ABSTRACT Title of dissertation: The functional importance and significance of ecdysteroids in molt-cycle regulation of the blue crab, Callinectes sapidus Sirinart Techa, Doctorate of Philosophy, 2014 Dissertation directed by Associate Professor J. Sook Chung Marine Estuarine Environmental Science This study aims to expand our understanding of how ecdysteroids and neuropeptide hormones (MIH/CHH) regulate molting in crustaceans using the blue crab Callinectes sapidus as a model animal. The hypothesis of this study is that ecdysteroids have a stimulatory effect on MIH/CHH production in eyestalks while generating both positive and negative feedback on ecdysteroidogenesis in Y-organs. Since ecdysteroids exert their signals through an ecdysteroid receptor complex, composed of an ecdysone receptor (EcR) and its partner, the retinoid-X receptor (RXR), the functional activity of ecdysteroids on tissues of interest is examined through EcR expression. Endogenous levels of ecdysteroids as well as expression in EcR, RXR and MIH/CHH are assayed in both embryonic and juvenile molt cycles. Similar patterns of changes in these two cycles suggest that molting control is driven by the same mechanisms in each. During embryonic molt, the correlation of ecdysteroid levels and MIH/CHH expression suggests that ecdysteroids are involved in MIH/CHH production. Using an in vitro system, stimulation of MIH expression occurs in eyestalks incubated with ponasterone A (PoA) and 20-hydroxyecdysone (20-HE) in the ratio found endogenously (3:1) and in ecdysteroid concentrations of 75 and 150 ng/ml. The in vivo response of MIH expression to depletion of ecdysteroid signals was examined by RNAi knockdown of an ecdysone receptor that was found to reduce the levels of MIH to those found by MIH knockdown, providing support for the hypothesis that ecdysteroids stimulate MIH expression through an ecdysone receptor. This study also found evidence suggesting that crustaceans have long-loop feedback for ecdysteroids in the neuroendocrine axis, which is similar to that found in insects. THE FUNCTIONAL IMPORTANCE AND SIGNIFICANCE OF ECDYSTEROIDS IN MOLT-CYCLE REGULATION OF THE BLUE CRAB, Callinectes sapidus By Sirinart Techa Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment Of the requirements for the degree of Doctorate of Philosophy 2014 Advisory Committee: Associate Prof. J. Sook Chung, Chair Associate Prof. Shaojun Du Associate Prof. Rosemary Jagus Associate Prof. Joseph Pitula Professor Allen Place © Copyright by Sirinart Techa 2014 ii ACKNOWLEDGEMENTS Firstly, I would like to thank Dr. Sook Chung, my supervisor and mentor, who inspired and extracted the best out of me. There were difficulties and obstacles that seemed impossible along the journey, but with her guidance, I was able to actually deal with these challenges. Dr. Chung gave me the chance to grow as a person and as an individual. I will always be humbly grateful. Next, I would like to thank my committee members: Drs. Jim Du, Rosemary Jagus, Joseph Pitula, and Allen Place for their suggestions, comments and support. I really appreciate all their time and effort to help me get through this process. I also thank Dr. David O’Brochta for his kindness in serving as the Dean’s Representative. Thanks also go to former and current members of the Chung laboratory: Dr. Nilli Zmora, Sarah Bembe, Javier Alvarez, Wilawan Thongda, and Dr. In Sook Ahn for their cooperation and collaboration during our work in the Chung laboratory. I appreciate the support of Oded Zmora and personnel from the hatchery that gave us young juvenile crabs. Everyone at the Aquaculture Research Center (ARC) at the Institute of Marine and Environmental Technology (IMET) deserve our thanks for maintenance of the crab systems. I am thankful for the tremendous support from my family, especially my aunt Kamonrat Poompramun, who raised me and made all this possible. Without her good heart and support, this would not have been possible to implement. I thank my husband, Weija Nyman, for all of his support and for always being on iii my side. I must also thank my dear friends, Pongpan Laksanalamai and Anchalee Jiemjit for their constant support, dedication and encouragement throughout my studies. Finally, I thank the Thai government for my scholarship (National Science and Technology Development Agency, NSTDA) and financial support during my seven years of study. Without it, I would never have had a chance to accomplish my goal. iv TABLE OF CONTENTS LIST OF TABLES………………………………………………………………………vii LIST OF FIGURES…………………………………………………………...............viii LIST OF ABBREVIATION……………………………………………………………...x 1. CHAPTER 1. Introduction ............................................................................... 1 1.1. Molting and its regulation ...................................................................... 1 1.1.1 Neuropeptide hormone family .................................................. 2 Insect................................................................................................2 Crustceans.......................................................................................2 1.1.2 Ecdysteroid hormones ............................................................. 4 Nuclear hormone receptor superfamily ........................................... 6 Ecdysteroid-responsive genes ........................................................ 7 1.2. The blue crab, Callinectes sapidus ....................................................... 8 1.2.1. Ovarian development .............................................................. 9 1.2.2. Embryonic development ....................................................... 10 1.2.3. Larval and post-larval development ...................................... 12 1.2.4. Molt stages and physilogical changes................................... 12 1.2.5. Ecdysteroids during a molt cycle............................................14 1.2.6. Ecdysteroid receptor complex in C. sapidus (CasEcR and CasRXR.................................................................................15 1.3. Hypothesis...........................................................................................18 2. CHAPTER 2. Changes in ecdysteroid levels and expression patterns of ecdysteroids-responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus ......................................... 20 2.1. Introduction ......................................................................................... 22 2.2. Materials and mathods ....................................................................... 24 2.2.1. Animals and sample collection .............................................. 24 2.2.2. Degenerate primers for cloning the full-length cDNA of C. sapidus BTBDP ............................................................ 25 2.2.3. PCR with degenerate primers of CasBTBDP with 5´and 3´ rapid amplification of cDNA ends (RACE) ......................... 25 2.2.4. Sequence analyses of CasBTBDP ....................................... 26 2.2.5. Total ecdysteroids during embryogenesis using ecdysteroid-radioimmunoassay (Ecd-RIA) ............................ 27 2.2.6. Expression levels of ecdysteroid-responsive factors and v neuropeptide hormones ........................................................ 27 2.2.7 Immunolocalization of CasMIH and CasCHH in C. sapidus embryos ................................................................................ 28 2.2.8. Statistical analysis.................................................................. 29 2.3. Results ............................................................................................... 29 2.3.1. Changes in the levels of ecdysteroids during ovarian and embryonic development.........................................................29 2.3.2. Expression levels of CasEcRs and CasRXRs during ovarian development and embryogenesis ............................ 