**6. Conclusions**

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 We also recently isolated a putative *gce* homolog from a Bombyliid, *Bombylius major* (A.A.B., unpublished).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

Molecular Evolution of Juvenile Hormone Signaling 347

fails to rescue the phenotypes of deficient oogenesis or reduced male courtship characteristic of *Met* adults, showing that *Met* has co-opted therole as the mediator of JH-regulated

RNAi-driven reduction of *gce* expression from either an *actin* or *tubulin* promoter demonstrates that unlike *Met*, *gce* is a vital gene. G*ce* underexpression in both *Met+* and *Met*  genetic backgrounds results in lethality. *Met+ ; UAS-gce-dsRNA / tubulin-GAL4* homo- /hemizygotes do not survive to adulthood, and die primarily at the pharate adult stage, while the same *gce* RNAi construct expressed in a *Met* mutant background shifts lethality to

Previously, USP was proposed as a candidate JH receptor (Jones, *et al*., 2001). Yet, USP only binds JH with micromolar affinity, requiring a hormone concentration that exceeds endogenous titers by orders of magnitude (Bownes & Rembold, 1987). It is now known that USP binds methyl farnesoate (MF), a precursor in the biological synthesis of JH III (Figure 1), with nanomolar affinity both in *D. melanogaster* and in *A. aegypti* (Jones *et al*., 2006; Jones *et al*., 2010). Recent studies on natural farnesoid derivatives including MF, JH III, and JHB3 (the main farnesoid secretion product of dipteran ring glands cultured *in vitro*) have teased out the relative activities of each of these compounds during development in a series of biological assays. Two recent studies have demonstrated that the activity series of these three compounds changes during development. Dietary MF and JH III (MF > JH III) were both more active than JHB3 in delaying larval attainment of the wandering stage. In contrast, JH III applied to prepupae (white puparial assay; Riddiford & Ashburner, 1991) showed much higher activity than MF or JHB3 in blocking adult eclosion (Jones *et al*., 2010;

Topical application or dietary provision of these compounds adds to endogenous hormone titers. Therefore, just as USP binds JH III at concentrations exceeding physiological levels, it is possible that MET nonspecifically binds MF or JHB3 under these conditions. It is an intriguing proposition that MET and USP, which interact both with each other and JHRE binding proteins (Bitra & Palli, 2009), may partner in a stage-specific manner throughout development in response to a fluctuating mélange of methyl farnesoids. Does GCE participate in the assembly of the molecular machinery that facilitates the crosstalk between JH and 20E signaling? This protein has been largely ignored in studies regarding the molecular interaction of these hormones. Further, it is unknown whether GCE binds any of the farnesoid products of the CA. There appears to be a correlation between the presence of paralogous *Met*-like genes and multiple JH isoforms in higher Diptera. If each of the farnesoids JH III, MF, and JHB3 indeed has a unique receptor protein, the possibility arises that GCE fills the role of JHB3 binder. Or perhaps in MET/GCE dimers, MET is the sole ligand binder, while GCE and MET are both necessary for target gene transcription. Clearly, further functional characterization of GCE is necessary to unravel the mechanisms through which JH signaling has evolved from the basal holometabola to the most evolutionarily

We extend thanks to Drs. John Freudenstein and H. Lisle Gibbs for their guidance in our various evolutionary analyses. Additionally, we would like to recognize Dr. Shaoli Wang

reproductive functions in *Drosophila*.

early pupae (Baumann *et al*., 2010a).

**6.1 Directions** 

Harshman *et al*., 2010).

diverged insects, the higher Diptera.

**7. Acknowledgments** 

genomic DNA templates, suggesting the possibility that the *Met/gce* duplication occurred within the Brachycera. *Met* function is evolutionarily conserved in Diptera; consistent with independent reports (Zhu *et al*., 2010) we observed that RNAi-driven reduction of *AaMet*  results in concomitant reduction of JH-inducible genes (Figure 4).

Fig. 4. Expression of three JH-inducible genes following RNAi-induced knockdown of *AaMet* (ds*Met*: orange bars) vs. controls (ds*B-gal*: red bars). *AaMet* reduction produced concomitant suppression of the *A. aegypti* homologs of *DmJHE*, *DmJhI-1*, and *DmJhI-26*.

Analysis of the nonsynonymous-to-synonymous substitution ratios (dN/dS) of *Met* and *gce*  orthologs within the genus *Drosophila* indicates a substantial relaxation of selective constraint on the C-terminal half of *gce*, downstream of the functional domains. Conversely, nonsynonymous substitutions in the N-terminal half are stringently selected against. Depressed dN/dS values across the *Met* coding sequence indicate strong selective constraint over the entire open reading frame (Baumann *et al*., 2010b).

