Juvenile Hormone Biosynthesis in the Cockroach, Diploptera

Juvenile hormone biosynthesis in the cockroach, Diploptera punctata: the characterization of the biosynthetic pathway and the regulatory roles of allatostatins and NMDA receptor

Juan Huang

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Cell and Systems Biology University of Toronto

Juvenile hormone biosynthesis in the cockroach, Diploptera punctata: the characterization of the biosynthetic pathway and the regulatory roles of allatostatins and NMDA receptor

Juan Huang

Doctor of Philosophy (2015), Department of Cell and Systems Biology, University of Toronto Abstract

The juvenile hormones (JH) play essential roles in regulating growth, development, metamorphosis, ageing, caste differentiation and reproduction in insects. Diploptera punctata, the only truly viviparous cockroach is a well-known model system in the study of JH biosynthesis and its regulation. The physiology of this animal is characterized by very stable and high rates of JH biosynthesis and precise and predictable reproductive events that correlate well with rates of JH production. Many studies have been performed on D. punctata to determine the function of JH. However, the pathway of JH biosynthesis has not been identified. In addition, although many factors are known to regulate JH biosynthesis, the exact mechanisms remain unclear. The aim of my research was to elucidate the JH biosynthetic pathway in D. punctata and study the mechanisms by which allatostatin (AST) and N-methyl-D-aspartate (NMDA) receptor regulate JH production. I have (1) identified genes in the JH biosynthetic pathway, and determined their roles in JH biosynthesis; (2) investigated the mode of action of AST by determining the signaling pathway of AstR and the target of AST action; (3) determined the role of the NMDA receptor in JH biosynthesis using RNA interference and treatment with an NMDA receptor antagonist. To validate the application value of my research, AST analogs with high JH inhibitory activity were designed and their activities on JH biosynthesis were measured by in vitro and in vivo bioassays. ii

Acknowledgements

It has been four and a half years since I began my studies at the University of Toronto. I still remember the first day in Toronto. I was excited and nervous. Starting from the first day I went to the lab, I was surrounded by friendly faces and help. My supervisor Stephen S. Tobe and his wife Martha Tobe helped me to get used to the culture on another continent. Jinrui Zhang helped me to find a place to live. And all the documents were handled in one day with the help of

Ekaterina F. Hult and Jane Linley. They successfully took away all my nervousness and made me feel that my life in Toronto would be exciting.

Before I came to Canada, I barely spoke English. Language has been troublesome for me. I was worried that no one would like to talk to me because of my poor English. But again, people in my lab, Ekaterina F. Hult, Jinrui Zhang, Koichiro J. Yagi, Shirley H. Tiu, Elisabeth Marchal and

Ilke van Hazel helped me to get over my trouble. They have been super patient, and always encouraged me to speak. My English was greatly improved with their help.

In regard to research, there are many people to thank. First, my supervisor Stephen S. Tobe; he has an interesting way to train his students. To start my project, Steve asked me to read papers and find a project I am interested in, instead of assigning me one. It was difficult in the beginning, but when I look back now, I find it was great training. Now I am able to start and complete a project independently, thanks to him. In addition, Steve always provided great suggestions for my projects, and he always encouraged me to try new things. He taught me: never to be afraid of failure, because that is part of PhD training. I would never have finished my

PhD without his support and supervision.

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And I would also like to thank Ekaterina F. Hult and Elisabeth Marchal for their great help during my PhD study. I was a chemist before I came to Toronto. I knew very little biology.

Ekaterina F. Hult not only helped me to start my project, but also taught me many techniques in biology. Most importantly, she has an ability to make me feel good about myself. She encouraged me many times when I was frustrated with my failed experiments. Elisabeth Marchal is the most kind and sweet person I have ever met. She is always very thoughtful and nice, and she always came up with great ideas. We were a great team and worked on several projects together. I learned a lot from her, not only her knowledge in biology, but also her attitude to research and life.

I would also like to acknowledge my other collaborators who contributed to my research projects: Jinrui Zhang, who taught me Radiochemical assay, and cockroach dissection; Koichiro

J. Yagi, who helped me set up HPLC and gave suggestions for my projects; Prof Barbara Stay, who taught me cockroach dissection and provided suggestions for my projects; Prof Jozef

Vanden Broeck, who provided the equipment and reagents to run the AstR functional assays;

Sven Zels, who taught me the technique of receptor functional assays; Ilke van Hazel, who helped me with the cell culture and the expression of NMDA receptors.

I would like to express my gratitude to my committee members: Belinda Chang, Ian Orchard,

William G. Bendena, David Lovejoy and Les Buck. I thank them for their great suggestions for my projects and my thesis.

I would like to thank all my friends in Toronto and in China. They brought so much happiness and joy to my life, and made my life in Toronto pleasant and colorful. Lastly, I would like to thank my parents and my brother for their support in the past four and a half years.

Table of Contents

Abstract ……………………………………………………………………………………….ii

Acknowledgement …………………………………………………………………………….iii

Table of Contents……………………………………………………………………………...v

Table of Figures………………………………………………………………………………..vii

Table of Tables…………………………………………………………………………………ix

Abbreviations…………………………………………………………………………………..x

Chapter 1: General Introduction

1.1 Juvenile hormones………………………………………………………………… 1 1.2 JH biosynthetic pathway…………………………………………………………...7 1.3 JH signaling pathway……………………………………………………………... 26 1.4 Diploptera punctata………………………………………………………………. 32 1.5 Regulation of JH titre……………………………………………………………... 38 1.6 Rational and objectives of my study……………………………………………... 49 1.7 References………………………………………………………………………….. 52

Chapter 2: Characterization of the Juvenile Hormone pathway in the viviparous cockroach, Diploptera punctata

2.1 Summary…………………………………………………………………………... 69 2.2 Introduction……………………………………………………………………….. 69 2.3 Materials and Methods…………………………………………………………… 73 2.4 Results…………………………………………………………………………….... 80 2.5 Discussion………………………………………………………………………….. 90 2.6 Supplementary data………………………………………………………………. 96 2.7 References………………………………………………………………………… 100

Chapter 3: Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata

3.1 Summary………………………………………………………………………….. 105

3.2 Introduction………………………………………………………………………. 105 3.3 Materials and Methods…………………………………………………………... 108 3.4 Results……………………………………………………………………………...114 3.5 Discussion………………………………………………………………………….126 3.6 Supplementary data……………………………………………………………….131 3.7 References………………………………………………………………………… 134

Chapter 4: Identification and characterization of the NMDA receptor and its role in regulating reproduction in the cockroach, Diploptera punctata

4.1 Summary………………………………………………………………………….. 138 4.2 Introduction………………………………………………………………………. 138 4.3 Materials and Methods…………………………………………………………... 141 4.4 Results……………………………………………………………………………...144 4.5 Discussion………………………………………………………………………….155 4.6 Supplementary data……………………………………………………………….160 4.7 References………………………………………………………………………… 166

Chapter 5: General discussion

5.1 Function of JH in reproduction...……………………………………………….. 169 5.2 Evolution of the JH biosynthetic pathway...……………………………………. 170 5.3 Regulation of JH biosynthesis….………………………………………………... 172 5.4 The value of my study in insect control…..……………………………………...175 5.5 Future perspective……………………………………………………………….. 176 5.6 References………………………………………………………………………….177

Chapter 6: Appendices……………………………………………………………………… 180

Table of Figures

Figure 1.1 Structures of the JH homologues in insects 2 Figure 1.2 The JH biosynthetic pathway 8 Figure 1.3 Dynamics of JH metabolism throughout the life cycle of D. punctata 35 Figure 2.1 Scheme of JH biosynthetic pathway 71 Figure 2.2 Tissue specific expression of genes encoding JH biosynthetic enzymes 82 Figure 2.3 Developmental expression of genes encoding JH biosynthetic enzymes 84 during the first gonadotrophic cycle of D. punctata Figure 2.4 The effect of JH precursors on JH biosynthesis by CA from mated female 86 D. punctata Figure 2.5 Efficiency of HMGR-JHAMT RNAi-mediated knockdown and the effect 86 of silencing on the transcription of the other genes encoding enzymes in the JH biosynthetic pathway in day 4 mated female D. punctata. Figure 2.6 JH regulates ovarian development 88 Figure 2.7 Transverse sections of the basal oocytes from day 4 control and HMGR- 89 JHAMT dsRNA-treated animals. Figure 3.1 Relative expression levels of Dippu-AstR and Dippu-AST mRNA in tissues 115 of day 4 males and mated females. Figure 3.2 Relative expression levels of Dippu-AstR and Dippu-AST mRNA during the 116 first gonadotrophic cycle Figure 3.3 The effect of Dippu-AST dsRNA on JH biosynthesis by the CA. 120 Figure 3.4 Dose-response curves for ASTs in CHO-WTA11 cells expressing Dippu- 121 AstR. Figure 3.5 Dose-response curves for the bioluminescence response induced in 122 CHOPAM28 and HEK293 cells expressing Dippu-AstR Figure 3.6 The effect of Dippu-AstR dsRNA on JH biosynthesis by the CA and on the 123 expression of genes encoding enzymes in the JH biosynthetic pathway of D. punctata Figure 3.7 The effect of AST on the expression levels of genes encoding enzymes in 124 the JH biosynthetic pathway in CA of day 6 mated female D. punctata Figure 3.8 JH precursors rescue the AST-induced JH inhibition. 124 Figure 4.1 Amino acid sequence alignment of the two Diploptera NR1 subunit 145 (DpNR1A, DpNR1B), and homologous receptors from D. melanogaster and T. castaneum Figure 4.2 Phylogram depicting the relationship between the NR1 subunits from 146 Diploptera and orthologues of this receptor from other insects. Figure 4.3 Molecular characterization of DpNR2. 149

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Figure 4.4 Graphic representation of the relative tissue distribution of (A) DpNR1A 150 transcript levels, (B) DpNR1B transcript levels and (C) DpNR2 transcript levels in tissues of day 4 adult male and mated female D. punctata. Figure 4.5 Relative transcript levels of DpNR1A, DpNR1B and DpNR2 in brains of 151 mated female D. punctata from day 0-day 7 after ecdysis. Figure 4.6 Relative transcript levels of DpNR1A, DpNR1B and DpNR2 in CA of of 151 mated female D. punctata from day 0-day 7 after ecdysis Figure 4.7 Relative transcript levels of DpNR1A, DpNR1B and DpNR2 in testes of 152 differentages of male D. punctata. Figure 4.8 The effect of DpNR2 dsRNA treatment on JH biosynthesis and basal 152 oocyte growth, and the interactions among these genes in mated female D. punctata. Figure 4.9 In vivo effect of MK-801 on JH biosynthesis, basal oocyte growth and 154 relative Vg mRNA levels. Figure 6.1 The effect of topical application of K15 and W206 on JH biosynthesis and 183 oocyte growth

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Table of Tables

Table 2.1 q-RT-PCR primer sequences and reaction efficiencies and correlation 77 coefficients in the q-RT-PCR assay Table 2.2 Primers for dsRNA construction 79 Table 3.1 Potency of Dippu-ASTs a: activation of AstR in CHO-WTA11 cells 125 (EC50) or inhibitory effect on JH release (IC50) Table 6.1 Structure of AST analogs 181 a Table 6.2 Potency of Dippu-AST analogs : inhibitory effect on JH release (IC50) 182 and activation of AstR in CHO-WTA11 cells (EC50)

Abbreviations

20E 20-hydroxyecdysone AC adenylate cyclase AST allatostatin AstR Allatostatin receptor AT Allatotropin CA corpora allata CHO Chinese hamster ovary CRE cAMP responsive element DMMP diphosphomevalonate DMPP dimethylallyl pyrophosphate FALD Farnesal dehydrogenase FOLD Farnesol dehydrogenase FPP farnesyl diphosphate FPPP Farnesyl diphosphate pyrophosphatase FPPS Farnesyl diphosphate synthase GnRH gonadotropin releasing hormone GPP Geranyl pyrophosphate HEK human embryonic kidney HMGR 3-hydroxy-3-methylglutaryl-CoA reductase HMGS 3-hydroxy-3-methylglutaryl-CoA synthase IPP isopentenyl pyrophosphate IPPI Isopentenyl diphosphate isomerase JH juvenile hormones JHMAT Juvenile hormone acid O-methyltransferase Kr-h1 Krüpel-homologues 1 LH luteinizing hormone MA mevalonic acid Met Methoprene-tolerant MF methyl farnesoate MK Mevalonate kinase NMDAR N-methyl-D-aspartate receptor PMK Phosphomevalonate kinase PPMD Diphosphomevalonate decarboxylase PTX pertussis toxin RXR retinoid X receptor Thiol Acetoacetyl-CoA thiolase USP Ultraspiracle Vg vitellogenin Vn vitellin

Chapter 1

General Introduction

1 Juvenile hormones

The juvenile hormones (JH), a family of acyclic sesquiterpenoids, play essential roles in regulating growth, development, metamorphosis, aging, caste differentiation and reproduction in insects. This family of hormones has been extensively studied because of its central role in insect development and reproduction and their potential value in pest control. This section will review the current knowledge of JHs, including the JH homologues, enzymes in the JH biosynthetic pathway and signal pathways of JH in insects.

1.1 JH homologues

JHs are synthesized and secreted by specialized, paired endocrine glands, the corpora allata (CA).

As early as 1934, Wigglesworth pointed out that insect metamorphosis was controlled by a hormone produced by CA, a gland near the insect brain (Wigglesworth, 1934). In 1956, a highly active extract, which produced anomalies in metamorphosis, was obtained from Cecropia Moth

Hyalophora cecropia (Williams, 1956). The structure of the first JH homologue was later elucidated by Röller et al (Röller et al., 1967), as methyl (2E,6E,10-cis)-10,11-epoxy-7-ethyl-3,

11-dimethyl-2,6-tridecadienoate. The structure was further confirmed as the 2E,6E,10-cis isomer

(Dahm et al., 1968), and the absolute configuration of the chiral centers (C10 and C11) was determined to be 10R,11S (Faulkner and Petersen, 1971; Meyer et al., 1971; Nakanishi et al.,

1971). This JH homologue was known as JH I (Fig. 1.1) (Goodman and Cusson, 2012).

1 11 5 COOCH 10 8 COOCH3 3 9 7 3 O 6 4 2 O O JH I JHB3

COOCH3 COOCH3 O O O

JH II JHSB3 HO

COOCH COOCH3 3 O O JH III 4'-Hydroxy JH III HO

COOCH3 COOCH3 O O JH 0 8'-Hydroxy JH III HO

COOCH3 COOCH3 O O iso-JH 0 12'-Hydroxy JH III

COOCH3

MF Figure 1.1 Structures of the JH homologues in insects. Figure adapted from Goodman and Cusson (2012)

JH I, which has only been identified in the Lepidoptera, not only plays important roles in regulating development, morphogenesis and reproduction in the Lepidoptera, but also has an effect on the development of other insects (Fisher and Mayer, 1982; Granger et al., 1979;

Granger et al., 1982; Shalaby et al., 1990; Steiner et al., 1999). In larval development of Corcyra,

JH I treatment on ligated early-last instar resulted in a stimulation on DNA synthesis with a consequent increase in DNA content and DNA concentration (Lakshmi and Dutta-Gupta, 1990).

During the last half of the larval molt of the tobacco hornworm, M. sexta, the presence of JH I at the peak of the ecdysteroid titer is important in inducing dopa decarboxylase (DDC), an enzyme which converts dopa to dopamine (Hiruma and Riddiford, 1985). In other insects, topical application of the synthetic JH I to adult Musca domestica vicina Macq resulted in a shortened gonoadotrophic cycle, decreased number of eggs and reduced hatching rate (Shalaby et al., 1990).

In addition, addition of JH I to the culture medium improved the development of single two-cell- stage embryos of a polyembryonic wasp Copidosoma floridanum (Iwabuchi, 1995).

A second JH homologue, JH II (methyl (2E, 6E, 10-cis)-10,11-epoxy-3,7,11-trimethyl-2,6- tridecadienoate) was identified in H. cecropia extracts (Meyer et al., 1970; Meyer et al., 1968).

JH II is the 2E, 6E, 10-cis isomer, as in JH I, but differs from JH I by a methyl group at C7 (Fig.

1.1). The absolute configuration of natural JH II at the C10, C11 positions has not yet been determined (Goodman and Cusson, 2012). Same as JH I, JH II has only been identified in the

Lepidoptera. Nevertheless, relatively little research has been performed on JH II. In Trichoplusia ni, the JH (JH I and JH II) and ecdysteroid titres were determined from the egg to the pupal molt

(Grossniklaus-Burgin and Lanzrein, 1990). Very little JH was detected in the freshly laid eggs of

T. ni, while in larval stages, JH II appeared to be the predominant or exclusive juvenile hormone to interact with ecdysteroids to regulate the larval development. JH II, which is the most

4 abundant JH in Sesamia nonagrioides, is involved both in diapause programming and diapause manifestation in this animal (Eizaguirre et al., 2005). In addition, JH II appears to be able to initiate male production followed by sexual reproduction in the water flea Daphnia magna

(Cladocera, Crustacea). Exposure of D. magna to either JH I or JH II reduces the reproduction rate, and induces parthenogenetically reproducing D. magna to produce male neonates (Oda et al., 2005).

JH III was first identified from organ cultures of CA of the tobacco hornworm moth, Manduca sexta (Judy et al., 1973). JH III displays the same E, E configuration at C2, C3 and C6, C7; however, it differs from other JH homologues, with methyl groups at the C3, C7, and C11 positions. This hormone only contains one chiral carbon (C10), which displays the 10R configuration in insects (Fig. 1.1). Of the juvenile hormone family, JH III is the most ubiquitous

JH homologue since it is the only JH biosynthesized and released in Orthoptera, Coleoptera,

Diptera, Hymenoptera, Dictyoptera, Lepidoptera, and the primitive ametamorphic Thysanura

(Baker et al., 1984; Tobe and Stay, 1985b). In larvae and adults of many insects, JH III is the principal or only JH homologue identified, such as in the cockroach, Nauphoeta cinerea,

Diploptera punctata and the firebrat, Thermobia domestica (Baker et al., 1984; Tobe et al., 1985).

JH 0 and its isomer 4-methyl JH I (iso-JH 0) were identified in M. sexta eggs (Bergot et al.,

1981). Differing from JH III, JH 0 contains ethyl groups at the C3, C7, and C11 positions (Fig.

1.1). To date, JH 0 and its isomer (iso-JH 0) have been identified only in the Lepidoptera and their functions in insects were unclear. JH III bisepoxide (JHB3), which contains a second epoxide substitution at C6, C7, was first identified in Drosophila melanogaster (Richard et al.,

1989b). JHB3 was determined to be the major in vitro JH product of larval ring glands and of adult CA-corpus cardiacum (CC) complexes of D. melanogaster. Later study identified JHB3 in

5 various dipteran species, such as Ceratitis capitata, Lucilia cuprina, Phormia regina,

Sarcophaga bullata (Bylemans et al., 1998; Lefevere et al., 1993; Moshitzky and Applebaum,

1995; Moshitzky et al., 2003). It has been demonstrated that the higher cyclorrhaphous Diptera produce JHB3 predominantly, and JHB3 is believed to be restricted to the higher Diptera

(Richard et al., 1989a; Richard et al., 1989b). In L. cuprina, JHB3 is the only juvenile hormone biosynthesized in vitro (Lefevere et al., 1993). Although it appeared that JHB3 production was restricted to higher Diptera, JHB3 was reported to be synthesized by CA and the male accessory glands of the mosquitoes, A. aegypti in vitro (Borovsky et al., 1994). However, recent work by Li et al. (Li et al., 2003) was unable to detect any JHB3 synthesized by CA complex of A. aegypti.

The existence of JHB3 in other orders has yet to be confirmed.

Even though JHs were identified and characterized in various species of insects, the structure of the JH in order Hemiptera has been a matter of controversy (Kotaki, 1993, 1996). Although JH

III and methyl farnesoate (MF) were reported as the products of CA in vitro in Dysdercus fasciatus (Bowers et al., 1983; Feldlaufer et al., 1982) and the presence of JH I in the hemolymph of Riptortus clavatus (Numata et al., 1992), there were no significant levels of the known JH or related compounds in the milkweed bug, Oncopeltus fasciatus (Baker et al., 1988).

The presence of an unknown Heteropteran JH was suggested (Miyawaki et al., 2006). The mystery regarding the JH in Hemiptera was not resolved until 2009, when Kotaki et al (2009) identified a new JH homologue, JH III skipped bisepoxide (JHSB3; Fig. 1.1) from Plautia stali, a member of the family Pentatomidae, suborder Heteroptera, order Hemiptera using a novel approach. The term “skipped” refers to a second epoxide substitution switching from C6, C7 as

in JHB3 to C2, C3. The absolute chemical structure of the novel skipped bisepoxide JH was characterized by the screening of a JH molecular library, and the juvenilizing activity of JHSB3

6 with different configurations on C2 and C3 and chirality on C10 was determined. Their result shows that the (2R, 3S) configuration is more important for biological activity than the chirality of C10, C11. JHSB3 with the 2R, 3S-configuration was more potent than those with the 2S, 3R- configuration and 2,3-double bond (Kotaki et al., 2011). The function of JHSB3 was determined in the last instars and adults of P. stali (Kotaki et al., 2011). Topical application of JHSB3 to last instar nymphs inhibited their metamorphosis, and JHSB3 application in allatectomized and diapausing adults stimulated the development of ovaries and ectadenia in females and males, respectively.

Another family of JH homologues, hydroxylated JHs (HJHs; Fig. 1.1) was identified from the

African locust Locusta migratoria (Mauchamp et al., 1999). JH III was identified as the main product released by the CA in vitro of L. migratoria (Mauchamp et al., 1985), while later studies discovered three different hydroxylated forms of JH III (4-OH, 8-OH, and 12-OH JH III) exhibited JH-like biological effects (Darrouzet et al., 1997; Mauchamp et al., 1999), in which 12-

OH JH III was found to be 100-fold more active than JH III.

Methyl farnesoate (MF; Fig. 1.1), a JH precursor without a C10, C11 epoxidation as in JH III, was first isolated to function as a JH from the hemolymph of the spider crab Libinia emarginata

(Laufer et al., 1987). Recent studies have shown multifunctional roles of MF in crustaceans, including reproduction, molting, larval development, morphogenesis, behaviour and general protein synthesis (Chang et al., 2001; Nagaraju, 2007). Studies in insects suggest that MF may also serve as a hormone in some insects. In the embryos of the cockroach N. cinerea, the predominant product released by the embryonic CA is MF until the stage of breaking of the chorion, and this substance circulates in embryonic haemolymph (Bürgin and Lanzrein, 1988;

Lanzrein et al., 1984). MF was also found to be biosynthesized by ring glands of larval D.

7 melanogaster (Richard et al., 1989a; Richard et al., 1989b), the embryonic CA of D. punctata

(Cusson et al., 1991b), the larval CA of Pseudaletia unipuncta (Cusson et al., 1991b) and the adult CA of Phormia regina (Yin et al., 1995). In addition, MF was also identiﬁed from the hemolymph of ﬁve orders of insects, including D. melanogaster (order Diptera), Schistocerca americana (order Orthoptera), three species of true bugs (order Hemiptera), worker honeybees,

Apis mellifera (order Hymenoptera), and three species of beetle (order Coleoptera) (Teal et al.,

2014). Based on the study on the activity of MF, Goodman and Cusson (2012) reviewed the in vivo biological role of MF in larvae and adults of D. melanogaster: During the larval stage, MF is more active in blocking adult development than JH III or JHB3, whereas JH III or JHB3 is more active once pupariation has been initiated.

2. JH biosynthetic pathway

The JH biosynthetic pathway, which comprises 13 discrete enzymatic steps, can be divided into two distinct biosynthetic parts: the mevalonate pathway and the JH-specific pathway (Fig. 1.2).

The mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. The isoprenoids produced by the mevalonate pathway are vital for diverse cellular functions, including the synthesis of cholesterol, haem A, ubiquinone, dilochol, and farnesylated proteins, growth control, and electron transport (Goldstein and Brown, 1990).

The most well-studied product of mevalonate pathway is cholesterol, because of its role in maintaining cell membranes and its implications for human cardiovascular diseases (Goldstein and Brown, 1990). The initial stages of biosynthesis of JH to the formation of farnesyl diphosphate (FPP) proceeds through the mevalonate pathway, which is shared in vertebrates and invertebrates (Belles et al., 2005; Goodman and Cusson, 2012).

O CoA Acetyl-CoA S Thiol O O CoA Acetoaacetyl-CoA S HMGS O OH O CoA HMG-CoA HO S HMGR O OH O Mevalonate HO OH MK O OH O

HO OP Mevolonate-P O OH O PMK

HO OPP Mevalonate-PP (MPP)

PPMD IPPI OPP Isopentenyl-PP (IPP) Dimethylallyl-PP (DMPP) OPP FPPS

OPP Geranyl-PP (GPP) FPPS

OPP Farnesyl-PP

FPPP

OH Farnesol

FOLD

Farnesal O O FALD

OH Farnesoic acid

CYP15C1 JHAMT O Methyl farnesoate (MF) O OMe OH O Orthopteran and Dictyopteran Juvenile hormone acid (JHA) CYP15A1 JHAMT Lepidoptera O

OMe O Figure 1.2 The JH biosynthetic pathway. The mevalonate pathway was shown before the dash line and the JH-specific pathway was after. Figure adapted from Goodman and Cusson (2012).

Insects and other arthropods, however, do not produce cholesterol as a final product of the mevalonate pathway, because they lack the enzymes squalene synthetase (farnesyl-diphosphate farnesyltransferase) and lanosterol synthase, which are required for the production of cholesterol

(Clark and Bloch, 1959). Thus, the second portion of the JH biosynthetic pathway comprises enzymatic steps unique to JH-producing organisms. Earlier studies on the JH biosynthetic pathway focused on the activity of enzymes in the mevalonate pathway (Casals et al., 1996;

Couillaud and Feyereisen, 1991; Feyereisen and Farnsworth, 1987a). Thanks to whole genome sequencing and CA trancriptomics studies, the identification and characterization of JH biosynthetic enzymes have greatly improved, especially for genes encoding enzymes in the JH- specific pathway (Consortium, 2006; Group, 2004; Mita et al., 2004; Noriega et al., 2006). Since

JH III is the most common JH homologs in insects, our review focuses on the study of enzymes directly involved in the biosynthesis of JH III.

2.1 Acetoacetyl-CoA thiolase (ACAT, Thiol)

The synthesis of JH begins with acetyl-CoA, which condenses with another acetyl-CoA through the catalysis of Thiol to form Acetoacetyl-CoA (Fig. 1.2). The gene encoding Thiol has been identified in the genome of D. melanogaster, B. mori, Anopheles gambiae and A. aegypti, A. mellifera and in an EST of the scolytid beetle Ips pini (Bomtorin et al., 2014; Eigenheer et al.,

2003; Keeling et al., 2004; Kinjoh et al., 2007; Nouzova et al., 2011). In vertebrates, the functional Thiol is comprised by a tetramer of identical subunits and has two cysteine residues at the active sites (Gehring and Harris, 1970), which are conserved in insects. In B. mori, Thiol was almost exclusively expressed in the CA-CC complex, wheras the transcription of Thiol in A. aegypti and A. mellifera was expressed in many tissues, including the ovary and fat body

(Bomtorin et al., 2014; Kinjoh et al., 2007; Nouzova et al., 2011).

2.2 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS)

The function of HMGS is to catalyze the condensation of acetyl-CoA and acetoacetyl-CoA to yield HMG-CoA (Fig. 1.2). The enzymatic activity of HMGS in the CA was determined in the adult female of D. punctata. The results show that enzymatic activity of HMGS has the same pattern as JH III biosynthesis, which suggests that HMGS influences the rate of JH biosynthesis.

However, the fact that the enzyme activity declined after the dramatic decrease of JH on day 6 indicates that changes in the activity of HMGS enzymes are apparently not responsible for the collapse in JH synthetic ability on day 6 (Couillaud and Feyereisen, 1991).

The gene encoding HMGS was first isolated in the cockroach B. germanica (Buesa et al., 1994;

Martinez-Gonzalez et al., 1993), and later in D. melanogaster (Spradling et al., 1999), the scolytid beetle Dendroctonus jeffreyi (Tittiger et al., 2000), B. mori (Kinjoh et al., 2007), A. aegypti (Nouzova et al., 2011), and A. mellifera (Bomtorin et al., 2014). HMGS predominantly expressed in CA-CC complex of B. mori (Kinjoh et al., 2007), A. aegypti (Nouzova et al., 2011), and A. mellifera (Bomtorin et al., 2014), and the transcript levels correspond to JH biosynthesis in the CA.

In B. germanica, two HMGS enzymes (HMGS-1 and HMGS-2) with 69% amino acid identity were demonstrated (Buesa et al., 1994), and the gene encoding HMGS-1 was considered to be a functional retrogene derived from HMGS-2 by retrotransposition (Buesa et al., 1994; Cabano et al., 1997; Casals et al., 2001). In mammals, two forms of HMGS (a mitochondrial form and a cytoplasmic form) have been detected, which are encoded by two different genes (Ayte et al.,

1990). In B. germanica, none of the two HMGS enzymes show any recognizable N-terminal leader peptide to target the protein to mitochondria, which suggests that the enzyme is cytosolic

11 in insects (Buesa et al., 1994). Both HMGS are highly expressed in the adult ovary, coordinately regulated in the ovary during the gonadotrophic cycle, but expressed differently throughout development (Ayte et al., 1990; Martinez-Gonzalez et al., 1993). The expression and enzymatic activities of both HMGS were also determined in the fat body of B. germanica (Casals et al.,

1996). HMGS-1 did not show any significant mRNA level or detectable protein level in the fat body, which indicates a limited role for HMGS-1 in the fat body. HMGS-2, on the other hand, shows a clear pattern in the fat body, which was consistent with that of vitellogenin production.

In D. jeffreyi, HMGS transcript localizes mainly in the metathorax and abdomen (Tittiger et al.,

2000). Topical application of JH III induced a dose- and time-dependent increase in HMGS transcripts in the male metathoracic-abdominal region, whereas no increase in the transcript levels was observed in the JH III-treated female. The JH III-mediated regulation of HMGS suggests that in addition to its function in the JH biosynthetic pathway, HMGS appears to control the isoprenoid pathway (Tittiger et al., 2000).

2.3 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)

HMGR catalyzes the first committed step of the isoprenoid biosynthetic pathway, the conversion of HMG-CoA to mevalonate (Fig. 1.2). It is believed to play an important role in the regulation of sterol synthesis and is generally referred to as the rate-limiting enzyme in cholesterol synthesis in vertebrates (Goldstein and Brown, 1990). Two distinct classes of HMGR have been identified

(Bochar et al., 1999): in eukaryotes, HMGR consist of a highly conserved C-terminal catalytic domain and a poorly conserved N-terminal membrane anchor domain, which contains two to eight inferred transmembrane helices. The second class of HMGR was discovered in the Archaea and the true bacterium Pseudomonas mevalonii, which lack the N-terminal membrane anchor domain. Both HMGR serve the same function in different species.

The presence of HMGR in the CA was first reported in M. sexta, which convert HMG-CoA to mevalonate (Bergot et al., 1979). The activity of HMGR was further studied in D. punctata

(Feyereisen and Farnsworth, 1987a) and the grasshopper, Schistocerca nitens (Baker and

Schooley, 1981). HMGR in the insect undergoes phosphorylation (inactive form) and dephosphorylation (active form), which could be altered by Mg-ATP or NaF (Monger and Law,

1982). In M. sexta, the activity of HMGR in the CA parallels, in most cases, the ability of the gland to synthesize JH (Baker and Schooley, 1981). In D. punctata, the activity of HMGR parallels JH biosynthesis until day 5 during the first gonadotrophic cycle. JH biosynthesis drops dramatically on day 6, while the activity of HMGR remains high till day 8. In addition, the half- life of HMGR was not related to the half-life of JH III biosynthesis (Feyereisen and Farnsworth,

1987a). The results suggest that HMGR is not the ‘the rate-limiting enzyme’ in JH biosynthesis in D. punctata.

To date, the gene encoding HMGR was cloned in many insect species, including D. melanogaster (Gertler et al., 1988), B. germanica (Martinezgonzalez et al., 1993), the I. pini

(Hall et al., 2002), Ips paraconfusus (Tittiger et al., 1999), D. jeffreyi (Tittiger et al., 2003), the moth Agrotis ipsilon (Duportets et al., 2000), B. mori (Kinjoh et al., 2007), A. aegypti (Nouzova et al., 2011), and A. mellifera (Bomtorin et al., 2014). The identification of HMGR genes has permitted further study of the function and the regulation of HMGR. For instance, a HMGR gene encoding 916 amino acid was identified in D. melanogaster. The 56% identity in C-terminal region to the hamster HMGR reflects the essential function of the C-terminal region. On the other hand, the high similarity in the membrane-spanning regions between the mammalian and

Drosophila HMGR suggests that the transmembrane domains may be essential for recognizing specific mevalonate derivatives or their binding proteins. In addition, the identification of

HMGR has allowed the expression of Drosophila HMGR in Schneider cells. Addition of mevalonate suppresses the transcript level and the enzymatic activity of Drosophila HMGR, which indicates a feedback regulation involved in the mevalonate pathway (Gertler et al., 1988).

The expression and the function of HMGR were also determined in many other insects. In B. germanica (Martinezgonzalez et al., 1993), A. ipsilon (Duportets et al., 2000) and A. mellifera

(Bomtorin et al., 2014), HMGR was found to express in many tissues, including fat body, ovary, muscle, brain and CA, whereas its expression in B. mori (Kinjoh et al., 2007) and A. aegypti

(Nouzova et al., 2011) was predominantly in the CA. Study of HMGR in Ips paraconfusus and D. jeffreyi suggests that HMGR is not only involved in the regulation of JH biosynthesis, but also the production of monoterpenoid pheromones in the males. (Tittiger et al., 2003; Tittiger et al.,

1999).

2.4 Mevalonate kinase (MK)

MK is responsible for the phosphorylation of mevalonate to produce the 5-phosphomevalonate

(mevalonate-P), which is metabolized in fungal, plant, and vertebrate systems to isopentenyl pyrophosphate (IPPI) (Cornforth et al., 1960) (Fig. 1.2). The isolation, purification, and characterization of MK was first reported in larval S. bullata by Goodfellow and Barnes (1971).

MK is distributed in the muscle and brain complex cytosol fractions of S. bullata (Cornforth et al., 1960). The gene encoding MK was identified in D. melanogaster and A. gambiae genomes, the EST database of D. punctata (Noriega et al., 2006), B. mori (Kinjoh et al., 2007), A. aegypti

(Nouzova et al., 2011), and A. mellifera (Bomtorin et al., 2014). In B. mori and A. aegypti, the transcript level of MK is highest in the CA, whereas in A. mellifera, the highest expression of

MK is in the brain (Bomtorin et al., 2014; Kinjoh et al., 2007; Nouzova et al., 2011). In addition,

14 the expression profile of MK in the CA of B. mori and A. aegypti corresponded to changes in JH biosynthesis.

2.5 Phosphomevalonate kinase (PMK)

Phosphomevalonate kinase (PMK) catalyzes the phosphorylation of mevalonate-P into 5- diphosphomevalonate (MPP), an essential step in isoprenoid biosynthesis (Fig. 1.2). Two nonorthologous genes encoding PMK have been identified: the Saccharomyces cerevisiae ERG8 gene, which is found in eubacteria, fungi and plants, and the human PMK gene, which is present only in animals (Houten and Waterham, 2001). In insects, the gene encoding PMK was identified in D. melanogaster and A. gambiae genomes, B. mori (Kinjoh et al., 2007), A. aegypti

(Nouzova et al., 2011), and A. mellifera (Bomtorin et al., 2014). In B. mori, PMK is expressed in multiple tissues, and the expression profile in the larval CA shows a similar pattern as that of the

JH biosynthesis (Kinjoh et al., 2007). In A. aegypti, the PMK was highly expressed in the CA, followed by ovary. The expression of PMK in the CA also coordinated with JH biosynthesis

(Nouzova et al., 2011).

2.6 Diphosphomevalonate decarboxylase (PPMD)

Mevalonate diphosphate decarboxylase (PPMD) is an enzyme in the mevalonate pathway that catalyzes the decarboxylation of the six-carbon MPP to the five-carbon isopentenyl diphosphate

(IPP). This reaction involves the dehydration of the substrate and the hydrolysis of one molecule of ATP, and Mg2+ is required (Jabalquinto et al., 1988). In rats, PPMD is a key enzyme in the

MVA pathway that is essential for the biosynthesis of the isoprenoids. However, there are not many studies on PPMD in insects. The gene encoding PPMD was identified from the genomes of D. melanogaster and A. gambiae, the EST database of I. pini (Keeling et al., 2004) and later in

B. mori (Kinjoh et al., 2007), and A. aegypti (Nouzova et al., 2011). In B. mori, PPMD was exclusively expressed in CC-CA complex of 4th instar larvae, and its expression paralleled the JH titre in 4th and 5th instars. In A. aegypti, PPMD was also exclusively expressed in the CA and its expression coordinated with JH biosynthesis by CA, which indicates that PPMD is involved in the regulation of JH biosynthesis in insects (Kinjoh et al., 2007; Nouzova et al., 2011).