31 a) Total CasEcRs...............................................................31 b) Total CasRXRs...............................................................33 2.3.3. Full-length cDNA sequence of BTB domain protein of C. sapidus (CasBTBDP).............................................................35 2.3.4 Expression levels of CasBTBDP during ovarian development and embryogenesis .........................................38 2.3.5 CasCHH and CasMIH expression.........................................39 2.3.6 Localization of CasCHH and CasMIH during eyestalk formation................................................................................42 2.4. Discussion .......................................................................................... 44 3. CHAPTER 3. Ecdysteroids induces the expression of molt-inhibiting hormone of the blue crab, Callinectes sapidus....................................................51 3.1 Introduction........................................................................................53 3.2. Materials and methods.....................................................................56 3.2.1 Animals and sample collections.............................................56 3.2.2 Temporal-spatial distributions of CasEcR isoforms in eyestalk ganglia and Y-organs at different molt stages........................56 3.2.3 Effects of in vitro incubations of ecdysteroids on CasMIH expression in eyestalk ganglia...............................................57 3.2.4 Total ecdysteroids in tissues using ecdysteroid- radioimmunoassay (Ecd-RIA)................................................58 3.2.5 MIH titers in hemolymph using RIA........................................58 A) Iodination........................................................................58 B) Hemolymph sample preparation.....................................59 3.2.6 Knockdown effects on CasMIH expression in eyestalk and molt interval using double-stranded RNA (dsRNA)................59 3.2.7 Expression levels of CasEcR and CasMIH using qRT-PCR assays...................................................................................60 3.2.8 Statistical analysis..................................................................61 3.3. Results..............................................................................................62 vi 3.3.1 Levels of CasMIH expression in eyestalk and of CasMIH in hemolymph during the molt cycle...........................................62 3.3.2 Endogenous changes in ecdysteroid concentrations in eyestalk and Y-organs at different molt stage......................................64 3.3.3 Effects of ecdysteroids on CasMIH expression......................65 3.3.4 Temporal-spatial distributions of CasEcR isoforms in eyestalk and Y-organs during the molt cycle........................................67 3.3.5 Levels of CasEcR expression in eyestalk during the molt cycle.......................................................................................68 3.3.6 Effects of CasMIH- and CasEcR1-dsRNA on CasMIH expression...............................................................................69 3.4 Discussion..........................................................................................72 3.5 Supplement figures............................................................................83 4. CHAPTER 4. General conclusions and future studies....................................86 5. REFERENCES ............................................................................................... 95 vii LIST OF TABLES Table 2-1 List of primers for CasBTBDP cloning and expression analysis ... .....26 Table 3-1 List of primers that were used for qRT-PCR assays and an end-point PCR assay..........................................................................................................61 viii LIST OF FIGURES Figure 1-1 Circulating ecdysteroid from a C. sapidus .........................................5 Figure 1-2 Structure organization of nuclear hormone receptor family................7 Figure 1-3 Schematic diagram illustrating ecdysteroids-responsive genes ........8 Figure 1-4 Life cycle of C. sapidus ................................................................. .....9 Figure 1-5 The developmental stages of C. sapidus embryos .......................... 11 Figure 1-6 A schematic diagram of the molt cycle of C. sapidus ...................... 13 Figure 1-7 Different forms of ecdysteroids circulating during mid premolt ....... 14 Figure 1-8 Isoform characterization of CasEcR and CasRXR ......................... 17 Figure 2-1 Levels of ecdysteroids during ovarian and embryonic development........................................................................................................30 Figure 2-2 Levels of total CasEcR expression during ovarian and embryonic development ....................................................................................................... 32 Figure 2-3 Levels of total CasRXR expression during ovarian and embryonic development ....................................................................................................... 34 Figure 2-4 Schematic diagram of BTB domain protein cDNA ........................... 35 Figure 2-5 Multiple alignment of BTBDP in C. sapidus and other arthropods............................................................................................................37 Figure 2-6 Expression levels of CasBTBDP during ovarian and embryonic development of C. sapidus ................................................................................. 39 Figure 2-7 Expression levels of CasCHH during eyestalk organogenesis in C. sapidus embryos ................................................................................................ 40 Figure 2-8 Expression levels of CasMIH during eyestalk organogenesis in C. sapidus embryos ................................................................................................ 41 Figure 2-9 Immunolocalization of CasCHH and CasMIH in the embryos at heart- beatand developing-eye stages. ......................................................................... 43 ix Figure 3-1 Expression levels of CasMIH in eyestalk ganglia during juvenile molt......................................................................................................................61 Figure 3-2 Storage levels of CasMIH in sinus gland ......................................... 63 Figure 3-3 Circulating levels in hemolymph of CasMIH during juvenile molt .... 64 Figure 3-4 Levels of ecdysteroid presence in eyestalk and Y-organs ............... 65 Figure 3-5 Expression levels of CasMIH in eyestalk ganglia after incubation with different concentrations of active ecdysteroids....................................................66 Figure 3-6 Spatial and temporal distributions of four CasEcR forms in eyestalk and Y-organs.......................................................................................................67 Figure 3-7 Expression levels of CasEcR1 in eyestalk during juvenile molt using isoform- specific primers......................................................................................68 Figure 3-8 Expression levels of CasMIH from a time course study of CasMIH- dsRNA injections in eyestalk ganglia...................................................................70 Figure 3-9 Molt interval after administered with dsRNAs...................................71 Figure 3-10 Proposed model of molting control in crustaceans.........................80 Figure S3-1 A time course study of CasMIH-dsRNA injections on the levels of CasMIH expression in eyestalk ganglia...............................................................83 Figure S3-2 Genomic arrangement of C. sapidus MIH......................................84 Figure S3-3 Expression levels of CasMIH in eyestalk ganglia after incubation with different ratios of active ecdysteroids by qRT-PCR