**5. Evolutionary conservation of JH signaling mechanisms**

Numerous JH target genes have been identified throughout Holometabola. Importantly, many of these genes are known components of the early 20E response. Table 1 lists some representative JH-inducible genes.


Table 1. Representative JH-inducible genes. Many of these genes have evolutionarily conserved roles in JH signaling in holometabolous insects. In addition, several are known components of the 20E transcriptional cascade.

The majority of the work done in our lab has been carried out on *D. melanogaster*, in which *DmMet* clearly plays a role in JH signaling: its absence both interferes with methoprene toxicity (Wilson & Fabian, 1986) and hinders JH-driven reproductive physiology (Wilson, 1992; Wilson *et al*., 2003). However, *Met* involvement in metamorphosis has been difficult to demonstrate in *Drosophila* (Riddiford, 2008). As previously stated, JH exposure has no effect on dipteran entry into metamorphosis, unlike other insects (Williams, 1961; Zhou & Riddiford, 2002). In recent years, researchers have turned to the model coleopteran, *T.* 

Differential intensity of transgene expression from *actin* and *tubulin* promoters was

*gce* underexpression had no observable effect on embryonic development, a stage during which no role for JH has been demonstrated. We have shown that *gce* transcription begins after about eight hours in early embryos, in contrast to *Met*, which is supplied as a maternal message (Baumann *et al*., 2010a). The importance of such divergence in temporal expression

Numerous JH target genes have been identified throughout Holometabola. Importantly, many of these genes are known components of the early 20E response. Table 1 lists some

transcription factor

transmembrane transporter

transmembrane transporter

RNA polymerase II transcription factor

Phosphoenolpyruvate carboxykinase (GTP)

*JHE* JH esterase JH-specific esterase Kethidi *et al.,* 2005

activity

activity

Table 1. Representative JH-inducible genes. Many of these genes have evolutionarily conserved roles in JH signaling in holometabolous insects. In addition, several are known

The majority of the work done in our lab has been carried out on *D. melanogaster*, in which *DmMet* clearly plays a role in JH signaling: its absence both interferes with methoprene toxicity (Wilson & Fabian, 1986) and hinders JH-driven reproductive physiology (Wilson, 1992; Wilson *et al*., 2003). However, *Met* involvement in metamorphosis has been difficult to demonstrate in *Drosophila* (Riddiford, 2008). As previously stated, JH exposure has no effect on dipteran entry into metamorphosis, unlike other insects (Williams, 1961; Zhou & Riddiford, 2002). In recent years, researchers have turned to the model coleopteran, *T.* 

Zhou *et al*., 1998;

Riddiford, 2002

Dubrovsky *et al.,*

Beckstead *et al.,*

Zhou &

2002

2004

2007

Heme binding Dubrovsky *et al.,*

Symbol Gene name Molecular function Reference *JhI-1* JH inducible protein 1 Endoribonuclease Dubrovsky *et al.,*

*JhI-26* JH inducible protein 26 Unknown 2000

previously reported in our lab (Barry *et al*., 2008).

**5. Evolutionary conservation of JH signaling mechanisms** 

*Br* Broad-Complex (BR-C) BTB POZ zinc finger

*mnd* Minidisks Amino acid

*JhI-21* JH inducible protein 21 Amino acid

profiles is unclear.

representative JH-inducible genes.

*E75A* Ecdysone-induced

*E74B* Ecdysone-induced

*pepck* Phosphoenolpyruvate

protein 75B

protein 74EF

carboxykinase

components of the 20E transcriptional cascade.

CG14949 CG14949 Unknown

*castaneum*. These beetles are both amenable to genetic manipulation and gene knockdown owing to the dramatic effects of systemic RNAi, and the larvae of this species are very sensitive to JH, unlike *D. melanogaster* larvae. Exposure to JH or a number of its chemical analogs precipitates supernumerary larval instars, similar to the effects of JH on the model lepidopteran, *Manduca sexta* (Parthasarathy & Palli, 2009). *T. castaneum*, like mosquitoes, has a single *Met-like* gene. In their seminal paper, Konopova and Jindra (2007) demonstrated that RNAi-mediated knockdown of *TcMet* results not only in a methoprene resistance phenotype, but also in the precocious metamorphosis of early instar larvae. A long soughtafter result, the genetic reduction of *TcMet* provided the phenotype frustratingly absent in *D. melanogaster*: metamorphic disruption. Reproductive roles for *TcMet* have also been shown; *TcMet* knockdown results in a substantial decrease in vitellogenin transcription, (Parthasarathy, *et al.*, 2010) consistent with *Met* deficiency in *D. melanogaster* females (Wilson & Ashok, 1998). These results demonstrate that the single *Met-*like genes in primitive Holometabola function in both development (metamorphosis) and reproduction. Further functional characterization of *TcMe*t (and the single *Met*-like gene of lower Diptera) could lead to a better understanding of how *DmMet* has apparently co-opted reproductive functional roles from a *gce*-like ancestor in higher Diptera