RT-PCR analysis of selected *D. melanogaster* tissues shows that *gce* is generally co-expressed with *Met* in known JH target tissues, including ovary and MAG. Overexpression of *gce* in a *Met* mutant background results in a dramatic enhancement of methoprene-conditional toxic and morphogenetic defects, similar to those seen in wild type (*Met+*) flies after methoprene exposure. *Met* mutant flies overexpressing *gce* show rescue of a non-conditional adult phenotype, that of defective development of posterior facets in the compound eye. Our results therefore support the notion of functional redundancy that has been hypothesized to account for *Met27* viability flies. On the other hand, we have also shown that these paralogs have undergone evolutionary subfunctionalization since their origin; *gce* overexpression fails to rescue the phenotypes of deficient oogenesis or reduced male courtship characteristic of *Met* adults, showing that *Met* has co-opted therole as the mediator of JH-regulated reproductive functions in *Drosophila*.

RNAi-driven reduction of *gce* expression from either an *actin* or *tubulin* promoter demonstrates that unlike *Met*, *gce* is a vital gene. G*ce* underexpression in both *Met+* and *Met*  genetic backgrounds results in lethality. *Met+ ; UAS-gce-dsRNA / tubulin-GAL4* homo- /hemizygotes do not survive to adulthood, and die primarily at the pharate adult stage, while the same *gce* RNAi construct expressed in a *Met* mutant background shifts lethality to early pupae (Baumann *et al*., 2010a).

### **6.1 Directions**

346 Gene Duplication

genomic DNA templates, suggesting the possibility that the *Met/gce* duplication occurred within the Brachycera. *Met* function is evolutionarily conserved in Diptera; consistent with independent reports (Zhu *et al*., 2010) we observed that RNAi-driven reduction of *AaMet* 

M et JHE (AAEL012886) JhI-1 (AAEL006600) JhI-26 (AAEL000516)

dsB-gal dsM et

Fig. 4. Expression of three JH-inducible genes following RNAi-induced knockdown of *AaMet* (ds*Met*: orange bars) vs. controls (ds*B-gal*: red bars). *AaMet* reduction produced concomitant suppression of the *A. aegypti* homologs of *DmJHE*, *DmJhI-1*, and *DmJhI-26*.

over the entire open reading frame (Baumann *et al*., 2010b).

Analysis of the nonsynonymous-to-synonymous substitution ratios (dN/dS) of *Met* and *gce*  orthologs within the genus *Drosophila* indicates a substantial relaxation of selective constraint on the C-terminal half of *gce*, downstream of the functional domains. Conversely, nonsynonymous substitutions in the N-terminal half are stringently selected against. Depressed dN/dS values across the *Met* coding sequence indicate strong selective constraint

RT-PCR analysis of selected *D. melanogaster* tissues shows that *gce* is generally co-expressed with *Met* in known JH target tissues, including ovary and MAG. Overexpression of *gce* in a *Met* mutant background results in a dramatic enhancement of methoprene-conditional toxic and morphogenetic defects, similar to those seen in wild type (*Met+*) flies after methoprene exposure. *Met* mutant flies overexpressing *gce* show rescue of a non-conditional adult phenotype, that of defective development of posterior facets in the compound eye. Our results therefore support the notion of functional redundancy that has been hypothesized to account for *Met27* viability flies. On the other hand, we have also shown that these paralogs have undergone evolutionary subfunctionalization since their origin; *gce* overexpression

results in concomitant reduction of JH-inducible genes (Figure 4).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Previously, USP was proposed as a candidate JH receptor (Jones, *et al*., 2001). Yet, USP only binds JH with micromolar affinity, requiring a hormone concentration that exceeds endogenous titers by orders of magnitude (Bownes & Rembold, 1987). It is now known that USP binds methyl farnesoate (MF), a precursor in the biological synthesis of JH III (Figure 1), with nanomolar affinity both in *D. melanogaster* and in *A. aegypti* (Jones *et al*., 2006; Jones *et al*., 2010). Recent studies on natural farnesoid derivatives including MF, JH III, and JHB3 (the main farnesoid secretion product of dipteran ring glands cultured *in vitro*) have teased out the relative activities of each of these compounds during development in a series of biological assays. Two recent studies have demonstrated that the activity series of these three compounds changes during development. Dietary MF and JH III (MF > JH III) were both more active than JHB3 in delaying larval attainment of the wandering stage. In contrast, JH III applied to prepupae (white puparial assay; Riddiford & Ashburner, 1991) showed much higher activity than MF or JHB3 in blocking adult eclosion (Jones *et al*., 2010; Harshman *et al*., 2010).

Topical application or dietary provision of these compounds adds to endogenous hormone titers. Therefore, just as USP binds JH III at concentrations exceeding physiological levels, it is possible that MET nonspecifically binds MF or JHB3 under these conditions. It is an intriguing proposition that MET and USP, which interact both with each other and JHRE binding proteins (Bitra & Palli, 2009), may partner in a stage-specific manner throughout development in response to a fluctuating mélange of methyl farnesoids. Does GCE participate in the assembly of the molecular machinery that facilitates the crosstalk between JH and 20E signaling? This protein has been largely ignored in studies regarding the molecular interaction of these hormones. Further, it is unknown whether GCE binds any of the farnesoid products of the CA. There appears to be a correlation between the presence of paralogous *Met*-like genes and multiple JH isoforms in higher Diptera. If each of the farnesoids JH III, MF, and JHB3 indeed has a unique receptor protein, the possibility arises that GCE fills the role of JHB3 binder. Or perhaps in MET/GCE dimers, MET is the sole ligand binder, while GCE and MET are both necessary for target gene transcription. Clearly, further functional characterization of GCE is necessary to unravel the mechanisms through which JH signaling has evolved from the basal holometabola to the most evolutionarily diverged insects, the higher Diptera.

