Phylogeographical evidence for historical long-distance dispersal in the flightless stick insect Ramulus mikado.

Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect Ramulus mikado across Japan, based on a multifaceted molecular approach [cytochrome oxidase subunit I (COI) haplotypes, nuclear simple sequence repeat markers and genome-wide single nucleotide polymorphisms]. Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.

Cell type specific polyploidization in the royal fat body of termite queens.

Tissue-specific endopolyploidy is widespread among plants and animals and its role in organ development and function has long been investigated. In insects, the fat body cells of sexually mature females produce substantial amounts of egg yolk precursor proteins (vitellogenins) and exhibit high polyploid levels, which is considered crucial for boosting egg production. Termites are social insects with a reproductive division of labor, and the fat bodies of mature termite queens exhibit higher ploidy levels than those of other females. The fat bodies of mature termite queens are known to be histologically and cytologically specialized in protein synthesis. However, the relationship between such modifications and polyploidization remains unknown. In this study, we investigated the relationship among cell type, queen maturation, and ploidy levels in the fat body of the termite Reticulitermes speratus. We first confirmed that the termite fat body consists of two types of cells, that is, adipocytes, metabolically active cells, and urocytes, urate-storing cells. Our ploidy analysis using flow cytometry has shown that the fat bodies of actively reproducing queens had more polyploid cells than those of newly emerged and pre-reproductive queens, regardless of the queen phenotype (adult or neotenic type). Using image-based analysis, we found that not urocytes, but adipocytes became polyploid during queen differentiation and subsequent sexual maturation. These results suggest that polyploidization in the termite queen fat body is associated with sexual maturation and is regulated in a cell type-specific manner. Our study findings have provided novel insights into the development of insect fat bodies and provide a basis for future studies to understand the functional importance of polyploidy in the fat bodies of termite queens.

Discovery of an underground chamber to protect kings and queens during winter in temperate termites.

Overwintering is a critical part of the annual cycle for species that live in temperate, polar, and alpine regions. Consequently, low-temperature biology is a key determinant of temperate species distribution. Termites are distributed predominantly in tropical regions, and a limited number of species are found in the temperate zone. Here, in the termite Reticulitermes speratus, we report the discovery of an underground chamber that protects kings and queens to survive the winter, which is separate from the one they used during the warmer breeding season. In the spring, the royals inhabited decayed logs on the ground, then moved to their underground chamber located in the roots of stumps in the fall. The winter minimum temperature measured in the royal chamber was higher than that in the logs on the ground. In overwintering termites, the kings and queens had higher cold tolerance than workers and soldiers. Air temperatures dropped below the critical temperature multiple times, as evidenced from the past 140 years of weather records in Kyoto. These results demonstrated the survival strategies of reproductives to overcome the environment at the latitudinal limits. This study helps further the understanding of the termite's seasonal phenology, long-term survivorship, and life cycle.

Genome analysis of "Candidatus Aschnera chinzeii," the bacterial endosymbiont of the blood-sucking bat fly Penicillidia jenynsii (Insecta: Diptera: Nycteribiidae).

Insect-microbe endosymbiotic associations are omnipresent in nature, wherein the symbiotic microbes often play pivotal biological roles for their host insects. In particular, insects utilizing nutritionally imbalanced food sources are dependent on specific microbial symbionts to compensate for the nutritional deficiency via provisioning of B vitamins in blood-feeding insects, such as tsetse flies, lice, and bedbugs. Bat flies of the family Nycteribiidae (Diptera) are blood-sucking ectoparasites of bats and shown to be associated with co-speciating bacterial endosymbiont "Candidatus Aschnera chinzeii," although functional aspects of the microbial symbiosis have been totally unknown. In this study, we report the first complete genome sequence of Aschnera from the bristled bat fly Penicillidia jenynsii. The Aschnera genome consisted of a 748,020 bp circular chromosome and a 18,747 bp circular plasmid. The chromosome encoded 603 protein coding genes (including 3 pseudogenes), 33 transfer RNAs, and 1 copy of 16S/23S/5S ribosomal RNA operon. The plasmid contained 10 protein coding genes, whose biological function was elusive. The genome size, 0.77 Mbp, was drastically reduced in comparison with 4-6 Mbp genomes of free-living γ-proteobacteria. Accordingly, the Aschnera genome was devoid of many important functional genes, such as synthetic pathway genes for purines, pyrimidines, and essential amino acids. On the other hand, the Aschnera genome retained complete or near-complete synthetic pathway genes for biotin (vitamin B7), tetrahydrofolate (vitamin B9), riboflavin (vitamin B2), and pyridoxal 5'-phosphate (vitamin B6), suggesting that Aschnera provides these vitamins and cofactors that are deficient in the blood meal of the host bat fly. Similar retention patterns of the synthetic pathway genes for vitamins and cofactors were also observed in the endosymbiont genomes of other blood-sucking insects, such as Riesia of human lice, Arsenophonus of louse flies, and Wigglesworthia of tsetse flies, which may be either due to convergent evolution in the blood-sucking host insects or reflecting the genomic architecture of Arsenophonus-allied bacteria.

