THE REGULATION OF JUVENILE HORMONE IN DICTYOPTERA: A FUNCTIONAL

AND EVOLUTIONARY STUDY OF USP/RXR AND ALLATOSTATIN

By

Ekaterina F. Hult

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Cell & Systems Biology

University of Toronto

© Copyright by Ekaterina F. Hult (2009)

ii

The regulation of juvenile hormone in Dictyoptera: A functional and evolutionary study of

USP/RXR and allatostatin

Ekaterina F. Hult, Master of Science Department of Cell & Systems Biology, University of Toronto, 2009

ABSTRACT

The objective of this study was to clarify the regulation of production and signal

transduction of juvenile hormone (JH) in insects by experimentally examining the function and

evolution of a putative receptor (USP/RXR) and a neuropeptide inhibitor (FGLamide

allatostatin). To examine the role of USP/RXR, the cDNA sequence of the receptor was obtained

from the cockroach Diploptera punctata. Transcript levels during developmentally critical

periods for JH sensitivity may suggest USP/RXR is JH responsive. Comparative sequence

analysis of evolutionary rates in the Mecopterida support current hypotheses which suggest some

gain in function along this lineage, although this acquisition may have occurred more gradually

than previously assumed. To examine allatostatin evolution within insects, ancestral peptides

inferred using maximum likelihood ancestral reconstruction methods were assayed for in vitro

inhibition of JH production in two cockroach species. Shifts in peptide potency in some ancestral

peptides reconstructed may be related to peptide copy number evolution.

iii

Acknowledgments

Major portions of Hult et al. (2008) have been reproduced in this thesis as Chapter Three.

Elsevier is acknowledged for the use of this material. I thank my coauthors for the permission to

reproduce this multiple author publication. In particular, I recognize C.J. Weadick who ran the

ancestral reconstruction analyses and contributed to the methods section of the paper. I am

grateful to all my coauthors, which include B.S.W. Chang and S.S. Tobe, for the fruitful

discussions that led to the final version of this publication. I also thank J.R. Zhang who provided

invaluable technical assistance with the radiochemical assays and dissections.

The staff members of the Cell and Systems Biology department, especially I. Buglass,

have been instrumental, providing guidance and friendly advice over the past few years. I convey

a heartfelt thanks to E.J. Linley who has been a source of constant encouragement. I thank all of

my labmates, past and present, with special thanks to J. Lam, N. Rahman, S.H.K. Tiu and F.

Martínez-Pérez for assisting me with my experimental work. I thank my fellow students in the

Chang lab, in particular I. van Hazel, who have offered both friendship and scientific advice. I

would also like to recognize J. Du for her assistance in estimating evolutionary rates.

I thank my supervisor S.S. Tobe, without whose guidance and attention to detail I would

never have learned to appreciate the joys and challenges of research. His support has enabled me

to grow professionally and intellectually, and I am especially appreciative of the opportunity to

attend scientific meetings and speak with key scholars in our field. Finally, I express sincere

gratitude to my advisory committee A.B. Lange and B.S.W. Chang. I have benefited greatly

from their insightful comments and continuing support throughout my studies.

iv

Table of Contents ABSTRACT................................................................................................................................... ii Acknowledgments ........................................................................................................................ iii Table of Contents ......................................................................................................................... iv List of Tables ................................................................................................................................ vi List of Figures............................................................................................................................... vi List of Abbreviations .................................................................................................................. vii Chapter One: Introduction: JH function, regulation of production, and signalling.............. 1

1.1 Production and functions ...................................................................................................... 2 1.2 Regulation of JH production................................................................................................. 6 1.3 Molecular mode of action of JH ........................................................................................... 9 1.4 Conservation of endocrine systems .................................................................................... 13 1.5 Objectives ........................................................................................................................... 15

Chapter Two: Molecular cloning and characterization of four RXR isoforms from the viviparous cockroach, Diploptera punctata ............................................................................... 18

Abstract ..................................................................................................................................... 19 2.1 Introduction......................................................................................................................... 19 2.2 Materials and methods ........................................................................................................ 23

2.2.1 Animals ........................................................................................................................ 23 2.2.2 Molecular cloning of DpRXR and Northern blot ........................................................ 24 2.2.3 Sequence comparison of functional domains .............................................................. 27 2.2.4 Estimation of evolutionary rates .................................................................................. 28 2.2.5 DpRXR expression ...................................................................................................... 29

2.3 Results................................................................................................................................. 31 2.3.1 Molecular cloning of DpRXR...................................................................................... 31 2.3.2 Sequence comparison of functional domains .............................................................. 36 2.3.3 Estimation of evolutionary rates .................................................................................. 47 2.3.4 DpRXR expression ...................................................................................................... 51

2.4 Discussion ........................................................................................................................... 58 2.5 Conclusions and future directions....................................................................................... 63

Chapter Three: Reconstruction of ancestral FGLamide-type insect allatostatins: A novel approach to the study of allatostatin function and evolution ................................................. 64

Abstract ..................................................................................................................................... 65 3.1 Introduction......................................................................................................................... 65 3.2 Materials and methods ........................................................................................................ 69

3.2.1 Ancestral reconstruction .............................................................................................. 69 3.2.2 Sequence collection and database analysis .................................................................. 71 3.2.3 Radiochemical assays of JH release in vitro................................................................ 72

3.2.3.1 Animals ................................................................................................................. 72 3.2.3.2 Peptides ................................................................................................................. 72 3.2.3.3 Radiochemical assay in vitro ................................................................................ 73

3.3 Results................................................................................................................................. 73 3.3.1 Ancestral reconstruction .............................................................................................. 73

v

3.3.2 Sequence and database analysis................................................................................... 81 3.3.3 Radiochemical assay for JH release............................................................................. 83

3.4 Discussion ........................................................................................................................... 83 3.5 Conclusions and future directions....................................................................................... 90

Chapter Four: Summary and Discussion ................................................................................. 91 Supplement to Chapter Two (S2) .............................................................................................. 98 Supplement to Chapter Three (S3) ......................................................................................... 104 References.................................................................................................................................. 124

vi

List of Tables Table 2.1 List of primers............................................................................................................... 25 Table 2.2 Percent identity and similarity by domain .................................................................... 37 Table 2.3 Percent identity and similarity for ORF........................................................................ 38 Table 2.4 Parameter estimates for RXR gene............................................................................... 49 Table 2.5 Likelihood ratio tests .................................................................................................... 50 Table 2.6 Stadium duration and staging accuracy of larvae ......................................................... 54 Table S2.1 Sequence data information ....................................................................................... 102 Table S3.1 Known FGLa-type AST sequences .......................................................................... 110 Table S3.2 Average posterior probabilities ................................................................................ 122 Table S3.3 Likelihood ratio tests ................................................................................................ 123

List of Figures Figure 1.1 The biosynthetic pathway of juvenile hormone.. .......................................................... 3 Figure 1.2 The structure of juvenile hormones and their precursors farnesoic acid and methyl

farnesoate. ............................................................................................................................... 4 Figure 2.1 Nucleotide and deduced amino acid sequence of D. punctata RXRA........................ 33 Figure 2.2 Nucleotide and deduced amino acid sequence of D. punctata RXRB........................ 34 Figure 2.3 Putative splice variants of DpRXR in adult female tissues......................................... 35 Figure 2.4 Multiple sequence alignment of USP/RXR A/B domain region where alternative

splicing occurs.. .................................................................................................................... 39 Figure 2.5 Multiple sequence alignment of USP/RXR LBD sequences.. .................................... 42 Figure 2.6 Phylogenetic tree of USP/RXR LBD sequences constructed in PhyML using WAG

substitution model with 100 bootstrap replicates.................................................................. 45 Figure 2.7 Phylogeny of species in USP/RXR data set used for PAML analysis.. ...................... 48 Figure 2.8 Differential expression of DpRXR.............................................................................. 52 Figure 2.9 Relative expression of overall DpRXR compared to β-actin internal control during

metamorphosis of female D. punctata.. ................................................................................ 55 Figure 2.10 Relative expression of overall DpRXR compared to β-actin internal control in mated

adult female D. punctata....................................................................................................... 57 Figure 3.1 Ancestral reconstruction of Dictyopteran ASTs.......................................................... 75 Figure 3.2 Map of amino acid changes across cockroach nodes for AST peptides inferred in the

reconstructed precursor genes............................................................................................... 77 Figure 3.3 Ancestral reconstruction of ancient insect ASTs......................................................... 79 Figure 3.4 Analysis of arthropod AST sequence data.. ................................................................ 82 Figure 3.5 Dose–response of individual corpora allata (CA) to ancestral peptides...................... 84 Figure S2.1 Multiple sequence alignment of USP/RXR LBD used in phylogenetic analyses..... 99 Figure S3.1 Alignment of extant hemimetabolous insect AST precursors and the results of

ancestral reconstruction using GASP and PAML software.. .............................................. 105 Figure S3.2 Alignment of conserved insect ASTs and the results of ancestral reconstruction using

PAML software................................................................................................................... 109

vii

List of Abbreviations

20E 20-hydroxyecdysone 9cRA 9-cis retinoic acid AF Activation function region AST Allatostatin ASTRs Allatostatin receptors AT Allatotropin Ba Blattidae ancestor BEB Bayes empirical Bayes Br Brain CA Corpora allata Ca Cockroach ancestor Ca-Truncated Truncated cockroach ancestor CC Corpora cardiaca cDNA Complementary DNA DBD DNA-binding domain DIG-labelled Digoxigenin-labelled Dippu-AST Diploptera punctata allatostatin dN/dS ratio of non-synonymous (dN) to synonymous (dS) substitutions DNA Deoxyribonucleic acid DpRXR Diploptera punctata retinoid X receptor EC50 half maximal effective concentration EcR Ecdysone receptor EcR-USP/RXR Ecdysone receptor-Ultraspiracle protein/Retinoid X receptor heterodimer EM Embryo ER Estrogen receptor FA Farnesoic acid FB Fatbody FGL/FGLa Phe-Gly-Leu/Phe-Gly-Leu amide GPCR G protein-coupled receptor GSP Gene Specific Primer H α-helix HNF4A Hepatocyte nuclear factor 4 alpha HR38 Hormone receptor 38 %I Percent sequence identity Ia Insect ancestor JH Juvenile hormone JHBP Juvenile hormone binding protein JHE Juvenile hormone esterase JHEH Juvenile hormone epoxide hydrolase JHRE Juvenile hormone response element LBD Ligand-binding domain LBP Ligand-binding pocket LRT Likelihood ratio test MET Methoprene-tolerant gene product Met Methoprene-tolerant gene MF Methyl farnesoate

viii

MG Midgut mRNA messenger ribonucleic acid NCA Nervi corporis allati NCC Nervi corporis cardiaci NR Nuclear hormone receptor ORF Open reading frame OV Ovary PCR Polymerase chain reaction PISCF Pro-Ile-Ser-Cys-Phe PPAR Peroxisome proliferator activated receptor RA Retinoic acid RACE Rapid amplification of cDNA ends RAR Retinoic acid receptor RNA Ribonucleic acid RT-PCR Reverse transcription polymerase chain reaction RXR Retinoid X receptor %S Percent sequence similarity S.E.M. Standard error of the mean SSC Sodium chloride, sodium citrate Svp Seven-up T3 L-3,5,3′-triiodothyronine TR Thyroid hormone receptor USP Ultraspiracle protein UTR Untranslated region VDR Vitamin D receptor W(X)6W-NH2 Trp-(any amino acid)6-Trp amide

1

CHAPTER ONE: INTRODUCTION: JH FUNCTION, REGULATION OF PRODUCTION, AND SIGNALLING

21.1 Production and functions

Juvenile hormone (JH), a highly pleiotropic sesquiterpenoid insect hormone, is

synthesized and released by the corpora allata (CA). The CA are ectodermally-derived endocrine

glands generally located in the posterior of the head (Tobe and Stay, 1985). The CA do not store

JH and consequently the rate of JH release is proportional to the rate of synthesis (Tobe and Stay,

1977). JH is produced through a biosynthetic pathway related to that of cholesterol (Fig 1.1).

Unlike the cholesterol pathway, the precursor, farnesyl pyrophosphate, is converted to farnesol,

not squalene, resulting instead in the synthesis of sesquiterpenoids. Farnesyl pyrophosphate is

converted to JH by a series of two dehydration steps, a methylation reaction and an epoxidation

reaction (Tobe and Bendena, 1999; Tobe and Stay, 1985).

The structure of JH was first resolved by Röller et al., (1967) and subsequently several

forms of JH, differing in the number of methyl and ethyl side chains, have been identified in

insects (Fig 1.2) (Tobe and Stay, 1985). In general, all JHs possess a methyl ester and an epoxide

group. JH III is the most widespread form, found in Orthoptera, Coleoptera, Diptera,

Hymenoptera, Dictyoptera, Lepidoptera, and the primitive ametamorphic Thysanura (Tobe and

Stay 1985; Baker et al., 1984). JH I and II are only found in the Lepidoptera, as are JH 0 and 4-

methyl JH I (Gilbert et al., 2000; Schooley and Baker, 1985). Additionally, JH III bisepoxide has

thus far only been identified in cyclorrhaphous Dipterans (Richard et al., 1989; Yin, 1994; Yin et

al., 1995). In some species where multiple forms of JH are synthesized, they may be released in

specific ratios (Yin et al., 1995). Although crustaceans do not produce JH, the mandibular organ,

an ectodermally-derived gland homologous to the CA, releases the JH precursors farnesoic acid

(FA) and methyl farnesoate (MF) (Fig. 1.1, 1.2) (Tobe et al., 1989; Cusson et al., 1991). In fact,

some insects also release MF from the CA (Cusson et al., 1991). JH has not been identified in all

3

Figure 1.1 The biosynthetic pathway of juvenile hormone. Figure is reproduced from Tobe and Bendena (1999). Instead of cholesterol, insects synthesize JHs from farnesyl pyrophosphate. Crustaceans synthesize the precursors of JH, farnesoic acid and methyl farnesoate.

4

Figure 1.2 The structure of juvenile hormones and their precursors farnesoic acid and methyl farnesoate. The vertebrate ligand for a candidate JH receptor, USP/RXR, 9-cis retinoic acid is shown at the bottom.

5insects. For example, the JH of basal insects in the Diplura and Protura is unknown, and the form

of JH in the Hemiptera remains unclear (Gilbert et al., 2000; Davey, 2000b). While attempts

have been made in ticks, JHs have not been isolated from non-insect arthropods of the

subphylum Chelicerata (Gilbert et al., 2000).

Among insects JH is involved in a broad range of physiological processes including,

pheromone production, caste determination of social insects, foraging behaviour, phase

polyphenism, diapause, vitellogenesis and metamorphosis (see Smith and Schal, 1990; Park and

Raina, 2004; Jassim et al., 2000; Dale and Tobe, 1986; Bruer et al., 2003; Shiga et al., 2003;

Glinka and Wyatt, 1996; Truman et al., 2006 for examples). In Crustacea, sesquiterpenoids are

involved in similar functions including metamorphosis and reproduction (Borst et al., 1987 and

Nagaraju, 2007 for review). While the role of JH in metamorphosis has been well described in

many insect groups, rates of JH biosynthesis during reproduction, set the Dictyoptera apart.

Here, there is often a correlation between CA activity and the gonadotrophic cycle. One species,

Diploptera punctata, is unique in that it is the only known viviparous cockroach, and as a

consequence of viviparity, JH production is tightly controlled during reproduction.

D. punctata is characterized by high rates of JH biosynthesis which are coordinated with

a precise and predictable order of reproductive events. In the adult female, mating stimulates

enhanced JH biosynthesis (Rüegg et al., 1983). During the subsequent gonadotrophic cycle both

JH production and oocyte growth rapidly increase, then as vitellin continues to accumulate in

oocytes, JH synthesis declines between days 5 and 6. After vitellin content reaches a maximum,

the chorion is formed and ovulation occurs between days 7 and 8. At this point the rate of JH

production has declined to day 1 levels and rates of JH biosynthesis remain low during the

gestation period (Tobe and Stay, 1977; Stay and Tobe, 1981). Furthermore, the precise

regulation of JH production is critical for this species as the presence of JH during pregnancy

6results in abortion (Stay and Lin, 1981). These features not only make D. punctata an interesting

physiological case, but also an excellent system for studying the regulation of JH production.

In D. punctata, JH is also necessary for developmental programming, i.e. determining

larval or imaginal pathways. In general, a high JH titre maintains a larval form, whereas the

absence of JH allows for imaginal differentiation (Kikukawa and Tobe, 1986a,b). Early in larval

stadia, JH is necessary to trigger release of prothoracicotropic hormone, and thereby ecdysteroid

secretion required for ecdysis. However, in final instars, ecdysteroid titre becomes elevated only

after JH release declines, suggesting continuously high JH titre may also block ecdysteroid

release (or synthesis) (Kikukawa and Tobe, 1986a). Not all stadia are JH-competent in D.

punctata. Allatectomy during the first 8 days of the penultimate stadium results in prolonged

stadium duration and precocious metamorphosis, whereas both first and second allatectomized

instars retain larval characteristics. Furthermore, allatectomy during the first 10 days of the final

stadium also increases stadium duration. However, allatectomized final instars still undergo

imaginal ecdysis (Kikukawa and Tobe, 1986b). This suggests JH is important for developmental

commitment which occurs prior to the final instar, at the point of the penultimate stadium

(Szibbo and Tobe 1983; Kikukawa and Tobe, 1986a,b). The identification of several genes,

induced by JH, which are required for maintenance of larval characteristics in holometabolous

insects, provides further evidence that JH not only suppresses ecdysteroid-mediated effects, but

also plays an active role in development (Parthasarathy et al., 2008; Minakuchi et al., 2008;

Konopova and Jindra, 2007).

1.2 Regulation of JH production

JH titre is a function not only of CA activity, but also other processes such as the

enzymatic degradation of JH. In the hemolymph, JH can be metabolized by JH esterases (JHE)

which covert JH to JH acid, and JH epoxide hydrolases (JHEH) which convert JH to JH diol.

The result of both is the degradation of JH into JH acid diol (see de Kort and Granger, 1996;

7Gilbert et al., 2000 for review). While JHE and JHEH likely work in conjunction, research

suggests that JHEH serves as the predominant route of JH metabolism in several insect species

(Jesudason et al., 1990). For example, the primary metabolite of JH II in Trichoplusia ni and JH

I in Manduca sexta, is JH diol (Kallapur et al., 1996; Halarnkar et al., 1993). Similarly, the in

vitro metabolism of JH III in the Dipteran Culex quinquefasciatus appears to occur primarily by

JHEH during the last larval stadium (Lassiter et al., 1995). There is also evidence to suggest that

the degradation of JH occurs mainly at tissues by tissue-bound hydrolases, whereas JHE plays a

more secondary role, active only at specific time points in development (de Kort and Granger,

1996). JH titre also represents interplay between JH degradation and the binding of JH to carrier

proteins in the hemolymph (see section 1. 3).

The direct regulation of JH production by the CA can occur through a diversity of

mechanisms. Intracellular calcium levels have been shown to play a role in both the release and

biosynthesis of JH III in D. punctata. Here, incubation of CA in medium lacking calcium, and

blockage of non-specific calcium channels, inhibits JH release (Kikukawa et al., 1987). Authors

suggest that because no build up of JH or MF occurs in the CA as a consequence of such

blockage, calcium affects overall JH biosynthesis. Neurotransmitters have also been shown to

affect the activity of the CA. For example, octopamine was found to stimulate JH biosynthesis in

both locusts and honey bees (Lafont-Cazal and Baehr, 1988; Kaatz et al., 1994; Rachinsky,

1994). However, in D. punctata and Gryllus bimaculatus octopamine inhibits JH biosynthesis

(Thompson et al., 1990; Woodring and Hoffmann, 1994). Another neurotransmitter, dopamine,

can also regulate JH biosynthesis. In M. sexta, dopamine either stimulates or inhibits JH

production depending on stadium and developmental timing (Granger et al., 1996). Furthermore,

serotonin has also been shown to stimulate JH biosynthesis in Apis mellifera (Rachinsky, 1994).

Both nervous and humoral inputs regulate JH biosynthesis. The innervation of the CA

can differ between insects. In cockroaches, the CA make nervous connections with the corpora

8cardiaca (CC) via nervi corporis allati (NCA) I, and in turn the CC makes connections with the

brain via the nervi corporis cardiaci (NCC) I and II (Tobe and Stay, 1985). In D. punctata,

humoral signals from the ovary can stimulate JH production, whereas nervous inputs inhibit JH.

For example, severance of the nerve connections between the CA and brain releases inhibition,

allowing an increase in JH biosynthesis thereby enabling male D. punctata to produce

vitellogenin. Conversely, the implantation of ovarioles with vitellogenic basal oocytes into male

animals with denervated CA also results in an increase of JH biosynthesis (Hass et al., 2003;

Rankin and Stay, 1984; Mundall et al. 1983). Implantation of vitellogenic ovarioles into

denervated males also decreases the sensitivity of the CA to some of the allatoregulatory

peptides discussed below (Fairbairn and Stay, 1995). Currently, the exact nature and composition

of the ovarian factors involved in this stimulation remain unknown.

CA activity is also controlled by allatoregulatory peptides. There are two general classes

of these peptides, allatotropins (ATs) which stimulate JH production in the CA, and allatostatins

(ASTs) which inhibit JH biosynthesis. First identified in M. sexta, ATs stimulate the production

of JH in adult, but not larval or pupal CA in this species (Kataoka et al., 1989). ATs have also

been shown to stimulate JH biosynthesis in several Lepidopteran species, Hymenoptera, and

Diptera (Audsley et al., 1999, 2000; Oeh et al., 2000; Rachinsky and Feldlaufer, 2000;

Rachinsky et al., 2000; Tu et al., 2001; Li et al., 2003). ATs also serve other functions among

insect lineages. ATs inhibit gut ion transport and stimulate foregut contractions in Lepidoptera,

whereas ATs accelerate heart rate in both cockroaches and Lepidoptera (Lee et al., 1998a; Duve

et al., 1999, 2000; Rudwall et al., 2000; Koladich et al., 2002; Veenstra et al., 1994).

ASTs, named for the ability of these peptides to inhibit JH production, fall into three

distinct families, the function of each being species and order specific (Tobe and Bendena,

2006). The first, and most widely distributed, is the FGLamide (FGLa)-type AST. FGLa ASTs

were first isolated, and later cloned from D. punctata (Woodhead et al., 1989; Donly et al.,

91993). FGLa ASTs possess a core C-terminal motif Y/FXFGL/I-NH2 and have been reported in

Insecta, Crustacea and nematodes, yet only inhibit JH biosynthesis in the Orthoptera, Isoptera,

and Dictyoptera (Tobe and Bendena, 2006). However, FGLa ASTs seem to function as

modulators of myogenic activity across taxa (see Chapter 3 for details; see Stay and Tobe, 2007

for review). The second family of ASTs, first identified in crickets, are characterized by the

consensus sequence W(X)6W-NH2 (Wang et al., 2004). W(X)6W-NH2 ASTs are found in the

orders Orthoptera, Dictyoptera, Lepidoptera, and Diptera but only inhibit JH production in

crickets (Wang et al., 2004; Schoofs et al., 1991; Lorenz et al., 2000; Predel et al., 2001;

Williamson et al., 2001). As with the other families of ASTs, the cricket-type ASTs have also

been shown to serve other functions such as myoinhibition in both Locusta migratoria and M.

sexta (Schoofs et al., 1991; Blackburn et al., 1995; 2001). Additionally, these peptides may act

as inhibitors of ecdysteroid synthesis by the prothoracic glands of Bombyx mori (Hua et al.,

1999). The third family, PISCF type ASTs, first identified in M. sexta, are highly conserved 15

amino acid peptides with unamidated C-termini, which occur primarily in the holometabolous

insects orders Diptera and Lepidoptera (Kramer et al., 1991). Recently, the identification of

PISCF-type ASTs in decapod crustaceans suggests that these peptides are not restricted to those

groups (Stemmler et al., 2009; Ma et al., 2009). PISCF-type ASTs inhibit JH biosynthesis in

Lepidoptera, and in some Diptera such as Aedes aegypti (Li et al., 2004, 2006). PISCF-type

ASTs also serve other functions. For example, in larval Drosophila PISCF-type ASTs inhibit

muscle contraction in the heart (Price et al., 2002). The inhibition of myogenic activity appears

to be a common thread for ASTs, and has lead many researchers to suggest that the regulation of

JH biosynthesis is not the original function of these peptides (Tobe and Bendena, 2006).

1.3 Molecular mode of action of JH

The means by which JHs, synthesized and released from the CA, move through the

hemolymph to target tissues and exert physiological effects is currently not well understood. The

10multiple processes in which JH is involved and the critical importance of JH in insect

development underscores the importance of elucidating the mechanism of JH action. Upon

release from the CA, JH is transported through the hemolymph by carrier proteins, or juvenile

hormone binding proteins (JHBPs). Several roles have been proposed for JHBPs: they allow the

lipophilic JHs to move into the aqueous hemolymph, prevent degradation, and may act as a

storage site for JH. The class of JHBP differs between insect orders. A low molecular weight,

high affinity, JH I and II JHBPs occur in the Lepidoptera (Whitmore and Gilbert, 1972; Dillwith

et al., 1985; Lerro and Prestwich, 1990). A high molecular weight JHBP, lipophorin, with

affinity for JH III serves as a JHBP in Blattodea, Isoptera, Hymenoptera, Diptera and Coleoptera

(de Bruijn et al., 1986; de Kort et al., 1987, de Kort and Koopmanschap, 1987; King and Tobe,

1992; Sevala et al., 1997; see Trowell, 1992 for review). In the Orthoptera, a very large

hexameric protein with 6 binding sites with high affinity for JH III acts as the JHBP

(Koopmanschap and de Kort, 1988; Braun and Wyatt, 1996). Currently, it is unclear what role

JHBPs play in transporting JH to cellular receptors (Trowell, 1992; Gilbert et al., 2000).

Once JH reaches target sites, JH is thought to move directly into the cell, as a

consequence of its lipophilicity, and subsequently moves into the nucleus, where binding to a

nuclear hormone receptor (NR) is believed to occur. However, this may not necessarily be the

case, and there is some evidence for cell surface receptors for JH (Davey, 2000a,b; 2007). Davey

draws parallels with vertebrate thyroid hormones (specifically L-3,5,3′-triiodothyronine or T3),

which undergo receptor-mediated endocytosis to enter target cells. First, the uptake of JH I has

been demonstrated in M. sexta epidermis where JH I accumulates at higher concentrations than

occur in the incubation media (Mitsui et al., 1979). A membrane bound JH binding protein has

been identified in L. migratoria, to which both T3 and JH III compete with equal ability for

binding (Kim et al., 1999). Furthermore, the uptake of rhodamine-conjugated T3 into the follicle

cells of L. migratoria appears to occur through the same receptor (Davey, 2000a). JH I in

11Rhodnius prolixus, and JH III in L. migratoria and Tenebrio molitor increase Na+/K+ ATPase

activity in follicle cell membranes, an effect likely mediated by a membrane JH receptor

(Ilenchuk and Davey, 1982; 1983; Webb et al., 1997; Sevala and Davey, 1993). There is also

some evidence of proteins which bind JH in the cytosol and nucleus. For example a 29 kDa JH I

binding protein has been isolated from the nuclei of M. sexta epidermal cells; this protein shows

little similarity to any known protein (Palli et al., 1994; Jones, 1995). How cell surface, cytosolic

or nuclear receptors are involved in the signal transduction which mediates various JH functions

remains unknown. Most recent research has focused on potential NRs of the superfamily of

steroid receptors.

