Hiroki Gotoh

Juvenile hormone titers and caste differentiation in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Termopsidae).

Termites are social insects, presenting morphologically distinct castes, performing specific tasks in the colony. The developmental processes underlying caste differentiation are mainly controlled by juvenile hormone (JH). Although many fragmentary data support this fact, there was no comparative work on JH titers during the caste differentiation processes. In this study, JH titer variation was investigated using a liquid chromatography-mass spectrometry (LC-MS) quantification method in all castes of the Japanese damp-wood termite Hodotermopsis sjostedti, especially focusing on the soldier caste differentiation pathway, which was induced by treatment with a JH analog. Hemolymph JH titers fluctuated between 20 and 720pg/microl. A peak of JH was observed during molting events for the pseudergate stationary molt and presoldier differentiation, but this peak was absent prior to the imaginal molt. Soldier caste differentiation was generally associated with high JH titers and nymph to alate differentiation with low JH titers. However, JH titer rose in females during alate maturation, probably in relation to vitellogenesis. In comparison, JH titer was surprisingly low in neotenics. On the basis of these results in both natural and artificial conditions, the current model for JH action on termite caste differentiation is discussed and re-appraised.

Juvenile Hormone Regulates Extreme Mandible Growth in Male Stag Beetles

The morphological diversity of insects is one of the most striking phenomena in biology. Evolutionary modifications to the relative sizes of body parts, including the evolution of traits with exaggerated proportions, are responsible for a vast range of body forms. Remarkable examples of an insect trait with exaggerated proportions are the mandibular weapons of stag beetles. Male stag beetles possess extremely enlarged mandibles which they use in combat with rival males over females. As with other sexually selected traits, stag beetle mandibles vary widely in size among males, and this variable growth results from differential larval nutrition. However, the mechanisms responsible for coupling nutrition with growth of stag beetle mandibles (or indeed any insect structure) remain largely unknown. Here, we demonstrate that during the development of male stag beetles (Cyclommatus metallifer), juvenile hormone (JH) titers are correlated with the extreme growth of an exaggerated weapon of sexual selection. We then investigate the putative role of JH in the development of the nutritionally-dependent, phenotypically plastic mandibles, by increasing hemolymph titers of JH with application of the JH analog fenoxycarb during larval and prepupal developmental periods. Increased JH signaling during the early prepupal period increased the proportional size of body parts, and this was especially pronounced in male mandibles, enhancing the exaggerated size of this trait. The direction of this response is consistent with the measured JH titers during this same period. Combined, our results support a role for JH in the nutrition-dependent regulation of extreme mandible growth in this species. In addition, they illuminate mechanisms underlying the evolution of trait proportion, the most salient feature of the evolutionary diversification of the insects.

Developmental Link between Sex and Nutrition; doublesex Regulates Sex-Specific Mandible Growth via Juvenile Hormone Signaling in Stag Beetles

Sexual dimorphisms in trait expression are widespread among animals and are especially pronounced in ornaments and weapons of sexual selection, which can attain exaggerated sizes. Expression of exaggerated traits is usually male-specific and nutrition sensitive. Consequently, the developmental mechanisms generating sexually dimorphic growth and nutrition-dependent phenotypic plasticity are each likely to regulate the expression of extreme structures. Yet we know little about how either of these mechanisms work, much less how they might interact with each other. We investigated the developmental mechanisms of sex-specific mandible growth in the stag beetle Cyclommatus metallifer, focusing on doublesex gene function and its interaction with juvenile hormone (JH) signaling. doublesex genes encode transcription factors that orchestrate male and female specific trait development, and JH acts as a mediator between nutrition and mandible growth. We found that the Cmdsx gene regulates sex differentiation in the stag beetle. Knockdown of Cmdsx by RNA-interference in both males and females produced intersex phenotypes, indicating a role for Cmdsx in sex-specific trait growth. By combining knockdown of Cmdsx with JH treatment, we showed that female-specific splice variants of Cmdsx contribute to the insensitivity of female mandibles to JH: knockdown of Cmdsx reversed this pattern, so that mandibles in knockdown females were stimulated to grow by JH treatment. In contrast, mandibles in knockdown males retained some sensitivity to JH, though mandibles in these individuals did not attain the full sizes of wild type males. We suggest that moderate JH sensitivity of mandibular cells may be the default developmental state for both sexes, with sex-specific Dsx protein decreasing sensitivity in females, and increasing it in males. This study is the first to demonstrate a causal link between the sex determination and JH signaling pathways, which clearly interact to determine the developmental fates and final sizes of nutrition-dependent secondary-sexual characters.

