wing morph induction. Our results indicate that the multitude of possible cues that trigger wing polyphenism can be mediated through different genetic pathways and that there are multiple genetic origins to wing polyphenism in insects.

Additive genetic variance for traits least related to fitness increases with environmental stress in the desert locust, Schistocerca gregaria.

Under environmental stress, previously hidden additive genetic variation can be unmasked and exposed to selection. The amount of hidden variation is expected to be higher for life history traits, which strongly correlate to individual fitness, than for morphological traits, in which fitness effects are more ambiguous. However, no consensual pattern has been recovered yet, and this idea is still debated in the literature. Here, we hypothesize that the classical categorization of traits (i.e., life history and morphology) may fail to capture their proximity to fitness. In the desert locust, Schistocerca gregaria, a model organism for the study of insect polyphenism, we quantified changes in additive genetic variation elicited by lifetime thermal stress for ten traits, in which evolutionary significance is known. Irrespective of their category, traits under strong stabilizing selection showed genetic invariance with environmental stress, while traits more loosely associated with fitness showed a marked increase in additive genetic variation in the stressful environment. Furthermore, traits involved in adaptive phenotypic plasticity (growth compensation) showed either no change in additive genetic variance or a change of moderate magnitude across thermal environments. We interpret this mitigated response of plastic traits in the context of integrated evolution to adjust the entire phenotype in heterogeneous environments (i.e., adaptiveness of initial plasticity, compromise of phenotypic compensation with stress, and shared developmental pathway). Altogether, our results indicate, in agreement with theoretical expectations, that environmental stress can increase available additive genetic variance in some desert locust traits, but those closely linked to fitness are largely unaffected. Our study also highlights the importance of assessing the proximity to fitness of a trait on a case‐by‐case basis and in an ecologically relevant context, as well as considering the processes of canalization and plasticity, involved in the control of phenotypic variation.

Contribution of Epigenetic Mechanisms in the Regulation of Environmentally-Induced Polyphenism in Insects.

Simple SummaryPolyphenism is a widespread phenomenon in insects that allows organisms to produce alternative and discrete phenotypes in response to environmental conditions. Epigenetic mechanisms, including histone post-translational modifications, DNA methylation and non-coding RNAs, are essential mechanisms that can promote rapid and flexible changes in the expression of transcriptional programs associated with the production of alternative phenotypes. This review summarizes knowledge regarding the contribution of those mechanisms in the regulation of the most-studied examples of polyphenism in insects.Many insect species display a remarkable ability to produce discrete phenotypes in response to changes in environmental conditions. Such phenotypic plasticity is referred to as polyphenism. Seasonal, dispersal and caste polyphenisms correspond to the most-studied examples that are environmentally-induced in insects. Cues that induce such dramatic phenotypic changes are very diverse, ranging from seasonal cues, habitat quality changes or differential larval nutrition. Once these signals are perceived, they are transduced by the neuroendocrine system towards their target tissues where gene expression reprogramming underlying phenotypic changes occur. Epigenetic mechanisms are key regulators that allow for genome expression plasticity associated with such developmental switches. These mechanisms include DNA methylation, chromatin remodelling and histone post-transcriptional modifications (PTMs) as well as non-coding RNAs and have been studied to various extents in insect polyphenism. Differential patterns of DNA methylation between phenotypes are usually correlated with changes in gene expression and alternative splicing events, especially in the cases of dispersal and caste polyphenism. Combinatorial patterns of histone PTMs provide phenotype-specific epigenomic landscape associated with the expression of specific transcriptional programs, as revealed during caste determination in honeybees and ants. Alternative phenotypes are also usually associated with specific non-coding RNA profiles. This review will provide a summary of the current knowledge of the epigenetic changes associated with polyphenism in insects and highlights the potential for these mechanisms to be key regulators of developmental transitions triggered by environmental cues.