2.7 Isopentenyl diphosphate isomerase (IPPI)

In the mevalonate pathway, IPP is the sole product of the ATP-dependent decarboxylation of

MPP and must be isomerized to DMPP to form Geranyl pyrophosphate (GPP) (Fig. 1.2). The enzyme isopentenyl diphosphate isomerase (IPPI), which catalyzes the isomerization of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMPP) (Fig. 1.2), plays a central role in isoprenoid biosynthesis (Ramos-Valdivia et al., 1997). Two isoforms of IPPI have been identified. Type 1 IPPI (IPPI-1) is a metalloprotein that is found in eukaryotes, and the optimal functioning of IPPI-1 requires a divalent metal cation (Mg2+ or Mn2+). Zinc has also been identified as an essential cofactor for the catalysis activity of IPPI-1 from E. coli (Carrigan and Poulter, 2003). The type 2 isoform (IPPI-2) is a flavoenzyme found in plant chloroplasts and bacteria; the isomerase activity requires not only a divalent metal cation, but also a reduced flavin coenzyme (de Ruyck et al., 2014).

In insect species, IPPI was first partially characterized from the extracts of B. mori, in which that the isomerase activity is dependent on the metal ions. Mn2+ was a better activator than Mg2+, especially at low concentrations (Koyama et al., 1985). Later study identified the sequence of

IPPI in the genome database of D. melanogaster and A. gambiae, the EST database of I. pini

(Keeling et al., 2004), B. mori (Kinjoh et al., 2007), A. aegypti (Diaz et al., 2012; Nouzova et al.,

2011), the spruce budworm, Choristoneura fumiferana (Sen et al., 2012), , M. sexta (Sen et al.,

2012) and A. mellifera (Bomtorin et al., 2014). The IPPI gene identified in A. aegypti (AaIPPI) encodes a 244 amino acid (aa) protein with high similarity to IPPI-1 in other organisms (Diaz et al., 2012). Two important motifs which are associated with the catalytic roles of IPPI-1 are well conserved: a TNACCSHPL motif containing a conserved cysteine residue and a WGEHEIDY motif that contains a conserved glutamate residue. The function of IPPI-1 in other organisms requires the binding of a divalent metal cation (Mg2+, Mn2+ or Zn2+) (Carrigan and Poulter, 2003), whereas the enzymatic assay shows that the full activity of AaIPPI requires Mg2+ or Mn2+ but not

Zn2+, and its activity can be completely inhibited by iodoacetamide (Diaz et al., 2012).

Insects in the order Lepidoptera produce five JH homologues (JH 0, JH I, 4-methyl JH I, JH II, and JH III) (See section 1.1). For the biosynthesis of different JH homologues, homologs of IPP and DMPP are involved into the mevalonate pathway, which requires the IPPI in Lepidoptera to catalyze the isomerization of homoisopentenyl diphosphate (HIPP) to homodimethylallyl diphosphate (HDMPP). Earlier studies in pig demonstrated that the isomerization of HIPP by porcine IPPI produced very little HDMPP. However, studies in insects demonstrated that CA homogenates of adult female M. sexta and purified IPPI from B. mori regiospecificly catalyzed the isomerization of HIPP to HDMPP, which suggests that the lepidopteran IPPI enzyme is structurally distinct from other isomerases (Baker et al., 1981; Koyama et al., 1985). Further study on IPPI in C. fumiferana and M. sexta confirmed the function of IPPI in catalyzing the isomerization of HIPP, and the homology models of the CfIPPI and HIPP isomerization study revealed that the lepidopteran IPPI enzyme has a larger active site cavity, to allow binding of larger substrates and to stabilize the high-energy intermediate formed during substrate isomerization (Sen et al., 2012).

The expression of IPPI mRNA varies in different tissues, but in most insects, the highest transcript level of IPPI was found in the CA. In B. mori, IPPI mRNA levels are expressed almost exclusively in the CA of 4th instar larvae, with relative low levels in other tissues (Kinjoh et al.,

2007). In A. mellifera and C. fumiferana, mRNA of IPPI was expressed in multiple tissues, with the highest transcript level in the CA (Bomtorin et al., 2014; Sen et al., 2012). IPPI mRNA of A. aegypti expressed in various tissues, including CA-CC, ovary, hindgut, brain, midgut, fat body of the female, and testis and accessory glands of the male (Diaz et al., 2012). The ubiquitous expression of IPPI suggests that IPPI might be involved in many metabolic pathways. The pattern of change of IPPI mRNA in the CA-CC during female pupal and adult development was consistent with the changes in JH biosynthesis, which suggests that the transcription of IPPI is partially responsible for JH biosynthesis (Diaz et al., 2012; Nouzova et al., 2011). Similar results were found in B. mori (Kinjoh et al., 2007). The expression of BmIPPI in the CA of 4th, 5th larvae, and pupae coordinate with the JH titre.

2.8 Farnesyl diphosphate synthase (FPPS)

The condensation of IPP and DMPP forms an intermediate compound GPP, which then undergoes a second condensation step to generate farnesyl diphosphate (FPP) (Fig. 1.2). This process is catalyzed by the enzyme Farnesyl diphosphate synthase (FPPS), a type of prenyltransferase. FPPS is a homodimeric protein, which is formed by tightly coupled subunits ranging from 32 to 44 kDa in size (Vandermoten et al., 2009a). The activity of FPPS requires divalent metal cations (Mg2+ or Mn2+). Sequence analysis of FPPSs revealed seven conserved regions, including two substrate binding regions, regions II and VI. Both regions contain an aspartate-rich motif, DDx(xx)xD (x represents any amino acid) (Liang et al., 2002). Region II, which includes the first aspartate-rich motif (FARM), is responsible for the determination of

18 chain-length, while region VI, which contains the second aspartate-rich motif (SARM), is considered to be the IPP binding site (Liang et al., 2002). The first FPPS genes in insects were cloned in A. ipsilon by Castillo-Gracia and Couillaud (1999) with high identity (about 40%) with other FPPSs and high transcript level in the CA. Additional insect FPPSs were identified in many insect orders, including Lepidoptera (Sen and Sperry, 2002), Diptera (Nouzova et al., 2011; Sen et al., 2007), Coleoptera (Taban et al., 2009), Hemiptera (Lewis et al., 2008; Sun and Li, 2012;

Zhang and Li, 2008), Hymenoptera (Bomtorin et al., 2014) and Blattodea (Noriega et al., 2006).

In Lepidoptera, two distinct forms of FPPS were identified, designated type-1 and type-2 FPPS

(FPPS-I and FPPS-II) (Cusson et al., 2006), while two slightly different isoforms of type-2 FPPS are present in B. mori (FPPS-2 and FPPS-3) (Kinjoh et al., 2007). Like the prenyltransferases in other organisms, FPPS in M. sexta requires the divalent cation (Mg2+ or Mn2+) for its activity.

The presence of detergent, glycerol, and non-specific protein-protein interactions improves the stability and catalytic activity of FPPS (Sen and Sperry, 2002). As described in section 1.1, the

Lepidoptera produce five JH homologues (Fig. 1.1), which requires the synthesis of ethyl/methyl-substituted FPP by FPPS. The preference of prenyltransferase to ethyl/methyl- substituted DMPP was determined using M. sexta CA homogenates, and the results suggest that the selectivity of the enzyme incline to the ethyl-substituted substrate (Sen et al., 1996).

Additional studies on the selectivity of this prenyltransferase were performed using different substrate analogs (Sen et al., 2006). Compared to pig liver FPPS, the lepidopteran enzyme derived from CA homologues displays greater steric latitude around the C-3 and C-7 alkyl positions of DMAPP and geranyl diphosphate (GPP). The enzymes generate more ethyl- branched geranyl/farnesyl diphosphate and the substrate specificity related to the enzyme localization (Sen et al., 2006). The structure analysis of FPPS-I and FPPS-II in C. fumiferana

19 revealed that FPPS-1 displays several unique active site substitutions, whereas FPPS-II has a more conventional catalytic cavity which indicates FPPS-I are better suited than FPPS-II for generating ethyl-substituted products. However, tissue distribution of FPPS mRNA showed that

FPPS-I is ubiquitous whereas FPPS-II is predominately expressed in the CA (Cusson et al.,

2006). The result is consistent with the distribution of FPPS 1-3 mRNA in 4th instar B. mori

(Kinjoh et al., 2007). These results suggest that FPPS-II may play a leading role in lepidopteran

JH biosynthesis despite its apparently more conventional catalytic cavity. In other species, FPPS forms a homodimeric protein. The recombinant C. fumiferana FPPS-2 was active in producing

FPP; However, expression of FPPS-1 (CfFPPS1, Pseudaletia unipuncta FPPS1, and A. ipsilon

FPPS1) in E. coli failed to display any FPPS activity in vitro. Surprisingly, the combination of

CfFPPS1 and CfFPP2 enhanced the enzyme activity, and an association between CfFPPS1 and

CfFPPS2 was observed, which suggests that FPPS-I and FPPS-II may derive from a heteromer to play a role in JH biosynthesis in moths. Whether these two enzymes form heterodimers in vivo has not yet been verified.

Two FPPS genes that encode proteins with about 80% identity were identified in the green peach aphid, Myzus persicae, and in the bird cherry-oat aphid Rhopalosiphum padi (Sun and Li, 2012;

Zhang and Li, 2008). Enzyme activity of FPPS in R. padi shows both enzymes could catalyze the formation of FPP from IPP and DMAPP (Sun and Li, 2012). However, the function of FPPS in

M. persicae was not determined. Without the feature of the FPPS in M. persicae, it is premature to conclude that these genes are involved JH biosynthesis. On the other hand, other prenyltransferase genes displaying dual geranyl diphosphate (GPP)/farnesyl diphosphate (FPP) synthase activity in vitro were identified in M. persicae. These two prenyltransferase genes encode very similar proteins, apart from the presence of a mitochondrial leader sequence (Lewis

20 et al., 2008). It is interesting to note that the prenyltransferase enzyme in aphid is not unique to the aphid species from which it was cloned. The molecular dynamics of the enzyme are responsible to maintain the balance between the production of GPP and FPP (Vandermoten et al.,

2009b).

Only single copies of the FPPS gene were identified in insects from other orders, such as Diptera

(Nouzova et al., 2011; Sen et al., 2007) and Coleoptera (Taban et al., 2009). The scanning of the

A. mellifera genome showed the presence of seven copies (Consortium, 2006). The expression of the first six copies of the FPPS gene revealed that FPPS3 is the bona fide gene involved in JH biosynthesis in honey bees (Bomtorin et al., 2014).

2.9 Farnesyl diphosphate pyrophosphatase (FPPP)

Farnesyl diphosphate pyrophosphatase (FPPP) catalyzes the hydrolysis of farnesyl diphosphate

(FPP) to farnesol (FOL). Relatively little was known about FPPP in insects. Cao et al. (2009) screened the D. melanogaster genome and identified the first FPPP genes in the insects. FPPP belongs to the haloalkanoic acid dehalogenase (HAD) super family that catalyzes phosphoryl transfer reactions (Allen and Dunaway-Mariano, 2004). Members of the HAD phosphatase superfamily have four conserved amino acid signature motifs, which are also well conserved in the DmFPPP. Nyati et al. (2013) identified 3 putative FPPP (AaFPPP-1, -2, and -3) paralogs through a search for orthologs of the DmFPPP in the CA of A. aegypti. Recombinant AaFPPP-1 and AaFPPP-2 displayed the features of FPPP in their ability to hydrolyze FPP into FOL, and the

FPPP activity of the CA extracts was found to be Mg2+-dependent. The determination of the function of FPPPs in JH biosynthesis using RNAi reveals that FPPP-1 plays the predominant function in JH biosynthesis. Unlike mRNA of enzymes in the mevalonate pathway which are

21 predominately expressed in the CA, AaFPPPs mRNA are expressed in various tissues, with

FPPP-1 highly expressed in midgut and Malpighian tubules, FPPP-2 in Malpighian tubules, and

FPPP-3 in brain and ovary. The ubiquitous expression of FPPP may result from the pleiotropic functions of farnesol and farnesal. In spite of the ubiquitous expression, the expression of FPPP-

1 and -2 in the CA correlated with JH biosynthesis in sugar-fed females, which suggests FPPP may play a role in the regulation of JH biosynthesis.

2.10 Farnesol dehydrogenase (FOLD)

A Farnesol dehydrogenase (FOLD) is responsible for the catalysis of the conversion of farnesol

(FOL) to farnesal (FAL) (Fig. 1.2). In vertebrates, plants, and fungi, the oxidation of FOL to

FAL is mediated by nicotinamide-dependent dehydrogenases (Chayet et al., 1973; Inoue et al.,

1984; Keung, 1991). Study of FOL oxidation using CA homogenates of the adult female M sexta, revealed that farnesol and/or farnesal dehydrogenase were NAD+-dependent enzymes (Baker et al., 1983). However, the conversion of FOL to FAL in larval M. sexta was not affected by nicotinamide. The enzyme, which oxidizes FOL to FAL in larval M. sexta, appears to be an oxygen-dependent enzyme, perhaps a flavin and/or iron-dependent oxidase (Sperry and Sen,

2001). A FOLD enzyme was identified and functionally characterized in the CA of adult A. aegypti (Mayoral et al., 2009a). In CA of adult female M sexta, the FOLD enzyme was ineffective in the addition of NADP+ (Baker et al., 1983). However, the FOLD in A. aegypti was characterized to be a NADP+-dependent farnesol-dehydrogenase. It is possible that moths utilize a different mechanism for FOL oxidation. In A. aegypti, FOLD is expressed in various tissues, and with a relatively low transcript level in the CA. On the other hand, the transcript levels of

FOLD in the CA coordinate with JH biosynthesis (Mayoral et al., 2009a).

2.11 Farnesal dehydrogenase (FALD)

Farnesal dehydrogenase (FALD), which catalyzes the oxidation of farnesal to FA, was one of the less understood steps in JH synthesis (Fig. 1.2). An early study using the CA homogenates of the adult female, M sexta predicted that FALD is an NAD+-dependent aldehyde dehydrogenase and this aldehyde dehydrogenase showed some substrate specificity for the 2E isomer (Baker et al.,

1983). Rivera-Perez et al. (2013) identified and characterized a FALD enzyme in female A. aegypti. Two FALD genes with 50% amino acid identity were identified, in which FALD-1 produces four different transcripts and FALD-2 produces one. All five FALD variants exhibit the activity to convert FAL to farnesoic acid (FA), and the oxidation is stimulated in the presence of

NAD+. mRNA of FALD variants have unique tissue distribution profiles, with each predominantly expressed in one unique tissue: FALD1-A in ovaries, FALD1-B in Malpighian tubes, FALD1-C in hindgut, FALD1-D in nervous tissue, while FALD2 are relatively low in transcript level comparing to FALD1. The reduction of FALD activity results in accumulation of farnesol, which subsequently converts back into farnesol, resulting in farnesol leaking out of the

CA. Oxidation of farnesal may be a rate limiting step in JH synthesis in mosquito after blood feeding.

2.12 Juvenile hormone acid O-methyltransferase (JHAMT)

In the final two steps of JH biosynthesis, FA is converted to JH III through a methyl transfer and an epoxidation (Fig. 1.2). The order of the final two steps is insect order dependent. In

Orthoptera, Dictyoptera, Coleoptera and Diptera, FA undergoes the methylation of the carboxyl group to produce methyl farnesoate (MF), which is epoxidized by a P450 monooxygenase at C10,

C11 position to generate JH III. In Lepidoptera, however, a reverse step order occurs:

23 epoxidation precedes methylation. Other ethyl-branched JH homologues are also synthesized through the same order, but with different precursors derived from homomevalonate and mevalonate (Shinoda and Itoyama, 2003). The modeling of A. gambiae, B. mori, D. melanogaster and T. castaneum JHAMTs and docking simulation shows that all insect JHAMTs are able to esterify both FA and JHA. The order of the methylation/epoxidation may be controlled by the specificity of the epoxidase. The epoxidase in Lepidoptera might have higher affinity than JHAMT for FA, which results in epoxidation precedes methylation. In other insects, however, the epoxidase can only selectively catalyze MF. Thus, esterification of FA to MF by

JHAMT occurs in earlier step (Defelipe et al., 2011).

The first JHAMT was cloned and functionally characterized in, B. mori (Shinoda and Itoyama,

2003). The sequence analysis reveals that JHAMT belongs to the SAM-dependent methyltransferases family, with a conserved S-adenosyl-L-methionine (SAM) binding motif. The protein encoded by the JHAMT gene was able to not only convert JH III acid to JH III, but also catalyze the conversion of JHA I, II, and FA to their cognate JH methyl esters in the presence of

S-adenosyl-L-methionine (SAM). Northern blot analysis shows that BmJHAMT mRNA is exclusively expressed in the CA. Expression of JHAMT correlates well with the JH biosynthetic activity of the CA in 4th and 5th instar larvae, pupae and adults, and especially in 5th instar larvae in which the shutdown of the expression of JHAMT appears to be the primary reason for the decline in JH biosynthesis. These results suggest that JHAMT is the rate limiting enzyme in the

JH biosynthesis in B. mori (Kinjoh et al., 2007; Shinoda and Itoyama, 2003). Orthologues of

JHAMT have also been cloned and characterized in other insect species, including T. castaneum

(Minakuchi et al., 2008a), D. melanogaster (Niwa et al., 2008), the Eri silkworm, Samia cynthia ricini (Sheng et al., 2008), A. aegypti (Mayoral et al., 2009b), the desert locust Schistocerca

24 gregaria (Marchal et al., 2011) and A. mellifera (Bomtorin et al., 2014). In all species, JHAMT is expressed predominately in the CA and the recombinant JHAMT protein from these insect can catalyze the methylation of FA into MF, as well as JHA into JH III. In T. castaneum, silencing the JHAMT gene using RNAi induced precocious metamorphosis, while in D. melanogaster

(Minakuchi et al., 2008a), JHAMT overexpression resulted in a pharate adult lethal phenotype

(Niwa et al., 2008). In S. gregaria, knockdown of JHAMT not only resulted in lower JH release, but also a suppression in FA-stimulated JH release. A delay in sexual maturation was also observed in JHAMT-silenced animals (Marchal et al., 2011). In many insects, such as T. castaneum (Minakuchi et al., 2008a), D. melanogaster (Niwa et al., 2008), S. cynthia ricini

(Sheng et al., 2008), S. gregaria (Marchal et al., 2011) and A. mellifera (Bomtorin et al., 2014), the expression of JHAMT in the CA correlates well with JH biosynthesis.

Farnesoic acid O-methyltransferase (FAMeT), which was first reported in a crustacean, was initially considered to be the enzyme converting FA to MF in crustaceans (Gunawardene et al.,

2001). And a FAMeT was cloned in D. melanogaster (Burtenshaw et al., 2008).

Immunohistochemical analysis shows the presence of FAMeT in the CA portion of the ring gland. However, recombinant FAMeT did not show any enzymatic activity in catalyzing the conversion of FA or JHA. In S. gregaria, FAMeT mRNA was expressed in several tissues, and its expression in the CA did not correlate with JH biosynthesis. In addition, silencing FAMeT has no effect on either JH release or MF content of the CA (Marchal et al., 2011). These results suggest that FAMeT does not encode a functional methyltransferase.

2.13 Juvenile hormone epoxidase (CYP15A1/CYP14C1)

Juvenile hormone epoxidase is involved in the epoxidation of FA in the Lepidotera or the epoxidation of MF in the Orthoptera, Dictyoptera, Coleoptera, and Diptera (Fig. 1.2). Early studies demonstrated that this epoxidase is a microsomal cytochrome P450 enzyme (Hammock,

1975). The first JH epoxidase, named CYP15A1, was cloned from D. punctata (Helvig et al.,

2004). This enzyme contains all the features of a typical microsomal P450, and its recombinant protein catalyzed the epoxidation of MF to JH III in the presence of NADPH. The product of epoxidation is mostly the (10R)-enantiomer. Studies on JHAMT reveal that the order of the final two steps is primarily determined by the substrate specificity of epoxidase (Section 1.2.12).

CYP15A1 from D. punctata showed strong substrate specificity to the natural substrate MF.

Substitution of natural MF with geometrical isomers of MF and other terpenoids, such as farnesol, farnesal, FA, and JH III showed little or no activity. DpCYP15A1 mRNA was exclusively expressed in the CA and the expression level is higher in CA with high JH biosynthetic activity. The expression and function of CYP15A1 has also been investigated in many other insects, including A. aegypti (Nouzova et al., 2011), S. gregaria (Marchal et al., 2011) and A. mellifera (Bomtorin et al., 2014). In all, CYP15A1 was selectively expressed in the CA.

In A. aegypti and A. mellifera, the expression of CYP15A1 does not correlate with JH biosynthesis, whereas in S. gregaria, CYP15A1 shows high levels of transcription in active CA.

Silencing CYP15A1 in S. gregaria resulted in a reduction in JH release and an accumulation of

MF within the CA.

CYP15C1, an ortholog of CYP15A1, was recently identified and characterized in the

Lepidoptera, B. mori (Daimon et al., 2012). And CYP15C1 shares high homology with the

CYP15A1 in D. punctata. The dimolting (mod) mutation, which causes precocious larval-pupal metamorphosis, results in a null mutation in the coding sequence of CYP15C1. CYP15C1 was

26 then expressed in Drosophila S2 cells and enzymological analysis revealed that CYP15C1 converts FA to JHA in a highly stereospecific manner. Further study showed that CYP15C1 is responsible for the mod mutant of B. mori and its molecular defect results in the absence of JHs

(JH I and JH II) in B. mori, indicating CYP15C1 plays essential roles in JH biosynthesis. In addition, CYP15C1 is specifically expressed in the CA. The expression of CYP15C1, on the other hand, did not show any change during development, which suggests that CYP15C1 is not the rate-limiting enzyme in the JH biosynthetic pathway. The CYP15 gene is not found in higher dipterans such as D. melanogaster, probably because the production of JHB3 requires a different epoxidase.

3. JH signaling pathway

3.1 Ultraspiracle (USP) as a JH receptor

Ultraspiracle (USP) is a homologue of the vertebrate retinoid X receptor (RXR), which can form heteromers with other nuclear receptors to bind with genomic response elements (Henrich et al.,

1994). In Lepidoptera and Diptera, USP displays high identity with RXR only in the DNA- binding domain, whereas the similarity in the ligand-binding domains is relatively low. In other insect orders and other arthropods, USP shows higher similarity to vertebrate (Iwema et al.,

2007). USP can interact with several nuclear receptors, such as EcR and DHR38 (Sutherland et al., 1995; Yao et al., 1992). The USP:EcR complex, which is formed by the heterodimerization between EcR and USP, is required for the binding of the steroid hormone 20-hydroxyecdysone

(Yao et al., 1992).

Using a fluorescence assay, Jones and Sharp (1997) demonstrated a protein-ligand interaction between the natural Drosophila JHs (JH III ester monoepoxide and bisepoxide, respectively) and

27 recombinant D. melanogaster USP. This interaction cannot be influenced by the addition of farnesol or 20E, which indicates the specificity of the binding. On the other hand, JH acid, which itself did not change the fluorescence of USP, affects the interaction between JH III ester and

USP. The action of JH acid indicates that JH acid may bind to USP in a different manner than JH

III ester. Further studies on D. melanogaster USP showed that it could also specifically bind to

JH III, which changes the conformation of USP and stabilizes the dimeric/oligomeric quaternary structure of USP. The JH III agonist methoprene shows a competitive inhibition in the the JH III-

USP interaction (Jones et al., 2001). Furthermore, the binding affinities between USP and other natural farnesoid products of the ring gland of D. melanogaster were determined (Jones et al.,

2006). MF exhibited a nanomolar affinity to USP, and the addition of an epoxide across a double bond or any substitution on C1 (Fig. 1.1) other than methyl ester, resulted in a decrease in affinity to USP. Mutational analysis showed that the binding of JH III to USP was strongly reduced by the mutation C472A/H475L.

Although USP has been demonstrated to interact with JHs, further studies on USP did not show its effect on JH action. The structure-based analysis of a Heliothis virescens USP protein shows that JHs could fit into the ligand binding pocket (LBP) of USP. However, the percentage of occupancy of LBP was relatively low, which raised concerns over the validity of USP as an JH receptor (Sasorith et al., 2002). In vivo USP activation assays in third instar Drosophila larvae demonstrated that neither natural JHs (JHI, JHII and JHIII) nor JH analogs (pyriproxifen and methoprene) were able to activate USP, whereas fenoxycarb, a carbamate insecticide that mimics the action of JH, induced a weak activation. Beck et al. (2009) determined the activation of USP by JHs and their analogs in transgenic animals expressing either the GAL4-TcUSP (T. castaneum) or the GAL4-DmUSP (D. melanogaster). Their results show that JHs and their

28 analogs were not able to activate USP. In particular, MF, which displayed a nanomolar affinity to

DmUSP (Jones et al., 2001), did not show any effect on the activation of USP. On the other hand, pre-incubation of organs with JH III lead to the repression of GAL4-TcUSP and the GAL4-

DmUSP activated by 20E. Thus, USP may not act as an JH receptor, but may interact with JH in the EcR/USP complex.

3.2 Methoprene-tolerant (Met) as a JH receptor

Methoprene-tolerant (Met) is a basic-helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) protein containing a HLH structure and two PAS domains (A and B). The Met gene was first discovered by an ethyl methane sulfonate mutagenesis screen (Wilson and Fabian, 1986). The Met mutation conferred a 100-fold-increased resistance to methoprene and JH III, and was resistant to methoprene-induced pseudotumor formation in larvae and to JH III- or methoprene-induced vitellogenic oocyte development in adult females, suggesting Met might be a JH receptor. The hydroxyapatite (HAP) binding assay of JH III to JH III-binding protein in fat body cells revealed a 10-fold lower binding affinity in the Drosophila Met strain (Shemshedini and Wilson, 1990).

In vitro synthesized Drosophila Met bound to JH III with high affinity (Kd = 5.3 ± 1.5 nM, mean

± SD). And the effectiveness of JHs in the activation of Met expressed in Drosophila S2 cells is

JH III>JH II>JH I>methoprene (Miura et al., 2005). A similar affinity was also found in the binding of Tribolium Met to JH III (Kd = 2.94 ± 0.68 nM, mean ± SD) (Charles et al., 2011). The function of Met and the high affinity of Met to JH suggest that Met may act as a JH receptor.

As a potential JH receptor, the ligand-binding properties of Met were determined (Charles et al.,

2011). To examine which part of the Tribolium Met protein is responsible for binding JH III, the truncated proteins with part of the conserved domain were synthesized for a ligand-binding assay.

The results show that Met specifically binds JH III through its C-terminal PAS domain (PAS-B domain plus C-terminal region). As a bHLH protein, Met requires either a homo- or heterodimer partner for its activity (Kewley et al., 2004). In A. aegypti, a Ftz-F1-interacting steroid receptor coactivator (FISC) was identified as a functional partner of Met in mediating JH-induced gene expression (Li et al., 2011). Microarray analysis and RNAi studies revealed an ortholog of FISC in T. castaneum, named steroid receptor co-activator (SRC), which was responsible for the formation of heterodimer with Met (Zhang et al., 2011). The closest relative of FISC/SRC in

Drosophila is Taiman (Tai) (Charles et al., 2011). The study on the interaction between Tai and

Met suggested a model of JH action on Met in Drosophila. Met forms a homodimer in the absence of JH, while the binding of JH to the PAS-B domain of Met results in conformational changes to release Met from the homophilic complex and allows it to bind Tai (Charles et al.,

2011). Another JH-dependent heterodimeric partner of Met, Cycle (CYC) was identified in A. aegypti (Shin et al., 2012). The binding between Met and CYC only occurs in the presence of JH

III, and is induced by JH III in a dose dependent manner. Both Met and CYC specifically binds to the E-box-like motif from the Kr-h1 gene promoter. Silencing CYC, Met or SRC/FISC using

RNAi, impaired the circadian activation of Kr-h1 and Hairy genes. Based on the previous studies, it currently appears that the JH receptor is composed of two DNA-binding bHLH/PAS transcription factors, in which Met is an obligatory component and the partner of Met varies in different insects.

In Drosophila, the Met mutant showed a high resistance to the toxic and morphogenetic effects of JHs and their analogs (Shemshedini et al., 1990; Wilson and Ashok, 1998; Wilson and Fabian,

1986). However, although Met-null mutants show reduced oogenesis, they are viable. The phenotype of complete absence of Met is too subtle to conclude that Met is a genuine JH

30 receptor (Wilson and Ashok, 1998). The possible reasons for the lack of an expected phenotype are: (1) JH has a weak effect on preadult Drosophila (Wilson and Ashok, 1998), (2) a paralogous of Met, germ-cell expressed (gce), exists in Drosophila. gce function as a JH receptor in the absence of Met (Abdou et al., 2011). To determine the clear role of Met, Met was studied in another insect model, T. castaneum, which only possess one Drosophila Met/gce gene. In this insect, silencing Met display a more clear effect. The loss of Met in early-instar larvae resulted in the production of premature pupae or heterochronic larva-pupa intermediates (Konopova and

Jindra, 2007). In addition, the knockdown of Met in the final larval instars disrupted the larval- pupal ecdysis and induced precocious development of adult structures (Parthasarathy et al.,

2008). In the true bug, Pyrrhocoris apterus, knockdown of Met also results in the similar phenotype as JH depletion, which causes precocious development of adult color pattern, wings and genitalia (Konopova et al., 2011). Met regulates premetamorphosis and metamorphosis in both holometabolous and hemimetabolous insects as in the JH signaling pathway.

JHs not only play important roles in insect metamorphosis, but also regulate the reproduction of insects. As a key factor in the JH signaling pathway, the function of Met in reproduction has also been determined. In the migratory locust, Locusta migratoria, silencing Met or Tai results in an arrest of ovarian development and a reduction in vitellogenin gene expression in the fat body

(Song et al., 2014). In D. punctata, the silencing of Met blocked basal oocyte development, suppressed the transcription of vitellogenin in the fat body and the uptake of vitellogenin by ovary. In addition, the typical profile of JH biosynthesis was disrupted in Met-knockdown animals, which results in the failure of patency (Marchal et al., 2014).

3.3 Krüpel-homologues 1 (Kr-h1) in downstream of JH signaling pathway

Kr-h1, a transcription factor with a DNA-binding motif of eight C2H2 zinc fingers, was shown to be the JH-inducible target of Met. In Drosophila, the expression of Kr-h1 is in JH-dependent manner and ectopic expression of Kr-h1 caused a phenotype similar to application of JH

(Minakuchi et al., 2008b). In a B. mori cell line, subnanomolar levels of natural JHs were able to induce BmKr-h1 rapidly (Kayukawa et al., 2012). This induction involves BmMet2 and BmSRC:

JH ligand to BmMet2 and interact with BmSRC to form a JH/BmMet2/BmSRC complex, which activates BmKr-h1 by interacting with a JH response element (kJHRE). The transcription of Kr- h1 demonstrates that the induction of BmKr-h1 by JH occurs only in the epidermis of penultimate-instars, but not in the prepupal stage (Kayukawa et al., 2014).

The role of Kr-h1 in conveying the JH signal to regulate metamorphosis has been studied in both hemimetabolous and holometabolous insects, including B. germanica (Lozano and Belles, 2011),

P. apterus (Konopova et al., 2011; Smykal et al., 2014b), R. prolixus (Konopova et al., 2011),

D. melanogaster (Minakuchi et al., 2008b), T. castaneum (Minakuchi et al., 2009) and B. mori (Kayukawa et al., 2014; Smykal et al., 2014b). Kr-h1 represses the adult morphogenesis. Knocking down Kr-h1 in the third instar or penultimate-instar larvae resulted in precocious adult development. The repressing action of Kr-h1 on morphogenesis involves the expression of the broad gene in holometabolous insects (Minakuchi et al., 2009), but not in hemimetabolous insects (Konopova et al., 2011; Smykal et al., 2014b). Even though the absence of Kr-h1 is necessary for adult morphogenesis, it is not required to maintain the larval program during the first two larval instars. The early stages of insect development appear to be initially independent of JH (Smykal et al., 2014b).

Aside from the function of Kr-h1 in metamorphosis, its roles in reproduction were also examined.

In the migratory locust, L. migratoria, Kr-h1 was demonstrated to convey the JH signal for the

32 induction of Vg. Depletion of Kr-h1 results in a drastic reduction in Vg expression in the fat body, and subsequently resulted in unsuccessful egg production (Song et al., 2014). However, in

P. apterus, knockdown of Kr-h1 did not block ovarian development or suppress Vg expression in the fat body(Smykal et al., 2014a). A similar result was also observed in

T. castaneum (Parthasarathy et al., 2010). Depletion of JHAMT or Met caused a significant reduction in Vg mRNA level, whereas the knockdown of Kr-h1 only caused a 30% reduction.

The downstream signal of JH appears to vary among stages and species.

4. Diploptera punctata

The Pacific beetle cockroach, D. punctata, has proven to be a valuable model insect in the study of the dynamics of regulation of juvenile hormone (JH) biosynthesis and metabolism, particularly during late nymph development and reproduction, as a consequence of several other unique physiological attributes: 1) strikingly high rates of JH biosynthesis compared to other insects, 2) maintenance of constant in vitro rates of JH biosynthesis, 3) precise and predictable reproductive events correlated with rates of JH production, 4) uniformity among colony members of the same age. In addition, Diploptera are easy to rear and handle in the laboratory, their CA are easily excised and the animals demonstrate high survival rates following surgical manipulations (Roth and Stay, 1961; Stay, 1999; Tobe and Stay, 1977). Thus, I chose D. punctata as my study model. In this section, the life cycle and the endocrine control of development and reproduction of D. punctata will be reviewed.

4.1 Nymphal development

On average, female D. punctata have four nymphal stages, whereas males have three;, however, the number of nymphal molts can vary depending on conditions (Holbrook and Schal, 1998,

2004). Based on observations of our laboratory colony of D. punctata, stadium durations of females are 13, 14, 16, and 21 days for the first, second, penultimate and final stages, respectively. Diploptera exhibit sexual dimorphism, and as adults, males are smaller and more slender than females. In contrast to 64 days of female nymphal life, males generally undergo imaginal ecdysis sooner, within approximately 50 days, with the first, penultimate and final stadia lasting 13, 16, and 22 days, respectively (Yagi et al., 1991).

During the growth and development of Diploptera, JH and ecdysteroids play critical roles in determining nymphal or imaginal developmental pathways. The circulating titer of JH and ecdysteroids in each stage are shown in Fig. 1.3A (Kikukawa and Tobe, 1986b). In the early nymphal stadia, higher rates of JH production are found in the latter half of the stadium. In final instars, JH release declines to undetectable rates after day 10, as hemolymph ecdysteroid titer starts to rise (Kikukawa and Tobe, 1986a, b). The decline in JH titer and the increase in circulating ecdysteroids in the final instars allow imaginal ecdysis to occur. During the first 8 days of the penultimate 3rd stadium, JH is critical for maintaining nymphal characters at the next molt. At this time, reduction in the JH titer results in precocious adult metamorphosis at the next ecdysis (Kikukawa and Tobe, 1986a). Furthermore, JH and ecdysteroids are critical to regulate the normal duration of the stadium (Kikukawa, 1989; Kikukawa and Tobe, 1986b). In final instars, ecdysteroid titer becomes elevated only after JH release declines. How JH interacts with ecdysteroids to regulate the development of D. punctata was investigated by the application of

JH analog prior to day 10 of last instar females. This resulted in the desynchronization of ecdysteroid release (Kikukawa, 1989; Kikukawa and Tobe, 1986b; Tobe et al., 1985), possibly because high JH titer blocks the release or synthesis of ecdysteroids. However, allatectomy on the first day of the last stadium resulted in a prolongation of low levels of ecdysteroids and the

34 low titer of JH did not promote the release of ecdysteroids (Kikukawa and Tobe, 1986b). Further investigation is needed to determine the interaction between JH and ecdysteroids in D. punctata.

4.2 Reproduction

After emergence, adult females mate immediately while still teneral, whereas adult males must be about a week old to successfully court and mate (Stay and Roth, 1958; Woodhead, 1986).

