Genomic and transcriptomic analyses of the subterranean termite Reticulitermes speratus: Gene duplication facilitates social evolution.

Termites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing a transitional taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome, and methylome of the Japanese subterranean termite Reticulitermes speratus Our analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression was prevalent in the R. speratus genome. The duplicated genes comprised diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense), and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but their expression patterns were highly variable, exhibiting caste biases. Some of the assayed duplicated genes were expressed in caste-specific organs, such as the accessory glands of the queen ovary and the frontal glands of soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification, leading to caste-biased expression and subfunctionalization and/or neofunctionalization conferring caste-specialized functions.

Evolution and functionalization of vitellogenin genes in the termite Reticulitermes speratus.

Eusociality has been commonly observed in distinct animal lineages. The reproductive division of labor is a particular feature, achieved by the coordination between fertile and sterile castes within the same nest. The sociogenomic approach in social hymenopteran insects indicates that vitellogenin (Vg) has undergone neo-functionalization in sterile castes. Here, to know whether Vgs have distinct roles in nonreproductive castes in termites, we investigated the unique characteristics of Vgs in the rhinotermitid termite Reticulitermes speratus. The four Vgs were identified from R. speratus (RsVg1-4), and RsVg3 sequences were newly identified using the RACE method. Molecular phylogenetic analysis supported the monophyly of the four termite Vgs. Moreover, the termites Vg1-3 and Vg4 were positioned in two different clades. The  dN/dS ratios indicated that the branch leading to the common ancestor of termite Vg4 was under weak purifying selection. Expression analyses among castes (reproductives, workers, and soldiers) and females (nymphs, winged alates, and queens) showed that RsVg1-3 was highly expressed in fertile queens. In contrast, RsVg4 was highly expressed in workers and female nonreproductives (nymphs and winged adults). Localization of RsVg4 messenger RNA was confirmed in the fat body of worker heads and abdomens. These results suggest that Vg genes are functionalized after gene duplication during termite eusocial transition and that Vg4 is involved in nonreproductive roles in termites.

TGFß signaling related genes are involved in hormonal mediation during termite soldier differentiation.

A working knowledge of the proximate factors intrinsic to sterile caste differentiation is necessary to understand the evolution of eusocial insects. Genomic and transcriptomic analyses in social hymenopteran insects have resulted in the hypothesis that sterile castes are generated by the novel function of co-opted or recruited universal gene networks found in solitary ancestors. However, transcriptome analysis during caste differentiation has not been tested in termites, and evolutionary processes associated with acquiring the caste are still unknown. Termites possess the soldier caste, which is regarded as the first acquired permanently sterile caste in the taxon. In this study, we performed a comparative transcriptome analysis in termite heads during 3 molting processes, i.e., worker, presoldier and soldier molts, under natural conditions in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Although similar expression patterns were observed during each molting process, more than 50 genes were shown to be highly expressed before the presoldier (intermediate stage of soldier) molt. We then performed RNA interference (RNAi) of the candidate 13 genes, including transcription factors and uncharacterized protein genes, during presoldier differentiation induced by juvenile hormone (JH) analog treatment. Presoldiers induced after RNAi of two genes related to TGFß (Transforming growth factor beta) signaling were extremely unusual and possessed soldier-like phenotypes. These individuals also displayed aggressive behaviors similar to natural soldiers when confronted with Formica ants as hypothetical enemies. These presoldiers never molted into the next instar, presumably due to the decreased expression levels of the molting hormone (20-hydroxyecdysone; 20E) signaling genes. These results suggest that TGFß signaling was acquired for the novel function of regulating between JH and 20E signaling during soldier differentiation in termites.

A lipocalin protein, Neural Lazarillo, is key to social interactions that promote termite soldier differentiation.

Social communication among castes is a crucial component of insect societies. However, the genes involved in soldier determination through the regulation of inter-individual interactions are largely unknown. In an incipient colony of the damp-wood termite Zootermopsis nevadensis, the first larva to develop into a third instar always differentiates into a soldier via frequent trophallactic feeding from the reproductives. Here, by performing RNA-seq analysis of third instar larvae, a homologue of Neural Lazarillo (named ZnNLaz1) was found to be the most differentially expressed gene in these soldier-destined larvae, compared with worker-destined larvae. This gene encodes a lipocalin protein related to the transport of small hydrophobic molecules. RNAi-induced knockdown of ZnNLaz1 significantly inhibited trophallactic interactions with the queen and decreased the soldier differentiation rates. This protein is localized in the gut, particularly in the internal wall, of soldier-destined larvae, suggesting that it is involved in the integration of social signals from the queen through frequent trophallactic behaviours. Based on molecular phylogenetic analysis, we suggest that a novel function of termite NLaz1 has contributed to social evolution from the cockroach ancestors of termites. These results indicated that a high larval NLaz1 expression is crucial for soldier determination through social communication in termites.