Ploidy dynamics in aphid host cells harboring bacterial symbionts.

Aphids have evolved bacteriocytes or symbiotic host cells that harbor the obligate mutualistic bacterium Buchnera aphidicola. Because of the large cell size (approximately 100 µm in diameter) of bacteriocytes and their pivotal role in nutritional symbiosis, researchers have considered that these cells are highly polyploid and assumed that bacteriocyte polyploidy may be essential for the symbiotic relationship between the aphid and the bacterium. However, little is known about the ploidy levels and dynamics of aphid bacteriocytes. Here, we quantitatively analyzed the ploidy levels in the bacteriocytes of the pea-aphid Acyrthosiphon pisum. Image-based fluorometry revealed the hyper polyploidy of the bacteriocytes ranging from 16- to 256-ploidy throughout the lifecycle. Bacteriocytes of adult parthenogenetic viviparous females were ranged between 64 and 128C DNA levels, while those of sexual morphs (oviparous females and males) were comprised of 64C, and 32-64C cells, respectively. During post-embryonic development of viviparous females, the ploidy level of bacteriocytes increased substantially, from 16 to 32C at birth to 128-256C in actively reproducing adults. These results suggest that the ploidy levels are dynamically regulated among phenotypes and during development. Our comprehensive and quantitative data provides a foundation for future studies to understand the functional roles and biological significance of the polyploidy of insect bacteriocytes.

Enhanced heterozygosity from male meiotic chromosome chains is superseded by hybrid female asexuality in termites.

Although males are a ubiquitous feature of animals, they have been lost repeatedly in diverse lineages. The tendency for obligate asexuality to evolve is thought to be reduced in animals whose males play a critical role beyond the contribution of gametes, for example, via care of offspring or provision of nuptial gifts. To our knowledge, the evolution of obligate asexuality in such species is unknown. In some species that undergo frequent inbreeding, males are hypothesized to play a key role in maintaining genetic heterozygosity through the possession of neo-sex chromosomes, although empirical evidence for this is lacking. Because inbreeding is a key feature of the life cycle of termites, we investigated the potential role of males in promoting heterozygosity within populations through karyotyping and genome-wide single-nucleotide polymorphism analyses of the drywood termite Glyptotermes nakajimai We showed that males possess up to 15 out of 17 of their chromosomes as sex-linked (sex and neo-sex) chromosomes and that they maintain significantly higher levels of heterozygosity than do females. Furthermore, we showed that two obligately asexual lineages of this species-representing the only known all-female termite populations-arose independently via intraspecific hybridization between sexual lineages with differing diploid chromosome numbers. Importantly, these asexual females have markedly higher heterozygosity than their conspecific males and appear to have replaced the sexual lineages in some populations. Our results indicate that asexuality has enabled females to supplant a key role of males.

Development of microsatellite markers for the geographically parthenogenetic stick insect Phraortes elongatus (Insecta: Phasmatodea).

Many plant and animal species exhibit geographic parthenogenesis, wherein unisexual (= parthenogenetic) lineages are more common in their marginal habitats such as high latitude or altitudes than their closely related bisexual counterparts. The Japanese stick insect, Phraortes elongatus (Thunberg) (Insecta: Phasmatodea), is known as a geographically parthenogenetic species due to the existence of both bisexual and unisexual populations. Here, we developed microsatellite markers to infer the genetic variation among populations of P. elongatus. Totally, 13 primer pairs were developed for the species, and they were tested on 47 samples collected from both a bisexual population and a unisexual population. All 13 loci were polymorphic in the bisexual population, whereas no loci were polymorphic in the unisexual population. The loss of variation in the unisexual population implies automixis with terminal fusion or gamete duplication as the mode of parthenogenesis. The markers developed in this study will be helpful for further comprehensive analysis of the genetic diversity and gene flow between bisexual and parthenogenetic lineages of P. elongatus.

Oocyte resorption in termite queens: Seasonal dynamics and controlling factors.