Mating and spermatophore transfer stimulate CA activity. Subsequently, rates of JH biosynthesis and oocyte growth increase rapidly (Roth and Stay, 1961; Ruegg et al., 1983; Stay and Tobe,

1977). By day 2 of the gonadotrophic cycle vitellogenesis commences (Fig. 1.3B) as Vg, the precursor of vitellin (Vn), becomes detectable in the hemolymph and fat body. At this time, oocytes reach about 0.8mm in length and begin to accumulate Vn (Mundall et al., 1981).

Elevated JH titer stimulates this synthesis and uptake, as the denervation of virgin female CA, or the implantation of mated female CA with ovaries into males, similarly results in Vg synthesis and uptake by the oocytes (Mundall et al., 1979; Stay and Tobe, 1977). As rates of JH biosynthesis rise, CA volume and cell number also increase in parallel, reaching a peak around day 4 to 5 (Szibbo and Tobe, 1981).

Figure 1.3. Dynamics of JH metabolism throughout the life cycle of D. punctata. (A) JH release during the development of female nymphs (as measured in vitro). Dashed lines indicate ecdysteroid titer in the penultimate and final instars. JH critical periods in the penultimate and final instar are indicated using bold lines. (B) JH release during the first gonadotrophic cycle of mated adult females. The inset indicates growth of the oocytes during development. (C) JH release by embryonic CA during development in the brood sac. MF release is indicated by a dashed line. Data based on (Kikukawa and Tobe, 1986b; Stay et al., 2002; Tobe et al., 1985)

As oocyte growth and Vn accumulation continues, rates of JH biosynthesis begin to decline around day 5. When oocytes reach approximately 1.6mm in length, the accumulation of Vn slows, the spaces between the follicle cells close and the cells begin to deposit the chorion (Stay et al., 1984; Woodhead et al., 2003). Hemolymph Vg titer also declines at this time (Mundall et al., 1981). As the oocytes approach their maximal length, 20-hydroxyecdysone becomes detectable in the oocytes, and levels rise during chorion formation (Stay et al., 1984). Ecdysone remains in the ovary where ecdysteroid conjugates are likely stored for use by the embryos during development (Stay unpublished). By day 7, the rate of JH production has declined to day

1 levels (Fig. 1.3B), choriogenesis is complete, and ovulation occurs. As the basal oocytes leave the common oviduct they are fertilized, pairs of fertilized eggs are then covered by a reduced oothecal membrane, and finally retracted into the brood sac where embryogenesis proceeds

(Roth, 1970; Woodhead et al., 2003).

Unlike members of many other insect orders, cockroaches demonstrate a clear correlation between cycles of JH biosynthetic activity by the CA, and patterns of oocyte growth and vitellogenesis. Oocyte maturation, fat body vitellogenin (Vg) production and the uptake of Vg by the ovaries are JH-dependent processes in this group (Engelmann, 1979; Rankin and Stay, 1984;

Roth and Stay, 1961; Stay and Tobe, 1978), whereas in higher orders, such as the Diptera, endocrine regulation of oocyte development requires both JH and ecdysteroids and synthesis of

Vg occurs in both the fat body and ovary (see (Raikhel et al., 2005; Valle, 1993)).

4.3 Embryo development

Embryos, usually 12 to 14, develop within the brood sac for approximately 63 days, and grow from 1.5mm at oviposition to 6.5mm by parturition. Dorsal closure of the body wall and

37 completion of the gut occurs relatively early, around 12 days after oviposition, when the embryos are 1.6mm in length. At this time, the CA begin to migrate to their final dorsal position (Stay and

Coop, 1973). Early embryonic CA produce and release the JH precursor, MF, in the virtual absence of JH, from day 16 until day 26 when embryos reach 2.6mm (Fig. 1.3C). During this time, allatostatins (ASTs), a family of regulatory neuropeptides (see below) which inhibit JH production in all post-embryonic developmental stages, stimulate MF and JH biosynthesis (Stay et al., 2002). Surprisingly, this is similar to the function of these peptides in some crustaceans

(Kwok et al., 2005). The mechanism by which this occurs merits further study in Diploptera as these finding may have important implications for the evolutionary history of these peptides

(Hult et al., 2008).

Unlike ovoviviparous blaberids, D. punctata is truly viviparous; females provide nourishment to the embryos via nutritive milk, rich in carbohydrates and lipocalin-like proteins that is secreted in large quantities by glandular cells in the brood sac epithelium. Secretion begins shortly after dorsal closure. Once the gut is completely formed, the embryos start to drink the fluid and begin to increase dramatically in dry weight (Stay and Coop, 1973; Stay and Coop, 1974; Williford et al., 2004). Elevated levels of JH can disrupt milk production and trigger a decline in production, but the humoral factor which promotes competence for milk production remains unidentified

(Evans and Stay, 1989, 1995; Stay and Lin, 1981). Currently, 25 Milk protein cDNAs have been identified; these genes exist in multiple copies at several loci. Further study of this gene family is critical, and may have implications for the evolution of viviparity in insects (Williford et al.,

2004).

Several days before parturition, a second cycle of oocyte growth begins, and re-mating is not required as females store sperm after copulation (Mundall et al., 1981; Rankin and Stay, 1985).

As a consequence of viviparity Diploptera oviposit fewer oocytes, less frequently than do oviparous species. However, nymphs are more developed at birth and reach reproductive maturity more quickly, with fewer nymphal molts, than other blaberids (Roth, 1970; Stay and

Coop, 1973).

5 Regulation of JH titre

As a hormone which plays essential roles in insects, JH is tightly regulated to maintain the development, metamorphosis and reproduction of insects. The circulating JH titer in the hemolymph is determined not only by the activity of the CA, but also by other processes, such as enzymatic degradation, binding to carrier proteins and uptake by target organs. In this section, the factors involved in the regulation of JH titre in the CA will be summarized, mainly focusing on the regulation in D. punctata.

CA are connected with the corpora cardiaca (CC) via nervi corporis allati (NCA) I, and in turn, through the CC with the brain via the nervi corporis cardiaci (NCC) I and II to medial and lateral neurosecretory cells of the brain respectively (Tobe and Stay, 1985a). JH, which is produced in the CA, is regulated by both neural and humoral inputs, including neuropeptides, neurotransmitters, ecdysteroids and even JH itself.

5.1 Allatostatins

ASTs are neuropeptides originally described to inhibit JH biosynthesis rapidly and reversibly in insects. Their existence was originally hypothesized by Tobe and Stay (1980) based on the relationship between JH biosynthesis and oocyte length. Aside from their ability to inhibit JH biosynthesis, ASTs have also been demonstrated to suppress muscular activity, inhibit vitellogenesis and modulate the activity of certain midgut digestive enzymes (Stay and Tobe,

2007). In insects, ASTs were initially grouped into three families, YXFGL-amide-allatostatins

(FLGa/ASTs), W2W9-allatostatins (MIP/ASTs) and PISCF-allatostatins (PISCF/ASTs) (Coast and Schooley, 2011; Stay and Tobe, 2007). However, MIP/AST was originally characterized to be a myoinhibitory peptide family, and therefore, the regulation of JH biosynthesis will be discussed only in FLGa/ASTs and PISCF/ASTs (Coast and Schooley, 2011).

The FLGa/AST was first discovered from brain extracts of D. punctata (Pratt et al., 1989; Stay and Tobe, 2007; Woodhead et al., 1989). In D. punctata, thirteen AST peptides are encoded by a single neuropeptide precursor (Donly et al., 1993). Members of the FGLa family of ASTs share a core C-terminal pentapeptide region (Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-NH2) which is the main functional region needed for inhibition of JH biosynthesis (Pratt et al., 1991; Stay et al., 1991).

Studies have demonstrated the pleiotropic nature of these peptides and their wide distribution.

ASTs have been found in many cells of the central nervous system, gut and ovary in D. punctata

(Garside et al., 2002; Stay et al., 1994b; Woodhead et al., 2003). A well-documented role for

FLGa/AST is the inhibitory effect on JH biosynthesis by the CA (Pratt et al., 1989; Stay et al.,

1994a; Woodhead et al., 1994; Woodhead et al., 1989), but they can also function as potent inhibitors of muscle contraction in the gut (Lange et al., 1995; Stay et al., 1994b), stimulate carbohydrate-metabolizing enzymes in the midgut and inhibit Vg production and cardiac activity

(Fuse et al., 1999; Garside et al., 2002; Martin et al., 1996; Vilaplana et al., 1999; Yu et al.,

1995b). Physiological and molecular work in D. punctata has confirmed the inhibitory function of FGLa AST (for review see (Stay and Tobe, 2007)). This effect occurs through paracrine release of FLGa/AST within the CA from cells in the pars lateralis of the brain sending axons in the NCC II, to the CA (Stay et al., 1992). The expression of the FLGa/AST precursor in the whole brain is negatively correlated with JH biosynthesis by the CA (Garside et al., 2003).

Furthermore, a negative correlation was also observed between sensitivity of CA to ASTs and JH biosynthesis during the first gonadotrophic cycle of mated adult females. The sensitivity of the

CA to ASTs declines during vitellogenesis and increases again before choriogenesis occurs (Pratt et al., 1990; Stay et al., 1991; Unnithan and Feyereisen, 1995).

Putative FLGa/AST receptors were characterised using affinity labeling and radio-ligand binding assays in the CA and the brain (Cusson et al., 1991a, 1992a; Yu et al., 1995a). More recently, a putative AST receptor was cloned belonging to the G-protein coupled receptor family of mammalian galanin receptors. Its relative expression level shows a steep rise on day 6 in the CA and the brain of the adult female, after which the level declines again. This downregulation is consistent with the loss of sensitivity of CA to AST following ovulation (Pratt et al., 1990; Stay et al., 1991; Unnithan and Feyereisen, 1995). The peak on day 6 suggests that AST and its receptor may be responsible for the decline of JH biosynthesis at this time. Initial RNAi experiments showed that silencing of the putative AST receptor results in an increase in JH production (Lungchukiet et al., 2008a; Lungchukiet et al., 2008b). The signal transduction pathway downstream of the FLGa/AST receptor likely involves the 1,4,5-inositol triphosphate(IP3)/Diacylglycerol (DAG) pathway, as well as protein kinase C (PKC) (Rachinsky et al., 1994). The IP3/DAG pathway is initiated upon binding of a neuropeptide to a G protein bound to the membrane. Increased IP3 induces release of intracellular Ca2+, whereas DAG activates PKC. Activators of PKC (phorbol esters) are potent inhibitors of JH III biosynthesis in

D. punctata (Feyereisen and Farnsworth, 1987b). Steps early in the JH pathway are likely the target of AST, more specifically the export of citrate from the mitochondria or the cytosolic conversion to acetyl-CoA, which will then enter the mevalonate pathway (Sutherland and

Feyereisen, 1996; Wang et al., 1994). However, more work is needed to further elucidate the

41 signal transduction pathway and to clearly identify the link between the AST receptor and the exact target of this neuropeptide in JH biosynthesis.

The insect PISCF/AST was first identified in the tobacco hornworm, M. sexta (Kramer et al.,

1991). PISCF/AST is a 15 amino acid peptide with the sequence qIRYRQCYFNPISCF, with a blocked N-terminus (pGlu), a free C-terminus, and a disulphide bridge linking two Cys residues at positions 7 and 14. Using HPLC- MALDI-TOF, PISCF/AST was isolated and characterized in the female mosquito A. aegypti (Li et al., 2006). AePISCF/AST inhibits JH biosynthesis in dose- dependent manner, with maximal inhibition in the nanomolar range. This inhibitory effect can be rescued by farnesoic acid, indicating that the target of AePISCF/AST is located before the formation of farnesoic acid. PISCF/AST plays important roles in regulating JH biosynthesis in A. aegypti. The levels of AePISCF/AST in the brain correlated with the sensitivity of CA to AST and the activity of CA in JH biosynthesis (Li et al., 2006). However, in other insects, such as D. melanogaster, PISCF/ASTs did not appear to regulate JH biosynthesis, but rather acts as a myotropin (Price et al., 2002). In A. pisum and M. persicae, PISCF/AST shows a significant dose-dependent feeding suppression effect, resulting in mortality, reduced growth and fecundity

(Down et al., 2010; Matthews et al., 2010).

Two PISCF/AST receptor (PISCF/AstR) genes were identified in D. melanogaster using a reverse pharmacological approach. They are G-protein-coupled receptors, which are the insect homologs of the mammalian opioid/somatostatin receptors. Site-directed mutagenesis studies demonstrated that a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding (Kreienkamp et al., 2002). Two PISCF/AstR paralogs were also isolated in A. aegypti (Mayoral et al., 2010). The tissue distribution of the two PISCF/AstR shows that both genes are expressed highest in the abdominal ganglia, whereas the expression

42 differs in other tissues, such as Malpighian tubules. In Malpighian tubules, PISCF/AstRB displays high expression, whereas the transcript level of PISCF/AstRA is relatively low.

Developmental expression of PISCF/AstR mRNA also displays different patterns, which suggests that the pleiotropic effects of PISCF/AST in mosquitoes might be mediated by the different receptor paralogs (Mayoral et al., 2010). The PISCF/AstR identified in T. castaneum could be activated by TcAST and T. castaneum allatostatin double C (Trica-ASTCC) as well as

M. sexta PISCF/AST in a dose-dependent manner (Audsley et al., 2013). TcPISCF/AstR is widely distributed, with the highest transcript level in the head and the gut. Whole mount immunocytochemistry localised TcPISCF/AstR in the median and lateral neurosecretory cells of the brain, in the CC, throughout the ventral nerve cord and in midgut neurosecretory cells, but not in Malpighian tubules, indicating that TcPISCF/AstR is close to PISCF/AstRA in A. aegypti.

5.2 Allatotropin

Allatotropin (AT) was first purified from the extracts of heads of pharate adult M. sexta (Kataoka et al., 1989). This 13-residue peptide (GFKNVEMMTARGF-NH2) was shown to stimulate JH biosynthesis in the CA of adult female M. sexta, but not in the CA of larval M. sexta, the mealworm beetle Tenebrio molitor, the grasshopper S. nitens and the cockroach Periplaneta americana (Kataoka et al., 1989). Further studies demonstrated that Manse-AT could also be active on the CA in other adult insects, including the Lepidoptera Heliothis virescens (Teal,

2002), Spodoptera frugiperda (Oeh et al., 2000), and Lacanobia oleracea (Audsley et al., 1999).

In addition, Manse-AT was also found to stimulate JH biosynthesis in the larvae of L. oleracea

(Audsley et al., 2000), in Diptera Phormia regina (Tu et al., 2001) and in Orthoptera Romalea microptera (Li et al., 2005).

Much work has attempted to identify other ATs. A putative AT has been extracted from the subesophageal ganglion of male cricket G. bimaculatus. The extract was able to stimulate JH biosynthesis in CA from G. bimaculatus and another cricket, Acheta domesticus (Lorenz and

Hoffmann, 1995). Extracts of the brain-subesophageal ganglion-CC-CA from P. apterus, also display activity in the stimulation of JH biosynthesis (Hodkova et al., 1996). A methanolic extract of the suboesophageal ganglia (SOG)-CC of the Mythimna loreyi virgin males stimulates the synthesis of JH III acid and iso-JH II, whereas synthetic Manse-AT had no significant effect

(Kou and Chen, 2000). Unfortunately, ATs in G. bimaculatus or P. apterus have not been isolated and identified. Another AT was isolated and identified from the abdominal ganglia of the mosquito A. aegypti (Veenstra and Costes, 1999). The sequence of this peptide was determined to be APFRNSEMMTARGF-NH2. The cDNA clone encoding this novel neuropeptide was shown to encode a single copy of this peptide. AT in A. aegypti (refer as AaAT) has a stimulatory effect on the JH biosynthesis of adult female CA, and it appears that the stimulation results from the increased ability of CA to convert FA to JH III induced by AaAT (Li et al., 2003). AaAT was also shown to induce JH biosynthesis in CA from newly emerged females (Li et al., 2003).

In M. sexta, Manse-AT was shown to stimulate JH biosynthesis in the CA of adult females, but not in larval or pupal CA of M. sexta. However, Manse-AT mRNA and immunoreactivity showed the expression of Manse-AT not only in the nervous system of adults but also in that of larvae, indicating that Manse-AT may play multiple roles in insects (Bhatt and Horodyski, 1999).

Later studies showed that in addition to regulating JH biosynthesis by the CA, AT are multifunction in different insects, including the stimulation of myoactivity (Bhatt and Horodyski,

1999; Paemen et al., 1991; Rudwall et al., 2000), inhibition of midgut ion transport (Lee et al.,

1998), stimulation of foregut movement (Duve et al., 2000; Duve et al., 1999), and cardioacceleratory effects (Koladich et al., 2002).

5.3 Neurotransmitters regulating JH biosynthesis

Several neurotransmitters are involved in the regulation of JH biosynthesis in insects.

Octopamine inhibits JH biosynthesis in the CA of D. punctata in vitro (Thompson et al., 1990).

In B. germanica, dopamine can have a stimulatory or inhibitory effect on the CA depending on the stage of the ovarian cycle (Pastor et al., 1991). In M. sexta, dopamine stimulates JH biosynthesis in CA from the first 2 days of the last larval stadium, but inhibits in CA from larvae in the beginning of the prepupal period. The stimulatory or inhibitory effect of dopamine is related to the the adenylyl cyclase system of CA (Granger et al., 1996). Ontogenetic differences in the control of JH biosynthesis by dopamine have also been demonstrated in Drosophila females in inhibiting or stimulating JH degradation (Gruntenko et al., 2005). The excitatory neurotransmitter L-glutamate, which acts through ionotropic receptors to raise intracellular Ca2+ concentration, stimulates JH biosynthesis in the CA of D. punctata through the action of NMDA-, kainate- and/or quisqualate-sensitive subtypes of ionotropic L-glutamate receptors (Chiang et al.,

2002b; Pszczolkowski et al., 1999).

5.4 Second messengers

The signal transduction pathways for neuropeptides and neurotransmitters usually involve intracellular reaction cascades regulating levels of second messengers. The effect on JH biosynthesis has been measured for several of these messengers.

Calcium is considered unique as a second messenger in the regulation of JH biosynthesis in D. punctata, since intracellular Ca2+ can function as a second messenger on its own, and, at the same

45 time, Ca2+ is essential for the function of cyclic nucleotides and the IP3/DAG second messenger system. Incubation of CA in medium lacking Ca2+, and blockage of non-specific Ca2+ channels, inhibits JH release. Because no buildup of JH or MF occurs in the CA as a consequence of such blockage, it seems likely that Ca2+ affects overall JH biosynthesis, instead of release (Kikukawa et al., 1987). JH biosynthesis rises as Ca2+ concentrations are increased in vitro. Moreover, elevated levels of extracellular Ca2+ can counteract the effect of brain extracts (containing AST) on JH biosynthesis (Aucoin et al., 1987). Two types of Ca2+ channels have been proposed for this action: 1) Ca2+ enters the CA cells through voltage-gated Ca2+ channels ((McQuiston et al., 1990;

Thompson and Tobe, 1986), as reviewed by (Rachinsky and Tobe, 1996)) or 2) there is a glutamate-induced Ca2+ influx via an ionotropic L-glutamate receptor, later reported to be the

NMDA receptor (Chiang et al., 2002a; Chiang et al., 2002b; Pszczolkowski et al., 1999). Release of Ca2+ from intracellular stores also appears to stimulate JH biosynthesis in vitro. The Ca2+-

ATPase inhibitor thapsigargin was shown to increase intracellular Ca2+ and induce JH biosynthesis (Rachinsky and Tobe, 1996; Rachinsky et al., 1994).

Compounds that increase intracellular cAMP concentrations were found to inhibit JH biosynthesis in an in vitro assay (Meller et al., 1985). The addition of brain extracts showed a dose-dependent elevation of cAMP levels (Aucoin et al., 1987). Together, these results suggest that cAMP could act as a second messenger for AST from the brain. A later study by Cusson et al. (Cusson et al., 1992b) measured levels of cGMP and cAMP in virgin and mated females following addition of AST. The mechanism of AST inhibition does not involve either cGMP or cAMP, suggesting that cyclic nucleotides are likely second messengers of another inhibitory signal affecting JH biosynthesis. A possible candidate is the neurotransmitter octopamine, which

46 in high concentrations can induce a rise in the level of cAMP in vitro (see above and (Thompson et al., 1990)).

5.5 Ovarian factor

The normal pattern of JH biosynthesis in the CA of D. punctata during the first gonadotrophic cycle of mated females requires the presence of the ovary (Rankin and Stay, 1984; Stay and

Tobe, 1978; Stay et al., 1983). Ovariectomy of females soon after the adult molt resulted in a low level of JH biosynthesis throughout the first gonadotrophic cycle (Stay and Tobe, 1978). The cyclic pattern in JH biosynthesis could be rescued following implantation into females of at least one-half an ovary from a day 0 mated female (Stay et al., 1983). Similarly, implantation of ovarioles with vitellogenic basal oocytes into male animals with denervated CA also resulted in an increase in JH biosynthesis (Hass et al., 2003; Rankin and Stay, 1984).

The effect of the ovary on JH biosynthesis appears to be stage-dependent. In a study by

Sutherland et al. (2000), young ovaries were found to have a dual role: stimulation of JH biosynthesis and repression of the mRNA levels of CYP4C7, a cytochrome P450 enzyme coding a gene involved in JH catabolism. These roles were reversed in post-vitellogenic ovaries. Rankin and Stay (1984) have shown that the size of the basal oocyte is a good indicator of the degree with which the ovary can stimulate JH biosynthesis. The ovary acquires this ability at the start of vitellogenesis but loses it post-vitellogenesis.

Currently, the exact nature of the ovarian factors involved in this modulation of JH production remains unknown. A study by Unnithan et al. (1998) suggested that a factor produced by ovaries in all stages can induce stable stimulation of JH synthesis. The effect of this ovarian factor is antagonized by a factor from the brain, distinct from ASTs (Unnithan et al., 1998). In contrast, a

47 study by Elliott et al. (Elliott et al., 2006) provided evidence for a different stage-specific peptidergic factor from the ovary that stimulates JH production. This ovarian factor is produced when basal oocyte lengths range from 0.76 to 1.15 mm (day 2 and 3 adult females). Ovaries with oocytes that are larger were no longer stimulatory in in vitro assays. This factor is considered the same as that described earlier by Rankin and Stay (1984), but to date, the exact peptidergic structure remains unknown.

An inhibitory peptidergic factor was reported from post-vitellogenic ovaries, which induced a rise in the cGMP content of CA in vitro (Chang et al., 2005). Elevated cGMP signaling was suggested to cause a long-term arrest on JH biosynthesis indirectly, through atrophy of the CA cells, reduction in protein content and depletion of the cellular machinery. This factor is thought to be the same post-vitellogenic ovary factor described by Sutherland et al. (2000) controlling the expression of CYP4C7. However, this peptide is not likely to be AST since the quantities of AST detected in the hemolymph are too low to exert a physiological effect on the CA (Stay et al.,

1994a). ASTs in the ovary probably act locally by facilitating ovulation or by preventing glycosylation of Vg and its release from the fat body (Garside et al., 2002; Rankin and Stay,

1984; Woodhead et al., 2003).

5.6 JH feedback

Negative feedback regulation of an endocrine gland, induced by its own product is a well-known endocrine control mechanism. A direct or indirect suppression of JH biosynthesis by the CA was first shown in a study assessing the effect of a JH analog. Topical application of this analog suppressed JH biosynthesis in a dose dependent fashion as did topical treatment with JH itself

(Tobe and Stay, 1979). A subsequent study suggested an indirect feedback of JH on the CA

48 through the brain, by comparing the effect of JH analog on males with CA connected or disconnected with the brain (Stay et al., 1994a). In both cases, the rates of JH biosynthesis were reduced compared to controls, intact CA more so than denervated ones. However, in animals in which the CA were disconnected from the brain, AST levels in the hemolymph were greatly elevated. These results indicate that the exogenous JH analog acts on the brain to inhibit JH biosynthesis, by paracrine release of AST in the intact CA and through the hemolymph in the disconnected CA (Ruegg et al., 1983; Stay et al., 1994a).

In vertebrates, the bulk end-product of mevalonate pathway, cholesterol, has a feedback regulation on the transcription and post-transcription of enzymes in the mevalonate pathway, which in turn mediates the production of cholesterol (Chang and Limanek, 1980; Clarke et al.,

1987; Goldstein and Brown, 1990). The addition of farnesol to cultured cells accelerated the degradation of HMGR, while the addition of mevalonate raised the activity of FPPP expressed in

CHO cells (Meigs et al., 1996; Meigs and Simoni, 1997). In insects, the accumulation of JH precursor or depletion of one enzyme in the pathway also results in a change in the activity of other enzymes. In CA with low FALD activity, farnesal accumulated and was converted back to farnesol that leaks from the CA, to balance the enzyme activity and the JH precursor pool

(Rivera-Perez et al., 2013). The expression and activity of enzymes in the JH biosynthetic pathway appears to be involved in JH feedback regulation of JH titre, since the rate of JH synthesis is regulated by the rate of flux of isoprenoids and the expression of genes in the pathway (Details seen Section 1.2). However, further investigation is required to prove this hypothesis.

5.7 Ecdysteroids

Injection of 20-hydroxyecdysone (20E) reduced oocyte growth and Vn content of basal oocytes

(Friedel et al., 1980) and inhibited JH biosynthesis in D. punctata (Stay et al., 1980). However, no inhibitory effect was observed following treatment of CA with 20E in vitro (Paulson and

Stay, 1987). In M. sexta, incubation of brain-CC-CA complexes with 20E resulted in a reduction in the production of JH acid (Granger and Janzen, 1987). The inhibitory effect of 20E in JH biosynthesis may be related to dopamine: 20E down regulates dopamine, which is responsible for the stimulation of JH biosynthesis (Granger et al., 1996). On the other hand, in D. punctata, a significant rise in DNA synthesis in CA from male adults treated with 20E suggested that ecdysteroids may affect JH biosynthesis by regulating proliferation of CA cells (Chiang et al.,

1995; Tsai et al., 1995). However, this conflicts with the observed 20E-induced decrease in CA cell number of ovariectomized D. punctata females (Tobe et al., 1984). The pathway through which ecdysteroids inhibit JH production by the CA is unclear.

6 Rationale and objectives of my study

The juvenile hormones are essential in regulating insect growth, development, metamorphosis, aging, caste differentiation and reproduction. The important roles of JH in insect development and reproduction and its value in pest control have led to multiple studies on the biosynthesis, regulation and function of JH. Among cockroach families, endocrine regulatory mechanisms are easier to discern in members of the Blaberidae. In these species, only the basal oocyte of each ovariole undergoes vitellogenesis during a given gonadotrophic cycle, and a long period of ovarian quiescence occurs while the oviposited fertilized eggs develop in the brood sac of the female (Roth, 1970). Among these species, D. punctata (Dictyoptera; Blattaria; Blaberidae), the only known truly viviparous species in that the brood sac provides nourishment to the embryos

(Roth, 1970), has become a model system for studies on reproduction and the regulation of JH production.

Recently, the JH biosynthetic pathway has been established in insects such as B. mori (Kinjoh et al., 2007), A. aegypti (Nouzova et al., 2011) and A. mellifera (Bomtorin et al., 2014). However, there are fewer studies on the JH biosynthetic pathway in D. punctata. Thus, in Chapter 2 of this thesis, the genes involved in the JH biosynthetic pathway were identified and characterized. This is the first report of the characterization of the JH biosynthetic pathway in hemimetabolous insects. The function of JH as a regulator of female cockroach reproduction, and the role of JH biosynthetic enzymes in regulating JH biosynthesis were established. In particular, our research revealed an interaction between the transcriptions of genes in the biosynthetic pathway i.e. a feedback mechanism is involved in the regulation of the expression of enzymes in the JH biosynthetic pathway.

In terms of the regulation of JH biosynthesis, ASTs have been demonstrated to be one of the essential inhibitory factors, particularly for D. punctata, in which no AT or other allatoregulatory neuropeptides have been discovered to date. Although the function of AST in the regulation of

JH has been well-studied, the mode of action of AST remains unclear. In Chapter 3, the mode of action of AST was studied by determining the signaling pathway in AST action, and the target of

AST action. DpAstR was expressed in multiple vertebrate cell lines, and the activation activities of the 13 ASTs were determined. Furthermore, my study showed that the activation of AstR results in the elevation of intracellular calcium and cAMP, which indicates that Ca2+ and cAMP can server as second messenger in the signaling pathway of AST action. In addition, the target of

AST action was studied using RNAi and the measurement of JH in the presence or absence of JH

51 precursors. Our result indicates that AST probably affects JH biosynthesis prior to the entry of

Acetyl-CoA into the JH biosynthetic pathway.

As a second messenger, Ca2+ has been demonstrated to modulate/regulate JH biosynthesis in D. punctata. NMDAR is an ionotropic receptor with a high Ca2+ permeability, and its activators glutamate and NMDA have been shown to stimulate JH biosynthesis, probably by activating the

NMDAR channel. Previous studies also showed that NMDA stimulates JH biosynthesis in vitro in the D. punctata (Chiang et al., 2002a). Thus, it was of interest to determine the function of

NMDAR in JH biosynthesis and reproduction. In chapter 4, the genes encoding NMDAR were identified in D. punctata and characterized in this thesis. The function of NMDAR in reproduction was also examined by knocking down the genes encoding NMDAR subunit 2, and by the application of NMDAR antagonist MK-801. However, neither JH biosynthesis nor oocyte growth was affected, suggesting that NMDAR does not play important roles in the regulation of

JH biosynthesis or reproduction in female D. punctata.

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Chapter 2

Characterization of the Juvenile Hormone pathway in the viviparous cockroach, Diploptera punctata

This chapter is an adapted reprint of my article: Juan Huang, Elisabeth Marchal, Ekaterina F. Hult, and Stephen S. Tobe, Characterization of the

Juvenile Hormone pathway in the viviparous cockroach, Diploptera punctata. PloS one, accepted.

Authors’ contribution:

E. Marchal performed part of the sequencing of the JH biosynthetic genes, and the tissue distribution and expression profiles. I performed the RNAi and RCA. The paper was written by

E. Marchal and me with editing by E.F. Hult and S.S. Tobe

Summary

Juvenile hormones (JHs) are key regulators of insect development and reproduction. The JH biosynthetic pathway is known to involve 13 discrete enzymatic steps. In the present study, we have characterized the JH biosynthetic pathway in the cockroach Diploptera punctata. The effect of exogenous JH precursors on JH biosynthesis was also determined. Based on sequence similarity, orthologs for the genes directly involved in the pathway were cloned, and their spatial and temporal transcript profiles were determined. The effect of shutting down the JH pathway in adult female cockroaches was studied by knocking down genes encoding HMG-CoA reductase

(HMGR) and Juvenile hormone acid methyltransferase (JHAMT). As a result, oocyte development slowed as a consequence of reduction in JH biosynthesis. Oocyte length, fat body transcription of Vg and ovarian vitellin content significantly decreased. In addition, silencing

HMGR and JHAMT resulted in a decrease in the transcript levels of other genes in the pathway.

Introduction

Juvenile hormones (JHs) play key roles in regulating growth, development, metamorphosis, aging, caste differentiation and reproduction in insects (as reviewed by Goodman and Cusson,

2012; Hartfelder, 2000). The multiple processes in which JH is involved and the critical role which JH plays in metamorphosis and reproduction emphasize the importance of elucidating the

JH biosynthetic pathway and the factors that regulate its biosynthesis.

JHs are sesquiterpenoid compounds that are synthesized and secreted by specialized, paired endocrine glands, the corpora allata (CA). The complete biosynthetic pathway of JH III (the most widespread and predominant JH homologue in insects) comprises 13 discrete enzymatic steps.

This pathway can be divided into two metabolic parts (Fig. 2.1): the early portion comprises the

70 mevalonate pathway to the formation of farnesyl diphosphate (FPP) and is conserved in both vertebrates and invertebrates; the later part of the pathway is specific to insects and other arthropods. In this later part, FPP is cleaved to farnesol, which is then oxidized to the carboxylic acid (farnesoic acid; FA), followed by methyl esterification, epoxidation and formation of JH

(Belles et al., 2005). The order in which these two final steps in JH biosynthesis occurs, appears to be insect order dependent. In orthopteran, coleopteran, dipteran and dictyopteran insects, FA is first methylated to MF, which in turn undergoes a C10, C11 epoxidation to the functional JH.

In Lepidoptera, however, the opposite situation prevails: epoxidation precedes methylation

(Defelipe et al., 2011; Marchal et al., 2011; Shinoda and Itoyama, 2003).

Recent studies have reported on the molecular elucidation of the JH pathway in several holometabolous insects such as the silkworm Bombyx mori, the mosquito, Aedes aegypti and the honeybee, Apis mellifera. In B. mori, all genes encoding enzymes involved in the mevalonate pathway (Kinjoh et al., 2007) and the isoprenoid branch of JH biosynthesis (Cheng et al., 2014;

Shinoda and Itoyama, 2003) have been isolated. Each enzyme in the mevalonate pathway is encoded by a single gene, except farnesyl diphosphate synthase (FPPS), which comprises three homologs. The genes encoding enzymes in the isoprenoid branch of JH biosynthesis, however, underwent gene duplication to create multiple copies. The transcripts for most JH enzymes are highly enriched or exclusively expressed in the CA (Ueda et al., 2009). The expression pattern of the genes encoding these enzymes in the CA correlates well with rates of JH biosynthesis

(Kinjoh et al., 2007). In A. aegypti, changes in the transcription of 11 of the enzymes are responsible in part for the dynamic changes in JH biosynthesis (Nouzova et al., 2011). The expression of genes in the JH biosynthetic pathway was also determined in female castes of A. mellifera and was found to correlate with the JH hemolymph titre in adult worker bees, but, not

Acetyl-CoA

Acetoacetyl-CoA thiolase (1- Thiol)

Acetoacetyl-CoA HMG-CoA synthase (2- HMGS) HMG-CoA HMG-CoA reductase (3- HMGR) Mevalonic acid Mevalonate kinase (4- MK) Phosphomevalonate Phosphomevalonate kinase (5- PMK)

Diphosphomevalonate

Diphosphomevalonate decarboxylase (6- PPMD) Isopentenyl diphosphate Dimethylallyl diphosphate Isophentenyl diphosphate isomerase (7- IPPI)

Farnesyl diphosphate synthase (8- FPPS)

Geranyl diphosphate Farnesyl diphosphate synthase (8- FPPS) Farnesyl diphosphate Farnesyl diphosphate pyrophosphatase (9- FPPP) Farnesol Farnesol dehydrogenase (10 – FOLD)

Farnesal Farnesal dehydrogenase (11 – FALD) Farnesoic acid Juvenile hormone acid methytransferase (12- JHAMT) Methyl farnesoate

Methyl farnesoate epoxidase (13- CYP15A1) Juvenile hormone III Fig. 2.1. Scheme of JH biosynthetic pathway (Adapted from Belles et al. (2005) and Nouzova et al. (2011)). The Insect- and Arthropod-specific pathway is represented in the dashed box. Precursors are in bold and connected by arrows. Enzymes are in italics. Abbreviations for the enzymes are given in brackets.

72 in larvae (Bomtorin et al., 2014). Until recently, no structural or molecular data were available on FPP pyrophosphatase (FPPP) or farnesal dehydrogenase (FALD). Current studies in the dipterans, the fly, Drosophila melanogaster and A. aegypti have characterized the gene encoding an FPP phosphatase (FPPP) (Cao et al., 2009; Nyati et al., 2013). Moreover, genes encoding an aldehyde dehydrogenase (FALD) have now been functionally characterized in A. aegypti

(Rivera-Perez et al., 2013).

Diploptera punctata, the only truly viviparous cockroach is a well-known model system in the study of JH biosynthesis and its regulation. The physiology of this animal is characterized by very stable and high rates of JH biosynthesis and precise and predictable reproductive events that correlate well with rates of JH production (see review by Marchal et al. (2013a)). In adult females, JH regulates oocyte maturation, fat body vitellogenin (Vg) production and the uptake of

Vg by the developing oocytes (Rankin and Stay, 1984; Stay and Tobe, 1978). Aside from the original molecular identification of CYP15A1 (Helvig et al., 2004), the gene encoding the epoxidase producing the functional JH, JH-related research in D. punctata has mainly focused on examining JH titre and physiological aspects of JH function (see Marchal et al. (2013a)).