### **5.1 JH regulation of the E-20 transcriptional cascade**

The molecular networks that link JH and 20E signaling pathways form the foundation of multiple aspects of insect physiology, as evidenced by the criticality of both hormones in development, reproduction, and diapause (Zhou & Riddiford, 2002; Soller *et al.,* 1999; Denlinger, 1985). *Broad Complex* (*Broad* or *BR-C*) is an early gene in the 20E cascade that encodes a family of alternatively spliced zinc finger transcription factors (four in *D. melanogaster*, Z1-Z4) fused to a common core protein. Certain *Broad* alleles phenocopy the morphogenetic defects incurred by methoprene exposure in *D. melanogaster*. Wilson *et al* (2006) showed phenotypic synergism in *Met* and *broad* double mutants, demonstrating JHsensitive MET and BROAD interaction (BROAD protein accumulation is comparable to that of wild type flies, suggesting physical interaction with, rather than transcriptional regulation by *Met*), and providing a link between JH and 20E signaling (Wilson *et al*., 2006).

In a hemimetabolous insect, *Oncopeltus fasciatus*, continuous *Broad* expression directs progressive development through nymphal instars (Erezyilmaz *et al.,* 2006). In Holometabola, *Broad* expression is confined to the prepupal stage, acting as a pupal specifier (Zhou & Riddiford, 2002). Loss of *Broad* expression, characteristic of the *npr1* mutant (*nonpupariating*; a deletion of the entire complementation group), results in the namesake phenotype of failure to enter the pupal program. Consequently, a restriction of *Broad*  expression during this developmental stage may have contributed to the evolution of complete metamorphosis. During larval development in *D. melanogaster*, JH represses *broad*. At pupariation, exogenous JH induces a second wave of *broad* expression in the abdominal epidermis, resulting in the deposition of a second pupal cuticle (Zhou & Riddiford, 2002), demonstrating that the networks underlying these signaling mechanisms are complex.

In *T. castaneum*, methoprene exposure induces *Broad* expression and this upregulation is ablated upon *TcMet* knockdown. Therefore, *TcMet* is upstream of *Broad* in JH signaling in these beetles (Konopova & Jindra, 2008). *Krüppel homolog 1* (*Kr-h1*) is upstream of *Broad* in *D. melanogaster* JH signaling, where its expression in abdominal epidermis produces sternal bristle disruption similar to that seen following low dose JHA exposure (Minakuchi *et al*.,

Molecular Evolution of Juvenile Hormone Signaling 345

(Chen, J.D., 2000; Zhu, *et al*., 2006), providing yet another link between 20E and JH signaling. The authors also report that coexpression of *DmMet* or *Dmgce* with *DmTaiman* (the *D. melanogaster AaFISC* ortholog) in the presence of JH III induced reporter gene expression in L57 cells (Li *et al*., 2011). Furthermore, this interaction has also been demonstrated in *T. castaneum* between *TcMet* and the FISC/TAI homolog, *TcSRC* (Steroid receptor coactivator; Zhang *et al*., 2011). This gene has previously been implicated in metamorphic activity. *T. castaneum* larvae treated with SRC RNAi fail to achieve critical weight and consequently die before the larval-pupal transition (Bitra *et al*., 2009). Therefore, MET interaction with FISC/SRC/TAIMAN underpins key transcriptional events of JH signaling throughout

