**1.1 JH and JHAs: Insecticidal use of hormone agonists**

Since the first chemical analysis resolved the sesquiterpenoid structure of endogenous JH (Röller *et al.,* 1967), several homologs have been identified, each bearing opposing, terminal epoxide and methyl ester functions. Variation in the degree and identity of alkyl group substitution at C3, C7, and C11 along the carbon skeleton defines the homologs. The evolutionary importance of multiple JH homologs is unclear. JH 0, I, II, and III have all been isolated from lepidopteran insects, whereas JH III, the presumed evolutionary precursor to the higher homologs, is found in all insects. JH bisepoxide (JHB3) has been identified as a product of the corpus allatum (CA) in higher Diptera including *Drosophila melanogaster* and *Sarcophaga bullata* (Richard *et al.,* 1989; Bylemans *et al.,* 1998)*.* Nearly identical in structure to JH III, JHB3 is distinguished by an additional epoxide group spanning C6-C7.

Molecular Evolution of Juvenile Hormone Signaling 335

polymorphic larval epidermis gives rise to pupal and adult structures. When flies are challenged with JHAs, the adult structures that differentiate from imaginal discs remain unaffected (Postlethwait, 1974). In *D. melanogaster*, only the abdominal histoblasts are JH sensitive; diagnostic (sublethal) doses of methoprene disrupt abdominal bristle formation in

The molecular events underlying 20E signaling are relatively well understood. Ecdysone released from the prothoracic glands is converted to its active metabolite 20E in target tissues, where it regulates transcription through a heterodimeric receptor complex comprised of *Ecdysone receptor* (EcR) and *Ultraspiracle* (USP) proteins. When bound with 20E, ECR-USP recognizes and binds ecdysone response elements located in the promoter region of target genes, inducing transcription of a hierarchical network of early and late genes. The early genes either repress their own expression or induce expression of late genes (Ashburner *et al.,* 1974). In this manner, the expression of genes involved in the 20E transcriptional cascade is tightly controlled. In contrast, the nature of JH signal transduction has been difficult to elucidate, largely due to the enigmatic nature of the JH receptor. A body of ever-increasing experimental evidence strongly supports the product of the *Methoprene tolerant* (*Met*) gene as the prime candidate for a JH receptor component (Wilson & Fabian,

*Met* was originally discovered by screening progeny of ethyl methanesulfonate (EMS) mutagenized *D. melanogaster* for resistance to methoprene (Wilson & Fabian, 1986). *Met* mutants show dramatically enhanced (~100 fold) resistance to both the toxicity and morphogenetic defects caused by methoprene exposure, but not to other classes of insecticides (Wilson & Fabian, 1986). Such resistance is not restricted to compounds with high structural similarity to JH; *Met* mutants are also resistant to the more potent,

Cloning and sequence analysis identified *Met* as a member of the basic Helix-Loop-Helix *Period Ahr Sim* (bHLH PAS) family of transcriptional regulators (Ashok *et al.,* 1998). PAS proteins function as dimers in a diverse array of functions in development, xenobiotic binding, and detection of environmental signals (Crews, 1993). Both the bHLH domain and the PAS repeats (PAS A and B) facilitate dimerization between PAS proteins (Huang, *et al*., 1993). Additionally, the PAS domains function in small molecule ligand binding and target gene specificity. Each dimerization partner recognizes and binds one half of a palindromic E-box consensus sequence CANNTG in the promoter region of target genes via the stretch of basic residues immediately N-terminal to the HLH motif. Examples of PAS proteins with ligand binding activity include the bacterial *photoreactive yellow protein* (*PYP*), and the

Genetic and biochemical data show that MET binds JH with nanomolar affinity (Shemshedini & Wilson, 1990) and that MET product is present in the nuclei of several known JH target tissues, including ovary, MAG, and larval fat body (Pursley *et al.,* 2000). In addition, MET can drive the expression of a reporter gene in a JH-sensitive manner (Miura

Analysis of the *Met27* null allele provided the first demonstration of insecticide resistance due to the absence of a target macromolecule (Wilson & Ashok, 1998). Even though *Met27* flies are viable, *Met* deficiency carries reproductive consequences, namely substantially

structurally distinct JHA pyriproxyfen (Riddiford & Ashburner, 1991).

