Higher levels of the lipophilic antioxidants coenzyme Q10 and vitamin E in long-lived termite queens than in short-lived workers.

Termite queens and kings live longer than nonreproductive workers. Several molecular mechanisms contributing to their long lifespan have been investigated; however, the underlying biochemical explanation remains unclear. Coenzyme Q (CoQ), a component of the mitochondrial electron transport chain, plays an essential role in the lipophilic antioxidant defense system. Its beneficial effects on health and longevity have been well studied in several organisms. Herein, we demonstrated that long-lived termite queens have significantly higher levels of the lipophilic antioxidant CoQ10 than workers. Liquid chromatography analysis revealed that the levels of the reduced form of CoQ10 were 4 fold higher in the queen's body than in the worker's body. In addition, queens showed 7 fold higher levels of vitamin E, which plays a role in antilipid peroxidation along with CoQ, than workers. Furthermore, the oral administration of CoQ10 to termites increased the CoQ10 redox state in the body and their survival rate under oxidative stress. These findings suggest that CoQ10 acts as an efficient lipophilic antioxidant along with vitamin E in long-lived termite queens. This study provides essential biochemical and evolutionary insights into the relationship between CoQ10 concentrations and termite lifespan extension.

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.

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.

A method for estimating colony size using queen fecundity in termites under field conditions.

Colony size in social insects is one of the most important factors in shaping their self-organized system. It affects a wide variety of traits such as foraging and defense strategies, social immune responses, the degree of polymorphism, and reproductive output. However, colony size estimation of subterranean termites in the field has been challenging, due to their extremely cryptic biology and multiple site-nesting behavior. Since natural selection favors workers that maximize the number of their siblings, the amount of egg production may reflect the number of workers in the colony. Here, we report a method for inferring colony size in the field using total egg production in each colony from a subterranean termite, Reticulitermes speratus. Our investigation of field colonies revealed that the body weight of queens reaches a peak and had the largest variance in June and July and accurately predicts the number of eggs laid by the queen per 24 h. Using laboratory-reared colonies, we found that the total egg production in each colony is proportional to the number of workers. We also estimated the colony size of 198 field colonies and found that the median and maximum colony size was 24,500 and 451,800 workers per colony. The method for inferring colony size presented here may also be applicable to termite species with a clear seasonality in egg production. The colony size estimate will contribute to understanding the life history strategies and social systems of termites.

Plastic brain structure changes associated with the division of labor and aging in termites.

Division of labor is a prominent feature of social insect societies, where different castes engage in different specialized tasks. As brain differences are associated with behavioral differences, brain anatomy may be linked to caste polymorphism. Here, we show that termite brain morphology changes markedly with caste differentiation and age in the termite, Reticulitermes speratus. Brain morphology was shown to be associated with reproductive division of labor, with reproductive individuals (alates and neotenic reproductives) having larger brains than nonreproductives (workers and soldiers). Micro-computed tomography (CT) imaging and dissection observations showed that the king's brain morphology changed markedly with shrinkage of the optic lobes during their long life in the dark. Behavioral experiments showed that mature primary kings lose visual function as a result of optic lobe shrinkage. These results suggested that termites restructure their nervous systems to perform necessary tasks as they undergo caste differentiation, and that they also show flexible changes in brain morphology even after the final molt. This study showed that brain morphology in social insects is linked to caste and aging, and that the evolution of the division of labor is underpinned by the development of diverse neural systems for specialized tasks.

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.

Heritable effects on caste determination and colony-level sex allocation in termites under field conditions.

The ecological success of social insects is attributed to the division of labor, where newly hatched offspring differentiate into either fertile progeny or functionally sterile worker castes. There is growing evidence for the heritable (genetic or epigenetic) effects on caste determination based on laboratory experiments. Here, we indirectly demonstrate that heritable factors have the principal role in caste determination and strongly affect colony-level production of both sexes of fertile dispersers (i.e., alates) in field colonies of the termite Reticulitermes speratus. An egg-fostering experiment suggests that the colony-dependent sex-specific caste fates were almost entirely determined before oviposition. Our investigation of field colonies revealed that such colony-dependent sex-specific caste fates result in the intercolonial variation in the numerical sex ratio of differentiated fertile offspring and, eventually, that of alates. This study contributes to better understanding the mechanisms underlying the division of labor and life-history traits in social insects.

