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

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

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

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

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

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

Molecular Evolution of Juvenile Hormone Signaling 337

of the genus *Drosophila,* some 63 million years ago (Tamura *et al*., 2004). The architecture of these genes is generally conserved in each species, with a few notable exceptions. A single conserved intron is present in *Met* in the PAS B domain of 11 species. In addition to this conserved intron, independent intron gains have occurred in the lineages leading to *D. simulans* and *D. willistoni*. A single *Met* ortholog exists in each *Drosophila* genome examined, but *D. persimilis* harbors two separate, consecutive loci on the X chromosome, currently called GL13106 and GL13107, that align to distinct regions of *DmMet*. The 5' putative gene GL13106 contains a complete PAS A domain followed by a severely truncated PAS B domain. We performed RT-PCR across these two genes and failed to obtain a single PCR product, suggesting that GL13106 and GL13107 indeed code for two distinct open reading frames. Eleven of the 12 representative *gce* orthologs contain at least six conserved introns, with independent intron gains evident in the lineages leading to *D. melanogaster*, *D. pseudoobscura*, and *D. mojavensis*, whereas a substantial deletion in *D. persimilis gce* has

In addition to the bHLH, PAS, and PAC domains, putative transactivation domains (TAD) are evident in *Met* and *gce* orthologs. TADs are glutamine and/or aspartic acid-rich motifs whose amino acid sequences are broadly defined and generally reside in the C-terminal region of PAS proteins (Ramadoss & Perdew, 2005). *Met* homologs show Q- and D-rich motifs between the PAS B and PAC domains, while alignments of *gce* homologs indicate a D-rich region C-terminal to the PAC domain. Miura *et al*. (2005) suggest the presence of a Cterminal TAD in recombinant MET protein, but this region has yet to be functionally

Using *DmMet* and *Dmgce* as query sequences, we conducted homology searches under tBLASTx criteria (translated nucleotide query to search a translated nucleotide database) against the publicly available EST library of *Glossina morsitans*, the tsetse fly. Our search recovered several clones, which were imported into the Sequencher program to produce two independent contigs. These composite nucleotide sequences were used to infer a gene tree with other holometabolan *Met* and *gce* orthologs, including those of two representative *Drosophila* species (Figure 2). This preliminary analysis reveals the presence of distinct *Met* and *gce* orthologs in the *G. morsitans* genome, indicating that the origin of *Met* predates the divergence of the Aschiza and Schizophora. These two taxonomic groups, which are estimated to have diverged more than 85 million years ago (Bertone & Wiegmann, 2009),

Based on an *a priori* hypothesis that *Met* and *gce* were subject to differential post-duplication selective constraint, we performed analyses of nonsynonymous-to-synonymous (dN/dS) substitution ratios on codon alignments of these *Drosophila* paralogs. Datasets were analyzed using the DataMonkey tool (Kosakovsky-Pond & Frost, 2005), a web-based implementation of the HyPhy package (Kosakovsky Pond *et al*., 2005). dN/dS analyses can be used to infer the relative selective pressure along entire coding sequences or in a site-specific manner. A substantially depressed dN/dS ratio (i.e. zero or close to zero) implies purifying (negative) selection. That is, nonsynonymous changes are stringently selected against. In contrast, when dN/dS is nearly one, neutral evolution is inferred. A dN/dS value far in excess of one implies positive selection, or adaptive evolution. In this case, nonsynonymous substitutions

eliminated the central portion of this gene, including the PAS repeats.

reside within the brachyceran infraorder Muscomorpha.

confer a selective advantage.

**3.2 Evidence for differential selective constraint imposed on** *Met* **and** *gce*

defined.

reduced oogenesis (~20% compared to *Met+*), consistent with a role for JH in this physiology. However, since absence of a JH receptor is expected to preclude normal development, the viability of *Met27* flies challenged the notion of *Met* as a *bona fide* JH receptor. Some evidence supports alternative mechanism(s) of JH signaling (see Flatt *et al.,* 2008; Riddiford *et.al.*, 2010). In this chapter, we review data that support the notion of *germ cell expressed* (*gce*), the paralog of *Met* in higher Diptera, as conferring at least partial functional redundancy.
