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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
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.
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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.
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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
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
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
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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
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
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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.
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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
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:
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.
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
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.
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
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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