Developmental Link between Sex and Nutrition; \u3ci\u3edoublesex\u3c/i\u3e 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 nutritiondependent 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

Expansion of presoldier cuticle contributes to head elongation during soldier differentiation in termites

In termites, the soldier caste possesses morphological features suitable for colony defence, despite some exceptions. Soldiers are differentiated via two moultings through a presoldier stage with dramatic morphogenesis. While a number of morphological modifications are known to occur during the presoldier moult, growth and morphogenesis seem to continue even after the moult. The present study, using the damp-wood termite Hodotermopsis sjostedti, carried out morphological and histological investigations on the developmental processes during the presoldier stage that is artificially induced by the application of a juvenile hormone analogue. Measurements of five body parameters indicated that head length significantly increased during the 14-day period after the presoldier moult, while it did not increase subsequently to the stationary moult (pseudergate moult as control). Histological observations also showed that the cuticular development played a role in the presoldier head elongation, suggesting that the soft and flexible presoldier cuticle contributed to the soldier morphogenesis in termites

Male neotenic reproductives accelerate additional differentiation of female reproductives by lowering JH titer in termites

Abstract Eusocial insects exhibit reproductive division of labor, in which only a fraction of colony members differentiate into reproductives. In termites, reproductives of both sexes are present in a colony and constantly engaged in reproduction. It has been suggested that the sex ratio of reproductives is maintained by social interactions. The presence of reproductives is known to inhibit the additional differentiation of same-sex reproductives, while it promotes the differentiation of opposite-sex reproductives. In this study, using the damp-wood termite Hodotermopsis sjostedti, physiological effects of male/female reproductives on the differentiation of supplementary reproductives (neotenics) were examined. The results showed that the only male-neotenic condition, i.e., the presence of male neotenics in the absence of female neotenics, accelerated the neotenic differentiation from female workers (i.e., pseudergates). Under this condition, the rise of juvenile hormone (JH) titer was repressed in females, and the application of a JH analog inhibited the female neotenic differentiation, indicating that the low JH titer leads to rapid differentiation. Thus, the only male-neotenic condition that actively promotes reproductive differentiation by manipulating physiological condition of females is suggested to be a mechanism underlying sexual asymmetry in reproductive function, which may lead the female-biased sex allocation of reproductives

The function of appendage patterning genes in mandible development of the sexually dimorphic stag beetle

One of the defining features of the evolutionary success of insects is the morphological diversification of their appendages, especially mouthparts. Although most insects share a common mouthpart ground plan, there is remarkable diversity in the relative size and shapes of these appendages among different insect lineages. One of the most prominent examples of mouthpart modification can be found in the enlargement of mandibles in stag beetles (Coleoptera, Insecta). In order to understand the proximate mechanisms of mouthpart modification, we investigated the function of appendage-patterning genes in mandibular enlargement during extreme growth of the sexually dimorphic mandibles of the stag beetle Cyclommatus metallifer. Based on knowledge from Drosophila and Tribolium studies, we focused on seven appendage patterning genes (Distal-less (Dll), aristaless (al), dachshund (dac), homothorax (hth), Epidermal growth factor receptor (Egfr), escargot (esg), and Keren (Krn). In order to characterize the developmental function of these genes, we performed functional analyses by using RNA interference (RNAi). Importantly, we found that RNAi knockdown of dac resulted in a significant mandible size reduction in males but not in female mandibles. In addition to reducing the size of mandibles, dac knockdown also resulted in a loss of the serrate teeth structures on the mandibles of males and females. We found that al and hilt play a significant role during morphogenesis of the large male specific inner mandibular tooth. On the other hand, knockdown of the distal selector gene Dll did not affect mandible development, supporting the hypothesis that mandibles likely do not contain the distal-most region of the ancestral appendage and therefore co-option of Dll expression is unlikely to be involved in mandible enlargement in stag beetles. In addition to mandible development, we explored possible roles of these genes in controlling the divergent antennal morphology of Coleoptera

Effects of JHA application on <i>GFP</i>^RNAi and <i>dsx</i>^RNAi individuals.

<p>The relationships between pupal weight and mandible length were described. Sex is indicated by color of symbols (male: blue and light blue, female: pink and orange). Hormone treatments are indicated by shape of symbols (acetone treatment: circle, JHA treatment: square). RNAi treatments are distinguished by closed (<i>GFP</i> dsRNA injection) or open (<i>dsx</i> dsRNA injection). Scale bars indicate 10 mm (in males) or 5 mm (in females).</p

Developmental staging chart of prepupal development from the larval-prepupal transition to pupation.

<p>Mandibular growth (cell proliferation) is indicated on the y-axis over developmental time in days on the x-axis (hashmark = 1 day). Three distinct forms are recognized - the last instar larva, the prepupa, and the pupa. Known landmarks of mandibular proliferation in large males are indicated by the red circles and shown in diagram form below the graph <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004098#pgen.1004098-Gotoh1" target="_blank">[5]</a>. The end of the larval period and the initiation of the prepupal period is indicated by Pupal Cell Construction (PCC) and is defined as the starting point of prepupal Stage 1. Outwardly the larva does not change its morphology. Stage 1 lasts approximately 2 days until the initiation of the first Gut Purge (GP) in which the prepupa begins to transform and empties out half of its gut contents. The time the prepupa spends in the first GP is known as Stage 2. Stage 3 is a quiescent phase where the prepupa undergoes massive adult imaginal tissue proliferation but outwardly appears suspended in the first GP. Stage 4 occurs over only a few hours and begins with the second GP in which the last remnants of the gut contents are egested and the prepupa completely metamorphoses into the pupa.</p