Female insects can resorb their oocytes that could not be oviposited. Oocyte resorption is proposed to be an adaptive mechanism to optimize fitness in hostile environments, recouping resources that might otherwise be lost. Social insects have developed reproductive division of labor, wherein a small number of queens are devoted to egg production. Matured queens are highly specialized in reproduction and are largely dependent on nestmate workers for their nourishment. Therefore, oocyte resorption in the queens should be influenced by social factors such as the amount of available workforce, as well as external and abiotic factors. In this study, we investigated the seasonal dynamics and regulation factors of oocyte resorption in actively reproducing termite queens. We continuously collected the field-nests of the subterranean termite Reticulitermes speratus and demonstrated that queens frequently resorbed their oocytes in late summer, even though it is one of the most productive seasons in this species. On the other hand, our laboratory experiment showed that oocyte resorption itself was strongly induced regardless of the season. We also found that the rate of oocyte resorption was influenced by colony size (the number of attending workers). These results suggest that termite queens seasonally resorb their oocytes, yet oocyte resorption itself is regulated by social factors rather than by seasonal factors. Our study provides a unique insight into the regulation of reproduction in social insects.

Transcriptomic analysis of epigenetic modification genes in the termite Reticulitermes speratus.

Eusocial insects display a caste system in which different castes are morphologically and physiologically specialized for different tasks. Recent studies have revealed that epigenetic modifications, including DNA methylation and histone modification, mediate caste determination and differentiation, longevity, and polyethism in eusocial insects. Although there has been a growing interest in the relationship between epigenetic mechanisms and phenotypic plasticity in termites, there is little information about differential expression levels among castes and expression sites for these genes in termites. Here we show royal-tissue-specific expression of epigenetic modification genes in the termite Reticulitermes speratus. Using RNA-seq, we identified 74 genes, including three DNA methyltransferases, seven sirtuins, 48 Trithorax group proteins, and 16 Polycomb group proteins. Among these genes, 15 showed king-specific expression, and 52 showed age-dependent differential expression in kings and queens. Quantitative real-time PCR revealed that DNA methyltransferase 3 is expressed specifically in the king's testis and fat body, whereas some histone modification genes are remarkably expressed in the king's testis and queen's ovary. These findings imply that epigenetic modification plays important roles in the gamete production process in termite kings and queens.

Evolutionary relationship of fat body endoreduplication and queen fecundity in termites.

Endoreduplication or nuclear genome replication without cell division is widely observed in the metabolically active tissues of plants and animals. The fat body cells of adult female insects produce abundant yolk proteins and become polyploid, which is assumed to accelerate egg production. Recently, it was reported that in termites, endopolyploidy in the fat body occurs only in queens but not in the other females; however, the relationship between the fecundity and ploidy level in the fat body remains unclear. Termite queens exhibit a huge variation in their egg-producing capacity among different species; queens in the species with a foraging lifestyle, in which workers leave the nest to forage outside, are much more fecund than those in the species living in a single piece of wood. In this study, we conducted ploidy analyses on three foragings and three wood-dwelling termites via flow cytometry. In all the species, the fat body of queens contained significantly more polyploid cells than that of other nonreproductive females, considering their body size effect. However, the male fat body, which is not involved in yolk production, did not show consistency in polyploid cell numbers among the species studied. Moreover, highly fecund queens in foraging termites exhibit higher levels of endopolyploidy in their fat body than those with less fecundity in wood-dwelling termites. These results suggest that endopolyploidy in the fat body of termite queens can boost their egg production, and the level of endopolyploidy in their fat body is linked to their fecundity. Our study provides a novel insight into the evolutionary relationship between endoreduplication and caste specialization in social insects.

High expression of the breast cancer susceptibility gene BRCA1 in long-lived termite kings.

Aging is associated with the accumulation of DNA damage. High expression of DNA repair genes has been suggested to contribute to prolonged lifespan in several organisms. However, the crucial DNA repair genes contributing to longevity remain unknown. Termite kings have an extraordinary long lifespan compared with that of non-reproductive individuals such as workers despite being derived from the same genome, thus providing a singular model for identifying longevity-related genes. In this study, we demonstrated that termite kings express higher levels of the breast cancer susceptibility gene BRCA1 than other castes. Using RNA sequencing, we identified 21 king-specific genes among 127 newly annotated DNA repair genes in the termite Reticulitermes speratus. Using quantitative PCR, we revealed that some of the highly expressed king-specific genes were significantly upregulated in reproductive tissue (testis) compared to their expression in somatic tissue (fat body). Notably, BRCA1 gene expression in the fat body was more than 4-fold higher in kings than in workers. These results suggest that BRCA1 partly contributes to DNA repair in somatic and reproductive tissues in termite kings. These findings provide important insights into the linkage between BRCA1 gene expression and the extraordinary lifespan of termite kings.