The documented effect of JH on gene transcription generally supports the presence of a

NR for JH. Nuclear run-on assays, a technique which allows changes in transcription rates to be

directly measured in isolated nuclei, have demonstrated that JH treatment affects transcription of

vitellogenin in L. migratoria, and juvenile hormone esterase (JHE) in the Lepidopteran T. ni

(Glinka and Wyatt, 1996; Venkataraman et al., 1994). Such JH-dependent gene transcription is

induced by the binding of protein complexes to cis regulatory elements in promoter regions

known as JH response elements (JHRE). JHRE have been identified in many species such as, the

Hymenopteran Apis mellifera, Dipteran Drosophila melanogaster, Lepidopteran Choristoneura

fumiferana, and Orthopteran L. migratoria (Li et al., 2007; Kethidi et al., 2004; Zhou et al.,

2002). Evidence also suggests there is no one common JHRE; some show no sequence

similarity, leading researchers to propose that JH mediated effects occur through a diverse array

of response elements (Li et al., 2007). The search for the proteins that bind at these sites,

including potential JH binding nuclear receptors, is ongoing.

Although the identity of the JH NR is currently unknown, there are several candidates.

One candidate, the product of the Methoprene-tolerant gene (Met) encodes a member of the

basic helix-loop-helix-PAS family of transcriptional regulators. Met was first identified in

12Drosophila mutants resistant to the JH analog pesticide methoprene (Ashok et al., 1998; Pursley

et al., 2000). Since that time, MET-like products have also been identified in Tribolium

castaneum and Aedes aegypti (Konopova and Jindra, 2007; Wang et al., 2007). In Drosophila

MET has been shown to bind JH III and activate reporter gene transcription (Miura et al., 2005).

MET affects metamorphosis in T. castaneum such that Met-deficient larvae pupate prematurely

(Konopova and Jindra, 2007, 2008). In Drosophila, double mutants of both Met and the 20-

hydroxyecdysone-response gene Broad-Complex result in more severe defects in viability and

oogenesis than expected with independent mutants (Wilson et al., 2006). While evidence for the

role of MET in JH action is growing, the majority of research has focused on another candidate,

ultraspiracle protein (USP).

USP is the insect homolog of the retinoid X receptor (RXR), a group II NR and a member

of the superfamily of steroid receptors. USP was first characterized for its role in the ecdysone

cascade, as the heterodimeric partner of the 20-hydroxyecdysone receptor (EcR) (Thomas et al.,

1993; Yao et al., 1992, 1993). Subsequently, USP was shown to bind JH III independently only

in D. melanogaster, but the physiological relevance of these results remains contentious (Jones

and Sharp, 1997; Jones et al., 2006, see Chapter 2 for details). Like its counterpart RXR, which

binds 9-cis retinoic acid (9cRA) in vertebrates, USP can form heterodimers with a number of

partner proteins (Levin et al., 1992; Heyman et al., 1992; Mangelsdorf et al., 1992; Zhang et al.,

1992; Mangelsdorf and Evans 1995). In higher insects such as A. aegypti two-hybrid and GST

pull-down assays demonstrated that USP/RXR may interact with EcR, hormone receptor 38

(HR38) and the transcription factor Seven-up (Svp). Similar studies in Drosophila have shown

that MET can also interact with EcR and USP, and that all three can interact with JHRE-binding

proteins Chd64 and FKBP39 (Zhu et al., 2003; Li et al., 2007). The mechanism of JH action is

complex, and likely involves several pathways and an array of proteins. It is also quite possible

13that there is no single JH receptor, and different forms of JH may employ many distinct receptors

or combinations of receptors (Yin et al., 1995; Davey, 2000b).

1.4 Conservation of endocrine systems

To gain the best understanding of endocrine systems in insects, we must also gain an

appreciation for the similarity of these systems across distant phyla. Endocrine systems in the

insects are in part neuroendocrine, characterized by neurosecretory cells which release

hormones. Neurosecretory activity has been demonstrated in many organisms across a diversity

of bilaterian taxa, including vertebrates. Furthermore, neurohormones are considered some of the

oldest blood-borne messengers (Scharrer, 1976; see Hartenstein, 2006 for review). Hormones

can be very broadly distributed. For example, insulin and insulin-related growth factors are

produced in vertebrate endocrine cells, yet have also been identified in neuroendocrine cells of

insects and molluscs (Froesch et al., 1985; Steiner et al., 1985; Li et al., 1992; Smit et al., 1996;

Lagueux et al., 1990; Kawakami et al., 1989). Even animals without nervous systems, such as

sponges, produce insulin (Robitzki et al., 1989).

Similarily, FGLa-type ASTs represent a family of regulatory neuropeptides which occur

across a diversity of taxa. AST precursors have been identified in a range of insects and

crustaceans, and neuropeptide-like sequences which resemble ASTs have also been found in

nematode genomes (Duve et al., 1997a; Billimoria et al., 2005; Huybrechts et al., 2003;

Dircksen et al., 1999; Yin et al., 2006; Duve et al., 2002; Yasuda-Kamatani and Yasuda, 2006;

Nathoo et al., 2001). While no peptides have yet been isolated, immunoreactivity to FGLa ASTs

has been described in molluscs (Bendena et al., 1999; Smart et al., 1994; see Chapter 3 for

details). While JHs are only known to be synthesized in Insecta and JH precursors in Crustacea,

sesquiterpenoid hormones show some degree of cross reactivity between taxa. For example, MF

can elicit metamorphosis in marine annelids, and at high concentrations ecdysis in nematodes

(Tobe and Bendena, 1999; Biggers and Laufer, 1999; Davey 1988). This has led some to suggest

14that JH originated, not for the regulation of reproduction, but as biotic cues for metamorphosis

(Sehnal et al., 1996; Tobe and Bendena, 1999; Davey, 2007). ASTs and JHs illustrate how

endocrine evolution can be characterized by the co-opting of existing structures for new and

diversified purposes (Niall, 1982).

Nuclear hormone receptors (NRs), like hormones, also display conservation across a vast

array of taxa. Traditionally, classical steroid receptors have been considered a ‘chordate

innovation’ (Baker, 2003). However recent molecular and phylogenetic approaches suggest a

wider diversity of NRs was present early in Bilateria. Bertrand et al., (2004) examined the

complete set of NRs present in the genomes of nine Metazoan species in a phylogenetic

framework to show that approximately 25 NR genes, including steroid and thyroid hormone

receptors, were present in the ancestor of bilaterians. This hypothesis is supported by the

identification of sequences similar to the estrogen receptor (ER) in Aplysia californica and

thyroid receptor (TR) in Schistosoma mansoni (Thornton et al., 2003; Bertrand et al., 2004).

Furthermore, estrogen-responsive ERs have recently been characterized in the annelids

Platynereis dumerilli and Capitella capitata (Keay and Thornton, 2009). NRs appear to be

specific to Metazoa and have not been found in plants or unicellular organisms (Escriva et al.,

1997). Gene duplication events and losses are also common in NR evolution. Several researchers

have identified two main periods of gene duplication, the first along the common bilaterian

ancestor, and the second in vertebrates after the split from arthropods. Periods of loss have also

been described in the nematodes, insects and chordates differentiating the NR complements in

those lineages (Escriva et al., 2000; Laudet et al., 1992; Bertrand et al., 2004).

The putative JH receptor USP/RXR is also widely distributed among Metazoa. USP/RXR

has been found in species ranging from vertebrates to sponges (Wiens et al., 2003). Similarly,

AST receptors (ASTRs), a type of G protein-coupled receptor (GPCR) identified in insects are

related to the receptor of the neuropeptide galanin in mammals. Recently, a receptor similar to

15ASTRs in insects has been identified in the nematode Caenorhabditis elegans (Bendena et al.,

2008). Given the commonalities between endocrine signalling systems, a clear comprehension of

their comparative function and regulation is vital.

The case of endocrine disruption, an effect which can be mediated by nuclear receptor

and coactivator modulation, highlights the value of comparative studies of hormones and

receptors (Tabb and Blumberg, 2006). For example, RXR appears to mediate the negative effects

of the organotin endocrine disruptor tributyltin chloride in amphibians, humans and molluscs

(Nishikawa et al., 2004; Iguchi and Katsu, 2008). Furthermore, JHs and JH analog-based

pesticides pyriproxyfen and fenoxycarb reduce number of offspring and affect sex ratio in the

water flea Daphnia magna (Tatarazako et al., 2003). AST mimics also have the potential to serve

as endocrine disruptors in pest management as a consequence of their ability to inhibit JH

biosynthesis, and thus influence insect development (Kai et al., 2009; Garside et al., 2000). The

safety and appropriateness of such targets cannot be determined without comparative studies of

endocrine hormones or receptors.

1.5 Objectives

JHs play a role in a diversity of biological processes in insects and both the function and

regulation of JH production can vary from species to species. However, despite the importance

of this hormone the molecular mode of action of JH remains unclear, and the majority of work

on this topic has been centered on the Holometabola. Furthermore, the evolutionary history of

the regulation of JH biosynthesis by allatoregulatory neuropeptides is also not well understood.

The general objective of this thesis is to close these gaps in our current understanding of this

system. To clarify these processes the putative JH receptor USP/RXR was isolated from D.

punctata and the functional history of FGLa-type ASTs was examined in cockroaches and

insects using ancestral reconstruction methods. These lines of research are outlined below.

16A. Molecular cloning and characterization of USP/RXR in D. punctata (Chapter 2)

Current data suggests USP/RXR may bind JHs only in the Mecopterida, a group of

higher insects that includes the Diptera, Trichoptera, Mecoptera, Siphonaptera and Lepidoptera.

It remains unclear if USP/RXR can bind JHs and functionally transactivate DNA independently

of the EcR heterodimer in all insects. Research on this topic has focused on Drosophila

melanogaster and little functional nor sequence data exists for the Hemimetabola. Therefore, we

sought to clone and sequence USP/RXR from the cockroach D. punctata (DpRXR) using RACE

PCR methods. Using this sequence it was possible to compare functional domains of DpRXR

with Mecopterida and vertebrate-type receptors.

Recent models of USP/RXR functional evolution suggest a loss of ligand-binding in

arthropods followed by a subsequent functional gain along the Mecopterida lineage which may

have led to JH binding. To test such hypotheses we estimated evolutionary rates, indicators of

selective constraint, across a dataset of invertebrate USP/RXR ligand-binding domain sequences,

using codon-based models of substitution.

The relationship between USP/RXR expression levels and rates of JH biosynthesis are

also not well characterized. To address this the relative expression of USP/RXR in D. punctata

was measured using semi-quantitative RT-PCR in various tissues during both developmentally

critical periods for JH sensitivity, and the first gonadotrophic cycle of mated adult females. We

expected to find higher levels of USP/RXR expression early in larval stadia, similar to the results

obtained from B. germanica (Maestro et al., 2005)

B. Reconstruction of ancestral FGLamide-type insect allatostatins (Chapter 3)

Many studies have been conducted in the insects with respect to the function of

individual FGLa-type AST peptides in specific insect groups. However, questions linger

regarding the origin and evolutionary history of AST function. A major goal within the field of

AST research is to establish the link between changes in genotype and peptide function. To begin

17to address this, we applied ancestral reconstruction methods to infer the sequence of the AST

precursor at all ancestral nodes within a phylogeny of AST sequences. This was done at two

separate scales, one encompassing only cockroaches, and another considering all insects. From

this, we mapped residue changes across the tree so as to determine when changes in AST

function and sequence coincided.

Given the sequence of the inferred ancestral peptides it is possible to recreate these

peptides in the lab and directly assay whether more ancient or recent peptides are potent

inhibitors of JH biosynthesis. This study represents the first time such ancestral reconstruction

methods have been applied to this system. In doing so, we were able to directly recreate and test

the evolutionary history of FGLa AST function with respect to the inhibition of JH production.

Several ancestral insect and cockroach peptides were synthesized and assayed in two different

species of cockroaches. Since the inhibition of JH biosynthesis by FGLa ASTs seems restricted

to the Orthoptera, Isoptera, and Dictyoptera we expected to see a gradual increase in peptide

potency along insect lineages leading to the cockroaches.

Additionally it is not well understood to what degree peptide sequences overlap between

insect groups. The lack of positional homology among peptide precursors has hampered the

overall analysis of FGLa ASTs across arthropods. To address this we constructed a searchable

list of all currently known peptides. With this we examined the number of unique peptides,

peptide frequency and overlap between Hemimetabola, Holometabola and Crustacea.

18

CHAPTER TWO: MOLECULAR CLONING AND CHARACTERIZATION OF FOUR RXR ISOFORMS FROM THE

VIVIPAROUS COCKROACH, DIPLOPTERA PUNCTATA

19Abstract

Although USP/RXR, a nuclear receptor and transcription factor, is typically considered a

component of a heterodimeric receptor complex with the ecdysone receptor (EcR), it has also

been hypothesized to be a putative juvenile hormone (JH) receptor. Evidence for the role of

USP/RXR in sesquiterpenoid signal transduction stems mainly from work conducted in

Drosophila melanogaster and other higher insects. Currently the function of USP/RXR in other

insect groups remains unclear. Using PCR-based cloning strategies, we isolated four putative

USP/RXR splice variants from the hemimetabolous insect D. punctata. The variants were

grouped into two types, DpRXRA and DpRXRB, each with a long (L) and a short (S) form

which differ by a 12 amino acid insertion/deletion in both cases. DpRXR isoforms vary only in

the N-terminal A/B domain, sharing identical DNA-binding (DBD) and ligand-binding (LBD)

domains. Evolutionary rates were estimated in codon-based substitution models using maximum

likelihood methods across a phylogeny of invertebrate USP/RXR LBD sequences. Elevated dN/dS

values support a gain in function along the Mecopterida lineage, a group of higher insects

proposed to have JH binding function. DpRXR isoforms are differentially expressed in mated

adult female Diploptera. Overall DpRXR expression is high in both the ovary and brain and low

in the corpora allata. A preliminary study of DpRXR expression during critical periods for JH

sensitivity suggests a possible role for DpRXR in JH signalling. However, further expression

profiles and ligand-binding assays are needed to characterize the function of DpRXR.

2.1 Introduction

USP/RXR, a group II nuclear receptor and transcription factor, belongs to the

superfamily of steroid receptors. USP/RXR is found across a diversity of Metazoan species

ranging from sponges to mammals (Wiens et al., 2003). RXR was first isolated by Mangelsdorf

et al. (1990) from human liver and kidney tissue. Subsequently 9-cis retinoic acid (9cRA), a

vitamin A derivative, was identified as the high affinity ligand of the vertebrate receptor (Levin

20et al., 1992; Heyman et al., 1992; Mangelsdorf et al., 1992). In vertebrates, RXR is involved in

an array of functions including development, differentiation and lipid metabolism. These

functions are mediated via the interaction of RXR with a multiple heterodimeric partner proteins

such as the retinoic acid receptor (RAR), vitamin D receptor (VDR), thyroid hormone receptor

(TR), and peroxisome proliferator-activated receptor (PPAR) (see Tate et al., 1994; Mangelsdorf

and Evans 1995; Chawla et al., 2001 for review). In addition, retinoid signalling can also be

transduced by RXR homodimers in vitro (Zhang et al., 1992).

USP, the insect homolog of RXR first isolated from Drosophila melanogaster, is

intimately linked to the process of metamorphosis (Oro et al., 1990). While the insect form was

originally referred to as USP, the terms USP and RXR are currently used interchangeably. In

insects, USP/RXR is a component of a heterodimeric receptor complex with the ecdysteroid

receptor (EcR-USP/RXR). In response to 20-hydroxyecdysone (20E) binding to EcR,

heterodimerization occurs and the complex then binds DNA and transactivates genes (Thomas et

al., 1993; Yao et al., 1992, 1993). Unlike the vertebrate receptor, no natural ligand has been

conclusively identified in the insects. In the invertebrates, USP/RXR is still considered an orphan

receptor because its function remains a matter of contention among researchers.

However, candidate ligands have been proposed, and in insects USP has been

hypothesized to be a putative juvenile hormone (JH) receptor (Jones and Sharp, 1997). JH is an

enticing candidate ligand as a consequence of the structural similarities of retinoids and

sesquiterpenoids. As a JH receptor, USP/RXR would conveniently tie together the major signal

transduction cascades involved in metamorphosis, ecdsyone and JH. Data supporting this

hypothesis comes from holometabolous insects, in particular D. melanogaster. In cell lines

expressing Drosophila USP, the application of JH III, and less so JH I and II, induces the

transcription of a transfected promoter suggesting that JHs bind USP and result in a functional

outcome (Wozniak et al., 2004; Xu et al., 2002). In vitro studies have also shown that, like

21vertebrate RXR, Drosophila USP can bind a ligand (JH III) as a homodimer independent of its

heterodimeric partner EcR (Xu et al., 2002; Fang et al., 2005). Recently, fluorescence-binding

assays have shown that Drosophila USP binds not only JH III but also the JH precursors

farnesol, farnesoic acid, and methyl farnesoate (MF) (Jones et al., 2006).

However, this JH-binding function has not been identified in other insect groups. Ligand-

binding assays in Locusta migratoria showed that whereas LmRXR was a functional component

of the ecdysone receptor heterodimer, neither the long nor short isoforms of LmRXR bound JH

III at the nanomolar range (Hayward et al., 2003). In Tribolium castaneum, a holometabolous

insect, USP acts as a silent partner of the EcR but is unable to transactivate DNA independently

in binding assays (Iwema et al., 2007). The species specificity of ligands is also unclear from

current data. Surprisingly, JH analogs such as methoprene, methoprene acid and hydroprene

acid, have been found to activate human RXR, whereas JH III does not (Harmon et al., 1995). In

general however, insect and non-insect arthropod USP/RXR does not appear to conversely bind

retinoids (Oro et al., 1990; Iwema et al., 2007; Guo et al., 1998).

A comparative analysis of structural data also serves to highlight differences in

USP/RXR among insect lineages. Crystallography experiments have shown that Dipteran and

Lepidopteran USP share a conserved LBD with a large hydrophobic cavity capable of accepting

a natural ligand (Clayton et al., 2001; Billas et al., 2001). These structural studies also revealed

that interactions between α-helices H1, H3 and H12 lock the protein into an inactive antagonistic

conformation, possibly preventing the binding of transcriptional coactivators (Clayton et al.,

2001; Billas et al., 2001). Unlike D. melanogaster and Heliothis virescens, the crystal structures

of two less derived insects, T. castaneum and the sweetpotato whitefly Bemisia tabaci, reveal

ligand-binding pockets (LBP) filled by residues from H6 and H11 (Iwema et al., 2007;

Carmichael et al., 2005). Structural studies also bring the proposed physiological relevance of

ligand-binding assays conducted in higher insects into question. Structure-based homology

22models of USP where features of vertebrate RXR are imposed on H. virescens USP, theoretically

demonstrate that JHs and JH analogs can dock into the LBP (Sasorith et al., 2002). However,

docking energies do not demonstrate any preference between acidic forms of JH I, II and III. In

fact, 9cRA, not considered functional in insect development, and JHs dock with similar scores

(Sasorith et al., 2002; Oro et al., 1990). Such data does not support high affinity ligand-binding

in USP. The interaction of 9cRA in vertebrate RXR clearly discriminates between structural

variants of RA, binding with 40-fold lower affinity to all-trans RA (Heyman et al., 1992;

Mangelsdorf et al., 1992).

Given current functional and structural data, what is the functional relationship between

USP/RXR in different insect lineages, and among vertebrate and invertebrate lineages? Based on

experimental data from Drosophila, crystallography work from H. virescens and relative

substitution rates among arthropods, a model for the functional evolution of USP/RXR function

has been proposed (Jones et al., 2006; Clayton et al., 2001; Billas et al., 2001; Sasorith et al.,

2002; Bonneton et al., 2003). Iwema et al. (2007) and Tocchini-Valentini et al. (2009) suggest

three groups of USP/RXR: 1. RXR type, 2. Mecopterida USP type and 3. non-Mecopterida USP

type. The RXR type evolved retinoid-binding function early, a function which is retained in the

Cnidaria, Mollusca, and Chordata. The non-Mecopterida type which includes insect groups such

as the Hymenoptera, Coleoptera, Orthoptera and Dictyoptera has, along with other non-insect

arthropods, lost ligand-binding function. However, the Mecopterida USP type, a group which

includes the Diptera, Trichoptera, Mecoptera, Siphonaptera and Lepidoptera, may have gained a

new USP function – JH binding.

The ability to assess such evolutionary hypotheses is hampered by the focus most

research has placed on Drosophila and the holometabolous insects. Currently sequence data is

only available for a handful of basal hemimetabolous species, and functional data exists only for

L. migratoria (Maestro et al., 2005; Hayward et al., 2003). Without an understanding of

23USP/RXR in these basal lineages, shifts in the structure and function of USP/RXR among the

insects will remain unclear and the JH signal transduction cascade unresolved. Here, we report

the sequence of USP/RXR in the viviparous cockroach D. punctata, adding data on the basal

insects. Multiple sequence alignments were used to compare structural domains across taxa at

functionally critical sites. To test current hypotheses of USP/RXR evolution, codon-based

likelihood methods were implemented to estimate evolutionary rates across an invertebrate

phylogeny and independently along the Mecopterida lineage. Finally, a preliminary analysis of

DpRXR developmental expression was used to examine the relationship between rates of JH

biosynthesis and USP/RXR levels.

2.2 Materials and methods

2.2.1 Animals

D. punctata were reared at 27°C and given lab chow and water ad libitum. Newly

ecdysed mated adult female D. punctata were selected, removed from the stock colony, placed in

containers and provided food and water. Adult females used for the collection of embryos, and

the isolation of newly emerged first larval instars, were maintained according to Stay and Coop

(1973), on a 12 hr light and 12 hr dark cycle, to ensure proper stadia number and length.

While ultimate instar females were picked directly from the stock colony, penultimate

female larvae were staged from birth. Batches of newborn first instar individuals were collected

from jars of pregnant females selected from the stock colony. Upon larval ecdysis, individuals

were moved to new containers, segregating individuals by stadia. Animals that did not molt to

subsequent stadia within the number of days described by Kikukawa and Tobe (1986a) were

discarded. After approximately four weeks penultimate larva were obtained, separated by sex

according to Szibbo (1982), and maintained under the conditions described above. On the

appropriate day, animals were immobilized on ice and dissected

242.2.2 Molecular cloning of DpRXR and Northern blot

Day 4 embryos were collected from staged female D. punctata and stored in RNAlater

(Ambion) until RNA extraction. Total RNA was extracted with the RNeasy Mini Kit (Qiagen)

according to manufacturer’s instructions using the mortar and pestle method of tissue disruption.

First strand cDNA was synthesized using the Superscript III First Strand Synthesis SuperMix Kit

(Invitrogen). Degenerate primers were designed using extant USP/RXR sequence data from L.

migratoria, Blattella germanica, D. melanogaster, and Periplaneta americana. Amplification

was carried out with Degenerate F and Degenerate R primers using JumpStart Taq polymerase

(Sigma) under the following conditions: 94°C for 2 min, six cycles at 94°C for 30 s, 55°C for 1

min, 68°C for 30 s, and 36 cycles at 94°C for 30 s, 60°C for 1 min, 68°C for 30 s, and 68°C for

10 min. The amplified fragment (222bp) was sub-cloned into the pJET1.2 cloning vector

(Fermentas) and sequenced. All primer sequences are listed in Table 2.1.

25

Table 2.1 List of primers Primer Name Sequence Degenerate F 5’-ATCCICCTAAYCATCCRCTSA-3’

Degenerate R 5’-ARRCAYTTYTGRTANCGRCA-3’

Oligo-d(T) anchor primer1

5'-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3'

Qo adaptor primer 5'-CCAGTGAGCAGAGTGACG-3'

Qi adaptor primer 5’-GAGGACTCGAGCTCAAGC-3’

Oligo-d(T) anchor primer2

5’-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3’

Oligo-d(T) anchored primer3

5’-NVTTTTTTTTTTTTTTTTTT-3’

USP GSP1 5’-TGAGCGGATCCAAGCACCTCTG-3’ USP GSP2 5’-GTGCGTAAAGATCTGTCCTACG-3’ PCR anchor primer 5’-GACCACGCGTATCGATGTCGAC-3’

USP 5RACES2 5’-TGACACCTGTTACGCTG-3’

USP 5RACE2 5’-CGTAGGACAGATCTTTACGCAC-3’

USP 5RACE4 5’-ACAGAGGTGCTTGGATCCGCTCAG-3’

DpRXR1F 5’-CCTGCCGAGAGGATAAGAACTGCA-3’

DpRXR1R 5’-GCTGCTATCAGCAGCTCATTCCA-3’

DpRXRB 1F 5’-CGGCCATGTTTGATACGAACAAAGT-3’

DpRXRB 2F 5’-CCTGAAATGGAAGGAAGCGAGAGA-3’

DpRXRA 1F 5’-ACCCATGATGTCTGTGACGGCCA-3’

DpRXR StopR 5’-TTAAGCATCACTGGACATGGGTGC-3’

B-actin F 5’-GGATGGTGTATCTCACACTGTACC-3’

B-actin R 5’-CTGCTGTAGTTGTGAAGCTGTAGC-3’

26For 3' RACE, first strand cDNA was synthesized from total day 4 embryonic RNA using

the Transcriptor High Fidelity cDNA synthesis Kit (Roche) with oligo-d(T) anchor primer1 at

50°C for 30min according to manufacturer’s instructions. Subsequently, 3' RACE PCR was

performed according to the 'classic' protocol described in Frohman (1994) using a series of two

nested PCR reactions with forward gene specific primers (USP GSP1, 2) and reverse adaptor

primers (Qo, Qi). For 5' RACE, first strand cDNA was synthesized with a gene-specific reverse

primer (USP 5RACES2) using the method described above. Subsequent RACE PCR was

performed according to 5’/3’RACE Kit, second generation (Roche) using a series of nested PCR

reactions using oligo-d(T) anchor primer2 and PCR anchor primer with gene-specific primers

USP 5RACE2, and USP 5RACE4. All RACE PCR reactions were conducted using the High

Fidelity PCR Enzyme Mix (Fermentas). Amplified fragments were sub-cloned into the pJET1.2

cloning vector (Fermentas) and sequenced.

5' RACE results revealed two alternative upstream DpRXR sequences, each with a long

and short form. The full open reading frame (ORF) was amplified with two separate forward

primers, DpRXRA 1F and DpRXRB 1F, and a common reverse primer DpRXR StopR, using

cDNA derived from day 4 embryos and day 1 mated adult female ovaries under the following

conditions: 94°C for 2 min, 35 cycles at 94°C for 30 s, 59/57°C for 1 min, 72°C for 2 min, and

72°C for 10 min. Both sets of cDNA yielded the same band pattern and amplified fragments

from the embryonic sample were sub-cloned and sequenced as above (Fig 2.1, 2.2). To ensure

that downstream variants, such as the 69bp insertion/deletion which occurs in the LBD of B.

germanica, were not overlooked in RACE PCR reactions primers DpRXR 1F and DpRXR 1R

were used to amplify the suspected region in D. punctata (Maestro et al., 2005). cDNAs derived

from total RNA isolated from mated adult female D. punctata were amplified using JumpStart

Taq polymerase (Sigma) under the following conditions: 94°C for 2 min, 35 cycles at 94°C for

30 s, 59°C for 1 min, 72°C for 30 s, and 72°C for 10 min (Fig 2.3 A).

