Morphological, histological and gene-expression analyses on stolonization in the Japanese Green Syllid, Megasyllis nipponica (Annelida, Syllidae).

Benthic annelids belonging to the family Syllidae (Annelida, Errantia, Phyllodocida) exhibit a unique reproduction mode called "schizogamy" or "stolonization", in which the posterior body part filled with gametes detaches from the original body, as a reproductive unit (stolon) that autonomously swims and spawns. In this study, morphological and histological observations on the developmental processes during stolonization were carried out in Megasyllis nipponica. Results suggest that the stolon formation started with maturation of gonads, followed by the formation of a head ganglion in the anteriormost segment of the developing stolon. Then, the detailed stolon-specific structures such as stolon eyes and notochaetae were formed. Furthermore, expression profiles of genes involved in the anterior-posterior identity (Hox genes), head determination, germ-line, and hormone regulation were compared between anterior and posterior body parts during the stolonization process. The results reveal that, in the posterior body part, genes for gonadal development were up-regulated, followed by hormone-related genes and head-determination genes. Unexpectedly, Hox genes known to identify body parts along the anterior-posterior axis showed no significant temporal expression changes. These findings suggest that during stolonization, gonad development induces the head formation of a stolon, without up-regulation of anterior Hox genes.

Gene expression profiles of chemosensory genes of termite soldier and worker antennae.

Termites have an elaborate social system that involves cooperation and division of labour among colony members. Although this social system is regulated by chemical signals produced in the colony, it remains unclear how these signals are perceived by other members. Signal transduction is well known to be triggered by the reception of odorant molecules by some binding proteins in the antennae, after which, a signal is transmitted to chemosensory receptors. However, there is insufficient information on the role of chemosensory genes involved in signal transduction in termites. Here, we identified the genes involved in chemosensory reception in the termite Reticulitermes speratus and performed a genome-wide comparative transcriptome analysis of worker and soldier antennae. First, we identified 31 odorant-binding proteins (OBPs), and three chemosensory protein A (CheA) from the genome data. Thereafter, we performed RNA sequencing to compare the expression levels of OBPs, CheAs, and previously identified chemosensory receptor genes between worker and soldier antennae. There were no receptor genes with significant differences in expression between castes. However, the expression levels of three non-receptor odorant-detection/binding proteins (OBP, CheA, and Sensory neuron membrane protein) were significantly different between castes. Real-time qPCR (RT-qPCR) analysis using antennae and other head parts confirmed that these genes were highly expressed in soldier antennae. Finally, independent RT-qPCR analysis showed that the expression patterns of these genes were altered in soldiers from different social contexts. Present results suggest that gene expression levels of some non-receptors are affected by both castes and behavioural interactions among colony members in termites.

Enhancement of receptor-mediated calcium responses by phenytoin through the suppression of calcium excretion in human gingival fibroblasts.

BACKGROUND AND OBJECTIVES: Gingival overgrowth caused by phenytoin is proposed to be associated with Ca2+ signaling; however, the mechanisms that increase the intracellular Ca2+ concentration ([Ca2+ ]i ) are controversial. The current study aimed to elucidate the mechanism underlying the phenytoin-induced increase in [Ca2+ ]i in human gingival fibroblasts (HGFs). METHODS: Effects of 100 µM phenytoin on [Ca2+ ]i in HGFs were examined at the single-cell level using fluorescence images of fura-2 captured by an imaging system consisting of an EM-CCD camera coupled to an inverted fluorescence microscope at room temperature. RESULTS: Exposure of HGFs to 100 µM phenytoin induced a transient increase in [Ca2+ ]i in the absence of extracellular Ca2+ , indicating that the phenytoin-induced increase in [Ca2+ ]i does not require an influx of extracellular Ca2+ . In addition, phenytoin increased [Ca2+ ]i in HGFs depleted of intracellular Ca2+ stores by thapsigargin, indicating that neither Ca2+ release from stores nor inhibition of Ca2+ uptake is involved. Furthermore, the phenytoin-induced [Ca2+ ]i elevation was reduced to 18.8% in the absence of extracellular Na+ , and [Ca2+ ]i elevation upon removal of extracellular Na+ was reduced to 25.9% in the presence of phenytoin. These results imply that phenytoin increases [Ca2+ ]i of HGFs by suppressing the Na+ /Ca2+ exchanger. Suppression of intracellular Ca2+ excretion is thought to enhance the Ca2+ responses induced by various stimuli. Analysis at the single-cell level showed that stimulation with 1 µM ATP or 3 µM histamine increased [Ca2+ ]i in 20-50% of cells, and [Ca2+ ]i increased in many unresponsive cells in the presence of phenytoin. CONCLUSION: Our findings demonstrate that phenytoin induced increase in [Ca2+ ]i by the inhibition of Ca2+ efflux in HGFs. It was also found that phenytoin strongly enhanced small Ca2+ responses induced by stimulation with a low concentration of ATP or histamine by inhibiting Ca2+ efflux. These findings suggest a possibility that phenytoin causes drug-induced gingival overgrowth by interacting with inflammatory bioactive substances in the gingiva.

