Toshihisa Yashiro

Queen Succession Through Asexual Reproduction in Termites

The evolution and maintenance of sexual reproduction may involve important tradeoffs because asexual reproduction can double an individuals contribution to the gene pool but reduces diversity. Moreover, in social insects the maintenance of genetic diversity among workers may be important for colony growth and survival. We identified a previously unknown termite breeding system in which both parthenogenesis and sexual reproduction are conditionally used. Queens produce their replacements asexually but use normal sexual reproduction to produce other colony members. These findings show how eusociality can lead to extraordinary reproductive systems and provide important insights into the advantages and disadvantages of sex.

The antibacterial protein lysozyme identified as the termite egg recognition pheromone

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus ‘termite-ball’ and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites.

Seasonal patterns of egg production in field colonies of the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

This is the first report on the annual egg production patterns in mature termite colonies in the field. Data on the seasonal patterns of egg production in field colonies are very important for understanding the annual colony growth schedule, resource allocation, and population dynamics of the termites. However, collecting the eggs from a sufficient number of colonies is extremely difficult in Reticulitermes termites because their multiple-site nesting makes it difficult to find the reproductive center of the colonies. Here, we first show the seasonal pattern of egg production in the subterranean termite Reticulitermes speratus by collecting the reproductive center of ten colonies each month from April through October. We had to destructively examine dozens of nests to find eggs from enough field colonies each month. Mature field colonies began to produce eggs in late May, soon after the swarming season, and the egg production rate (EPR) reached its maximum in early July. The eggs hatched until late October. The EPR was significantly correlated with the average monthly temperature. Additional investigation of the egg distributions in the nests showed that most eggs were kept around the royal cell, which contained the reproductives. The largest colony had 109 supplemental queens and 94,023 eggs, suggesting that each queen produced an average of 24.7 eggs per day, based on the known mean hatching period of an inseminated egg of 34.95±0.12 (SE) days.

Distribution and Phylogenetic Analysis of Termite Egg-Mimicking Fungi “Termite Balls” in Reticulitermes Termites

Abstract To understand the evolution of parasitism, a fundamental question is what influences the distribution of globally distributed parasites among different hosts. Reticulitermes termites often harbor brown fungal balls, called “termite balls,” alongside their eggs. The termite ball was identified as the sclerotium of a parasitic corticioid fungus, an undescribed species of genus Fibularhizoctonia, which mimics termite eggs physically and chemically so as to be protected in termite nests. This phenomenon has been found for Reticulitermes species in Japan and the United States, but not all the Reticulitermes species in these countries have termite balls. To identify factors influencing the distribution of termite balls among their host species, we conducted wide-range sampling in the temperate zone and in subtropical zones in Japan and Taiwan. We also conducted phylogenetic analyses of termite ball fungi and their host termites based on molecular data. In Amami-Oshima Island, R. amamianus Morimoto colonies had termite balls only in temperate highland regions, but no termite balls were found in its subtropical lowland region. The introduced termite R. kanmonensis Takematsu had termite balls as well as sympatrically occurring R. speratus (Kolbe). The termite ball fungi isolated from these two species showed no significant molecular difference, suggesting no host race formation. Phylogenetic analysis showed that R. kanmonensis in Japan did not diverge from R. flaviceps (Oshima), which has no termite balls, in Taiwan. Analyses of termite ball distribution among nine Reticulitermes species suggested that climate is the most important factor restricting the distribution of the termite ball fungi.

Termite queens close the sperm gates of eggs to switch from sexual to asexual reproduction

Significance To clarify the evolution of parthenogenesis, given the potential sexual conflict over genetic transmission, identifying the mechanism regulating egg fertilization in females is essential. In the termite Reticulitermes speratus, queens produce their replacements (neotenic queens) parthenogenetically but use sexual reproduction to produce other colony members. We discovered that queens of the termite close micropyles (openings for sperm entry) of their eggs to produce parthenogenetic offspring in the presence of kings. Furthermore, we found that queens control the proportion of micropyleless eggs by regulating of the number of micropyles over time. This study describes a novel route of the evolution of parthenogenesis in favor of females’ interests without interference from males. Males and females are in conflict over genetic transmission in the evolution of parthenogenesis, because it enhances female reproductive output but deprives the males’ genetic contribution. For males, any trait that coerces females into sexual reproduction should increase their fitness. However, in the termite Reticulitermes speratus, queens produce their replacements (neotenic queens) parthenogenetically while using normal sexual reproduction to produce other colony members. Here, we show that termite queens produce parthenogenetic offspring in the presence of kings by closing the micropyles (sperm gates; i.e., openings for sperm entry) of their eggs. Our field survey showed that termite eggs show large variation in numbers of micropyles, with some having none. Microsatellite analysis showed that embryos of micropyleless eggs develop parthenogenetically, whereas those of eggs with micropyles are fertilized and develop sexually. Surveys of eggs among queens of different age groups showed that queens begin to lay micropyleless eggs when they are older and thus, need to produce their replacements parthenogenetically. In addition, we found clear seasonality in new neotenic queen differentiation and micropyleless egg production. This micropyle-dependent parthenogenesis is the first identification, to our knowledge, of the mechanism through which females control egg fertilization over time in diploid animals, implying a novel route of the evolution of parthenogenesis in favor of female interests without interference from males.