27Total RNA used for Northern blot was isolated from day 1 mated adult female midgut

(n=10) and ovary (n=40) with TRIzol (Invitrogen) according to manufacturer’s instructions.

30ug of midgut and ovary RNA was electrophoretically separated on a 1.2% denaturing agarose

gel and transferred to a positively-charged nylon membrane (Roche) by capillary action using

10X sodium chloride, sodium citrate (SSC). The blot was then ultraviolet cross-linked, and

hybridized to a 508bp DIG-labelled cDNA probe (15ng/ml) in high-SDS hybridization buffer

(50% deionized formamide, 7% SDS, 5X SSC, 2% blocking reagent, 0.1% N-lauroylsarcosine,

50mM sodium phosphate, pH 7.0) for 48 hr at 50°C. The labelled probe corresponds to amino

acids 123 to 298 (according to DpRXRA-L), a region common to all DpRXR variants. The blot

was then washed twice with 2X SSC, 0.1% SDS at RT, and twice with 0.5X SSC, 0.1% SDS at

55°C. Immunological detection was carried out with anti-DIG antibody (Roche) diluted 1:10,000

in 1X blocking buffer followed by chemiluminescent detection with 0.1ml CDP-Star substrate

(Roche). The blot was exposed to X-ray film (Agfa) for 10 min and developed to visualize the

hybridization signal (Fig 2.3 B).

2.2.3 Sequence comparison of functional domains

Known USP/RXR sequences were collected from literature, GenBank, and FlyBase using

a combination of BLAST and keyword searches (Table S2.1). Percent identity and similarity of

amino acid sequences were calculated for each domain of USP/RXR in the pairwise alignment

tool EMBOSS (http://www.ebi.ac.uk/Tools/emboss/align/index.html) using the "needle" or

global alignment method with the Blosum62 matrix under default parameters. Amino acid

multiple sequence alignments of the LBD and N-terminal A/B domain were constructed using

ClustalW (Thompson et al., 1994) as implemented in MEGA 4 (Tamura et al., 2007) and

adjusted by eye to ensure structural motifs were maintained in the alignment. BOXSHADE 3.21

was used to shade conserved and semi-conserved residues in the alignments. As in Maestro et al.

(2005), a truncated amino acid alignment, limited to the LBD region, was used to construct a

28phylogeny of USP/RXR sequences. Poorly aligned regions and major gaps were deleted (see Fig

S2.2). The resulting 260 residues were used to construct the phylogenetic tree by maximum

likelihood methods in PhyML (Guindon et al., 2005) using the WAG substitution model

(Whelan and Goldman, 2001). Four substitution rate categories were used to estimate the gamma

parameter shape (Yang, 1994) with 100 bootstrap replicates (Felsenstein, 1985) to assess branch

support.

2.2.4 Estimation of evolutionary rates

To test current hypotheses of USP/RXR evolution, maximum likelihood methods were

used to estimate the ratio of non-synonymous (dN) to synonymous (dS) substitutions or dN/dS (ω)

often used as an indicator of selective constraint operating on a gene. Under no selective

pressure, sequences evolve neutrally, and this is indicated by ω =1, whereas ω <1 indicates

purifying selection, and ω >1 positive selection (Kimura, 1977; Yang and Bielawski, 2000). To

accomplish this, we implemented a range of codon-based substitution models using the codeml

program of the PAML software package, version 4.2b (Yang, 1997). A nucleotide version of the

amino acid alignment used for phylogenetic analysis above was modified to include only

sequences from the protostome groups Mollusca, Chelicerata, and Insecta (see Fig. S2.2, and

Table S2.1). A tree reflecting current understanding among major insect and arthropod lineages

was used in the analysis (Grande et al., 2008; Giribet et al., 2001; Ahyong et al., 2007; Tsang et

al., 2008; Jeyaprakash and Hoy, 2009; Whiting et al., 1997; Hunt et al., 2007; Weller et al.,

1992; Yeates and Wiegmann, 1999). Several branch (Yang, 1998;Yang and Nielsen, 1998) and

branch-site (Yang and Nielsen, 2002) models were implemented in order to test for positive

selection. Codon frequencies were estimated using the F3x4 method, and when possible models

were run from several starting ω values ranging above and below 1 in order to test for

convergence. Likelihood ratio tests (LRTs; Felsenstein, 1981) were then conducted to determine

statistical significance.

29To investigate the gain in function hypothesized to have occurred in the Mecopterida,

branch models were implemented which allowed for an additional ω parameter along the

lineages leading to the Mecopterida (M1m), or alternatively along the lineage leading to the

ancestor of Mecopterida and Hymenoptera (M1m+h). These models were compared against M0

where only one ω value was estimated across the phylogeny. To examine selective pressures

among codon sites in the Mecopterida, branch-site models were applied to the dataset. Branch-

site models allow the use of a Bayes empirical Bayes (BEB) analysis (Yang et al., 2005) to

identify specific positively selected sites within the gene along a given branch. A model with

Mecopterida designated as foreground, MA, was compared against a stringent model for positive

selection, MA1, and a less stringent model, M1a. Positive selection is detected if MA is a better

fit than MA1 where classes of positively selected sites have ω set to 1, whereas relaxed purifying

selection (possibly suggestive of positive selection) is indicated if MA is a better fit than M1a

which does not allow a class of sites with ω >1.

2.2.5 DpRXR expression

Tissues collected from animals were either stored in RNAlater (Ambion) or directly

extracted. Total RNA was extracted using RNeasy Mini Kit (Qiagen) for adult brain and ovary,

and TRIzol (Invitrogen) for all larval tissues, and adult CA. RNA from ovary and brain were

treated with DNAse I (Invitrogen) for 15 min at RT to eliminate genomic DNA contamination.

However, RNA yields from CA and some larval extracts were too low to perform this treatment.

cDNA was synthesized with oligo-d(T) anchored primer3 with MMLV-RT (NEB) using 1μg

RNA from ovary and brain, and 250 ng from the corpora allata (CA). For larval samples used in

tissue-tissue comparison, 250ng were used for brain, CA and ovary.

To compare the differential expression of putative splice variants, equal amounts of

cDNA from day 1 mated adult female ovary, and day 5 brain were amplified using either

DpRXRA 1F or DpRXRB 2F with a common reverse primer USP 5RACE4 under the following

30conditions: 94°C 2min, 35 cycles of 94°C 30 s, 61.1/58.4°C 1 min, 72°C 30 s followed by 72°C

for 10 min. As a control, β-actin was amplified with primers B-actin F and B-actin R under the

same conditions with an annealing temperature of 62°C. To eliminate the possibility of

preferential amplification of long or short isoforms, a PCR was performed with both sets of

DpRXR primers using equal amounts of purified plasmids containing each DpRXR isoform as a

template. For both primer sets, amplification profiles were identical for each isoform at

increasingly non-saturated cycle numbers (data not shown). To compare tissue-to-tissue

expression of DpRXR, equal amounts of cDNA from day 8-10 penultimate female brain, CA and

ovary were amplified with USP GSP1 and DpRXR 1R under the same conditions as above with

an annealing temperature of 58.4°C. The same reaction conditions described above for the

control were used to amplify β-actin for 30 cycles. For semi-quantitative RT-PCR samples were

amplified with the same DpRXR and β-actin specific primers under the same conditions with

modified cycle number, 32 cycles and 24 cycles respectively. Each sample used in semi-

quantitative RT-PCR represents RNA extracted from pooled group of tissues from between 16

and 40 animals. Two sets of such samples were run in two duplicate reactions. All PCR reactions

were analyzed on 1.5% agarose gels stained with ethidium bromide. The resulting gel images

were quantified with ImageJ (NIH) for semi-quantitative analysis. Relative expression, RXR

band intensity divided by β-actin intensity, was plotted with standard error. For penultimate

instar females, only one set of pooled samples was analyzed (n=1) for each data point, and are

therefore plotted without error.

312.3 Results

2.3.1 Molecular cloning of DpRXR

Degenerate primers amplified a 222bp fragment from day 4 embryonic (EM) D. punctata

cDNA. The deduced amino acid sequence of the fragment was identical to the DNA-binding

domain of B. germanica and L. migratoria RXR. 3'RACE isolated a single downstream sequence

and untranslated region (UTR). Although a portion of the 3'UTR was obtained, the

polyadenylation signal was absent, suggesting the data for that region is incomplete. 5'RACE

resulted in the identification of four sequence variants. The four putative splice variants were

designated DpRXRA-Long, DpRXRA-Short, DpRXRB-Long and DpRXRB-Short and encode

proteins of 449, 437, 427 and 415 amino acids respectively (Fig 2.1, 2.2). DpRXR variants

occurred in the region corresponding the A/B domain and 5' UTR. The open reading frame

(ORF) of DpRXRA and B vary only in the N-terminus of the domain, and differ in length by 22

amino acids. Both DpRXRA and B have long and short forms that differ by a 12 amino acid

insertion/deletion (Fig 2.1 B, 2.2B). All isoforms share identical downstream domains, a DBD or

C domain (66 amino acids), a hinge domain D (23 amino acids), and a LBD or E/F domain (231

amino acids).

In both B. germanica and L. migratoria, splice variants occur in the LBD between α-

Helix 1 (H1) and H3, not the A/B domain. To determine if such splice variants were present in

D. punctata, but not identified during 3'RACE, primers were designed flanking the position of

the possible insert. A longer LBD similar to that of B. germanica would have resulted in an

amplified fragment approximately 469bp in length, whereas a LBD without the insert would

yield a 400bp fragment. PCR amplification of cDNA obtained from mated female D. punctata

ovary, midgut, fatbody and brain yielded only the shorter 400bp fragment (Fig 2.3 A). However,

while not identified here, LBD splice variants may exist in tissues or developmental stages not

screened in this study.

32To determine if four separate mRNAs give rise to the four variants of DpRXR, a

Northern blot was conducted using a 508bp DIG-labelled probe which hybridized to the C

domain, D domain and a portion of the E/F domain, a region common to all forms of DpRXR

(Fig 2.3 B). A single yet smeared band occurred at 2kb, consistent with the length cDNA

sequenced given the incomplete untranslated regions described above. No signal was obtained

from mated female midgut tissue, suggesting either low tissue specific expression of DpRXR

and/or sample degradation. The hybridization pattern did not reveal separate bands

corresponding to each alternative cDNA. However, because the four putative splice variants

differ by as little as 36bp, it is likely not possible to resolve them on an agarose gel. In addition,

any RNA degradation at the 5' end would also make resolution of similarly sized splice variants

difficult. Genomic DNA sequencing should be performed in the future to verify the gene

structure of DpRXR and the location of introns and splice sites.

33A 1 GTCGAGACGCGGCTGTTGTGCGGTGCGGTGGTACGTGATGTCACAATGCTCAAGAAGGAGAAACCCATGATGTCTGTGACGGCCATC 87 1 M L K K E K P M M S V T A I 29 88 ATCCAGGGCGCTCAGGCCCAGCAACAGCAGCACTGGGGCCGAGTTGCAGGACTGACCCTGGAGAACAGTCTGCCTATCAGTTCAATG 174 30 I Q G A Q A Q Q Q Q H W G R V A G L T L E N S L P I S S M 58 175 GAGCCACAGTCACCTCTCGACATGAAGCCAGACACTGCCAGTCTCCTGGGGTCCGGAAGCTTCAGTCCGACAGGAGGAGGAGGTGGA 261 59 E P Q S P L D M K P D T A S L L G S G S F S P T G G G G G 87 262 CCAAACAGTCCTGGGTCGTTCAGTATTGGTCACAGCAGTGTGCTGAACAACTCAACGAGCAGTTCACAGTCTAAAAGTACATCGAGC 348 88 P N S P G S F S I G H S S V L N N S T S S S Q S K S T S S 116 349 TCCTCTTCATACCCCCCCAACCACCCACTCAGCGGATCCAAGCACCTCTGTTCCATCTGTGGAGACAGAGCCAGCGGCAAACACTAT 435 117 S S S Y P P N H P L S G S K H L C S I C G D R A S G K H Y 145 436 GGCGTGTACAGCTGTGAAGGATGTAAGGGCTTCTTCAAGAGAACTGTGCGTAAAGATCTGTCCTACGCCTGCCGAGAGGATAAGAAC 522 146 G V Y S C E G C K G F F K R T V R K D L S Y A C R E D K N 174 523 TGCATCATTGACAAAAGACAGCGTAACAGGTGTCAGTACTGTCGCTACCAGAAATGTCTTGGCATGGGCATGAAGAGAGAAGCAGTT 609 175 C I I D K R Q R N R C Q Y C R Y Q K C L G M G M K R E A V 203 610 CAGGAGGAGAGACAGCGAACCAAGGAGCGAGACCAGAATGAAGTAGAGTCTACAAGCAGCCTGCACACAGACATGCCAGTGGAGCGC 696 204 Q E E R Q R T K E R D Q N E V E S T S S L H T D M P V E R 232 697 ATTCTAGAGGCGGAGAAGAGAGTGGACTGCAGACCCGAGCAGCAAGTAGAGATAGAGTCTGCAGTGACCAACATCTGTCAGGCAACC 783 233 I L E A E K R V D C R P E Q Q V E I E S A V T N I C Q A T 261 784 AACAAACAGTTGTTCCAGCTGGTGGAGTGGGCAAAGCACATCCCACACTTCACCAGTTTGCCCCTCAGCGACCAGGTGCTGCTCCTA 870 262 N K Q L F Q L V E W A K H I P H F T S L P L S D Q V L L L 290 871 CGGGCCGGTTGGAATGAGCTGCTGATAGCAGCTTTCTCCCATCGCTCCGTTGAGGTTAAGGATGGCATTGTGTTAGCCACTGGACTG 957 291 R A G W N E L L I A A F S H R S V E V K D G I V L A T G L 319 958 ACAGTGCATCGTAACTCAGCGCACCAGGCCGGTGTGGGTGCCATATTTGATCGTGTTCTTACTGAACTCGTCGCCAAGATGCGAGAA 1044 320 T V H R N S A H Q A G V G A I F D R V L T E L V A K M R E 348 1045 ATGAAAATGGACAAAACAGAACTCGGCTGTCTGCGTTCCATCATCCTGTTTAACCCAGATGTACGTGGCCTCAAGTCGTCGCAGGAC 1131 349 M K M D K T E L G C L R S I I L F N P D V R G L K S S Q D 377 1132 GTCGAGGTGCTGAGGGAGAAGGTGTATGCAGCTCTTGAAGAATACACTCGCACCACTTACCCCGATGAACCCGGCCGCTTCGCCAAG 1218 378 V E V L R E K V Y A A L E E Y T R T T Y P D E P G R F A K 406 1219 CTGCTGCTGCGCCTTCCCTCCCTGCGCTCCATCAGTCTAAAGTGTCTGGAGTATCTCTTCTTCTTCAGACTCATCGGAAACGTACCC 1305 407 L L L R L P S L R S I S L K C L E Y L F F F R L I G N V P 435 1306 ATCGACGAGTTCCTCATGGAAATGCTAGAAGCACCCATGTCCAGTGATGCTTAATTCACTGTAAAACACAACACTGCAGCCCTCGTT 1392 436 I D E F L M E M L E A P M S S D A * 452 1393 CTCTCTAGGATTCTAGGATTTGGTTCCCTGTAGGTACCCGTGATAGAGCAGACACAGGTCCTGGAAGGACTGCAGCGCCATAGAATT 1479 1480 CAGTACTGGTAATTA 1494

B CAGCAGCACTGGGGCCGA------------------------------------GTTGCAGGACTGACCCTG CAGCAGCACTGGGGCCGAGGTATGTTGCACGTGTCAGTACCTCGCCCTGCCGGCTTTGCAGGACTGACCCTG Q Q H W G R G M L H V S V P R P A G F A G L T L

Figure 2.1 Nucleotide and deduced amino acid sequence of D. punctata RXRA. (A) The solid arrow indicates the position of the insertion/deletion that distinguishes long and short isoforms. The DNA-binding domain is double underlined and the dashed arrow shows the position where the E/F domain begins. (B) The sequence of the DpRXRA-L insertion is given by the bold underlined letters.

34A 1 TCGGCCATGTTTGATACGAACAAAGTGGGGTGAAAAAGGTTTATTTTGTTAAATCTAATCTGACATTTGGTGCTAATTAGTTAGTAA 87 88 GTGATTAAAAGTGGAGATTATCCTGAAATGGAAGGAAGCGAGAGAGTTGCAGGACTGACCCTGGAGAACAGTCTGCCTATCAGTTCA 174 30 M E G S E R V A G L T L E N S L P I S S 58 175 ATGGAGCCACAGTCACCTCTCGACATGAAGCCAGACACTGCCAGTCTCCTGGGGTCCGGAAGCTTCAGTCCGACAGGAGGAGGAGGT 261 59 M E P Q S P L D M K P D T A S L L G S G S F S P T G G G G 87 262 GGACCAAACAGTCCTGGGTCGTTCAGTATTGGTCACAGCAGTGTGCTGAACAACTCAACGAGCAGTTCACAGTCTAAAAGTACATCG 348 88 G P N S P G S F S I G H S S V L N N S T S S S Q S K S T S 116 349 AGCTCCTCTTCATACCCCCCCAACCACCCACTCAGCGGATCCAAGCACCTCTGTTCCATCTGTGGAGACAGAGCCAGCGGCAAACAC 435 117 S S S S Y P P N H P L S G S K H L C S I C G D R A S G K H 145 436 TATGGCGTGTACAGCTGTGAAGGATGTAAGGGCTTCTTCAAGAGAACTGTGCGTAAAGATCTGTCCTACGCCTGCCGAGAGGATAAG 522 146 Y G V Y S C E G C K G F F K R T V R K D L S Y A C R E D K 174 523 AACTGCATCATTGACAAAAGACAGCGTAACAGGTGTCAGTACTGTCGCTACCAGAAATGTCTTGGCATGGGCATGAAGAGAGAAGCA 609 175 N C I I D K R Q R N R C Q Y C R Y Q K C L G M G M K R E A 203 610 GTTCAGGAGGAGAGACAGCGAACCAAGGAGCGAGACCAGAATGAAGTAGAGTCTACAAGCAGCCTGCACACAGACATGCCAGTGGAG 696 204 V Q E E R Q R T K E R D Q N E V E S T S S L H T D M P V E 232 697 CGCATTCTAGAGGCGGAGAAGAGAGTGGACTGCAGACCCGAGCAGCAAGTAGAGATAGAGTCTGCAGTGACCAACATCTGTCAGGCA 783 233 R I L E A E K R V D C R P E Q Q V E I E S A V T N I C Q A 261 784 ACCAACAAACAGTTGTTCCAGCTGGTGGAGTGGGCAAAGCACATCCCACACTTCACCAGTTTGCCCCTCAGCGACCAGGTGCTGCTC 870 262 T N K Q L F Q L V E W A K H I P H F T S L P L S D Q V L L 290 871 CTACGGGCCGGTTGGAATGAGCTGCTGATAGCAGCTTTCTCCCATCGCTCCGTTGAGGTTAAGGATGGCATTGTGTTAGCCACTGGA 957 291 L R A G W N E L L I A A F S H R S V E V K D G I V L A T G 319 958 CTGACAGTGCATCGTAACTCAGCGCACCAGGCCGGTGTGGGTGCCATATTTGATCGTGTTCTTACTGAACTCGTCGCCAAGATGCGA 1044 320 L T V H R N S A H Q A G V G A I F D R V L T E L V A K M R 348 1045 GAAATGAAAATGGACAAAACAGAACTCGGCTGTCTGCGTTCCATCATCCTGTTTAACCCAGATGTACGTGGCCTCAAGTCGTCGCAG 1131 349 E M K M D K T E L G C L R S I I L F N P D V R G L K S S Q 377 1132 GACGTCGAGGTGCTGAGGGAGAAGGTGTATGCAGCTCTTGAAGAATACACTCGCACCACTTACCCCGATGAACCCGGCCGCTTCGCC 1218 378 D V E V L R E K V Y A A L E E Y T R T T Y P D E P G R F A 406 1219 AAGCTGCTGCTGCGCCTTCCCTCCCTGCGCTCCATCAGTCTAAAGTGTCTGGAGTATCTCTTCTTCTTCAGACTCATCGGAAACGTA 1305 407 K L L L R L P S L R S I S L K C L E Y L F F F R L I G N V 435 1306 CCCATCGACGAGTTCCTCATGGAAATGCTAGAAGCACCCATGTCCAGTGATGCTTAATTCACTGTAAAACACAACACTGCAGCCCTC 1392 436 P I D E F L M E M L E A P M S S D A * 453 1393 GTTCTCTCTAGGATTCTAGGATTTGGTTCCCTGTAGGTACCCGTGATAGAGCAGACACAGGTCCTGGAAGGACTGCAGCGCCATAGA 1479 1480 ATTCAGTACTGGTAATTA 1497

B ATGGAAGGAAGCGAGAGA------------------------------------GTTGCAGGACTGACCCTG ATGGAAGGAAGCGAGAGAGGTATGTTGCACGTGTCAGTACCTCGCCCTGCCGGCTTTGCAGGACTGACCCTG M E G S E R G M L H V S V P R P A G F A G L T L

Figure 2.2 Nucleotide and deduced amino acid sequence of D. punctata RXRB. (A) As in figure 2.1, the solid arrow indicates the position of the insertion/deletion that distinguishes long and short isoforms. The DNA-binding domain is double underlined and the dashed arrow shows the position where the E/F domain begins. (B) The sequence of the DpRXRB-L insertion is given by the bold underlined letters.

35

Figure 2.3 Putative splice variants of DpRXR in adult female tissues. (A) Amplification of LBD region where splice variation is known to occur in other lower insects. Only the short form of region (400bp) was isolated from D. punctata. (B) Northern blot hybridized to 508bp DIG-labelled probe common to all DpRXR isoforms. One band corresponding to 2kb in size was seen in the early ovary. The age of animals in days is shown above tissue labels. Abbreviations are as follows: ovary (OV), brain (Br), midgut (MG), and fatbody (FB).

362.3.2 Sequence comparison of functional domains

To compare the DpRXR sequence to those of other species percent sequence identity (I)

and similarity (S) were calculated for each domain and across the full length of the protein

(Table 2.2, 2.3). In general, the A/B domain tends to be quite variable across species. The A/B

domain of DpRXRB had the highest score, sharing 88.4% I and 94.7% S with the cockroach B.

germanica. The N-terminal splice variation of DpRXR affected scores for this region of the

protein. For example, DpRXRA had a higher % I with Aedes aegypti USPA than USPB, 46.2%

compared to 35.0%. DpRXRB, on the other hand, had a higher % I with A. aegypti USPB than

USPA, 42.4% and 35.5%, respectively. In contrast, the DBD is highly conserved with a score

>81% I across all species. The similarity and identity of the hinge or D domain tends to be more

variable across taxa. The 23 amino acid D domain of DpRXR shares 100% I with B. germanica

but only about 20% to the 54 amino acids D. melanogaster sequence. Analysis of the LBD

revealed that DpRXR shares >61% I and >73.1% S with chordates, crustaceans, molluscs, and

non-Mecopterida insects but only <45.2% I and <65.1% S with Mecopterida-type USPs.

A multiple sequence alignment of the N-terminal A/B domain was constructed for both

DpRXRA and B (Fig. 2.4). The 12 amino acid insertion/deletion that differentiates the long and

short forms of DpRXR did not align well with any other sequences. Vertebrate RXRs did not

align well within the A/B region and were excluded. However, among the invertebrates,

sequences could be separated into two groups based on the splice variants of DpRXR. USP/RXR

sequences from the Hymenoptera, Nematocera, Lepidoptera, Crustacea, and Chelicerata aligned

with the N-terminal portion of the A/B domain of DpRXRA. Similarly, sequences that aligned

with DpRXRB included the Nematocera and Lepidoptera but also included the Orthoptera,

Blattaria, Coleoptera, Brachycera and Mollusca. Neither type seemed restricted to certain taxa,

37

38

39

Figure 2.4 Multiple sequence alignment of USP/RXR A/B domain region where alternative splicing occurs. Dashed lines indicate the region of the A/B domain in the D. punctata RXR schematic which has been aligned. Amino acid residues identical to DpRXR are shown in black. The 12 amino acid insertion/deletion which differentiates long and short forms is highlighted in blue letters. The N-terminal regions of the domain which differs between the A and B type DpRXR are highlighted in dark green and light green boxes respectively. Abbreviations are as follows: Diploptera punctata (Dpu), Apis mellifera (Ame), Aedes aegypti (Aae), Chironomus tentans (Cte), Manduca sexta (Mse), Celuca pugilator (Cpu), Marsupenaeus japonicus (Mja), Liocheles australasiae (Lau), B. germanica (Bge), L. migratoria (Lmi), T. castaneum (Tca), D. melanogaster (Dme), Biomphalaria glabrata (Bgl).

40

41which suggests that both A and B variants may commonly occur in a given species but have yet

to be isolated or reported.

An alignment of the LBD across a diverse range of taxa demonstrated that in general the

domain is well conserved (Fig. 2.5). Species from the Mecopterida group share two divergent

regions, not present in DpRXR, that do not align well with other species; these are highlighted in

large blue boxes in figure 2.5. These regions form two loops in the protein; the first connects α-

helices H1 to H3, and the second connects H5 to β-sheet 1. Structurally the first loop creates

contacts with the last α-helix H12 locking it in an inactive position (Billas et al., 2001; Clayton et

al., 2001). However, the role of the second loop is unclear from current crystallography work. Of

particular interest was the conservation of the LBP. 16 of 20 sites implicated in ligand-binding in

H. sapiens RXRα are identical in DpRXR (Egea et al., 2000). 8 of 17 ligand-binding sites in H.

virescens USP are identical in D. punctata with an additional three semi-conserved sites (Billas

et al., 2001). DpRXR also shares 16 identical and 4 semi-conserved residues with the 31 ligand-

binding amino acids in D. melanogaster USP (Clayton et al., 2001). The AF-2 transcription

activation domain located at the C-terminus of H12 (FLMEMLE) of DpRXR is 100% identical

to that of vertebrates. It is also conserved in Mollusca and Orthoptera, but somewhat divergent in

the non-insect arthropod sequences shown and highly divergent in the higher insects. Upon

ligand-binding in vertebrates, H12 moves over the LBP to create a surface for the interaction of

cofactors which mediate the activation of transcription. The two glutamic acid residues critical

for this function are conserved in DpRXR, but in higher insects, one or both of these sites are

modified (Wurtz et al., 1996). In addition, 10 of 11 (AKLLLRLPALR) key sites which mediate

the dimerization of partner proteins in H10 of H. sapiens RXRα are conserved in DpRXR (Lee et

al., 1998b).

42

Figure 2.5 Multiple sequence alignment of USP/RXR LBD sequences. Amino acids identical to D. punctata RXR are shown in black. Amino acid sites implicated in ligand-binding for Human, Heliothis, and Drosophila are shown above the alignment in orange, blue and green respectively. Loop regions which occur between α-helices only in the higher insects are indicated by a shaded blue field and the location of the AF-2 region is indicated by an un-shaded green box. The position of α-helices H1-12 and β-sheets S1-2 in Human RXRα are shown below the alignment. Abbreviations are as follows: D. melanogaster (Dme), A. aegypti (Aae), C. tentans (Cte), Bombyx mori (Bmo), M. sexta (Mse), H. virescens (Hvi), Chimarra marginata (Cma), A. mellifera (Ame), T. castaneum (Tca), B. tabaci (Bta), D. punctata (Dpu), B. germanica (Bge), L. migratoria (Lmi), Amblyomma americanum (Aam), C. pugilator (Cpu), M. japonicus (Mja), Thais clavigera (Tcl), B. glabrata (Bgl), Branchiostoma floridae (Bfl), Polyandrocarpa misakiensis (Pmi), Danio rerio (Dre), Xenopus laevis (Xla), Homo sapiens (Hsa).