A general mechanism for conditional expression of exaggerated sexually-selected traits

Sexually‐selected exaggerated traits tend to be unusually reliable signals of individual condition, as their expression tends to be more sensitive to nutritional history and physiological circumstance than that of other phenotypes. As such, these traits are the foundation for many models of sexual selection and animal communication, such as “handicap” and “good genes” models. Exactly how expression of these traits is linked to the bearers condition has been a central yet unresolved question, in part because the underlying physiological mechanisms regulating their development have remained largely unknown. Recent discoveries across animals as diverse as deer, beetles, and flies now implicate the widely conserved insulin‐like signaling pathway, as a common physiological mechanism regulating condition‐sensitive structures with extreme growth. This raises the exciting possibility that one highly conserved pathway may underlie the evolution of trait exaggeration in a multitude of sexually‐selected signal traits across the animal kingdom.

Juvenile Hormone titre and vitellogenin gene expression related to ovarian development in primary reproductives compared with nymphs and nymphoid reproductives of the termite Reticulitermes speratus

To elucidate the reproductive cycle of termite queens, incipient colonies of Reticulitemes speratus (Isoptera: Rhinotermitidae) are established under laboratory conditions, and the transition of colony development is observed at 0.5, 1.5, 2.5, 3.5, and 7.5 months (stages I–V, respectively) after colony foundation. Ovarian development, vitellogenin gene expression and Juvenile Hormone (JH) titres are examined in the queens and in nonphysogastric nymphoids collected from natural colonies. A reproductive cycle in queens is observed, in which the oviposition rate is relatively higher during stages I and II, and then decreases during stages III and IV. Vitellogenic oocytes are not observed in the ovaries during stages III and IV, and the expression level of the vitellogenin gene is low, suggesting that egg production in queens is repressed during these stages. However, vitellogenin gene expression and egg deposition in queens resumes during stage V. Juvenile Hormone levels rise during the transition from nymphs to stage I queens, and elevated JH titres are observed also during stages III and IV. The decrease in JH titre in queens at stage II precedes the decline in vitellogenesis at stages III and IV. Thus, JH titre and vitellogenesis are correlated in an offset pattern. However, nonphysogastric nymphoid reproductives do not have vitellogenic oocytes in their ovaries, and their JH titre is two‐fold higher than that of queens, suggesting that elevated JH titre precedes vitellogenesis, as in queens.

Juvenile hormone titre and related gene expression during the change of reproductive modes in the pea aphid

Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive‐mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive‐mode change. Using liquid chromatography‐mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short‐day conditions than in aphids producing parthenogenetic morphs under long‐day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real‐time quantitative reverse‐transcription‐PCR across the reproductive‐mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short‐day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short‐days, leading to the production of sexual morphs.

Exaggerated Trait Growth in Insects

Animal structures occasionally attain extreme proportions, eclipsing in size the surrounding body parts. We review insect examples of exaggerated traits, such as the mandibles of stag beetles (Lucanidae), the claspers of praying mantids (Mantidae), the elongated hindlimbs of grasshoppers (Orthoptera: Caelifera), and the giant heads of soldier ants (Formicidae) and termites (Isoptera). Developmentally, disproportionate growth can arise through trait-specific modifications to the activity of at least four pathways: the sex determination pathway, the appendage patterning pathway, the insulin/IGF signaling pathway, and the juvenile hormone/ecdysteroid pathway. Although most exaggerated traits have not been studied mechanistically, it is already apparent that distinct developmental mechanisms underlie the evolution of the different types of exaggerated traits. We suggest this reflects the nature of selection in each instance, revealing an exciting link between mechanism, form, and function. We use this information to make explicit predictions for the types of regulatory pathways likely to underlie each type of exaggerated trait.