Genetic diversity of termite-egg mimicking fungi “termite balls” within the nests of termites

Antagonistic or mutualistic interactions between insects and fungi are well-known, and the mutualistic interactions of fungus-growing ants, fungus-growing termites, and fungus-gardening beetles with their respective fungal mutualists are model examples of coevolution. However, our understanding of coevolutionary interactions between insects and fungi has been based on a few model systems. Fungal mimicry of termite eggs is one of the most striking evolutionary consequences of insect–fungus associations. This novel termite–fungus interaction is a good model system to compare with the relatively well-studied systems of fungus-growing ants and termites because termite egg-mimicking fungi are protected in the nests of social insects, as are fungi cultivated by fungus-growing ants and termites. Recently, among systems of fungus-growing ants and termites, much attention has been focused on common factors including monoculture system for the ultimate evolutionary stability of mutualism. We examined the genetic diversity of termite egg-mimicking fungi within host termite nests. RFLP analysis demonstrated that termite nests were often infected by multiple strains of termite egg-mimicking fungi, in contrast to single-strain monocultures in fungus combs of fungus-growing ants and termites. Additionally, phylogenetic analyses indicated the existence of a free-living stage of the termite egg-mimicking fungus as well as frequent long-distance gene flow by spores and subsequent horizontal transmission. Comparisons of these results with previous studies of fungus-growing ants and termites suggest that the level of genetic diversity of fungal symbionts within social insect nests may be important in shaping the outcome of the coevolutionary interaction, despite the fact that the mechanism for achieving genetic diversity varies with the evolutionary histories of the component species.

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.

Male same-sex pairing as an adaptive strategy for future reproduction in termites

A wide variety of animals display same-sex behaviours, including courtship, copulation and pairing. However, these behaviours create a paradox, as selection seemingly acts on maladaptive traits, and they have often been regarded as cases of mistaken identity, especially in invertebrates. We show that termite males show nest establishment and pairing formation that usually occur in monogamous colony foundation and demonstrate how this contributes to their fitness. We found that pairs of male dealates stopped searching for females and established nests without females, although single males rarely ceased searching for mates. Males in these male–male pairings had much higher survival than single males. Our colony fusion experiment showed that a male in a surviving same-sex pair can replace a male in an incipient colony and produce offspring. A mathematical model demonstrated that the observed strategy of establishing a male–male pairing instead of searching for females is advantageous when the risk of predation is high, even when colony fusion is very rare. These results indicate that, under certain ecological conditions, a cooperative same-sex pairing with a potential rival for reproduction can be adaptive. Our study implies the existence of various possibilities for explaining the adaptive significance of same-sex sexual behaviours.

Loss of males from mixed-sex societies in termites

BackgroundSexual 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.ResultsHere, 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.ConclusionsOur 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 new species of Trichogramma (Hymenoptera: Trichogrammatidae) parasitic on eggs of the alderfly Sialis melania (Neuroptera: Sialidae) from Japan, with comments on its phylogeny and male wing polymorphism

A new species of Trichogramma that parasitizes Sialis melania eggs is described as Trichogramma tajimaense Yashiro, Hirose and Honda, sp. nov. from Japan. Its phylogenetic position is based on a DNA‐based analysis, and data regarding its male wing polymorphism are also presented. The view that T. tajimaense is closely related to T. semblidis, another parasitoid of Sialis eggs, is supported by the results of a phylogenetic analysis, as well as by the biological and morphological similarities between both species. Trichogramma tajimaense is also similar in male wing polymorphism to T. kurosuae, a gregarious egg parasitoid of the lepidopteran Ivela auripes, as both Trichogramma species exhibit male wing trimorphism (fully alate, brachypterous and apterous forms) in contrast to the male wing dimorphism (fully alate and apterous forms) of T. semblidis. However, no phylogenetic analysis reveals a close relationship between T. tajimaense and T. kurosuae, and a difference exists between these two species in the mean percentage of flightless (brachypterous and apterous) males that emerge from a host egg mass; 96% of T. tajimaense males are incapable of flight, whereas about 50% of T. kurosuae males are flightless. Because all or almost all males of T. semblidis parasitizing Sialis eggs are apterous, T. tajimaense is more similar to T. semblidis than to T. kurosuae in the proportion of flightless males. In addition, male wing polymorphisms of Trichogramma in relation to mating systems could also show a similarity between T. tajimaense and T. semblidis when considering both species as quasi‐gregarious parasitoids of Sialis eggs.