*et al.,* 2005). All of the above data satisfy criteria for a hormone receptor.

female flies (Madhavan, 1973).

**2. Molecular mechanism of JH signal transduction** 

1986; Konopova & Jindra, 2007; Yang *et al*., 2011).

vertebrate *aryl hydrocarbon receptor* (*Ahr*).

The major JHs and some juvenile hormone analogs (JHAs) are presented in Figure 1.

Fig. 1. Structures of endogenous JH molecules and two synthetic JHAs, methoprene and pyriproxyfen.

The physiology and chemistry of JH prompted intense research into the synthesis and commercial-scale production of JH analogs, or juvenoids, for agricultural use. The allure of these compounds was at least twofold. First, juvenoids exhibit extremely low nontarget (in particular, mammalian) toxicity. Second, it was originally thought that insect resistance to JHAs would be unlikely, since an insect was not likely to become refractory to an endogenous hormone (Williams, 1967). Methoprene, a juvenoid structurally similar to endogenous JH, has enjoyed success in the management of larval mosquito populations. However, JHAs need not mimic the chemical structure of endogenous JH, as exemplified by the pyridine-based pyriproxyfen, whose activity exceeds JH by two orders of magnitude in dipteran white puparial and larval assays (Riddiford and Ashburner, 1991).

Exogenous JH exposure can elicit classic antimetamorphic activity in both Lepidoptera and Coleoptera (Srivastava & Srivastava, 1983; Konopova & Jindra, 2007), extending larval development through one or more supernumerary instars. Also in these insects, exposure to exogenous JH or to its chemical analogs (JHA) can result in the deposition of a second pupal cuticle (Zhou & Riddiford, 2002). Thus, in Lepidoptera and Coleoptera, JH exposure at an inappropriate time inhibits 20E-directed developmental progression.

In Diptera, treatment with exogenous JH produces dose-dependent lethality at the pharate adult stage. All adult structures arise from imaginal discs in flies, and these discs are insensitive to JH during development, unlike Lepidoptera and Coleoptera, in which the

The major JHs and some juvenile hormone analogs (JHAs) are presented in Figure 1.

Methyl farnesoate JH III

JHB3 JH II

JH I JH 0

Methoprene Pyriproxyfen

The physiology and chemistry of JH prompted intense research into the synthesis and commercial-scale production of JH analogs, or juvenoids, for agricultural use. The allure of these compounds was at least twofold. First, juvenoids exhibit extremely low nontarget (in particular, mammalian) toxicity. Second, it was originally thought that insect resistance to JHAs would be unlikely, since an insect was not likely to become refractory to an endogenous hormone (Williams, 1967). Methoprene, a juvenoid structurally similar to endogenous JH, has enjoyed success in the management of larval mosquito populations. However, JHAs need not mimic the chemical structure of endogenous JH, as exemplified by the pyridine-based pyriproxyfen, whose activity exceeds JH by two orders of magnitude in dipteran white puparial and larval assays (Riddiford and Ashburner,

Exogenous JH exposure can elicit classic antimetamorphic activity in both Lepidoptera and Coleoptera (Srivastava & Srivastava, 1983; Konopova & Jindra, 2007), extending larval development through one or more supernumerary instars. Also in these insects, exposure to exogenous JH or to its chemical analogs (JHA) can result in the deposition of a second pupal cuticle (Zhou & Riddiford, 2002). Thus, in Lepidoptera and Coleoptera, JH exposure at an

In Diptera, treatment with exogenous JH produces dose-dependent lethality at the pharate adult stage. All adult structures arise from imaginal discs in flies, and these discs are insensitive to JH during development, unlike Lepidoptera and Coleoptera, in which the

inappropriate time inhibits 20E-directed developmental progression.

Fig. 1. Structures of endogenous JH molecules and two synthetic JHAs, methoprene and

pyriproxyfen.

1991).

polymorphic larval epidermis gives rise to pupal and adult structures. When flies are challenged with JHAs, the adult structures that differentiate from imaginal discs remain unaffected (Postlethwait, 1974). In *D. melanogaster*, only the abdominal histoblasts are JH sensitive; diagnostic (sublethal) doses of methoprene disrupt abdominal bristle formation in female flies (Madhavan, 1973).