43

44A phylogenetic tree was constructed using maximum likelihood methods to demonstrate

the relationship between DpRXR and other species in the LBD (Fig. 2.6). Neither of the two

commonly utilized outgroups yielded a properly rooted and reliable phylogeny. Using the

cnidarian Tripedalia cystophora RXR as an outgroup rooted the tree between arthropods and

molluscs, resulting in a topology where protostomes group with deuterostomes (‘X’ in Fig 2.6).

However, rooting with sequences from a related nuclear receptor, hepatocyte nuclear factor 4α

(HNF4A), generated a tree rooted at the midpoint splitting insect lineages non-parsimoniously

(‘*’ in Fig. 2.6). Such a topology would imply an ancestral duplication lead to Mecopterida and

non-Mecopterida type USP/RXRs. Instead an unrooted radial tree is shown which clearly

demonstrates the long branch length leading to the taxa contained in the Mecopterida group. In

contrast, all other taxa cluster relatively close together. In general, all species fell within the

appropriate broad taxonomic group (Crustacea, Insecta, etc) with the exception of the hemipteran

B. tabaci which surprisingly grouped with the Chelicerata. As expected, DpRXR grouped with

the other Hemimetabola B. germanica and L. migratoria.

45

Figure 2.6 Phylogenetic tree of USP/RXR LBD sequences constructed in PhyML using WAG substitution model with 100 bootstrap replicates. Poorly aligned regions were eliminated by eye resulting in 260 positions (see Fig S2.1). Blue arrow shows the position of DpRXR, the ‘*’ indicates the position of the root if the tree is constructed using HNF4A sequences as the outgroup, and the ‘X’ indicates the position of the root if cnidarian RXR is used as the outgroup. Open circles at nodes indicate bootstrap values >70, and solid circles values >90.

46

47

2.3.3 Estimation of evolutionary rates

The current theory of USP/RXR functional evolution proposes that the ligand-binding

function was lost in some arthropod lineage followed by a subsequent gain in function along the

Mecopterida lineage which enabled JH binding. This hypothesis, of functional gain, was tested

by estimating evolutionary rates across a dataset of invertebrate USP/RXR LBD sequences,

using codon-based models of substitution (Fig 2.7). Likelihood scores and ω values, as

calculated by PAML, are shown in Table 2.4. Branch models implemented in PAML allow ω to

be freely estimated along specified foreground branches while all other background branches are

constrained to the same ω across the phylogeny. A branch model analysis, for which the

Mecopterida lineage was set as foreground, demonstrated an elevated value, ω=999 (Table 2.4).

However, LRT indicated that the additional parameter did not yield a significantly better fit for

the data than M0 where one ω is estimated across the entire phylogeny (p=0.186, p-value <0.05

significant) (Table 2.5). The lineage leading to the ancestor of Hymenoptera and Mecopterida

was also freely estimated, in a separate analysis, but in this case ω was not found to be elevated

(ω=0.012). Similarly, the added parameter along that branch also did not yield a statistically

better fit than M0 (p=0.344) (Table 2.4, 2.5).

48

Figure 2.7 Phylogeny of species in USP/RXR data set used for PAML analysis. The Mecopterida, highlighted in red, is the lineage proposed to be under positive selection.

49

Table 2.4 Parameter estimates for RXR gene Model lnL Parameter values Positively selected sites Branch Models:

M0 -19203.8 ω0=0.0413 - M1m -19202 ω0=0.0414, ω1=999 - M1m+h -19203.4 ω0=0.0420, ω1=0.012 -

Branch-Site Models: - M1a -19161.1 ω0=0.0406, ω1=1.0000 - MA1 -19144.8 ω0=0.0402, ω1=1.0000,

ω2a=1.0000, ω2b=1.0000 p0=0.7770, p1=0.0209, p2a=0.1969, p2b=0.0053

-

MA -19142.5 ω0=0.0408, ω1=1.0000, ω2a=5.3806, ω2b=5.3806 p0=0.7671, p1=0.0206, p2a=0.2068, p2b=0.0056

T16, F25, R27, V48, V49, Q68, L98, D216 (at P>0.95) V28, W92, S97, F129, C203, C208 (at P>0.99)

p is the proportion of sites

50

Table 2.5 Likelihood ratio tests Model d.f. p-value M1m-M0 1 0.186 M1m+h-M0 1 0.344 MA-M1a 2 9.351E-05 MA-MA1 1 0.127

d.f. degrees of freedom

51Positive selection may not act on all sites in the gene so branch-site models were

implemented to estimate ω for each site in the gene along the specified foreground lineage.

Branch-site model MA demonstrated that a proportion of sites in the USP/RXR gene have ω>1

in the Mecopterida lineage (Table 2.4). However, when compared to branch-site model MA1, a

more stringent test for positive selection, LRT showed that this result was not statistically

significant (p=0.127). When compared to M1a, a model that is a less stringent test for positive

selection, LRT showed that the result was highly statistically significant (p=9.351 x 10-5). Using

a BEB analysis in MA, a class of sites with ω>1 was identified (Table 2.4). Eight sites showed

ω>1 at a posterior probability P>0.95, and six at P>0.99. Of those 14 sites, only two sites, V48

and V49, are implicated in ligand-binding (refer to Fig. S2.1, amino acids according to D.

melanogaster). Several sites F25, R27, V28 lie within Loop H1-H3. W92, S97 and L98 lie in the

region immediately N-terminal to loop H5-S1. Overall, these results indicate that there are

significantly elevated ω values along the Mecopterida lineage, suggestive of relaxed constraint

and possibly positive selection.

2.3.4 DpRXR expression

To examine tissue- and stage-specific expression profiles, RT-PCR was used to amplify

DpRXR. The following data represent a preliminary analysis that must be repeated and expanded

to be conclusive. Current RT-PCR results suggest that putative DpRXR alternative splice

variants are differentially expressed in mated adult female D. punctata. Both DpRXRB-L and S

are relatively equivalent in the day 1 ovary, but the short form predominates in the day 5 brain

(Fig 2.8 A). For DpRXRA, the long form predominates in the ovary but DpRXRA-L and S are

more equivalent in the brain (Fig. 2.8 A).The expression of overall DpRXR was also examined

in day 8-10 penultimate female larvae. Results show that expression is low in the CA, but high in

52

Figure 2.8 Differential expression of DpRXR. (A) RT-PCR amplification of splice variants in the 5’ region of DpRXR and β-actin internal control from mated adult female tissue. Each of the four expected DpRXR products are visible. (B) RT-PCR amplification of overall DpRXR and β-actin internal control from female penultimate instar tissue. The age of animals in days is shown above tissue labels. Abbreviations are as follows: brain (Br), corpora allata (CA), ovary (OV).

A

53the ovary and brain (Fig. 2.8 B). A greater sampling of tissues across stages will be necessary to

understand the role of DpRXR in the future and in particular, the function of putative splice

variants.

To examine the relationship between overall DpRXR expression, rates of JH biosynthesis

and ecdysone titre during larval development, semi-quantitative RT-PCR was used to measure

RXR tissue expression. Average stadium duration and staging accuracy for this study are shown

in Table 2.6. Preliminary data from penultimate larvae (n=1) suggest that during the critical

period for JH, days 0-8, DpRXR expression falls in the CA and brain, but remains relatively

constant in the ovary of penultimate female larvae (Fig. 2.9). After day 10, as ecdysone titre rises

and rates of JH biosynthesis remain high, DpRXR expression rises in the brain and ovary (Fig

2.9 C, D). The relationship of DpRXR with JH III and ecdysone cannot be resolved only from

data in the penultimate instar because both JH and ecdysone fluctuate simultaneously. However,

JH III and ecdysone are uncoupled in the ultimate instar in which rates of JH biosynthesis fall

before ecdysone titre rises. Preliminary data from last instar females (n=2) suggest that DpRXR

levels are higher earlier in the stadium, during the JH critical period from days 0-10, compared to

levels of expression later in the stadium (Fig 2.9). However, paired two-tailed t-tests showed that

these differences are not statistically significant. An initial analysis of DpRXR expression in the

CA of adult females (n=2) during the first gonadotrophic cycle showed no discernable trend in

expression levels (Fig. 2.10 B). Several data points showed large error in the expression in both

final instar and adult females, possibly as a result of small sample size and RNA degradation in

one set of samples. Additional rounds of collection, RNA extraction and RT-PCR analysis

should clarify the trends of DpRXR expression during development.

54

Table 2.6 Stadium duration and staging accuracy of larvae Penultimate female Ultimate female Duration (Days) 16.28 ± 0.88* n = 65 21.06 ± 0.74 n=34 Accuracy (%) 96.92 n = 40 100 n=34

* Average stadium duration ± standard deviation

55

Figure 2.9 Relative expression of overall DpRXR compared to β-actin internal control during metamorphosis of female D. punctata. (A) Rates of JH III biosynthesis and ecdysone titre for penultimate and ultimate larval instars from Kikukawa and Tobe (1986a). Relative expression of DpRXR in (B) corpora allata, (C) brain and (D) ovary as determined by semi-quantitative PCR analysis. For penultimate tissues, one set of pooled tissue samples was run in duplicate (n=1), whereas two sets of pooled tissue samples were run in duplicate for ultimate instars (n=2). Error bars indicate S.E.M.

56

57

0 1 2 3 4 5 60

50

100

150

200

Age (Days)

JH B

iosy

nthe

sis

(pm

ol.h

-1.p

erpa

ir)

0 1 2 3 4 5 60.5

0.6

0.7

0.8

0.9

1.0

1.1

Age (Days)

RXR/

B-a

ctin

(per

pai

r)

Figure 2.10 Relative expression of overall DpRXR compared to β-actin internal control in mated adult female D. punctata. (A) Rates of JH biosynthesis during the first gonadotrophic cycle of mated female D. punctata, adapted from Lenkic et al. (2009). (B) Relative expression of DpRXR in corpora allata of mated adult females as determined by semi-quantitative PCR analysis. Two sets of pooled tissue samples were run in duplicate (n=2), and error bars indicate S.E.M.

A

B

58

2.4 Discussion

Our results demonstrate that whereas DpRXR shares a great deal of similarity with basal

Hemimetabola sequences, several striking differences were also revealed. Most surprising is the

isolation of four putative N-terminal A/B domain splice variants. While splice variants of this

nature have been reported in A. aegypti, Chironomus tentans, Manduca sexta, and T. castaneum,

this is the first report of such a splicing pattern in a hemimetabolous insect (Kapitskaya et al.,

1996; Vogtli et al., 1999; Jindra et al., 1997; Tan and Palli, 2008). Although the role of the A/B

domain has not been well characterized in insects, in vertebrates, the A/B domain of RXR

functions in transcriptional activation, both modulating ligand-dependant activation and ligand-

independent constitutive activation of transcription in synergism with the C-terminal AF-2

region (Nagpal et al., 1992; 1993). As a result, alternative splice variants possess different

transcriptional activation efficiencies (Brocard et al., 1996). N-terminal splice variants have also

been reported in the vertebrates. RXR α, β, γ all exhibit alternative splice variants with distinct

N-termini in the mouse (Liu and Linney, 1993; Nagata et al., 1994; Brocard et al., 1996). In the

mouse, both RXRβ and γ variants are generated from separate exons by two different promoters

(Liu and Linney, 1993; Nagata et al., 1994). This is consistent with our results which describe

two 5' UTRs for each DpRXRA and DpRXRB. The characterization of the DpRXR genomic

sequence will be required to confirm this gene structure.

In A. Aegypti, variants are alternatively expressed, USPa predominates in the fat body

whereas USPb is predominant in the ovary. Furthermore, A. aegypti USP is elevated in the ovary

during the previtellogenic period and after the onset of vitellogenesis (Kapitskaya et al., 1996).

Isoforms of USP/RXR are also differentially expressed during metamorphosis in M. sexta and, to

some extent, in T. castaneum (Jindra et al., 1997; Tan and Palli, 2008). Here, we demonstrate

that DpRXRA and DpRXRB long and short forms are differentially expressed in mated adult

59female brain and ovary. However, the role of putative splice variants in the developmental and

reproductive processes of Diploptera remains unclear as a consequence of the limited scope of

our tissue analysis. A more in-depth exploration of isoforms-specific expression should be

conducted in the future.

The lack of splice variation in the LBD was unexpected given the presence of such

isoforms in both B. germanica and L. migratoria (Maestro et al., 2005; Hayward et al., 1999;

2003). RXRs with alternative LBDs have also been reported in the crustaceans Celuca

pugilator, Gecarcinus lateralis, and Carcinus maenas (Chung et al., 1998; Durica et al., 2002;

Kim et al., 2005; accession # EU683889). The location of these insertions/deletions in B.

germanica, L. migratoria and C. pugilator corresponds to the location of loop H1-H3 in the

Mecopterida (Fig. 2.5, Durica et al., 2002). This region is critical in influencing the folding of

USP/RXR, as residues in loop H1-H3 make contacts with H12, resulting in the aforementioned

inactive antagonist conformation of the receptor in Diptera and Lepidoptera (Billas et al., 2001;

Clayton et al., 2001). Although the effect of such insertion/deletions on ligand-binding function

is unknown, residues implicated in ligand-binding lie within loop H1-H3 in both D.

melanogaster and H. virescens (Clayton et al., 2001; Billas et al., 2001). If indeed these residues

are required for JH binding, DpRXR is lacking this region rendering it unable to assume the

conformation of Mecopterida USP and therefore is unlikely to bind JH in the same manner. Our

analysis of evolutionary rates also revealed a class of sites under positive selection in the loop

H1-H3 region. It is possible the longer LBD variants occur in D. punctata and a more extensive

tissue distribution analysis may yield additional isoforms.

Comparisons of LBD sequences showed that DpRXR retains many of the features in

lower insects and vertebrates. Of particular note are the residues important for dimerization and

coactivator binding; many of these sites are conserved in DpRXR. Little effort has been made to

understand the role of heterodimeric partners in ligand-binding among the insects. Several

60potential partners such as MET and HR38 have been identified, yet assays are generally

conducted with USP/RXR alone, or in conjunction only with EcR (Zhu et al., 2003; Li et al.,

2007). Given that the presence of USP/RXR significantly affects the ability of EcR to

transactivate DNA in the presence of ecdysone, an understanding of heterodimeric interactions is

critical for ligand identification (Yao et al., 1993; Ogura et al., 2005; Nakagawa et al., 2007).

52% and 47% of sites implicated in D. melanogaster and H. virescens USP ligand-binding are

conserved in DpRXR. 80% of the sites involved in mediating 9cRA binding in human RXRα are

maintained in DpRXR. In terms of DpRXR functionality, the conservation of sites does not

necessarily imply ligand binding. In T. castaneum, many functional motifs are also conserved in

terms of sequence, yet no independent ligand-binding was observed in this species (Iwema et al.,

2007). The mollusc Biomphalaria glabrata and cephalochordate Branchiostoma floridae both

show high sequences similarity with vertebrate type RXR, yet have a greatly reduced ability to

transactivate transcription by retinoid binding (Bouton et al., 2005; Tocchini-Valentini et al.,

2009).

The degree of sequence similarity among vertebrate, lower insect and non-insect

arthropod sequences is puzzling. It is currently unclear if USP/RXR serves some common

function in these groups which results in selective constraint. Some have suggested USP in

higher insects is the result of gene duplication and subsequent divergence. However, no insect

has been identified with both vertebrate-like RXR and Mecopterida-like USP. Using a probe

targeting the highly conserved DBD, southern blots in L. migratoria only yield a single band

(Hayward et al., 1999). Hayward et al. (1999) suggests the conservation of functional

heterodimerization with EcR among arthropod USP/RXR proteins is evidence that

Hemimetabola USP/RXR is a true homolog of Drosophila USP. However, such data does not

completely rule out the possibility that gene duplication and subsequent loss of RXR led to USP

in the Mecopterida.

61Our phylogenetic analyses are consistent with the work of other researchers,

demonstrating the long branch length leading to USP/RXR sequences within the Mecopterida.

While it has been shown that the Mecopterida have higher relative substitution rates, our study is

the first time codon-based likelihood phylogenetic methods have been used to estimate dN/dS

ratios for this data gene (Bonneton et al., 2003; Iwema et al., 2007). Our findings, of

significantly elevated ω values indicate that the Mecopterida lineage is under relaxed constraint,

and are suggestive of positive selection. Bonneton et al., (2003) postulated that high substitution

rates are unique to Mecopterida. Our results confirm this, as elevated ω values were not found

along the branch leading to the ancestor of Mecopterida and Hymenoptera. Elevated ω in the

Mecopterida supports the development of a new function along that branch, but it is important to

note that the reason for such an elevated ω cannot be drawn from such an analysis. Whether

functional changes generated by these substitutions lead to the ability to bind JHs remains

unclear.

DpRXR expression in the ovary and brain is consistent with a role for USP in JH

signalling. JH biosynthesis in the CA, at least in mated female D. punctata, is regulated by

feedback loops which involve direct nervous and indirect humoral stimuli. The brain and ovary

both serve as components of this neuroendocrine axis (Stay and Tobe, 1978; Stay and

Woodhead, 1990; Stay et al., 1983; Lenkic et al., 2009). In this scheme, expression of DpRXR is

not necessarily expected in the CA, but rather at JH target sites that mediate such feedback

mechanisms. Although our preliminary expression profile only examined the CA of mated

females, future analyses should be conducted in additional tissues with a focus on the ovary

because USP/RXR expression is associated with oogenesis and vitellogenesis in many species

(Hayward et al., 2003; Durica et al., 2002; Maestro et al., 2005; Kapitskaya et al., 1996;

Horigane et al., 2008; Tiu and Tobe, unpublished data). We expected to see changes in DpRXR

expression to occur during metamorphosis, particularly during critical periods for JH sensitivity

62in penultimate larvae in which allatectomy results in both prolonged stadium length and

precocious metamorphosis unlike final instar larvae (Kikukawa and Tobe, 1986b). However, the

relationship between JH and DpRXR in this stadium was unclear. Our results do suggest that

DpRXR levels, like rates of JH biosynthesis, are high early in final instar females (Kikukawa and

Tobe, 1986a). Similarly, initial RXR expression is high in the fatbody and prothoracic glands of

final instar B. germanica but then falls after the second day of the stadium (Maestro et al., 2005).

These findings are suggestive of a relationship between DpRXR and JH, but to assess the

significance and reliability of the results, these experiments will need to be repeated.

Given our data, and the somewhat conflicting current understanding for USP/RXR, is

there common function of RXR? Recently it has been suggested that USP/RXR may not be a

high affinity receptor at all in insects, serving a modulatory role instead, and that JH itself may

act as a modulator of other signalling pathways (Li et al., 2007). Both retinoids and JHs have

been shown to affect RXR levels in arthropods, yet do not seem to act directly as ligands in all

groups (Durica et al., 1999; Barchuk et al., 2004; Chung et al., 1998; Hiruma et al., 1999). This

is consistent with the recent findings of Beck et al., (2009); using an in vivo approach, the

authors demonstrated that JH III did not directly activate Drosophila USP, but repressed the

activation of the ecdysone heterodimer. In fact, even the high affinity binding of 9cRA with

vertebrate RXR may not be physiologically relevant. The concentration of 9cRA required to

elicit binding is not present in vivo and only all-trans RA, not 9cRA, is required for embryonic

development in mice. Instead, RXR may act to influence the specificity and activity of its

heterodimeric partners (Mic et al., 2003). Additionally, some chordate systems, which appear to

lack the ability to synthesize retinoids, and do not rely on these compounds for development, still

express RXR (Cañestro and Postlethwait, 2007). In this capacity, USP/RXR may not be as

different in vertebrates and insects as it may appear. A modulatory role would also be consistent

63with our expression data and evolutionary divergences may allow USP/RXR to interact with a

varied, perhaps overlapping, collection of pathways in distinctive lineages.

2.5 Conclusions and future directions

Ultimately, ligand-binding assays will be needed to determine the functional

characteristics of DpRXR. Ideally, such tests should be conducted with multiple alternative

USP/RXR partner proteins. Currently, no crystal structures have been resolved for USP/RXR in

basal insects or non-insect arthropods, and consequently the structure of the LBP in these groups

is also unknown. Additionally, a greater diversity of sequences from these groups is needed to

answer the lingering questions about USP/RXR function. Partial sequence data have been

reported from Collembola and Myriapoda (Bonneton et al., 2003). Data from non-arthropod

invertebrates such as onychophora will be important in understanding functional shifts over

evolutionary time. RXR has proved to be a very plastic nuclear receptor in terms of function and

structure and such studies will eventually clarify its role in insects while adding to our

understanding of endocrine and developmental control in both vertebrates and invertebrates.

64

CHAPTER THREE: RECONSTRUCTION OF ANCESTRAL FGLamide-TYPE INSECT ALLATOSTATINS: A NOVEL

APPROACH TO THE STUDY OF ALLATOSTATIN FUNCTION AND EVOLUTION

65Abstract

Allatostatins (ASTs) are a class of regulatory neuropeptides, with diverse functions,

found in an array of invertebrate phyla. ASTs have complex gene structure, in which individual

ASTs are cleaved from a precursor peptide. Little is known about the molecular evolution of

AST structure and function, even in extensively studied groups such as cockroaches. This paper

presents the application of a novel technique for the analysis of this system, that of ancestral

reconstruction, whereby ancestral amino acid sequences are resurrected in the laboratory. We

inferred the ancestral sequences of a well-characterized peptide, AST 7, for the insect ancestor,

as well as several cockroach ancestors. Peptides were assayed for in vitro inhibition of JH

production in Diploptera punctata and Periplaneta americana. Our results surprisingly, indicate

a decrease in potency of the ancestral cockroach AST 7 peptide in comparison with more ancient

ones such as the ancestral insect peptide, as well as more recently evolved cockroach peptides.

We propose that this unexpected decrease in peptide potency at the cockroach ancestor may be

related to the concurrent increase in peptide copy number in the lineages leading to cockroaches.

This model is consistent with current physiological data, and may be linked to the increased role

of ASTs in the regulation of reproductive processes in the cockroaches.

3.1 Introduction

Allatostatins (ASTs) are a class of regulatory neuropeptides found in a diversity of

invertebrate phyla. The cockroach type AST, or FGLamide (FGLa) types, were first discovered

in the viviparous cockroach Diploptera punctata (Woodhead et al., 1989; Pratt et al., 1989).

FGLa ASTs share the C-terminal motif (Y/F)XFG(L/I)-NH2 which forms the core active region

of the peptide (Tobe and Bendena, 2006). However, not all AST-like peptides possess the same

C-terminal sequence; for example, nematode sequences terminate in MGL/FGF/MGF (Nathoo et

al., 2001; Husson et al., 2005). Whereas ASTs were named and characterized as a consequence

of their ability to inhibit juvenile hormone (JH) biosynthesis by the corpora allata (CA), these

66peptides have also been found to serve many other functions. ASTs act as myomodulators across

a wide variety of invertebrate phyla including helminths, Crustacea, and the insect orders

Diptera, Lepidoptera, Dictyoptera and Orthoptera (for example see Jorge-Rivera and Marder,

1997; Mousley et al., 2005; Bendena et al., 2008; Lange et al., 1995; Aguilar et al., 2003; Predel

et al., 2001; Duve et al., 1995 and Duve et al., 1996; Veelaert et al., 1996; Vanden Broeck et al.,

1996). The FGLa ASTs also inhibit vitellogenin production in the fat body of the German

cockroach Blattella germanica, stimulate the activity of carbohydrate-metabolizing enzymes in

the midgut of D. punctata and inhibit cardiac activity in B. germanica (Martin et al., 1996; Fusé

et al., 1999; Vilaplana et al., 1999).

From a molecular evolutionary standpoint, ASTs are fascinating as a consequence of

their complex gene structure, and striking diversity of function. ASTs arise from

preproallatostatin, the precursor peptide, in which multiple ASTs are cleaved at dibasic

KR/RR/KK/RK endoproteolytic cleavage sites (Tobe and Bendena, 2006). These repeats are

assumed to be the result of duplication events, and both the amino acid sequence of the precursor

peptide and its structural organization can vary greatly across extant species (Bellés et al., 1999;

Bendena et al., 1999). In terms of molecular evolution, this presents an interesting situation

where peptide sequences within a gene can diverge and acquire new functions over time, yet are

regulated together as part of the same precursor.

The evolutionary history of ASTs and their diversity of function are of particular interest

in insects. Many studies have been conducted in insects but the functional differences in AST

peptides with respect to phylogeny remain poorly understood. Although the myomodulatory role

of ASTs appears to be conserved across invertebrate groups, this is not the case for other AST

functions. Physiological studies examining the effect of ASTs on JH production by insect

corpora allata have been performed in many species. Although FGLa ASTs are present in both

hemimetabolous and holometabolous insects, the ability to regulate JH biosynthesis appears to

67be limited to only some of the hemimetabolous insect orders—Orthoptera, Dictyoptera and

Isoptera (for a review see Stay and Tobe, 2007). Thus far, other functions of ASTs, such as the

aforementioned regulation of digestive enzyme activity, have also only been described in the

Hemimetabola, and in particular only in the Dictyoptera (Martin et al., 1996; Fusé et al., 1999;

Vilaplana et al., 1999).

Even within the Hemimetabola, the relationship between primary structure of a peptide

and its function remains unclear. Although Bellés et al. (1999) demonstrated that in cockroaches

AST peptides are highly conserved in terms of sequence, the potency of a given peptide is not

necessarily conserved across species. The seventh AST (AST 7) of the cockroaches B.

germanica and D. punctata both inhibit JH biosynthesis but with different potencies; AST 7 is

more potent in D. punctata than in B. germanica, by several orders of magnitude (Bellés et al.,

1994; Tobe et al., 2000). ASTs do not necessarily serve the same functions in all closely related

species; ASTs inhibit JH biosynthesis in the cricket Gryllus bimaculatus but have no effect in

another Orthopteran, the locust Schistocerca gregaria (Lorenz et al., 1995 and Lorenz et al.,

1999; Veelaert et al., 1996). Molecular studies examining the relationships between individual

ASTs and among AST precursor genes have attempted to address questions regarding function

and phylogeny (Bellés et al., 1999; Bendena et al., 1999). However, there are still no conclusive

answers regarding the origin and evolutionary history of AST function in Hemimetabola or any

other group.

Among insects, cockroaches are of particular interest because of their diversity of

reproductive modes. In cockroaches, three basic modes of reproduction occur, oviparity in which

fertilized eggs develop outside of the body, ovoviviparity in which fertilized eggs are carried in a

brood sac, and viviparity in which fertilized eggs are carried within the brood sac and are

nurtured by the female (Roth, 1970). Reproductive processes such as vitellogenesis and oocyte

maturation are linked to the well-coordinated regulation of JH production by the corpora allata

68during the reproductive cycle (Tobe and Stay, 1977). This requirement for the regulation of JH

production is likely to be linked to the increased number of functional roles and potency of ASTs

in cockroaches. Additionally, AST peptides have also been well studied in the Dictyoptera and

much comparative data are available. The cockroach AST peptides have been well characterized

in terms of regulation of JH production in the greatest number of hemimetabolous species, and

thus lend themselves well to further study (Tobe et al., 2000; Bellés et al., 1994; Weaver et al.,

1994; Lorenz et al., 1999; Yagi et al., 2005).

