Our research focuses on the social system and evolution of termites. We are primarily interesting in understanding the reproductive mechanism and evolution of chemical communication and defensive strategies against various parasites and predators. We have been characterizing the molecular, physiological, behavioral, and ecological factors that regulate these processes. On the basis of these basic findings of termite biology, we are also developing a novel technology to control termites most effectively by using their social behavior.
■Asexual Queen Succession (AQS)
The Japanese subterranean termite Reticulitermes speratus is classified as multiple-site nesters based on feeding and nesting habits, whereby nests of a single colony are interconnected by belowground tunnels and aboveground shelter tubes (the tunnel made of wood pieces, soil and termite excretions). In this type of termites, each colony has cryptic nesting habits with a transient, hidden royal chamber found underground or deep inside wood, making it difficult to reliably collect kings and queens.
In previous studies, the breeding system of subterranean termites has been estimated by genotyping workers or culturing laboratory colonies rather than from census data from field colonies. And it was long believed that the inbreeding cycles of generations of neotenic reproductives (fertile individuals newly differentiated in the colony) propagated the colony after the death of the primary king and queen.
However, microsatellite genotyping on wild kings and queens revealed that secondary queens have only the genes derived from a primary queen. In other words, a primary queen produces secondary queens by using parthenogenesis, but uses normal sexual reproduction to produce other colony members (workers, soldiers and alates). Moreover, it is revealed that a primary king lives much longer than a primary queen.
This amazing reproduction system was named asexually queen succession (AQS), and it was clarified for the first time in 2009 by Matuura et al.. This system permits termites not only to avoid inbreeding in the colony but also to increase the number of queens, resulting in producing a large number of eggs. Because supplemental queens have only the genes from the former queens, the genetic diversity of workers, soldiers and alates will be maintained.
(Matsuura et al. 2009, Science)
■Micropyle-dependent Parthenogenesis in Termite
Many animals on the planet propagate sexually by fertilization between an oocyte and a sperm. However, sexual reproduction has lower proliferation efficiency than parthenogenesis because sexual reproduction must produce the males that do not lay eggs. Also, parthenogenesis is an efficient reproductive system in increasing the females’ gene contribution. Therefore, the near ubiquity of sexual reproduction is one of the most enduring puzzles in evolutionary biology.
One factor that contributes to the difficulty of evolution of parthenogenesis may be the males’ enforced fertilization. In diploid insects, it has been considered that the regulation of fertilization by females is impossible, because eggs are automatically fertilized by sperms pumped out from spermatheca at the time of oviposition.
On the surface of insect eggs, there are the pores that permit sperms to enter in egg (micropyle). Observing micropyles of a total of 6,000 eggs in the Japanese subterranean termite R. speratus, we revealed that the number of micropyles varies by egg, and some eggs have no micropyle. Comparing micorsatellite genotypes of the embryos of eggs with and without micropyles, the embryos of micropyleless eggs had only matternal alleles, indicating parthenogenetic development, whereas those of eggs that had micropyles (even only a singe micropyle) had both paternal and maternal alleles, indicating sexual development. Moreover, it is also revealed that the number of micropyles varies according to the age of queen, and older queens lay more micropyleless eggs than younger queens. In addition, queens regulate the number of micropyles seasonally. That is, although queens of R. speratus produce alates, soldiers and workers by sexual reproduction, they produce micropyleless eggs before their deaths, and only such eggs develop parthenogenetically and can differentiate into supplemental queens.
This micropyle-dependent parthenogenesis is the first identification of mechanism through which females control egg fertilization over time in diploid animals, implying a novel route of the evolution of parthenogenesis in favor of females’ interests without interference from males.
(Yashiro and Matsuura 2014, PNAS)
(Above) Electron microscope picture of an egg. There are the sperm gates (micropyle) on the egg surface. (Bottom) The number of micropyles. Some eggs have no micropyle (MP: micropyle).