## **7. Acknowledgments**

We extend thanks to Drs. John Freudenstein and H. Lisle Gibbs for their guidance in our various evolutionary analyses. Additionally, we would like to recognize Dr. Shaoli Wang

Molecular Evolution of Juvenile Hormone Signaling 349

Crews, S.T., 2003. PAS Proteins, Regulators and Sensors of Development and Physiology.

Dame, D.A., Wichterman, G.J., Hornby, J.A., 1998. Mosquito *Aedes taeniorhynchus* resistance

Davis, R.E., Kelly, T.J., Masler, E.P., Fescemyer, H.W., Thyagaraja, B.S., Borkovec, A.B. 1990.

Denlinger, D.L., 1985. Hormonal control of diapause. In: Kerkut, G.A., Gilbert, L.I. (Eds.),

Dubrovsky E.B., Dubrovskaya ,V.A., Bilderback, A. L., Berger, E.M. 2000. The isolation of

Dubrovsky, E.B., Dubrovskaya, V.A., Berger, E.M., 2002. Juvenile hormone signaling during oogenesis in *Drosophila*. Insect Biochemistry and Molecular Biology. 32:1555-1565. Dubrovsky, E.B., Dubrovskaya V.A., Berger E.M. 2004. Hormonal regulation and functional

Erezyilmaz, D.F., Riddiford, L.M., Truman, J.W. 2006. The pupal specifier *broad* directs

Flatt, T., Heyland, A., Rus, F., Porpiglia, E., Sherlock, C., Yamamoto, R., Garbuzov, A., Palli,

Flaveny, C.A., Murray, I.A., Perdew, G.H., 2010. Differential gene regulation by the human and mouse *aryl hydrocarbon receptor*. Toxicological Sciences. 114:217-225. Godlewski, J., Wang, S., and Wilson, T.G., 2006. Interaction of bHLH-PAS proteins involved

Harshman, L.G., Song, K.D., Casas, J., Schuurmans, A., Kuwano, E., Kachman, S.D.,

Huang, Z. J., Edery, I., Rosbash, M. l993. PAS is a dimerization domain common to *Drosophila Period* and several transcription factors. Nature. 364: 259-262. Juhász, G., Puskás, L.G., Komonyi, O., Erdi, B., Maróy, P., Neufeld, T.P., Sass, M. 2007. Gene

Kethidi, D.R., Xi, Z., Palli, S.R. 2005. Developmental and hormonal regulation of juvenile

Konopova, B., Jindra, M., 2007. Juvenile hormone resistance gene *Methoprene-tolerant*

*Drosophila* fat body. Cell Death and Differentiation. 14:1181-1190.

the National Academy of Sciences, USA. 10419:10488-10493.

methoprene. Journal of Insect Physiology, 36:231-238.

pathway. Developmental Biology. 268:258-270.

Communications. 3424:1305-1311.

National Academy of Sciences, USA. 103:6925-6930.

to methoprene in an isolated habitat. Journal of the American Mosquito Control

Hormonal control of vitellogenesis in the gypsy moth, *Lymantria dispar* (L.): suppression of haemolymph vitellogenein by the juvenile hormone analogue,

Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 8.

two juvenile hormone-inducible genes in *Drosophila melanogaster*. Developmental

role of *Drosophila* E75A orphan nuclear receptor in the juvenile hormone signaling

progressive morphogenesis in a direct-developing insect. Proceedings of the

S.R., Tatar, M., Silverman, N. 2008. Hormonal regulation of the humoral innate immune response in *Drosophila melanogaster*. Journal of Experimental Biology.

in juvenile hormone reception in *Drosophila*. Biochemical and Biophysical Research

Riddiford, L.M., Hammock, B.D. 2010. Bioassays of compounds with potential juvenoid activity on Drosophila melanogaster: juvenile hormone III, bisepoxide juvenile hormone III and methyl farnesoates. Journal of Insect Physiology. 56:1465-

expression profiling identifies FKBP39 as an inhibitor of autophagy in larval

hormone esterase gene in *Drosophila melanogaster*. Journal of Insect Physiology.

controls entry into metamorphosis in the beetle *Tribolium castaneum*. Proceedings of

Boston, Kluwer.

Association. 14:200-203.

Pergamon, New York. 37-84.

Biology. 224:486-495.

211:2712-24.

1470.

51:393-400.

for her expertise and contributions to much of the work presented herein, and for her mentorship of AAB. Work presented in this text was supported by NIH grant AI052290 to T.G.W.