In recent years, the scope of species in which ASTs have been identified has grown

considerably. FGLa immunoreactivity has been described in many lower invertebrates such as

Trematodes and Hydrozoa, as well as in Gastropoda and Cephalopoda, but no AST-like

sequences have been identified from any of these groups to date (Bendena et al., 1999; Smart et

al., 1994). The available AST sequence data have also expanded. In addition to insects, FGLa

AST precursors have been described in several crustacean genera and neuropeptide-like-protein

encoding sequences with sequences similar to FGLa have been identified in nematodes via

genome project searches (Duve et al., 1997a; Billimoria et al., 2005; Huybrechts et al., 2003;

Dircksen et al., 1999; Yin et al., 2006; Duve et al., 2002; Yasuda-Kamatani and Yasuda, 2006;

Nathoo et al., 2001). This expansion in sequence knowledge has enabled more sophisticated

molecular evolutionary studies, including the application of ancestral reconstruction techniques

to the analysis of AST function.

Experimental ancestral reconstruction approaches use phylogenetic statistical methods of

sequence analysis to infer sequences that existed in the past; these inferred sequences are then

synthesized in the laboratory and studied in functional assays (Thornton, 2004). Ancestral

reconstruction methods can provide information about the evolutionary history of functional and

biochemical characteristics of proteins and peptides which could not otherwise be experimentally

studied (Chang and Donoghue, 2000). These methods have been successful in previous studies

69experimentally reconstructing chymases, visual pigments and hormone receptors, among others,

as well as for studying the paleobiology of extinct species (Chandrasekharan et al., 1996; Chang

et al., 1995 and Chang et al., 2002; Thornton et al., 2003; Gaucher et al., 2008). However,

ancestral reconstruction methods have never been before applied to ASTs or any other complex

family of invertebrate peptide hormones.

Genes such as those coding for the FGLa ASTs present special challenges to ancestral

reconstruction methodologies as a consequence of peptide shuffling and the expansion and

contraction of peptide number. Here, we present a reconstruction of cockroach full-length AST

precursor genes and a reconstruction of highly conserved peptides from insect ASTs for ancestral

nodes within cockroach lineages, as well as the insect ancestor. Problems with positional

homology of AST peptides prevented the inclusion in our analyses of the crustacean sequences;

instead a frequency analysis of ASTs in arthropod groups was used to understand the pattern of

AST occurrence in different taxa. To examine the role of amino acid changes in terms of AST

activity, we have performed assays of ancestral peptide AST 7 at reconstructed nodes to show

their potency in inhibition of JH biosynthesis in vitro.

3.2 Materials and methods

3.2.1 Ancestral reconstruction

To reconstruct the ancestral FGLa AST precursor gene, we first constructed an alignment

of all currently known hemimetabolous insect AST precursor gene sequences. FGLa AST

precursor genes of six cockroaches, and two Orthopteran insects were used (GenBank/EMBL

accession nos. D. punctata U00444, Periplaneta americana X91029, B. germanica AF068061,

Blaberus craniifer F068062, Supella longipalpa AF068063, Blatta orientalis AF068064, G.

bimaculatus AJ302036, S. gregaria Z58819). The sequences were translated to amino acids,

aligned using ClustalX 2.0 (Thompson et al., 1997) and adjusted by eye to ensure that known

structure/functional motifs in the active peptide domains of the AST gene were in alignment. The

70amino acid alignment (termed ‘hemimetabolous alignment’) is presented in Fig. S3.1. Although

several holometabolous insect AST precursor genes are known, they were found to be too

divergent from the hemimetabolous insect sequences to align reliably across the whole gene. We

therefore constructed a second, shorter alignment (termed ‘insect alignment’, Fig. 3.3) that

comprised only of relatively conserved regions of several holometabolous and hemimetabolous

insect AST genes. Identical sequences within a family were removed to allow more rapid

analysis (species names and GenBank/EMBL accession nos. D. punctata U00444, P. americana

X91029, B. germanica AF068061, B. craniifer AF068062, G. bimaculatus AJ302036, S.

gregaria Z58819, Spodoptera frugiperda AJ555184, Helicoverpa armigera AF015296, Bombyx

mori NM_001043571, Drosophila melanogaster AF263923, Drosophila grimshawi (see Bowser

and Tobe, 2007), Apis mellifera XM_001120780, Calliphora vomitoria (see East et al., 1996),

Aedes aegypti U66841, Anopheles gambiae XM_313511, Calanus finmarchicus EU000307).

Ancestral reconstructions were carried out using both the codeml (for amino acid and

codon data) and baseml (for nucleotide data) programs of the PAML software package, version

3.15 (Yang, 1997), and the rje_ancseq module of the GASP software package (Edwards and

Shields, 2004). Maximum likelihood/Bayesian ancestral reconstruction methods infer the most

likely ancestral sequence reconstructions for nodes on a given phylogeny, according to a

specified model of evolution. Most probable ancestral character states are inferred for all sites in

the alignment, for any internal node (Yang et al., 1995; Yang, 2006). While a variety of

substitution models can be considered in codeml and baseml, the methods these programs

implement either ignore gaps or treat gaps as ambiguous character states. The module

rje_ancseq, while not as accurate at inferring ancestral states as codeml, explicitly considers the

historical pattern of insertions and deletions in the gene of interest, using parsimony to assign

gaps prior to sequence reconstruction (Edwards and Shields, 2004). Since the hemimetabolous

alignment contained many gaps (Fig. S3.1), we chose to combine both approaches, and

71employed the rje_ancseq program to infer the ancestral gap pattern, and both rje_ancseq and

codeml/baseml to infer the ancestral sequence data. The shorter insect alignment, which

contained only areas of relatively high sequence similarity, was not analyzed using rje_ancseq as

it contained relatively few gaps.

The JTT (Jones et al., 1992) amino acid substitution matrix was used to infer the

ancestral sequence data in rje_ancseq, while the JTT (Jones et al., 1992) and WAG (Whelan and

Goldman, 2001) amino acid substitution models, the M0 and M3 (Goldman and Yang, 1994;

Yang et al., 2000) codon substitution models, and the HKY (Hasegawa et al., 1985) nucleotide

substitution model, were used to estimate ancestral sequence data in codeml/baseml. The

analyses carried out in codeml/baseml were performed both with and without the addition of a

gamma parameter, which allows the overall substitution rate in the maximum likelihood analysis

to vary across sites (Yang, 1996). In all cases, the addition of the gamma parameter led to a

significantly better fitting model (P-values <0.001) as determined by likelihood ratio tests

(Felsenstein, 1981; Yang, 2006), so only those results are presented (see Table S3.3). Trees

reflecting current understanding of insect phylogenetics (Fig. 3.1 and Fig. 3.2; Kambhampati,

1995 and Kambhampati, 1996) (Fig. 3.3; Wheeler et al., 2001; Kristensen, 1991; Pashley et al.,

1993; Boudreaux, 1979; Regier et al., 2005) were used in the ancestral reconstruction analyses.

The ancestral sequence data, along with posterior probability estimates for each amino acid at

each site, were extracted from the codeml/baseml output files using a customized Perl script.

3.2.2 Sequence collection and database analysis

A sequence database of all known FGLa allatostatin precursors and peptide sequences

was collected from literature, GenBank and EMBL (Table S3.1). This database was similar to

AST data from Liu et al. (2006); however, our dataset includes peptides isolated by protein

methods as well (http://signalling.peptides.be). Two nematode species (Caenorhabditis elegans

and Caenorhabditis briggsae) are listed but were not used for analysis. The compiled list was

72entered into a Microsoft access database and searched using a visual basic executable. Each entry

was tagged with one of the following classifications: Crustacea, Holometabola or Hemimetabola.

Using search parameters, the number of total sequences within each groups were counted and the

number ASTs shared between groups was determined (Fig. 3.4). A degenerate search was used

to determine the frequency of AST sequences within the dataset. Sequences of interest were

input into the search executable and all sequences identical to the input sequence or containing

the input sequence were called up (i.e., zero amino acid differences). In degenerate searches,

either one or two amino acid sites were allowed to differ from the input sequence, all resulting

hits containing the sequence were counted.

3.2.3 Radiochemical assays of JH release in vitro

3.2.3.1 Animals

Assays were conducted on D. punctata and P. americana. D. punctata were kept at 27 °C

in constant darkness and given lab chow and water ad libitum. Newly ecdysed mated adult

female D. punctata were selected, removed from the colony, placed in containers and provided

food and water for 7 days at which point the animals were dissected. P. americana were kept at

27 °C on a 12:12 light:dark cycle with lab chow and water available ad libitum. Last instar

females were generously provided by the animal physiology teaching labs at the Department of

Cell and Systems Biology at the University of Toronto (Toronto, Canada). Newly molted adult

females were isolated from this group and dissected on day 4.

3.2.3.2 Peptides

Peptides predicted by ancestral reconstruction were synthesized by GL Biochem Ltd.

(Shanghai, China) at >95% purity. The reconstruction of the ancestral cockroach AST 7

predicted at nodes 12 and 14 were synthesized—the Blattidae ancestor (Ba)

SPSGMQRLYGFGL-NH2 and the cockroach ancestor (Ca) APSGMQRLYGFGL-NH2,

respectively (Fig. 3.1). For the reconstruction of conserved regions of both hemimetabolous and

73holometabolous insects, AST 7 from nodes 1 and 3 were synthesized—the insect ancestor (Ia)

SRLYSFGL-NH2 and the cockroach ancestor, an N-terminally truncated version of Ca (Ca-

truncated) QRLYGFGL-NH2, respectively (Fig. 3.3). All peptide stocks were prepared in

ddH2O.

3.2.3.3 Radiochemical assay in vitro

Day 7 mated adult female D. punctata and virgin day 4 P. americana were immobilized

on ice and corpora allata dissected under sterile conditions. Only oviposited D. punctata females

were used, to ensure uniform staging. A short-term in vitro assay of JH release in T199 medium

(GIBCO) [2% Ficoll, 1.3 mM CaCl2·2H2O and 3 μCi/ml l-[methyl-14C]methionine

(2.07 GBq/mmol; Amersham)] followed by rapid partition was conducted on individual corpora

allata according to Feyereisen and Tobe (1981) and Tobe and Clarke (1985). Dose–response

curves were generated using the percent inhibition of JH release and analyzed using non-linear

regression.

3.3 Results

3.3.1 Ancestral reconstruction

For both the cockroach and insect datasets, ancestral sequences were estimated using a

variety of codon, nucleotide and amino acid-based likelihood models of substitution (Yang,

1997). Since these methods do not explicitly consider gaps in the alignment, where necessary,

we also estimated ancestral sequences using a method that does consider gaps (Edwards and

Shields, 2004). A reduced alignment consisting only of highly conserved regions of the AST

precursor gene was also analyzed, and this truncated alignment contained considerably fewer

gaps. Within datasets, the ancestral reconstruction results are generally similar regardless of the

method or substitution model used. Likelihood/Bayesian methods of ancestral reconstruction

include posterior probability values, an indication of the reliability of the reconstruction given a

specific substitution model. These posterior probabilities, as calculated in PAML, were generally

74high across the different methods, particularly for the nodes reconstructed (Figs. 3.1B and 3.3C;

Table S3.2). Ancestral reconstructions for the different models are contained in the

supplementary data (Figs. S3.1 and S3.2).

For the hemimetabolous dataset, the inferred amino acid changes across the phylogeny

are shown in Fig. 3.1 and Fig. 3.2. In particular, the reconstruction of AST 7 showed several

interesting amino acid substitutions. The first substitution occurred along the branch connecting

nodes 14 (the parent node, representing the ancestor of all cockroaches) and node 12, and

involved substituting a non-polar alanine for a polar serine residue. The second substitution

occurred along the branch-connecting node 14 (again, the node representing the ancestor of all

cockroaches) and node 11, and involved substituting a methionine for an alanine. In the ‘insect’

dataset, we also noted two interesting substitutions in AST 7 (Fig. 3.3B). Both of these

substitutions appear to have arisen at node 3 (representing the ancestor of the cockroaches). The

first substitution involved substituting a serine for a glutamine (both of which are polar, non-

charged residues), whereas the second involved substituting a polar serine for a non-polar

glycine. Interestingly, the maximum likelihood ancestral sequence of AST 7 was identical at the

nodes representing the ancestor of the hemimetabolous insects and the ancestor of all insects.

Several reconstructed peptides, present at nodes where changes in peptide copy number are

thought to have occurred, were chosen for further analysis. Peptides were selected based on the

presence of amino acid substitutions and the availability of comparative physiological data.

Therefore, the inferred AST 7 peptides from node 14 and 12 from the cockroach dataset, and

nodes 1 and 3 from the insect dataset, were synthesized and assayed for biological activity (Figs.

3.1A and 3.3A).

75

Figure 3.1 Ancestral reconstruction of Dictyopteran ASTs. (A) Phylogeny of hemimetabolous insect groups used in our analysis. Shaded circles indicate the nodes where an AST peptide was experimentally resurrected. Species where ASTs were tested are underlined. Mapped on to this phylogeny are the inferred ancestral amino acid sequences of AST 7, underlined red letters show changes which occur across all nodes and bold letter show amino acids that change across extant taxa. The EC50 values for AST 7 inhibition of JH release in the extant peptides are shown on the right, ND indicates a species for which no data are available (Weaver et al., 1994; Bellés et al., 1994; Tobe et al., 2000; Lorenz et al., 1999). (B) Distribution of posterior probabilities across sites at node 14 for different models of ancestral reconstruction as implemented in PAML. Most sites show probabilities >0.95, indicating that the amino acid reconstructions are likely to be correct. Sites inferred under the nucleotide model HKY+G have relatively lower probabilities due to third position variability in codons.

76

77

Figure 3.2 Map of amino acid changes across cockroach nodes for AST peptides inferred in the reconstructed precursor genes. Each box represents an AST peptide, the first number within the box denotes the AST followed by the amino acid and N-terminal site number at which the change occurs. For example, 7-M5A represents a change in the fifth site of AST 7 from methionine to alanine. Deletions and insertions are shown using the abbreviations del and ins, respectively. For complete alignment, see supplementary data (Fig. S3.1).

78

79

Figure 3.3 Ancestral reconstruction of ancient insect ASTs. (A) Insect phylogeny of insect orders used in our analysis. The nodes where an AST was resurrected are indicated by arrows and shaded circles. All peptides were tested in D. punctata, which is shown in underlined text in the phylogeny. (B) Alignment of conserved insect ASTs created using ClustalX and modified by eye. Ancestral sequences for each node reflect a consensus of all models used for reconstruction as reported in Fig. S3.2 and resurrected peptides are indicated with a gray box. Variable sites are indicated by a question mark (?). (C) Distribution of posterior probabilities across sites at node 1 for different models of ancestral reconstruction used.

80

813.3.2 Sequence and database analysis

The collection of sequence data for all known FGLa ASTs revealed a great number of

ASTs. In total, cockroach type AST-like sequences have been reported in 43 species; 10

Hemimetabola, 23 Holometabola, 8 Crustacea and 2 Nematoda. AST sequences have been

obtained using peptide isolation and identification as well as molecular methods so not all

species have known precursor genes. Of the 43 species listed, genetic sequence information is

known for only 31.

Within the Arthropoda, a total of 431 FGLa AST sequences have been identified, and 233

different sequences and of those, 168 are specific to a species. Little overlap occurs between the

ASTs found in Insecta and Crustacea. The Hemimetabola share four sequences with the

Crustacea and only two with the Holometabola (Fig. 3.4A). Because a lack of positional

homology restricted our reconstruction only to the conserved regions of insect precursor genes, a

search strategy was implemented to analyze peptide patterns within the entire dataset. This

method allowed us to find a sequence of interest even when embedded within a longer AST.

Database searches showed that D. punctata (Dippu) AST 2 (amino acids 11–18) and AST 6

occurred most frequently. However, using a degenerate search changed peptide frequency. When

one amino acid site was allowed to vary from the input sequence, Dippu-ASTs 2- and 6-like

sequences were the more frequent (Fig. 3.4B). When two amino acids were allowed to vary,

Dippu-AST 6-like sequences are found in all but two insect species and all but one crustacean

species (data not shown). The most frequent of all ASTs using the degenerate search method was

Dippu-AST 1; this peptide appears in 372 of the 431 ASTs when two sites vary from the input.

This likely reflects the length of the sequence; Dippu-AST 1 (LYDFGL) would be contained in

nearly any AST as its sequence is within two amino acids of the core AST motif (Y/F)XFG(L/I).

82

Figure 3.4 Analysis of arthropod AST sequence data. (A) Proportional Venn diagram showing the number of different FGLa-type AST-like peptides known in arthropod groups. The diameter of each circle is proportional to the size of the dataset for each group; overlapping regions indicate identical ASTs shared between groups. (B) AST frequency analysis using degenerate database searches of Dippu-AST sequences. The number of sequences within the dataset that match the input sequence are shown for identical sequences [0], sequences with one amino acid variation from the input [1], and sequences with two amino acid variations from the input [2].

833.3.3 Radiochemical assay for JH release

To ascertain if the ancestral reconstruction approach to AST analysis had physiological

relevance, several peptides were synthesized and assayed for their ability to inhibit JH release by

the corpora allata. AST 7 from ancestral cockroach nodes 12 and 14 were tested in D. punctata

and P. americana. In both species, the cockroach ancestor, the ancestral peptide from node 14,

was less potent than the extant AST 7 (EC50=1.57×10−8 M and 3.41×10−9 M for D. punctata and

P. americana, respectively) (Fig. 3.5A). The Blattidae ancestor, the ancestral peptide at node 12,

demonstrated potency equivalent to that of the extant AST 7 of D. punctata with an EC50 of

1.81×10−9 M (Fig. 3.5A). The Blattidae ancestor is identical to the extant AST 7 in P. americana

and was not tested in this species as it has been tested previously (Weaver et al., 1994). For the

insect dataset, peptides corresponding to AST 7 inferred for the insect and cockroach ancestors

were synthesized and tested only in D. punctata. Here, the insect ancestor, from node 1, was a far

more potent inhibitor of in vitro JH release than the truncated cockroach ancestor at node 3, with

EC50 values of 1.85×10−9 and 2.63×10−8 M, respectively (Fig. 3.5C).

3.4 Discussion

Our results demonstrate that in experimental assays of inhibition of JH release, our

reconstructed ancestral peptides (that are homologs of AST 7) are highly potent at the ancestral

insect node, less potent at the ancestral cockroach node and show increased potency again in

more recent cockroach ancestors. To ensure that our results were not simply artefacts of the

experimental system, we assayed the ancestral peptides in two different species of cockroaches

(D. punctata and P. americana). The trend of high potency for the more recent cockroach and

most ancient insect ancestral nodes, with decreased potency in the cockroach ancestor, was

found for assays in both species. This trend, of increased potency in terms of the inhibition of JH

84

Figure 3.5 Dose–response of individual corpora allata (CA) to ancestral peptides. (A) Inhibition of JH release from CA of day 7 mated female D. punctata following incubation with Dictyopteran ancestral peptides: Blattidae ancestor (Ba) from node 12 (N=10) and cockroach ancestor (Ca) from node 14 (N=10–18). (B) Effect of Ca on the JH release from day 4 virgin female P. americana CA (N=5–10). (C) The effect of ancestral insect peptides on JH release from the CA of day 7 mated female D. punctata, Insect ancestor (Ia) from node 1 (N=13–41) and truncated cockroach ancestor (Ca-Truncated) from node 3 (N=16–41). Dippu-ASTs (5 or 7) were used as positive controls and all error bars indicate standard error.

85

86production, appears to have occurred by two different pathways in cockroaches, with different

sets of amino acid substitutions occurring in the two lineages (Fig. 3.1).

The question then arises as to why there is a decrease and subsequent increase in the

potency of ASTs in the cockroach ancestors? Generally speaking, ASTs are thought to have

increased dramatically in number, particularly within hemimetabolous insects, and diversified

from a much smaller set of ancestral sequences (Bellés et al., 1999; Bendena et al., 1999; Tobe

and Bendena, 1999). Although duplicated AST peptides have obvious differences from

duplicated genes in that they are transcribed as one unit, once processed, the peptides may be

involved in separate physiological pathways, and therefore it may be useful to consider their

evolution in light of current theories of gene duplication. A classic prediction of gene duplication

theory is that duplicated genes will confer redundancy, and thus allow for the accumulation of

deleterious mutations (Ohno, 1970; Prince and Pickett, 2002). The early history of cockroaches

appears to have been accompanied by a dramatic increase in AST peptide copy number;

cockroach precursors contain 13–14 ASTs whereas holometabolous precursors only contain

between four and nine ASTs and the crustacean outgroup, C. finmarchicus, contains seven (see

Table S3.1). The newfound redundancy at this point in cockroach evolutionary history may

explain the decreased potency of the cockroach ancestor AST 7. It is also important to note that

downstream effectors of AST peptides such as the AST receptor are thought to exist in multiple

forms, and thus may have contributed to the expansion of AST copy number, but these receptors

have only recently been identified, and little is known of their structure and function (Tobe and

Bendena, 2006; Bendena et al., 2008; Auerswald et al., 2001; Lungchukiet et al., 2008).

Gene duplication theory also predicts that duplicates of multifunctional genes may be

preserved, as different duplicates specialize for different functions over time (Wistow, 1993;

Force et al., 1999), and recent surveys suggest that multifunctional genes are quite common

(Piatigorsky, 2007). Experimental studies have shown that additional functions for ASTs occur

87in the Hemimetabola, and coincide with changes in peptide copy number. There is general

agreement that myomodulatory functions of FGLa ASTs are the most widespread and likely the

most ancient (for review see Bendena et al., 1999; Stay and Tobe, 2007; Tobe and Bendena,

1999). In hemimetabolous groups, which possess a large number of ASTs, ASTs serve several

other functions such as the regulation of JH production, gut enzyme activity and vitellogenin

production (Stay and Tobe, 2007; Martin et al., 1996; Fusé et al., 1999). This model is also

supported by the situation in S. gregaria, a hemimetabolous insect with a lower peptide copy

number of 10, in which ASTs do not regulate JH production (Vanden Broeck et al., 1996;

Veelaert et al., 1996). It is also of interest that recent genomic studies of the holometabolous

insect, Tribolium castaneum, have demonstrated that both the AST precursor and receptor genes

are absent. Accordingly, gains and losses in neuropeptide genes may be fairly common (Li et al.,

2008). Future work on this system should assay the capacity of ancestrally reconstructed ASTs to

efficiently perform these other functions; the decreased potency of AST 7 at the cockroach

ancestor node could possibly reflect its specialization on another function at that point in

evolutionary history. Ideally, these studies would be performed in conjunction with

reconstructions of the ancestral AST receptor in order to investigate the evolutionary history of

interactions between ASTs and their receptors. Other G protein-coupled receptors have been

successfully reconstructed and studied in this manner (Chang et al., 2002; Kuang et al., 2006).

Although function and copy number support the observed decrease in peptide potency,

the question remains as to why the ASTs gained functional importance over evolutionary time in

the cockroach lineages. This increased potency of ancestral cockroach ASTs may reflect the

changes in reproductive biology within cockroach lineages. In these species, the timing of JH

production is known to be well coordinated with reproductive events and the regulation of JH

production is required for this timing (Tobe and Stay, 1977). In cockroaches, there is often a

correlation between corpora allata activity and the gonadotrophic cycle whereby cycles of JH

88biosynthesis occur during vitellogenesis, although these cycles differ in pattern between species

(Tobe and Stay, 1977). In contrast to the role of ASTs in the regulation of JH biosynthesis in

cockroaches, a clear correlation of corpora allata activity with vitellogenesis is not observed in S.

gregaria (Tobe and Pratt, 1975). In D. punctata, the only known viviparous cockroach species,

the situation is exaggerated because here, the precise regulation of JH production is critical; the

presence of JH during pregnancy results in abortion (Stay and Lin, 1981). This shift towards

more complex reproductive modes in cockroaches could explain the corresponding shift in the

importance of the ASTs as regulators of JH biosynthesis with respect to reproductive success and

may account for the increased potency of more recent peptide ancestors.

Our database analysis of ASTs demonstrates that there is a far greater number of ASTs

than previously thought. Previous estimates range from 50 to 150, whereas we show here that

over 431 sequences are known (Kai et al., 2006; Bendena et al., 1999; Mousley et al., 2005; Liu

et al., 2006). Interestingly, the peptides we were able to align within the insect precursor genes,

according to positional homology, correspond to the peptides with the highest frequency found

using our database searches. Of particular interest is the copepod C. finmarchicus; its AST

precursor is unlike all other crustaceans previously sequenced, and is the most basal arthropod

AST sequenced to date. It contains very few peptides, nearly all of which bear sequence

similarity to the ASTs conserved in our insect alignments and the peptides with greatest

frequency in our database. While this type of frequency-based analysis cannot determine which

set of ASTs was present in the ancestral condition, it is ideal for finding peptide patterns when

positional homology is lacking. This approach will be valuable as a starting point for future

molecular and physiological studies.

There are several caveats which must be applied to the present work. First, there is the

importance of downstream effectors in the signal cascade of ASTs. Assay of the extant D.

punctata AST 7 in P. americana demonstrated that the peptide lost potency, with the EC50

89decreasing from 1.94×10−9 to 6.9×10−8 M (Tobe et al., 2000; Weaver, 1991). Such differences in

activity for a given peptide must be the result of downstream signalling events or differences in

receptor specificity and represent a great unknown in AST research. Few FGLa AST receptors

are known: in Drosophila, two receptors specific to ASTs have been identified, but they share

only 47% overall sequence similarity (Birgül et al., 1999; Lenz et al., 2001). Orthologs of the

Drosophila receptors have been identified in P. americana, B. mori, A. mellifera, A. gambiae, C.

elegans and recently in D. punctata (Auerswald et al., 2001; Gäde et al., 2008; Tobe and

Bendena, 2006; Bendena et al., 2008; Lungchukiet et al., 2008). Aside from these receptors,

little is known with regard to the signal transduction cascade for the AST signal, and given the

multiple actions of ASTs, there are likely to be multiple pathways. While it is clear that amino

acid changes in peptide sequence would affect activity, this study highlights the importance of

these other signalling events. Our tests were primarily conducted in D. punctata, a highly derived

species in which precise regulation of JH biosynthesis is particularly critical, and the ancestral

AST peptides assayed here may in fact have different effects in other species.

Second, there is some contention concerning the evolutionary history of the role of ASTs

in the regulation of JH biosynthesis. JH is not present in all groups in which ASTs occur; for

example in Crustacea, the pathway for sesquiterpenoid biosynthesis terminates with methyl

farnesoate, the immediate precursor of JH (Tobe and Bendena, 1999). Despite the altered

pathway, and the absence of JH, studies have shown that AST peptides have a role in this

system. Kwok et al. (2005) demonstrated that ASTs stimulate the production of methyl

farnesoate by the mandibular organ, the endocrine gland homologous to insect corpora allata, in

the crayfish P. clarkii. This is similar to the action of ASTs in early embryonic development of

D. punctata, which has led to the suggestion that sesquiterpenoid regulation may have been an

early function of ASTs in arthropods (Stay et al., 2002). However, no other crustacean species

have been assayed to date (Stay and Tobe, 2007).