Time-course transcriptome data of silk glands in day 0-7 last-instar larvae of Bombyx mori (w1 pnd strain).

Time-course transcriptome expression data were constructed for four parts of the silk gland (anterior, middle, and posterior parts of the middle silk gland, along with the posterior silk gland) in the domestic silkworm, Bombyx mori, from days 0 to 7 of the last-instar larvae. For sample preparation, silk glands were extracted from one female and one male larva every 24 hours accurately after the fourth ecdysis. The reliability of these transcriptome data was confirmed by comparing the transcripts per million (TPM) values of the silk gene and quantitative reverse transcription PCR results. Hierarchical cluster analysis results supported the reliability of transcriptome data. These data are likely to contribute to the progress in molecular biology and genetic research using B. mori, such as elucidating the mechanism underlying the massive production of silk proteins, conducting entomological research using a meta-analysis as a model for lepidopteran insect species, and exploring medical research using B. mori as a model for disease species by utilising transcriptome data.

Efficient RNA interference method during caste differentiation with hormone treatment in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae).

Unveiling the proximate mechanism of caste differentiation is crucial for understanding insect social evolution, and gene function analysis is an important tool in this endeavor. The RNA interference (RNAi) technique is useful in termites, but its knockdown effects may differ among species. One of the most important model species in the field of termite sociogenomics is Reticulitermes speratus Kolbe (Isoptera: Rhinotermitidae). Presoldier and worker differentiation of this species can be artificially induced by juvenile hormone and 20-hydroxyecdysone application, respectively. However, appropriate RNAi technique of genes expressed during caste differentiation has never been considered. To clarify this issue, first, we injected nine different volumes of nuclease-free water (NFW, 0-404.8 nL) into workers and found that survival and caste differentiation rates were strongly reduced by the application of the top three largest volumes. Second, we injected double-stranded (ds) RNA of ecdysone receptor homolog (RsEcR) (2.0 µg/151.8 nL NFW) into workers with hormone treatments. The expression levels of RsEcR were significantly reduced at 9 days after dsRNA injection. RsEcR RNAi strongly affected both molting events during presoldier and worker differentiation induced by hormone treatments. The present results highlight the need for caution regarding injection volumes for RNAi experiments using hormone treatments. We suggest that the injection of dsRNA solution (2 µg; approximately 100-200 nL) is suitable for RNAi experiments during caste differentiation induced by hormone application in R. speratus.

Co-Expression Network and Time-Course Expression Analyses to Identify Silk Protein Regulatory Factors in Bombyx mori.

Bombyx mori is an important economic insect and an animal model in pharmacomedical research. Although its physiology has been studied for many years, the mechanism via which silk protein genes are regulated remains unclear. In this study, we performed two-step expression screening, namely co-expression network and time-course expression analyses to screen silk protein regulation factors. A co-expression network analysis using RNA-seq data that were obtained from various tissues, including the silk glands of B. mori, was performed to identify novel silk protein regulatory factors. Overall, 91 transcription factors, including some known ones, were found to be co-expressed with silk protein genes. Furthermore, time-course expression analysis during the fifth instar larvae stage revealed that the expression pattern of 13 novel transcription factors was highly relevant to that of silk protein genes and their known regulatory factor genes. In particular, the expression peak of several transcription factors (TFs) was detected before the expression of silk protein genes peak. These results indicated that a larger number of genes than expected may be involved in silk protein regulation in B. mori. Functional analyses of function-unknown transcription factors should enhance our understanding of this system.

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.

Reproductive Soldier Development Is Controlled by Direct Physical Interactions with Reproductive and Soldier Termites.