An earlier study predicted 4 genes encoding enzymes in JH biosynthetic pathway in D. punctata based on sequence similarity with Drosophila and Anopheles gambiae genes (Noriega et al.,

2006). To further characterize the JH biosynthetic pathway in this important model system on a molecular level, we have confirmed and identified 11 out of 13 genes encoding the JH biosynthetic enzymes, and have also analyzed the tissue distribution and developmental transcript profile of these genes during the first gonadotropic cycle of the female cockroach. The predominant expression of these genes in the CA and the correlation between their transcript levels and the rates of JH biosynthesis suggests that the 11 genes cloned in our study are

73 encoding functional enzymes. In addition, the transcript levels of the 11 genes indicate that the expression of these genes partly regulate JH biosynthesis. Addition of exogenous JH precursors was able to stimulate JH biosynthesis in CA with low activity suggesting that JH production was also regulated by flux of substrates in the pathway.

We also used RNA interference (RNAi) to study the effect of silencing genes in the pathway on rates of JH biosynthesis and ovarian development. HMGR and JHAMT were chosen as the target genes for the RNAi, because both are well-studied genes whose expression patterns clearly correlate with rates of JH biosynthesis (Goodman and Cusson, 2012). Our results show that JH biosynthesis decreased and ovarian development slowed in HMGR-JHAMT dsRNA treated animals. Of particular interest is our discovery that manipulation of individual genes encoding the JH biosynthetic enzymes using dsRNA technology resulted in a significant decrease in the transcript levels of other genes, which indicates a feedback mechanism is involved in the regulation of the expression of enzymes in the JH biosynthetic pathway.

Materials and methods

Animals - The D. punctata colony was maintained at 27 °C in constant darkness and animals were fed lab chow and water at libitum. To obtain pools of synchronised animals, newly molted female adult cockroaches were picked from the colony, placed in separate containers and provided with water and lab chow. Mated status was confirmed by the presence of a spermatophore.

Tissue collection and RNA extraction - D. punctata were dissected in modified cockroach ringer solution (150 mM NaCl, 12 mM KCl, 10 mM CaCl2.2H2O, 3 mM MgCl2.6H2O, 10 mM HEPES,

40 mM glucose, pH 7.2) using a dissecting microscope. Samples were flash-frozen in liquid N2

74 to prevent RNA degradation and were stored at -80 ºC until further processing. For each dissected female, basal oocyte lengths were measured to determine the physiological age. CA samples were taken from day 0 to 7 of adult females for the developmental profiles of genes encoding enzymes in the JH biosynthetic pathway. For each time point, three biologically independent pools of 10 animals each were collected. To determine the tissue distribution of the genes of interest, the following tissues were dissected from 3 independent pools of 10 animals each: brain (Br), nerve cord (NC), corpora allata (CA), fat body (FB), midgut (MG), Malpighian tubules (MT), ovary (Ov) from females, and accessory gland (AG) and testes (Te) from males.

For the RNAi experiments (§2.6), 3 biologically independent pools comprising CA from 7 animals were collected. Pooled samples were homogenised with RNase-free pestles and total

RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. An additional DNase treatment (RNase-free DNase set, Qiagen) was performed to eliminate potential genomic DNA contamination. Because of the small size of the CA, RNA from this tissue was extracted using the RNAqueous®-Micro Kit (Ambion), followed by the recommended DNase step. Quality and concentration of the resulting RNA samples were measured using a Nanodrop spectrophotometer (Thermo Scientific.).

Sequencing genes involved in the JH biosynthetic pathway - Since no genome or full transcriptome sequence data are currently available for D. punctata, a first set of degenerate primers was developed based on a multiple sequence alignment of known orthologous sequences from different insects. Such an alignment was made for Acetoacetyl-CoA thiolase (Thiol),

HMG-CoA reductase (HMGR), Phosphomevalonate kinase (PK), Diphosphomevalonate decarboxylase (PPMD), Isopentenyl diphosphate isomerase (IPPI), Juvenile hormone acid methyltransferase (JHAMT) and the JH target vitellogenin (Vg). Degenerate primers are listed in

Supplementary table S1. For Methyl farnesoate epoxidase (CYP15A1) the full sequence was already characterised (GenBank accession number AY509244) (Helvig et al., 2004). For HMG-

CoA synthase (HMGS), Mevalonate kinase (MK), Farnesyl diphosphate synthase (FPPS) and

Farnesol dehydrogenase (FOLD), a partial sequence was found in the D. punctata CA EST database (Noriega et al., 2006). Their sequence was partially confirmed using gene-specific primers (Table S1). A temperature-gradient polymerase chain reaction (PCR) was run using Taq

DNA polymerase (Sigma-Aldrich Co.) with D. punctata day 4 CA cDNA as a template. Bands of the expected size were cut out and further purified using the GenEluteTM Gel extraction Kit

(Sigma-Aldrich Co.). The resulting DNA fragments were subcloned into a CloneJET™ cloning vector using the CloneJET™ PCR Cloning Kit (Fermentas) and sequenced. Sequences for PMK,

PPMD and JHAMT identified using degenerate primers, were too short to submit to NCBI’s

GenBank; therefore, RACE (Rapid Amplification of cDNA Ends) was performed. Their sequences were confirmed using primers listed in Table S1.

Radiochemical assay (RCA) - The in vitro radiochemical assay (RCA) for JH biosynthesis was performed (Feyereisen and Tobe, 1981; Tobe and Clarke, 1985). CA were incubated in TC199 medium for 3h, then transferred to new medium containing JH precursors for another 3h incubation. JH biosynthesis in both incubations was measured, and first incubation measurements were used as a control. JH precursors acetyl CoA, mevalonic acid (MA), diphosphomevalonate (DPPM) and farnesol (Sigma-Aldrich, Canada) were dissolved in water before use. cDNA synthesis and quantitative real-time PCR (q-RT-PCR) - cDNA was transcribed from an equal amount of RNA using the SuperScript™ III First-Strand Synthesis SuperMix for q-RT-

PCR in a final volume of 20 µl following the manufacturer’s instructions (Invitrogen Life

Technologies). All samples were reverse transcribed together in a single run. The resulting cDNA samples were diluted 10-fold with PCR grade water. A calibrator sample was prepared by pooling 5 µl of each cDNA sample. In the same run, negative control reactions were set up without reverse transcriptase enzyme to test for genomic DNA contamination. q-RT-PCR primers were designed using IDT’s (Integrated DNA Technologies) PrimeQuest design tool (http://eu.idtdna.com/PrimerQuest/Home/Index). Primer sets were subsequently validated by determining relative standard curves for each gene transcript using a five-fold serial dilution of the calibrator cDNA sample. Efficiency and correlation coefficients (R²) were determined for each primer pair. Primers used for q-RT-PCR profiling are listed in Table 2.1. q-RT-PCR reactions were carried out in triplicate in a total volume of 10 µl containing 5 µl of

IQ™ SYBR® Green Supermix (Bio-Rad), 1 µl forward and reverse primer (5 µM), 2 µl of MQ- water and 1 µl of cDNA. All reactions were performed using Bio-Rad’s CFX384 Touch ™ Real-

Time PCR Detection System using a two-step thermal cycling profile: 95ºC for 3 min, followed by 40 cycles of 95ºC for 10 s and 59ºC for 30 s. Upon completion of every run, a dissociation protocol (melt curve analysis) was performed to check for formation of primer dimers. A few representative PCR products were run on a 1.2% agarose gel containing GelRedTM (Biotium) and visualised under UV to confirm target specificity. Prior to target gene profiling, previously described housekeeping genes were tested for their stability in the designed tissue distribution, temporal profiling and RNAi experiments. The optimal housekeeping genes were selected using the geNorm and Normfinder software as described previously (Marchal et al., 2013b). For each tested cDNA sample, the normalization factor for the reference genes relative to the calibrator samples was calculated and used to determine the normalized expression levels of the target genes relative to the calibrator (Vandesompele et al., 2002).

Table 2.1. q-RT-PCR primer sequences and reaction efficiencies and correlation coefficients in the q-RT-PCR assay.

Correlation Efficiency (%) Gene name F primers R primers coefficient (R2)

Thiol 5’-TGCCTTCCAAAAGGAGAATG-3’ 5’- ACATCACCTGCCATCAACAC-3’ 90 0.97

HMGS 5’- TGCTGGGAAGTACACAGCAGG-3’ 5’- CTCCACGAGCTTGCTGACTG-3’ 83 0.994

HMGR 5’-TGGGAGCATGTTGTGAAAAT-3’ 5’-ACCAAGCAGCCCTCAGTAGT-3’ 95 0.988

MK 5’- TACGGCAAAACTGCCCTTGC-3’ 5’- AATGGAGGAGGTTCGGCGA-3’ 93 0.996

PMK 5’- TACGAAAACAACGAGGATGG-3’ 5’- TTCTGCATCATCTACACCTTCA-3’ 100 0.985

PPMD 5’- TGGAAGGTGACATAACAGCAA-3’ 5’- ATCCTTGATGCCAGTGAACA-3’ 90 0.967

IPPI 5’- CCTTCCCCAACCATGTAACT-3’ 5’- ACCAACGCCATTTGTCTCTT-3’ 100 0.992

FPPS 5’-TGCTTTGGAGATCCTGAGGT-3’ 5’- TGTTCAGGAGTGGTTCGTTG-3’ 96 0.987

FOLD 5’- TGGCGCGTAGGGTAGACAA-3’ 5’- GACCCATTTGAAAGCCTCCTTGA-3’ 93 0.993

JHAMT 5’- ATCCAGGTGCTGGAAGGAGAG-3’ 5’- CTGCCCAGAGTCGAACAGG-3’ 99 0.984

CYP15A1 5’- GTTGGGATCTCGGAGCATGG-3’ 5’- CGAACACGTCATGCATCGGT-3’ 100 0.992

Vg AAAGGTGTCCTCAGCCAGC TCCTCCATCTCGGATTGGGA 95 0.998

RNA interference (RNAi) - CA cDNA was used as a template to amplify fragments of genes to be used in dsRNA construction. These fragments were subcloned and sequenced as described above.

Two separate constructs were designed in different regions of the genes to eliminate off-target effects. Primers used are given in Table 2.2. dsRNA was synthesized using the MEGAscript

RNAi kit (Ambion). The procedure is based on the high-yield transcription reaction of a user- provided linear transcript with a T7 promoter sequence. Transcription was carried out at 37 °C overnight. The reaction mixture was treated with DNase I and RNase, and then purified by phase-solid phase adsorption purification, according to the manufacturer’s instructions (Ambion).

The dsRNA concentration was determined using a Nanodrop spectrophotometer (Thermo

Scientific.). Diluted dsRNA was run on a 1.2% agarose gel to examine integrity of the construct and efficiency of duplex formation. The negative control construct (-pJET) was designed in a non-coding region of the pJET 1.2/blunt cloning vector (CloneJet PCR Cloning Kit, Thermo

Scientific).

Several RNAi trials with different injection timings and dsRNA construct concentrations were investigated to obtain an efficient gene silencing (data not shown). Newly molted mated females were injected with 3 μg HMGR dsRNA or control dsRNA on day 0 and day 2, and with 3 μg

JHAMT dsRNA or control dsRNA on day 1 and day 3. CA, ovary and fat body samples were taken on day 4 as described above. Fat body was immediately stored in liquid N2 prior to RNA extraction. CA were dissected and cleaned in TC199 medium (GIBCO; 1.3 mM Ca2+, 2% Ficoll, methionine-free) for use in the RCA or flash frozen in liquid N2 to prevent RNA degradation.

Basal oocyte length was measured and the ovaries were collected for vitellogenin measurements

(§ 2.7) and histology (§ 2.8).

Table 2.2. Primers for dsRNA construction Name F Primer(5'-3') R Primer(5'-3') HMGR dsRNA ACATGGACAGTTCTGTGCCT CCCAACTTTTGCAGATGACAG CACTTCTCGCATTGTGGCT CAGTACCCTTGGAGAGC JHAMT dsRNA AAAAGAGACGCAGCCCACGCA CGATCCTCGTGGGAACAGATG GTACAGCACGCCACCTCCA AACTACGGCACTCTGGAGC Control dsRNA TTGCGCTCACTGCCAATTGC CTGGCCTTTTGCTCACATGTT *The T7 promoter sequence was added at the 5' end of each dsRNA primer.

Vitellogenin ELISA - Single dissected ovaries were homogenized and extracted twice in 50 µl

PBS according to Mundall et al. (1981). Total ovary protein content was determined using a

Bradford assay (Bradford, 1976) performed according to the manufacturer’s (Sigma-Aldrich) 96 well plate protocol.

Vitellin was quantified using an indirect enzyme-linked immunosorbent assay (ELISA) following the protocol described in Marchal et al. (2014). A rabbit polyclonal antibody made against D. punctata mature egg homogenate was used as the first antibody (Stoltzman and Stay,

1997). Goat anti-rabbit IgG, HRP-linked antibody (1:3000 in 1% BSA) was then added as the second antibody. Wells were treated with 100ul of TMB for 10 min. Absorbance was measured at 650 nm with a Molecular Devices SpectraMax Pus 384 microplate reader.

Histology and microscopy - To view overall structure, a subset of oocytes was fixed for 3 days in

3% glutaraldehyde in 0.1 M phosphate buffer, post-fixed for 1 hr with 1% OsO4 in 0.1 M phosphate buffer, dehydrated in ascending ethanol series, and embedded in Spurr’s resin.

Sections (1 µm) were cut with a Leica EM UC6 ultramicrotome, then mounted and stained with methylene blue and toluidine blue. All light microscopy was conducted using a Leica DMI3000 inverted microscope.

Results

Identification of genes encoding JH biosynthetic enzymes and Vg in D. punctata.

CYP15A1 was previously identified in D. punctata and the genes encoding HMGS, MK, FPPS and FOLD were present in a CA EST database of D. punctata (Helvig et al., 2004; Noriega et al.,

2006). We confirmed the sequence of HMGS, MK, FPPS and FOLD. For the remaining enzymes in the JH biosynthetic pathway, we aligned (predicted) orthologous sequences from different

81 insect orders and designed degenerate primers based on conserved domains. We succeeded in cloning an additional five (partial) sequences for enzymes in the conserved mevalonate pathway:

Thiol, HMGR, PMK, PPMD and IPPI and one extra sequence encoding an enzyme in the JH- specific part of the pathway, JHAMT. The complete sequences for HMGR and JHAMT were obtained. The ORFs of HMGR and JHAMT encode proteins of 825 and 274 amino acids in length, respectively. Amino acid alignments of these proteins with known insect orthologs are given in Supplementary Fig. S2.1. Moreover, a partial sequence of Vg was cloned from D. punctata adult female fat body. The sequences for the genes were deposited in NCBI’s GenBank with accession numbers shown in Table S2.1.

Tissue specificity and developmental transcript profiles during the first gonadotropic cycle are consistent with roles of these genes in JH biosynthesis.

Using q-RT-PCR, the relative transcript levels of 11 genes in the JH biosynthetic pathway were determined in several tissues of day 4 mated females and males. Relative transcript levels for the

Diploptera orthologs of genes in the JH pathway were normalized to transcript levels of the reference genes EF1a and Tubulin (Marchal et al., 2013b). To represent the tissue distribution data for the 11 enzyme-encoding genes in one figure, the transcript levels were normalized to the transcript level of PMK measured in the calibrator sample. The orthologous genes encoding enzymes in the JH biosynthetic pathway were most highly expressed in the CA, which is consistent with the functions of the enzymes (Fig. 2.2). Most genes appear to be exclusively expressed in the day 4 female CA: Thiol, HMGS, HMGR, MK, PPMD, FPPS, JHAMT and

CYP15A1; whereas a few show a broader tissue distribution: PMK, IPPI and FOLD. These latter

3 appear to be expressed not only in nervous tissues but also in peripheral tissues such as the ovary, fat body and Malpighian tubules. Moreover, there are 1000-fold differences in relative

Figure 2.2. Tissue specific expression of genes encoding JH biosynthetic enzymes. All tissues were dissected from day 4 mated adult females, except accessory glands (AG) and testes (Test) were from day 4 adult male. Abbreviations on the X-axis: Brain (Br), corpora allata (CA), nerve cord (NC), midgut (Mg), Malpighian tubules (MT), fat body (Fb), and ovary (Ov). Bars represent the mean of three biologically independent pools of ten animals run in triplicate and normalized to Tubulin and EF1α. Vertical error bars indicate SEM.

83 mRNA levels of the enzyme-encoding genes, with PMK, PPMD, IPPI and FOLD being present in low abundance, Thiol, HMGS and HMGR being of intermediate abundance and MK, FPPS,

JHAMT and CYP15A1 being represented in high abundance (Fig. S2.2).

Our next step was to follow the relative transcript levels in the CA of mated females throughout the first gonadotropic cycle. Target gene expression was normalized to transcript levels of EF1a and Armadillo according to a previous study (Marchal et al., 2013b). In general, the expression of the 11 genes is correlated with the in vitro rates of JH biosynthesis in the CA and relative mRNA levels for DippuVg measured in the female fat body (Fig. 2.3). The relative mRNA levels in the CA for most of the genes of interest were low at the beginning of the adult female stage when JH biosynthesis is low; rose during the vitellogenic portion of the first gonadotropic cycle reaching a peak on day 3-4 and thereafter began to decline on day 5, attaining a low level during oviposition on day 7. However, there appear to be three exceptions to this pattern: PMK, IPPI and FPPS. Relative mRNA quantities for PMK and IPPI were very low compared to other genes and did not display dramatic changes during the first gonadotropic cycle. FPPS transcripts were high on day 0, remained high until day 4 and then declined on day 5 when vitellogenesis slows.

Addition of JH precursors stimulates JH biosynthesis in CA with low JH biosynthetic activity in vitro.

Previous studies suggested that JH synthesis is controlled by the rate of flux of isoprenoids in A. aegypti (Nouzova et al., 2011). To determine the role of other JH precursors in regulating JH biosynthesis, we tested the rate of JH biosynthesis with the addition of JH precursors in the early steps of mevalonate pathway or the addition of farnesol. The addition of acetyl CoA, DPPM or farnesol to the incubation medium had a significant stimulatory effect on JH biosynthesis,

Figure 2.3. Developmental expression of genes encoding JH biosynthetic enzymes during the first gonadotrophic cycle of D. punctata. Measurements were taken every day during the cycle (day 0 to day 7 after the final molt). Bars represent the mean of three biologically independent pools of ten animals run in triplicate and normalized to Armadillo and EF1α. Vertical error bars indicate SEM. Inset at the right bottom shows JH biosynthesis per individual CA (n=12) and the transcript level of DpVg in fat body during the first gonadotrophic cycle (Marchal et al., 2014). Vertical error bars indicate SEM.

85 whereas MA did not (Fig. 2.4A). For the first time, we demonstrated that acetyl CoA, as the first precursor in the JH biosynthetic pathway, was able to stimulate JH biosynthesis. The rank order of the stimulatory effects of the different JH precursors on JH biosynthesis is as follows: farnesol > acetyl CoA > DMMP > MA.

We also evaluated the sensitivity of CA to farnesol during the first gonadotrophic cycle. CA were dissected from day 0, 3, 4, 5, and 7, and incubated with medium containing 40 µM farnesol.

On days 3 and 4, when the CA show high JH biosynthetic activity, the addition of farnesol had no effect on in vitro JH biosynthesis (Fig. 2.4B). On the other hand, JH biosynthesis in CA of day 0, 5 and 7 mated females was significantly increased.

Injection of HMGR-JHAMT dsRNA resulted in a significant downregulation of the target genes but also of other genes in the JH biosynthetic pathway.

A systemic RNAi response was observed following injection of HMGR- and JHAMT dsRNA into animals every other day during the first gonadotropic cycle. Relative transcript levels were measured in the CA of day 4 animals using q-RT-PCR. A significant knockdown of 64% and 94% was measured for HMGR and JHAMT, respectively. Off-target effects were investigated by checking the Ct value of housekeeping genes . Moreover, two different dsRNA constructs were used to eliminate off-target effects (primers listed in Table 2). Both constructs yielded a similar phenotype. q-RT-PCR was used to determine the relative mRNA levels of the other genes involved in the JH biosynthetic pathway using the control and treated CA described above. A significant reduction in relative expression levels was observed for all genes encoding the enzymes directly involved in the pathway, with the exception of the genes encoding FOLD and the epoxidase, CYP15A1

(Fig. 2.5).

Figure 2.4. The effect of JH precursors on JH biosynthesis by CA from mated female D. punctata. JH biosynthesis was determined in CA that were first incubated in medium TC199 (control), and then in medium with JH precursor (treatment). (A) JH precursors stimulate JH biosynthesis by CA from day 7 mated female cockroach, D. punctata. 100µM of JH precursor was added to the medium during the second incubation. (B) The sensitivity of CA to JH precursors during the first gonadotrophic cycle. 40µM farnesol was added during the second incubation. Values represent mean ± SEM (n≥10). Significant differences are indicated ***p < 0.001.

Figure 2.5. Efficiency of HMGR-JHAMT RNAi-mediated knockdown and the effect of silencing on the transcription of the other genes encoding enzymes in the JH biosynthetic pathway in day 4 mated female D. punctata. The data represent averages of 3 pools (7 pairs of CA per pool), run in triplicate using q-RT-PCR and normalised to Tubulin and EF1a transcript levels. Values represent mean ± SEM. Significant differences are indicated by asterisks (*p < 0.05, **P < 0.005, ***p < 0.001, ****p < 0.0001).

Silencing HMGR and JHAMT resulted in a significant reduction in rates of JH biosynthesis and slows ovarian development.

The downregulation of HMGR and JHAMT resulted in a 73% reduction in the rates of JH biosynthesis in the CA of day 4 adult females as measured using the in vitro RCA (Fig. 2.6A).

To confirm the role of JH in inducing Vg transcription in the fat body and uptake in the developing basal oocytes, fat body and ovaries were dissected from four day old control and treated adult cockroaches. The basal oocyte length was significantly decreased following silencing of HMGR and JHAMT. The average oocyte length in the HMGR-JHAMT RNAi animals measured 0.8 mm compared to the control of 1.21 mm (Fig. 2.6B). The transcript level of the Vg in the fat body of the HMGR-JHAMT dsRNA-treated animals was reduced 80% (Fig.

2.6C). In addition, the knockdown of HMGR-JHAMT mRNA resulted in a significant reduction in vitellin content compared to the controls (Fig. 2.6D).

We also studied the histology of the developing basal oocytes in control and treated females.

Control oocytes were fully patent on day 4, showing large intercellular spaces between the follicle cells and the clear presence of yolk spheres. In the HMGR-JHAMT RNAi animals, on the other hand, patency was not fully induced and as a result, yolk was not deposited in these oocytes (Fig. 2.7).

Figure 2.6. JH regulates ovarian development: (A) The application of HMGR-JHAMT dsRNA results in a dramatic decrease of JH biosynthesis (n=16). (B) Oocyte length in HMGR, JHAMT dsRNA-treated animals compared to control animals (n≥27). (C) Relative Vg mRNA levels in HMGR-JHAMT dsRNA-treated animals compared to control animals. The transcription data represent averages of 3 pools (5 ovaries per pool), run in triplicate using q-RT-PCR and normalised to Tubulin and EF1a transcript levels. (D) Vitellin content measured by ELISA in HMGR-JHAMT dsRNA-treated animals compared to controls (n=9). Animals were dissected on day 4 after the final molt in control and treated groups. Values represent mean ± SEM. Significant differences are indicated by asterisks (**P < 0.005, ***p < 0.001, ****p < 0.0001).

Figure 2.7. Transverse sections of the basal oocytes from day 4 control and HMGR-JHAMT dsRNA-treated animals. Follicle cells (Fc), intercellular gaps (arrows), yolk spheres (arrowheads) and lipid spheres (L) are indicated. Scale bars represent 200 µm.

Discussion

Sequence of genes encoding enzymes in the JH biosynthetic pathway - Through in silico data mining and degenerate primer PCR and RACE, we have successfully characterized 11 of the 13 enzyme-encoding genes in the JH biosynthetic pathway of D. punctata. Cheng et al (2014) showed that enzymes in the early steps of the mevalonate pathway were generated by single copy genes in many insects, whereas genes encoding FPPS and enzymes in the JH-specific pathway probably underwent gene duplication in Lepidoptera. In D. punctata, only a single copy of these genes was identified. However, at this point, the existence of multiple copies of several of these genes cannot be ruled out and this issue will have to await the assembly of the

Diploptera genome.

The full-length HMGR and JHAMT were cloned from D. punctata CA. HMGR encodes a protein of 825 amino acids in length. Following a conserved domain search, HMGR was observed to contain the typical conserved motifs found in members of the HMG-CoA reductase superfamily of proteins. JHAMT encodes a protein of 274 amino acids in length and contains the motifs typically found in AdoMet-dependent methyltransferases. Still missing are sequences for farnesyl diphosphate pyrophosphatase (FPPP) and farnesal dehydrogenase (FALD). Molecular analysis of these genes was long hampered as a consequence of the minute size of the CA but using a newly developed sensitive assay for measuring JH precursor pools employing fluorescent tags, the genes encoding FPPP and FALD were recently characterized in A. aegypti (Nyati et al.,

2013; Rivera-Perez et al., 2013; Rivera-Perez et al., 2012). These were, however, not included in our study because the availability of only dipteran sequences made their characterization in the phylogenetic basal D. punctata problematic and therefore remain uncharacterized in this species to date.

Transcription of enzymes in the JH biosynthetic pathway partly regulates JH biosynthesis - The

11 orthologs of JH biosynthesis genes cloned in our model D. punctata are predominately expressed in the CA, and their relative transcript levels correlate well with the rates of JH biosynthesis (Fig. 2.2 and 2.3). Our results suggest that CA are highly specialized tissues for the biosynthesis of JH, and genes identified in our study are encoding functional enzymes in the JH pathway. Enzymes in the mevalonate pathway are responsible for not only JH production but also for the production of other terpenoids such as defensive secretions and pheromones (as reviewed by (Belles et al., 2005; Goldstein and Brown, 1990)). It is therefore of interest that all mevalonate enzyme-encoding genes except PMK and IPPI are exclusively expressed in the CA with trace amounts in other tissues. This can be explained by the strikingly high rates of JH biosynthesis in the CA of day 4 adult female D. punctata. For the later enzymes involved in the

JH specific steps of the pathway, the transcription of JHAMT and CYP15A1 is CA-specific, whereas FOLD is expressed in many tissues. Similar results were observed in A. aegypti and A. mellifera (Bomtorin et al., 2014; Mayoral et al., 2009a; Nouzova et al., 2011). Because of the multiple functions of farnesol, the oxidation of farnesol to farnesal does not appear to be a JH biosynthesis-specific reaction (Mayoral et al., 2009a).

Changes in rates of JH biosynthesis during the first gonadotropic cycle of D. punctata are dynamic, corresponding to specific reproductive events (see below) and are therefore tightly regulated. The transcript levels of enzyme-encoding genes in JH biosynthesis are highly coordinated with the rates of JH biosynthesis (Fig. 2.3), suggesting that at least part of the regulation of JH biosynthesis involves coordinated changes in the transcription of the genes in the biosynthetic pathway, in agreement with studies performed in A. aegypti (Nouzova et al.,

2011) and B. mori (Kinjoh et al., 2007), although the ability of each gene to regulate JH

92 biosynthesis appears to differ. In addition, the expression patterns of HMGS and HMGR mRNA were similar to the patterns of their enzyme activities (Couillaud and Feyereisen, 1991;

Feyereisen and Farnsworth, 1987). Although the transcription of most genes in the biosynthetic pathway showed a correlation with the JH biosynthesis, there are a few exceptions, including

PMK, IPPI and FPPS. PMK and IPPI, which are expressed in multiple tissues (Fig. 2.2), and may not be specifically regulated in the CA. For FPPS, 3 FPPS homologs were found in B. mori

(Ueda et al., 2009), and 7 in A. mellifera (Bomtorin et al., 2014). Although we currently have identified only 1 FPPS gene in D. punctata, additional FPPS genes may exist in the CA that regulate JH biosynthesis.

Effect of exogenous JH precursors on rates of JH biosynthesis - We have found that JH precursors are able to stimulate JH biosynthesis (Fig. 2.4A), which suggests that the rate of JH biosynthesis is not only controlled by transcription of the genes in the pathway, but also by the flux of substrates in the pathway. Farnesol stimulates JH biosynthesis by CA from day 0, 5 and 7 mated females. However, application of farnesol to CA showing high JH biosynthetic activity

(day 3 and 4) does not result in a significant change in JH biosynthesis (see Fig. 2.4B). These results suggest that during the first gonadotrophic cycle of D. punctata, the supply of this precursor is rate-limiting in CA with showing low JH biosynthetic activity, whereas there are other factors, including nutrients and neurotransmitters that control JH production at high rates of

JH biosynthesis. Nevertheless, 14 JH precursors are involved in the JH biosynthetic pathway, and each JH precursor connects to the ‘upstream’ and ‘downstream’ enzymes. Many factors could affect the stimulatory effect of JH precursor in JH production, including cell permeability of the added compounds, the activity of upstream/downstream enzymes, and the size of other JH precursor pools.

A feedback mechanism involved in the regulation of the transcription of JH biosynthetic genes -

HMGR and JHAMT have been studied in the JH pathway since they are representative enzymes involved in the well-conserved mevalonate pathway and in the JH-specific portion of the pathway respectively (Debernard et al., 1994; Li et al., 2013; Marchal et al., 2011;

Martinezgonzalez et al., 1993; Mayoral et al., 2009c; Monger et al., 1982; Wang et al., 2013).

We therefore chose to silence HMGR and JHAMT on a posttranscriptional level using RNAi during the first gonadotropic cycle of D. punctata. RNAi trials have shown that fewer injections of HMGR or JHAMT dsRNA and with lower concentrations of construct, did not result in a significant silencing of our target gene mRNA levels (data not shown). We therefore conclude that the RNAi response in D. punctata appears to be systemic, but is not as effective as in another hemimetabolous insect model, the desert locust, Schistocerca gregaria in which smaller quantities of the JHAMT dsRNA construct induced a longer downregulation of the same target gene during the first gonadotropic cycle (Marchal et al., 2011). A further in-depth study on the

RNAi machinery D. punctata should be performed to explain the differences in sensitivity compared to other hemimetabolous insect species.

JH biosynthesis is controlled by many factors including neuropeptides (allatostatins, allatotropins, short neuropeptide F) (Kaneko and Hiruma, 2014; Weaver and Audsley, 2009), neurotransmitters (octopamine, dopamine and glutamate) (Granger et al., 1996; Pszczolkowski et al., 1999; Thompson et al., 1990) and JH itself (Goodman and Cusson, 2012; Marchal et al.,

2013a). Topical application of JH or a JH analog resulted in a suppression of JH biosynthesis, which suggests a negative feedback regulation of CA in D. punctata (Tobe and Stay, 1979). In higher animals, cholesterol, the bulk end-product of mevalonate pathway, shows a feedback regulation on the transcription and post-transcription of enzymes in the mevalonate pathway

(Chang and Limanek, 1980; Clarke et al., 1987; Goldstein and Brown, 1990). Later studies showed that farnesol, an intermediate product in the JH biosynthetic pathway, is able to accelerate the degradation of HMGR, and the increase of mevalonate flux raises the activity of

FPPP expressed in CHO cells (Meigs et al., 1996; Meigs and Simoni, 1997). In our study, regulating transcription of the JH biosynthetic enzymes seems to affect the entire biosynthetic pathway, rather than individual steps. The silencing of HMGR and JHAMT resulted in a significant decrease in the transcript level of several other genes in the pathway (Fig. 2.5). A possible explanation is that the accumulation of JH precursors as a result of RNAi treatment resulted in a feedback on the expression of other genes in the JH biosynthetic pathway to balance the size of JH precursor pools and the enzyme activity.

The essential role of JH in reproduction - Our results clearly show that select silencing of genes encoding enzymes in the JH biosynthetic pathway effectively reduces JH biosynthesis in vitro

(Fig. 2.6). As a well-known model for the study of the regulation of JH biosynthesis, D. punctata displays a consistent characteristic profile of JH production during the first gonadotropic cyclethat is closely correlated with specific reproductive events such as vitellogenesis and chorionation. Once JH production rises on day 2, vitellogenin synthesis in the fat body commences, and vitellogenin is taken up from the hemolymph and is incorporated into the developing basal oocytes. The elevated JH titer results in patency in the follicular epithelium of the basal oocytes, permitting the uptake of Vg and subsequent yolk formation (Marchal et al.,

2014; Pratt and Davey, 1972). On day 5, the spaces in the follicular epithelium close as rates of

JH biosynthesis decline and Vg transcription in the fat body and circulating Vg levels decrease.

At this point, choriogenesis begins and JH biosynthesis remains low until oviposition on day 7

(as reviewed by Marchal et al., 2013a). Our RNAi study has focused on day 4 of the first

95 gonadotropic cycle, a time when JH titre is high in controls and animals are vitellogenic (Tobe et al., 1985) as characterized by high DippuVg mRNA transcript levels in the fat body and JH- dependent induction of patency in the maturing basal oocytes. Following the reduction in JH production by the silencing of DippuHMGR and DippuJHAMT, mRNA levels of DippuVg in the fat body are significantly reduced, incorporation of vitellin into the oocytes is impaired and patency does not occur (Fig. 2.6 and 2.7). Previous reports have shown that in S. gregaria, oocyte length is significantly affected following treatment of females with JHAMT dsRNA

(Marchal et al., 2011). RNAi was also used in the cotton bollworm Helicoverpa armigera to effectively silence HMGR, resulting in reduced Vg expression (Wang et al., 2013). Each of these studies highlights specific aspects of JH-regulated reproductive events. The results described in the current paper confirm the central role played by JH during the female reproductive cycle of D. punctata.

JH biosynthesis is a fundamental process in regulating insect development, metamorphosis and reproduction (Goodman and Cusson, 2012). Although the genes directly involved in JH biosynthesis have been characterized using molecular techniques in several insect species, there was no similar study in the hemimetabolous insects. Thus, our study is the first to characterize the majority of the genes directly involved in JH biosynthesis in a hemimetabolous insect and a well- known model for studying the physiology of JH. This paper now provides the molecular tools to study the regulatory mechanisms of JH production in D. punctata (for a first study, see (Huang et al., 2014)). In addition, the role of JH biosynthetic enzymes and the JH precursor supplies in regulating JH biosynthesis have been demonstrated, as has the critical function of JH as the master regulator of cockroach female reproduction.