Transcriptome Analysis of the Regulatory Mechanism of FoxO on Wing Dimorphism in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

Simple SummaryThe brown planthopper (BPH) Nilaparvata lugens can develop into either long-winged or short-winged adults depending on environmental stimuli received during larval stages. The transcription factor NlFoxO serves as a key regulator determining alternative wing morphs in BPH, but the underlying molecular mechanism is largely unknown. Here, we investigated the transcriptomic profile of forewing and hindwing buds across the 5th-instar stage, the wing-morph decision stage. Our results indicated that NlFoxO modulated the developmental plasticity of wing buds mainly by regulating the expression of cell proliferation-associated genes.The brown planthopper (BPH), Nilaparvata lugens, can develop into either short-winged (SW) or long-winged (LW) adults according to environmental conditions, and has long served as a model organism for exploring the mechanisms of wing polyphenism in insects. The transcription factor NlFoxO acts as a master regulator that directs the development of either SW or LW morphs, but the underlying molecular mechanism is largely unknown. Here, we microinjected SW-destined morphs with double stranded-RNA (dsRNA) targeting NlFoxO (dsNlFoxO) to change them into LW-winged morphs. In parallel, SW-destined morphs microinjected with dsRNA targeting the gene encoding green fluorescence protein (dsGfp) served as a negative control. The forewing and hindwing buds of 5th-instar nymphs collected at 24, 36, and 48 h after eclosion (hAE) were used for RNA sequencing. We obtained a minimum of 43.4 million clean reads from forewing and hindwing buds at a single developmental time. Differentially expressed genes (DEGs) were significantly enriched in various Gene Ontology (GO) terms, including cellular process, binding, and cell part. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis showed that up-regulated genes in dsNlFoxO-treated forewing and hindwing buds were largely associated with the cell cycle and DNA replication. Furthermore, most up-regulated genes displayed higher expression at 24-, and 36-hAE relative to 48 hAE, indicating that wing cells in LW-destined wings might actively proliferate during the first 36 h in 5th-instar nymphs. Our findings indicated that LW development in BPH was likely dependent on the duration of cell proliferation in the 5th-instar stage, which sheds light on the molecular basis of wing polymorphism in insects.

Epigenetic variation in animal populations: Sources, extent, phenotypic implications, and ecological and evolutionary relevance

Laboratory experiments and fieldwork with asexually reproducing invertebrates and vertebrates clearly revealed that animal populations can produce substantial phenotypic variation despite genetic identity. This epigenetically caused phenotypic variation comes from two different sources, namely directional environmental induction and bed-hedging developmental stochasticity. Both occur together and are mediated by molecular epigenetic mechanisms like DNA methylation, histone modifications and microRNAs. These epigenetic mechanisms are also involved in insect polyphenism, phenotypic changes in early domestication, and gene expression change and chromatin rearrangement during speciation. Epigenetic variation is particularly important for asexual populations helping them to stay in the game of life when the environmental conditions change. However, it is also relevant for sexually reproducing populations, as shown for genetically impoverished invasive groups, cave animals and sessile taxa that cannot evade unfavourable environmental conditions. Experiments revealed that epigenetic marks can be transgenerationally inherited and persist for several generations. First evidence suggests that inherited epimutations with phenotypic effects may end-up in phenotype-fixing genetic mutations by accelerated mutation of methylated nucleotides. Refined concepts, suitable animal models, fast and affordable new omics techniques that require only small tissue samples, and appropriate data interpretation tools are now available enabling future investigations in ecological and evolutionary epigenetics with high accuracy.

Special Significance of Non-Drosophila Insects in Aging.