903.5 Conclusions and future directions

Ancestral reconstruction methods serve as powerful tools for investigating protein

structure and function. Here, we have shown that these methods can be successfully applied to a

peptide hormone system, an approach that has never been used in this context. However, to

resolve broad questions about the origin of ASTs, more data will be needed both in terms of

sequence and physiology. We were not able to assay any of the reconstructed peptides in terms

of myoinhibition, nor were we able to test their action in higher insects. Such studies will be

essential in the future. In particular, more data from primitive arthropod groups will be needed

before we can attempt to determine which ASTs were part of the ancient complement of

peptides. As the dataset of known ASTs grows in size, so too will the accuracy of reconstruction

methods. They will no doubt prove invaluable for the future study of AST evolution.

91

CHAPTER FOUR: SUMMARY AND DISCUSSION

92 In general, both juvenile hormone and the regulatory neuropeptides of the allatostatin

family represent functionally diverse compounds. The physiological systems where JH plays a

role also differ between insect groups. For example, in cockroaches rates of JH biosynthesis are

closely correlated with reproductive events. In contrast, a clear correlation of CA activity and

vitellogenesis is not observed in the locust S. gregaria (Tobe and Stay, 1977; Tobe and Pratt,

1975). The role of putative JH receptors also appears to vary between insects. The functional

activation of gene transcription by JH III USP/RXR binding has only been demonstrated in D.

melanogaster (Jones and Sharp, 1997; Wozniak et al., 2004; Xu et al., 2002; Fang et al., 2005).

In contrast, the lower insect L. migratoria has not been shown to bind JH III via USP/RXR

(Hayward et al., 2003). Similarly, FGLa ASTs have only been shown to inhibit JH biosynthesis

in the orders Orthoptera, Dictyoptera and Isoptera (for a review see Stay and Tobe, 2007). Thus,

neither the molecular mode of JH action nor the regulation of JH biosynthesis can be generalized

across insects. Therefore, we sought to examine both a putative JH receptor (USP/RXR) and a

neuropeptide inhibitor of JH biosynthesis (FGLa ASTs) from a comparative perspective using

both experimental and evolutionary approaches.

In our investigation of USP/RXR, we sought to demonstrate whether DpRXR shares

functional motifs with either Meopterida or vertebrate-type sequences, whether codon-based

maximum likelihood methods of estimating evolutionary rates could confirm current hypotheses

of USP/RXR's functional evolution, and, finally, how levels of DpRXR expression correlate with

the critical periods for JH sensitivity during larval development.

Our results revealed four DpRXR variants with a pattern of N-terminal A/B domain

splice variation thus far only described in the Holometabola (Kapitskaya et al., 1996; Vogtli et

al., 1999; Jindra et al., 1997; Tan and Palli, 2008). Functional motifs implicated in ligand

binding, dimerization and transcriptional activation demonstrated more similarity with vertebrate

sequences (Egea et al., 2000; Lee et al., 1998b; Wurtz et al., 1996). Furthermore, loop regions

93within Mecopterida-type USP/RXR known to confer unique antagonistic LBD conformations in

these species were not present in DpRXR (Billas et al., 2001; Clayton et al., 2001). Estimation of

evolutionary rates showed significantly elevated ω values along the Mecopterida, supporting

current evolutionary models which suggest a functional shift in ligand-binding along this lineage

(Iwema et al., 2007; Tocchini-Valentini et al., 2009; Bonneton et al., 2003). Our preliminary

analysis of DpRXR expression showed higher levels of expression at target tissues, such as brain

and ovary, for JH. Elevated DpRXR expression levels occurred during the JH critical period in

final instar larvae, suggesting either a direct or indirect role for DpRXR in JH dependent larval

development.

Expression levels of DpRXR reported here, and the upregulation of USP/RXR in A.

mellifera by JH, may suggest that USP/RXR responsiveness to JH occurred earlier than the

Mecopterida (Barchuk et al., 2004). However, it is also important to recall that USP/RXR is a

functional component of the heterodimeric ecdysteroid receptor complex (Thomas et al., 1993;

Yao et al., 1992, 1993). Even in species where USP/RXR does not appear to independently bind

JHs, the role in ecdysteroid signalling is maintained. For example, USP/RXR-EcR is responsive

to ecdysone and ecdysone analogs in both L. migratoria and T. castaneum (Hayward et al., 2003;

Iwema et al., 2007). Thus we cannot consider USP/RXR function in isolation, as amino acid

substitutions in functional domains would affect the role of the receptor in its capacity as a

heterodimeric partner. In the Diptera and Lepidoptera, relative substitution rates demonstrate that

both USP/RXR and EcR are divergent from other insect and arthropod sequences (Bonneton et

al., 2003). In higher insects, EcR is divergent in regions implicated in dimerization and

transcriptional activation located outside of the LBD. Bonneton et al. (2003 and 2006) suggest

that changes along holometabolous lineages are indicative of both the co-evolution of USP/RXR-

EcR as well as the acquisition of new partner proteins. Thus, differences in the USP/RXR

sequences may not necessarily suggest changes in ligand-binding function but rather,

94dimerization. In our study, sites identified as under positive selection within the LBD of

USP/RXR along the Mecopterida branch did not occur at sites implicated in ligand-binding

(Table 2.4). In the context of heterodimer formation, elevated ω values may indicate the gain of

dimerization partners, and not only a gain in ligand binding. This underscores the importance of

screening USP/RXR ligand-binding function in conjunction with putative partner proteins in

future assays.

Our discussion in Chapter Two highlighted the need for future ligand-binding assays to

determine the functional activity of DpRXR. Comparison of amino acid sequence data itself is

not enough to draw definitive conclusions about ligand-binding function. Overall, we concluded

that dissimilarities in the LBP and structural loop regions do not suggest similar functionality in

Mecopterida and Diploptera LBDs. Recent in vivo studies in Drosophila, which suggest

USP/RXR does not directly mediate JH-dependent transcription, make it impossible to determine

what the implication of these sequence differences are in terms of JH binding function (Beck et

al., 2009). Given the observed sequence similarities with vertebrate type RXR reported here, it is

tempting to suggest that retinoid-binding function occurs in insects. Recently, displacement

binding experiments have shown that L. migratoria RXR binds both 9cRA and all-trans RA in

the high nanomolar range. Although the authors suggest a morphogenic role for retinoids in

Locusta embryogenesis, the total concentration of RA in whole embryos was determined to fall

within the low nanomolar range (Nowickyj et al., 2008). Given the small quantity of RA found

within Locusta embryos, the physiological significance of these in vitro findings is unclear. In

fact, recent findings suggest levels of 9cRA are far too low to elicit RXR binding in mouse

embryos, calling into question the physiological relevance of in vitro vertebrate RXR ligand-

binding assays (Mic et al., 2003). Thus the role of both insect and vertebrate USP/RXR in vivo

remains open at this point. In the future, in vivo based approaches will be necessary for the

elucidation of endocrine signal transduction pathways in insects.

95 Our investigation of FGLa ASTs sought to examine what amino acid substitutions have

occurred within AST precursor genes across ancestral insect and cockroach nodes, what the

implication of such substitutions are on the potency of ancient AST peptides in terms of the

inhibition of JH biosynthesis, and what patterns exist in peptide frequency throughout insect and

crustacean groups.

Results of our ancestral reconstruction revealed that AST 7 in particular had several

interesting amino acid substitutions. In the cockroach dataset, two different substitutions were

detected along two separate lineages. When considering the dataset of all insects, AST 7 was

found to have two substitutions which arose at the ancestor of the cockroaches. Assays of

reconstructed AST 7 peptides for the inhibition of JH biosynthesis showed that ancient insects

and recent cockroach peptides were much more potent than expected, whereas peptides at the

cockroach ancestor showed decreased potency. Searches of our compiled FGLa AST peptide

database showed that very few peptides are shared across insects and crustaceans. Using

degenerate searches we were able to demonstrate that Dippu-AST 2 (amino acids 11–18) and

AST 6 were the most frequent peptides across the entire dataset. Furthermore, these more

frequent peptides corresponded with the highly conserved insect peptides we were able to align

according to positional homology within the AST precursor.

Our discussion in Chapter Three emphasized the importance AST gene structure in

influencing the functional history of AST peptides. We concluded that increased copy number of

AST peptides, within the precursor, early in the history of cockroaches might explain decreased

potency as a consequence of newfound peptide redundancy (Bellés et al., 1999; Bendena et al.,

1999; Tobe and Bendena, 1999; Ohno, 1970; Prince and Pickett, 2002). However, a full analysis

of non-insect arthropod ASTs is necessary for a more complete understanding of AST

evolutionary history. Unfortunately, our dataset was forced to exclude the AST precursor genes

of decapod crustaceans. All decapod precursors isolated to date lack positional homology with

96insect sequences and contain 35 or more peptides (Yin et al., 2006). These species are critical for

our understanding AST function. Similarities in the action of ASTs in the early embryonic

development of D. punctata and the action of ASTs in the decapod P. clarkii has led to the

suggestion that regulation of sesquiterpenoid production may have been an early function of

ASTs in arthropods (Kwok et al., 2005; Stay et al., 2002). The need for additional non-insect

arthropod data is clear and since the publication of this work several researches have identified

AST precursor genes in a greater diversity of invertebrates.

Recently, genome databases have been used to identify FGLa AST genes in the

branchiopod Daphnia pulex, the tardigrade Hypsibius dujardini and the arachnid Ixodes

scapularis, all of which demonstrate low peptide copy number (Martínez-Pérez et al., 2009;

Gard et al., 2009; Weaver and Audsley, 2009). FGLa AST precursors have also been identified

in a greater diversity of insects such as the body louse Pediculus humanus, the pea aphid

Acyrthosiphon pisum and the parasitoid wasp Nasonia vitripennis (Martínez-Pérez et al., 2009;

Weaver and Audsley, 2009). Similarly, these non-Dictyopteran insect AST precursor genes also

demonstrate relatively low peptide copy number. However, the functions of these newly

identified peptides have yet to be assayed. Genomic data available through the ‘Ecdysozoan

Sequencing Project’ underway at the Broad Institute will also further our understanding of

neuropeptide evolution in the future (Weaver and Audsley, 2009).

Increasing availability of genetic data for FGLa ASTs has enabled more sophisticated

molecular evolutionary studies such as our own. Martínez-Pérez et al. (2009) used a distribution

of AST precursor sequences to examine the gene structure and intron position across insect and

arthropod lineages. The results of this study suggest that introns are preferentially inserted near

codons for the dibasic proteolytic cleavage site Lys-Arg and that some regulation of splicing is

required in forming the mRNA encoding preproallatostatin. It is clear that in the coming years

more FGLa AST precursor genes will be identified and, more importantly, they are likely to be

97found more widely distributed across taxa. Such data will make it easier to clarify the functional

evolution of ASTs.

Given these two lines of research on FGLa ASTs and USP/RXR, can we draw any

conclusions about JH, JH signal transduction or the regulation of JH production? Currently, our

data lack the scope to address these issues in such a broad sense. However, our results provide

clues as to how to address some of the fundamental questions in insect endocrinology. Our

USP/RXR results stress the need to identify other putative components of protein complexes

involved in JH signalling. Knowledge of these proteins is required in order to resolve the

relationship between ligand-binding and heterodimerization. To address this, less stringent

degenerate primers could be used to flush out other members of the D. punctata NR

complement. Currently, work is underway in our laboratory to sub-clone and sequence another

putative JH receptor, MET. To date, MET has never been characterized in any hemimetabolous

insects. In vivo approaches will also be critical; unfortunately many refined genetic methods are

only available in Drosophila. Given our data, it is clear that holometabolous insects belonging to

the Mecopterida are not appropriate models for generalizing endocrine systems in insects. Gene

silencing using RNA interference has recently been applied in the cockroach B. germanica

(Martín et al., 2006). The knock down of DpRXR during the gonadotrophic cycle of adult female

D. punctata may demonstrate an in vivo role for USP/RXR in vitellogenesis. Overall, our study

suggests that answers regarding the function and evolution of JH signal transduction and the

regulation of JH production will come from comparative molecular and phylogenetic studies of

more basal insect lineages and non-insect ecdysozoans.

98

SUPPLEMENT TO CHAPTER TWO (S2)

99

Figure S2.1 Multiple sequence alignment of USP/RXR LBD used in phylogenetic analyses. PAML dataset did not include last 12 sequences, leaving only protostome species in the alignment. For accession numbers see table S2.1.

100

T_clavigera DMPVEQILEAEIAVEPKIDT----YIDAQ---------KEPVTNICQAADKQLFTLVDWAKRIPHFVELPLEDQVILLRAGWNELLIGGFSHRSTQVTDG---------------------------ILL

B_glabrata DMPVEQILEAELAVDPKIDT----YIDAQ---------KDPVTNICQAADKQLFTLVEWAKRIPHFTELPLEDQVILLRAGWNELLIAGFSHRSIMAKDG---------------------------ILL

L_stagnalis DMPVEQILEAELAVDPKIDT----YIDAQ---------KDPVTNICQAADKQLFTLVEWAKRIPHFTELPLEDQVILLRAGWNELLIAGFSHRSIMAKDG---------------------------ILL

L_australasiae DMPIERILEAELYIEPKRDD----SDPDH---------KDPVANICQAADHQLYQLVEWAKHVPHFTDLPLDDQMVLLKAGWNELLIAAFSHRSIDVKDG---------------------------IVL

O_moubata EMPIDRILEAELRVEPKAED----LEVNALADLTMPPEKDPVTNICQAADRQLHQLVEWAKHIPHFTELPLEDRMVLLKAGWNELLIAAFSHRSM?VKDG---------------------------IVL

I_scapularis EMPLERILEAELRVEPKGGS----APES----------QDPVSSICQAADRQLHQLVEWAKHIPHFVELPLEDRMVLLKAGWNELLIAAFSHRSIGVRDG---------------------------IVL

A_americanum1 EMPLERILEAELRVESQTGT----LSESAQQ-------QDPVSSICQAADRQLHQLVQWAKHIPHFEELPLEDRMVLLKAGWNELLIAAFSHRSVDVRDG---------------------------IVL

A_americanum2 DMPLERILEAEMRVEQPAPS----VLAQTAG-------RDPVNSMCQAAP-PLHELVQWARRIPHFEELPIEDRTALLKAGWNELLIAAFSHRSVAVRDG---------------------------IVL

M_japonicus DMPISQIRDAELNSDPTDDL----LFEEG----------DAVTHICQAADRHLVQLVEWAKHIPHFTDLPVDDQVILLKAGWNELLIASFSHRSMGVKDG---------------------------IVL

C_maenas DMPIASIREAELSVDPIDEQ----PLDQGVEVTNVNPLQDVVSNICQAADRHLVQLVEWAKHIPHFTDLPIEDQVVLLKAGWNELLIASFSHRSMGVEDG---------------------------IVL

G_lateralis DMPIASIREAELSVDPIDEQ----PLDQGVEVPCANPLQDVVSNICQAADRHLVQLVEWAKHIPHFTDLPIEDQVVLLKAGWNELLIASFSHRSMGVEDG---------------------------IVL

C_pugilator DMPIASIREAELSVDPIDEQ----PLDQGVEVSCANPLQDVVSNICQAADRHLVQLVEWAKHIPHFTDLPIEDQVVLLKAGWNELLIASFSHRSMGVEDG---------------------------IVL

L_migratoria DMPVERILEAEKRVECKAEN----QVEYES--------ICQAANICQATNKQLFQLVEWAKHIPHFTSLPLEDQVLLLRAGWNELLIAAFSHRSVDVKDG---------------------------IVL

B_germanica DMPVERILEAEKRVECKSEQ----QVEFELELNGVGP-KSAVTNICQATNKQLFQLVEWAKHIPHFTTLPLSDQVLLLRAGWNELLIAAFSHRSVEVKDG---------------------------IVL

D_punctata DMPVERILEAEKRVDCRPEQ----QVEIE----------SAVTNICQATNKQLFQLVEWAKHIPHFTSLPLSDQVLLLRAGWNELLIAAFSHRSVEVKDG---------------------------IVL

P_humanus DMPVERILEAEKRVECKVEN----QNEYEN----------AVANICQATNTQLYQLVEWAKHIPHFSSLPIEDQVLLLRAGWNELLIAAFSHRSVEVRDG---------------------------IVL

A_pisum DMPVELILRAENKADAIKTE----QQYIEQQH------QHTVGAICQATDKQLIQLVEWAKHIPHFKNLPLGDQVLLLRAGWNELMIAAFSHRSISVKDG---------------------------IVL

B_tabaci DMPIERILEAELRVEPKNED----IDS-----------RDPVSDICQAADRQLYQLIEWAKHIPHFTELPVEDQVILLKSGWNELLIAGFSHRSMSVKDG---------------------------IML

X_pecki DMPIERILEAERRIDCKIEF----PVEFEN----------SVSNFCQATNTQLFQIIDWAKHIPYFTSLPVADQVVLLKASWNELLITNFSYRSIDARDA---------------------------IVL

L_decemlineata EMSIERLLEAEKRVECNDP--------PVALE-------NAVTNICQATNKQLLQLVEWAKLIPHFTSLPVSDQVLLLRAGWNELLIASFSHRSMQTQEG---------------------------IIL

T_molitor EMPLDRIIEAEKRIECTPAG----GSGGVGEQ-------DGVNNICQATNKQLFQLVQWAKLIPHFTSLPMSDQVLLLRAGWNELLIAAFSHRSIQAQDA---------------------------IVL

T_castaneum DMPLERIIEAEKRVECNDPL----VALVVNEN-------TTVNNICQATHKQLFQLVQWAKLVPHFTSLPLTDQVQLLRAGWNELLIAAFSHRSMQAQDA---------------------------IVL

N_vitripennis DMPVELILEAEKRFEYLAEN----QASYEQ--------------FDNHNNKQMRYMVEWAKRLPQFTSLPLEDQARLLRAGWNELQIAAFSHRSIDIEDG---------------------------IIL

P_fuscatus DMPIERILEAEKRVDCKVEH----DGNYE-----------------------LFQLVTWAKHIPHFTSLPLEDQVLLLRGGWNELLIASFSHRSIGIKDG---------------------------IVL

A_mellifera DMPIERILEAEKRVECKMEQ----QGNYEN----------AVSHICNATNKQLFQLVAWAKHIPHFTSLPLEDQVLLLRAGWNELLIASFSHRSIDVKDG---------------------------IVL

M_scutellaris DMPIERILEAEKRVECKMEQ----QGNYEN----------AVSHICNATNKQLFQLVAWAKHIPHFTSLPLEDQVLLLRAGWNELLIASFSHRSIDVKDG---------------------------IVL

S_depilis DMPIERILEAEKRVECKMEQ----QGNYEN----------AVSHICNATNKQLFQLVAWAKHIPHFTSLPLEDQVLLLRAGWNELLIASFSHRSIDVKDG---------------------------IVL

C_marginata ELSIERLIEIESSPTESQSE--LQYLRVSPNSVVPTRYRGPVSSLCQIGNRQLRALVDWARCLPHFNRLQLSDQVLLLKSSWNELLIIAIAWRSIEYLE---NEREND-NGNDKT--NNKTIPTPQLMCL

C_fumiferana ELSIERLTEMESLVADPSEE--FQFLRVGPDSNVPPRYRAPVSSLCQIGNKQIAALVVWARDIPHFGQLELDDQVVLIKASWNELLLFAIAWRSMEYLE---DERE----NGDG----TRSTTQPQLMCL

B_mori ELSIERLLELEALVADSAEE--LQILRVGPESGVPAKYRAPVSSLCQIGNKQIAALIVWARDIPHFGQLEIDDQILLIKGSWNELLLFAIAWRSMEFLN---DERE----NVD-----SRNTAPPQLICL

M_sexta ELSIERLLEIESLVADPPEE--FQFLRVGPESGVPAKYRAPVSSLCQIGNKQIAALVVWARDIPHFGQLELEDQILLIKNSWNELLLFAIAWRSMEYLT---DERE----NVD-----SRSTAPPQLMCL

C_suppressalis ELSIERLLEMESLVADTSEE--CQFLRVGPESNVPPKFRAPVSSLCQIGNKQIAALVVWARDIPHFSQLEMDDQVLLIKGAWNELLLFAIAWRSMEFLN---DERE----NMD----GSRTTSPPQLMCL

P_interpunctella ELSIERLLEMEALVADTSEE--FQFLRVGPDSNVPPKFRAPVSSLCQIGNKQIAALVVWARDIPHFSQLELEDQVTLIKASWNELLLFAIAWRSMEYLT---DERD----NVD----GSRTTSPPQLMCL

H_virescens ELSIERLLEMESLVADPSEE--FQFLRVGPDSNVPPKFRAPVSSLCQIGNKQIAALVVWARDIPHFSQLEMEDQILLIKGSWNELLLFAIAWRSMEFLT---EERD----GVDGT--GNRTTSPPQLMCL

H_armigera ELSIERLLEMESLVADPSEE--FQFLRVGPDSNVPPKFRAPVSSLCQIGNKQIAALVVWARDIPHFSQLELEDQILLIKGSWNELLLFAIAWRSMEYLT---EERD----GVDGT--GNRTTSPPQLMCL

S_litura ELSIERLLEMESMVADPTEE--YQFLRVGPDSNVPPKFRAPVSSLCQIGNKQIAALVVWARDIPHFNSLHLEDQMLLIKASWNELLLFAIAWRSMEYLT---EERE----VVDSS--GNRSTSPPQLMCL

S_exigua ELSIERLLEMESLVADPSEE--YQFLRVGPDSNVPPKFRAPVSSLCQIGNKQIAALVVWARDIPHFSSLELEDQTLLIKSSWNELLLFAIAWRSMEYLT---EERE----VVDSS--GNRTTSPPQLMCL

D_melanagaster DFSIERIIEAEQRAETQCGDRALTFLRVGPYSTVQPDYKGAVSALCQVVNKQLFQMVEYARMMPHFAQVPLDDQVILLKAAWIELLIANVAWCSIVSLDDGGAGGGGGGLGHDGSFERRSPGLQPQQLFL

D_psuedoobscura DFTIERLLDAEQRAEAQSGDRALAFLRVGPNSTVQPDYKGAVSALCQVVNKQLYQMVEYARLMPHFAQLPLDDQVILLKAAWNELLIANVAWCSIVSLDDAMATG----MGHDSAFERRSPVLQPQQLFL

L_cuprina DLTIERIIEAEQKAESLSGDNVLPFLRVGNNSMVQHDYKGAVSHLCQMVNKQLYQMVEYARRTPHFTHLQREDQILLLKAGWNELLIANVAWCSIESLDAEYASPG---TVHDGSFGRRSPVRQPQQLFL

C_tentans DITVERLMEADQMSEARCGDKSIQYLRVAANTMIPPEYRAPVSAICAMVNKQVFQHMDFCRRLPHFTKLPLNDQMYLLKQSLNELLILNIAYMSIQYVE---PDRR----NADG---SLERRQISQQMCL

A_gambiae DVVVDRFLEAEQIGEQKSGDNAIPYLRVGQNSMIPSEYKGAVSHLCQMVNKQIYQLIEFARRLPNFSNLPREDQVTLLRSGWNEMLIASVAWRSMEYIE---TER-----PPDGRNDGRVTIRQPQLMCL

A_albopictus DVTIERITAAEQLSEQKSGDNAIPYLRVGSNSMIPPEYKGAVSHLCQMVNKQIYQLIDFARRLPHFTNLHRDDQVMLLRCGWNEMLIAAVAWRSMEYIE---TER-----SPDG---SRISIRQPQLMCL

A_aegypti DVTIERIHEAEQLSEQKSGDNAIPYLRVGSNSMIPPEYKGAVSHLCQMVNKQIYQLIDFARRVPHFINLPRDDQVMLLRCGWNEMLIAAVAWRSMEYIE---TER-----SSDG---SRITVRQPQLMCL

B_floridae DMPVEKIQEAEMAVEPKDGN---MVEQ----------PNDPVTNICQAADKQLVTLVEWAKRIPHFSDLPIDDQVILLRAGWNELLIAAFSHRSIDVKDG---------------------------ILL

P_misakiensis DMPVDKILEAELISDPKVEQ---VVPFE------QVNENDPVSNICKAADRQLVTLVEWAKRIPHFSSLPLEDQVILLRAGWNELLIASFSHRSIDVKDS---------------------------ILL

C_intestinalis DMPVDKILQAELASDPKMEE---LINMQ------EPID----TSVCKAADHQLFTLVEWAKRVPMFGTLPLDDQVTLLRAGWNELLIASFSHRSIEIPDG---------------------------IIL

M_musculusa DMPVEKILEAELAVEPKTET---YVEAN--MGLNPSSPNDPVTNICQAADKQLFTLVEWAKRIPHFSELPLDDQVILLRAGWNELLIASFSHRSIAVKDG---------------------------ILL

M_musculusb EMPVDRILEAELAVEQKSDQ---GVEGPGATGGGGSSPNDPVTNICQAADKQLFTLVEWAKRIPHFSSLPLDDQVILLRAGWNELLIASFSHRSIDVRDG---------------------------ILL

M_musculusg DMPVERILEAELAVEPKTES---YGDMN-----VENSTNDPVTNICHAADKQLFTLVEWAKRIPHFSDLTLEDQVILLRAGWNELLIASFSHRSVSVQDG---------------------------ILL

X_laevisa DMPVEKILEAEHAVEPKTET---YTEAN--MGLAPNSPSDPVTNICQAADKQLFTLVEWAKRIPHFSELPLDDQVILLRAGWNELLIASFSHRSIAVKDG---------------------------ILL

X_laevisb EMPVEKILEAELAVEQKSDQ---SLEG-------GGSPSDPVTNICQAADKQLFTLVEWAKRIPHFSELALDDQVILLRAGWNELLIASFSHRSISVKDG---------------------------ILL

X_laevisg EMPVERILEAELAVDPKIEA---FGDAG-----LPNSTNDPVTNICHAADKQLFTLVEWAKRIPYFSDLPLEDQVILLRAGWNELLIASFSHRSVSVQDG---------------------------ILL

H_sapiensa DMPVERILEAELAVEPKTET---YVEAN--MGLNPSSPNDPVTNICQAADKQLFTLVEWAKRIPHFSELPLDDQVILLRAGWNELLIASFSHRSIAVKDG---------------------------ILL

H_sapiensb EMPVDRILEAELAVEQKSDQ---GVEGPGGTGGSGSSPNDPVTNICQAADKQLFTLVEWAKRIPHFSSLPLDDQVILLRAGWNELLIASFSHRSIDVRDG---------------------------ILL

H_sapiensg DMPVERILEAELAVEPKTES---YGDMN-----MENSTNDPVTNICHAADKQLFTLVEWAKRIPHFSDLTLEDQVILLRAGWNELLIASFSHRSVSVQDG---------------------------ILL

101

T_clavigera ATGLHVHRSSAHQAGVGTIFDRVLTELVAKMREMKMDKTELGCLRAIVLFNPDAKGLQSVQEVEQLREKVYASLEEYCKQRYPDEPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGQTPIDTFLMEMLE

B_glabrata ATGLHVHRSSAHQAGVGTIFDRVLTELVAKMRDMKMDKTELGCLRAVVLFNPDAKGLTAVQEVEQLREKVYASLEEYTKSRYPEEPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDQPIDTFLMEMLE

L_stagnalis ATGLHVHRSSAHQAGVGTIFDRVLTELVAKMREMKMDKTELGCLRAVVLFNPDAKGLTAVQEVEQLREKVYASLEEYTKTRYPEEPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDQPIDTFLMEMLE

