Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix-loophelix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met-Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met-Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors. structure modeling | insecticide action | metamorphosis | Tribolium | Drosophila J uvenile hormone (JH) prevents adult transition (metamorphosis) of insect larvae until they have attained an appropriate stage (1, 2), and it typically stimulates oogenesis in adult females (3). How JH achieves its function remains unclear, mainly because a JH receptor has long eluded identification (4). The lipophilic nature of the sesquiterpene JH suggests an intracellular receptor, yet none of the known insect nuclear hormone receptors have been linked with the biological function of JH. A screen for Drosophila mutants resistant to methoprene (5), a JH mimic and a widely used insecticide (6), uncovered the Methoprene-tolerant (Met) protein containing a basic helix-loop-helix (bHLH) motif followed by two Per-Arnt-Sim (PAS) domains (7). Recombinant Drosophila Met was shown to bind JH at physiological (nanomolar) concentrations and to mediate a weak JH-and methoprene-dependent transcriptional activation in vitro (8). However, Met-null mutant flies were viable and fertile (5), leaving the notion that Met is a putative JH receptor unsupported with an anticipated developmental phenotype. Latest reports show that, in Drosophila, Met might functionally overlap with its paralog, encoded by the germ cell-expressed (gce) gene. Gce can increase sensitivity of Met-null mutants to methoprene (9), and only simultaneous loss of both Met and Gce is lethal (10). However, the actual mode of interaction between JH/methoprene and Met or Gce still remains unclear.Knockdown of the single Met gene in the flour beetle Tribolium castaneum induced beetle larvae to pupate before reaching their final instar (11), producing a precocious metamorphosis phenotype similar to that caused by loss of JH itself (12).Conversely, removal of Met precluded inhibition of adult ...

No abstract

Drosophila egg production depends upon the nutritional available to females. When food is in short supply, oogenesis is arrested and apoptosis of the nurse cells is induced at mid-oogenesis via a mechanism that is probably controlled by ecdysteroid hormone. We have shown that expression of some ecdysone-response genes is correlated with apoptosis of egg chambers. Moreover, ecdysteroid injection and application of juvenile hormone induces and suppresses the apoptosis, respectively. In this study, we investigated which tissues show increases in the concentration of ecdysteroids under nutritional shortage to begin to link together nutrient intake, hormone regulation and the choice between egg development or apoptosis made within egg chambers. We measured ecdysteroid levels in the whole body, ovaries and haemolymph samples by RIA and found that the concentration of ecdysteroid increased in all samples. This contributes to the idea that nutritional shortage leads to a rapid high ecdysteroid concentration within the fly and that the high concentration induces apoptosis. Low concentrations of ecdysteroid are essential for normal oogenesis. We suggest there is threshold concentration in the egg chambers and that apoptosis at mid-oogenesis is induced when the ecdysteroid levels exceed the threshold. Starvation causes the ovary to retain the ecdysteroid it produces, thus enabling individual egg chambers to undergo apoptosis and thus control the number of eggs produced in relation to food intake.

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

Significance Specific gene functions have been successfully suppressed by gene silencing or editing in many organisms. However, genetic manipulation to suppress the function of a target tissue has not been achieved using cytotoxin genes. We established transgenic silkworms with posterior silk glands (PSGs) that express the enzymatic domain of the cytotoxin pierisin-1A (P1A). The larvae with the modified PSGs produced the sericin cocoons with potential utilities in tissue engineering. The targeted P1A expression was found to cause site-specific repression of certain protein synthesis that appeared to have no impact on the developmental stages of individuals. Thus, the new approach through targeted P1A expression could be applicable to the development of biologically useful model organisms with tissue-specific dysfunction.