assay...........................85 x LIST OF ABBREVIATION AF Activation function CasAK C. sapidus Arginine Kinase CasBTBDP C. sapidus BTB domain protein CasCHH C. sapidus Crustacean Hyperglycemic Hormone CasEcR C. sapidus Ecdysone Receptor CasMIH C. sapidus Molt-Inhibiting Hormone CasRXR C. sapidus Retinoid-X receptor DBD DNA-binding domain dsRNA double-stranded RNA Ecd-RIA Ecdysteroid-radioimmunoassay IRES Internal ribosome entry site LBD Ligand-binding domain LBP Ligand-binding pocket PoA Ponasterone A PTTH Prothroracicotropic hormone uORF Upstream open reading frame 20-HE 20-hydroxyecdysone 1 CHAPTER 1. INTRODUCTION 1.1 Molting and its regulation Growth and the developmental processes of all animals involves an increase in body size and changes in body-plan formation. However, these changes in arthropods are limited to their exoskeleton. In order to grow, the old shell must be shed and the new one synthesized. This process is called molting. Since molting is essential for growth and development in arthropods, the mechanisms that regulate these processes must be better understood. It has been well documented that molting is stimulated by ecdysteroids and the circulating levels of ecdysteroids in all arthropods highly correlate with the molt stages [1-9]. As a comparison, the regulation of ecdysteroidogenesis in insects and crustaceans is different. In insects, prothoracicotropic hormone (PTTH) is produced in the brain and secreted from the corpora allata to stimulate prothoracic glands that then produce ecdysteroids [10-12]. Alternatively, neuropeptides, produced in the eyestalks of crustaceans, suppress ecdysteroidogenesis in the Y-organs, which is equivalent to the prothoracic gland [5,13-23]. It has been reported that ecdysteroids have stimulatory effects on the production of PTTH in Manduca sexta [24-27]. Moreover, it has been proposed that ecdysteroids may have a positive effect on neuropeptide production in the eyestalks of crustaceans [14,28]. 2 1.1.1 Neuropeptide hormones Insects In insects, PTTH is produced in the brain and secreted by the corpora allata to stimulate the prothoracic gland. PTTH targets the prothroracic gland and triggers ecdysteroidogenesis through a signaling cascade involving Ca2+, cAMP and a MAP kinase [11,29]. In addition to physiological and environmental cues such as weight gain and photoperiod, PTTH is positively regulated by ecdysteroids as reported in M. sexta [24-27]. As reported in M. sexta and Pieris brassicae, ecdysteroids also generate a short-loop feedback on the prothoracic gland. Lower levels stimulate, while higher titers inhibit the action of the prothoracic gland [30-32]. Crustaceans The finding that the molting progression is induced by eyestalk ablation [33] has led to the discovery that molting in crustaceans is suppressed by eyestalk factors, which were later attributed to neuropeptides. Neuropeptide hormones belong to the crustacean hyperglycemic hormone (CHH) family and include CHH, molt-inhibiting hormone (MIH), gonad-stimulating and -inhibiting hormone (GS/IH), and mandibular organ-inhibiting hormone (MOIH) [34-38]. MIH and CHH are synthesized in neurosecretory cells in the X-organ located within the eyestalk ganglia. These neuropeptides are transported through the axonal tract, stored in the sinus gland, and secreted into the hemolymph to suppress ecdysteroidogenesis in Y-organs [18-21,37,39-43]. Inhibitory actions of 3 MIH and CHH on the Y-organs have been reported in several crustaceans including Carcinus maenas, Callinectes sapidus, Gecarcinus lateralis, Procambarus clarkii, and Uca pugilator through in vitro incubation studies [5,13- 22,44,45]. In addition, only two in vivo studies confirm the intrinsic hormonal signaling of MIH and CHH on the activity of Y-organs [37,46]. To date, regulation in the levels of MIH expression during the molt cycle has not been well understood. A few reports indicate that ecdysteroids may have an effect on the levels of MIH in the eyestalks. Administering ecdysteroids in Cancer anternarius increased neuropeptide production in eyestalk ganglia [28]. Moreover, there is a report in Uca pugilator that a concentration of ecdysteroids in the eyestalk ganglia was higher than the hemolymph [14], implicating a possible accumulation of ecdysteroids in this tissue. Recent studies have shown that the upstream promoter regions of MIH genes, isolated from C. pagurus and Charybdis feriatus, contain the binding sites for ecdysteroid-responsive factors [45,47]. Furthermore, hemolymph titers of MIH and CHH during the molt cycle of C. maenas show that MIH levels are significantly higher during the late premolt compared to the other stages [48]. These lines of evidence suggest that ecdysteroids may have a positive effect on the expression of neuropeptides in the eyestalk ganglia. 4 1.1.2 Ecdysteroid hormones Ecdysteroids are a class of steroid hormones that are derived from cholesterol and found in animals, plants and fungi [1,2]. Ecdysteroids play important roles in several processes in arthropods, most significantly in molting. Ecdysteroidogenesis in the prothroracic gland or Y-organ is similar, but the crustacean Y-organ produces a greater diversity of ecdysteroids [49-52]. According to the review by Mykles [52], the synthetic pathway of ecdysteroids can be divided into two stages: 1) conversion of cholesterol to 5-diketol, and 2) conversion of 5-diketol to secreted products. The former stage is common to all decapod species, while the latter one has four pathways that are named according to the secreted ecdysteroids by the Y-organ: 1) ecdysone, 2) 3- dehydroecdysone, 3) 25-deoxyecdysone, and 4) 3-dehydro-25-deoxyecdysone [52]. Among the four forms, crustacean ecdysteroids are usually secreted in three forms (ecdysone, 3-dehydroxyecdysone, 25-deoxyecdysone, and 3- dehydro-25-deoxyecdysone) depending on the species [50,52-55]. In peripheral tissues, these compounds are modified to an active form. It is well known that molting is induced by ecdysteroids. Molt stages are defined by the levels of hemolymphatic ecdysteroids and setogenesis [3-9,56]. Within a molt cycle, ecdysteroid titers fluctuate: low during postmolt and intermolt, increasing at early premolt, peaking at midpremolt and returning to basal levels prior to ecdysis, as reported in C. magister, C. maenas, C. sapidus, Daphnia magna, Hyas araneu, Metopograpsus messor, and P. clarkii (Fig. 1-1) [3-9]. 5 The clearance of ecdysteroids in crustaceans is reported in Homarus americanus, and the rate is molt-stage dependent: lowest at early premolt, followed by the mid premolt stage, and highest during the postmolt and intermolt stages [57]. Ecdysteroids were hydrolyzed into high polarity metabolites including 20-hydroxyecdysonoic acid and 20, 26-dihydroxyecdysone and were excreted in urine (95%), while the apolar metabolites were found in feces (5%) [58,59]. Therefore, it is suggested that titers of circulating ecdysteroids during each molt stage are determined by the difference between the rate of synthesis and clearance [60]. Figure 1-1. Circulating ecdysteroid from a C. sapidus female during the last two molts: pre-pubertal molt and adult molt assayed by ecdysteroid-RIA. Numbers on the X-axis represents days before each molt. C4= intermolt, D0= early molt, D1-D2= mid premolt, D3/4=late premolt and E= ecdysis. The p value: *, p < 0.05; **, p < 0.005 0 50 100 150 200 250 300 350 Days to adult To ta l e cd ys te ro id s (n g) /m lh em o ly m pg h -----------C4------------D0-D1D2D3/4------------C4----------D0--D1-D2-D3/4-- Molt stages E E * ** ** ** ** ** 6 Nuclear hormone receptor superfamily Nuclear receptors are phylogenetically related proteins clustered into a large superfamily, including receptors for hydrophobic molecules such as steroid hormones, retinoic acids (all trans and 9-cis isoforms), thyroid hormones, and fatty acids [61]. Nuclear receptors are commonly found in sponges and Metazoans including echinoderms, tunicates, arthropods and vertebrate [62]. The nuclear hormone receptor family is characterized by 5-independent functional domains (domains A-F as originally defined by Krust): 1) A/B or N- terminal domains or activation function-1 (AF1): exhibiting ligand-independent trans-activation; 2) C or DNA-binding domain (DBD): binding to regulatory elements of the target genes; 3) D or hinge region (HR) domain: providing flexibility within the molecule; 4) E or ligand-binding domain (LBD): serving as docking sites for its cognate ligands and transcriptional regulators (co-activator or co-repressor); and 5) F or C-terminal or AF2 domain: trans-activating with ligand- dependent manner [63-65] as shown in Fig. 1-2. Since the DBD and AF reside within the same molecule, the nuclear hormone receptor can directly trans-activate the target genes when its cognate ligands bind to the LBD [66]. In general, binding of the ligands to the LBD stimulates conformational changes in the LBD and AF2 which strengthens partner dimerization or releases a co-repressor. Eventually, the pre-initiation complex is recruited to the promoter, and transcription initiation is started. 