King- and queen-specific degradation of uric acid contributes to reproduction in termites.

Caste-based reproductive division of labour in social insects is built on asymmetries in resource allocation within colonies. Kings and queens dominantly consume limited resources for reproduction, while non-reproductive castes such as workers and soldiers help reproductive castes. Studying the regulation of such asymmetries in resource allocation is crucial for understanding the maintenance of sociality in insects, although the molecular background is poorly understood. We focused on uric acid, which is reserved and used as a valuable nitrogen source in wood-eating termites. We found that king- and queen-specific degradation of uric acid contributes to reproduction in the subterranean termite Reticulitermes speratus. The urate oxidase gene (RsUAOX), which catalyses the first step of nitrogen recycling from stored uric acid, was highly expressed in mature kings and queens, and upregulated with differentiation into neotenic kings/queens. Suppression of uric acid degradation decreased the number of eggs laid per queen. Uric acid was shown to be provided by workers to reproductive castes. Our results suggest that the capacity to use nitrogen, which is essential for the protein synthesis required for reproduction, maintains colony cohesion expressed as the reproductive monopoly held by kings and queens.

Why and how do termite kings and queens live so long?

Lifespan varies greatly across the tree of life. Of the various explanations for this phenomenon, those that involve trade-offs between reproduction and longevity have gained considerable support. There is an important exception: social insect reproductives (queens and in termites, also kings) exhibit both high reproductive outputs and extraordinarily long lives. As both the ultimate and proximate mechanisms underlying the absence of the fecundity/longevity trade-off could shed light on the unexpected dynamics and molecular mechanisms of extended longevity, reproductives of social insects have attracted much attention in the field of ageing research. Here, we highlight current ecological and physiological studies on ageing and discuss the various possible evolutionary and molecular explanations of the extended lifespans of termite reproductives. We integrate these findings into a coherent framework revealing the evolution of longevity in these reproductives. Studies on termites may explain why and how ageing is shaped by natural selection. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns?'

Reproduction deep inside wood: a low O2 and high CO2 environment promotes egg production by termite queens.

Extreme conditions are normal for animals living in harsh environments. These animals adapt to their habitats and can use difficult conditions by default. Organisms living in enclosed spaces, notably termites in decaying wood, experience low O2 and high CO2 gas conditions due to limited gas exchange and high insect density. Termite queens, in particular, reproduce in royal chambers deep inside the wood, wherein tens of thousands of individuals engage in social labour. Here, we demonstrate that royal chambers in termite nests have low O2 and high CO2 gas concentrations, which enhance egg production by queens. We identified a unique gas condition of royal chambers in the nest of the subterranean termite Reticulitermes speratus, which is characterized by low O2 (15.75%) and high CO2 (4.99%) concentrations. Queens showed significantly greater fecundity under the low O2 and high CO2 gas conditions in the royal chambers than under ambient gas conditions. Quantitative PCR analysis revealed that the royal chamber gas conditions significantly promoted the expression levels of the vitellogenin genes RsVg1, RsVg2 and RsVg3 in queens compared with ambient gas conditions. This study highlights the adaptation of animals that live in closed habitats, which are hypoxic and hypercapnic as the result of their own metabolism, so as to have a high fitness in such environmental conditions.

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.

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.

Long-Lived Termite Queens Exhibit High Cu/Zn-Superoxide Dismutase Activity.

In most organisms, superoxide dismutases (SODs) are among the most effective antioxidant enzymes that regulate the reactive oxygen species (ROS) generated by oxidative energy metabolism. ROS are considered main proximate causes of aging. However, it remains unclear if SOD activities are associated with organismal longevity. The queens of eusocial insects, such as termites, ants, and honeybees, exhibit extraordinary longevity in comparison with the nonreproductive castes, such as workers. Therefore, the queens are promising candidates to study the underlying mechanisms of aging. Here, we found that queens have higher Cu/Zn-SOD activity than nonreproductive individuals of the termite Reticulitermes speratus. We identified three Cu/Zn-SOD sequences and one Mn-SOD sequence by RNA sequencing in R. speratus. Although the queens showed higher Cu/Zn-SOD activity than the nonreproductive individuals, there were no differences in their expression levels of the Cu/Zn-SOD genes RsSOD1 and RsSOD3A. Copper (Cu2+ and Cu+) is an essential cofactor for Cu/Zn-SOD enzyme activity, and the queens had higher concentrations of copper than the workers. These results suggest that the high Cu/Zn-SOD activity of termite queens is related to their high levels of the cofactor rather than gene expression. This study highlights that Cu/Zn-SOD activity contributes to extraordinary longevity in termites.