Aging is the leading risk factor of human chronic diseases. Understanding of aging process and mechanisms facilitates drug development and the prevention of aging-related diseases. Although many aging studies focus on fruit fly as a canonical insect system, minimal attention is paid to the potentially significant roles of other insects in aging research. As the most diverse group of animals, insects provide many aging types and important complementary systems for aging studies. Insect polyphenism represents a striking example of the natural variation in longevity and aging rate. The extreme intraspecific variations in the lifespan of social insects offer an opportunity to study how aging is differentially regulated by social factors. Insect flight, as an extremely high-intensity physical activity, is suitable for the investigation of the complex relationship between metabolic rate, oxidative stress, and aging. Moreover, as a “non-aging” state, insect diapause not only slows aging process during diapause phase but also affects adult longevity during/after diapause. In the past two decades, considerable progress has been made in understanding the molecular basis of aging regulation in insects. Herein, the recent research progress in non-Drosophila insect aging was reviewed, and its potential utilization in aging in the future was discussed.

Nutrition-responsive gene expression and the developmental evolution of insect polyphenism.

Nutrition-responsive development is a ubiquitous and highly diversified example of phenotypic plasticity, yet its underlying molecular and developmental mechanisms and modes of evolutionary diversification remain poorly understood. We measured genome-wide transcription in three closely related species of horned beetles exhibiting strikingly diverse degrees of nutrition responsiveness in the development of male weaponry. We show that (1) counts of differentially expressed genes between low- and high-nutritional backgrounds mirror species-specific degrees of morphological nutrition responsiveness; (2) evolutionary exaggeration of morphological responsiveness is underlain by both amplification of ancestral nutrition-responsive gene expression and recruitment of formerly low nutritionally responsive genes; and (3) secondary loss of morphological responsiveness to nutrition coincides with a dramatic reduction in gene expression plasticity. Our results further implicate genetic accommodation of ancestrally high variability of gene expression plasticity in both exaggeration and loss of nutritional plasticity, yet reject a major role of taxon-restricted genes in the developmental regulation and evolution of nutritional plasticity.

Mutations in NlInR1 affect normal growth and lifespan in the brown planthopper Nilaparvata lugens

The brown planthopper (BPH) Nilaparvata lugens contains two insulin receptor homologues, designated NlInR1 and NlInR2. NlInR1 is strikingly homologous to the typical InR in insects and vertebrates, containing a ligand-activated intracellular tyrosine kinase catalytic domain. Herein, we report an optimized CRISPR/Cas9 system to induce mutations in the NlInR1 locus in BPH, consisting of a Cas9 plasmid that is specifically expressed in the germline via the Nlvasa promoter and versatile sgRNA expression plasmids under the control of the U6 promoter. We systematically evaluated the efficiency of injection mix compositions and demonstrated an appropriate combination of Cas9/sgRNA to target essential genes. Furthermore, we showed that homozygous mutants for the NlInR1 gene are early embryonic lethal, whereas heterozygous mutants grow more slowly, exhibit a severe reduction in body weight and wing size and live longer than the wild type. Interestingly, the severity of the mutant phenotype was different when targeting distinct important domains of the NlInR1 locus. The severity of the mutant phenotype is similar to that of insulin/insulin-like growth factor (IGF) signaling pathway deficiencies in vertebrates, suggesting a conserved function of NlInR1 in the regulation of development and longevity. Global expression profiling suggests that NlInR1 regulates many cellular processes in BPH, including insulin resistance, phototransduction, metabolism, endocytosis, longevity, biosynthesis and protein processing. Our results also pave the way for understanding the precise molecular mechanism of insulin signaling in wing polyphenism in insects.