7 Figure 1-2. Schematic illustration of the structural and functional organization of a nuclear hormone receptor family. The evolutionarily conserved regions C and E are indicated as white and grey boxes, respectively, while the line represents the divergent regions A/B, D, and F. Two transcription activation functions (AFs) have been described: a constitutively active AF1 in the A/B region and a ligand-inducible AF2 in region E or F. DBD: DNA binding domain, LBD: ligand binding domain, and NLS: nuclear localization signal [67]. Ecdysteroid-responsive genes After receiving the ecdysteroid signals, the EcR-RXR complex, a master transcription factor, will trigger the expression of downstream transcription factors including EcR-RXR itself. In Drosophila, the EcR/USP heterodimer induces the expression of early ecdysteroid-responsive genes such as Broad complex, E74, and E75 which are required during development and metamorphosis [68-75]. The Broad complex gene is located in the 2B5 locus and encodes three alternate pairs of C2H2 zinc finger motifs for binding to regulatory elements of late ecdysteroid-responsive genes such as E71 [70,72,76]. E74 genes encode an ETS DNA-binding domain [73,77-80], while E75 genes located in 75B locus encode a nuclear hormone receptor [72,81-83]. Although these transcriptional factors are downstream of ecdysteroid responses, their transcription is directly induced by 20-hydroxyecdysone [72,82,83] C A/B E D F AF1 DBD hinge LBD AF2 NLS Helix H12 NLS (ligand-dependent) Dimerization (strong)Dimerization (weak) 8 Figure 1-3. Schematic diagram illustrating the differential effects of ecdysteroids titers on the expressions of their responsive genes in Drosophila: higher levels of ecdysteroids trigger early ecdysteroid- responsive genes including of the Broad complex (BR-C), E74A, E75A and EcR. 2Bc and br are alleles of Broad complex genes [73]. 1.2. The blue crab, Callinectes sapidus Blue crabs, Callinectes sapidus, are classified into the Phylum Arthropoda and Subphylum Crustacea. C. sapidus belongs to the family known as Portunidae and is distributed across two hemispheres from the north Atlantic coast of the United States to the coast of Uruguay in South America [84]. Blue crabs are important to the ecosystem and commercial fishery along the Chesapeake Bay where the brood stock density has drastically declined in the last few decades. Therefore, a better understanding of how their growth is regulated is necessary. Similar to other arthropods, blue crabs undergo growth, development, and reproduction through molting. Molting in C. sapidus is repeated 27 to 29 times during their lifecycle starting from hatching and until adulthood (Fig.1-4) [60]. As a result, the body size increases 20-50% in each molt cycle, which is referred to as a molt increment. Females undergo a terminal-pubertal molt during the active reproductive period, while males continue to molt during their adulthood [85]. 9 Figure 1-4. Lifecycle of C. sapidus starting from (1) a sponge female carrying the fertilized eggs, (2) developing embryo inside embryonic envelopes, (3) hatching larva, (4) megalops, (5) juvenile and (6) adult crab. 1.2.1. Ovarian development Similar to other arthropods, reproduction in the adult female crab includes nuclear replication, oocyte differentiation, and yolk (or vitellin) deposition [86]. The early phase of oocyte development, called previtellogenesis, occurs during both sexual rest and reproductive activity. During this phase, the cellular organelles are synthesized and the glycoproteins are accumulated [87]. The latter phase is vitellogenesis and only occurs during reproduction. Vitellogenesis is involved in the synthesis of vitellogenin, a precursor yolk protein, in the fat body of insects or hepatopancreas of crustaceans [23,88,89]. After being processed from vitellogenin, vitellin, a lipoprotein complex, is circulated in 1 4 5 6 2 3 10 the hemolymph and endocytosed into the oocytes [87]. The differentiated oocytes continue accumulation of vitellin, maternal mRNAs, and other cytoplasmic proteins, resulting in a significant increase in oocyte diameter [23,87,90]. Moreover, vitellin is known as a carrier of the ecdysteroids that are required for early embryogenesis in insects and crustaceans [91-96]. 1.2.2 Embryonic development In C. sapidus, the fertilized eggs are extruded from the gonopores of the females and attached on the inner-branch hairs (ovigerous hairs) of swimmerets, and this egg mass is referred to as sponge [97]. The developmental stages of decapod embryos such as C. maenas, C. sapidus, H. americanus and Palaemon serratus are classified according to the size of remaining yolk, together with the levels of development of eyes, appendages, and heart [98-101]. In C. sapidus, embryogenesis is divided into five stages based on the criteria reported in C. maenas, as shown in Fig. 1-5 [98]. The yolk stage includes embryos from zygotic formation until the end of gastrulation. Organogenesis occurs at the limb-formation stage when the divided cells are differentiated and migrate to one pole of the egg, forming a clear cluster of these cells. The organogenesis of the embryos continues and can be observed by the presence of eye pigment at the eye-anlage stage. While the eyes continue to develop, the heart starts to function at the heart-beat stage. Eye development is completed at the developing-eye stage and the fully-developed embryos are referred to as hatching imminent. In C. sapidus, these processes are completed in 14-19 days depending on water temperature zoeae (Zoeal stage 1). Figure 1-5. The developmental stages of the criteria as stated [98]. During crustacean which results in the release of ecdysteroids the levels of ecdysteroids during suggest that zygotic ecdysteroidogenesis [102] and the larvae are released as the first C. sapidus embryos classified according to embryogenesis, vitellin is being continuously utilized [101,103,104]. However, changes in embryogenesis of decapod crustaceans may occur during this period 11 [100,105]. 12 1.2.3. Larval and post-larval development After hatching, the released larva are called zoea and have a cylindrical shape, a long and sharp beak, two pairs of antennae, and four pairs of leg-like appendages [97,106]. The zoea undergoes 7 or 8 cycles of molting and finally becomes a post-larval form called a megalopa. The megalopa has a flattened body with a shorter and wider abdomen than that of the zoea. In C. sapidus, the megalopa metamorphoses into the first juvenile crab (crab 1) [97]. Development of zoeae and megalopae in the Chesapeake Bay might be affected by endocrine disruptors, a consideration due to the transport of larvae and megalopae to lower salinity in the upper bay. During this period, the bay watersheds peak in contamination with pesticides that are considered endocrine disruptors, such as atrazine, chlordane and endosulfan [107-111]. In particular, endosulfan sulphate promotes embryonic abnormalities, reduces molting frequency, and interferes with molting processes through blocking of the binding of ecdysteroids and EcR as reported in D. magna and Macrobrachium malcolmsoii [110-112]. Therefore, it is possible that the blue crab population might be affected by these endocrine disruptors that interfere with molting. 