Structure-function analyses performed using site-directed mutagenesis identified regions of MET that are necessary for homodimerization and GCE binding. Point mutations in the bHLH and PAS A domains (*Met1* and *Metw3* alleles, respectively) had no effect on partner binding, whereas N- and C-terminal truncations, deletions in the HLH or PAS A domains, and a point mutation in the PAS B domain (*Met128* allele) all inhibited dimerization (Godlewski, *et al.,* 2006). Structure-function data for AaMET and AaFISC binding illustrates that the criticality of PAS domains for protein-protein interaction. Interestingly, two-hybrid assays showed that MET/FISC interaction increased when AaMET lacked a bHLH domain (Li *et al*., 2011). Therefore, this domain is unnecessary for MET-FISC interaction, suggesting that the sole function of the *AaMet* bHLH may be in DNA binding. In contrast, deletion of

Mixespression of *DmTaiman* in a variety of *Met*/*gce* genetic backgrounds will be valuable from both physiological and evolutionary perspectives. How do these proteins interact in the context of hormonal control of *D. melanogaster* development? Presumably, during larval development JH secreted from the CA inhibits MET and GCE interaction while promoting MET and GCE binding with TAIMAN. Are MET:TAI and GCE:TAI dimers functionally congruent in *D. melanogaster* or do these complexes preferentially regulate disparate target genes? Is the *Met*-like gene in *A. aegypti* and *T. castaneum* functionally analogous to *Met*/*gce* or are other, unidentified proteins involved? How has the interaction of these proteins

RT-PCR analysis with degenerate primers identified a single *Met*-like homolog in the genome of each of the three mosquito species, *Aedes aegypti, Anopheles gambiae*, and *Culex pipiens*. Likewise, a single *Met*-like ortholog exists in the beetle, *T. castaneum*, (Konopva & Jindra, 2007). Phylogenetic analysis and comparison of intron number and position in each of the identified mosquito genes indicates that the mosquito *Met* orthologs share higher sequence identity with *Dmgce* than *DmMet*, suggesting that *DmMet* arose from the duplication of an ancestral, *gce-*like gene in lower Diptera. To examine the evolutionary history of *Met* and *gce* within the Diptera, we mined the public *G. morsitans* EST library, recovering unique putative *Met* and *gce* orthologs in this fly, showing conservation of a *Met*  homolog within the Schizophora. We also recently isolated a putative *gce* homolog from a Bombyliid, *Bombylius major* (A.A.B., unpublished). Taxonomically, this group of flies exists in the Asilomorpha, a paraphyletic sister taxon to the Muscomorpha within the dipteran infraorder, Brachycera. While this study is in its preliminary stages, only a single *Met*-like gene has thus far been obtained from this fly using degenerate PCR with cDNA and

the bHLH domain in FISC hindered the JH-induced interaction with AaMET.

changed during dipteran evolution following the origin of *Met*?

holometabolous insects.

**6. Conclusions** 

2008). Genetic suppression of *TcKr-h1* induces precocious metamorphosis, similar to *TcMet* deficiency; *TcMet* knockdown in combination with JHA treatment demonstrated that *TcKrh1* exists downstream of *TcMet* and upstream of *TcBroad* (Minakuchi *et al*., 2009). Similarly, *Kr-h1* upregulation in newly eclosed *A. aegypti* females depends on *AaMet* expression (Zhu *et al.,* 2010). Therefore, the relationships among these genes are generally conserved within holometabolan evolution.

While *Kr-h1* also has demonstrated roles in JH-influenced social behavior of honeybees, it has not been reported whether *AmKr-h1* is under the transcriptional control of an *A. mellifera Met*-like protein. However, there is evidence for conservation in the sets of genes regulated by JH between flies and bees. This is perhaps unsurprising given the deep evolutionary conservation of these genetic mechanisms; *Kr-h1* and *Broad* expression profiles in two species of hemimetabolous thrips, whose life histories involve pupa-like, quiescent or nonfeeding stages, are compatible with the expression profiles of *Broad* and *Kr-h1* in holometabolous insects (Minakuchi *et al*., 2011).