Supplementary data

A. BlageHMGR 1 M-VGRLFRAHGQFCASHPWEVIVATLTLTVCMLTVDQ------RP-LGLPPGWGHNC------ITLEEYNAADMIVMTLIRCVAVLYSYYQFCHLQKLGSKYILGI DippuHMGR 1 M-VGWLFLAHGQFCASHPWEVIVATFTLTACMLTVDP------HP-LGLPPGWRKNC------ISLEEYNAADVIVMTLIRCVAVLYSYYQFCHLQKLGSKYILGI ApimeHMGR 1 M-LTRLFEIHGRFCAGHPLEVIVTTFTLTACILNMETG------NGHPSVPLTAHCGPGR------CNSDDLNAADVIVMTIIRCLAILFTYHQFRNLQKLGSKYILGI TricaHMGR 1 M-TTRLFRAHGEFCATHPWEVIVATLTLAACMLTVDQQ------HPAPPPKPTLRYCAEC------LQEAEYNAADLIVMTLIRCLAVLYCYYQFCNLEKLGSKYILGI AedaeHMGR 1 MKVGRLFRAHGEFCASHPWEVIVALVTLTACIITFDKG------SADPFQQSSSRSGSGRPC------PSWRFSSVSPDEAFRCEEVEQNGIDVILMTIVRCSAILYCYYHFCNLQKLGSKYILGI DromeHMGR 1 M-IGRLFRAHGEFCASHPWEVIVALLTITACMLTVDKNNTLDASSGLGTATASAAAAGGSGSGAGSGASGTIPPSSMGGSATSSRHRPCHGWSQSCDGLEAEYNAADVILMTIVRCTAVLYCYYQFCSLHRLGSKYVLGI BommoHMGR 1 M---KVWGAHGEFCARHQWEVIVATLALLACAASVERHG------PGNRSEHCAGWARACP------GLEAEYQAADAVIMTFVRCAALLYAYYQVLNLHKIASKYLLII

BlageHMGR 93 AGLFTVFSSFVFSSSVINFLGSDVSDLKDALFFFLLLIDLSKATVLAQFALSSRSQDEVKHNIARGIAMLGPTITLDTVVETLVIGVGMLSGVRRLEVLCCFACMSVIVNYVVFMTFYPACLSLILELSRSGESGRPAWH DippuHMGR 93 AGLFTVFSSFVFSSSVINFLGSDISDLKDALFFFLLLIDLSKATLLAQFALSSCSQDEVKHNIARGIAMLGPTITLDTVVETLVIGVGTLSGVRRLEVLCCFACMSVIVNYIVFMTFYPAFLSLILELARSGEGGRPAWH ApimeHMGR 97 AGLFTVFSSVVFTSSVVNFGRSDISDLKDALFFFLLIIDLSKAAVLAQLALSSRNKEEVRANIARGMSLLGPTITLDTLVETLLISIGSLSGVRRLEILCSFACLGVVVNYIVFMTFYPACLSLILELSRETNTMKPLSA TricaHMGR 97 AGLFTVFSSFVFTSTVLNLLWIDVSDLKDALFFFLLLIDLSKAAMLAQSALSASNQEEVKSNIARGMAVLGPTITLDTIVETLVIGVGTLSGVHRLEMLSYFACLSVLVNYIVFMTFYPACLSLILELSRTTN---IYGN AedaeHMGR 115 AGLFTVFSSFIFTSTVINFLGSEVSDLKDALFFFLLLIDLSKAAILAQLALCGSSQSEVTMNIARGMEILGPAISLDTLVETLVIGVGTLSGVQRLELLSGFAVLSAIVNYIVFMTFYPACLSLILDLSRNAGNLIQKNK DromeHMGR 140 AGLFTVFSSFIFTTAIIKFLGSDISELKDALFFLLLVIDLSNSGRLAQLALSGSNQAEVTQNIARGLELLGPAISLDTIVEVLLVGVGTLSGVQRLEVLCMFAVLSVLVNYVVFMTFYPACLSLIFDLSRSGVDMSVVRE BommoHMGR 96 AGVFSTFASFIFTSALASLFWSELASIKDAPFLFLLVADVARGARMAKAGWSAG--EDQGKGVGRALSLLGPTATLDTLLAILLVGVGALSGVPRLEHMCTFACLALLVDYIVFITFYPACLSLVADFATNRK---EIAH

BlageHMGR 233 DKS--LIIKALHEEDQKPNPVVQRVKVIMSAGLMLVHAHRWVRCLSIALWPDLTSLRYFCTHCDTGVSYSRWSFASEGEELPTVKLVTG--DSVVNSNST----DDAQLHYYIMRWLTVSADHIVILILLLALAVKFVFF DippuHMGR 233 DRS--LIMKALHEEDQKPNPVVQRVKVIMSAGLMLVHAH------SRWSFASEGEELPIVNLVTG--DSVVITNST----QDAQIHDYIMRWLTVSADHIVILILLLALAVKFVFF ApimeHMGR 237 DKI--FMMHPLDEEDQKPNPVVERVKLIMIAGLFVVHAN------SRFKSE-ESEDTVEGKVSST--NSHVIVNSYNETEDSSEVKGYLMNWLSVSADNIVILILLLALAIKFIFF TricaHMGR 234 KQS--LIARALKEEDHKSNPVVQRVKLIMSAGLMLVHAR------SRWPFK--EDDVENIRPLVV--EQHMTLNRT----EDTTLHEYIMKWVTVSADHIVILILLLALVVKFVFF AedaeHMGR 255 KEN--LLARVLTEEDQKPNPVVQRVKLIMSSGLMIVHVL------SRLAISEKDSDTAEHIIASHSHEHLAAMNKT----EPNEISEFIMRWLSISTEQIVTYILIIALGVKFVFF DromeHMGR 280 KAKGSLLLKSLTEEEQKANPVLQRVKLIMTTGLMAVHIY------SRVAFSGSDYDAVDKTLTPT-LSLNVSNNRT----ESGEIADIIIKWLTMSADHIVISIVLIALVVKFICF BommoHMGR 231 DSP------FSEEDLKPNPVVQRVKMIMAAGLLCVHLT------SRWPWS------ANHGIIEG--PIDASIPVP----HDNILLHSYVKWFSVSADYIVIATLLCALIIKFIFF

BlageHMGR 365 ETRDELTTTRGMDGWVEVSSP------VEHKYVQTE------QPSCSAP------EQPLEEPPASN------RSIDECLSVCK-SDVGAQ--ALSDCEVMALVTS- DippuHMGR 335 EVKDELNTTRGMDGWVEVSSP------VEHKFVQTD------QVCWELPSSS---QEPDEQPLPCD------RPVQECLAICK-SDAGAN--SLSDSEVMSLVSS- ApimeHMGR 342 EDKEDIAKQLQFKVEDDTEEK------IENEYMKEKKFEIEYVKDKENENEDMNFSFLTTKMPFKLSFAKMQTISIPWIDGKEEQIDEKQCTVNTSNDKNLFSQIPRSVEECLKIYK-SELGAN--GLTDEEVIQLVKN- TricaHMGR 334 ENKEELAEQLRAHISTESVDPGKKDNRFKMPLIKTQSFFLTNNTKED------SACEDKEVQTDIGRLESEFEKLPAGN-----DVSVKTCRSLEECLKIYNDSNLGGA--ALSDDEVILLVKN- AedaeHMGR 359 D-RHNLSDQILLSVANEAAAAAAAAAAAAQKQKQLELELERPQPVATFT------LAETVPVEEKATQSELCLLPGRGQRAKSMGADDELNLDDLEDELVEREPRPLEMCLKILNETDDGAV--GLTDEEIKMIVRAG DromeHMGR 385 DNRDPLPDQLRQSGPVAIAAKASQTTPIDEEHVEQEKDTENS------AAVRTLLFTIEDQSSANASTQTDLLPLRHRLVGPIKPPRPVQECLDILNSTEEGSGPAALSDEEIVSIVHAG BommoHMGR 322 EEQRNWVYDMDDMTVKEVINDTDLS--RKPKFSVGD------DSNSEVSTQTDEAGNVEDMEWPTLSPSSSASKLNAKKRPMVECLELYR---SGACT-SLSDEEVIMLVEQ-

BlageHMGR 443 -GHIAGYQLEKVVRNPERGVGIRRQILTKTADL-KDALDNLPYKNYDYLKVMGACCENVIGYMPVPVGVAGPLNLDGRLVHVPLATTEGCLVASTNRGMRALMRCGVTSRIVADGMTRGPVVRFPNIDRASEAMLWMQVP DippuHMGR 416 -GHVAAYQLEKMVGDPERGVGIRRKILTQKADL-KDALDNLPYKNYDYTKVMGACCENVIGYMPVPVGVAGPLKLDGCLVYVPLATTEGCLVASTNRGSRALMRCGVTSRIVADGMTRGPVVRFPNIERASEAMLWMQAA ApimeHMGR 472 -NHIAAYQLEKAVGDMERGVEIRRFIIGEAGNF-LDYLSNLPYKDYDYSKVLGACCENVMGYVPVPLGIAGPLLLDGELYYVPMATTEGCLVASTNRGSRALLKCGVTSRVVADGMTRGPVVRFPNIVRASEAMAWMQDP TricaHMGR 445 -KHIPAYQIEKAVDDPERGVGIRRKILAREGNF-SEALTDLPFRNYDYAKVMGACCENVIGYMPVPVGYAGPLNLDGRHVYVPMATTEGCLVASTNRGCRALLDCGVTSRVVSDGMTRGPVVRFPSITKASEAMSWMKCS AedaeHMGR 488 NGYCPLYKIETVIGDAERGVKIRRDMIQKEANLPANAFKHLSYKNYDYSKVMNACCENVLGYVQIPVGYAGPLILDGVRYYVPMATTEGALVASTNRGCKAISTRGVTSFVEDIGMTRAPCIKFPNVLRAAQAKRWMETP DromeHMGR 499 GTHCPLHKIESVLDDPERGVRIRRQIIGSRAKMPVGRLDVLPYEHFDYRKVLNACCENVLGYVPIPVGYAGPLLLDGETYYVPMATTEGALVASTNRGCKALSVRGVRSVVEDVGMTRAPCVRFPSVARAAEAKSWIEND BommoHMGR 422 -SHIPMHRLEAVLEDPLRGVRLRRRVIASRFNN-ETAIKQLPYLNYDYSKVLNACCENVIGYIGVPVGYAGPLVVDGKPYMIPMATTEGALVASTNRGAKAIGSRGVTSVVEDVGMTRAPAVKLPNVVRAHECRQWIDNK

BlageHMGR 581 YNFEQIKKNFDSTSRFARLSKIHIRVAGRHLFIRFIATTGDAMGMNMLSKGTEVALAYVQQVYPDMEILSLSGNFCTDKKPAAVNWIEGRGKSVVCEAIVPADIIKSVLKTSVQALMDVNITKNLIGSAVAGSIGGFNAH DippuHMGR 554 QNFEAMKKHFDSTSRYARLSKIHIRVAGRHLFIRFVATTGDAMGMNMLSKGTEVALSFVQQMFPDMEILSLSGNFCTDKKPAAVNWIEGRGKSVVCEAVVPADVVSSVLKTSVQALVDLNITKNFYGSAIAGSVGGNNAH ApimeHMGR 610 DNFKEMKNSFNLTSRFARLTKINIRIAGRHLFIRFVATTGDAMGMNMLSKGTEKSLNTVKEHFPDMEILSLSGNFCTDKKPAAVNWVCGRGKSVVCEAVVPADIVTNVLKTSVHALVDVNISKNMIGSAIAGSIGGFNAH TricaHMGR 583 QNFEAMKQQFDSSSRFARLSKLLIKIAGRHLFVRFEAKTGDAMGMNMVSKGTEMSLKYVQKQFPEMEILSLSGNFCTDKKPAAVNWIEGRGKSVVCEAIIPSEIVKKVLKTSTPALVDVNNSKNMIGSAVAGSIGGFNAH AedaeHMGR 628 ENFAVIKKAFDSTSRFARLQELHIAMDGPILYARFRALTGDAMGMNMVSKGSEMALREVHRSFPDMQIISLSGNFCSDKKPAAINWIKGRGKRVICEAIVPADKLRTILKTNARTLVQCNKLKNMTGSAVAGSIGGNNAH DromeHMGR 639 ENYRVVKTEFDSTSRFGRLKDCHIAMDGPQLYIRFVAITGDAMGMNMVSKGAEMALRRIQLQFPDMQIISLSGNFCCDKKPAAINWIKGRGKRVVTECTISAATLRSVLKTDAKTLVECNKLKNMGGSAMAGSIGGNNAH BommoHMGR 560 ENYALLKEAFDSTSRFARLQEIHVGVDGATLYLRFRATTGDAMGMNMVSKGAENALKLLKTFFRDMEVISLSGNYCSDKKAAAINWIKGRGKRVVCETVISSENLRTIFKTDAKTLSRCNKIKNLSGSALAGSIGGNNAH

BlageHMGR 721 AANIVTAIFIATGQDPAQNVGSSNCMTLMEPWGEDGKDLYVSCTMPSIEIGTIGGGTVLPPQAACLDMLGVRGANEMCPGENANTLARIVCGTVLAGELSLMSALAAGHLVKSHMRHNRSSVSTS------GSEPS DippuHMGR 694 AANIVTAIFIATGQDPAQNVGSSNCITIMEPWGDDKKDLYVSCSMPSIEIGTVGGGTILPPQAACLDMLGVKGANAVCPGENANMLARIVCGTVLAGELSLMSALAAGHLVKSHLRHNRSSVTTS------GSEPS ApimeHMGR 750 AANIVTAIFIATGQDPAQNVGSSNCMTLMEPWGTDGSDLYVSCTMPSIEIGTVGGGTILPAQGACLSILGVKGAHSDEPGENASRLARIVCATVLAGELSLMAALTAGHLVKSHLRHNRSSTTVTNAMSVPQKYTGTKLS TricaHMGR 723 AANIVTAIYIATGQDPAQNIGSSNCMTLMEPWGETGEDLYVSCTMPSIEIGTVGGGTVLPAQSSCLEMLRVKGSHPDCPGENASQLARIVCGTVLAGELSLMAALTAGHLVRSHLRHNRSTTTIP------EEFS AedaeHMGR 768 AANMVTAIYIATGQDPAQNVTSSNCSTNMEPYGDNGEDLYMTCTMPSLEVGTVGGGTGLPGQGACLDMLGVRGAHPTHPAENSKQLARVICATVMAGELSLMAALVNSDLVKSHMRHNRSSVAVNPG----LPATAQLQS DromeHMGR 779 AANMVTAVFLATGQDPAQNVTSSNCSTAMECWAENSEDLYMTCTMPSLEVGTVGGGTGLPGQSACLEMLGVRGAHATRPGDNAKKLAQIVCATVMAGELSLMAALVNSDLVKSHMRHNRSSIAVN------S BommoHMGR 700 AANMVTAIFIATGQDPAQNVTSSNCSTNMEAYGENGEDLYVTCTMPSLEVGTVGGGTVLTGQGACLEILGVKGAG-TRPAENSARLASLICATVLAGELSLMAALVNSDLVKSHMRHNRSTLNVQ------TAN BlageHMGR 851 TP------ACKS------DippuHMGR 824 KS------

ApimeHMGR 890 VPNLLQPVQNVCKGLLEKS------TricaHMGR 852 QNRYHIP---PCKDI------AedaeHMGR 904 APSLLTACNSSSSGSSSSIGTSLSAKQ DromeHMGR 905 ANNPLNVTVSSCSTIS------BommoHMGR 827 VEPYTVALKVPPS------

B. SchgrJHAMT 1 MDKAELYSRSNGLQRWEASAALEAAWPALRWPAPP-LRVLDVGCGAGDVTVDLLLPRLPP-HTQLVGTDVSAAMVEHAAELYGAAHPGLSFQLLDIADPDIDASPVYQLAPFDKIFSFFCLHWVPEQRQAAENLHRLLKP DippuJHAMT 1 MHKAELYSSSHGLQKRDAAHALTEYLDHMTWR-PG-DRVLDVGCGPGFVTAQELMPRLPQDFAILVGTDVSHAMVQHATSTY--VQPKLKFAHLDISSTHIDK-ELWEPG-FDKIFSFYCLHWIPDQRTAVNNIYHLLRP TricaJHAMT 1 MNKASLYSKYSGLQKNDASFVIDNYLRLIKWK-PN-ANILDIGSGDGNVIFELLLPKIPKHFAKFVGTDISEEMVLFAKNQCN--DPKIDFLQMDIS----ATIPPEFHEYFDHIFSFYCLHWVVEQRQAMKNIFDMLKP DromeJHAMT 1 MNQASLYQHANQVQRHDAKLILDEFASTMQWRSDGEDALLDVGSGSGNVLMDFVKPLLP-IRGQLVGTDISSQMVHYASKHYQR-EERTRFQVLDIGCER-LPEELS--GRFDHVTSFYCLHWVQNLKGALGNIYNLLKP AedaeJHAMT 1 MNKPNLYHRANGVQRRDAKEILDEHGHLLRWKEENEDSLLDIGCGSGDVLIDFVIPMVPPKRARVLGTDVSEQMVRFARKVHSD-VENLFFETLDIEGD--ISSFLNKWGCFDHITSFYCLHWVRSQRSAFSNIYNLMAP BommoJHAMT 1 MNNADLYRKSNSLQKRDALRCLEEHANKIKWKKIG-DRVIDLGCADG-SVTDILKVYMPKNYGRLVGCDISEEMVKYANKHHG--FGRTSFRVLDIEGD--LTADLK--QGFDHVFSFYTLHWIRDQERAFRNIFNLLGD ApimeJHAMT 1 MFLVEEYVKASTIQYRDAADIIGEFAEEMSEMKGK---CLDIGCGPGIVTKELILPNLSP-EAKLVGMDISRPMIEYAKNMYHD-EERLSFQLLDIET---MDLPKDTFDQFNNVLSFYCLHWCQNFRKAFDNIYKLLRP

SchgrJHAMT 139 GG-EVVLSLLAHCPIFSVYEGLAHKPQWKEYMEDARRFISPYHHSEDPAREMNELLCRAGFRVTLCTRQQRSFTFPGHSALIEAVTAVNPFVERLPETLQQEFLEDCMKEVLRQKLVSIEDDADSNNNSNGSNRSGNNAV DippuJHAMT 135 AG-EALVLLMAKCPVFNVYTAQSNKPKWQQYMKDASRYISPYHQLKDPKSEFINIVEDVGFHVVDCDCRQNKFNYGTLERLKDAIKAVNPFMDRIPEELQEEYLNDCLSEARRIKCT------ESNNN------V TricaJHAMT 133 GG-EMLLTFLASNPIYDIYERMAKSNKWGPYMNNLKKYISPYHHSEDPETELENLLKKEGFITHLCRVENRSYTFPSFSVLSKSVSAVNPFIKKLPENEIDTYIEDYLKEVRKLKTIT----IETCNN------DromeJHAMT 136 EGGDCLLAFLASNPVYEVYKILKTNDKWSTFMQDVENFISPLHYSLSPGEEFSQLLNDVGFVQHNVEIRNEVFVYEGVRTLKDNVKAICPFLERMPADLHEQFLDDFIDIVISMNLQQ------GEN------AedaeJHAMT 138 NG-DCLLGFLARNPIFDIYDQLSNSAKWSMYMTDVDKYISPYQYCENPVGEIEEILSSVGFTKYKIHIADKIYVYEGIDSLKKAVQAVNPFSERMPLDLQEDFLNDYIAVVRRMSLSEN-----CCGN------BommoJHAMT 133 EG-DCLLLFLGHTPIFDVYRTLSHTEKWHSWLEHVDRFISPYHDNEDPEKEVKKIMERVGFSNIEVQCKTLFYVYDDLDVLKKSVAAINPFN--IPKDILEDFLEDYIDVVREMRLLDR-----CNNN------ApimeJHAMT 133 GG-KGLFMLLSWNDGFDVYKKLYANPRYRPYMQEPERFIPIFHECKDRRVNLRKILETTGFEILHCSEREKSYIYKNSEIMKKHIMAINPFISRIPNSLKKEFEDEITREIVNMKIQLLN----KDEN------

SchgrJHAMT 278 DANSKKLTTRYSVLTVVAAKAAAQNGVRTVR------DippuJHAMT 257 EEVT---TVSYDIIVAHIKKP------TricaJHAMT 256 NDNEEKIHVPYKLFVTFASKPV------DromeJHAMT 257 NEDQKFLSP-YKLVVAYARKTPEFVNNVFLEPTHQNLVKGIN AedaeJHAMT 260 ENDYKFITP-YKLVVVYAVK------BommoJHAMT 253 VGESVSIKFNYKVISVYARKLCLSLM------ApimeJHAMT 256 GEQEYNILDRYQIFVTYIRKPVC------

Figure S2.1. Amino acid sequence alignment of Diploptera proteins with several functionally characterized ortholog insect proteins. (A) DippuHMGR with HMGR from the German cockroach Blattella germanica (GenBank accession number: CAA49628.1 (Martinezgonzalez et al., 1993), the honeybee Apis mellifera (GenBank accession number: XP_623118.1), the red flour beetle Tribolium castaneum (GenBank accession number: XP_973850.1), the mosquito Aedes aegypti (GenBank accession number: XP_001659923.1), the fly Drosophila melanogaster (GenBank accession number: NP_732900.1) and the silkworm Bombyx mori (GenBank accession number: BAF62108.1 (Kinjoh et al., 2007)) (B) DippuJHAMT with JHAMT from the desert locust, S. gregaria (GenBank accession number: ADV17350.1 (Marchal et al., 2011)), the red flour beetle T. castaneum (GenBank accession number: NP_001120783.1 (Minakuchi et al., 2008)), the fly D. melanogaster (GenBank accession number: AB113579.1 (Niwa et al., 2008)), the mosquito A. aegypti (GenBank accesson number: ABD65474.1 (Mayoral et al., 2009b)), the silkworm B. mori (GenBank accession number: NP_001036901.1 (Shinoda and Itoyama, 2003)) and the honeybee A. mellifiera (GenBank accession number: AGG79412.1 (Li et al., 2013)).

3 5 0 0

y 3 0 0 0

t i

t 2 5 0 0

n 2 0 0 0 a

u 1 5 0 0 q 1 0 0 0

A 5 0 0 N

2 0 0 R

1 5 0

e 1 0 0 v

i 5 0

t a

l 2 0 e

R 1 0

0 I l K D D S K S T 1 P o R A L i G P M M M IP h G M 5 P P O M P A 1 F T M F P H H H P J Y C

Figure S2.2. Comparison of transcript levels of genes encoding JH biosynthetic enzymes in CA of day 4 mated female. Bars represent the mean of three biologically independent pools of ten animals run in triplicate and normalized to Tubulin and EF1α. Vertical error bars indicate SEM. Enzyme abbreviations are as in Fig. 2.1.

Table S2.1. (Degenerate) primer sequences for cloning of (partial) sequences for orthologous genes encoding JH biosynthesis enzymes in D. punctata. Gene name abbreviations were shown in Fig. 2.1.

Gene name F Primer(5'-3') R Primer(5'-3') Accession number (NCBI) Thiol GGDCARAATCCWGCDAGRCARGC TTGHGCWGCRAANGCTTCRTT KJ188021 HMGS AGAGTGAACTGGAAGTGCA TATGAAGCACGCACTCCTC KJ188022 HMGR AATGGTAGGGTGGCTGTTTCTA GTATTTCATGATTTGCTGGGTTC KJ188023 MK CTGCCCCCGGTAAAGTTATC GATGCTTGACTTCCTGCACC KJ188024 PMK ATGGAAGTAAACAAAATTTCAG CACTCTGTTTCTGCATCATCTAC KJ188025 PPMD TGTTCAGAAAAYAATTTYCCNAC TGATTNSWRTCYTTCATTGT KJ188026 IPPI CAYMGDGCVTTYAGTKTDTTY TCDAWYTCRTGTTCKCCCCA KJ188027 FPPS TGGCGCGTAGGGTAGACAA GATCCATAACATTCTGCAAACATTG KJ188028 FOLD ATGCAGCGTTGGACTGGA TCCAGCATTGTTGATGAGAA KJ188029 JHAMT ATGCACAAAGCAGAACTGTATTC TCAAGGCTTTTTAATATGAGCGACAAT KJ188030 CYP15A1 ATGGTCATCGCTCTTATTGTCATC ACCATTCATTTCCTTGGAATCAACT AY509244 Vg TGGAACRCDCTICTCTGYTGYCT TTKAKSAYGTTRAYTTCCCAG KJ188031

100

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Chapter 3

Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata

This chapter is an adapted reprint of my article:

Juan Huang, Elisabeth Marchal, Ekaterina F. Hult, Sven Zels, Jozef Vanden Broeck, Stephen S.

Tobe, Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata, Insect Biochem Mol Biol, 2014, 54, 61-68.

Authors’ contribution:

I designed and performed all experiments in this study and wrote the paper with editing by E.

Marchal, E.F. Hult, and S.S. Tobe.

105

Summary

The FGLamide allatostatins (FGL/ASTs) are a family of neuropeptides with pleiotropic functions, including the inhibition of juvenile hormone (JH) biosynthesis, vitellogenesis and muscle contraction. In the cockroach, Diploptera punctata, thirteen FGLa/ASTs and one allatostatin receptor (AstR) have been identified. However, the mode of action of ASTs in regulation of JH biosynthesis remains unclear. Here, we determined the tissue distribution of

Dippu-AstR and Dippu-AST. The transcript level of Dippu-AstR in the CA corresponds to the JH biosynthesis, while the expression of Dippu-AST mRNA in the brain did not show any significant change during the first gonadotrophic cycle. In addition, silencing Dippu-AstR results in a significantly increase of JH biosynthesis, while the knockdown of Dippu-AST has no effect.

These results suggest that it is the change of AST receptor in the CA, instead of AST levels in the brain that control the AST-mediated inhibition of JH production.

To determine the signal pathway of Dippu-AstR, we expressed Dippu-AstR in vertebrate cell lines, and activated the receptor with the Dippu-ASTs. Our results show that all thirteen ASTs activated Dippu-AstR in a dose dependent manner, albeit with different potencies. Functional analysis of AstR in multiple cell lines demonstrated that activation of the AstR receptor resulted

2+ in elevated levels of Ca and cAMP, which suggests that Dippu-AstR can act through the Gαq and Gαs protein pathways. The study on the target of AST action reveals that FGL/AST affects

JH biosynthesis prior to the entry of acetyl-CoA into the JH biosynthetic pathway.

Introduction

Allatostatins (ASTs), a family of pleiotropic neuropeptides, were originally named for their ability to inhibit juvenile hormone (JH) biosynthesis by corpora allata (CA) rapidly and 106 reversibly (Bendena et al., 1999). Three families of ASTs have been identified in insects and named as: FGLa/ASTs (A-type), MIP/ASTs (B-type) and the PISCF/ASTs (C-type) (Coast and

Schooley, 2011). The distribution and function of the three families of ASTs has been reviewed by Stay and Tobe (Stay and Tobe, 2007). The best documented role of FGLa/AST is their ability to inhibit juvenile hormone (JH) biosynthesis by corpora allata (CA) (Stay and Tobe, 2007).

Later studies demonstrated other functions of FGLa/ASTs, including regulation of myotropic activity in gut tissues (Duve et al., 1995; Lange et al., 1995) and cardiac rhythm (Vilaplana et al.,

1999), inhibition of vitellogenin synthesis in the fat body (Martin et al., 1996), and stimulation of enzyme activity in the lumen of the midgut (Fuse et al., 1999).

As neuropeptides, ASTs exert their effects by binding to a G protein-coupled receptor, AstR.

FGLa/AST receptors, which are structurally related to the mammalian galanin receptor, were first identified in the fly Drosophila melanogaster DAR-1 (Birgul et al., 1999; Lenz et al., 2000) and DAR-2 (Birgul et al., 1999; Lenz et al., 2000), and later in the silkworm, Bombyx mori

(Secher et al., 2001) and the stick insect, Carausius morosus (Auerswald et al., 2001). In D. punctata, putative AstRs have previously been partially characterized using photoaffinity labeling and a radioligand-binding assay in the CA and brain (Cusson et al., 1991, 1992; Yu et al., 1995). Lungchukiet et al (Lungchukiet et al., 2008b) identified a putative FGLa/AST receptor gene in D. punctata. Silencing this gene resulted in a significant increase in JH biosynthesis (Lungchukiet et al., 2008a; Lungchukiet et al., 2008b).

The responses of FGLa/AST receptor to FGLa/ASTs have been assayed in D. melanogaster

(DAR-1 and DAR-2) (Birgul et al., 1999; Larsen et al., 2001), B. mori (Secher et al., 2001) and the cockroach, Periplaneta americana (Gade et al., 2008). The studies in B. mori and P. americana focused on the response of AstR to exogenous ASTs. The signaling pathway of AstR 107 was, however, not completely elucidated. Larsen et al (Larsen et al., 2001) expressed Drosophila

FGLa/AST receptors, DAR-1 and DAR-2 in CHO cells and activated them with four putative

FGLa/ASTs from D. melanogaster AST and four FGLa/ASTs from D. punctata in the presence or absence of pertussis toxin (PTX). They found that PTX caused a complete loss of Ca2+ signal in cells expressing DAR-1, and a decreased Ca2+ signal in cells expressing DAR-2. Their results suggested that the activation of DAR-1 and DAR-2 by FGLa/ASTs coupled to multiple signaling pathways, including Gi/o protein and other, PTX-insensitive G-proteins.

Although the function of AST has been well-studied, little is known about the precise target of

AST action. Previous studies focused on select enzymes in the JH biosynthetic pathway. It is now known that thirteen enzymes are involved in the JH biosynthetic pathway (Fig. 2.1) (Belles et al., 2005; Nouzova et al., 2011). The potential targets of action of ASTs were originally studied by employing different known JH precursors (Pratt et al., 1991; Pratt et al., 1989). The results suggested that the inhibitory action of AST on JH biosynthesis resides in step(s) prior to mevalonate. However, neither HMG-CoA synthase nor HMG-CoA reductase activity was affected by ASTs (Sutherland and Feyereisen, 1996). These authors proposed that the target of

AST action on JH biosynthesis is in step(s) prior to the JH biosynthetic pathway, and may be related to the transport of citrate from mitochondria to cytosol and/or to the cleavage of citrate to yield acetyl-CoA.

Our study focuses on the viviparous cockroach, Diploptera punctata, in which ASTs were first characterized. The first gonadotropic cycle is characterized by a precise regulation of JH biosynthesis necessary to coordinate a specific series of reproductive events closely correlated with oocyte growth. This makes D. punctata an ideal model for a more in depth study of the mode of action of AST and its signal transduction pathway. We have analyzed the spatial 108 expression pattern of the AST precursor and AstR which is consistent with their roles in regulating JH biosynthesis. Moreover, by using an aequorin-based assay and expressing the receptor in a mammalian system, we have unambiguously identified that AST is a ligand for the candidate AstR (Lungchukiet et al., 2008a; Lungchukiet et al., 2008b) and that this receptor can couple to Ca2+ and cAMP.

Thirteen FGLa/ASTs have been identified in D. punctata. These peptides share a conserved C- terminal Tyr (Phe)- Xaa-Phe-Gly-Leu-NH2, which is believed to be the main functional region for the inhibition of JH biosynthesis (Donly et al., 1993; Marchal et al., 2013a). The FGLa/ASTs inhibit JH biosynthesis by CA at low concentrations in vitro but with different potencies (Tobe et al., 2000). Our study has also examined the relationship between binding affinity and potency with a view to understanding the sites of action of the peptides.

The precise target of AST action remains unclear so far. Our study did not find any significant changes in the transcript level of genes encoding enzymes in JH biosynthetic pathway. The rescue of AST-induced JH inhibition by JH precursors suggest that the target of AST action is prior to the entry of Acetyl-CoA into the JH biosynthetic pathway.

Materials and Methods

Insects - D. punctata were reared in cages and fed with lab chow and water at libitum at 27 ºC in a dark room. Newly molted female adult cockroaches were picked from the colony and raised in separate containers. Mated status was confirmed by the presence of a spermatophore.

Tissue collection - Cockroach tissues were dissected under a dissecting microscope. Basal oocyte length was measured to determine the physiological age of cockroaches. Selected tissues were dissected and cleaned in sterile cockroach ringer solution (150 mM NaCl, 12 mM KCl, 10 mM 109

CaCl2.2H2O, 3 mM MgCl2.6H2O, 10 mM HEPES, 40 mM Glucose, pH 7.2), and stored at -80 ºC to prevent degradation.

RNA extraction and cDNA synthesis - Pooled samples were homogenized using a plastic pestle and RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. An additional DNase treatment (RNase-free DNase set, Qiagen) was performed to eliminate potential genomic DNA contamination. RNA of CA was extracted using the

RNAqueous®-Micro Kit (Ambion). DNase treatment was performed to eliminate genomic DNA contamination. The quantity and quality of RNA was determined using a Nanodrop spectrophotometer (Thermo Scientific). An equal amount of RNA was transcribed with

Superscript III reverse transcriptase (Invitrogen Life Technologies) utilizing random hexamers as described in the protocol. The resulting cDNA was diluted tenfold.

Quantitative Real Time-PCR (q-RT-PCR) - Prior to target gene profiling, Tubulin and EF1a were chosen as the optimal housekeeping genes according to a previous study (Marchal et al., 2013b).

The q-RT-PCR reactions were performed in triplicate on a CFX384 Touch ™ Real-Time PCR

Detection System (Bio-Rad) in a final volume of 10 µl, containing 1 µl of cDNA, 5 µl IQ™

SYBR® Green Supermix (Bio-Rad), 1 µl forward and reverse primer (5 µM) and 2 µl of MQ- water. The reaction was incubated for 3 min in 95ºC, followed by 40 cycles with following thermal profile: 95ºC, 10 s; 59ºC, 30 s. Target specificity was confirmed by performing a dissociation protocol (melt curve analysis) and running a few representative q-RT-PCR products on an agarose gel containing GelRed™ (Biotium). Realtime primers used for q-RT-PCR are listed in Table S3.1. Primer sets were validated by determining relative standard curves for each gene transcript using a five-fold serial dilution of a calibrator cDNA sample. Efficiency and correlation coefficient (R²) are shown in Table S1. The primer sets for genes in the JH 110 biosynthetic pathway were chosen as in Chapter 2. The quantity of mRNA for each tested gene relative to reference genes was determined as described by Vandesompele et al. (Vandesompele et al., 2002).

Peptides and substrates - Thirteen Dippu-ASTs were custom synthesized by GL Biochemical

Ltd., Shanghai (China). Peptides were purified by high performance liquid chromatography

(HPLC) (purity ≥95%). Peptides were dissolved in water to obtain a concentration of 1mM.

Peptide solutions were stored at -80 ºC prior to further processing and dilution.

Acetyl-CoA, mevalonic acid (MA), diphosphomevalonate (DPPM) and farnesol were purchased from Sigma-Aldrich Canada. Substrates were dissolved in water before use.

Cell culture and transfection - Chinese hamster ovary (CHO) WTA11 and PAM28 cells stably expressing apoaequorin (Euroscreen, Belgium) and human embryonic kidney (HEK) 293 cells were cultured in monolayers in Dulbecco’s Modified Eagles Medium nutrient mixture F12-Ham

(DMEM/F12) (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen),

100 IU/ml penicillin and 100 µg/ml streptomycin (Invitrogen). An additional 250 µg/ml Zeocin

(Invitrogen) was added to the medium for CHO-WTA11 cells, and an additional 5 µg/ml

Puromycin (Sigma) was added to the medium for PAM cells. The cells were cultured at 37ºC with a constant supply of 5% CO2.

Transfections with pcDNA3.1D-Dippu-AstR or empty pcDNA3.1D vector were carried out in

T75 flasks at 60 to 80% confluency. Transfection medium for CHO cells was prepared using the

Lipofectamine LTX kit (Invitrogen) with 3.75ml Opti-MEM, 7.5 µg vector construct and 18.75

µl Plus™ Reagent in a 5 ml polystyrene round-bottom tube. After 5 min incubation at room temperature, 45 µl LTX (Invitrogen) was added. Transfection medium was added dropwise to the cells after 30 min incubation at room temperature. The transfection medium of the HEK293 111 cells was similar to that of the CHO cells, except that 9 µg DNA construct (6 µg of vector construct and 3 µg of reporter gene plasmid) was added to the medium. The luciferase ORF, downstream of a cAMP responsive element (CRE) served as the reporter gene. After transfection, cells were incubated overnight and an additional 10 ml of culture medium was added. The cells were allowed to grow for another night prior to intracellular Ca2+ or cAMP measurements.

Aequorin assay - CHO cells were detached using 1× Phosphate Buffered Saline (PBS) containing 0.2% EDTA (pH 8.0), collected and pelleted by centrifugation in DMEM/F12 medium. The number of viable and nonviable cells was determined using a NucleoCounter NC-

100™ (Chemometic). The cells were then resuspended to a density of 5×106 cells/ml in sterile filtered DMEM/bovine serum albumin (BSA) medium (DMEM/F12 with L-glutamine and 15 mM HEPES, without Phenol red, supplemented with 0.1% BSA). A concentration of 5 µM coelenterazine h (Invitrogen) was added and the cells were incubated for 4 h in the dark at room temperature with gentle shaking to reconstitute the holo-enzyme aequorin. The cells were then diluted 10-fold in BSA medium and incubated for another 30 min in the dark with gentle shaking.

Peptides were dissolved in BSA medium and dispensed in 50 µl aliquots into the wells of a white

96-well plate. Fifty µl of the cell suspension was injected into each well and light emission was recorded using a Mithras LB 940 multimode microplate reader (Berthold Technologies) over 30 sec. The cells were lysed by injection of 50 µl 0.3% Triton X-100 and light emission was monitored for an additional 8 sec. The total response (ligand + Triton-X100) is the representative for the quantity of viable cells present in the well. BSA medium was used as a negative control in each row of the plate, and 1 µM ATP served as a positive control. The negative response was subtracted from the luminescence measured in wells of the same row. Calculations were made 112 using the output file from Microwin software (Berthold Technologies) in Excel (Microsoft).

Further analysis was done in Excel and GraphPad Prism 6. cAMP reporter assay - HEK cells were detached using 1× PBS containing 0.2% EDTA (pH 8.0), collected and pelleted by centrifugation in DMEM/F12 medium. The number of viable and nonviable cells was determined using a NucleoCounter NC-100™ (Chemometic). The cells were then resuspended to a concentration of 1×106 cell/ml in DMEM/F12 containing 200 µM 3- isobutyl-1-methylxanthine (IBMX, Sigma) to prevent cAMP breakdown. Peptides were dissolved in IBMX medium in the presence or absence of 20 µM forskolin, and were dispensed in 50 µl aliquots into the wells of a white 96-well plate. Fifty µl of cell suspension was added into each well and the plate was incubated in a CO2 incubator (5% CO2) at 37ºC for 3.5 h. Fifty

µl of Steadylite Plus substrate (PerkinElmer) was then added to each well and the plate was incubated in the dark for 15 min while gently shaking. Light emission resulting from the luciferase enzymatic activity was recorded for 5 s/well using a Mithras LB940. Results were analyzed using the output of MicroWin and further processed by Excel and GraphPad Prism 6.

