The royal food of termites shows king and queen specificity.

Society in eusocial insects is based on the reproductive division of labor, with a small number of reproductive individuals supported by a large number of nonreproductive individuals. Because inclusive fitness of all colony members depends on the survival and fertility of reproductive members, sterile members provide royals with special treatment. Here, we show that termite kings and queens each receive special food of a different composition from workers. Sequential analysis of feeding processes demonstrated that workers exhibit discriminative trophallaxis, indicating their decision-making capacity in allocating food to the kings and queens. Liquid chromatography tandem-mass spectrometry analyses of the stomodeal food and midgut contents revealed king- and queen-specific compounds, including diacylglycerols and short-chain peptides. Desorption electrospray ionization mass spectrometry imaging analyses of 13C-labeled termites identified phosphatidylinositol and acetyl-l-carnitine in the royal food. Comparison of the digestive tract structure showed remarkable differences in the volume ratio of the midgut-to-hindgut among castes, indicating that digestive division of labor underlies reproductive division of labor. Our demonstration of king- and queen-specific foods in termites provides insight into the nutritional system that underpins the extraordinary reproduction and longevity of royals in eusocial insects.

Age-Dependent Increase in Soldier Pheromone of the Termite Reticulitermes speratus.

Pheromone communication helps maintaining the sophisticated colony organization in social insects. In the termite Reticulitermes speratus, there are two functionally distinct soldier groups: royal guards and entrance guards. Royal guards protect kings and queens in the innermost part of the nest, whereas entrance guards prevent predators from intruding into the nest at the periphery. A recent study revealed that younger and older soldiers work as royal and entrance guards, respectively. This age-dependent distribution is thought to help workers to recognize where in the nest they are located. However, it is not known whether workers can discriminate the age of soldiers. Here, we show that the abundance of soldier pheromone changes with age and that workers discriminate a soldier's age by recognizing the pheromone abundance. Gas chromatography-mass spectrometry analysis revealed that the chemical profiles of extracts in three soldier groups of different ages (newly differentiated, royal guard, and entrance guard soldiers) are markedly different. Entrance guard soldiers have the most soldier pheromone among the three age classes. Furthermore, our bioassays suggested that the worker's movement from chamber to chamber is inhibited only when a soldier with less soldier pheromone is located at the chamber entrance. These results suggest that the soldier pheromone functions as a soldier age indicator and that workers change their behavior depending on the age of the soldier defending the chamber entrance. This study contributes to our understanding of the relationship between aging and pheromone communication in social insects.

Chemical identification of an aggregation pheromone in the termite Reticulitermes speratus.

Social behaviours in termites are regulated by sophisticated chemical communication systems. The majority of subterranean termites continuously forage for new wood resources to expand their nesting areas; an aggregation pheromone is presumed to regulate this process. However, the chemical components of this pheromone have never been determined. We identified the chemical properties of the aggregation pheromone that signals nestmate presence and induces arrest in the termite Reticulitermes speratus. The results of gas chromatography-mass spectrometry analyses and bioassays indicated that R. speratus worker release the pheromone to their nesting site. The pheromone consists of an aromatic compound (2-phenylundecane), cuticular hydrocarbons (pentacosane and heptacosane), fatty acids (palmitic acid and trans-vaccenic acid), and cholesterol; the pheromone induces long-term aggregation at new nesting and feeding sites. Although 2-phenylundecane alone attracted workers, the combination of all six compounds showed greater arrestant activity than 2-phenylundecane alone. This suggests that 2-phenylundecane functions as an attractant, whereas the remaining five components function as arrestants. Our results indicate that foraging worker termites produce a multi-component aggregation pheromone by combining a volatile hydrocarbon and non-volatile lipids with cuticular hydrocarbons. This pheromone enables rapid, long-lasting aggregation of termite workers, which contributes to efficient feeding and colonisation of new wood. Our work furthers the understanding of chemical communication systems underlying social assembly 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.

A Parental Volatile Pheromone Triggers Offspring Begging in a Burying Beetle.