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.

Construction of a massive genetic resource by transcriptome sequencing and genetic characterization of Megasyllis nipponica (Annelida: Syllidae).

Understanding the processes and consequences of the morphological diversity of organisms is one of the major goals of evolutionary biology. Studies on the evolution of developmental mechanisms of morphologies, or evo-devo, have been extensively conducted in many taxa and have revealed many interesting phenomena at the molecular level. However, many other taxa exhibiting intriguing morphological diversity remain unexplored in the field of evo-devo. Although the annelid family Syllidae shows spectacular diversity in morphological development associated with reproduction, its evo-devo study, especially on molecular development, has progressed slowly. In this study, we focused on Megasyllis nipponica as a new model species for evo-devo in syllids and performed transcriptome sequencing to develop a massive genetic resource, which will be useful for future molecular studies. From the transcriptome data, we identified candidate genes that are likely involved in morphogenesis, including genes involved in hormone regulation, sex determination and appendage development. Furthermore, a computational analysis of the transcriptome sequence data indicated the occurrence of DNA methylation in coding regions of the M. nipponica genome. In addition, flow cytometry analysis showed that the genome size of M. nipponica was approximately 524 megabases. These results facilitate the study of morphogenesis in molecular terms and contribute to our understanding of the morphological diversity in syllids.

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.

Thoracic crop formation is spatiotemporally coordinated with flight muscle histolysis during claustral colony foundation in Lasius japonicus queens.

In a majority of ants, a newly mated queen independently founds a colony and claustrally raises her first brood without foraging outside the nest. During claustral independent colony foundation (ICF) in several ants, the esophagus of the founding queen expands and develops into a "thoracic crop," which is then filled with a liquid substrate for larval feeding. It has been suggested that these substrates are converted from the founding queen's body reserves (e.g., histolyzed flight muscles) or redistributed from a gastral crop. Here, we describe thoracic crop development in Lasius japonicus queens during claustral ICF. The foundresses claustrally feed their larvae from weeks 2-5 after ICF onset, and the first worker emerges at week 6. The development proceeds as follows: in week 0, the foundress' dorsal esophageal wall is pleated and thickened. Then, from weeks 2-5, the esophagus expands toward a dorsal space previously occupied by flight muscles, following flight muscle histolysis. Gastral crop expansion follows esophageal expansion. Thus, thoracic crop formation may be spatiotemporally coordinated with flight muscle histolysis in Lasius japonicus queens, and similar developmental regulations might be common in other claustral ICF ants.

Termite sociogenomics: evolution and regulation of caste-specific expressed genes.

Termite genomes have been sequenced in at least five species from four different families. Genome-based transcriptome analyses have identified large numbers of protein-coding genes with caste-specific expression patterns. These genes include those involved in caste-specific morphologies and roles, for example high fecundity and longevity in reproductives. Some caste-specific expressed genes belong to multi-gene families, and their genetic architecture and expression profiles indicate they have evolved via tandem gene duplication. Candidate regulatory mechanisms of caste-specific expression include epigenetic regulation (e.g. histone modification and non-coding RNA) and diversification of transcription factors and cis-regulatory elements. We review current knowledge in the area of termite sociogenomics, focussing on the evolution and regulation of caste-specific expressed genes, and discuss future research directions.

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.

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.

Male neotenic reproductives accelerate additional differentiation of female reproductives by lowering JH titer in termites.