Radiochemical assay (RCA) - The in vitro RCA for JH biosynthesis was performed as described by Feyereisen and Tobe (Feyereisen and Tobe, 1981) and modified by Tobe and Clarke (Tobe and Clarke, 1985). Two incubations were conducted in the JH biosynthesis rescue experiments.

CA were incubated in TC199 medium with 10-6 M AST7 for 3h, and then transferred to fresh medium with 10-6 M AST7 and JH precursors for a second 3h incubation. JH biosynthesis was determined after each incubation. The rate of JH biosynthesis during the first incubation was used as the control value.

RNA interference (RNAi) - Dippu-AstR and Dippu-AST dsRNA constructs were prepared using the MEGAscript® RNAi Kit (Ambion). Primers used are given in Table S3.2. The Fragments 113 were amplified by PCR using forward and reverse primers with T7 promoters

(TAATACGACTCACTATAGGGAGA) attached to the 5’ end. Fragments were then subcloned and sequenced to verify the presence of the T7 promoter. The amplified fragment was used in an

RNA transcription reaction and incubated overnight to yield annealed dsRNA transcripts. A nuclease digestion was subsequently performed to remove ssRNA and DNA remaining in the product. The dsRNA was further purified according to the manufacturer’s instructions (Ambion).

Concentration of the dsRNA construct was determined using a nanodrop instrument (Thermo

Fisher Scientific Inc.). Five-fold diluted dsRNA was run on a 1.2% agarose gel to examine the quality and integrity of the construct. The control dsRNA was prepared using a PCR fragment amplified from a non-coding region of pJET1.2 cloning vector (Thermo Scientific).

Dippu-AstR dsRNA was diluted in cockroach saline to a concentration of 250ng/µl. Each adult female was injected with 4 µl of dsRNA solution on day 0, 2 and 4 after the final moult. CA were dissected on day 6 and stored in liquid nitrogen prior to RNA extraction. A second set of cockroaches was injected with Dippu-AstR dsRNA in the same scheme as described above. CA were dissected on day 6 and cleaned in TC199 medium (GIBCO; 1.3 mM Ca2+, 2% Ficoll, methionine-free) for their use in the radiochemical assay (RCA) determining the effect of AST on JH biosynthesis. Basal oocyte length was measured during dissection.

Dippu-AST dsRNA was diluted in cockroach saline to a concentration of 250ng/µl. Each adult female was injected with 4 µl of dsRNA solution on day 0, 2, 4 and 5 after the final moult. Brain were dissected on day 6 and stored in liquid nitrogen prior to RNA extraction. CA were dissected on day 6 and cleaned in TC199 medium (GIBCO; 1.3 mM Ca2+, 2% Ficoll, methionine-free) for their use in the radiochemical assay (RCA) determining the effect of AST on JH biosynthesis.

Basal oocyte length was measured during dissection. 114

Data analysis – Data set were analyzed by T-test to determine the significant difference. All results were expressed as mean ± SEM, and considered significantly different at P ≤ 0.05.

Results

Tissue distribution of Dippu-AST and Dippu-AstR

The tissue distribution of Dippu-AST and Dippu-AstR was determined in adult male and female cockroaches using q-RT-PCR (Fig. 3.1). Nine tissues were used to examine the tissue specificity of Dippu-AST and Dippu-AstR: brain (Br), nerve cord (NC), corpora allata (CA), fat body (Fb), ovary (Ov), midgut (MG) and Malpighian tubules (MT) from females and accessory gland (AG) and testes (Te) from males. The Dippu-AST gene is expressed in brain, nerve cord and midgut, which is consistent with the pleiotropic functions of ASTs (Fig. 3.1). Dippu-AstR shows the highest transcription levels in the CA, followed by nerve cord, brain and fat body. All the other tissues tested showed either negligible or undetectable levels of Dippu-AstR mRNA.

Developmental expression during the first gonadotrophic cycle of females

We determined the developmental profile of Dippu-AstR in the CA, brain and fat body of mated females during their first gonadotrophic cycle (Fig. 3.2). In CA, the Dippu-AstR mRNA declined on days 2-4 and then increased significantly on day 6. The transcript level of Dippu-AstR in the

CA was inversely correlated with the rate of JH biosynthesis (Fig. 3.2A). In brain, on the other hand, transcript levels of Dippu-AstR and Dippu-AST showed only minor variations (Fig. 3.2B).

The transcript level of Dippu-AstR in fat body was relatively low, with the highest level of mRNA expression on day 4, and negligible on all other days (data not shown).

115

t i

t D ip p u -A s tR n a D ip p u -A S T

u 4

e 2

e R

0 B r N C C A F b O v T e AG M G M T

Figure 3.1. Relative expression levels of Dippu-AstR (black) and Dippu-AST (gray) mRNA in tissues of day 4 males and mated females. The data represent averages of 3 pools (10 animals per pool), run in triplicate. Tissues tested are brain (Br), nerve cord (NC), corpora allata (CA), fat body (FB), ovary (Ov), midgut (MG) and Malpighian tubules (MT) from females and testes (Te) and accessory gland (AG) from males. Values represent mean ± SEM.

116

D ip p u -A s tR R

J H b io s y n th e s is J

t 2 0 2 5

H s

o o

2 0

s y

1 5 y

t i

n t

t n

h a

1 5 e u

i q

1 0

( A

1 0 m

N R

l m

5 h e

5 /

i t

A a

) l

e 0 0 R D 0 D 1 D 2 D 3 D 4 D 5 D 6 D 7 B

R R

t 2 .0 D ip p u -A s tR 4

a s

t A

f D ip p u -A S T

y 1 .5 3

t i

R t

n a

A u

q q

1 .0 2

u A

n N

t R

0 .5 1

o e

f v

t a

S l

e 0 .0 0 R D 0 D 1 D 2 D 3 D 4 D 5 D 6 D 7

Figure 3.2. Relative expression levels of Dippu-AstR and Dippu-AST mRNA during the first gonadotrophic cycle. (A) Dippu-AstR mRNA expression and JH biosynthesis in CA. Dippu- AstR mRNA levels were quantified by q-RT-PCR and normalized against levels of Armadillo and EF1a mRNA (Marchal et al., 2013b). The data represent the average of 3 biologically independent pools (10 animals per pool), run in triplicate. Rate of JH biosynthesis was measured using the RCA, n≥10. (B) Dippu-AstR and Dippu-AST mRNA expression in brain. Dippu-AstR and Dippu-AST mRNA levels were normalized against levels of Tubulin and EF1a mRNA (Marchal et al., 2013b). Values represent mean ± SEM.

117

Effect of Dippu-AST dsRNA on JH biosynthesis

To determine the controlling role of AST in JH biosynthesis, we knocked down Dippu-AST using RNAi. Injecting Dippu-AST dsRNA results in a 91% decrease of Dippu-AST mRNA in the brain. However, JH biosynthesis of CA did not show any significant change in the dsRNA treated animals (Fig. 3.3).

Functional activation of Dippu-AstR with ASTs

We initially expressed Dippu-AstR in CHO-WTA11 cells. This cell line expresses the Ca2+ reporter apoaequorin and Gα16, a promiscuous G protein that couples to most GPCRs with subsequent mobilization of intracellular Ca2+ (Stables et al., 1997). As shown in Fig. 3.4, all 13 tested ASTs induced clear dose-dependent bioluminescence responses in AstR expressing cells.

Most of the peptides showed similar degrees of biological efficacy (ability to activate AstR) but differed considerably in potency, with EC50 values ranging from 0.2 nM for AST6 to 30 nM, in the case of AST13 (Table 3.1). In general, the abilities of ASTs to activate AstR corresponded to their potencies as inhibitors of JH production by the CA, with the exception of AST1, AST5 and

AST6 (Tobe et al., 2000) (Table 3.1).

To determine the second messenger pathways involved in Dippu-AstR activation, we first expressed Dippu-AstR in CHO-PAM28 cells (lacking the promiscuous Gα16). The expression of apoaequorin in the cell allows testing whether the receptor can couple naturally through intracellular Ca2+. Two ASTs (AST5 and 6) were chosen because of their high potency in inhibiting JH biosynthesis or in activating Dippu-AstR. As shown in Fig. 3.5A, AST5 and 6 induced dose-dependent intracellular Ca2+ responses in AstR-transfected CHO-PAM28 cells, with EC50 values of 21.4 nM and 1.1 nM, respectively. 118

We subsequently tested whether the receptor coupled with cAMP in the signal transduction pathway. We expressed Dippu-AstR in HEK293 cells, which contain the luciferase gene under the control of a cAMP response element. We assayed different concentrations of AST5 and

AST6 in the presence or absence of forskolin. Forskolin activates adenylate cyclase (AC), which increases intracellular levels of cAMP. If AstR would couple negatively to AC, we would expect lower levels of cAMP following application of ASTs. This was not the case. Application of

AST5 or AST6 to transfected HEK cells did not cause any significant change in cAMP level.

However, in the absence of forskolin, AST5 and AST6 treatments resulted in a dose-dependent increase in cAMP concentration, as measured by assay of luciferase activity (Fig. 3.5B). The

EC50 values were 287.8 nM for AST5 and 24.3 nM for AST6. The EC50 values for the cAMP- based luciferase assay in HEK293 cells were higher than for the Ca2+ responses detected in either of the two CHO cell lines, but the relative order of ligand potency was maintained. CHO-

WTA11, CHO-PAM28 and HEK293 cells transfected with pcDNA3.1D (empty vector) did not show any response to ASTs.

AST does not affect the transcript level of enzymes in the JH biosynthetic pathway

The injection of AstR dsRNA on day 0, 2 and 4 resulted in a 67% knockdown of AstR mRNA levels in CA of day 6 females (Fig. 3.6A). The JH biosynthetic activity of CA was measured using the RCA. In AstR knockdown animals, JH production increased by 60% and the response of CA to AST decreased by 28% (Fig. 3.6B and 3.6C).

Eleven of 13 genes encoding enzymes in the JH biosynthetic pathway have been identified

(Chapter 2). To determine the target of AST action in the JH biosynthetic pathway, we measured the mRNA levels of the 11 genes encoding enzymes catalyzing different steps in the JH biosynthetic pathway. Although AstR dsRNA treatment resulted in an increase in the rate of JH 119 biosynthesis, none of the relative transcript levels showed significant changes (Fig. 3.6D). The high variation of the transcript level of JHAMT on day 6 can be explained by previous results that show a great degree of fluctuation in JHAMT mRNA levels at times when rates of JH biosynthesis are highly dynamic (see Chapter 2).

To confirm our results, we incubated CA in medium containing 10-7 M AST7 for 3 hours, which results in a 60 to 70% decrease in JH biosynthesis (Tobe et al., 2000), and determined the transcript level of the enzymes in the CA. As in the AstR RNAi experiments, the mRNA levels of the 11 genes tested show no significant changes (Fig. 3.7).

JH precursors reverse AST-induced inhibition of JH biosynthesis

Earlier studies showed that mevalonate and farnesol were able to partially reverse AST-induced inhibition of JH biosynthesis (Pratt et al., 1991; Pratt et al., 1989). To determine which enzyme(s) is/are affected by ASTs in the JH biosynthetic pathway, we treated CA with select JH precursors in the presence of 10-6 M AST7. If the activity of enzymes was inhibited by AST, the JH precursors prior to the enzyme will not be able to reverse the inhibition of AST. As shown in Fig.

3.8, all exogenous JH precursors acetyl-CoA, mevalonic acid, diphosphomevalonate and farnesol significantly stimulated the rate of JH biosynthesis in the presence of AST7.

120

A B T

1 .5 ) C o n tro l S

A 2 5

f D ip p u -A S T

/ d sR N A

/ l

y 2 0

t m

n 1 .0

p (

u 1 5

e N

h 1 0 R

0 .5 t

s v

i 5

i a

*** b

0 .0 H

R 0 J c o n tro l D ip p u -A S T d s R N A C o n tro l D ip p u -A S T d s R N A

Figure 3.3. The effect of Dippu-AST dsRNA on JH biosynthesis by the CA. (A) Efficiency of Dippu-AstR RNAi-mediated knockdown in the brain of day 6 mated females. Relative quantity of Dippu-AstR mRNA levels in CA was compared between control and AST dsRNA treated animals. (B) JH biosynthesis by CA from Dippu-AST dsRNA-treated animals. Glands were taken from day 6 mated females, n≥10. The quantity of mRNA data represent averages of 3 pools (8 pairs of CA per pool) run in triplicates using q-RT-PCR and normalised to Tubulin and EF1a mRNA (Marchal et al., 2013b) . Vertical bars indicate S.E.M. . Significant differences are indicated by asterisks (*P < 0.05) 121

A S T 1 A S T 2 A S T 3 A S T 4 E C 5 0 = 1 .2 9 5 e -0 0 9 1 2 0 1 2 0 E C 5 0 = 2 .0 3 3 e -0 0 9 1 2 0 E C 5 0 = 2 .8 2 9 e -0 0 9 1 2 0 E C 5 0 = 2 .4 8 9 e -0 0 9

1 0 0 1 0 0 1 0 0 1 0 0

e e e e

c c c c

n n n

n 8 0 8 0 8 0 8 0

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c c c c

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m 4 0 4 0 4 0 4 0

u u u u

l l l l

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i i i

i 2 0 2 0 2 0 2 0

b b b b

% % % % 0 0 0 0 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -2 0 lo g [A S T 1 ] (M ) -2 0 lo g [A S T 2 ] (M ) -2 0 lo g [A S T 3 ] (M ) -2 0 lo g [A S T 4 ] (M )

A S T 5 A S T 6 A S T 7 A S T 8

1 2 0 E C 5 0 = 4 .2 1 4 e -0 0 9 1 2 0 E C 5 0 = 1 .9 2 9 e -0 1 0 1 2 0 E C 5 0 = 2 .1 3 5 e -0 0 9 1 2 0 E C 5 0 = 4 .0 8 7 e -0 0 9

1 0 0 1 0 0 1 0 0 1 0 0

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c c c

n n

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b b b

% % % % 0 0 0 0 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -2 0 lo g [A S T 5 ] (M ) -2 0 lo g [A S T 6 ] (M ) -2 0 lo g [A S T 7 ] (M ) -2 0 lo g [A S T 8 ] (M )

A S T 9 A S T 1 0 A S T 1 1 A S T 1 2

1 2 0 E C 5 0 = 5 .6 5 4 e -0 0 9 1 2 0 E C 5 0 = 7 .7 1 7 e -0 1 0 1 2 0 E C 5 0 = 2 .7 4 9 e -0 0 9 1 2 0 E C 5 0 = 1 .0 4 4 e -0 0 8

1 0 0 1 0 0 1 0 0 1 0 0

e e e

c c c

n n

n 8 0 8 0 8 0 8 0

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c c c

s s s

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e 6 0 6 0 6 0 6 0

n n n

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m m

m 4 0 4 0 4 0 4 0

u u u

l l l

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i i

i 2 0 2 0 2 0 2 0

b b b

% % % 0 0 0 0 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -2 0 lo g [A S T 9 ] (M ) -2 0 lo g [A S T 1 0 ] (M ) -2 0 lo g [A S T 1 1 ] (M ) -2 0 lo g [A S T 1 2 ] (M )

A S T 1 3

1 2 0 E C 5 0 = 2 .9 9 9 e -0 0 8

1 0 0

e c

n 8 0

c s

e 6 0

n i

m 4 0

l o

i 2 0

% 0 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -2 0 lo g [A S T 1 3 ] (M )

Figure 3.4. Dose-response curves for ASTs in CHO-WTA11 cells expressing Dippu-AstR. Data points represent the average ± SEM of three independent measurements performed in duplicate and are expressed as percentage of the maximal response. The zero response level corresponds to treatment with BSA buffer only. 122

A D ip p u -A s tR in C H O -P A M 2 8 B D ip p u -A s tR in H E K 2 9 3

1 2 0 1 2 0

A S T 5 A S T 5 e

1 0 0 e 1 0 0

c n

A S T 6 n A S T 6 e

8 0 e 8 0

s e

6 0 e 6 0

i m

4 0 m 4 0

o i

2 0 i 2 0

% % 0 0

-2 0 -2 0 -1 4 -1 3 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 -1 2 -1 1 -1 0 -9 -8 -7 -6 -5 -4 C o n c e n t r a t io n lo g [ M ] C o n c e n t r a t io n lo g [ M ]

Figure 3.5. Dose-response curves for the bioluminescence response induced in (A) CHO-

PAM28 and (B) HEK293 cells expressing Dippu-AstR. Data points represent the average ±

SEM of three independent measurements performed in duplicate and are expressed as a percentage of the maximal response. The zero response level corresponds to treatment with BSA buffer only. Peptides tested are AST5 (squares) and AST6 (triangles).

123

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l y

1 .5 h t

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i m

1 0

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0 .5 2 0

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e 0 .0 0 0 R c o n tro l A s tR d s R N A c o n tro l A s tR d s R N A c o n tro l A s tR d s R N A

D c o n tro l

5 y

t A s tR d s R N A

i t

n 4

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m 2

i t

a 1

e R 0 T h io l H M G S H M G R M K P M K P P -M e v D IP P I F P P S F O L D J H A M T C Y P 1 5

Figure 3.6. The effect of Dippu-AstR dsRNA on JH biosynthesis by the CA and on the expression of genes encoding enzymes in the JH biosynthetic pathway of D. punctata. (A) Efficiency of Dippu-AstR RNAi-mediated knockdown in mated females. Relative quantity of Dippu-AstR mRNA levels in CA was compared between control and AstR dsRNA treated animals. (B) JH biosynthesis by CA from Dippu-AstR dsRNA-treated animals. Glands were taken from day 6 mated females, n≥10. (C) Effect of 10-7 M Dippu-AST7 on JH biosynthesis by CA from control and AstR knockdown animals. CA were incubated first in normal medium for 3h and subsequently incubated in medium with 10-7 M AST7 for another 3h. The percentage of inhibition was calculated using the rate of JH biosynthesis during the first and second incubation. (D) The effect of silencing Dippu-AstR on the transcript levels of genes encoding enzymes in the JH biosynthetic pathway. Enzyme abbreviations are as described in Figure S1. The mRNA quantity data represent averages of 3 pools (8 pairs of CA per pool) run in triplicates using q-RT- PCR. Vertical bars indicate S.E.M.. Significant differences are indicated by asterisks (*P < 0.05).

124

2 0 y

t c o n tro l

i t

n tre a tm e n t

a 1 5

N 1 0

v i

t 5

e R 0 AC AT H M G S H M G R M K P M K P P M e v D IP P I F P P S F AL D J H AM T E P O X

Figure 3.7. The effect of AST on the expression levels of genes encoding enzymes in the JH biosynthetic pathway in CA of day 6 mated female D. punctata. CA were incubated for 3h in normal (control) medium or medium supplemented with 10-7 M AST7 (treatment) and then washed with cockroach saline prior to the RNA extraction. RNA was extracted from CA in control and treatment groups to determine the transcript level of genes encoding enzymes in JH biosynthetic pathway. Enzyme abbreviations are as described in Figure S1. The data represent averages of 3 pools (8 pairs of CA per pool) run in triplicate using q-RT-PCR. Vertical bars indicate S.E.M.

1 5

) h / c o n tr o l

A ***

C /

l J H p re c u rs o r

o 1 0

(

s ***

a 5 e

l ***

e ***

H J 0 Ac e tyl C o A M A M AP T F a r n e s o l

Figure 3.8. JH precursors rescue the AST-induced JH inhibition. JH biosynthesis was evaluated in CA that were first incubated in medium TC199 with 10-6 M AST7 (control), and then in medium with 10-6 M AST7 together with the individual precursors: Acetyl-CoA (100 µM), MA (100 µM), DPPM (100 µM) or farnesol (40 µM). CA were dissected from day 7 mated females. Each data point represents mean ± SEM (n ≥ 10) Significant differences are indicated by asterisks (***P < 0.001).

125

a Table 3.1. Potency of Dippu-ASTs : activation of AstR in CHO-WTA11 cells (EC50) or inhibitory effect on JH release (IC50)

Rank1 Rank2 1 2 EC50 IC50 Allatostatin order order Sequence /nM /nM (EC50) (IC50) AST1 1.295 3 9.566 11 Leu-Tyr-Asp-Phe-Gly-Leu-NH2 AST2 2.033 4 0.4254 3 Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val- Tyr-Asn-Phe-Gly-Leu-NH2 AST3 2.829 8 12.54 12 Ser-Lys-Met-Tyr-Gly-Phe-Gly-Leu-NH2 AST4 2.489 6 1.139 5 Asp-Gly-Arg-Met-Tyr-Ser-Phe-Gly-Leu-NH2 AST5 4.214 10 0.1063 1 Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 AST6 0.1929 1 2.725 8 Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 AST7 2.135 5 0.4118 2 Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2 AST8 4.087 9 3.275 10 Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 AST9 5.654 11 2 6 Gly-Asp-Gly-Arg-Leu-Tyr-Ala-Phe-Gly-Leu-NH2 AST10 0.7717 2 0.9225 4 Pro-Val-Asn-Ser-Gly-Arg-Ser-Ser-Gly-Ser-Arg-Phe-Asn-Phe-Gly-Leu-NH2 AST11 2.749 7 2.795 9 Tyr-Pro-Gln-Glu-His-Arg-Phe-Ser-Phe-Gly-Leu-NH2 AST12 10.44 12 2.011 7 Pro-Phe-Asn-Phe-Gly-Leu-NH2 AST13 29.99 13 12.84 13 Ile-Pro-Met-Tyr-Asp-Phe-Gly-Ile-NH2 a Potency is defined as the dose required to achieve a given level of activation of AstR or 1 inhibition of JH biosynthesis, and are listed in rank order. EC50 was determined by AstR 2 activation assay in CHO-WTA11 cells (details seen Fig. 1). IC50 values are as reported by (Tobe et al., 2000) (CA from day 2 virgin female).

126

Discussion

Our observations on the expression of the Dippu-AstR and Dippu-AST provide insights into the tissue-specific interaction between the ligands and the receptor. In brain and nerve cord, both

Dippu-AstR and Dippu-AST are expressed, whereas in CA, only the receptor is expressed. This is consistent with observations that AST is delivered to the CA by nerves from neurosecretory cells in the brain (Stay et al., 1992). In the midgut, however, Dippu-AST is expressed, but its receptor is not (Fig. 3.1); tissue distribution of Dippu-AstR and Dippu-AST in day 7 animals provided similar results (Fig. S3.1). Previous studies suggest that ASTs can induce myotropic activity in gut tissues (Duve et al., 1995; Lange et al., 1995). Furthermore, putative receptors for ASTs in midgut were partially characterized using a radioligand-binding assay (Bowser and Tobe, 2000).

Therefore, the undetectable transcript level of Dippu-AstR in midgut suggests that one or more additional AstR in D. punctata may exist, just as two receptors for allatostatins have been identified in Drosophila melanogaster (Birgul et al., 1999; Lenz et al., 2000).

The sensitivity of CA to ASTs differs between animals of different ages (Pratt et al., 1990; Stay et al., 1991). CA appear more sensitive to AST treatment when JH biosynthetic activity is low than at stages of high JH production. In the present study, we have shown that the transcript levels of Dippu-AstR in the CA correlate with changes in the sensitivity of the CA to ASTs (Fig.

3.2A). It is therefore reasonable to suggest that the changing transcript levels of Dippu-AstR are partly responsible for the changes in JH biosynthesis during the first gonadotropic cycle. The ability of ASTs to inhibit JH biosynthesis depends not only on the sensitivity of the CA, but also on the concentration of the ASTs (Stay et al., 1996). q-RT-PCR of the AST precursor gene showed that there was no significant change in the transcript level of ASTs (Fig. 3.2B). Lloyd et al (2000) determined the quantity of AST in CA and brain by ELISA using antibody against

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Dippu-AST7, and found no significant change in the content of AST7 during the first gonadotropic cycle. Meanwhile, silencing the AST precursor gene did not cause any significant increase of JH production (Fig. 3.3). Same result was observed in the German cockroach, B. germanica (Maestro and Belles, 2006). These results suggest that the AST-inhibition of JH biosynthesis by AST is controlled by the change of expression of the AST receptor.

To functionally analyze AstR, we expressed the Dippu-AstR in mammalian cells and activated it with Dippu-AST. We first expressed Dippu-AstR in CHO cells, demonstrating that it is indeed a

Diploptera allatostatin receptor. All thirteen ASTs are very potent in activating AstR expressed in CHO-WTA11 cells with EC50 values in the low nanomolar range (Fig. 3.4). Despite the structural similarities, there were some differences in the response to individual peptides. AST13, which contains isoleucine at the C-terminus, exhibited the highest EC50. This result reinforces the importance of the C-terminal pentapeptide motif Y/FXFGL-NH2. Moreover, the amino acids outside of the C-terminal pentapeptide motif appear to determine the affinity of AST for AstR as well. AST5 and AST6 have the same pentapeptide motif (Table 3.1), whereas their ability to activate the receptor is significantly different. The change of Pro to Leu, and Ala to Asp decreases AST affinity of AstR by 20-fold.

A P. americana allatostatin receptor was expressed in Xenopus oocytes, and the potency of different Periplaneta ASTs in activating Peram-AstR was determined (Gade et al., 2008). Five

Dippu-ASTs (AST 1, 2, 3, 6 and 13) have the same structure as in P. americana (AST 1, 2, 3, 6 and 14). And the rank order of potency of these ASTs in activating Dippu-AstR expressed in

CHO-WTA11 cells is similar to that of P. americana receptor (Gade et al., 2008). However, the potencies of AST 1, 5 and 6 in activating AstR differ from their capacities to inhibit JH production in vitro (Table 1). AST 5, which showed the highest inhibitory activity, demonstrated

128 only a moderate potency in activating the expressed Dippu-AstR, ranking 10 out of 13. In contrast, AST1, which was one of the weakest inhibitors of JH biosynthesis in vitro (ranking 11 out of 13), appears to be one of the most potent peptides in receptor activation. AST6, the structure of which is conserved in several insect orders, showed the highest potency in activating expressed Dippu-AstR (Bendena et al., 1999). Its ability to inhibit JH biosynthesis, on the other hand, was moderate. Different reasons could contribute to the differences between potency in activating the receptor and inhibiting JH biosynthesis in vitro. The susceptibility of ASTs to degradation by enzymes in the CA differs between ASTs, which could result in different potencies in inhibiting JH biosynthesis. AST5, which showed high resistance to the degradation by hemolymph and membrane preparations, presented the highest activity in inhibiting JH biosynthesis (Garside et al., 1997a, b). Furthermore, tests with cloned receptors in mammalian cells might not fully reflect the actual situation in insects. The posttranslational modifications of

AstR or the interacting proteins present in receptor-expressing cells could affect the conformational and functional properties of this receptor. Nevertheless, we here chose mammalian cell lines because there may be fewer interacting proteins in this heterologous system than in insect cell lines.

The inhibition of JH biosynthesis by Dippu-ASTs is a complex process, probably involving more than one second messenger. Previous studies showed that ASTs regulate JH biosynthesis by acting through the inositol trisphosphate (IP3)/diglyceride (DAG) second messenger systems, in which protein kinase C (PKC) and Ca2+ are involved (Feyereisen and Farnsworth, 1987b;

Rachinsky and Tobe, 1996; Rachinsky et al., 1994). However, the role of Ca2+ in regulation of

JH biosynthesis is confounding. JH production shows dose-dependency on extracellular Ca2+ in the medium, whereas the Ca2+ ionophore A23187 caused a rapid decline in JH release

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(Kikukawa et al., 1987). Inhibition of JH biosynthesis by brain extracts was antagonized by inorganic Ca2+channel blockers (Feyereisen and Farnsworth, 1987a). However, elevation of intracellular Ca2+ levels by treating CA with the Ca2+-mobilizing drug thapsigargin diminished the inhibitory effect of ASTs (Rachinsky et al., 1994). The activation of Dippu-AstR induced a dose-dependent intracellular Ca2+ response in CHO-PAM28 cells (Fig. 3.5A), which supports the involvement of Ca2+ as a second messenger of AST. Aucoin et al (1987) suggested that there may be both a Ca2+-dependent and a Ca2+-independent pathway (cAMP) involved in the inhibition of JH biosynthesis. Incubating CA with brain extracts resulted in an increase in the level of cAMP and a decrease in JH biosynthesis. Forskolin, which causes a dose-dependent accumulation of cAMP, led to a rapid and dose-dependent inhibition of JH biosynthesis.

Moreover, the sensitivity of CA to forskolin shows the same pattern as its sensitivity to AST during the first gonadotrophic cycle (Feyereisen and Farnsworth, 1987a; Meller et al., 1985). In contrast, another study suggest that levels of cAMP do not increase following treatment of CA with ASTs. Our findings demonstrated that the activation of Dippu-AstR induced a dose- dependent cAMP response in HEK293 cells, which suggests that cAMP may be involved in the signaling pathway of AST cells (Fig. 3.5B). Dippu-AstR appears to dually couple through the

2+ Gαs and Gαq pathways and triggers the release of intracellular Ca and the increase in cAMP levels. Studies on Drosophila AstRs suggested that DAR-1 and DAR-2 couple to Gi/o mediated signaling and other G-proteins (Birgul et al., 1999; Larsen et al., 2001). However, activation of

AstR in the presence of forskolin did not significantly influence the cAMP signal. This result suggests that there is no involvement of Gαi in the Dippu-AstR pathway.

AST regulates JH biosynthesis by affecting either enzymes directly involved in the JH biosynthetic pathway or steps prior to the production of acetyl-CoA. We have studied the effect

130 of AST on the transcript levels of genes in the JH biosynthetic pathway. The inhibitory effect of

AST on JH biosynthesis was rapid and reversible, which suggests that AST may not regulate JH biosynthesis through transcript levels ((Pratt et al., 1991; Pratt et al., 1989). Our study confirmed this earlier assumption by revealing that the transcript levels of genes in the JH biosynthetic pathway were not affected by AstR silencing or AST treatment in vitro (Fig. 3.6 and 3.7).

Previous studies indicate that ASTs regulate JH biosynthesis through steps(s) prior to the production of mevalonate (Pratt et al., 1991; Pratt et al., 1989). However, the enzyme activities of HMG-CoA synthase or HMG-CoA reductase were not affected by AST (Sutherland and

Feyereisen, 1996). To determine which enzyme in the JH biosynthetic pathway was influenced by AST, we used several JH precursors to reverse the AST-induced inhibition of JH biosynthesis.

All the JH precursors (including acetyl-CoA) were able to partially restore JH biosynthesis, thereby suggesting that the enzyme activities were not affected by AST. The target of AST action probably lies prior to the start of the JH biosynthetic pathway in regulating the production of precursors of acetyl-CoA such as the transport of citrate from mitochondria to cytosol and/or the cleavage of citrate to yield acetyl-CoA (Sutherland and Feyereisen, 1996). Further investigation is needed to identify the exact target of AST.

In D. punctata, ASTs play an important role in the regulation of JH biosynthesis. The potency of the 13 ASTs in activating AstR reveals the structure-activity relationship of AST action. The activation of AstR in CHO and HEK cells suggests that both Ca2+ and cAMP can be involved in the signal transduction of AST. Our results show that AST does not affect the transcript levels and that the activities of enzymes in the JH biosynthetic pathway remain normal following treatment with AST. AST probably affects JH biosynthesis prior to the entry of precursors into the JH biosynthetic pathway. The exact target of AST action remains an open question.

131

Supplementary data

Table S3.1 Oligonucleotide sequences for primers used in q-RT-PCR for reference and target genes. Efficiencies and R² values are indicated.

Reference genes F-primer R-primer Efficiency (%) R2

Tubulin 5'-AAATTACCAACGCTTGCTTTGAA-3' 5'-TGGCGAGGATCGCATTTT-3' 95.1 0.993

EF1α 5'-TCGTCTTCCTCTGCAGGATGTCT-3' 5'-GGGTGCAAATGTCACAACCATACC-3' 99.2 0.994

Armadillo 5'-GCTACTGCACCACTCACAGAATTATT-3' 5'-CTGCAGCATACGTTGCAACA-3' 94.5 0.980

Target genes F-primer R-primer Efficiency (%) R2

AstR 5’-GCCATTTGGAGAAATCTGGT-3’ 5’-GAACCGACATGGAAGTGATG-3’ 95.0 0.974

AST 5’-GGCAAAAGAGCAAGACCTTACAG-3’ 5’-CCTCCTCGCTTGCCAAGTC-3’ 95.6 0.997

Thiol 5’-TGCCTTCCAAAAGGAGAATG-3’ 5’- ACATCACCTGCCATCAACAC-3’ 90 0.970

HMGS 5’- TGCTGGGAAGTACACAGCAGG-3’ 5’- CTCCACGAGCTTGCTGACTG-3’ 83 0.994

HMGR 5’-TGGGAGCATGTTGTGAAAAT-3’ 5’-ACCAAGCAGCCCTCAGTAGT-3’ 95 0.988

MK 5’- TACGGCAAAACTGCCCTTGC-3’ 5’- AATGGAGGAGGTTCGGCGA-3’ 93 0.996

PMK 5’- TACGAAAACAACGAGGATGG-3’ 5’- TTCTGCATCATCTACACCTTCA-3’ 100 0.985

PP-MevD 5’- TGGAAGGTGACATAACAGCAA-3’ 5’- ATCCTTGATGCCAGTGAACA-3’ 90 0.967

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Table S3.1 (Continue) Oligonucleotide sequences for primers used in q-RT-PCR for reference and target genes. Efficiencies and R² values are indicated.

Target genes F-primer R-primer Efficiency (%) R2

IPPI 5’- CCTTCCCCAACCATGTAACT-3’ 5’- ACCAACGCCATTTGTCTCTT-3’ 100 0.992

FPPS 5’-TGCTTTGGAGATCCTGAGGT-3’ 5’- TGTTCAGGAGTGGTTCGTTG-3’ 96 0.987

FOLD 5’- TGGCGCGTAGGGTAGACAA-3’ 5’- GACCCATTTGAAAGCCTCCTTGA-3’ 93 0.993

JHAMT 5’- ATCCAGGTGCTGGAAGGAGAG-3’ 5’- CTGCCCAGAGTCGAACAGG-3’ 99 0.984

CYP15 5’- GTTGGGATCTCGGAGCATGG-3’ 5’- CGAACACGTCATGCATCGGT-3’ 100 0.992

Table S3.2. Primers for dsRNA construction Name F Primer(5'-3') R Primer(5'-3') AstR dsRNA TGTAATCATACGGCTAACGGATC AATGGTAGAACGTAGTCTGTTGC

AST dsRNA GTCTACCGTCGGCTCTTGTA TGCTTCTCTTGCCGAGACC

Control dsRNA TTGCGCTCACTGCCAATTGC CTGGCCTTTTGCTCACATGTT *The T7 promoter sequence was added at the 5' end of each dsRNA primer.

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1 5

D ip p u -A s tR

t i

t D ip p u -A S T

n a

u 1 0

e 5

e R

0 B r N C C A F b O v T e AG M G M T

Figure S3.1. Relative transcript levels of Dippu-AstR (black) and Dippu-AST (gray) mRNA in tissues of day 7 male and mated female D. punctata. Dippu-AstR/AST mRNA were quantified by q-RT-PCR. The data represent the average of 3 biologically independent pools (10 animals per pool), run in triplicate. Tissue abbreviations are as described in Figure 2.1. Values represent mean ± SEM.

134

References

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Chapter 4

Identification and characterization of the NMDA receptor and its role in regulating reproduction in the cockroach, Diploptera punctata

This chapter is an adapted reprint of my article:

Juan Huang, Ekaterina F. Hult, Elisabeth Marchal, Stephen S. Tobe, Identification and characterization of the NMDA receptor and its role in regulating reproduction in the cockroach, Diploptera punctata, 2014, paper submitted to ‘Journal of experimental biology’

Authors’ contribution:

I designed and performed all experiments in this study and wrote the paper with editing by E. Marchal, E.F. Hult, and S.S. Tobe.

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Summary

The NMDA receptor (NMDAR) plays important roles in excitatory neurotransmission and in the regulation of reproduction in mammals. NMDAR in insects comprises two subunits, NR1 and

NR2. In this study, we identified two NR1 paralogs and eleven NR2 alternative splicing variants in D. punctata. This is the first report of NR1 paralogs in insects. The tissue distribution and expression profile of DpNR1A, DpNR1B and DpNR2 in different tissues were also investigated.

Previous studies have demonstrated NMDA-stimulated JH biosynthesis in the corpora allata (CA) through the influx of extracellular Ca2+ in Diploptera punctata. However, our data show that the transcript levels of DpNR1A, DpNR1B and DpNR2 were low in the CA. In addition, neither partial knockdown of DpNR2 nor in vivo treatment with a physiologically relevant dose of MK-

801 resulted in any significant change in JH biosynthesis by CA or basal oocyte growth.