L_australasiae ASGLIVHRNSAHGAGVGTIFDRVLTELVAKMREMNMDKTELGCLRAIVLFNPEAKGLKSVTHVENLRERVYSALEDYCRQNYFDQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDNFLMSMLE

O_moubata ATGLVVQRHSAHSAGVGAIFDRVLTELVAKMRELRMDRTELGCLRAIVLFNPEARGLRCSAQVEALRERVYAALEDHCRQQYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDNFLLSMLE

I_scapularis ATGLVVQRHSAHGAGVGAIFDRVLTELVAKMREMKMDRTELGCLRAVVLFNPEAKGLRSTAQVEALREKVYAALEEHCRQQYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDNFLLSMLE

A_americanum1 ATGLVVQRHSAHGAGVGAIFDRVLTELVAKMREMKMDRTELGCLLAVVLFNPEAKGLR-TCPSGGPEGESVSALEEHCRQQYPDQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDNFLLSMLE

A_americanum2 ATGLVVQRHSAHGAGVGDIFDRVLAELVAKMRDMKMDKTELGCLRAVVLFNPDAKGLRNATRVEALREKVYAALEEHCRRHHPDQPGRFGKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDSFLLNMLE

M_japonicus ATGLVVHRSSAHHAGVGDIFDRVLSELVAKMKEMKMDKTELGCLRSIVLFNPDVKGLSACDTIEVLREKVYATLEEYTRTSYPDQPGRFAKLLLRLPALRSIGLKCLEYLFLFKLLGDTPLDNYLMKMLV

C_maenas ATGLVVHRSSAHQAGVGAIFDRVLSELVAKVKEMKMDKTELGCLRAIVLFNPDAKGVTCCSDVEILREKVYAALEESTRTTYPGEPGRFPKLLLRLPSLRSIGLKCLEYLFFFKLIGDTPLDSYSMKMLV

G_lateralis ATGLVIHRSSAHQAGVGAIFDRVLSELVAKMKEMKIDKTELGCLRSIVLFNPDAKGLNCCNDVEILREKVYAALEEYTRTTYPDEPGRFAKLLLRLPALRSIGLKCLEYLFLFKLIGDTPLDSYLMKMLV

C_pugilator ATGLVIHRSSAHQAGVGAIFDRVLSELVAKMKEMKIDKTELGCLRSIVLFNPDAKGLNCVNDVEILREKVYAALEEYTRTTYPDEPGRFAKLLLRLPALRSIGLKCLEYLFLFKLIGDTPLDSYLMKMLV

L_migratoria ATGLTVHRNSAHQAGVGTIFDRVLTELVAKMREMKMDKTELGCLRSVILFNPEVRGLKSAQEVELLREKVYAALEEYTRTTHPDEPGRFAKLLLRLPSLRSIGLKCLEHLFFFRLIGDVPIDTFLMEMLE

B_germanica ATGLTVHRNSAHQAGVGAIFDRVLTELVAKMREMKMDKTELGCLRSVILFNPDVRGLKSSQEVELLREKVYAALEEYTRTTYPDEPGRFAKLLLRLPSLRSISLKCLEYLFFFRLIGNVPIDEFLMEMLE

D_punctata ATGLTVHRNSAHQAGVGAIFDRVLTELVAKMREMKMDKTELGCLRSIILFNPDVRGLKSSQDVEVLREKVYAALEEYTRTTYPDEPGRFAKLLLRLPSLRSISLKCLEYLFFFRLIGNVPIDEFLMEMLE

P_humanus GAGITVHRNSAHQAGVGTIFDRVLTELVAKMRDMNMDRTELGCLRSIILFNPEVRGLKSGQEVELLREKVYAALEEYTRVTRPEEPGRFAKLLLRLPALRSIGLKCLEHLFFFRLIGDIPIDTFLMDMLG

A_pisum ATGLTVDRDSAHQAGVEAIFDRVLTELVAKMRDMGMDRTELGCLRTIILFNPGSKGLQSVNEVEVLRDKVYVALEEYCRTTHPEEPGRFAKLLLRLPSLRSIGLKCLEHLFFYKLIGDSPIDTFLMEVLE

B_tabaci ATGLVVHRNCAHQAGVGAIFDRVLTELVAKMREMKMDKTELGCLRSIVLFNPEAKGLKSTQQVENLREKVYAILEEYCRQTYPDQSGRFAKLLLRLPALRSIGLKCLEHLFFFKLVGNTSIDSFLLSMLE

X_pecki ATGYAVNKNSAHQAGLEAIFDRVLTEVVYKMREIRMDKTEIGCLKCITLFNSEIKGLKSAQEVESLREKVFCVPDEHTRINYPNEQGRFAKLLLRLPPVRSIALKCTDYLFFCRLV--LPIDAFLREMLE

L_decemlineata ATGLTINKSTAQAVGVGNIYDRVLSELVNKMKEMRMDKTELGCLRAIILYNPDVRGLQSTQEVEILREKIYENLEEYTRTTHPNEPGRFAKLLLRLPALRSIGLKCLEHLFFFRLIGDVTIDTFITEMLE

T_molitor ATGLTVNKTSAHAVGVGNIYDRVLSELVNKMKEMKMDKTELGCLRAIILYNPTCRGIKSVQEVEMLREKIYGVLEEYTRTTHPNEPGRFAKLLLRLPALRSIGLKCSEHLFFFKLIGDVPIDTFLMEMLE

T_castaneum ATGLTVNKSTAHAVGVGNIYDRVLSELVNKMKEMKMDKTELGCLRAIILYNPDVRGIKSVQEVEMLREKIYGVLEEYTRTTHPNEPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDVPIDTFLMEMLE

N_vitripennis MTGFTLHKNSAQQAGVGAIFERVLTELVHKMKSMKMDKTELGCLRAIILFNPEVRGLKAHQEVDMLREKVYVALDEYTRLHRPDEPGRFAKLLLRLPALRSIGLKCTEHLFFFRLLGDLPLNELLTDMLE

P_fuscatus ATGITVYRSSAQQAGVGTIFDHVLSELVTKMRDMKMDKTELGCLRSIILFNPDVRGLKSMQEVSLLREKIYAALEEYTRVSCPNDSGRFAKLLLRLPSIRSIGLKCLEHLFFYKLIGDVPIDEFIMELLE

A_mellifera ATGITVHRNSAQQAGVGTIFDRVLSELVSKMREMKMDRTELGCLRSIILFNPEVRGLKSIQEVTLLREKIYGALEGYCRVAWPDDAGRFAKLLLRLPAIRSIGLKCLEYLFFFKMIGDVPIDDFLVEMLE

M_scutellaris ATGITVHRNSAQQAGVGTIFDRVLSELVSKMREMKMDRTELGCLRSIILFNPEVRGLKSIQEVTLLREKIYAALEGYCRVAWPDDAGRFAKLLLRLPAIRSIGLKCLEYLFFFKMIGDVPIDDFLVEMLE

S_depilis ATGITVHRNSAQQAGVGTIFDRVLSELVSKMREMKMDRTELGCLRSIILFNPEVRGLKSIQEVTLLREKIYAALEGYCRVAWPDDAGRFAKLLLRLPAIRSIGLKCLEYLFFFKMIGDVPIDDFLVEMLE

C_marginata MPGMTLHRNSALLAGVGVMFDRILSELSLKMRQMRVDQAELACLKAVILFNPDLRGVKGRQEIDAIRDKVYALLEDHCRTRRAGEEGRFASLLLRLPALRSISLKCFEHLFFFRLFGDVSIETCLLEVWQ

C_fumiferana MPGMTLHRNSAQQAGVGAIFDRVLSELSLKMRTLRMDQAEYVALKAIVLLNPDVKGLKNRQEVDVLREKMFSCLDDYCRRSRSNEEGRFASLLLRLPALRSISLKSFEHLYFFHLVAEGSISGYIREALR

B_mori MPGMTLHRNSALQAGVGQIFDRVLSELSLKMRSLRMDQAECVALKAIILLNPDVKGLKNKQEVDVLREKMFLCLDEYCRRSRGGEEGRFAALLLRLPALRSISLKSFEHLYLFHLVAEGSVSSYIRDALC

M_sexta MPGMTLHRNSALQAGVGQIFDRVLSELSLKMRTLRMDQAEYVALKAIILLNPDVKGLKNKPEVVVLREKMFSCLDEYVRRSRCAEEGRFAALLLRLPALRSISLKCFEHLYFFHLVADTSIASYIHDALR

C_suppressalis MPGMTLHRNSALQAGVGQIFDRVLSELSLKMRHLRMDQAEYVALKAIILLNPDIKGLGNRQEVEVLREKMYSCLDEYCRRVRVSEEGRFASLLLRLPALRSISLKSFEHLFFFHLVADSSIAGYIRDLLR

P_interpunctella MPGMTLHRNSALQAGVGQIFDRVLSELALKMRSLPVDQAEYVALKAVILLNPDVKGLNSRQEVEVLREKMYSCLDEYCRRSRGSEEGRFASLLLRLPALRSISLKSFEHLFFFHLVADGSIPGYIRDALR

H_virescens MPGMTLHRNSALQAGVGQIFDRVLSELSLKMRTLRVDQAEYVALKAIILLNPDVKGLKNRQEVEVLREKMFLCLDEYCRRSRSSEEGRFAALLLRLPALRSISLKSFEHLFFFHLVADTSIAGYIRDALR

H_armigera MPGMTLHRNSALQAGVGQIFDRVLSELSLKMRSLRVDQAEYVALKAIILLNPDVKGLKNRQEVEVLREKMFLCLDEYCRRSRGSEEGRFAALLLRLPALRSISLKSFEHLFFFHLVADTSIAGYIRDALR

S_litura MPGMTLHRNSALQAGVGQIFDRVLSELSLKMRALRFDQAEYVALKAIILLNPDVKGLKNRLDVELLREKMFSCLDEYVRRSRGGEEGRFAALLLRLPALRSISLKSFEHLFFFHLVADTSIASYIRDALR

S_exigua MPGMTLHRNSALQAGVGQIFDRVLSELSLKMRALRVDQAEYVALKAIILLNPDVKGLKNRAEVEILREKMFSCLDEYVRRSRGGEEGRFAALLLRLPALRSISLKSFEHLFFFHLVADTSIATYIREALR

D_melanagaster NQSFSYHRNSAIKAGVSAIFDRILSELSVKMKRLNLDRRELSCLKAIILYNPDIRGIKSRAEIEMCREKVYACLDEHCRLEHPGDDGRFAQLLLRLPALRSISLKCQDHLFLFRITSDRPLEELFLEQLE

D_psuedoobscura NQSFSYHRNSAIKAGVSTIFDRILSELSVKMKRLNLDRRELACLKAIILYNPDMRGIKNRAEIEICREKVYACLDEHCRVEHPGDDGRFAQLLLRLPALRSISLKCLDHLFFFRIISDRPLEELFIEQLE

L_cuprina NQNFSYHRNSAIKANVVSIFDRILSELSIKMKRLNIDRSELSCLKAIILFNPDIRGLKCRADVEVCREKIYACLDEHCRTEHPGDDGRFAQLLLRLPALRSISLKCLDHLFFFRLIGERALEELIAEQLE

C_tentans SRNYTLGRNMAVQAGVVQIFDRILSELSVKMKRLDLDATELCLLKSIVVFNPDVRTLDDRKSIDLLRSRIYASLDEYCRQKHPNEDGRFAQLLLRLPALRSISLKCLDHLFYFQLIDDKNVENSVIEEFH

A_gambiae GPNFTLHRNSAQQAGVDSLFDRILCELAIKMKRLDVNRAELGILKAIILFNSDIRGLKCRKEIDQMREKIYACLDEYCKTQHPSEDGRFAQLLLRLPALRSISLKCIDHLNFLRLLGDKQLDNFIIEMLD

A_albopictus GPNFTLHRNSAQQAGVDTLFDRILCELGIKMKRLDVTRAELGVLKAIILFNPDIRGLKCQNGDDGMREKIYACLDEHCKQQHPSEDGRFAQLLLRLPALRSISLKCLDHLNFIRLLSDKHLDNFIIEMLD

A_aegypti GPNFTLHRNSAQQAGVDTLFDRILCELGIKMKRLDVTRAELGVLKAIILFNPDIRGLKCQKEIDGMREKIYACLDEHCKQQHPSEDGRFAQLLLRLPALRSISLKCLDHLNFIRLLSDKHLDSFIVEMLD

B_floridae ASGLHVHRSSAHQAGVGTIFDRVLTELVAKMRDMKMDKTELGCLRAIVLFNPDAKGLTDPSLVESLREKVYASLEEYCKQQYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

P_misakiensis ASGLHVHRHSAHQAGVGPIFDRVLTELVSKMRDMMMDKTELGCLRAIVLFNPDVKNLSDSAHIESLREKVYASLEAYCRSKYPDQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDKFLMNMLE

C_intestinalis ASGLRVYRQSAHSAGVGAIFDRVLTELIAKMRDMSMDRTELGCLRAIVLFNPDAKDLTDPAYIETLREKVYASLEVYCKSKYPDQAGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGNTPIDQFLMDKLA

M_musculusa ATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMQMDKTELGCLRAIVLFNPDSKGLSNPAEVEALREKVYASLEAYCKHKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

M_musculusb ATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMRMDKTELGCLRAIILFNPDAKGLSNPGEVEILREKVYASLETYCKQKYPEQQGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

M_musculusg ATGLHVHRSSAHSRGVGSIFDRVLTELVSKMKDMQMDKSELGCLRAIVLFNPDAKGLSNPSEVETLREKVYATLEAYTKQKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDSFLMEMLE

X_laevisa ATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMQMDKTELGCLRAIVLFNPDSKGLSNPLEVEALREKVYASLEAYCKQKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

X_laevisb ATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMRMDKTELGCLRAIILFNPDAKGLSNPGDVEVLREKVYASLESYCKQKYPDQQGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

X_laevisg ATGLHVHRSSAHNAGVGSIFDRVLTELVSKMKDMDMDKSELGCLRAIVLFNPDAKGLSNAAEVEALREKVYATLESYTKQKYPDQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

H_sapiensa ATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMQMDKTELGCLRAIVLFNPDSKGLSNPAEVEALREKVYASLEAYCKHKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

H_sapiensb ATGLHVHRNSAHSAGVGAIFDRVLTELVSKMRDMRMDKTELGCLRAIILFNPDAKGLSNPSEVEVLREKVYASLETYCKQKYPEQQGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

H_sapiensg ATGLHVHRSSAHSAGVGSIFDRVLTELVSKMKDMQMDKSELGCLRAIVLFNPDAKGLSNPSEVETLREKVYATLEAYTKQKYPEQPGRFAKLLLRLPALRSIGLKCLEHLFFFKLIGDTPIDTFLMEMLE

102

Table S2.1 Sequence data information Used in Analysis

Group Species Accession No. Seq comp.

Phylogeny PAML

Dictyoptera Blattella germanica RXRL AJ854490 * *

Blattella germanica RXRS AJ854489 *

Orthoptera Locusta migratoria RXRL AY348873 * *

Locusta migratoria RXRS AF136372 *

Hemiptera Bemisia tabaci EF174330 * * *

Acyrthosiphon pisum XM_001947055 *

Phthiraptera Pediculus humanus DS235123 *

Coleoptera Tenebrio molitor AJ251542 * *

Leptinotarsa decemlineata AB211193 * *

Tribolium castaneum (San Bernardino)

AM295014 * * *

Strepsiptera Xenos pecki AY827155 * *

Hymenoptera Scaptotrigona depilis DQ190542 * *

Melipona scutellaris AY840093 * *

Polistes fuscatus AY827156 * *

Apis mellifera AY273778 * * *

Nasonia Vitripennis RXR1 XM_001605769 *

Diptera Lucilia cuprina AY007213 * *

Drosophila melanogaster X53417 * * *

Drosophila pseudoobscura FBgn0078154 * *

Aedes albopictus AF210734 * *

Aedes aegypti USPA AF305213 * * *

Aedes aegypti USPB AF305214 *

Chironomus tentans USP1 AF045891 * * *

Chironomus tentans USP2 See Vogtli et al., 1999

*

Anopheles gambiae XM_320944 *

Trichoptera Chimarra marginata DQ083513 * * *

Lepidoptera Chilo suppressalis AB081840 * *

Bombyx mori U06073 * * *

Heliothis virescens AX383958 * * *

Choristoneura fumiferana AF016368 * *

Manduca sexta USP1 U44837 * * *

Manduca sexta USP2 U57921 *

Plodia interpunctella AY619987 * *

103 Spodoptera exigua EU642475 * *

Spodoptera litura EU180022 * *

Helicoverpa armigora EU526832 * *

Crustacea Celuca pugilator AF032983 * * *

Gecarcinus lateralis RXRa DQ067280 * *

Marsupenaeus japonicus AB295493 * * *

Carcinus maenas RXRII EU683889 * *

Chelicerata Ornithodoros moubata AB353290 * *

Amblyomma americanum RXR1 AF035577 * * *

Amblyomma americanum RXR2 AF035578 * * *

Liocheles australasiae AB297930 * * *

Ixodes scapularis DS749069 *

Mollusca Lymnaea stagnalis AY846875 * *

Thais clavigera AY704160 * * *

Biomphalaria glabrata AY048663 * * *

Cephalochordata Branchiostoma floridae AF378829 * *

Tunicata Polyandrocarpa misakiensis AB030318 * *

Ciona intestinalis AB210673 *

Vertebrata Mus musculus RXRa X66223 *

Mus musculus RXRb X66224 *

Mus musculus RXRg X66225 *

Xenopus laevis RXRa L11446 * *

Xenopus laevis RXRb X87366 *

Xenopus laevis RXRg L11443 *

Danio rerio RXRa U29940 *

Homo sapiens RXRa X52773 * *

Homo sapiens RXRb M84820 * *

Homo sapiens RXRg U38480 * *

Cnidaria Tripedalia cystophora AF091121

104

SUPPLEMENT TO CHAPTER THREE (S3)

105

Figure S3.1 Alignment of extant hemimetabolous insect AST precursors and the results of ancestral reconstruction using GASP and PAML software. Numbers indicate AST with respect to occurrence in the precursor sequence. Amino acid changes across ancestral nodes are highlighted in red for AST peptides, in the spacer regions amino acid changes are not highlighted.

106 B. orientalis ------------------------------------------------------------ P. americana ---------MGFLQLILLSIILLHLSTGSLATAPANSGHNGAPEETPSGAATGSGLLPHL B. germanica ---MPGPRTWYSLQAALVLSLLLKLSSSAFATTTS-AGTHAVQEESSAG--GGAEILPRL S. longipalpa ---MPDSRTCISLQAVLLLALLLQLANSAFGTATAPAG---SPEEASSN--AGSELLSHL D. punctata ---MSGPRTCFCLPSALVL-VLLSLSTSALGTAPEPSG---VHEESPAG--GGTDLLPHP B. craniifer ---MPGPRTYITLPAALLL-VLLSLSTTALGTATEPSG---VHEESPAG--GGAELLPHP G. bimaculatus ------PASDAAAAQEAAGELLERL-------------------ENEAG--SG------- S. gregaria MGMTSRSSSSEAARLPLPALVLLLLCTSP-ATPQEVPG------DAMTG--GGPASAPVS Node 14 GASP ---MPGPRTCGFLQLALLLILLLKLSTSALATAPEPSG---VPEESPAG--GGSGLLPHL Node 14 JTT+G ---MPGPRTCGSLQLALLSIILLHLSTSALATAPAPSG---VPEESPAG--GGSELLPHL Node 11 GASP ---MPGPRTCYSLQAALLLSLLLKLSTSALGTAPEPSG---VPEESPAG--GGSELLPHL Node 11 JTT+G ---MPGPRTCISLQAALLLSVLLQLSTSALGTATAPSG---VPEESPAG--GGAELLPHL Node 12 GASP ---------MGFLQLILLSIILLHLSTGSLATAPANSG---APEETPSG--TGSGLLPHL Node 12 JTT+G ---------MGFLQLILLSIILLHLSTGSLATAPANSG---APEETPSG--TGSGLLPHL Node 10 GASP ---MPGPRTCYSLQAALLLSLLLKLSSSAFGTATSPAG---VPEESSAG--GGSELLPHL Node 10 JTT+G ---MPGPRTCISLQAALLLSLLLQLSTSAFGTATAPAG---VPEESSAG--GGAELLPHL Node 9 GASP ---MPGPRTCICLPAALLL-VLLSLSTSALGTAPEPSG---VHEESPAG--GGAELLPHP Node 9 JTT+G ---MPGPRTCISLPAALLL-VLLSLSTSALGTATEPSG---VHEESPAG--GGAELLPHP 1 2 B. orientalis --------------------VNDLSELDFIKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG P. americana EES----------------SVNDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG B. germanica EEL------------------ADNSELDLVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG S. longipalpa ED-------------------SENPELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG D. punctata EDL----------------SASDNPDLEFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG B. craniifer EEL----------------SASDNSDLEFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG G. bimaculatus --------------------ATPDDELEFYKRLYDFGVGKRAYSYVSEYKRLPVYNFGLG S. gregaria TASEAAAASPPGSASTGAAPMDAESEYDLYKRLCDFGVGKRAYTYVSEYKRLPVYNFGLG Node 14 GASP EES----------------SANDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 14 JTT+G EES----------------SASDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 11 GASP EEL----------------SASDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 11 JTT+G EEL----------------SASDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 12 GASP EES----------------SVNDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 12 JTT+G EES----------------SVNDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 10 GASP EEL------------------SDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 10 JTT+G EEL------------------SDNSELDFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 9 GASP EEL----------------SASDNSDLEFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG Node 9 JTT+G EEL----------------SASDNSDLEFVKRLYDFGLGKRAYSYVSEYKRLPVYNFGLG 3 4 B. orientalis KRS----KMYGFGLGKRSGNDGRLYSFGLGKR---DYDDYIQE----------------- P. americana KRS----KMYGFGLGKRSGNDGRLYSFGLGKR---DYDDYIQE----------------- B. germanica KRS----KMYGFGLGKRAGSDGRLYSFGLGKR---DYDDYYGDD---------------- S. longipalpa KRS----KMYGFGLGKRAGSDSRLYSFGLGKR---DYDDYYEE----------------- D. punctata KRS----KMYGFGLGKR---DGRMYSFGLGKR---DYD-YYGEE---------------- B. craniifer KRS----KMYGFGLGKR---DGRMYSFGLGKR---DYD-YYGE----------------- G. bimaculatus KRAGG--RQYGFGLGKRA--GGRQYGFGLGKRTPGDEDDYYFPD---------------- S. gregaria KRATGAASLYSFGLGKR---GPRTYSFGLGKRGDDEPNDYSEQELFADVDGDSEDALPVA Node 14 GASP KRS----KMYGFGLGKRAGSDGRLYSFGLGKR---DYDDYYGED---------------- Node 14 JTT+G KRS----KMYGFGLGKRAGSDGRLYSFGLGKR---DYDDYYGED---------------- Node 11 GASP KRS----KMYGFGLGKRAGSDGRLYSFGLGKR---DYDDYYGED---------------- Node 11 JTT+G KRS----KMYGFGLGKRAGSDGRLYSFGLGKR---DYDDYYGEE---------------- Node 12 GASP KRS----KMYGFGLGKRSGNDGRLYSFGLGKR---DYDDYIQE----------------- Node 12 JTT+G KRS----KMYGFGLGKRSGNDGRLYSFGLGKR---DYDDYIQED---------------- Node 10 GASP KRS----KMYGFGLGKRAGSDGRLYSFGLGKR---DYDDYYGED---------------- Node 10 JTT+G KRS----KMYGFGLGKRAGSDGRLYSFGLGKR---DYDDYYGED---------------- Node 9 GASP KRS----KMYGFGLGKR---DGRMYSFGLGKR---DYD-YYGEE---------------- Node 9 JTT+G KRS----KMYGFGLGKR---DGRMYSFGLGKR---DYDDYYGEE----------------