1.2.4. Molt stages and physiological changes During the molt cycle, physiological and structural changes are closely related to molt stages, and crustacean molting is classified into four different stages: intermolt (C4), premolt (D0-4), ecdysis (E), and postmolt (A-C3). Premolt is 13 further characterized by three stages: early premolt (D0), mid premolt (D1-D2), and late premolt (D3-D4) [113,114], as shown in Fig.1-6. Figure 1-6. A schematic diagram of the molt cycle of C. sapidus with associated phenotypic changes of the swimming leg. Inner ring: blue=premolt, yellow=ecdysis, and light blue=postmolt; outer ring: color represents the progression of molting: blue=intermolt (C4), magenta=premolt (D0-D4), red=ecdysis (E), purple=postmolt (A-B). (http://www.serc.si.edu/education/resources/bluecrab/molting.aspx) Normal activities such as eating and swimming occur during intermolt when the exoskeleton is rigid, and the membranous tissue is attached to the hypodermis and the cuticle [115]. During premolt, the old cuticle is detached from the hypodermis by digestive enzymes (chitinases) secreted by epithelial cells [116], while the new cuticles composed of an organic matrix are laid. At the late midpremolt (D2) stage, organic materials from the old shell are reabsorbed, resulting in a breakable shell. Ecdysis begins through the breakage of the ecdyseal line assisted by the hydrostatic pressure that is generated by the water 14 uptake induced by the release of gut-borne CHH [117,118]. After tanning and calicification during postmolt (A, B, C1-C3 stages), the animals resume normal activities. 1.2.5 Ecdysteroids during a molt cycle In C. sapidus, a recent study reported that during the mid premolt (D2) stage, Ponasterone A (PoA) is the major active form followed by 20- hydroxyecdysone (20-HE) as shown in Fig. 1-7, and they are circulated with a 3:1 ratio [60]. Interestingly, ratios of the two major forms in the last three molts seem variable when the ratios of 20-HE to PoA are increased when the animals become mature. Figure 1-7. Different forms of ecdysteroids circulating during mid premolt (D2) from juvenile (black bar), pre-pubertal (light-grey bar), and adult (grey bar) molts, assayed by HPLC-RIA. % Ecdysteroid represents percentages of 20-hydroxyecdysone (20-OH E), ecdysone (E), 3-dehydroxyecdysone (3 dehydro E), Ponasterone A (PoA), 25- deoxyecdysone (25 deoxy E), and unknown [60]. Juvenile Pre-pubertal Adult 15 1.2.6 Ecdysteroid receptor complex in C. sapidus (CasEcR and CasRXR) In a previous study, cDNA sequences encoding for EcR and RXR in C. sapidus (CasEcR-CasRXR) were isolated and characterized [119]. The 5’ untranslated region (5’UTR) of both the CasEcR and CasRXR cDNA sequences contain translational regulatory sites: an upstream open reading frame (uORF) and an internal ribosome entry site (IRES). Messenger RNAs containing uORFs are often up-regulated in their translations under stress or a starvation condition, while the translations of other mRNAs are down-regulated by phosphorylated Eukaryotic Initiation Factor 2 (eIF2) [120]. Reduction in the levels of ternary complexes (Met-tRNAiMet and eIF2-GTP) will slow the reinitiation of uORFs and allow the 40S preinitiation complex to scan through the encoding ORF. On the contrary, IRES provides an alternative pathway for translation initiation. Specific mRNAs containing IRES can be continuously translated when the cap-dependent mechanism is impaired [121-123]. Four putative isoforms of both CasEcR and CasRXR are found in this species. CasEcR is especially important because it binds directly to ecdysteroids [124,125]. Two putative CasEcR isoforms (CasEcR1a/CasEcR2a) have an insert in the HR domain that lies between the LBD and the DBD. This insert (HRi) contains four residues of Pro that may generate steric hindrance and interfere with conformational changes, resulting in an inefficiency inof ecdysteroid transduction. In addition, there are two types of ligand-binding pocket (LBP): type-1 (CasEcR1/CasEcR1a) and type-2 (CasEcR2/CasEcR2a) that differ in 16 hydrophobic fields (Fig. 1-8), suggesting that these differences may be responsible for binding with several forms of circulating ecdysteroids (Fig. 1-7). This notion seems to agree with the finding that during the mid premolt, two active ecdysteriods, PoA and 20-HE, are found in the hemolymph. PoA is more hydrophobic than the 20-HE [60]. Considered together, this suggests that type-1 CasEcR would be stronger for binding with 20-HE than PoA, whereas type-2 CasEcR would prefer to bind with the PoA. However, the specific binding to ecdysteroids and significant roles of four CasEcR isoforms in molt-related tissues (eyestalks and Y-organs) still remain unknown. CasRXR appears to have variations from inserted fragment(s) at the T-box within the DBD and/or at the anterior of the LBD. Both inserts are predicted to have effects on the dimerization of CasRXR to its CasEcR partner as suggested in U. pugilator [126-128]. 17 Figure 1-8. Isoform characterization of CasEcR (A) and CasRXR (B) from multiple alignment of putative encoding protein. Putative protein structures of CasEcR1 (A´) and CasEcR2 (A´´) analyzed with PyMol program based on the X-ray structure of Heliothis virescens EcR-USP complex bound to Ponasterone A. Variations in CasEcR1 and CasEcR2 are labeled in the ball-and-stick model: green and red representing hydrophobic and hydrophilic residues, respectively. Small legends represent surface model differences between CasEcR1 and CasEcR2, respectively [119]. AF1 DBD HR LBD AF2 CasEcR1 CasEcR1a AF1 DBD HRi LBD AF2 AF1 DBD HR LBD AF2CasEcR2 AF1 HRi LBD AF2CasEcR2a DBD (A) (A´) (A´´) AF1 DBD HR LBD AF2 CasRXR1 CasRXR1a AF1 DBDT HR LBD AF2 AF1 DBD HR LBDL AF2 CasRXR2 AF1 HR LBDL AF2 CasRXR2a DBDT (B) 18 1.3. Hypothesis This study aims to expand the knowledge of how ecdysteroids and neuropeptide hormones regulate molting in crustaceans, using blue crab C. sapidus as a model animal. The hypothesis of this study is that ecdysteroids have a stimulatory effect on neuropeptide production in eyestalks while generating both positive and negative feedback on ecdysteroidogenesis in Y- organs. Since ecdysteroids exert their signals through the ecdysteroid receptor complex composed of EcR and RXR, the functional activity of ecdysteroids on tissues of interest can be examined through EcR expression. Two main objectives that guided the effort to determine whether ecdysteroids have a direct effect on activities of eyestalk ganglia included: 1) An aim to determine the expression levels of endogenous neuropeptides (CHH and MIH), ecdysteroid-responsive factors (EcR, RXR, and BTBDP) and ecdysteroid levels during C. sapidus embryogenesis. As proposed in research on arthropods, the first cycle of molting occurs during embryogenesis (referred to as embryonic molt). Due to the times and patterns of their expressions, it has been suggested that there is an interaction between ecdysteroids and neuropeptides. By obtaining these results, expressions of neuropeptides and ecdysteroids can be compared to determine whether the embryonic ecdysteroids are correlated with the newly produced neuropeptides. 