Microarray data from *D. melanogaster* and *A. mellifera* identified a subset of conserved, JHinducible genes (Li *et al.,* 2007). In the promoter region of 16 of the *D. melanogaster* orthologs, a conserved JH response element (JHRE) was identified. RNAi-driven reduction of the expression of two proteins identified as JHRE binders, *FKBP39* and *Chd64*, inhibits JHIIIinduced expression of a reporter construct, suggesting their involvement in JH-dependent transcriptional machinery. Bitra and Palli (2009) demonstrated physical interaction of MET with both ECR and USP. Furthermore, column pulldown assays showed FKBP39 and CHD64 as binding partners of *D. melanogaster* ECR, USP, and MET, providing a more robust framework for a protein complex involving constituents of both JH and 20E signaling pathways (Li *et al*.*,* 2007). *FKBP39*, which is present at the onset of metamorphosis (Riddiford, 2008), is an inhibitor of autophagy in *D. melanogaster*; *FKBP39* overexpression precludes the developmental autolysis of larval fat body cells in wandering third instar larvae (Juhász *et al*.*,* 2007), a physiology shown to be partially dependent on MET/GCE regulation of caspase gene expression (Liu *et al*., 2009). A role for GCE in any of these protein complexes has yet to be reported. *Chd64* is expressed during larval molts, but not in the third instar or during metamorphosis (Riddiford, 2008). Accordingly, putative regulatory complexes consisting of different combinations of these elements may assemble in a stage- or tissue-specific manner. Assembly of differential protein complexes in response to JH, 20E, or both could be a strategy for the tight regulation of the activities of these counteracting hormones.

The *Met*-like genes of *Tribolium* and *Drosophila* appear to act in similar genetic environments to regulate the expression of members of the 20E induced transcriptional cascade, including EcR (Riddiford *et al*., 2010) and USP (Xu *et al*., 2010), the heterodimeric components of the ecdysone receptor, various orphan nuclear receptors involved in 20E activity, and 20E-induced caspase genes involved in PCD (Liu *et al*., 2009). Knockdown of seven nuclear receptors (E75, HR3, EcR, USP, SVP, FTZ-F1, and HR4) results in a significant reduction of vitellogenin prouction in *T. castaneum* (Xu *et al*., 2010), a phenotype similar to that obtained via *TcMet* knockdown (Parthasarathy & Palli, 2009). The data presented in this section therefore strongly support for the action of *Met*-like genes as crucial to 20E/JH crosstalk.

### **5.2 Discovery of an evolutionarily conserved** *Met* **binding partner**

Recent biochemical data from *A. aegypti* indicate that *AaMet* binds another bHLH PAS gene, *AaFISC*, and that this interaction requires a high JH titer. FISC is a coactivator of EcR/USP

2008). Genetic suppression of *TcKr-h1* induces precocious metamorphosis, similar to *TcMet* deficiency; *TcMet* knockdown in combination with JHA treatment demonstrated that *TcKrh1* exists downstream of *TcMet* and upstream of *TcBroad* (Minakuchi *et al*., 2009). Similarly, *Kr-h1* upregulation in newly eclosed *A. aegypti* females depends on *AaMet* expression (Zhu *et al.,* 2010). Therefore, the relationships among these genes are generally conserved within

While *Kr-h1* also has demonstrated roles in JH-influenced social behavior of honeybees, it has not been reported whether *AmKr-h1* is under the transcriptional control of an *A. mellifera Met*-like protein. However, there is evidence for conservation in the sets of genes regulated by JH between flies and bees. This is perhaps unsurprising given the deep evolutionary conservation of these genetic mechanisms; *Kr-h1* and *Broad* expression profiles in two species of hemimetabolous thrips, whose life histories involve pupa-like, quiescent or nonfeeding stages, are compatible with the expression profiles of *Broad* and *Kr-h1* in

Microarray data from *D. melanogaster* and *A. mellifera* identified a subset of conserved, JHinducible genes (Li *et al.,* 2007). In the promoter region of 16 of the *D. melanogaster* orthologs, a conserved JH response element (JHRE) was identified. RNAi-driven reduction of the expression of two proteins identified as JHRE binders, *FKBP39* and *Chd64*, inhibits JHIIIinduced expression of a reporter construct, suggesting their involvement in JH-dependent transcriptional machinery. Bitra and Palli (2009) demonstrated physical interaction of MET with both ECR and USP. Furthermore, column pulldown assays showed FKBP39 and CHD64 as binding partners of *D. melanogaster* ECR, USP, and MET, providing a more robust framework for a protein complex involving constituents of both JH and 20E signaling pathways (Li *et al*.*,* 2007). *FKBP39*, which is present at the onset of metamorphosis (Riddiford, 2008), is an inhibitor of autophagy in *D. melanogaster*; *FKBP39* overexpression precludes the developmental autolysis of larval fat body cells in wandering third instar larvae (Juhász *et al*.*,* 2007), a physiology shown to be partially dependent on MET/GCE regulation of caspase gene expression (Liu *et al*., 2009). A role for GCE in any of these protein complexes has yet to be reported. *Chd64* is expressed during larval molts, but not in the third instar or during metamorphosis (Riddiford, 2008). Accordingly, putative regulatory complexes consisting of different combinations of these elements may assemble in a stage- or tissue-specific manner. Assembly of differential protein complexes in response to JH, 20E, or both could be a strategy