■Kin selection in Termites
■Termite Queen Pheromone
The division of labor is one of the most prominent features of colony behavior in termites; reproduction is primarily monopolized by queens, whereas workers specialized in the other tasks required for colony growth and survival. When queens are dead or their ability of egg production is not to be equal to the labor power of workers, other female individuals differentiate into supplemental queens and start reproduction in place of old queens. Conversely, it means that reining queens inhibit the differentiation of other females into new queens using some kind of method. Although volatile pheromones produced by reining queens have long been believed to be the prime factor inhibiting the differentiation into new queens, it has not been identified for more than five decades because of an extremely small amount of the pheromone and the difficulty of collecting wild mature queens.
However, we succeed the identification of the volatile queen pheromone in R. speratus and revealed it is composed of n-butyl-n-butyrate and 2-metheyl-1-butanol in a 2:1 ratio. And an artificial pheromone blend consisting of these two compounds had a strong inhibitory effect similar to living queens. Moreover, the same compounds are also emitted by eggs, acting both as an attractant to workers and an inhibitor of new queen differentiation (Matsuura et al. 2010, PNAS).
Recently, it has been revealed that this volatile queen pheromone has the promoting effect on the salivary lysozyme production of workers (Suehiro and Matsuura 2015, Insectes Sociaux) and the antifungal activities (Matsuura and Matsunaga 2015, Ecological Research). So the multifunctional roles of termite queen pheromone continue to be become clear.
■Termite Egg Recognition Pheromone
Termite workers recognize the eggs laid by queens and pile these eggs in nursery cells to care for them. Workers groom eggs frequently and smear their saliva on the egg surfaces to protect them from desiccation and pathogenic infection. Termites recognize their eggs based on the morphology (shape, size and surface texture) and the chemical. Therefore, the identification of the pheromone evoking the egg-piling and -protection behaviors is very important not only to understand the evolution of social behaviors but also to develop termite control agents.
Using the MALDI-TOF MS analysis, we revealed that the termite egg recognition pheromone (TERP) consists of lysozyme (the protein decomposing the bacterial cell wall), and eggs start to express the lysozyme gene when they are in the queen ovary.
(Matsuura et al. 2007, PLOS ONE)
Egg grooming of R. speratus
Workers transporting glass beads coated with egg extract
Because the TERP induces the egg-piling behavior strongly, it enables the dummy eggs to be transported by workers to the center of termite nest. Therefore, if the termite pesticide is enclosed with the dummy eggs, we can effectively destroy the center of termite nest. To realize this innovative technology, our laboratory progress with that development in collaboration with public sector.
■The Evolution of Termite Ball
Termite workers transport eggs to nursery cells and smear their saliva containing antimicrobial agents. Such egg-protection behavior is important and basic social behavior because eggs cannot live without grooming by workers. However, in nursery cells, some brown spheres (not eggs) are often found in egg piles.
These brown spheres are the sclerotia of atheloid fungi of genus Fibularhizoctonia, and they are called “termite balls.” Surprisingly, termite balls mimic termite eggs both chemically by producing the β-glucosidase (another component of TERP) and morphologically by making sclerotia with diameters that exactly match termite egg size and curvature (Matsuura et al. 2009).
(Left) Termite balls transported into the egg pile. (Right) Propagation of termite balls in the termite nest.
Fluorescence detection of β-glucosidase Responses of β-glucosidase were detected in salivary gland (a,e), egg (b,f), hindgut (c,g) of termites, and termite balls (d,h). The eggs of Argentine ant were used as control (bottom-left in b, f).
Termite balls generally exist in the colony of Reticulitermes termites, and they are widespread in Japan and USA (Matsuura et al. 2000, 2005; Yashiro and Matsuura 2007, 2009; Yashiro et al. 2011). Also, the new species of termite ball has been recently found in the nest of higher termite Nasutitermes takasagoensis in Iriomote Island (Matsuura and Yashiro 2010). Our laboratory advance research related to the life cycle of termite balls.