2) An aim to assess the direct effects of ecdysteroids on MIH expression in eyestalk ganglia where neuropeptides are specifically produced. Endogenous expressions of MIH and EcR1, the major isoform in eyestalks, were examined 19 from eyestalk ganglia during the preceding juvenile molt. In addition, expression of three isoforms of EcR in Y-organs was compared to determine whether the expression patterns were similar with those of the eyestalks. Induction of ecdysteroids on MIH expression was performed by in vitro incubation of the intermolt eyestalks with different ecdysteroid concentrations and ratios that mimic endogenous ecdysteroids during the C. sapidus premolt. To confirm direct interactions between ecdysteroids and MIH expression, knockdown of EcR1 using double-stranded RNA (dsRNA) was carried out to test its functional significance in vivo. It was expected that the dsRNA specific for EcR1 and MIH would experience a reduction in MIH expression levels. 20 2. CHAPTER 2 Changes in ecdysteroid levels and expression patterns of ecdysteroids- responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus Abstract Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1-4 have expression levels of maternal CasEcR and CasRXR 10-50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full- length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is 21 followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus. 22 2.1 Introduction Embryogenesis is a developmental process that entails a complex network of regulatory genes [129]. The embryo of many arthropod species starts with a large amount of yolk protein (vitellins= VT) at fertilization. Extensive research reports that for crustaceans, their embryos undergo molting within an embryonic envelope during a relatively long period of embryogenesis [95,99,130- 134]. Molting is essential for growth and development that recapitulates throughout the life cycle of each given arthropod species. During embryogenesis of decapod crustaceans, such as Carcinus maenas, Callinectes sapidus, Homarus americanus, and Palaemon serratus, the first molt occurs within embryonic envelopes [98-101]. Hatching coincides with the true first molt accompanied by shedding of the exuvium. The crustacean molt is known to be regulated mainly through the interplay between ecdysteroids and neuropeptide hormones [34,35,37,38,98,135-137] However, molecular mechanism(s) of the endocrine system by which the growth of embryos occur, still remains less well understood. The hormonal mechanism of hatching that accompanies shedding of the exuvium is equivalent to that of the ecdysis recapitulating events in juvenile and adult stages. Both of these processes are likely driven largely by hormonal cascades and characterized by ecdysial behavior and a significant amount of water uptake [98,118]. During embryogenesis, eyestalk-originated neuropeptides, including crustacean hyperglycemic hormone (CHH) and molt- 23 inhibiting hormone (MIH), are expressed early at the eye-anlage stage, with levels of these hormones continuing to increase until completion of embryogenesis [98]. Ecdysteroids conjugated to VT and transported into oocytes play a critical role in the early embryogenesis of arthropods [91-96]. During embryogenesis, the utilization of VT often coincides with the release of ecdysteroids. Therefore, embryogenesis is staged with the size of the remaining yolk, together with the development of eye, appendage, and heart tissues [98-101]. The continuous utilization of VT during crustacean embryogenesis may reflect the companion levels of ecdysteroids [101,103,104]. However, changes in the levels of ecdysteroids during embryogenesis of decapod crustaceans [100,105] suggest that the newly formed Y-organs are active for the production of zygotic ecdysteroids during this period. The functional importance of ecdysteroids involves the presence of ecdysteroids receptors (EcRs) and other ecdysteroid-responsive transcriptional proteins such as Broad complex (BR-C) and Broad-Complex-Tramtrack-Bric-a brac (BTB) domain protein (BTBDP) [70,138]. In C. sapidus, the presence of four different cDNAs of EcRs and RXRs suggests that there may be putative structural isoforms of these receptors [119]. BR-C, an early ecdysteroid- responsive factor, is involved in reproduction, embryogenesis, and molting of both insects and crustaceans such as Drosophila melanogaster, Tribolium castaneum, and Penaeus monodon [139-144]. The functional role of BTBDP has not yet been examined during the embryogenesis of any crustaceans. 24 We aim to profile changes in eyestalk neurohormones and ecdysteroids during embryogenesis. Herein, we report for the first time in crustaceans that expression levels of ecdysteroid-responsive factors (including CasEcR, CasRXR, and CasBTBDP) and CasMIH and CasCHH during the embryogenesis of the blue crab C. sapidus, correspond to the ecdysteroid profiles. 2.2 Materials and methods 2.2.1 Animal and sample collection Study batches of adult females (C. sapidus) were obtained from a Chesapeake Bay watershed spawning ground by the annual winter dredge survey of the Virginia Institute of Marine Science (VIMS, Virginia) and screened for diseases [145,146]. Disease-free animals were maintained in a closed- recirculatory system holding at 30 ppt artificial seawater at 22°-23°C (8L:16D), fed daily with chopped squid and shrimp, and monitored daily for the extrusion of eggs (= spawning). Ovarian samples were collected from adult females and staged according to the criteria as described [147]. Upon spawning, embryos were biopsied at every two days until they hatched. These embryos were examined for developmental stages under a dissecting microscope following the criteria described [98], collected, and stored at -80oC until analysis. 25 2.2.2 Degenerate primers for cloning the full-length cDNA of C. sapidus BTBDP Degenerate primers of CasBTBDP (Table 2-1) were generated based on conserved regions identified from the multiple sequence alignment (www.genome.jp/ClustalW) of the putative BR-C isolated in insects including A. aegypti (AY499537.1, AY499538.1, AY499539.1, AY499540.1), B. mori (NM_001111334.1, NM_001111333.1, NM_001043511.), and D. melanogaster (NM_001144672.3, NM_166897.2). The identified conserved amino acid (aa) sequences were reverse translated (http://www.bioinformatics.org/sms2/rev_trans.html) using C. sapidus codon usage (http://www.kazusa.or.jp/codon/). 2.2.3 PCR with degenerate primers of CasBTBDP with 5´and 3´ rapid amplification of cDNA ends (RACE) The cDNAs for 5´and 3´ rapid amplification of cDNA ends (RACE) were generated with total RNA extracted from Y-organs of a juvenile female at intermolt stage as described [119]. The initial BTBDP sequence was obtained from a two-step PCR assay with degenerate primers [119,148-153]. Touch-down (TD) PCR was carried out with 3´RACE cDNA of Y-organ and primers of BTBDP- dF1 and universal primers (UPM) using Advantage Taq Polymerase (BD Biosciences) under the PCR conditions as described [119,150-153]. A nested PCR was performed as described [119] with two primers: BTBDP-dF2 and Nested Universal Primer (NUP). 26 Table 2-1 The list of primers for CasBTBDP cloning and expression analysis Forward 5´ to 3´ Reverse 5´ to 3´ dF1 ATHACNWSYGCYTTYGARAA dR1 TGRTGNACRTTVACYTCDCC dF2 TTYGTNGAYGTSACYYTSGCYTG dR2 ACYTCNCCRTGRTARATRAA F2 CACTCAAGTGGAACAATCACAAGTCC R1 CATCCTTCATGAAGACAATGGGGTG F3 GTCGCCTCATCACGAACCCAACAGCC R2 or qR GTAGTCGCTGCATGTTGACAGCACG qF CAGGAGGTATTCATTGACGTCACAC “d” represents degenerate primers. 'q' primers are for qRT-PCR assays. Forward primers (F1, F2, and F3) were used for 3' RACE and reverse for CasBTBDP (R1, and R2) were used for 5´RACE. A band was identified with the expected size of 700 bp on a 1.5% agarose gel. The remaining procedures for cloning, sequencing, and sequence analysis were described [149-153]. This initial sequence was used for generating the gene-specific primers (BTBDP-5R1, -5R2, -3F2 and -3F3 listed in Table 2-1) that were used for obtaining the full-length CasBTBDP using 5´ and 3´RACE, respectively. 