Temperature‐ and food‐dependent foraging gene expression in foragers of the red imported fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae)

AbstractThe behavioural plasticity and adaptability of some insects, allowing them to adapt to diverse habitats, is strongly associated with foraging genes (for). The role of the foraging gene is closely related to insect polyphenism and some abiotic and biotic factors. The red imported fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae) has successfully expanded its global range and is considered as a model insect because of its adaptability to a range of environmental factors. We use a quantitative reverse transcriptase‐polymerase chain reaction to study levels of S. invicta foraging (Sifor) gene expression in foragers after changes in temperature and food availability. Sifor expression levels are down‐regulated in colonies kept at 18 °C and up‐regulated in colonies kept at 35 °C after transferring individuals from a colony kept at 26 °C. Moreover, Sifor expression levels decrease when food is deprived and increase when food is restored in comparison with the control. This shows that Sifor is linked to temperature to a great extent and to food transformations to some extent, and suggests that Sifor may explain the ability of fire ant foragers to acclimatize to daily temperature fluctuations. Further research on for gene functions will generate more data with respect to eliciting its role in ecological adaptation to environmental change.

Epigenetics and insect polyphenism: mechanisms and climate change impacts

Phenotypic plasticity is a ubiquitous process found in all living organisms. Polyphenism is an extreme case of phenotypic plasticity which shares a common scheme in insects such as honeybees, locusts or aphids: an initial perception of environmental stimuli, a neuroendocrine transmission of these signals to the target tissues, the activation of epigenetic mechanisms allowing the setup of alternative transcriptional programs responsible for the establishment of discrete phenotypes. Climate change can modulate the environmental stimuli triggering polyphenisms, and/or some epigenetics marks, thus modifying on the short and long terms the discrete phenotype proportions within populations. This might result in critical ecosystem changes.

MiR-34 modulates wing polyphenism in planthopper.

Polyphenism is a successful strategy adopted by organisms to adapt to environmental changes. Brown planthoppers (BPH, Nilaparvata lugens) develop two wing phenotypes, including long-winged (LW) and short-winged (SW) morphs. Though insulin receptor (InR) and juvenile hormone (JH) have been known to regulate wing polyphenism in BPH, the interaction between these regulators remains largely elusive. Here, we discovered that a conserved microRNA, miR-34, modulates a positive autoregulatory feedback loop of JH and insulin/IGF signaling (IIS) pathway to control wing polyphenism in BPH. Nlu-miR-34 is abundant in SW BPHs and suppresses NlInR1 by targeting at two binding sites in the 3’UTR of NlInR1. Overexpressing miR-34 in LW BPHs by injecting agomir-34 induces the development towards SW BPHs, whereas knocking down miR-34 in SW BPHs by injecting antagomir-34 induces more LW BPHs when another NlInR1 suppressor, NlInR2, is also suppressed simultaneously. A cis-response element of Broad Complex (Br-C) is found in the promoter region of Nlu-miR-34, suggesting that 20-hydroxyecdysone (20E) might be involved in wing polyphenism regulation. Topic application of 20E downregulates miR-34 expression but does not change wing morphs. On the other hand, JH application upregulates miR-34 expression and induces more SW BPHs. Moreover, knocking down genes in IIS pathway changes JH titers and miR-34 abundance. In all, we showed that miRNA mediates the cross talk between JH, 20E and IIS pathway by forming a positive feedback loop, uncovering a comprehensive regulation mechanism which integrates almost all known regulators controlling wing polyphenism in insects.

Juvenile hormone as a physiological regulator mediating phenotypic plasticity in pancrustaceans.

Phenotypic plasticity and polyphenism, in which phenotypes can be changed depending on environmental conditions, are common in insects. Several studies focusing on physiological, developmental, and molecular processes underlying the plastic responses have revealed that similar endocrine mechanisms using juvenile hormone (JH) are used to coordinate the flexible developmental processes. This review discusses accumulated knowledge on the caste polyphenism in social insects (especially termites), the wing and the reproductive polyphenisms in aphids, and the nutritional polyphenism and sexual dimorphism in stag beetles. For the comparison with non-insect arthropods, extensive studies on the inducible defense (and reproductive polyphenism) in daphnids (crustacean) are also addressed. In all the cases, JH (and methyl farnesoate in daphnids) plays a central role in mediating environmental stimuli with morphogenetic processes. Since the synthetic pathways for juvenoids, i.e., the mevalonate pathway and downstream pathways to sesquiterpenoids, are conserved across pancrustacean lineages (crustaceans and hexapods including insects), the evolution of developmental regulation by juvenoids that control molting (ecdysis) and metamorphosis is suggested to have occurred in the ancestral arthropods. The discontinuous postembryonic development (i.e., molting) and the regulatory physiological factors (juvenoids) would have enabled plastic developmental systems observed in many arthropod lineages.