Injection of animals with a high dose of MK-801 (30 µg/animal/injection), which paralyzed the animals for 4-5 h, resulted in a significant decrease in JH biosynthesis on days 4 and 5. However, the reproductive events during the first gonadotrophic cycle in female D. punctata were unaffected. Thus, NMDAR does not appear to play important roles in the regulation of JH biosynthesis or mediate reproduction of female D. punctata.

Introduction

L-glutamate (Glu), a major excitatory amino acid transmitter, mediates diverse physiological functions in the vertebrate nervous system (Mahesh and Brann, 2005). The Glu receptors (GluR) have been classified into three major subtypes: the α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptor, N-methyl-D-aspartate (NMDA) receptor and kainate receptor (Madden, 2002). The NMDA receptors (NMDAR) are distinguished from other

139 ionotropic receptors by their unique properties, including selective agonists and antagonists, high

Ca2+ permeability, and voltage-dependent Mg2+ blockade (McBain and Mayer, 1994). The unique properties of NMDAR allow it to play key roles in excitatory neurotransmission and important neurological processes, including learning, memory and behavior (Mussig et al., 2010;

Newcomer and Krystal, 2001; Xia et al., 2005).

NMDAR in vertebrates is composed of two subunits, NR1 and NR2, and in some cases NR3 subunits (Madden, 2002). The NR1 subunit is essential for the basic channel activity of NMDAR, whereas the NR2 subunit contributes to enhance and modulate the receptor function (Sydow et al., 1996). Although much is known about the function of NMDAR in vertebrates, little information is available on NMDARs in insects. Thus far, two subunits, NR1 and NR2, have been identified in insects. The NR1 and NR2 subunits were previously reported to be distributed throughout the brain of Drosophila melanogaster and Apis mellifera (Wu et al., 2007; Xia et al.,

2005; Zannat et al., 2006).

The importance of NMDAR in the regulation of reproduction of mammals is well-known.

NMDAR mediates reproduction through the regulation of pulse and surge gonadotropin- releasing hormone (GnRH)/luteinizing hormone (LH) secretion (Maffucci et al., 2009; Mahesh and Brann, 2005). In insects, juvenile hormones (JHs) are key regulators of growth, development, metamorphosis, aging, caste differentiation and reproduction (Goodman and Granger, 2005;

Hartfelder, 2000). As a result of the importance of JH in physiological processes, its biosynthesis is tightly regulated by many factors, including neuropeptides (allatostatins, allatotropins) (Stay and Tobe, 2007) and neurotransmitters (octopamine, dopamine and glutamate) (Granger et al.,

1996; Pszczolkowski et al., 1999; Thompson et al., 1990). Chiang et al (2002) demonstrated that

JH biosynthesis by corpora allata (CA) of the cockroach, Diploptera punctata, is stimulated by

140 an NMDA-induced influx of Ca2+ ions and this elevation was significantly reduced by NMDAR antagonists including Mg2+, MK-801 or conantokin T. Furthermore, a Drosophila larval mutant for NMDAR1 showed reduced mRNA levels of the gene encoding JH acid methyltransferase

(JHAMT), a key regulatory enzyme of JH biosynthesis in this species (Huang et al., 2011).

These studies suggest that NMDAR plays a role in the regulation of JH biosynthesis.

As a high-affinity antagonist of NMDARs, MK-801 has been used to study the function of

NMDAR in both vertebrates and invertebrates (Rawls et al., 2009; Sircar et al., 1987; Troncoso and Maldonado, 2002). In addition to the inhibitory effect of MK-801 in NMDA-stimulated JH biosynthesis in D. punctata, MK-801 was found to influence ovarian development and vitellogenesis in the flesh fly Neobellieria bullata and the locust Schistocerca gregaria (Begum et al., 2004; Chiang et al., 2002). In S. gregaria, the inhibition of vitellogenesis was overcome by treatment with JH. A later study on the butterfly Bicyclus anynana and the cricket Gryllus bimaculatus showed that MK-801 affects JH biosynthesis in vitro and JH titres in both species, and subsequently regulates insect reproduction (Geister et al., 2008).

D. punctata is a well-known model in studying the physiology of JH biosynthesis and regulation; in this animal, JH biosynthesis is high and stable, and the reproductive events correlate very well with rates of JH production (see review by Marchal et al. (2013a)). In this study, we chose D. punctata as our model to determine the role of NMDAR in the regulation of JH biosynthesis and reproduction. We identified the genes encoding the subunits of NMDAR in D. punctata, and examined the expression of NMDAR in several tissues. In addition, we investigated the roles of

NMDAR in the regulation of JH biosynthesis, vitellogenesis and oocyte growth in vivo using

RNA interference (RNAi) and MK-801 treatments.

141

Materials and Methods

Insects - D. punctata were reared in cages and fed with lab chow and water at libitum at 27 -28

ºC in a dark room. Newly molted male and female adult cockroaches were picked from the colony and raised in separate containers. Mated status in the females was confirmed by the presence of a spermatophore.

Tissue collection - Cockroach tissues were dissected under a dissecting microscope. Basal oocyte length was measured to determine the physiological age of female cockroaches. Selected tissues were dissected and cleaned in sterile cockroach ringer solution (150 mM NaCl, 12 mM KCl, 10 mM CaCl2.2H2O, 3 mM MgCl2.6H2O, 10 mM HEPES, 40 mM Glucose, pH 7.2), flash-frozen in liquid nitrogen to prevent RNA degradation and stored at -80 ºC until further processing.

RNA extraction and cDNA synthesis - Selected tissues were collected from adult females and male for gene sequence, tissue distribution and developmental profiling. RNA extraction and cDNA synthesis were performed as described by Marchal et al. (2013b). For the tissue distribution and developmental profiling, three biologically independent pools of 10 animals each were collected. For RNAi and MK-801 treatment experiments, 3 biologically independent pools of brain and fat body were collected, each pool containing tissue from 5 animals.

Sequencing of DpNR1A, DpNR1B and DpNR2 - Degenerate primer sequences were designed for

DpNR1A, DpNR1B and DpNR2 based on conserved amino acid sequences of several insect orthologs. Primers used for degenerate PCR are listed in Table S4.1. Partial sequences were obtained using these primers in a standard T-gradient PCR using Taq DNA polymerase (Sigma-

Aldrich) and a D. punctata brain cDNA sample. After purification, the resulting DNA fragments were subcloned into a pJET 1.2/blunt cloning vector (CloneJet PCR Cloning Kit, Thermo

142

Scientific) and sequenced following the protocol outlined in the ABI PRISM BigDye Terminator

Ready Reaction Cycle Sequencing Kit (Applied Biosystems). The complete sequence of

DpNR1A, DpNR1B and DpNR2 was obtained using 5’-RACE (Rapid Amplification of cDNA

Ends) and 3’-RACE strategies, following the protocol outlined in the Roche 5'/3' RACE Kit.

Primers used for RACE are listed in Table S4.1.

Phylogenetic analysis - For the phylogenetic analysis of DpNR1 genes, the NR1 sequences of 15 insect species (identified or predicted) were used. These sequences were aligned using ClustalW as implemented within MEGA 6.06 (Tamura et al., 2013). Poorly aligned positions and gaps were removed, which resulted in 854 amino acid residues. The obtained alignment was used to construct a phylogenetic tree in PhyML 3.0 (Guindon and Gascuel, 2003) based on the maximum-likelihood principle, using the WAG substitution model (Whelan and Goldman, 2001).

Four substitution rate categories were used to estimate the gamma parameter shape with 100 bootstrap replicates to assess branch support (Felsenstein, 1985; Yang, 1994). The resulting tree was then rooted using the sea slug, Aplysia californica as an outgroup.

Quantitative Real Time-PCR (q-RT-PCR) - Primers used for q-RT-PCR are shown in Table S4.2.

Primer sets were validated by determining relative standard curves for each gene transcript using a five-fold serial dilution of a calibrator cDNA sample. Efficiency and correlation coefficient (R²) can be found in Table S2. Reactions were performed in triplicate on a CFX384 Touch ™ Real-

Time PCR Detection System (Bio-Rad) as described previously by Marchal et al. (2013b).

Target specificity was confirmed by running a few representative q-RT-PCR products on an agarose gel containing GelRed™ (Biotium). The optimal housekeeping genes for target gene profiling and RNAi experiments were chosen according to a previous study (Marchal et al.,

143

2013b). The quantity of mRNA for each tested gene relative to reference genes was determined as described by Vandesompele et al. (2002).

RNA interference (RNAi) - Primers for DpNR2 and control pJET dsRNA constructs with T7 promoters are shown in Table S4.3. Double-stranded RNA (dsRNA) constructs were prepared using the MEGAscript® RNAi Kit (Ambion). A PCR using forward and reverse primers with attached T7 promoters was performed to amplify the fragment, which was subcloned and sequenced to verify the presence of the T7 promoter. The amplified fragment was used in an

RNA transcription reaction which was incubated overnight to obtain a high yield of annealed dsRNA construct. A nuclease digestion was subsequently performed to remove ssRNA and DNA remaining in the product. The dsRNA was further purified according to the manufacturer’s instructions (Ambion). Concentration of the dsRNA construct was determined using a Nanodrop instrument (Thermo Fisher Scientific Inc., Canada). Five-fold diluted dsRNA was run on a 1.2% agarose gel to examine the quality and integrity of the construct.

Newly molted adult female cockroaches (day 0) were injected with 2 µg of either DpNR2 dsRNA or pJET dsRNA diluted in 5 µl of cockroach saline. This treatment was repeated on days

1, 2 and 3, and the effect of DpNR2 dsRNA was determined on day 4. Brains were dissected and stored in liquid nitrogen prior to RNA extraction. CA were dissected and cleaned in TC199 medium (GIBCO; 1.3 mM Ca2+, 2% Ficoll, methionine-free) for use in the radiochemical assay

(RCA) (see below). Basal oocyte length was measured during dissection.

MK-801 in vivo assay - MK-801 was dissolved in ddH2O to a concentration of 6 µg/µl and injected into animals using a Hamilton syringe. Newly molted adult females were injected with

30 µg of MK-801 on days 0, 1 and 3, and the effect of MK-801 was determined from day 4 to day 8. Fat body was dissected from day 4 animals and stored in liquid nitrogen prior to RNA

144 extraction. CA were dissected and cleaned in TC199 medium (GIBCO; 1.3 mM Ca2+, 2% Ficoll, methionine-free) for use in the RCA (see below). Basal oocyte length was also measured.

Results

Identification of DpNR1 subunits

Two DpNR1 were identified with open reading frames of 2871bp (DpNR1A) and 2703bp

(DpNR1B). This was accomplished employing a degenerate PCR approach with adult brain cDNA. 5’-RACE and 3’-RACE experiments were performed to complete the sequences. The two

DpNR1 sequences were deposited in the NCBI GenBank and received accession numbers:

KJ747198 and KJ747199. Unlike the alternative splicing variants of NR1 genes in other insects, the differences between the two variants in D. punctata are distributed throughout the whole gene (Fig. 4.1) and may be the result of gene duplication. A phylogenetic tree of NR1 was constructed by maximum likelihood methods (Fig. 4.2). Generally, the sequences of NR1 were grouped based on insect orders, and the two DpNR1s (Blattodea) cluster together with confidence. On the other hand, the relationship between orders is not well-resolved as indicated by low bootstrap values at deeper nodes. The lack of power to resolve these nodes could be explained by insufficient sampling among hemimetabolous insects.

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Fig. 4.1. Amino acid sequence alignment of the two Diploptera NR1 subunit (DpNR1A, DpNR1B), and homologous receptors from D. melanogaster (DNR1, GenBank acc. no. NP_730940.1) and T. castaneum (TNR1, GenBank acc. no. XP_969654.1, predicted sequence). Conservatively substituted residues are highlighted in yellow, and the different residues between DpNR1A and DpNR1B in green. Three putative hydrophobic transmembrane regions (TM1, TM3, and TM4) and one hydrophobic pore-forming segment (TM2) are highlighted in the boxes. Agonist-binding domain (S1 and S2 domains) are underlined.

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Fig. 4.2. Phylogram depicting the relationship between the NR1 subunits from Diploptera and orthologues of this receptor from other insects. Phylogenetic analysis was conducted in PhyML 3.0 using WAG substitution model with 100 bootstrap replicates. Poorly aligned amino acid were eliminated by eye resulting in 854 positions. The bar represents 0.2 substitutions per site. The sea slug, Aplysia californica was used as outgroup to root the tree.

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Identification of DpNR2 subunits

DpNR2 undergoes alternative splicing, mostly at the 3’ end, generating eleven different transcripts, which may encode nine different proteins. Full-length cDNAs for all eleven variants have been isolated and their sequences were deposited in the NCBI GenBank with accession numbers: KJ747200 to KJ747210. The deduced amino acid sequence and nucleotide sequence of

DpNR2 subunits was shown in Supplementary Fig. S4.1. In the eleven transcripts, there are two

5’ untranslated region, two insertions, and four different 3’ ends (Fig. 4.3A). The sequence of insertions, deletions and different 3’-ends are shown in Fig. 4.3B. All eleven DpNR2 variants contain the domain structure, four hydrophobic regions (TM1-TM4) and two ligand binding domains. Amino acid sequence comparisons between DpNR2A-1 and NR2 subunits from D. melanogaster, A. mellifera, and A. aegypti were determined (Table S4.4). DpNR2A-1 shares the highest similarity with NR2 subunits from A. mellifera.

Expression of DpNR1A, DpNR1B and DpNR2

Previous studies of NMDAR in insects have focused on its function in brain (Wu et al., 2007;

Xia et al., 2005; Zannat et al., 2006). In the present study, we were interested in assessing the role of NMDAR in other tissues. We therefore determined the localization and relative abundance of DpNR1A, DpNR1B and DpNR2 mRNA in day 4 adult male and female cockroaches using q-RT-PCR (Fig. 4.4). Specific q-RT-PCR primers for each DpNR1 variant were designed to determine whether transcripts for the different DpNR1 paralogs are present in the same tissue. Q-RT-PCR primers for DpNR2 are located in the conserved region of DpNR2 common to all putative splice variants. In mated female cockroaches, DpNR1A, DpNR1B and

DpNR2 were highly expressed in brain, followed by nerve cord. Previous study has shown that

NMDA stimulated JH biosynthesis in the CA of Diploptera (Chiang et al., 2002). However, the

148 transcript level of DpNR1A, DpNR1B and DpNR2 in the CA was relatively low compared to other tissues. In male cockroaches, the highest transcript levels of DpNR1A, DpNR1B and

DpNR2 were measured in the brain (Fig. 4.4). It is interesting to see that the transcript level of

DpNR2 in testes was very low whereas both genes encoding NR1 were high.

To further study the function of NMDAR in D. punctata, we determined the developmental profile of DpNR1A, DpNR1B and DpNR2 in the brain, CA and testes. DpNR1A and DpNR1B were stably transcribed in brain of day 0 to day 7 post-emergence mated female D. punctata.

DpNR2 mRNA in the brain showed a slight increase on days 2, 3 and 7, but none of the changes were significant (Fig. 4.5). In the CA, the transcript level of DpNR1B and DpNR2 remained very low and did not show any significant change during the first gonadotrophic cycle (Fig. 6).

DpNR1A mRNA levels exhibited a significant increase on day 6. In male cockroaches, we studied the expression of NMDAR in testes of males of differing ages. The transcript level of

DpNR2 was very low throughout all the ages tested (Fig. 4.7).

Effect of DpNR2 dsRNA on JH biosynthesis and oocyte growth

To investigate the role of NMDAR in reproduction, DpNR2 was silenced using RNAi. The injection of 2 µg of DpNR2 dsRNA on days 0, 1, 2 and 3 resulted in a 48.7% knockdown of

DpNR2 mRNA levels in brains of day 4 females (Fig. 4.8a). Knockdown of DpNR2 also resulted in a decrease in the transcript levels of DpNR1A and DpNR1B. However, the JH biosynthetic activity of CA and basal oocyte lengths in dsRNA-treated animals did not show any significant difference from control animals (Fig. 4.8b, 4.8c). A similar result was also observed in DpNR1B dsRNA- treated animals (Fig. S4.2).

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Fig. 4.3. Molecular characterization of DpNR2. (A) The schematic structures of the 11 variants of the NR2. NR2 variants are generated by alternative splicing, including two 5’ untranslated region, two insertions, and four different 3’ ends. The 5’ end untranslated region is shown with black or dash line. The position of insertion 1 is shown by the black arrow and insertion 2 by a gray arrow. The alternated carboxyl-terminal sequences are indicated by colored boxes: A1 (Green), A2 (Red), B (Gray), and C (Purple). Four putative transmembrane segments (TM I – IV) are shown by bold black lines. The agonist-binding domains S1 and S2 are indicated by the hatched boxes. The accession number is displayed under the gene number. (B) The sequence of insertion, deletion and the alternative created carboxyl-terminal.

150

1 N

R 2

n l

a 1

R q

0 B r N C C A F b M G M T O v B r T e A G F e m a le M ale B

2 .5

2 .0

R N

1 .5

i t

t 1 .0

u R q 0 .5

0 .0 B r N C C A F b M G M T O v B r T e A G F e m a le M ale C

f 3

i t

t 2

R q 1

0 B r N C C A F b M G M T O v B r T e A G F e m a le M ale

Fig. 4.4. Graphic representation of the relative tissue distribution of (A) DpNR1A transcript levels, (B) DpNR1B transcript levels and (C) DpNR2 transcript levels in tissues of day 4 adult male and mated female D. punctata. Relative mRNA quantity was normalized against levels of Tubulin and EF1α mRNA (Marchal et al., 2013b). The data represents an average of 3 pools (10 animals per pool), run in triplicate. Abbreviations used: Br brain, NC nerve cord, CA corpora allata, Fb fat body, Ov ovary, MG midgut, MT Malpighian tubules, AG accessory gland and Te testes. Values represent mean ± SEM.

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1 5

y N R 1A

i t

n N R 1B a

u N R 2

q 1 0

e 5

e R 0 D 0 D 1 D 2 D 3 D 4 D 5 D 6 D 7

Fig. 4.5. Relative transcript levels of DpNR1A, DpNR1B and DpNR2 in brains of mated female D. punctata from day 0-day 7 after ecdysis. mRNA levels were normalized against levels of Tubulin and EF1a mRNA (Marchal et al., 2013b). The data represent the average of 3 biologically independent pools (10 animals per pool), run in triplicate. Values represent mean ± SEM.

0 .5 y

t N R 1A

i t

n 0 .4 N R 1B

a u

q N R 2

0 .3

N R

m 0 .2

i t

a 0 .1

e R 0 .0 D 0 D 1 D 2 D 3 D 4 D 5 D 6 D 7

Fig. 4.6. Relative transcript levels of DpNR1A, DpNR1B and DpNR2 in CA of of mated female D. punctata from day 0-day 7 after ecdysis. mRNA levels were normalized against levels of Armadillo and EF1a mRNA (Marchal et al., 2013b). The data represent the average of 3 biologically independent pools (10 animals per pool), run in triplicate. Values represent mean ± SEM.

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N R 1A

4 N R 1B

y t

i N R 2

t n

a 3

N 2

v i

t 1

e R 0 D 0 D 2 D 4 D 6 D 8 D 1 0 D 1 5 D 2 5

Fig. 4.7. Relative transcript levels of DpNR1A, DpNR1B and DpNR2 in testes of different ages of male D. punctata. mRNA levels were normalized against levels of Tubulin and EF1a mRNA (Marchal et al., 2013b). The data represent the average of 3 biologically independent pools (10 animals per pool), run in triplicate. Values represent mean ± SEM.

B A C

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4 0 1 .5 A

N R 2 d sR N A C

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)

o n

0 .8 3 0 m

(

u 1 .0

(

g s

A 0 .6

n N s 2 0

* e

e m

0 .4 t

t 0 .5

y c

i 1 0

s o

a 0 .2

0 .0 H 0 0 .0 N R 1 A N R 1 B N R 2 J c o n tro l N R 2 d s R N A c o n tro l N R 2 d s R N A

Fig. 4.8. The effect of DpNR2 dsRNA treatment on JH biosynthesis and basal oocyte growth, and the interactions among these genes in mated female D. punctata. (A) Relative quantity of DippuNR1A, DippuNR1B and DippuNR2 mRNA levels in brain between control and dsRNA treated animals. mRNA levels were normalized against levels of Tubulin and EF1a mRNA (Marchal et al., 2013b). The data represent the average of 3 biologically independent pools (5 animals each pool), run in triplicate. (B) JH biosynthesis by CA from control and dsRNA-treated animals. (C) Basal oocyte length in control and dsRNA-treated animals. Values represent mean ± SEM. Levels of significance to the control are indicated with the asterisk symbol: *P < 0.05

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In vivo effect of NMDAR antagonist MK-801 on JH biosynthesis and ovarian development

The effect of NMDAR on JH biosynthesis and ovarian development was also determined by injecting the NMDAR non-competitive antagonist MK-801 into the animals. To determine the optimal dose of injection, newly molted adult females were injected with 0 (control), 3, 12 and

30 µg MK-801 on days 0, 1 and 3. JH biosynthesis and basal oocyte length were determined on day 4. As shown in Fig. S4.3, there was no significant effect on JH biosynthesis in animals treated with 3 µg or 12 µg MK-801.

To further study the effect of MK-801 on reproduction, mated female cockroaches were injected with 30 µg MK-801 on days 0, 1, and 3 following ecdysis. Rates of JH biosynthesis and basal oocyte lengths in control and treated animals were determined from day 4 to day 8. As shown in

Fig. 4.9A, application of MK-801 resulted in a 34% and 26% decrease of JH biosynthesis in day

4, and 5 animals, respectively. On day 6, however, MK-801 treated animals showed higher rates of JH biosynthesis than control animals. On days 7 and 8, there was no significant difference in

JH biosynthesis between control and MK-801 treated animals. Furthermore, MK-801 did not have any effect on basal oocyte length (Fig. 4.9B). All animals oviposited on day 8. To study the effect of MK-801 on the Vg synthesis, we determined the transcript level of DpVg in the fat body of day 4 animal. No significant change was found in the transcription of DpVg in MK-801- treated animals (Fig. 4.9C).

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5 0 t m

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n 1 .8

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0 1 .2 R 0 .0 J d a y 4 d a y 5 d a y 6 d a y 7 d a y 8 d a y 4 d a y 5 d a y 6 d a y 7 c o n tro l M K -8 0 1 A G E A G E

Figure 4.9. In vivo effect of MK-801 on JH biosynthesis (A), basal oocyte growth (B) and relative Vg mRNA levels (C). Females were injected with MK-801 on days 0, 1 and 3 following ecdysis (30µg/animal). Control was injected with ddH2O. JH biosynthesis (A) and oocyte length (B) were determined on day 4 to day 8. All animals oviposited on day 8. The mRNA level of Vg was determined in the fat body of day 4 animals. mRNA levels were normalized to levels of Tubulin and EF1a mRNA (Marchal et al., 2013b). The data represent the average of 3 biologically independent pools (5 animals each pool), run in triplicate. Values represent mean ± SEM. (A) N≥15; (B) N≥10. Levels of significance to the control are indicated with an asterisk: *P < 0.05, **P < 0.01, ***P < 0.001

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Discussion

We have identified two distinct NR1 genes and eleven NR2 variants in D. punctata. The major functional domains appear to be well conserved in both DpNR1 and DpNR2 amino acid sequences (Fig. 4.1 and 4.3). The protein contains three hydrophobic transmembrane regions

(TM1, 3-4), a hydrophobic pore-forming segment (TM2), and two ligand binding domains (S1 and S2) (Fig. 4.1 and 4.3) (Dingledine et al., 1999; Kuryatov et al., 1994; Stern-Bach et al.,

1994). The asparagine residue, which was predicted to control Ca2+ permeability and voltage- dependent Mg2+ blockade, is present in the TM2 domain of DpNR1 (N630 in DpNR1A and

N608 in DpNR1B) (Burnashev et al., 1992). The overall amino acid sequence identity between

DpNR1, DpNR2 and NMDAR subunits in other species is shown in Table S4.4. DpNR1A has higher sequence identity to NR1 in other species than DpNR1B. Both DpNR1 and DpNR2 subunits show highest homology to NMDAR of A. mellifera.

The two NR1 subunits are 65.6% identical at the amino acid level and the differences are distributed throughout the entire gene, which suggests that the two NR1 paralogs result from gene duplication (Fig. 4.1). The other NR1 paralogs were isolated from the Zebrafish (Cox et al.,

2005). They encode the same length of protein with only a few amino acid differences. No NR1 paralogs other than in Zebrafish have been identified in vertebrate or invertebrate species.

Phylogenetic analysis of DpNR1 with NR1s from other insect species shows that the two

DpNR1s are grouped together, which suggests that these are paralogs resulting from a gene duplication event in D. punctata that did not occur in other insects. However, given that our dataset was composed primarily of higher insects, and the small number of insect species in which NR1 has been identified, it is difficult to make definitive conclusions about the origin of the paralogs. DpNR2 undergoes alternative splicing, generating eleven different transcripts that

156 may encode nine protein isoforms (Fig. 4.3). Unlike NR2 in Drosophila in which alternative splicing occurs mainly at the 5’ untranslated region, DpNR2 undergoes alternative splicing principally at the 3’-end (Xia et al., 2005).

Tissue distribution shows that both DpNR1 and DpNR2 mRNA accumulated in brain and nerve cord, which is consistent with the role of NMDAR in learning and memory (Xia et al., 2005).

The NR1 subunit is essential for the basic channel activity of NMDAR, whereas the NR2 subunit is regarded as the rate-limiting molecule in controlling the optimal channel properties of

NMDAR (Monyer et al., 1992; Sprengel et al., 1998; Tang et al., 1999). In D. punctata, we observed that both DpNR1 paralogs are stably expressed in brain, whereas the relative DpNR2 mRNA level underwent a slight change, probably to regulate the activity of NMDAR (Fig. 4.5).

In rats, D-aspartic acid (D-Asp) can induce testosterone synthesis through the activation of

NMDAR in testis (Santillo et al., 2014). A high transcript level of DpNR1 was observed in adult

D. punctata testes, indicating that NMDAR may also play a role in regulation of reproduction in our model insect (Fig. 4.6). However, the transcript level of DpNR2 in testes was negligible compared to that of DpNR1, which suggests that NR1 may form a homomeric functional channel, as was observed in an earlier study in which expression of Drosophila NR1 alone produced a weak but significant NMDA response (Ultsch et al., 1993; Xia et al., 2005). An alternative explanation for the low expression of DpNR2 in the testes could be the existence of one or more additional DpNR2 subunits in D. punctata. Four distinct NR2 subunits (A-D) were identified in mammalian species. All four NR2 subunits are expressed in brain (Cull-Candy et al., 2001). In testis, however, the expression of NR2 subunits varies (Hu et al., 2004; Santillo et al., 2014). In the rat testis, only NR2A and NR2D are strongly expressed, whereas NR2B and NR2C are undetectable (Santillo et al., 2014). In mouse testis, on the other hand, only NR2B subunit is

157 highly expressed (Hu et al., 2004). Thus, it is possible that an additional NR2 gene is expressed in the testis of D. punctata.

The function of NMDAR in reproduction is well-studied in mammals (Mahesh and Brann, 2005).

However, clear evidence linking NMDAR to reproduction in insects remains limited. The possible role of NMDAR in reproduction of insects was described by Chiang et al. (2002). In that study, NMDA stimulates JH biosynthesis by inducing a Ca2+-influx from the extracellular environment in the CA of D. punctata (Chiang et al., 2002). However, our study demonstrates that the transcript levels of DpNR1 and DpNR2 in the CA are very low throughout the first gonadotrophic cycle.

Treatment with MK-801 in vivo blocked vitellogenesis in S. gregaria and resulted in a reduction in JH titre in G. bimaculatus (Begum et al., 2004; Geister et al., 2008). For this reason, the role of NMDAR in JH biosynthesis and reproduction was further examined by the administration of

MK-801 in vivo in D. punctata. Administration of a high dose of MK-801 results in a significant decrease in JH biosynthesis by day 4 and 5 CA, indicating that MK-801 does have an effect on

JH biosynthesis. However, the effect of MK-801 on JH biosynthesis is not apparent at doses less than 12 µg/animal/injection (about 60 µg/g body mass) (Fig. S4.3). The result of administration of MK-801 in S. gregaria suggests that the synthesis of Vg in the fat body was affected by MK-

801 injection (Begum et al., 2004). In D. punctata, the production of Vg in fat body and the uptake of Vg by the basal oocytes are JH-dependent events (Marchal et al., 2013a; Rankin and

Stay, 1984; Stay and Tobe, 1978). Although rates of JH biosynthesis on days 4 and 5 in MK-

801-treated animals are lower than in controls, the change in transcript level of DpVg was not significant relative to controls (Fig. 4.9C), and the pattern of JH biosynthesis during the first gonadotrophic cycle is similar to that in control animals (Fig. 4.9A). Oocyte growth in treated

158 animals is also similar to the controls (Fig. 4.9B). All females oviposited on day 8. Overall, administration of MK-801 does not have a significant effect on reproduction in female D. punctata.

Treatment of animals with an extremely high dose of MK-801 in vivo significantly reduced JH biosynthesis on days 4 and 5, even though MK-801 did not show any significant effect on JH biosynthesis in vitro. Partial knockdown of DpNR2 did not show any effect on JH biosynthesis or on basal oocyte growth (Fig. 4.8). To date, there is no clear evidence showing that the effect of

MK-801 on JH biosynthesis is mediated through NMDAR. In rats, injection of 0.2 µg/g MK-801 resulted in a failure to elevate LH, FSH, or progesterone (Luderer et al., 1993). In rats and mice, a dose of 0.1 µg/g MK-801 was the maximum dose that could be used without causing sensorimotor impairments and/or signs of intoxication (Van der Staay et al., 2011). Injection of

30 µg/animal of MK-801 paralyzed the cockroaches for 4-5 h. Therefore, it is possible that the significant decrease in JH biosynthesis on days 4 and 5 in D. punctata was the result of physiological stress induced by MK-801 treatment, rather than the action of NMDAR.

Accordingly, NMDAR does not appear to play important roles in the regulation of JH biosynthesis or reproduction in female D. punctata.

In conclusion, two NR1 paralogs and eleven NR2 alternative splicing variants have been identified in D. punctata. The expression of NMDAR subunits suggests that NMDAR may play a role in the reproduction of male cockroaches. However, in the female cockroach, although a previous study suggested that NMDAR mediates JH biosynthesis in D. punctata, our data reveal a different story. Neither in vivo treatment of MK-801 nor partial knockdown of DpNR2 have any effect on JH biosynthesis. The decrease in JH biosynthesis at a high dose in MK-801-treated animals appears to result from physiological stress, rather than the direct action of NMDAR. In

159 addition, no reproductive events were affected following the blocking of NMDAR activity through RNAi or MK-801 treatment. A reexamination of the function of NMDA and receptors in the reproduction of insects now appears to be appropriate and timely.

160

Supplementary data

Table S4.1 Degenerate primer and RACE primer sequences for cloning DpNR1A, DpNR1B and DpNR2 cDNAs.

Name Symbol Degenerate/RACE primer sequences NMDA receptor NR1A F 5'- CTRTCGCCCGATGGTCARTTYGG -3' subunit 1 variant R 5'- CAGTATGAAYACYCCTGCCATRT -3' A Fn 5'- ACGTATTTCAACATYGGYGGHGT-3' Rn 5'- CCAAATTGGCCATCCGGCGACAA-3' 3’Race 5'- GGATTCGCCATGATCATTGTGGCA -3' 5’Race 5'- GGATCCATTGAAATCGCTGTGGA -3' NMDA receptor NR1B F 5'- CTRTCGCCCGATGGTCARTTYGG -3' subunit 1 variant R 5'- CAGTATGAAYACYCCTGCCATRT -3' B Fn 5'- ATCCACAGCTCCGACACDGAYGG -3' Rn 5'- GTACCTTCTCCTATGCCACTATT-3' 3’Race 5'- TAAATGATGCGAGACTGCGTA-3' 5’Race 5'- CCACACATCAGCTTGATGGGA -3' NMDA receptor NR2 F 5'- ATACCGGTCATCKCVTGGAAYGC -3' subunit 2 R 5'- AACATCCAAGARGCYGTRTCRAA -3' Fn 5' - CTCGGAGAGGGAAGCAGTTGTGG -3’ Rn 5' -TCGAGCAGTCGYTTRTTRAACAT - 3' 3’Race 5'- CCAGAACCGATCCACTGTAGCA -3' 5’Race 5'- CTCTCCGCTCAAGGCCGGAATT -3'

161

Table S4.2 Oligonucleotide sequences for primers used in q-RT-PCR for reference and target genes. Efficiencies and R² values are

indicated.

Reference Amplicon Efficiency F-primer R-primer R2 genes size (bp) (%)

Tubulin 5'-AAATTACCAACGCTTGCTTTGAA-3' 5'-TGGCGAGGATCGCATTTT-3' 58 95.1 0.993

EF1α 5'-TCGTCTTCCTCTGCAGGATGTCT-3' 5'-GGGTGCAAATGTCACAACCATACC-3' 109 99.2 0.994

5'-GCTACTGCACCACTCACAGAATTATT- Armadillo 5'-CTGCAGCATACGTTGCAACA-3' 64 94.5 0.980 3'

Amplicon Efficiency Target genes F-primer R-primer R2 size (bp) (%)

DpNR1A 5’- ATCGAGAAGCGGAAAACACT -3’ 5’- GTTGCTGGATCATTGACACC -3’ 80 90.2 0.984

DpNR1B 5’- GCACACTTTGGGACTCAAGA -3’ 5’- CCTCCAGCAACTAGCATGAA -3’ 54 100 0.983

DpNR2 5’- AAGAACCAGAACCGATCCAC -3’ 5’- GGCCACTAGGAAATCCAAAA -3’ 105 91.7 0.974

DpVg 5’-AAAGGTGTCCTCAGCCAGC-3’ 5’-TCCTCCATCTCGGATTGGGA-3’ 105 95.1 0.998

162

Table S4.3 Nucleotide sequences of primers used in making the dsRNA constructs.

Gene F-primer (5'-3') R-primer (5'-3')

DpNR2 ACAGGATATGGTATCGCCTTTAGC TCTTGAGCTTCGAAAACTGCAC

DpNR12 TAACTGGGGACAGTCCACAC TCAGGTGAGAGTCCAACATGG pJET TTGCGCTCACTGCCAATTGC CTGGCCTTTTGCTCACATGTT

Table S4.4 Amino acid identities (in %) between NMDAR subunits in D. punctata and other insects. DpNR1 was compared with NR1 in other species, and DpNR2A-1 was compared with NR2 in other species.