Protective role of testis-specific peroxiredoxin 4 against cellular oxidative stress.

Peroxiredoxin (PRDX), a newly discovered antioxidant enzyme, has an important role in hydrogen peroxide reduction. Among six PRDX genes (PRDX1-6) in mammals, PRDX4 gene is alternatively spliced to produce the somatic cell form (PRDX4) and the testis specific form (PRDX4t). In our previous study, PRDX4 knockout mice displayed testicular atrophy with an increase in cell death due to oxidative stress. However, the antioxidant function of PRDX4t is unknown. In this study, we demonstrate that PRDX4t plays a protective role against oxidative stress in the mammalian cell line HEK293T. The PRDX4t-EGFP plasmid was transferred into HEK293T cells; protein expression was confirmed in the cytoplasm. To determine the protective role of PRDX4t in cells, we performed image-based analysis of PRDX4t-EGFP expressed cells exposed to UV irradiation and hydrogen peroxide using fluorescent probe CellROX. Our results suggested that PRDX4t-EGFP expressed cells had reduced levels of oxidative stress compared with cells that express only EGFP. This study highlights that PRDX4t plays an important role in cellular antioxidant defense.

Uric acid, an important antioxidant contributing to survival in termites.

Reactive oxygen species (ROS) are generated spontaneously in all organisms and cause oxidative damage to biomolecules when present in excess. Accumulated oxidative damage accelerates aging; enhanced antioxidant capacity may be a positive factor for longevity. Recently, numerous studies of aging and longevity have been performed using short-lived animals, however, longevity mechanisms remain unknown. Here we show that a termite Reticulitermes speratus that is thought to be long-lived eusocial insect than other solitary insects uses large quantities of uric acid as an antioxidant against ROS. We demonstrated that the accumulation of uric acid considerably increases the free radical-scavenging activity and resistance against ultraviolet-induced oxidative stress in laboratory-maintained termites. In addition, we found that externally administered uric acid aided termite survival under highly oxidative conditions. The present data demonstrates that in addition to nutritional and metabolic roles, uric acid is an essential antioxidant for survival and contributes significantly to longevity. Uric acid also plays important roles in primates but causes gout when present in excess in humans. Further longevity studies of long-lived organisms may provide important breakthroughs with human health applications.

An Efficient Antioxidant System in a Long-Lived Termite Queen.

The trade-off between reproduction and longevity is known in wide variety of animals. Social insect queens are rare organisms that can achieve a long lifespan without sacrificing fecundity. The extended longevity of social insect queens, which contradicts the trade-off, has attracted much attention because it implies the existence of an extraordinary anti-aging mechanism. Here, we show that queens of the termite Reticulitermes speratus incur significantly lower oxidative damage to DNA, protein and lipid and have higher activity of antioxidant enzymes than non-reproductive individuals (workers and soldiers). The levels of 8-hydroxy-2'-deoxyguanosine (oxidative damage marker of DNA) were lower in queens than in workers after UV irradiation. Queens also showed lower levels of protein carbonyls and malondialdehyde (oxidative damage markers of protein and lipid, respectively). The antioxidant enzymes of insects are generally composed of catalase (CAT) and peroxiredoxin (Prx). Queens showed more than two times higher CAT activity and more than seven times higher expression levels of the CAT gene RsCAT1 than workers. The CAT activity of termite queens was also markedly higher in comparison with other solitary insects and the queens of eusocial Hymenoptera. In addition, queens showed higher expression levels of the Prx gene RsPRX6. These results suggested that this efficient antioxidant system can partly explain why termite queens achieve long life. This study provides important insights into the evolutionary linkage of reproductive division of labor and the development of queens' oxidative stress resistance in social insects.