2.2.4 Sequence analyses of CasBTBDP The open reading frame (ORF) of the full-length cDNA sequence of CasBTBDP was identified with ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). The Conserved Domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd) remarked the conserved domains. The sequence similarity of the deduced aa sequences between CasBTBDP and other arthropod BR-C was analyzed using ClustalW (http://www.ebi.ac.uk) and Boxshade server (http://www.ch.embnet.org/software/BOX_form.html). The 5´ and 3´ UTR sequences of CasBTBDP were further analyzed to examine whether 27 or not they contained putative translational regulatory elements using RegRNA (http://regrna.mbc.nctu.edu.tw/html/prediction.html) [154]. 2.2.5 Total ecdysteroids during embryogenesis using ecdysteroid- radioimmunoassay (Ecd-RIA) Staged ovaries and embryos (~30 mg) were homogenized in ice-cold DEPC-treated PBS and dissolved in 0.1 M borate buffer (pH 8.0). Our preliminary data showed that the same amounts of total ecdysteroids were measured in both the crude extract and the samples treated with esterase and protease (unpublished results). Therefore, the samples homogenized in the borate buffer were estimated for the levels of ecdysteroids using the same protocol as described [60]. The amounts of ecdysteroids were calculated using AssayZap program (BIOSOFT) and presented in pg/oocyte or pg/embryo. 2.2.6 Expression levels of ecdysteroid-responsive factors (CasEcRs, CasRXRs, and CasBTBDP) and neuropeptide hormones (CasMIH and CasCHH) using qRT-PCR assays Embryos at various developmental stages (10-20 mg wet weight/ sample) were homogenized in 150 µl of ice-cold DEPC-treated PBS. Two thirds of each of these homogenized samples was used for the total RNA by following the procedures described [153]. After treating with DNase I (Fermentas), the total RNAs (1.5 µg) were subjected to cDNA synthesis using RevertAid Reverse Transcriptase (Fermentas) and finally diluted to 12.5 ng total RNA/µl. The sample cDNAs (25 ng of total RNA eqv.) of ovaries, embryos, and zoeae were 28 estimated in duplicate for levels of CasBTBDP, CasMIH, and CasCHH expression, using Fast SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems 7500 instrument. In particular in this study, the expression of CasCHH only in the eyestalk, not in the pericardial organ was measured [153]. For measuring total levels of CasEcRs and CasRXRs, the primers were designed from the region that was conserved and shared in all four isoforms. Arginine kinase (AK) was measured in the same sample cDNAs as a reference gene. The standards for qRT-PCR assays were generated similarly as described [149,150,152,153]. The expression levels are noted as copies/µg total RNA. 2.2.7 Immunolocalization of CasMIH and CasCHH in C. sapidus embryos Whole-mount immunohistochemistry was carried out for the localization of CasCHH and CasMIH in the embryos at heart-beat and developing-eye stages by following the procedures as described [98]. Embryos were fixed in a Drosophila fixative overnight [150] and their embryonic envelopes were removed for antisera penetration. After washing with PTX containing 0.05% sodium azide, the embryos were incubated with specific anti-rabbit-CasMIH serum [23] or - CasCHH serum that recognized CHH present only in the eyestalk ganglia [149] at a final dilution of 1:2,000 for 3 days at room temperature. After washing as above, the embryos were incubated with an Alexa 488 conjugated goat anti- rabbit IgG (Life Technologies) as described [150]. The embryo samples that were obtained from at least 3-4 adult females were examined using a confocal microscope (Zeiss), with the pictures collected using Z projection (BioRad). The 29 staining intensity and area of CasCHH and CasMIH were determined and compared using ImageJ (http://imagej.nih.gov/ij). 2.2.8 Statistical analysis All results represent mean ± 1SE (n), in which n is the number of replicates. The data were subjected to the normality test using the Shapiro-Wilk test (SigmaPlot) prior to all statistical tests SigmaPlot (version 12.3). The data that did not show a normal distribution were analyzed using nonparametric ANOVA (Kruskal-Wallis Test) or unpaired t test. In all cases, statistical significance was accepted at P<0.05 and noted with letters or asterisks (* or **) . 2.3. Results 2.3.1 Changes in the levels of ecdysteroids during ovarian and embryonic development Significant changes in amounts of total ecdysteroids are observed during ovarian development and embryogenesis (Fig. 2-1). The stage-1 ovary shows undetectable amounts of total ecdysteroids, while the stage-2 ovary contains 0.3 ± 0.1 pg/oocyte (n=7), the similar amount as observed in the stage-3 ovary. The levels of ecdysteroids are positively co-related with ovarian development and significantly (P<0.001) increased from 0.3 ± 0.1 pg/oocyte (n=5) at stage 3 to 0.9 ± 0.1 pg/oocyte (n=6) at stage 4 (Fig. 2-1, inset). 30 Figure 2-1. Levels of ecdysteroids by Ecd-RIA during ovarian (inset) and embryonic development The data are presented as mean ± SE pg/oocyte or embryo. All data were subjected to a normal distribution using the Shapiro-Wilk test (SigmaPlot). When the data did not show the normal distribution, a nonparametric test (Mann-Whitney Rank Sum test) was employed. Statistical significance was accepted at P < 0.05 and noted with letters at each developmental stage. n.d.= not detectable in At the yolk stage, the ecdysteroid levels observed are statistically significant (P<0.001) reduced to 0.3 ± 0.1 pg/embryo (n=6) from 0.9 ± 0.1 pg/oocyte at ovarian stage 4. At the limb-formation stage, the ecdysteroid levels start to rise (1.0 ± 0.4 pg/embryo, n=12) and continue to increase significantly (P<0.001) to 7.9 ± 0.1 pg/embryo (n=9) at the eye-anlage stage and 11.1 ± 1.5 pg/embryo (n=6) at the heart-beat stage. Total ecdysteroid levels reach a peak at the developing-eye stage (21.3 ± 4.4 pg/embryo, n=9). With hatching- 31 imminent embryos, these levels then precipitously drop to 7.8 ± 1.5 pg/embryo (n=7), similar to the levels observed at the eye-anlage and heart-beat stages, and continue to decrease significantly (P<0.05) to 2.3 ± 0.5 pg/embryo (n=5) at zoeal stage (Fig. 2-1). 2.3.2 Expression levels of CasEcRs and CasRXRs during ovarian development and embryogenesis a) Total CasEcRs Ovary and embryo samples contain different levels of CasEcRs expression: ovary tissue shows 10-50 times higher levels than those of embryo tissue (Fig. 2-2). During the ovarian development from stage 1 to 4, there is no marked difference in the CasEcRs expression: 8.4 ± 0.5 x105 copies/µg total RNA (n=3) at stage 1; 10.9 ±1.8 x105 copies/µg total RNA (n=6) at stage 2; 7.1 ± 1.2 x105 copies/µg total RNA (n=7) at stage 3; and 6.2 ± 2.8 x 105 copies/µg total RNA (n=2) at stage 4. 32 Figure 2-2. Levels of total CasEcR expression during ovarian and embryonic development of C. sapidus by qRT-PCR assays. Sample cDNAs (25 ng total RNA eqv.) of ovaries (n=4) and embryos (n= 5-10). The data are presented as mean ± SE copies/µg total RNA. All data were subjected to a normal distribution using the Shapiro- Wilk test (SigmaPlot). When the data did not show the normal distribution, a nonparametric test (Mann-Whitney Rank Sum test) was employed. Statistical significance was accepted at P < 0.05 and noted with letters at each developmental stage. N.D= no statistical difference The levels of CasEcRs change during embryogenesis. At the yolk stage, levels of CasEcRs were measured at 2.1 ± 0.4 x104 copies/µg total RNA (n=11) and start to rise at the limb-formation stage (0.7 ± 0.2 x105 copies/µg total RNA, n=12). At the eye-anlage stage, the expression of CasEcRs is elevated to 1.1 ± 0.3 x105 copies/µg total RNA (n=9). However, these levels begin to decline to 0.9 ± 0.5 x105 copies/µg total RNA (n=7) at the heart-beat stage, continuing to 33 drop to 0.4 ± 0.2 x105 copies/µg total RNA (n=5) and 0.1 ± 0.1 x105 copies/µg total RNA (n=5) at both the developing-eye and hatching-imminent stage, respectively. In zoeae, the expression of CasEcRs is measured at 0.2 ± 0.1 x105 copies/µg total RNA (n=5). b) Total CasRXRs Similar to the expression of CasEcRs, the levels of CasRXRs in ovaries are ~10 times higher than the embryos (Fig. 2-3). The expression of CasRXRs during ovarian development remains constant, ranging from 6.3 ± 0.9 (x105 copies/µg total RNA, n=3) at stage 1; 6.9 ± 1.1 x105 copies/µg total RNA (n=6) at stage 2; 5.5 ± 0.7 x105 copies/µg total RNA (n=7) at stage 3; and 6.2 ± 1.1 x105 copies/µg total RNA (n=2) at stage 4. 