The *Met*-like genes of *Tribolium* and *Drosophila* appear to act in similar genetic environments to regulate the expression of members of the 20E induced transcriptional cascade, including EcR (Riddiford *et al*., 2010) and USP (Xu *et al*., 2010), the heterodimeric components of the ecdysone receptor, various orphan nuclear receptors involved in 20E activity, and 20E-induced caspase genes involved in PCD (Liu *et al*., 2009). Knockdown of seven nuclear receptors (E75, HR3, EcR, USP, SVP, FTZ-F1, and HR4) results in a significant reduction of vitellogenin prouction in *T. castaneum* (Xu *et al*., 2010), a phenotype similar to that obtained via *TcMet* knockdown (Parthasarathy & Palli, 2009). The data presented in this section therefore strongly support for the action of *Met*-like

Recent biochemical data from *A. aegypti* indicate that *AaMet* binds another bHLH PAS gene, *AaFISC*, and that this interaction requires a high JH titer. FISC is a coactivator of EcR/USP

for the tight regulation of the activities of these counteracting hormones.

**5.2 Discovery of an evolutionarily conserved** *Met* **binding partner** 

holometabolan evolution.

holometabolous insects (Minakuchi *et al*., 2011).

genes as crucial to 20E/JH crosstalk.

(Chen, J.D., 2000; Zhu, *et al*., 2006), providing yet another link between 20E and JH signaling. The authors also report that coexpression of *DmMet* or *Dmgce* with *DmTaiman* (the *D. melanogaster AaFISC* ortholog) in the presence of JH III induced reporter gene expression in L57 cells (Li *et al*., 2011). Furthermore, this interaction has also been demonstrated in *T. castaneum* between *TcMet* and the FISC/TAI homolog, *TcSRC* (Steroid receptor coactivator; Zhang *et al*., 2011). This gene has previously been implicated in metamorphic activity. *T. castaneum* larvae treated with SRC RNAi fail to achieve critical weight and consequently die before the larval-pupal transition (Bitra *et al*., 2009). Therefore, MET interaction with FISC/SRC/TAIMAN underpins key transcriptional events of JH signaling throughout holometabolous insects.

Structure-function analyses performed using site-directed mutagenesis identified regions of MET that are necessary for homodimerization and GCE binding. Point mutations in the bHLH and PAS A domains (*Met1* and *Metw3* alleles, respectively) had no effect on partner binding, whereas N- and C-terminal truncations, deletions in the HLH or PAS A domains, and a point mutation in the PAS B domain (*Met128* allele) all inhibited dimerization (Godlewski, *et al.,* 2006). Structure-function data for AaMET and AaFISC binding illustrates that the criticality of PAS domains for protein-protein interaction. Interestingly, two-hybrid assays showed that MET/FISC interaction increased when AaMET lacked a bHLH domain (Li *et al*., 2011). Therefore, this domain is unnecessary for MET-FISC interaction, suggesting that the sole function of the *AaMet* bHLH may be in DNA binding. In contrast, deletion of the bHLH domain in FISC hindered the JH-induced interaction with AaMET.

Mixespression of *DmTaiman* in a variety of *Met*/*gce* genetic backgrounds will be valuable from both physiological and evolutionary perspectives. How do these proteins interact in the context of hormonal control of *D. melanogaster* development? Presumably, during larval development JH secreted from the CA inhibits MET and GCE interaction while promoting MET and GCE binding with TAIMAN. Are MET:TAI and GCE:TAI dimers functionally congruent in *D. melanogaster* or do these complexes preferentially regulate disparate target genes? Is the *Met*-like gene in *A. aegypti* and *T. castaneum* functionally analogous to *Met*/*gce* or are other, unidentified proteins involved? How has the interaction of these proteins changed during dipteran evolution following the origin of *Met*?