Parental care is a notable aspect of reproductive effort in many animals. The interaction between offspring begging and the parental feeding response is an important communication mechanism that regulates offspring food supply, and reducing the cost of superfluous begging is beneficial to both parents and offspring. Here we concluded that parents of the burying beetle Nicrophorus quadripunctatus inform their offspring of their preparation for provisioning by emitting "provisioning pheromone." Female parents emitted an antimicrobial aromatic compound, 2-phenoxyethanol, in their regurgitation before provisioning, and this compound elicits begging behavior from their offspring. Furthermore, begging incurs growth and survival costs, and parents spent more than 85% of their time in close proximity to their offspring without provisioning. Therefore, it is suggested that limiting offspring begging during provisioning is beneficial to both parents and offspring. We report here a novel aspect of parent-offspring communication in family life.

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.

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.

Age-based soldier polyethism: old termite soldiers take more risks than young soldiers.

Who should take on risky tasks in an age-heterogeneous society? Life-history theory predicts that, in social insects, riskier tasks should be undertaken by sterile individuals with a shorter life expectancy. The loss of individuals with shorter life expectancy is less costly for colony reproductive success than the loss of individuals with longer life expectancy. Termite colonies have a sterile soldier caste, specialized defenders engaged in the most risky tasks. Here we show that termite soldiers exhibit age-dependent polyethism, as old soldiers are engaged in front-line defence more than young soldiers. Our nest defence experiment showed that old soldiers went to the front line and blocked the nest opening against approaching predatory ants more often than young soldiers. We also found that young soldiers were more biased toward choosing central nest defence as royal guards than old soldiers. These results demonstrate that termite soldiers have age-based task allocation, by which ageing predisposes soldiers to switch to more dangerous tasks. This age-dependent soldier task allocation increases the life expectancy of soldiers, allowing them to promote their lifetime contribution to colony reproductive success.

Multi-functional roles of a soldier-specific volatile as a worker arrestant, primer pheromone and an antimicrobial agent in a termite.

Division of labour in eusocial insects is characterized by efficient communication systems based on pheromones. Among such insects, termites have evolved specialized sterile defenders, called soldiers. Because they are incapable of feeding themselves, it has been suggested that soldiers are sustained by workers and emit the pheromone arresting workers. However, such a soldier pheromone has not been identified in any termite species, and the details of the soldier-worker interaction remain to be explored. Here, we identified a soldier-specific volatile sesquiterpene as a worker arrestant, which also acts as a primer pheromone regulating soldier differentiation and fungistatic agent in a termite Reticulitermes speratus Chemical analyses revealed that (-)-ß-elemene is the major component of soldier extract, and its authentic standard exhibited arrestant activity to workers and inhibited the differentiation from workers to soldiers. This compound also showed fungistatic activity against entomopathogenic fungi. These suggest that (-)-ß-elemene secreted by soldiers acts not only as a worker arrestant but also as one component of inhibitory primer pheromone and an anti-pathogenic agent. Our study provides novel evidence supporting the multi-functionality of termite soldier pheromone and provides new insights into the role of soldiers and the evolutionary mechanisms of pheromone compounds.

Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus.

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole.

Caste-Specific and Sex-Specific Expression of Chemoreceptor Genes in a Termite.

The sophisticated colony organization of eusocial insects is primarily maintained through the utilization of pheromones. The regulation of these complex social interactions requires intricate chemoreception systems. The recent publication of the genome of Zootermopsis nevadensis opened a new avenue to study molecular basis of termite caste systems. Although there has been a growing interest in the termite chemoreception system that regulates their sophisticated caste system, the relationship between division of labor and expression of chemoreceptor genes remains to be explored. Using high-throughput mRNA sequencing (RNA-seq), we found several chemoreceptors that are differentially expressed among castes and between sexes in a subterranean termite Reticulitermes speratus. In total, 53 chemoreception-related genes were annotated, including 22 odorant receptors, 7 gustatory receptors, 12 ionotropic receptors, 9 odorant-binding proteins, and 3 chemosensory proteins. Most of the chemoreception-related genes had caste-related and sex-related expression patterns; in particular, some chemoreception genes showed king-biased or queen-biased expression patterns. Moreover, more than half of the genes showed significant age-dependent differences in their expression in female and/or male reproductives. These results reveal a strong relationship between the evolution of the division of labor and the regulation of chemoreceptor gene expression, thereby demonstrating the chemical communication and underlining chemoreception mechanism in social insects.