■Caste-specific Expression of Termite Chemoreceptors
Eusocial insects have the specialized behavioral and morphological groups within a colony, which are also called castes, and individuals from each caste engage in caste-specific tasks. In termite societies, the king and queen concentrate on reproduction, soldiers defend their colony, and workers take charge of multiple tasks such as foraging, hygiene management and caring for queen and broods. Termite castes consist of both sexes, and both male and female are engaged in works together. Accordingly, it is predicted that different castes (or sexes) have different repertories of chemoreceptors, which receive the chemical substances required for their tasks. However, there was no information on the differential expression of chemoreception-related genes among castes and between sexes in termites.
Insect chemoreceptors are distributed not only on the antennae but also on the mouth, and some species have the receptors on the leg tip. Although the olfaction-related receptors exist mainly on antennae, the gustation-related receptors are known to exist in various parts. Accordingly, we extracted and analyzed total RNA from the whole body of each caste in R. speratus using high-throughput mRNA sequencing (RNA-seq), which is the method that comprehensively and quantitatively analyzes the all genes expressed in the living organism. Subsequently, the putative genes coding “chemoreceptors” and “transporter proteins that carries a chemical to a receptor” were explored. Finally, we performed the differential expression analyses of these genes among castes and between sexes. As a result, we revealed that the repertories of expressed chemoreceptor genes vary by caste, sex and age. This suggests not only adaptation of chemoreception systems for efficient task allocation but also sexual division of labor.
(Mitaka et al. 2016, PLOS ONE)
Differential expression of chemoreceptor genes among castes and between sexes. For each gene, the more cell is white, the higher expression level is in the caste than others.
Age-dependent alternation of gene expression levels. A male alate becomes a young primary king after establishing new colony, and then it takes long time to become mature. The expression level of this chemoreceptor rises with age.
■Fat Body in Termite Queen
Increases in DNA content caused by endoreduplication are widely observed in the metabolically active tissues of plants and animals. During egg production, insect females synthesize very large amounts of vitellogenin (a precursor protein of egg yolk) in their fat bodies, and female fat bodies of some insects become polyploid to accelerate vitellogenin production.
Eusocial insects have developed reproductive division of labor, wherein queens lay most of the eggs while other individuals have reduced fertility and perform tasks required for maintenance of the colony. Accordingly, in eusocial insects, the activity level of vitellogenin synthesis for egg production should be higher only in queens than other castes.
Our study revealed that termite queens have disproportionately more DNA in their fat body cells. DNA content analysis using flow cytometry demonstrated that more cells contained 4C-DNA than 2C-DNA in the fat bodies of R. speratus queens. This high level of endoreduplication was not found in the fat body cells of other castes. This caste-dependent doubling of DNA content in fat body cells suggests that termites exploit endoreduplication to boost egg production, in conjunction with the development of reproductive division of labor.
(Nozaki and Matsuura 2015, Entomological Science)
■Intercolonial Variation in Termite Shelter Tube Patterns
Building behaviors occur in various organisms from bacteria to humans. Social insects build various structures such as large nests and underground galleries, achieved by self-organization. Structures built by social insects have recently been demonstrated to vary widely in size and shape within a species, even under the same environmental conditions (Mizumoto and Matsuura 2013, Insectes Sociaux). However, little is known about how intraspecific variation in structures emerges from collective behaviors.
Workers of Reticulitermes speratus constructing the shelter tube
We revealed that the colony variation of structures can be generated by simply changing two behavioral parameters of group members, even with the same building algorithm. Laboratory experiments of termite shelter tube construction demonstrated clear intercolonial variation, and a two-dimensional lattice model showed that it can be attributed to the extent of positive feedback and the number of individuals engaged in building. This study contributes to explaining the great diversity of structures emerging from collective building in social insects.
(Mizumoto et al. 2015, Royal Society Open Science)
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