Loss of males from mixed-sex societies in termites.

BACKGROUND: Sexual reproduction is the norm in almost all animal species, and in many advanced animal societies, both males and females participate in social activities. To date, the complete loss of males from advanced social animal lineages has been reported only in ants and honey bees (Hymenoptera), whose workers are always female and whose males display no helping behaviors even in normal sexual species. Asexuality has not previously been observed in colonies of another major group of social insects, the termites, where the ubiquitous presence of both male and female workers and soldiers indicate that males play a critical role beyond that of reproduction. RESULTS: Here, we report asexual societies in a lineage of the termite Glyptotermes nakajimai. We investigated the composition of mature colonies from ten distinct populations in Japan, finding six asexual populations characterized by a lack of any males in the reproductive, soldier, and worker castes of their colonies, an absence of sperm in the spermathecae of their queens, and the development of unfertilized eggs at a level comparable to that for the development of fertilized eggs in sexual populations of this species. Phylogenetic analyses indicated a single evolutionary origin of the asexual populations, with divergence from sampled sexual populations occurring about 14 million years ago. Asexual colonies differ from sexual colonies in having a more uniform head size in their all-female soldier caste, and fewer soldiers in proportion to other individuals, suggesting increased defensive efficiencies arising from uniform soldier morphology. Such efficiencies may have contributed to the persistence and spread of the asexual lineage. Cooperative colony foundation by multiple queens, the single-site nesting life history common to both the asexual and sexual lineages, and the occasional development of eggs without fertilization even in the sexual lineage are traits likely to have been present in the ancestors of the asexual lineage that may have facilitated the transition to asexuality. CONCLUSIONS: Our findings demonstrate that completely asexual social lineages can evolve from mixed-sex termite societies, providing evidence that males are dispensable for the maintenance of advanced animal societies in which they previously played an active social role.

A Genomic Imprinting Model of Termite Caste Determination: Not Genetic but Epigenetic Inheritance Influences Offspring Caste Fate.

Eusocial insects exhibit the most striking example of phenotypic plasticity. There has been a long controversy over the factors determining caste development of individuals in social insects. Here we demonstrate that parental phenotypes influence the social status of offspring not through genetic inheritance but through genomic imprinting in termites. Our extensive field survey and genetic analysis of the termite Reticulitermes speratus show that its breeding system is inconsistent with a genetic caste determination model. We therefore developed a genomic imprinting model, in which queen- and king-specific epigenetic marks antagonistically influence sexual development of offspring. The model accounts for all known empirical data on caste differentiation of R. speratus and other related species. By conducting colony-founding experiments and additively incorporating relevant socio-environmental factors into our genomic imprinting model, we show the relative importance of genomic imprinting and environmental factors in caste determination. The idea of epigenetic inheritance of sexual phenotypes solves the puzzle of why parthenogenetically produced daughters carrying only maternal chromosomes exclusively develop into queens and why parental phenotypes (nymph- or worker-derived reproductives) strongly influence caste differentiation of offspring. According to our model, the worker caste is seen as a "neuter" caste whose sexual development is suppressed due to counterbalanced maternal and paternal imprinting and opens new avenues for understanding the evolution of caste systems in social insects.

The first report of gynandromorphy in termites (Isoptera; Kalotermitidae; Neotermes koshunensis).

This is the first report of gynandromorphy in Isoptera. An Asian dry-wood termite, Neotermes koshunensis (Shiraki) [Kalotermitidae], possessing both male and female phenotypic characteristics, was found on Okinawa Island, Japan. This deformed individual showed morphological and anatomical hermaphroditism in the abdomen. The right side of the seventh sternite was the female form and contained an ovary, while the left side was the male form and contained a testis. Genotypic analysis revealed that this individual was a genotypic bilateral chimera. These results suggested that the termite was a bilateral gynandromorph with a male left side and a female right side. As reported previously in other insects, double fertilization (by two sperms, one with an X and one with a Y chromosome) of a binucleate egg is the most likely mechanism that generated this genotypic bilateral chimera. N. koshunensis has the ability to reproduce through parthenogenesis, in which the secondary polar body is likely to be used for nuclear phase recovery. If the second polar body in this mechanism has high fertility and healthy embryogenic potential, like an egg nucleus, some of gynandromorphs might be produced by a side effect of parthenogenetic ability.