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

Abstract

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.

Author Summary

Sexual dimorphisms such as the exaggerated antlers of deer, the enormous clawed chelae of crabs, and the horns and mandibles of beetles, are widespread across animal taxa and have fascinated biologists for centuries. Much recent work has uncovered the importance of the role of the sex-determination pathway in the expression of sexually dimorphic traits. However, critical interactions between this pathway and other growth regulatory mechanisms – for example, the physiological mechanisms involved in nutrition-dependent expression of these traits – are less well understood. In this study, we provide evidence of a developmental link between nutrition-sensitivity and sexual differentiation in the giant mandibles of the sexually dimorphic stag beetle, Cyclommatus metallifer. We examined the regulation and function of a key sex determination gene in animals, doublesex ( dsx), and its interaction with juvenile hormone (JH), an important insect hormone known to regulate insect polyphenisms including the regulation of the disproportionate growth of male stag beetle mandibles. We found that Cmdsx changes mandibular responsiveness to JH in a sex-specific pattern. Based on these results, we hypothesize that sex-specific regulation of JH responsiveness is a developmental link between nutrition and sexual differentiation in stag beetles.