Synaptic organization and division of labor in the exceptionally polymorphic ant Pheidole rhea

Social insect polyphenisms provide models to examine the neural basis of division of labor and anatomy of the invertebrate social brain. Worker size-related behavior is hypothesized to enhance task performance, raising questions concerning the integration of morphology, behavior, and cellular neuroarchitecture, and how variation in sensory inputs and cognitive demands of behaviorally differentiated workers is reflected in higher-order processing ability. We used the highly polymorphic ant Pheidole rhea, which has three distinct worker size classes – minors, soldiers, and supersoldiers – to examine variation in synaptic circuitry across worker size and social role. We hypothesized that the density and size of synaptic complexes (microglomeruli, MG) would be positively associated with behavioral repertoire and the relative size of the mushroom bodies (MB). Supersoldiers had significantly larger and less dense MG in the lip (olfactory region) of the MB calyx (MBC), and larger MG in the collar (visual region) compared to minors. Soldiers were intermediate in synaptic phenotype: they did not differ significantly in MG density from minors and supersoldiers, had MG of similar size to minors in the lip, and did not differ from these two worker groups in MG size in the collar. Results suggest a complex relationship between MG density, size, behavior, and worker body size involving a conserved and plastic neurobiological development plan, although workers show strong variation in size and social role.

Advances on polyphenism in insects.

Polyphenism denotes that one genome produces two or more distinct phenotypes due to environmental inductions. Many cases have been reported in insects, for example, metamorphosis, seasonal polyphenism, the caste of eusocial insects and so on. Polyphenism is one of the most important reasons for insects to survive and thrive, because insects can adapt and use the environmental cues around them in order to avoid predators and reproduce by changing their phenotypes. Polyphenism has received growing attentions, ranging from the earlier description of this phenomenon to the exploration of possible inducing factors. With the recent advent of the genomic era, more and more studies based on next generation sequencing, gene knockout and RNA interference have been reported to reveal the molecular mechanism of polyphenism. In this review, we summarize the progresses of the polyphenism in insects and envision prospects of future researches.

Transcriptome sequencing and estimation of DNA methylation level in the subsocial wood-feeding cockroach Cryptocercus punctulatus (Blattodea: Cryptocercidae)

The wood-feeding cockroach genus Cryptocercus is a subsocial and sister group of the eusocial cockroaches, i.e., termites. Although Cryptocercus is a key taxon for understanding the evolution of eusociality in the Blattodea (cockroaches and termites), few genetic resources are available for comparative genetic analyses. In this study, we conducted transcriptome sequencing of Cryptocercus punctulatus Scudder using next-generation sequencing technology to generate a massive genetic resource. By transcriptome sequencing and subsequent de novo transcriptome assembly, we obtained 132,191 contigs. The assembled transcriptome contained almost all of the conserved core eukaryote or insect genes. Furthermore, juvenile hormone- and DNA methylation-related genes that are considered to be key genes for the caste polyphenism in eusocial insects, were identified from the transcriptome assembly. In addition, the ratio of the observed (O) cytosine-phosphate-guanine (CpG) content to the expected (E) one (CpG O/E), which is a proxy for the DNA methylation level, was calculated for coding regions extracted from the assembly. The CpG O/E values were less than 1 in most of the coding regions, and the frequency distribution of the CpG O/E values was bimodal rather than unimodal. These results indicate that most C. punctulatus genes are involved in DNA methylation.