In eusocial insects (e.g., ants, bees, and termites), the roles of different castes are assigned to different individuals. These castes possess unique phenotypes that are specialized for specific tasks. The acquisition of sterile individuals with specific roles is considered a requirement for social evolution. In termites, the soldier is a sterile caste. In primitive taxa (family Archotermopsidae and Stolotermitidae), however, secondary reproductives (neotenic reproductives) with their mandibles developed into weapons (so-called reproductive soldiers, also termed as soldier-headed reproductives or soldier neotenics) have been reported. To understand the developmental mechanism of this unique caste, it is necessary to understand the environmental cues and developmental processes of reproductive soldiers under natural conditions. Here, we established efficient conditions to induce reproductive soldiers in Zootermopsis nevadensis. Male reproductive soldiers frequently developed after the removal of both the king and soldiers from an incipient colony. Similarly, high differentiation rates of male reproductive soldiers were observed after king-and-soldier separation treatment using wire mesh. However, no male reproductive soldiers were produced without direct interaction with the queen. These results suggest that male reproductive soldier development is repressed by direct physical interactions with both the king and soldiers and facilitated by direct physical interaction with the queen.

Evolutionary transition of doublesex regulation from sex-specific splicing to male-specific transcription in termites.

The sex determination gene doublesex (dsx) encodes a transcription factor with two domains, oligomerization domain 1 (OD1) and OD2, and is present throughout insects. Sex-specific Dsx splicing isoforms regulate the transcription of target genes and trigger sex differentiation in all Holometabola examined to date. However, in some hemimetabolous insects, dsx is not spliced sexually and its sequence is less conserved. Here, to elucidate evolutionary changes in dsx in domain organisation and regulation in termites, we searched genome and/or transcriptome databases for the dsx OD1 and OD2 in seven termite species and their sister group (Cryptocercus woodroaches). Molecular phylogenetic and synteny analyses identified OD1 sequences of termites and C. punctulatus that clustered with dsx of Holometabola and regarded them as dsx orthologues. The Cryptocercus dsx orthologue containing OD2 was spliced sexually, as previously shown in other insects. However, OD2 was not found in all termite dsx orthologues. These orthologues were encoded by a single exon in three termites for which genome information is available; they were not alternatively spliced but transcribed in a male-specific manner in two examined species. Evolution of dsx regulation from sex-specific splicing to male-specific transcription may have occurred at an early stage of social evolution in termites.

A Crucial Caste Regulation Gene Detected by Comparing Termites and Sister Group Cockroaches.

Sterile castes are a defining criterion of eusociality; investigating their evolutionary origins can critically advance theory. In termites, the soldier caste is regarded as the first acquired permanently sterile caste. Previous studies showed that juvenile hormone (JH) is the primary factor inducing soldier differentiation, and treatment of workers with artificial JH can generate presoldier differentiation. It follows that a shift from a typical hemimetabolous JH response might be required for soldier formation during the course of termite evolution within the cockroach clade. To address this possibility, analysis of the role of JH and its signaling pathway was performed in the termite Zootermopsis nevadensis and compared with the wood roach Cryptocercus punctulatus, a member of the sister group of termites. Treatment with a JH analog (JHA) induced a nymphal molt in C. punctulatus RNA interference (RNAi) of JH receptor Methoprene tolerant (Met) was then performed, and it inhibited the presoldier molt in Z. nevadensis and the nymphal molt in C. punctulatus Knockdown of Met in both species inhibited expression of 20-hydroxyecdysone (20E; the active form of ecdysone) synthesis genes. However, in Z. nevadensis, several 20E signaling genes were specifically inhibited by Met RNAi. Consequently, RNAi of these genes were performed in JHA-treated termite individuals. Knockdown of 20E signaling and nuclear receptor gene, Hormone receptor 39 (HR39/FTZ-F1ß) resulted in newly molted individuals with normal worker phenotypes. This is the first report of the JH-Met signaling feature in termites and Cryptocercus JH-dependent molting activation is shared by both taxa and mediation between JH receptor and 20E signalings for soldier morphogenesis is specific to termites.

Ecdysone signaling regulates soldier-specific cuticular pigmentation in the termite Zootermopsis nevadensis.

Termite caste differentiation requires hormonal regulation, but understanding of the role of ecdysone is limited. Here, we investigated the expression and function of ecdysone-related genes during soldier differentiation in the damp-wood termite Zootermopsis nevadensis. Ecdysone receptor gene (EcR) was highly expressed in the head just after the presoldier molt. Knockdown of EcR expression in the early presoldier period inhibited the molts into soldiers. However, knockdown in the middle period affected tyrosine metabolic gene expression and inhibited soldier-specific cuticular tanning. These results suggest that ecdysone activation is involved in soldier-specific cuticle formation.

Gene expression changes in the tyrosine metabolic pathway regulate caste-specific cuticular pigmentation of termites.