Eusocial insects exhibit reproductive division of labor, in which only a fraction of colony members differentiate into reproductives. In termites, reproductives of both sexes are present in a colony and constantly engaged in reproduction. It has been suggested that the sex ratio of reproductives is maintained by social interactions. The presence of reproductives is known to inhibit the additional differentiation of same-sex reproductives, while it promotes the differentiation of opposite-sex reproductives. In this study, using the damp-wood termite Hodotermopsis sjostedti, physiological effects of male/female reproductives on the differentiation of supplementary reproductives (neotenics) were examined. The results showed that the only male-neotenic condition, i.e., the presence of male neotenics in the absence of female neotenics, accelerated the neotenic differentiation from female workers (i.e., pseudergates). Under this condition, the rise of juvenile hormone (JH) titer was repressed in females, and the application of a JH analog inhibited the female neotenic differentiation, indicating that the low JH titer leads to rapid differentiation. Thus, the only male-neotenic condition that actively promotes reproductive differentiation by manipulating physiological condition of females is suggested to be a mechanism underlying sexual asymmetry in reproductive function, which may lead the female-biased sex allocation of reproductives.

Evaluation of the population structure and phylogeography of the Japanese Genji firefly, Luciola cruciata, at the nuclear DNA level using RAD-Seq analysis.

The Genji firefly, Luciola cruciata, is widely distributed throughout the major Japanese islands (Honshu, Shikoku, and Kyushu) and distinguished into two ecological types on the basis of the flash interval of the mate-seeking males (4-sec slow-flash or 2-sec fast-flash intervals). The boundary of the ecological types corresponds to the Fossa Magna, a great rupture zone that separates eastern and western Japan. Although the degree of genetic differentiation of the two types has been evaluated using allozyme and mitochondrial DNA sequence data, it has not been evaluated using genome-wide data. Based on the genome-wide data obtained using single-end restriction-site-associated DNA (RAD-Seq), principal component, gene-level phylogenetic tree, admixture, and Wright's fixation index analyses, we identified three phylogenetic groups in L. cruciata: East-Honshu, West-Honshu, and Kyushu. This grouping corresponds to the ecological types: East-Honshu to the slow-flash type and West-Honshu and Kyushu to the fast-flash type. Although introgression was exceptionally observed around adjacent or artificially transplanted areas, gene flow among the groups was almost absent in the natural populations. The phylogenetic tree under the coalescent model also evaluated differentiation among the East-Honshu, West-Honshu and Kyushu groups. Furthermore, because the distribution patterns of the three groups are consistent with the geological history of Japanese islands, a vicariant speciation scenario of L. cruciata is concluded. In addition, we identified genetic markers that can be used to distinguish the three genetic groups for genetic management of firefly transplantation in nature conservation and regeneration.

Termite soldier mandibles are elongated by dachshund under hormonal and Hox gene controls.

In social insects, interactions among colony members trigger caste differentiation with morphological modifications. In termite soldier differentiation, the mandible size considerably increases through two moltings (via the presoldier stage) under the control of juvenile hormone (JH). Regulatory genes are predicted to provide patterning information that induces the mandible-specific cell proliferation. To identify factors responsible for the mandibular enlargement, expression analyses of 18 candidate genes were carried out in the termite Hodotermopsis sjostedti Among those, dachshund (dac), which identifies the intermediate domain along the proximodistal appendage axis, showed mandible-specific upregulation prior to the molt into presoldiers, which can explain the pattern of cell proliferation for the mandibular elongation. Knockdown of dac by RNAi reduced the mandibular length and distorted its morphology. Furthermore, the epistatic relationships among Methoprene tolerant, Insulin receptor, Deformed (Dfd) and dac were revealed by combined RNAi and qRT-PCR analyses, suggesting that dac is regulated by Dfd, downstream of the JH and insulin signaling pathways. Thus, caste-specific morphogenesis is controlled by interactions between the factors that provide spatial information and physiological status.

Life Cycle of the Japanese Green Syllid, Megasyllis nipponica (Annelida: Syllidae): Field Collection and Establishment of Rearing System.