DpNR1B D. melanogaster A. mellifera A. aegypti DpNR1A 65.6 65.4 75.4 68.2 DpNR1B --- 55.6 62.7 58.8 DpNR2A-1 --- 67.3% 75.6% 71.0%

163

AGCTCGACCTTTGGGACTATGATTGTTGGGCAAAGTTGCATCGTAATTCTCTTGTTTACGGTTACCATGGTGACTACTTCGAGGTCTCCTTGGTTGGGTGTG ACTCCAGCGGTATCCAACCTTTCGTCGAGTTCGCCCAGATTGTCAGCTTGGAGAGAAAATAACTCTGGGCCGCACCATCACCATCATCAAGGGGAGAAGAAA GGAAGAGAAGGAAACTTGTCCATTGGACTTATTGTGCCGTACACTAACTTTGGCGTAAGGGACTATATTCGGGCAGTGAAGAGTGCTGTGGAGAAATTGGCG AAACCAAGAGGCAGGAGACTCAACTTTTTCAAGAAGTACAATTTCTCTCCCAACGAAGTTCACAGCGTCATGATGTCACTAACTCCAAGCCCCACTGCCATT CTTAACTCACTCTGCAAGGAGTTTCTCTCTGTGAACGTCTCCGCCATATTGTACCTG 1 - ATGAACTATGAGAAATATGGAAGGAGTACGGCGTCTGCGCAATATTTCTTGCAGCTTGCGGGCTACTTGGGAATCCCGGTTATCGCCTGG - 90 1 - M N Y E K Y G R S T A S A Q Y F L Q L A G Y L G I P V I A W - 30

91 - AACGCGGATAATTCCGGCCTTGAGCGGAGAGCGTCTCAATCAAGCCTTCAGCTGCAGTTAGCACCATCTCTGGAGCACCAGACGGCCGCC - 180 31 - N A D N S G L E R R A S Q S S L Q L Q L A P S L E H Q T A A - 60

181 - ATGTTGAGTATATTGGAGAGATACAAGTGGCATCAGTTCTCTGTGGTCACCAGTCAGATAGCGGGTCACGATGACTTCCTACAGGCGGTC - 270 61 - M L S I L E R Y K W H Q F S V V T S Q I A G H D D F L Q A V - 90

271 - AGAGAGAGGATCACCGAAGTGCAAGATAGATTCAAGTTCACGATCCTGAATCAAGTGTTGGTCACAAAGCCGGTAGATTTACTAGACCTG - 360 91 - R E R I T E V Q D R F K F T I L N Q V L V T K P V D L L D L - 120

361 - GTCAACTCTGAGTCTCGAGTGATGTTACTCTACGCCACCAGAGAAGAGGCCATACACATCTTGAAAGCCGCCAGAGATTACCAGATCACT - 450 121 - V N S E S R V M L L Y A T R E E A I H I L K A A R D Y Q I T - 150

451 - GGAGAGAACTACGTATGGGTCGTCACCCAGAGCGTCATGGAGAACCTCCAGACACCTTTCGGTTTCCCTGTCGGCATGCTCGGTGTCCAT - 540 151 - G E N Y V W V V T Q S V M E N L Q T P F G F P V G M L G V H - 180

541 - TTCGATACAAGCAGCACATCCCTTGTGAACGAAATCACCACCGCCATTAGGGTGTACGCATACGGGGTAGAGGATTTTGTGAATGATCCC - 630 181 - F D T S S T S L V N E I T T A I R V Y A Y G V E D F V N D P - 210

631 - AGAAATGTCAATCTCTCTCTCAGCACACAATTATCTTGTGAAGGGATGGGAGACTCCAGATGGAAAACAGGAGACAGATTCTTCAGGTAC - 720 211 - R N V N L S L S T Q L S C E G M G D S R W K T G D R F F R Y - 240

721 - CTCCGGAACGTGAGTGTTGAGGGGGATACAGGAAAGCCACATGTAGAATTCACACCGGAAGGAGTTCTGAAGGCAGCAGAGTTGAAAATA - 810 241 - L R N V S V E G D T G K P H V E F T P E G V L K A A E L K I - 270

811 - ATGAATTTGAGGCCTGGTGTTAGCAAGCAGCTTGTGTGGGAAGAGATCGGAGTGTGGAAATCTTGGGAGAAGGAAGGTCTGGACATCAAA - 900 271 - M N L R P G V S K Q L V W E E I G V W K S W E K E G L D I K - 300

901 - GACATCGTGTGGCCTGGGAACAGTCACACTCCGCCTCAGGGAGTTCCTGAAAAGTTCCACCTGAAGATAACTTTCCTGGAAGAACCTCCA - 990 301 - D I V W P G N S H T P P Q G V P E K F H L K I T F L E E P P - 330

991 - TATATCAACCTTGCGCCCCCGGATCCCGTCACCGGAAAATGCAGCATGAACAGGGGCGTTCTCTGCCGGGTGGCCAAGGAAGAAGAAATG - 1080 331 - Y I N L A P P D P V T G K C S M N R G V L C R V A K E E E M - 360

1081 - GAAAAGGTGGATGTACCGATGGCACATAAAAACGGCAGTTTCTACCAATGCTGCTCTGGGTTCTGCATAGACCTGCTAGAAAAATTCGCA - 1170 361 - E K V D V P M A H K N G S F Y Q C C S G F C I D L L E K F A - 390 S1 1171 - GAAGAACTTGGGTTCACATACGAACTCGTGAGGGTGGAAGATGGAAAATGGGGGACTCTGGAGAACGGGAAGTGGAACGGATTGATAGCG - 1260 391 - E E L G F T Y E L V R V E D G K W G T L E N G K W N G L I A - 420

1261 - GATCTCGTCAACCGGAAGACAGACATGGTGATGACGTCACTGATGATAAACTCGGAGAGGGAAGCAGTTGTGGACTTCACTGTGCCCTTT - 1350 421 - D L V N R K T D M V M T S L M I N S E R E A V V D F T V P F - 450

1351 - ATGGAGACTGGCATTGCCATTTTGGTGGCCAAGAGAACTGGTATCATATCTCCCACAGCTTTTCTTGAACCGTTTGACACGGCCTCCTGG - 1440 451 - M E T G I A I L V A K R T G I I S P T A F L E P F D T A S W - 480

1441 - ATGCTGGTAGGTGTAGTAGCGATCCAGGCAGCCACTTTTACTATTTTCCTTTTCGAATGGCTGAGTCCTTCAGGATTCGACATGAAGAAT - 1530 481 - M L V G V V A I Q A A T F T I F L F E W L S P S G F D M K N - 510 TM 1 1531 - GTGGATGGTTTCCAGCAAGTGTCTCCGTCCCCAAATCACCGGTTTTCATTGTTCAGGACCTATTGGTTAGTGTGGGCTGTCCTCTTCCAA - 1620 511 - V D G F Q Q V S P S P N H R F S L F R T Y W L V W A V L F Q - 540 TM 2 1621 - GCAGCCGTACATGTAGATTCCCCAAGAGGCTTCACTGCCAGGTTCATGACCAATGTGTGGGCCATGTTTGCAGTGGTGTTCCTTGCTATT - 1710 541 - A A V H V D S P R G F T A R F M T N V W A M F A V V F L A I - 570 TM 3 1711 - TACACTGCCAACCTGGCTGCCTTCATGATCACAAGAGAAGAATTTCATGAATTTTCTGGCCTCGACGATTCTAGGCTGTCAAAACCATTC - 1800 571 - Y T A N L A A F M I T R E E F H E F S G L D D S R L S K P F - 600

1801 - AGTCACAAACCCATGTACAGGTTTGGTACTATCCCATGGAGCCACACCGACTCAACTCTTAGTAAATATTTTGCCCCCATGCATGCCTAC - 1890 601 - S H K P M Y R F G T I P W S H T D S T L S K Y F A P M H A Y - 630 S2 1891 - ATGAAGAACCAGAACCGATCCACTGTAGCAGAGGGAATAGAAGCTGTTCTTAGTGGTGAACTGGATGCTTTCATTTATGATGGCACAGTT - 1980 631 - M K N Q N R S T V A E G I E A V L S G E L D A F I Y D G T V - 660

1981 - TTGGATTTCCTAGTGGCCCAGGATGAGGACTGCCGTCTGCTCACTGTGGGATCATGGTATGCCATGACAGGATATGGTATCGCCTTTAGC - 2070 661 - L D F L V A Q D E D C R L L T V G S W Y A M T G Y G I A F S - 690

2071 - CGCAACTCTAAATATGTTCAAATGTTCAACAAGCAAATGCTTGATTTTCGGGAAAATGGGGACTTGGAGAGACTACGGAGGTATTGGATG - 2160 691 - R N S K Y V Q M F N K Q M L D F R E N G D L E R L R R Y W M - 720

2161 - ACCGGGACATGTAAGCCTGGTAAACAAGAACATAAATCCAGTGACCCTCTAGCCCTGGAGCAGTTTTTATCTGCATTTTTGCTGCTCATG - 2250 721 - T G T C K P G K Q E H K S S D P L A L E Q F L S A F L L L M - 750 TM 4 2251 - TCTGGTATCCTACTAGCTGCAGTACTCTTAGCACTAGAACATGTGTATTTCAAATATGTTAGAAAACATTTGGCCAAAACAGACAGAGGA - 2340 751 - S G I L L A A V L L A L E H V Y F K Y V R K H L A K T D R G - 780

2341 - GGTTGCTGTGCTCTAATAAGTTTGAGCATGGGCAAATCATTAACGTTCCGCGGTGCAGTTTTCGAAGCTCAAGATATATTAAGACATCAT - 2430 781 - G C C A L I S L S M G K S L T F R G A V F E A Q D I L R H H - 810

2431 - CGTTGTCGGGATCCTATATGTGATACACATTTATGGAAAGTGAAACACGAGTTGGATCTCGCCCGTATGAAGATTCGACAATTGCAGAAA - 2520 811 - R C R D P I C D T H L W K V K H E L D L A R M K I R Q L Q K - 840

2521 - GAACTTGAAGCTCACGGCATCAAGCCAAGTAGAAGGAAGAAGAAGAAACGCGTCCCGTCCTGCTGGGTCTCCTGCTTCACACGAAACCAG - 2610 841 - E L E A H G I K P S R R K K K K R V P S C W V S C F T R N Q - 870

164

2611 - CCCGCGGGAGAGCAGGCGTCCCAATTGTTGAATGCAGATGACGTCCTCAAACCGCGTGATATGACAAGAAATCACGTGACATCTACAGAA - 2700 871 - P A G E Q A S Q L L N A D D V L K P R D M T R N H V T S T E - 900

2701 - TTCTCCGGCCGTTTTCACAGTGCGGGCCAGTTATACAGGTAA - 2742 901 - F S G R F H S A G Q L Y R * - 930

Fig S4.1. The nucleotide sequence and deduced amino acid sequence of DpNR2B-2. Agonist- binding domains (S1 and S2 domains) are underlined; three hydrophobic transmembrane regions (TM1, TM3, and TM4) and one hydrophobic pore-forming segment (TM2) are highlighted in the boxes. Two insertions, and alternative 3’-end are highlighted in yellow, green and purple, respectively.

A B C )

2 .0 8 0 ) 1 .5

c o n tro l A

t m

N R 1 2 d sR N A h

(

a o

1 .5 6 0 h

m g

q 1 .0

(

1 .0 s 4 0

e y

m *

t c

e 0 .5 n

** o

o i

0 .5 2 0

R H

0 .0 0 B 0 .0 J N R 1 1 N R 1 2 N R 2 C o n tro l N R 1 2 d s R N A C o n tro l N R 1 2 d s R N A

Fig. S4.2. The effect of DpNR1B dsRNA treatment on JH biosynthesis and basal oocyte growth, and the interactions among these genes in mated female D. punctata. (A) Relative quantity of DippuNR1A, DippuNR1B and DippuNR2 mRNA levels in brain between control and dsRNA treated animals. mRNA levels were normalized against levels of Tubulin and EF1a mRNA. The data represent the average of 3 biologically independent pools (5 animals each pool), run in triplicate. (B) JH biosynthesis by CA from control and dsRNA-treated animals. (C) Basal oocyte length in control and dsRNA-treated animals. Values represent mean ± SEM. Levels of significance to the control are indicated with the asterisk symbol: *P < 0.05, **P < 0.01

165

) 6 0 c o n tro l A

C M K -8 0 1

l o

m 4 0

(

s i

s ***

h t

n 2 0

H J 0 3 µ g 1 2 µ g 3 0 µ g

Fig. S4.3. JH biosynthesis of CA in day 4 mated female D. punctata injected with different doses of MK-801 on days 0, 1 and 3. Control was injected with ddH2O. Values represent mean ± SEM (n ≥17). Levels of significance to the control are indicated with the asterisk symbol: ***P < 0.001.

166

References

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Chapter 5

General Discussion

1. Function of JH in reproduction

The original function of JH in insects was considered to be a regulator of reproduction (Tobe and

Bendena, 1999). Several different types of hormones are involved in the endocrine control of female reproduction, of which JHs and ecdysteroids are most studied. In most hemimetabolous insects, the major events of reproduction are controlled by JH, while in higher insects, both JH and ecdysteroids were involved (Raikhel et al., 2005). In D. punctata, allatectomy or implantation of CA with ovaries into male animals showed that JH stimulates Vg synthesis and

Vg uptake (Mundall et al., 1979; Stay and Tobe, 1977). Moreover, in several other cockroaches species, treatment with JH or JH analogs achieved the same result (Comas et al., 1999; Don-

Wheeler and Engelmann, 1997; Weaver and Edwards, 1990). In this thesis, the function of JH in regulating female reproduction was further investigated by repressing JH biosynthesis using

RNAi. As expected, the lowered JH biosynthesis on day 4 in the CA resulted in a significant reduction of Vg mRNA levels, uptake of Vn into the oocytes, and in blocking patency (Chapter

2). The increase of JH biosynthesis on day 3 and 4 is essential for the development of oocytes.

On the other hand, the decrease in JH production starting on day 5 is required to complete the regular growth of oocytes. An abnormal high titre of JH after day 5 resulted in a failure in oviposition (Hult et al, data unpublished). It is obvious that JH plays critical functions in regulating cockroach female reproduction and therefore must be tightly regulated.

170

2. Evolution of the JH biosynthetic pathway

2.1 The loss of sterols biosynthesis in arthropods

In most eukaryotic animal and plant systems, cholesterol and related sterols, which are essential component of cell membranes, are biosynthesized through the mevalonate pathway. Arthropods, however, do not appear to synthesize cholesterol, but rather produce sesquiterpenoids. The final product of the mevalonate pathway is JH, rather than cholesterol, as a consequence of the absence of genes encoding squalene synthase and other subsequent enzymes to produce sterols

(Belles et al., 2005). The biosynthesis of JH and cholesterol share the mevalonate pathway until the production of FPP. In Arthropoda, FPP is converted to FOL and thereafter to FA (Figure

1.1), while it is catalyzed to squalene in animals producing cholesterol. In some plants, on the other hand, cholesterol and JH III and their related compounds are synthesized, suggesting that both the JH and cholesterol biosynthetic pathway are present in plant system (Yang et al., 2013).

Tobe and Bendena (1999) suggest that the ability to biosynthesize cholesterol was maintained in the ancestor of the arthropod/annelid lineage, but lost in the arthropod line, probably because of the abundance of cholesterol in the diet. The biosynthesis of JH in some plants is the result of convergence, which is unrelated to its occurrence in arthropods. Nevertheless, the reason for losing the ability to biosynthesize cholesterol de novo in arthropods remains unknown.

2.2 Enzymes in the JH biosynthetic pathway controlling the end product of the JH

biosynthetic pathway

As shown in Figure 1.1, various JHs are produced in insects. These JHs shares the main carbon skeleton, but with different methyl/ethyl substitutions and carbon-sites for epoxidation. Most ethyl substituted JH III products occur in the order of the Lepidoptera (Details seen Chapter 1

171 section 1). The reason for the various end products of the JH biosynthetic pathway in

Lepidoptera can be found in the catalytic selectivity of enzymes in the pathway. Unlike in other insects, both acetyl-CoA and propionyl-CoA are involved in the early steps of JH biosynthesis to produce ethyl-branched JHs in M. sexta (Brindle et al., 1987; Brindle et al., 1988). The branched-chain amino acid transaminase, which generates propionyl-CoA by the metabolism of leucine and valine, is found only in the CA of Lepidoptera (Brindle et al., 1988). In addition, enzymes in the JH biosynthetic pathway have evolved to utilize different substrates for JH biosynthesis. For instance, IPPI in M. sexta and C. fumiferana contains an active site cavity, which allows it to bind to larger substrates (ethyl-substituted IPP) and to stabilize the high- energy intermediates to form ethyl- substituted DMPP (Sen et al., 2012). FPPS in M. sexta is also adapted to catalyze both ethyl- and methyl-substituted DMPP and GPP, and the selectivity of the enzyme inclines to the ethyl-substituted substrate (Sen et al., 1996; Sen et al., 2006).

As noted in Chapter 1, section 1.2.13, CYP15 genes have been characterized to encode cytochrome P450 (CYP) enzymes that epoxidize MF to JH in Orthoptera, Dictyoptera and

Coleoptera, or FA to JHA in Lepidotera. However, in higher Diptera that produce bisepoxy forms of JH III, no CYP15 gene has been found (Daimon and Shinoda, 2013). Different CYP enzymes that could catalyze the bisepoxidization of MF may be present in the higher Diptera to compensate the loss of CYP15, resulting in the production of JHB3. However, in P. stali that produces JHSB3, a CYP15 ortholog was identified. The function of this CYP15 ortholog has not yet been determined. Whether there are other epoxidases involved in the synthesis of JHSB3 remains unclear. In addition, in the crustacean D. pulex, no CYP15 ortholog was found in the genome. The lack of CYP15 genes may be the reason for crustaceans using MF as their JH-like bioactive compound.

172

The catalytic selectivity of the enzymes in the JH biosynthetic pathway determines not only the structure of the JH products, but also the order of steps in the JH biosynthetic pathway. As shown in Figure 1.2, the last two steps (methylation and epoxidation) of the JH biosynthetic pathways differ between Lepidoptera and other insects. Two different epoxidase enzymes,

CYP15A1 and CYP15C1 are involved in the last steps of JH biosynthesis in different insect orders. The order of the methylation and epoxidation steps appears to be controlled by the selectivity of the epoxidase. CYP15C1, the functional epoxidase in Lepidoptera, specifically converts FA to JHA (Daimon et al., 2012). Therefore, epoxidation occurs before methylation in

Lepidoptera. However, in other insect orders, CYP15A1 has been characterized as the active epoxidase. The substrate selectivity of CYP15A1 to MF determines that methylation precedes epoxidation (Helvig et al., 2004).

D. punctata, as a more basal species in the Insecta, did not appear to undergo any significant modification of the enzymes in the JH biosynthetic pathway. Thus, only JH III has been identified as the JH biosynthetic product of CA.

3. Regulation of JH biosynthesis

3.1 Enzymes in the biosynthetic pathway

Regulation of JH biosynthesis is a complicated process involving multiple regulators. The best- characterized regulators are the neuropeptides, including ATs and ASTs. Enzymes in the JH biosynthetic pathway were also shown to regulate JH biosynthesis (Couillaud and Feyereisen,

1991; Feyereisen and Farnsworth, 1987; Kinjoh et al., 2007; Nouzova et al., 2011). Some enzymes have been proposed to catalyze rate-limiting steps in the pathway (e.g. FALD in A. aegypti (Rivera-Perez et al., 2013); JHAMT in B. mori (Shinoda and Itoyama, 2003)). In this

173 thesis, the transcript levels of genes in the JH biosynthetic pathway were determined in relation to the JH biosynthesis in D. punctata. Similar to other species, the expression of most genes is well-correlated with the JH biosynthetic activity, suggesting that JH biosynthesis is in part controlled by the transcription of enzymes. However, no rate-limiting enzyme could be identified in D. punctata. My study has also shown that precursor supply mediates JH biosynthesis. In addition, silencing individual genes encoding the JH biosynthetic enzymes resulted in a significant decrease in the transcript levels of other genes. Therefore, a feedback among genes in

JH biosynthetic pathway was proposed: the expression of enzymes in the pathway is correlated with other enzymes to obtain the balance between enzyme activity and JH precursor supply.

Further experiments to determine the amount of JH precursors in the CA after silencing individual genes would provide evidence to support the hypothesis.

3.2 Neuropeptides

In cockroaches, ASTs compose a family of 13 to 14 neuropeptides with the conserved C- terminal sequence (Y/FXFGL/I-NH2) and variable N-terminal sequences. All the peptides are encoded by a single gene. Evolutionary analysis of this gene indicates that the AST sequences were generated by a gene duplication event before the species diverged (Belles et al., 1999).

Structure-activity studies show that all ASTs are able to inhibit JH biosynthesis effectively, albeit with different potencies (Tobe et al., 2000). This result suggests that AstRs in cockroaches are relatively unselective, binding ASTs with different structures. In my thesis, the selectivity of

DpAstR to its ligand was determined by the activation of DpAstR expressed in CHO cells using

13 DpASTs and AST analogs. All ASTs and AST analogs were able to activate DpAstR, and the selectivity of DpAstR to DpASTs was shown to be structure-dependent (Table 3.1 and Table 6.1).

174

The core sequence (Y/FXFGL/I-NH2) is essential for binding, while the N-terminal region regulates binding affinity.

In general, the ability of AST to bind AstR correlates well with its potency to inhibit JH production by the CA (Table 3.1). AST is a pleiotropic neuropeptide, mediating different physiological effects, such as midgut enzyme activity and hindgut muscle contraction. However, in these functions, binding of AST to its receptor does not seem to correlate with its activity

(Lange et al., 1995). In addition, the AstR identified in the CA of D. punctata was not found to be expressed in the midgut, where the precursor of its ligand AST was highly expressed. My study supports the hypothesis proposed by Tobe and Bendena (1999) that there are different AST receptor subtypes in different tissues. Unfortunately, no second AstR could be identified in D. punctata.

As mentioned before (Chapter 1, section 3.1), there are different families of allatostatic neuropeptides. The FGLa/ASTs which inhibits JH biosynthesis in cockroaches, do not show the allato-inhibitory effect on the CA of Lepidoptera or Diptera. Instead, the PISCF/AST family is involved to regulate JH biosynthesis in these insect orders. Unlike in cockroaches where 14

FGLa/ASTs with very high sequence homology are encoded by a single gene, the PISCF/AST precursor gene only encodes one AST peptide in Lepidoptera and Diptera (Kramer et al., 1991;

Li et al., 2006). In addition to PISCF/AST, another type of neuropeptide, termed allatotropin

(AT), which stimulates JH biosynthesis in the CA, was also identified in Lepidoptera and Diptera.

3.3 Second messenger (Ca2+)

Calcium, an intracellular second messenger, is a critical factor in controlling JH biosynthesis.

However, the role of calcium in the JH biosynthesis seems to vary depending on species and

175 stage (Allen et al., 1992; Dale and Tobe, 1988; Kikukawa et al., 1987). To make it more complicated, calcium was determined to act as a second messenger in the signal pathway of AST action to inhibit JH biosynthesis (Chapter 3). Calcium can also act as the second messenger of

Manse-AT to enhance CA activity (Horodyski et al., 2011). In this thesis, the function of

NMDAR as an ionotropic calcium channel was investigated. A previous study demonstrated that

NMDA stimulates JH biosynthesis in the CA through the influx of Ca2+ into the CA cells

(Chiang et al., 2002). However, our study, using RNAi and in vivo treatment with NMDAR antagonists, revealed that NMDAR does not appear to regulate JH biosynthesis (Chapter 4).

Clearly, a complex mechanism is involved in the regulation of JH biosynthesis by calcium.

4. The value of my study in insect control

As an essential hormone controlling many physiological processes in insects, JH production is tightly regulated to allow normal development. This characteristic makes JH a potential target for insect control. Thousands of JH analogs have been synthesized and bioassayed to discover potential insecticides (Goodman and Cusson, 2012). In addition to JH analogs, ASTs which inhibit JH biosynthesis at low doses, have been employed in the design of potential insect growth regulators (IGRs). Previous studies have determined the distribution, activity and structure- activity relationship of the thirteen ASTs in D. punctata, which has permitted the development of analogs of the naturally occurring ASTs (Donly et al., 1993; Rankin and Stay, 1987; Tobe et al.,

2000). The development of AST analogs as potential insecticides need to overcome two major problems: (1) maintaining the high allato-inhibitory activity being a shorter peptide; (2) having a high resistance to the peptidases in the CA and hemolymph (Marchal et al., 2013). In this thesis, two series of AST analogs were designed and synthesized based on the previous rules. Some

AST analogs not only exhibited similar potencies in inhibiting JH biosynthesis in vitro to the

176 naturally occurring AST (Table 6.2), but were also able to regulate JH biosynthesis and oocyte growth in an in vivo bioassay (Fig. 6.1). Preliminary degradation results show that the AST analogs have higher resistance to peptidases in hemolymph than natural AST. These AST analogs can be considered as potential IGRs. Further modification of these AST analgos is now required to obtain higher allato-inhibitory activity and higher resistance to peptidases in the cockroach.

5. Future perspectives

This thesis aimed to characterize the JH biosynthetic pathway and determine factors regulating

JH biosynthesis in D. punctata. Genes in the JH biosynthetic pathways were identified, functionally characterized, and regulatory factors including AST and NMDAR were studied.

However, many questions still remain unanswered. The exact target of AST action remains unknown. The role of AST in regulating JH biosynthesis is well-established, while the roles of other regulatory factors including second messengers, JH feedback and ecdysteroids remain unclear. In addition to the regulation of JH biosynthesis, the JH action and cross-talking with other hormone also require further investigate.

D. punctata is a great model in studying the endocrinology of arthropods. In this animal the JH titre is relatively high and stable, only JH III is is synthesized, and reproductive events are well- correlated with JH biosynthesis. The genome of D. punctata is currently being assembled which will allow high-throughput and more in depth studies aimed at identifying the regulatory mechanisms targeting JH biosynthesis. A first study is currently ongoing involving differential transcriptomics of CA with low versus high JH biosynthetic activity.

177

References

Allen, C.U., Janzen, W.P., Granger, N.A., 1992. Manipulation of intracellular calcium affects in vitro juvenile hormone synthesis by larval corpora allata of Manduca sexta. Mol Cell Endocrinol 84, 227-241. Belles, X., Graham, L.A., Bendena, W.G., Ding, Q., Edwards, J.P., Weaver, R.J., Tobe, S.S., 1999. The molecular evolution of the allatostatin precursor in cockroaches. Peptides 20, 11-22. Belles, X., Martin, D., Piulachs, M.D., 2005. The mevalonate pathway and the synthesis of juvenile hormone in insects. Annu Rev Entomol 50, 181-199. Brindle, P.A., Baker, F.C., Tsai, L.W., Reuter, C.C., Schooley, D.A., 1987. Sources of propionate for the biogenesis of ethyl-branched insect juvenile hormones: Role of isoleucine and valine. Proc Natl Acad Sci U S A 84, 7906-7910. Brindle, P.A., Schooley, D.A., Tsai, L.W., Baker, F.C., 1988. Comparative metabolism of branched-chain amino acids to precursors of juvenile hormone biogenesis in corpora allata of lepidopterous versus nonlepidopterous insects. J Biol Chem 263, 10653-10657. Chiang, A.S., Lin, W.Y., Liu, H.P., Pszczolkowski, M.A., Fu, T.F., Chiu, S.L., Holbrook, G.L., 2002. Insect NMDA receptors mediate juvenile hormone biosynthesis. Proc Natl Acad Sci U S A 99, 37-42. Comas, D., Piulachs, M.D., Belles, X., 1999. Fast induction of vitellogenin gene expression by juvenile hormone III in the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae). Insect Biochem Mol Biol 29, 821-827. Couillaud, F., Feyereisen, R., 1991. Assay of HMG-CoA synthase in Diploptera punctata corpora allata. Insect Biochem 21, 131-135. Daimon, T., Kozaki, T., Niwa, R., Kobayashi, I., Furuta, K., Namiki, T., Uchino, K., Banno, Y., Katsuma, S., Tamura, T., Mita, K., Sezutsu, H., Nakayama, M., Itoyama, K., Shimada, T., Shinoda, T., 2012. Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori. PLoS Genet 8, e1002486. Daimon, T., Shinoda, T., 2013. Function, diversity, and application of insect juvenile hormone epoxidases (CYP15). Biotechnol Appl Biochem 60, 82-91. Dale, J.F., Tobe, S.S., 1988. Differences in the stimulation by calcium ionophore of juvenile hormone III release from corpora allata of solitarious and gregarious Locusta migratoria. Experientia 44, 240-242. Don-Wheeler, G., Engelmann, F., 1997. The biosynthesis and processing of vitellogenin in the fat bodies of females and males of the cockroach Leucophaea maderae. Insect Biochem Mol Biol 27, 901-918. Donly, B.C., Ding, Q., Tobe, S.S., Bendena, W.G., 1993. Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata. Proc Natl Acad Sci U S A 90, 8807-8811. Feyereisen, R., Farnsworth, D.E., 1987. Characterization and regulation of HMG-CoA Reductase during a cycle of Juvenile Hormone synthesis. Mol Cell Endocrinol 53, 227-238. Goodman, W.G., Cusson, M., 2012. The juvenile hormones, in: Gilbert, L.I. (Ed.), Insect Endocrinology. Academic Press, London, p. 55. Helvig, C., Koener, J.F., Unnithan, G.C., Feyereisen, R., 2004. CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata. Proc Natl Acad Sci U S A 101, 4024-4029. Horodyski, F.M., Verlinden, H., Filkin, N., Vandersmissen, H.P., Fleury, C., Reynolds, S.E., Kai, Z.P., Broeck, J.V., 2011. Isolation and functional characterization of an allatotropin receptor from Manduca sexta. Insect Biochem Mol Biol 41, 804-814.

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Kikukawa, S., Tobe, S.S., Solowiej, S., Rankin, S.M., Stay, B., 1987. Calcium as a regulator of juvenile hormone biosynthesis and release in the cockroach Diploptera punctata. Insect Biochem 17, 179-187. Kinjoh, T., Kaneko, Y., Itoyama, K., Mita, K., Hiruma, K., Shinoda, T., 2007. Control of juvenile hormone biosynthesis in Bombyx mori: Cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochem Mol Biol 37, 808-818. Kramer, S.J., Toschi, A., Miller, C.A., Kataoka, H., Quistad, G.B., Li, J.P., Carney, R.L., Schooley, D.A., 1991. Identification of an allatostatin from the tobacco hornworm Manduca sexta. Proc Natl Acad Sci U S A 88, 9458-9462. Lange, A.B., Bendena, W.G., Tobe, S.S., 1995. The effect of the thirteen Dip-Allatostatins on myogenic and induced contractions of the cockroach (Diploptera Punctanta) hindgut. J Insect Physiol 41, 581-588. Li, Y., Hernandez-Martinez, S., Fernandez, F., Mayoral, J.G., Topalis, P., Priestap, H., Perez, M., Navare, A., Noriega, F.G., 2006. Biochemical, molecular, and functional characterization of PISCF-allatostatin, a regulator of juvenile hormone biosynthesis in the mosquito Aedes aegypti. J Biol Chem 281, 34048-34055. Marchal, E., Hult, E.F., Huang, J., Stay, B., Tobe, S.S., 2013. Diploptera punctata as a model for studying the endocrinology of arthropod reproduction and development. Gen Comp Endocr 188, 85-93. Mundall, E.C., Tobe, S.S., Stay, B., 1979. Induction of vitellogenin and growth of implanted oocytes in male cockroaches. Nature 282, 97-98. Nouzova, M., Edwards, M.J., Mayoral, J.G., Noriega, F.G., 2011. A coordinated expression of biosynthetic enzymes controls the flux of juvenile hormone precursors in the corpora allata of mosquitoes. Insect Biochem Mol Biol 41, 660-669. Raikhel, A.S., Brown, M.R., Belles, X., 2005. Hormonal control of reproductive processes, in: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive molecular insect science. Elsevier, Oxford, pp. 433-491. Rankin, S.M., Stay, B., 1987. Distribution of allatostatin in the adult cockroach, Diploptera punctata and effects on corpora allata in vitro. Journal of Insect Physiology 33, 551-558. Rivera-Perez, C., Nouzova, M., Clifton, M.E., Garcia, E.M., LeBlanc, E., Noriega, F.G., 2013. Aldehyde dehydrogenase 3 converts farnesal into farnesoic acid in the corpora allata of mosquitoes. Insect Biochem Mol Biol 43, 675-682. Sen, S.E., Ewing, G.J., Thurston, N., 1996. Characterization of lepidopteran prenyltransferase in Manduca sexta corpora allata. . Arch Insect Biochem Physiol 32, 315–332. Sen, S.E., Hitchcock, J.R., Jordan, J.L., Richard, T., 2006. Juvenile hormone biosynthesis in M. sexta: substrate specificity of insect prenyltransferase utilizing homologous diphosphate analogs. Insect Biochem Mol Biol 36, 827-834. Sen, S.E., Tomasello, A., Grasso, M., Denton, R., Macor, J., Beliveau, C., Cusson, M., Crowell, D.N., 2012. Cloning, expression and characterization of lepidopteran isopentenyl diphosphate isomerase. Insect Biochem Mol Biol 42, 739-750. Shinoda, T., Itoyama, K., 2003. Juvenile hormone acid methyltransferase: a key regulatory enzyme for insect metamorphosis. Proc Natl Acad Sci U S A 100, 11986-11991. Stay, B., Tobe, S.S., 1977. Control of juvenile hormone biosynthesis during the reproductive cycle of a viviparous cockroach 1. Activation and inhibition of corpora allata. Gen Comp Endocrinol 33, 531-540. Tobe, S.S., Bendena, W.G., 1999. The regulation of juvenile hormone production in arthropods. Functional and evolutionary perspectives. Ann N Y Acad Sci 897, 300-310. Tobe, S.S., Zhang, J.R., Bowser, P.R.F., Donly, B.C., Bendena, W.G., 2000. Biological activities of the allatostatin family of peptides in the cockroach, Diploptera punctata, and potential interactions with receptors. Journal of Insect Physiology 46, 231-242.

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Weaver, R.J., Edwards, J.P., 1990. The role of the corpora allata and associated nerves in the regulation of ovarian cycles in the oviparous cockroach Periplaneta americana. J Insect Physiol 36, 51-59. Yang, H., Kim, H.S., Jeong, E.J., Khiev, P., Chin, Y.W., Sung, S.H., 2013. Plant-derived juvenile hormone III analogues and other sesquiterpenes from the stem bark of Cananga latifolia. Phytochemistry 94, 277-283.

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Chapter 6

Appendices

ASTs which inhibit JH biosynthesis at low doses, have been employed in the design of potential insecticides. Furthermore, molecular cloning, structure determination, distribution and activity of the thirteen ASTs in D. punctata, have permitted the development of analogs of the naturally occurring ASTs (Donly et al., 1993; Rankin and Stay, 1987; Tobe et al., 2000). Although ASTs inhibit JH biosynthesis effectively in vitro, they have some shortcomings as potential pesticides.

Firstly, the longer the peptide, the more unwanted side-products that are produced during chemical synthesis. For ASTs, even the shortest AST peptide has 6 amino acids (Tobe et al.,

2000). Secondly, ASTs are susceptible to metabolic inactivation by peptidases in hemolymph and midgut (Garside et al., 1997a, b). For example the half-life of Dippu-AST 5 (AST5: Asp-

Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide) is less than 1 hour when incubated with membrane preparations of brain and midgut (Garside et al., 1997b). Therefore, much effort has been expended to overcome this problem and create better AST pseudopeptides. In this thesis, two series of AST anaologs were designed and their activities in regulating JH biosynthesis in vitro and in vivo in D. punctata were shown in this chapter.

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Table 6.1. Structure of AST analogs

Analogs Structure

Gly Phe Gly Leu NH2

W201 O

Gly Phe Gly Leu NH2 W202 O

Gly Phe Gly Leu NH2

W203 O

Gly Phe Gly Leu NH2 W204 O

Gly Phe Gly Leu NH2

W205

W206 O

Gly Phe Gly Leu NH2 Br W70 O

Gly Phe Gly Leu NH2

Br W71 O

Gly Phe Gly Leu NH2 Br K15

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a Table 6.2. Potency of Dippu-AST analogs : inhibitory effect on JH release (IC50) and activation of AstR in CHO-WTA11 cells (EC50)

Sample Group (R1) IC50 /nM Rank (IC50) EC50 /nM Rank (EC50) AST6 1.45 2 0.2 1 W201 CH 2 957 10 2952 10 W202 (CH2)2 93.5 8 472.7 9 W203 (CH2)3 77.0 7 153.3 7 W204 (CH2)4 69.5 5 443.3 8 W205 (CH2)5 37.8 4 62.6 2 W206 (CH2)6 27.2 3 65.5 4 Sample Group (R2) IC50 /nM Rank (IC50) EC50 /nM Rank (EC50) W70 1-Br 128 9 147.4 6 W71 2-Br 78.4 6 95.6 5 K15 3-Br 6.98 1 62.8 3

a Potency is defined as the dose required to achieve a given level of inhibition of JH biosynthesis or activation of AstR. 1 EC50 was determined by AstR activation assay in CHO-WTA11 cells (details seen Fig. 2). 2 IC50 values are measured using RCA (CA from day 7 mated female).

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Fig. 6.1. The effect of topical application of K15 and W206 on JH biosynthesis (A) and oocyte growth (B). AST analogs were diluted in 20% DMSO and 80% acetone to a concentration of 10-3 M, and applied 5 µl to the dorsal abdomen of each D. punctata female on day 0. The effect of AST analogs on JH biosynthesis and basal oocyte growth was determined from day 4 to day 8.

184

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185

The author would like to thank:

Elisabeth Marchal, Ekaterina F. Hult, and Stephen S. Tobe for permitting to use the following material: (1) Diploptera punctata as a model for studying the endocrinology of arthropod reproduction and development. Gen Comp Endocr , 2013, 188, 85-93. (2) Characterization of the

Juvenile Hormone pathway in the viviparous cockroach, Diploptera punctata. PloS one, accepted. (3) Identification and characterization of the NMDA receptor and its role in regulating reproduction in the cockroach, Diploptera punctata, 2014, paper submitted to ‘Journal of experimental biology’

Elisabeth Marchal, Ekaterina F. Hult, Sven Zels, Jozef Vanden Broeck, and Stephen S. Tobe for permit to use the following material: Mode of action of allatostatins in the regulation of juvenile hormone biosynthesis in the cockroach, Diploptera punctata, Insect Biochem Mol Biol, 2014, 54,

61-68.

And Journal: ‘Insect biochemistry and molecular biology’ for allowing the author to reprint the article in the institutions open scholarly website (institutional repository).