In social insects, all castes have characteristic phenotypes suitable for their own tasks and to engage in social behavior. The acquisition of caste-specific phenotypes was a key event in the course of social insect evolution. However, understanding of the genetic basis and the developmental mechanisms that produce these phenotypes is still very limited. In particular, termites normally possess more than two castes with specific phenotypes (i.e. workers, soldiers, and reproductives), but proximate developmental mechanisms are far from being fully understood. In this study, we focused on the pigmentation of the cuticle as a model trait for caste-specific phenotypes, during the molts of each caste; workers, soldiers, presoldiers (intermediate stage of soldiers), and alates (primary reproductives) in Zootermopsis nevadensis. Expression patterns of cuticular tanning genes (members of the tyrosine metabolic pathway) were different among each molt, and high expression levels of several "key genes" were observed during each caste differentiation. For the differentiation of castes with well-tanned cuticles (i.e. soldiers and alates), all focal genes except DDC in the former were highly expressed. On the other hand, high expression levels of yellow and aaNAT were observed during worker and presoldier molts, respectively, but most other genes in the pathway were expressed at low levels. RNA interference (RNAi) of these key genes affected caste-specific cuticular pigmentation, leading to soldiers with yellowish-white heads and pigmented mandibular tips, presoldiers with partly pigmented head cuticles, and alates with the yellow head capsules. These results suggest that the pigmentation of caste-specific cuticles is achieved by the regulation of gene expression in the tyrosine metabolic pathway.

Knockdown of the juvenile hormone receptor gene inhibits soldier-specific morphogenesis in the damp-wood termite Zootermopsis nevadensis (Isoptera: Archotermopsidae).

The Methoprene-tolerant (Met) protein has been established as a juvenile hormone (JH) receptor. Knockdown of the Met gene caused precocious metamorphosis and suppression of ovarian development. However, the function of Met in caste development of social insects is unclear. In termites, JH acts as a central factor for caste development, especially for soldier differentiation, which involves two molts from workers via a presoldier stage. Increased JH titer in workers is needed for the presoldier molt, and the high JH titer is maintained throughout the presoldier period. Although presoldiers have the fundamental morphological features of soldiers, the nature of the cuticle is completely different from that of soldiers. We expected that JH signals via Met are involved in soldier-specific morphogenesis of the head and mandibles during soldier differentiation, especially in the presoldier period, in natural conditions. To test this hypothesis, we focused on soldier differentiation in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Met homolog (ZnMet) expression in heads increased just after the presoldier molt. This high expression was reduced by ZnMet double stranded (dsRNA) injection before the presoldier molt. Although this treatment did not cause any morphological changes in presoldiers, it caused strong effects on soldiers, their mandibles being significantly shorter and head capsules smaller than those of control soldiers. Injection of ZnMet dsRNA throughout the presoldier stage did not affect the formation of soldier morphology, including cuticle formation. These results suggested that the rapid increase in ZnMet expression and subsequent activation of JH signaling just after the presoldier molt are needed for the formation of soldier-specific weapons. Therefore, besides its established role in insect metamorphosis, the JH receptor signaling also underlies soldier development in termites.

High Laccase2 expression is likely involved in the formation of specific cuticular structures during soldier differentiation of the termite Reticulitermes speratus.

Termite soldiers are morphologically specialized for colony defense. Analysis of the mechanisms of soldier differentiation is important for understanding the establishment of termite societies. Soldiers differentiate from workers through a presoldier stage and have well-sclerotized and pigmented cuticles. These characteristics are important for nest defense and are likely to be caused by soldier-specific mechanisms of cuticular tanning. The molecular mechanisms leading to cuticular tanning have not been elucidated. Laccase2 (Lac2) plays important roles in this process in insects, and we hypothesized that Lac2 expression may be involved in soldier-specific cuticular tanning. We observed inner and outer head cuticle changes and compared the Lac2 expression patterns among three molts (worker-worker, worker-presoldier and presoldier-soldier) in the termite Reticulitermes speratus. Quantitative analyses of head cuticle colors showed that the color properties changed more conspicuously in presoldier-soldier molts than in the other two molts. Histological observations showed that the exocuticles of soldier heads were substantially thicker than those of worker and presoldier heads, underwent tanning before or just after ecdysis, and were pigmented at earlier time points than other molts. Finally, markedly higher Lac2 expression levels were observed just before and after ecdysis only in the presoldier-soldier molt. These results suggest that specific cuticular formation occurs in the exocuticles during soldier differentiation, and that the high level of Lac2 expression during the presoldier-soldier molt is related to soldier-specific cuticular tanning. We speculate that evolution of the regulatory mechanisms of Lac2 expression were important for the acquisition of soldier-specific cuticles.