Some polychaete species in the family Syllidae exhibit distinctive life cycles, in which a posterior part of the body of an individual detaches as a reproductive individual called a "stolon". This type of reproductive mode is known as stolonization or schizogamy. Although a number of observations have been reported, and techniques using molecular markers have recently been applied to characterize this phenomenon, little is known about the developmental and physiological mechanisms underlying stolonization. In the present study, Megasyllis nipponica, a common syllid species distributed throughout Japan, is proposed as a model to reveal the developmental and physiological mechanism of stolonization, and the rearing system to maintain it in laboratory conditions is described. This species was repeatedly sampled around Hokkaido, where more dense populations were found from August to October. The animals were maintained in the laboratory under stable long-day condition (20°C, 16L:8D), and fed mainly with spinach powder. Stolonization processes, spawning, embryonic and postembryonic development were observed and documented, and the required period of time for each developmental stage was recorded. The complete generation time was around two months under the rearing condition. The information provided is valuable to maintain this and other syllid species in the laboratory, and hence contributes to the establishment of new evolutionary and developmental research lines in this group of annelids.

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.

The evolutionary history of termites as inferred from 66 mitochondrial genomes.

Termites have colonized many habitats and are among the most abundant animals in tropical ecosystems, which they modify considerably through their actions. The timing of their rise in abundance and of the dispersal events that gave rise to modern termite lineages is not well understood. To shed light on termite origins and diversification, we sequenced the mitochondrial genome of 48 termite species and combined them with 18 previously sequenced termite mitochondrial genomes for phylogenetic and molecular clock analyses using multiple fossil calibrations. The 66 genomes represent most major clades of termites. Unlike previous phylogenetic studies based on fewer molecular data, our phylogenetic tree is fully resolved for the lower termites. The phylogenetic positions of Macrotermitinae and Apicotermitinae are also resolved as the basal groups in the higher termites, but in the crown termitid groups, including Termitinae + Syntermitinae + Nasutitermitinae + Cubitermitinae, the position of some nodes remains uncertain. Our molecular clock tree indicates that the lineages leading to termites and Cryptocercus roaches diverged 170 Ma (153-196 Ma 95% confidence interval [CI]), that modern Termitidae arose 54 Ma (46-66 Ma 95% CI), and that the crown termitid group arose 40 Ma (35-49 Ma 95% CI). This indicates that the distribution of basal termite clades was influenced by the final stages of the breakup of Pangaea. Our inference of ancestral geographic ranges shows that the Termitidae, which includes more than 75% of extant termite species, most likely originated in Africa or Asia, and acquired their pantropical distribution after a series of dispersal and subsequent diversification events.

Intracolonial genetic variation affects reproductive skew and colony productivity during colony foundation in a parthenogenetic termite.

BACKGROUND: In insect societies, intracolonial genetic variation is predicted to affect both colony efficiency and reproductive skew. However, because the effects of genetic variation on these two colony characteristics have been tested independently, it remains unclear whether they are affected by genetic variation independently or in a related manner. Here we test the effect of genetic variation on colony efficiency and reproductive skew in a rhinotermitid termite, Reticulitermes speratus, a species in which female-female pairs can facultatively found colonies. We established colonies using two types of female-female pairs: colonies founded by sisters (i.e., sister-pair colonies) and those founded by females from different colonies (i.e., unrelated-pair colonies). Colony growth and reproductive skew were then compared between the two types of incipient colonies. RESULTS: At 15 months after colony foundation, unrelated-pair colonies were larger than sister-pair colonies, although the caste ratio between workers and nymphs, which were alternatively differentiated from young larvae, did not differ significantly. Microsatellite DNA analyses of both founders and their parthenogenetically produced offspring indicated that, in both sister-pair and unrelated-pair colonies, there was no significant skew in the production of eggs, larvae, workers and soldiers. Nymph production, however, was significantly more skewed in the sister-pair colonies than in unrelated-pair colonies. Because nymphs can develop into winged adults (alates) or nymphoid reproductives, they have a higher chance of direct reproduction than workers in this species. CONCLUSIONS: Our results support the idea that higher genetic variation among colony members could provide an increase in colony productivity, as shown in hymenopteran social insects. Moreover, this study suggests that low genetic variation (high relatedness) between founding females increases reproductive skew via one female preferentially channeling her relatives along the reproductive track. This study thus demonstrated that, in social insects, intracolonial genetic variation can simultaneously affect both colony efficiency and reproductive skew.

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, Reticulitermes speratus and Nasutitermes takasagoensis. 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.