Social interactions affecting caste development through physiological actions in termites

A colony of social insects is not only an aggregation of individuals but also a functional unit. To achieve adaptive social behavior in fluctuating environmental conditions, in addition to coordination of physiological status in each individual, the whole colony is coordinated by interactions among colony members. The study on the regulation of social-insect colonies is termed “social physiology.” Termites, a major group of social insects, exhibit many interesting phenomena related to social physiology, such as mechanisms of caste regulation in a colony. In their colonies, there are different types of individuals, i.e., castes, which show distinctive phenotypes specialized in specific colony tasks. Termite castes comprise reproductives, soldiers and workers, and the caste composition can be altered depending on circumstances. For the regulation of caste compositions, interactions among individuals, i.e., social interactions, are thought to be important. In this article, we review previous studies on the adaptive meanings and those on the proximate mechanisms of the caste regulation in termites, and try to understand those comprehensively in terms of social physiology. Firstly, we summarize classical studies on the social interactions. Secondly, previous studies on the pheromone substances that mediate the caste regulatory mechanisms are overviewed. Then, we discuss the roles of a physiological factor, juvenile hormone (JH) in the regulation of caste differentiation. Finally, we introduce the achievements of molecular studies on the animal sociality (i.e., sociogenomics) in terms of social physiology. By comparing the proximate mechanisms of social physiology in termites with those in hymenopterans, we try to get insights into the general principles of social physiology in social animals.

Soldier presence suppresses presoldier differentiation through a rapid decrease of JH in the termite Reticulitermes speratus

The regulation of caste differentiation is essential to insect eusociality. Termite soldiers are sterile and cannot eat by themselves because they have specialized mouth morphology. Almost all termite species have a soldier caste, and the soldier ratio per colony is maintained at a low level, probably by elaborate regulatory mechanisms. Although the soldier presence is considered to negatively affect soldier differentiation in all examined species, the detailed mechanism remains unclear. Presoldier differentiation can be induced artificially by juvenile hormone (JH) application to workers, showing that JH is a key factor underlying the regulation of soldier differentiation. In this study, to elucidate physiological changes in workers because of the soldier presence during the molt into presoldiers, JH III applications and JH titer quantifications were carried out in the rhinotermitid termite Reticulitermes speratus. Firstly, the effects of soldier presence before the molt into presoldiers induced by JH III application to workers were investigated. The rates of presoldier molt induced by the treatments with soldiers were significantly lower than those without soldiers. Secondly, worker JH titers in the presence or absence of soldiers were quantified by LC-MS on day 0, 5, 10, and 15 after JH application. Results indicated that the worker JH titers (endogenous+applied JH III) in the presence of soldiers were significantly lower than those without soldiers on day 5 after the JH treatment. On days 10 and 15, such soldier effects were not observed. Finally, the effective duration of soldier presence after the JH application was elucidated. A 4 day period of co-existence with soldiers suppressed presoldier differentiation, suggesting that the soldier presence rapidly decreased the JH titer in other colony members (i.e., workers), resulting in the inhibition of presoldier production.

Juvenile hormone mediates developmental integration between exaggerated traits and supportive traits in the horned flour beetle Gnatocerus cornutus

Sexually selected exaggerated traits are often coupled with modifications in other nontarget traits. In insects with weapons, enlargements of nontarget characters that functionally support the weapon often occur (i.e. supportive traits). The support of sexual traits requires developmental coordination among functionally related multiple traits—an explicit example of morphological integration. The genetic theory predicts that developmental integration among different body modules, for which development is regulated via different sets of genes, is likely to be coordinated by pleiotropic factors. However, the developmental backgrounds of morphological integrations are largely unknown. We tested the hypothesis that the juvenile hormone (JH), as a pleiotropic factor, mediates the integration between exaggerated and supportive traits in an armed beetle Gnatocerus cornutus. During combat, males of this beetle use exaggerated mandibles to lift up their opponents with the supportive traits, that is, the head and prothoracic body parts. Application of methoprene, a JH analog (JHA), during the larval to prepupal period, induced the formation of large mandibles relative to the body sizes in males. Morphometric examination of nontarget traits elucidated an increase in the relative sizes of supportive traits, including the head and prothoracic body parts. In addition, reductions in the hind wing area and elytra length, which correspond to flight and reproductive abilities, were detected. Our findings are consistent with the genetic theory and support the idea that JH is a key pleiotropic factor that coordinates the developmental integration of exaggerated traits and supportive characters, as well as resource allocation trade‐offs.