107 5 6 7 B. orientalis --DEDEDISSGDDDVDNSEYEDLMDKRDRMYSFGLGKRARPYSFGLGKRSP-SG-M-QRL P. americana --DEDEDISSGDDDVDNSEYEDLMDKRDRMYSFGLGKRARPYSFGLGKRSP-SG-M-QRL B. germanica --DEEDHQTSADEDIEDADSVDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SS-A-QRL S. longipalpa --DEDEDQQSSGEDIDDSDAVDLVDKRERLYSFGLGKRARPYSFGLGKRAPSSG-V-QRL D. punctata --DEDDQQAIGDEDIEESDVGDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-A-QRL B. craniifer --DEDDQLANGDEDIEDSEVGDLIDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-T-QRL G. bimaculatus --EEEEDVP--EDNLDDS---DSVDKRDRLYSFGLGKRSRPFGFGLGKRA---G-M---- S. gregaria VEADERELPEAAEEEMPGVFTELMDKRGRLYSFGLGKRARPYSFGLGKRA---GPAPSRL Node 14 GASP --DEDEDQASGDDDIDDSDYGDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-M-QRL Node 14 JTT+G --DEDEDISSGDEDIDDSDYGDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-M-QRL Node 11 GASP --DEDDDQASGDEDIEDSDVGDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-A-QRL Node 11 JTT+G --DEDDDQASGDEDIEDSDVGDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-A-QRL Node 12 GASP --DEDEDISSGDDDVDNSEYEDLMDKRDRMYSFGLGKRARPYSFGLGKRSP-SG-M-QRL Node 12 JTT+G --DEDEDISSGDDDVDNSEYEDLMDKRDRMYSFGLGKRARPYSFGLGKRSP-SG-M-QRL Node 10 GASP --DEDDDQTSADEDIEDSDSVDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-A-QRL Node 10 JTT+G --DEDDDQASGDEDIEDSDAVDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-A-QRL Node 9 GASP --DEDDQQAIGDEDIEDSDVGDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-A-QRL Node 9 JTT+G --DEDDQQANGDEDIEDSDVGDLMDKRDRLYSFGLGKRARPYSFGLGKRAP-SG-A-QRL 8 9 10 B. orientalis YGFGLGKR-GGSMYSFGLGKRADGRLYAFGLGKRPVSSA-RQTGSRFNFGLGKRSDEIDL P. americana YGFGLGKR-GGSMYSFGLGKRADGRLYAFGLGKRPVSSA-RQTGSRFNFGLGKRSDEIDL B. germanica YGFGLGKR---ALYSFGLGKRAGGRLYSFGLGKRPVNSG-RQSGSRFNFGLGKRSDDFDI S. longipalpa YGFGLGKR-GGSLYSFGLGKRGGGRLYAFGLGKRPVNSG-RQTGSRFNFGLGKRSEDFDL D. punctata YGFGLGKR-GGSLYSFGLGKRGDGRLYAFGLGKRPVNSG-RSSGSRFNFGLGKRSDDIDF B. craniifer YAFGLGKRAGSSLYSFGLGKRGEGRLYGFGLGKRPVNSG-RSSGSRFNFGLGKRSEDIDI G. bimaculatus YSFGLGKR-AQHQYSFGLGKRGEGRMYSFGLGKRPNY--ERMAGSRFNFGLGKR---ADA S. gregaria YSFGLGKR--------------EGRMYSFGLGKRPLYGGDR----RFSFGLGKR---APA Node 14 GASP YGFGLGKR-GGSLYSFGLGKRGDGRLYAFGLGKRPVNSG-RQSGSRFNFGLGKRSDDIDL Node 14 JTT+G YGFGLGKR-GGSLYSFGLGKRGDGRLYAFGLGKRPVNSG-RQSGSRFNFGLGKRSDDIDI Node 11 GASP YGFGLGKR-GGSLYSFGLGKRGDGRLYAFGLGKRPVNSG-RQSGSRFNFGLGKRSDDIDL Node 11 JTT+G YGFGLGKR-GGSLYSFGLGKRGDGRLYAFGLGKRPVNSG-RQSGSRFNFGLGKRSDDIDI Node 12 GASP YGFGLGKR-GGSMYSFGLGKRADGRLYAFGLGKRPVSSA-RQTGSRFNFGLGKRSDEIDL Node 12 JTT+G YGFGLGKR-GGSMYSFGLGKRADGRLYAFGLGKRPVSSA-RQTGSRFNFGLGKRSDEIDL Node 10 GASP YGFGLGKR-GGSLYSFGLGKRGGGRLYAFGLGKRPVNSG-RQSGSRFNFGLGKRSDDFDL Node 10 JTT+G YGFGLGKR-GGSLYSFGLGKRGGGRLYAFGLGKRPVNSG-RQSGSRFNFGLGKRSDDFDI Node 9 GASP YGFGLGKR-GGSLYSFGLGKRGDGRLYAFGLGKRPVNSG-RSSGSRFNFGLGKRSDDIDI Node 9 JTT+G YGFGLGKR-GGSLYSFGLGKRGDGRLYAFGLGKRPVNSG-RSSGSRFNFGLGKRSDDIDI 11 B. orientalis KEIEEEIA-EEGKRSPQSHRFSFGLGKREVAPSELEAVRNE--------ERDKGKHQDET P. americana KEIEEEIA-EEGKRSPQGHRFSFGLGKREVAPSELEAVRNE--------ERDKGKHQDET B. germanica RELEGKFA-EEDKRSPQEHRFSFGLGKREVAPSELEAVKNE--------EKDSVSNQE-- S. longipalpa ---------EEDKRFPQDHRFAFGLGKRKLHPVSIEAVRDE--------EKDNESESKDV D. punctata RELEEKFA--EDKRYPQEHRFSFGLGKREVEPSELEAVRNE--------EKDNSSVHD-- B. craniifer RDLEEKFA-EEEKRYPQEHRFAFGLGKREVAPSELEAVKNE--------ERDSASVHD-- G. bimaculatus NPAYLLSDLGEEKRGP-DHRFAFGLGKREVSPNELEAVREEQLHHDKEAQQHELAEAAPA S. gregaria -----------------EHRFSFGLGKRDARSADSQ------------------------ Node 14 GASP RELEEKFA-EEDKRSPQEHRFSFGLGKREVAPSELEAVRNE--------EKDKGSEQDDT Node 14 JTT+G RELEEKFA-EEDKRSPQEHRFSFGLGKREVAPSELEAVRNE--------EKDNASNQDET Node 11 GASP RELEEKFA-EEDKRSPQEHRFSFGLGKREVAPSELEAVRNE--------EKDSVSEQDDV Node 11 JTT+G RELEEKFA-EEDKRSPQEHRFSFGLGKREVAPSELEAVRNE--------EKDNASNQDDV Node 12 GASP KEIEEEIA-EEGKRSPQGHRFSFGLGKREVAPSELEAVRNE--------ERDKGKHQDET Node 12 JTT+G KEIEEEIA-EEGKRSPQGHRFSFGLGKREVAPSELEAVRNE--------ERDKGKHQDET Node 10 GASP RELEGKFA-EEDKRSPQEHRFSFGLGKREVAPSELEAVRNE--------EKDSVSEQEDV Node 10 JTT+G RELEEKFA-EEDKRSPQEHRFSFGLGKREVAPSELEAVRNE--------EKDNASNQEDV Node 9 GASP RELEEKFA-EEDKRYPQEHRFSFGLGKREVAPSELEAVRNE--------EKDSSSVHD-- Node 9 JTT+G RELEEKFA-EEDKRYPQEHRFSFGLGKREVAPSELEAVRNE--------EKDNASVHD--

108 12 B. orientalis RKNGTYE-YHHTGERVKRSLHYAFGLGKRGASPYDFESSPSFESDEDEEMGTDEFSRLIR P. americana RKNGTSESYHHTGERVKRSLHYAFGLGKRGGSPYDFESSPSFESDEDEEMGTDEFSRLIR B. germanica KKNNTNDAHIHNGERVKRSLHYPFGFGK-QDSGFDLHS-SSLSSEENDDIGPEEFARMVR S. longipalpa SVQEKKN--STTGERVKRSLS---------ASPYDTS-----ASEED----VDEFARLIR D. punctata KKNNTND--MHSGERIKRSLHYPFGI-RKLESSYDLNSASSLNSEENDDITPEEFSRMVR B. craniifer KRNNTND--LHSGERIKRSLHYPFGI-RKQESSYDLNSASSLNSEENDDITPEEFSRMIR G. bimaculatus PEREPNDAHANGKHAVKRSLHYGFGIGKRTSDAFGLDVDPE---EDDRDAISEDFTRYIR S. gregaria ------------------------------------------------------------ Node 14 GASP KKNGTND-HHHTGERVKRSLHYPFGIGKRGASPYDLESAPSLESEEDDDIGPEEFSRLIR Node 14 JTT+G KKNNTND-HMHSGERVKRSLHYPFGIGKRQGSPYDLDSAPSLNSEEDDDIGPEEFSRMIR Node 11 GASP KKNNTND-HIHTGERVKRSLHYPFGIGKKQASPYDLNSASSLNSEENDDIGPEEFSRMIR Node 11 JTT+G KKNNTND-HMHSGERVKRSLHYPFGIGKKQESSYDLNSASSLNSEENDDIGPEEFSRMIR Node 12 GASP RKNGTSE-YHHTGERVKRSLHYAFGLGKRGASPYDFESSPSFESDEDEEMGTDEFSRLIR Node 12 JTT+G RKNGTSE-YHHTGERVKRSLHYAFGLGKRGGSPYDFESSPSFESDEDEEMGTDEFSRLIR Node 10 GASP KKNNTND-HIHTGERVKRSLHYPFGFGK-QASPYDLHS-SSLSSEENDDIGPEEFARMIR Node 10 JTT+G KKNNTND-HIHSGERVKRSLHYPFGIGK-QESSYDLNS-SSLSSEENDDIGPEEFARMIR Node 9 GASP KKNNTND--LHSGERIKRSLHYPFGI-RKQESSYDLNSASSLNSEENDDITPEEFSRMIR Node 9 JTT+G KKNNTND--MHSGERIKRSLHYPFGI-RKQESSYDLNSASSLNSEENDDITPEEFSRMIR 13 14 B. orientalis RPYNFGLGKRIPMYDFG??????? P. americana RPYNFGLGKRIPMYDFGIGKRSEH B. germanica RPFEYARQKQVPMYDFGIGKRSER S. longipalpa RPFNFGLGKRIPMYDFGIGKRSER D. punctata RPFNFGLGKRIPMYDFGIGKRSER B. craniifer RPFNFGLGKRIPMYDFGIGKRSER G. bimaculatus RPYSFGLGKRVPMYDFGIGKRADR S. gregaria ------------------------ Node 14 GASP RPYNFGLGKRIPMYDFGIGKRSER Node 14 JTT+G RPYNFGLGKRIPMYDFGIGKRSER Node 11 GASP RPFNFGLGKRIPMYDFGIGKRSER Node 11 JTT+G RPFNFGLGKRIPMYDFGIGKRSER Node 12 GASP RPYNFGLGKRIPMYDFGIGKRSEH Node 12 JTT+G RPYNFGLGKRIPMYDFGIGKRSEH Node 10 GASP RPFNFGLGKRIPMYDFGIGKRSER Node 10 JTT+G RPFNFGLGKRIPMYDFGIGKRSER Node 9 GASP RPFNFGLGKRIPMYDFGIGKRSER Node 9 JTT+G RPFNFGLGKRIPMYDFGIGKRSER

109

1 2 6 7 S. frugiperda LEKRSPHYDFGLGKRAYSYVSE---YKRLPVYNFGLGKRSRPYSFGLGKRARPYSFGLGKR H. armigera LAKRSPHYDFGLGKRAYSYVSE---YKRLPVYNFGLGKRSRPYSFGLGKRARPYSFGLGKR B. mori LEKRSPQYDFGLGKRAYSYVSE---YKRLPVYNFGLGKRSRPYLFGLGKRARPYSFGLGKR A. mellifera -------------KRAYTYVSE---YKRLPVYNFGIGKR-RQYSFGLGKRRQPYSFGLGKR A. aegypti LAVRSPKYNFGLGKRRY-IIEDVPGAKRLPHYNFGLGKRAYRYHFGLGKRPNRYNFGLGKR A. gambiae LAVRSPKYNFGLGKRRY-IIEDVPGAKRLPHYNFGLGKRAYRYHFGLGKRPNRYNFGLGKR L. cuprina YDKRVERYAFGLGRRAYTYTNGGNGIKRLPVYNFGLGKRARPYSFGLGKRNRPYSFGLGKR D. melanogaster IDKRVERYAFGLGRRAYMYTNGGPGMKRLPVYNFGLGKRSRPYSFGLGKR----------- D. grimshawi IDKRMERYAFGLGRRAYMYSNGGAGMKRLPVYNFGLGKRSRPYSFGLGKR----------- D. punctata FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR P. Americana FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR B. germanica LVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR B. craniifer FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYAFGLGKR G. bimaculatus FYKRL--YDFGVGKRAYSYVSE---YKRLPVYNFGLGKRSRPFGFGLGKR--MYSFGLGKR S. gregaria LYKRL--CDFGVGKRAYTYVSE---YKRLPVYNFGLGKRARPYSFGLGKRSRLYSFGLGKR C. finmarchicus VSKR-EPYNFGIGKR--SQMWG----KRQP-YNFGVGKRA-PYGFGIGKRA-LYGFGIGKR Node 1 HKY+G LDKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRSRPYSFGLGKRSRLYSFGLGKR Node 1 JTT+G FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRSRLYSFGLGKR Node 1 M0+G FDKRL--YDFGLGKRAYTYVSE---YKRLPVYNFGLGKRARPYSFGLGKRPRLYSFGLGKR Node 1 M3 LDKRL--YDFGLGKRAYTYVSE---YKRLPVYNFGLGKRARPYSFGLGKRPRLYSFGLGKR Node 1 G Consensus FDKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRSRLYSFGLGKR Node 2 HKY+G LDKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRSRPYSFGLGKRSRLYSFGLGKR Node 2 JTT+G FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRSRLYSFGLGKR Node 2 M0+G FDKRL--YDFGLGKRAYTYVSE---YKRLPVYNFGLGKRARPYSFGLGKRPRLYSFGLGKR Node 2 M3 LDKRL--YDFGLGKRAYTYVSE---YKRLPVYNFGLGKRARPYSFGLGKRPRLYSFGLGKR Node 2 G Consensus FDKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRSRLYSFGLGKR Node 3 HKY+G FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR Node 3 JTT+G FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR Node 3 M0+G FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR Node 3 M3 FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR Node 3 G Consensus FVKRL--YDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRQRLYGFGLGKR Node 4 HKY+G LDKRLPQYDFGLGKRAYSYVSE---YKRLPVYNFGLGKRSRPYSFGLGKRARPYSFGLGKR Node 4 JTT+G FEKRLPHYDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKRSRPYSFGLGKR Node 4 M0+G FDKRLPHYDFGLGKRAYTYVSE---YKRLPVYNFGLGKRARPYSFGLGKRPRPYSFGLGKR Node 4 M3 LDKRLPQYDFGLGKRAYTYVSE---YKRLPVYNFGLGKRARPYSFGLGKRPRPYSFGLGKR Node 4 G Consensus* FDKRLPHYDFGLGKRAYSYVSE---YKRLPVYNFGLGKRARPYSFGLGKR?RPYSFGLGKR

Figure S3.2 Alignment of conserved insect ASTs and the results of ancestral reconstruction using PAML software. Numbers indicate AST with respect to occurrence in the precursor sequence of cockroaches and the sequences that we were able to align to those peptides. Consensus sequences were generated using only the models with the G parameter. Amino acid changes across ancestral nodes are highlighted in red for AST peptides, in the spacer regions amino acid changes are not highlighted. In this dataset gaps in the reconstruction were inferred by eye according to the extant sequence data. An * indicates a consensus with an ambiguous site and a ? shows where there is an ambiguous site.

110

Table S3.1 Known FGLa-type AST sequences collected from literature, GenBank and EMBL databases. Peptides that are shared in two or more species are listed in column three; ASTs that occur only in a single known species are listed in column two. *Peptides with internal cleavage sites are not listed separately unless done so by the reference from which they were taken. E.g. in G. Bimaculatus only AYSYVSEYKRLPVYNFGL is listed, not both AYSYVSEYKRLPVYNFGL and LPVYNFGL. MET-Callatostatins are not listed in this table.

111

Table S3.1 Known FGLa-type AST sequences Genus Species AST unique to Species AST shared with other Species References Orhtoptera Gryllus bimaculatus 2 COPIES AGGRQYGFGL *AYSYVSEYKRLPVYNFGL (Meyering-Vos et al., 2001) SRPFGFGL DRLYSFGL

AGMYSFGL VPMYDFGI

AQHQYSFGL

GEGRMYSFGL

PNYERMAGSRFNFGL

GPDHRFAFGL

SLHYGFGI

PYSFGL

Schistocerca gregaria AYTYVSEYKRLPVYNFGL ARPYSFGL (Vanden Broeck et al., 1996) ATGAASLYSFGL

GPRTYSFGL

GRLYSFGL

AGPAPSRLYSFGL

EGRMYSFGL

PLYGGDRRFSFGL

APAEHRFSFGL

Carausius morosus GRQYSFGL LYDFGL (Lorenz et al., 2000) ADGRTYAFGL

IPMYDFGL

TSSLYSFGL

Dictyoptera

Diploptera punctata APSGAQRLYGFGL LYDFGL (Donly et al., 1993) YPQEHRFSFGL AYSYVSEYKRLPVYNFGL

SKMYGFGL

DRLYSFGL

ARPYSFGL

112

GGSLYSFGL

PFNFGL

IPMYDFGI

DGRMYSFGL

PVNSGRSSGSRFNFGL

GDGRLYAFGL

Periplaneta americana SPQGHRFSFGL LYDFGL (Ding et al., 1995) AYSYVSEYKRLPVYNFGL

SKMYGFGL

SGNDGRLYSFGL

DRMYSFGL

ARPYSFGL

SPSGMQRLYGFGL

GGSMYSFGL

ADGRLYAFGL

PVSSARQTGSRFNFGL SLHYAFGL

PYNFGL

IPMYDFGI

Blattella germanica AGSDGRLYSFGL LYDFGL (Bellés et al., 1999) APSSAQRLYGFGL AYSYVSEYKRLPVYNFGL

ALYSFGL SKMYGFGL

AGGRLYSFGL DRLYSFGL

PVNSGRQSGSRFNFGL ARPYSFGL

SPQEHRFSFGL VPMYDFGI

Blaberus craniifer APSGTQRLYAFGL LYDFGL (Bellés et al., 1999) AGSSLYSFGL AYSYVSEYKRLPVYNFGL

GEGRLYGFGL SKMYGFGL

YPQEHRFAFGL DGRMYSFGL

DRLYSFGL

113

ARPYSFGL

PVNSGRSSGSRFNFGL

PFNFGL

IPMYDFGI

Blatta orientalis SPQSHRFSFGL LYDFGL (Bellés et al., 1999) AYSYVSEYKRLPVYNFGL

SKMYGFGL

SGNDGRLYSFGL

DRMYSFGL

ARPYSFGL

SPSGMQRLYGFGL

GGSMYSFGL

ADGRLYAFGL

PVSSARQTGSRFNFGL

SLHYAFGL

PYNFGL

Supella longipalpa AGSDSRLYSFGL LYDFGL (Bellés et al., 1999) ERLYSFGL AYSYVSEYKRLPVYNFGL

APSSGVQRLYGFGL SKMYGFGL

GGGRLYAFGL ARPYSFGL

PVNSGRQTGSRFNFGL GGSLYSFGL

FPQDHRFAFGL PFNFGL

IPMYDFGI

Isoptera

Reticulitermes flavipes LYDFGL (Yagi et al., 2008) AYSYVSEYKRLPVYNFGL

DRLYSFGL

GGSLYSFGL

SLHYAFGL

Lepidoptera

Heliothis virescens AYSYVSEYKRLPVYNFGL (Berg et al., 2007)

114

SRPYSFGL

ARPYSFGL

ERDMHRFSFGL

Galleria mellonella SRPYLFGL (Huybrechts et al., 2005) LSSKFNFGL

SPHYDFGL

ARPYSFGL

SRPYSFGL

Helicoverpa armigera YSKFNFGL SPHYDFGL (Davey et al., 1999) AYSYVSEYKRLPVYNFGL

ARPYSFGL

ARAYDFGL

LPMYNFGL

ARSYNFGL

SRPYSFGL

ERDMHRFSFGL

Cydia pomonella SPHYNFGL AYSYVSEYKRLPVYNFGL (Duve et al., 1997b) ARGYDFGL SRPYSFGL

LPLYNFGL ARPYSFGL

KMYDFGL

Lacanobia oleracea AYSYVSEYKRLPVYNFGL (Audsley and Weaver, 2003) SRPYSFGL (Audsley et al., 2005) ARPYSFGL

ARAYDFGL

SPHYDFGL

ARSYNFGL

ERDMHRFSFGL

Spodoptera littoralis AYSYVSEYKRLPVYNFGL (Audsley et al., 2005)

115

SRPYSFGL

ARPYSFGL

SPHYDFGL

ARAYDFGL

LPMYNFGL

Manduca sexta AKSYNFGL ARPYSFGL (Audsley et al., 2005) SRPYSFGL (Audsley and Weaver, 2003) SPHYDFGL (Davis et al., 1997)

Bombyx mori SPQYDFGL AYSYVSEYKRLPVYNFGL (Secher et al., 2001) ARMYSFGL ARPYSFGL

ARSYSFGL SRPYLFGL

QRDMHRFSFGL LSSKFNFGL

Spodoptera frugiperda ERDMHGFSFGL SPHYDFGL (Abdel-Latief et al., 2004) AYSYVSEYKRLPVYNFGL

SRPYSFGL

ARPYSFGL

ARAYDFGL

LPMYNFGL

ARSYNFGL

LSSKFNFGL

Diptera

Calliphora vomitoria LNEERRANRYGFGL VERYAFGL (East et al., 1996) AYTYTNGGNGIKRLPVYNFGL

ARPYSFGL

NRPYSFGL

DPLNEERRANRYGFGL

ANRYGFGL

Lucilia cuprina VERYAFGL (East et al., 1996) AYTYTNGGNGIKRLPVYNFGL

116

ARPYSFGL

NRPYSFGL

DPLNEERRANRYGFGL

ANRYGFGL

Drosophila melanogaster VERYAFGL (Lenz et al., 2000) AYMYTNGGPGMKRLPVYNFGL

SRPYSFGL

TTRPQPFNFGL

Drosophila subobscura VERYAFGL (Munté et al., 2005) AYMYNNGGPGMKRLPVYNFGL

SRPYSFGL

Drosophila pseudoobscura VERYAFGL (Bowser and Tobe, 2007) AYMYNNGGPGMKRLPVYNFGL

SRPYSFGL

TTRPQPFNFGL

Drosophila madeirensis VERYAFGL (Munté et al., 2005) AYMYNNGGPGMKRLPVYNFGL

SRPYSFGL

Drosophila simulans VERYAFGL (Bowser and Tobe, 2007) AYMYTNGGPGMKRLPVYNFGL

SRPYSFGL

TTRPQPFNFGL

Drosophila yakuba VERYAFGL (Bowser and Tobe, 2007) AYMYTNGGPGMKRLPVYNFGL

SRPYSFGL

TTRPQPFNFGL

117

Drosophila Ananassae AYMYTNGGAGMKRLPVYNFGL MERYAFGL (Bowser and Tobe, 2007) SRPYSFGL

TTRPQPFNFGL

Drosophila grimshawi AYMYSNGGAGMKRLPVYNFGL MERYAFGL (Bowser and Tobe, 2007) SRPYSFGL

TTRPQPFNFGL

Drosophila mojavensis AYMYNNGGPGMKRLPVYNFGL (Bowser and Tobe, 2007) MERYAFGL

SRPYSFGL

TTRPQPFNFGL

Aedes aegypti ASAYRYHFGL SPKYNFGL (Veenstra et al., 1997) RVYDFGL LPHYNFGL

LPNRYNFGL

Anopheles gambiae TASGNGAGSAYRYHFGL SPKYNFGL GenBank accession no. XP_313511

RAYDFGL LPHYNFGL

LPNRYNFGL

Hymenoptera

Apis mellifera AYTYVSEYKRLPVYNFGI GenBank accession no. XP_001120780

GRDYSFGL (Audsley and Weaver, 2006) RQYSFGL

GRQPYSFGL

PNDMLSQRYHFGL

Decapoda

Carcinus maenas EPYAFGL YAFGL (Duve et al., 1997a) DPYAFGL AGPYAFGL

SPYAFGL GGPYAFGL

ASPYAFGL YSFGL

118

APQPYAFGL AGPYSFGL

ATGQYAFGL SGQYSFGL

PDMYAFGL EAYAFGL

EYDDMYTEKRPKVYAFGL NPYAFGL

SDMYSFGL APTDMYSFGL

GYEDEDEDRPFYALGLGKRPRTYSFGL

Cancer borealis SKMYGFGL (Billimoria et al., 2005) GGSLYSFGL (Huybrechts et al., 2003) DRLYSFGL

GDGRLYAFGL

EAYAFGL

NPYAFGL

AGPYAFGL

GGPYAFGL

APTDMYSFGL

SDYAFGL

GHYNFGL

AAPYEFGL

PQNMYSFGL

Orconectes limosus SAGPYAFGL (Dircksen et al., 1999) PRVYGFGL

AGPYAFGL

Macrobrachium rosenbergii HNDYVFGL DRTYSFGL (Yin et al., 2006) SPGYSFGL GGPYAFGL

EGLYAFGL AGPYAFGL

SGTYNFGL AGQYAFGL

GQYAFGL AGHYSFGL

SKTFSFGL SGSYSFGL

DRSYSFGL

119

SQQYAFGL

PRHYAFGL

PSSYAFGL

PKNYAFGL

DSDIQTRPGQYAFGL

5 COPIES PQHYAFGL

PQQYAFGL

AQQYAFGL

ASSYSFGL

SPYSFGL

PDAYSFGL

SRTYQFGL

ENQYAFGL

3 COPIES SSPYAFGL

SRPYAFGL

VPGSYGFGL

Penaeus monodon ANEDEDAASLFAFGL SAGPYAFGL (Duve et al., 2002) PDAEESNKRDRLYAFGL AGPYAFGL

DRLYAFGL AGQYAFGL

TGGPYAFGL YAFGL

SGHYAFGL AGHYSFGL

ANQYAFGL DRTYSFGL

TPSYAFGL YSFGL

PQRDYAFGL SDYAFGL

ANQYTFGL GHYNFGL

ASQYTFGL AAPYEFGL

SQYTFGL PQNMYSFGL

YTFGL

SGHYNFGL

AGPYEFGL

GGPYEFGL

GPYEFGL

120

SPYEFGL

NPYEFGL

NEVPDPETERNSYDFGL

EVPDPETERNSYDFGL

PETERNSYDFGL

NSYDFGL

YDFGL

PSAYSFGL

DARGALDLDQSPAYASDLGKRIGSAYSFGL

TARGALDLDQSPAYASDLGKRIGSAYSFGL

SVAYGFGL

TVAYGFGL

(X)GIYGFGL

Procambarus clarkii QNNYGFGL ADLYSFGL (Yasuda-Kamatani and Yasuda, 2006)

TPNYAFGL SGNYNFGL

QGMYSFGL SGQYSFGL

PDMYSFGL PRNYAFGL

PDLYSFGL SYDFGL

ADMYSFGL PRVYGFGL

SRQYSFGL 4 COPIES AGPYAFGL

TAGPYAFGL 3 COPIES SGPYAFGL

TGPYAFGL ADPYAFGL

PNPYAFGL AGQYSFGL

DGMYSFGL AGPYSFGL

SGPYSFGL

SGAYSFGL

Panulirus interruptus HNNYAFGL SGNYNFGL GenBank accession no. BAF64528

TPDYAFGL 5 COPIES PRNYAFGL

EGMYSFGL SYDFGL

121

ADLFSFGL SGPYAFGL

SQYAFGL ADLYSFGL

NRQYSFGL 20 COPIES ADPYAFGL

PNNYAFGL AGQYSFGL

SRQYAFGL SGSYSFGL

TSDEKVSDLDAFGL AGPYSFGL

PTTYSFGL

TASYGFGL

DGMYAFGL

ADPYSFGL

Copepoda

Calanus finmarchicus EPYGFGI GenBank accession no. ABS29318

3 COPIES APYGFGI

ALYGFGI

EPYNFGI

Nematoda

Caenorhabditis elegans AAMRSFNMGF MAAPKQMVFGF (Nathoo et al., 2001) LIMGL YKPRSFAMGF

SVSQLNQYAGFDTLGGMGL

ALSTFDSLGGMGL

ALQHFSSLDTLGGMGF

Caenorhabditis briggsae SYSQLNQYAGFDTLGGMGF MAAPKQMVFGF GenBank accession nos. XP_001678632

AALGTFDSIGGMGL YKPRSFAMGF XP_001667388 APLQMTSLDTLGGMGF

ASMRSFNMGF

RLIMGL

122

Table S3.2 Average posterior probabilities Ancestral Node Model Average peptide

Posterior Probability Insect Reconstruction: Node 1 JTT+G 0.907 HKY+G 0.899 M0+G 0.875 M3 0.876 Node 3 JTT+G 1 HKY+G 0.979 M0+G 1 M3 0.999 Cockroach Reconstruction: Node 12 JTT+G 1 HKY+G 0.999 M0+G 1 WAG+G 1 Node 14 JTT+G 0.954 HKY+G 0.895 M0+G 0.940 WAG+G 0.961

Table S3.2 Average posterior probabilities for reconstructed AST 7 (across all AST 7 amino acid sites) peptides tested for biological activity in this study.

123

Table S3.3 Likelihood ratio tests Models lnL P-value Degrees of

freedom Insect Reconstruction: HKY -2097.3801 HKY+G -1921.4947 vs. HKY 1.74389E-78 1 JTT -613.73919 JTT+G -591.60326 vs. JTT 2.85795E-11 1 M0 -1656.7312 M3 -1619.8421 vs. M0 3.61239E-15 4 M0+G -1627.4584 vs. M0 1.98643E-14 1 Cockroach Reconstruction: HKY -7215.0261 HKY+G -7052.3371 vs. HKY 9.76036E-73 1 JTT -3522.2652 JTT+G -3449.1294 vs. JTT 1.13233E-33 1 M0 -6628.3612 M0+G -6515.0015 vs. M0 3.09597E-51 1 WAG -3532.6551 WAG+G -3467.3401 vs. WAG 2.98341E-30 1

Table S3.3 Likelihood ratio tests. For the insect reconstruction the addition of the gamma distribution (G) significantly improves HKY, JTT, and M0. For M0, we also get a significant improvement if we instead use the M3 model. For the cockroach reconstruction G improves the fit in all cases.

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