34 Figure 2-3. Levels of total CasRXR expression during ovarian and embryonic development of C. sapidus by qRT-PCR assays. Sample cDNAs (25 ng total RNA eqv.) of ovaries (n=4) and embryos (n= 5-10). The data are presented as mean ± SE copies/µg total RNA. All data were subjected to a normal distribution using the Shapiro- Wilk test (SigmaPlot). When the data did not show the normal distribution, a nonparametric test (Mann-Whitney Rank Sum test) was employed. Statistical significance was accepted at P < 0.05 and noted with letters at each developmental stage. N.D= no statistical difference Similar to the expression pattern of CasEcRs, the levels of CasRXRs are as low as 0.2 ± 0.1 x105 copies/µg total RNA (n=8) at the yolk stage. At the limb- formation and eye-anlage stages, levels of CasRXRs are significantly (P<0.05) increased to 0.6 ± 0.1 x105 copies/µg total RNA (n=8) and peaked to 0.7 ± 0.1 x105 copies/µg total RNA (n=10), respectively. After the heart-beat stage, levels of CasRXRs are reduced from 0.5 ± 0.2 x 105 copies/µg total RNA (n=7), to 0.2 ± 0.1 x 105 copies/µg total RNA (n=5) at the developing x 105 copies/µg total RNA (n=5) at the hatching contained levels of CasRXRs 2.3.3 Full-length cDNA sequence of (CasBTBDP) The full-length cDNA sequence (1,650 nt) of accession no. KC736588) includes 204 nt of 5´untranslated region (UTR), 948 nt of the open reading frame (ORF), and 495 nt of 3´UTR Figure 2-4. Schematic diagram of (CasBTBDP: accession no. KC736588) and its putative regulatory elements analyzed using Regulatory RNA analysis program in 5´UTR. The putative protein encoded in ORF contains BTB domain and is identified as a member of BTB superfamily. The putative protein (CasBTBDP) shows the highest sequence identity (72%) with BR crustacean species P. monodon, mori, Apis mellifera, and putative CasBTBDP does not contain a zinc -eye stage, and to 0.4 ± 0.2 -imminent stage. Hatching zoeae with 0.1 ± 0.1 x 105 copies/µg total RNA (n=4) BTB domain protein of C. sapidus CasBTBDP (GenBank (Fig. 2-4). BTB domain protein cDNA isolated in [155]. Post transcriptional control is predicted C. sapidus and followed by those from insect species: T. castaneum with 51, 50, and 47%, respectively. The -finger motif at the C-termin 35 . C. sapidus BTB domain -C of a B. us (Figs. 36 2-4 and 2-5). As shown in Fig. 2-4, two putative translational, regulatory elements are predicted in the 5´UTR: internal ribosome entry site (IRES: C4-G99 and G167-G258) and an upstream ORF (uORF: A54-A146). 37 Trc 1 --MVDTQHFCLRWNNYQSSITSAFENLRDDEDFVDVTLACDGKSLKAHRVVLSACSPYFRELLKSTPCKHPVIVLQDVAW Apm 1 --MVDTQHFCLRWNNYQSSITSAFENLRDDEDFVDVTLACDGRSLKAHRVVLSACSPYFRELLKSTPCKHPVIVLQDVAF Bom_Z1 1 MVDSQTQHFCLRWNNYQRSITSAFENLRDDEDFVDVTLACDGKSLKAHRVVLSACSPYFRELLKSTPCKHPVIVLQDVAY Pem_6.2 1 ---MEEGYLALRWNNHNTIFTKILTLLREQEAYVDVSLACAGRLYPAHKFVLSTCSEYFKEMFSKNPCKHPIVFMKDVST Pem_6.1 1 ---MEEGYLALRWNNHNTIFTKILTLLREQEAYVDVSLACAGRLYPAHKFVLSTCSEYFKEMFSKNPCKHPIVFMKDVST Cas 1 ---MTDELLSLKWNNHKSTFADILTILRDQEVFIDVTLACGGKQYPAHKFVLSTCSDYFKEMFTKNPCKHPIVFMKDVSA Pem_4 1 ---MEDGLLSLKWNNHKTTFFEILRVLREKANYTDATIAVDGKFYPVHKLVMSTCSEYFSEIFEKTPCKSPVIVLKDVRS Trc 79 TDLHALVEFIYHGEVNVHQRSLSSFLKTAEVLRVSGLTQQHGD-DREQLAQVQSLVRSQQSTPTSNHHPSFTEKLVEDAL Apm 79 SDLHALVEFIYHGEVNVHQRSLSSFLKTAEVLRVSGLTQQADQTDRDELSHVRALAAGGNHLP---FHEKLVESFPRGGS Bom_Z1 81 TDLHALVEFIYHGEVNVHQHSLSSFLKTAEVLRVSGLTHNDAA-QGPLLQTMRSSAAPSPHTPPHPAHSTHIPQPYPDKL Pem_6.2 78 KDMEALLDFMYKGEVHVPQSELGSLLRTAEGLQVKGLAVPDDSPRGSSTTPIVPSASSVPRSPPPSLMAPMHMRGKRKRP Pem_6.1 78 KDMEALLDFMYKGEVHVPQSELGSLLRTAEGLQVKGLAVPDDSPRGSSTTPIVPSASSVPRSPPPSLMAPMHMRGKRKRP Cas 78 RDLEALLDFMYRGEVNVPHHNLASLIKTAEGLQVKGLAVPDD---------------------------PVQLRKAEPRS Pem_4 78 QDMEALLDYMYLGEVNVNQNDLASLLKTAECLRIKGLAVPDED------TTKVRKAPPDDRQESPPPKRRRNEDNPSSAP Trc 158 FTSPSSPPHGATVNQLLRRAALQHRRERRISSDPEGDHKRPRPDHIIGNNNNNELNLSHTQPPQTQPADFSPSAMKNNAL Apm 156 LPTPVTPTP--TTVQQLLRRAQIRRNERRTPDPHDETAKKPRVLSPPLNNND---------------------------- Bom_Z1 160 EEALLQPSS--SIPPMMRRIPLPPPRRMSSTDNSPDVIKRPRHDNNNTESPQ---------------------------- Pem_6.2 158 PESAKKDDPPKLTLRPDLGPPATNRSRMRNRPPGMPELKRIKREERNAANVG---------------------------- Pem_6.1 158 PESAKKDDPPKLTLRPDLGPPATNRSRMRNRPSGMPELKRIKREEHSAANVG---------------------------- Cas 131 RE-LRETHTPRPPPEPSASPP--------------PKKKRTREPEHVSERMT---------------------------- Pem 152 RPVSPSVNAPSKTTTPSVTPPVQSAS--------LPGSQSQDGIQDSSLDVP---------------------------- Trc 238 NLNMNSSSTTTTQNEANNGITSTERENSPCSPSPTLNSRLNEENVKSEPMELLCSTNQEENSNDSGDVANDNGPNNLPQG Apm 206 -----ATPTDFSMGVKNNHVSSKVEGNGVHEENSPLEDNIKCEPLELTGGNSGNAAGNNEDSSDSGAAASDRPP------ Bom_Z1 210 -MHAADFSTKNHSILNSRGHEQGNNGNGISNSSSSPSPRLMDEVKNEPVDMICPSNPDIDRCTDDTPPHTHHRPIGGPPS Pem_6.2 210 ---------TIEPGEVPGSPSPTPSSHNSDEQSQNLAHKIKTERSEYSKEEAEDLDEDEGVAGEVMSGEEEQEQEPEEEE Pem_6.1 210 ---------TIEPGEVPGSPSPTPSSHNSDEQSQNLAHKIKTERSEYSKEEAEDLDEDEGVAGEVMSGEEEQEQEPEEEE Cas 168 ---------TLYGNMSPHHEPNSP-------------YDLSQKSSSSSVSQSDNLHNDTAAPNPMSPDQPPSHQHPVHHH Pem 196 ---------PMVKVEMQEADDPDDYRKDNSYEGGSVNEGEMGSDFGAELSKAEHDPDSYGSGSYAGPSIQPGGDLPWDEG Trc 318 PHSGSSAG--------------------DHDEHDSPIGPYLTPSESKLFATAAGSFNFSMAALAADPTGLGGLN--QSLQ Apm 275 --ASASSN--------------------EHEAESEHTSTPNFLSEAKIFPPTPGSFNFSMAALATEHTPLSVKFPGMGHG Bom_Z1 289 RSSSADAE--------------------DRTPPPQLPPSPFIPPPDTKLFNPSNSYNFTMEALAQHSTLAGLQSPLASDG Pem_6.2 281 EEEEEEEEEGALGEGEGLSHGVLSDVEDGYEQSNSSLPNSDISTTELLQVDLSEDGTQFIIGPGGFGDMMSRTSSLAGDD Pem_6.1 281 EEEEEEEE-GALGEGEGLSHGVLSDVEDGYEQSNSSLPNSDISTTELLQVDLSEDGTQFIIGPGGFGDMMSRTSSLAGDD Cas 226 QQHLANKE-------------------------------------------------------------------NSPDD Pem 267 DSSSFPQE--------------------GFSGDLPAGQQPQGDWDHVRPAALIPVVEIRQPVAASTPTSIAQLMTGACPK Trc 376 ANADSLAGTSQESSGGG-----------------RPGRTAFCDICNKTFSRFWSLQRHLSDTHFYTPQNLQCNVCGRSYR Apm 333 LQPPDLAGTSQGSAGLPSAILAMRLSHPLHGNLLPPGVCYTCDVCGKTLSTKLTLKRHKEQQHFQPLNSAVCALCHKVFR Bom_Z1 349 MASTSQGGAR------------------------TPQEDYRCDPCNKSLSSLTRLKRHIQNVHMRPSREPVCNICQRVYS Pem_6.2 361 ERNGDKEQKKPFVCPLCGQSFTRRDNLANHIKTHTGDRPFMCQYCHKCFSRKDYLKQHERI--HTGEKPYPCDICGRAFT Pem_6.1 360 ERNGDKEQKKPFVCPLCGQSFTRRDNLANHIKTHTGDRPFMCQYCHKCFSRKDYLKQHERI--HTGEKPYPCDICGRAFT Cas 239 TRRVSSTPQPPTPQPLPPKSPLP--------------------------LASSEVNCQEKS--HEEADPMPGPSWRPVAT Pem 327 TAAIPMGPASGAFTPYTASTSPRGSTPDLAQVAPADKQSFMCPVCGKQFGQPYNLRRHLTT--HTGERPYRCPHCNYAAS Trc 439 SRNSLVSHRSQYHREGNEIKFENDCFI--------------- Apm 413 TLNSLNNHKSIYHRRQK------------------------- Bom_Z1 405 SLNSLRNHKSIYHRKQPAPPAPGPFYPVN------------- Pem_6.2 439 RKEGLTDHMRCHSDFRAFSCETCGKSFKQKCGPRFHKRNYKH Pem_6.1 438 RKGGLTDHMRCHSDFRAFSCETCGKSFKQKCGLRFHKRNYKH Cas 291 KPEDEVVS---------------------------------- Pem 405 QNVHLEKHIRRIHLNNGQNETPTGPAVTWAPAATAVTP---- Figure 2-5. Multiple alignment of BTBDP in C. sapidus (Cas), and BTB domain protein from other arthropods: A. mellifera (Apm: NP_001035356.1), B. mori (Bom _Z1: NP_001104804.1), P. monodon (PemBR-C: Pem_4: JN638741.1; Pem_6.1: JN638739.1; Pem_6.2: JN638740.1), and T. castanaum (Trc: NP_001104734.1) using ClustalW (http://www.ebi.ac.uk) website and Boxshade server (http://www.ch.embnet.org/software/BOX_form.html). The gray and black shades Zf-H2C2 Zf-H2C2 38 represent the highly conserved residues among the analyzed sequences. Putative zinc finger domain (Zf-H2C2) of PemBR-C isoforms and common BTB domain are bracketed. 2.3.4 Expression levels of CasBTBDP during ovarian development and embryogenesis As shown in Fig. 2-6, during ovarian development, levels of CasBTBDP expression are high and remain constant ranging from 6.3 ± 0.9 x105 copies/µg total RNA (n=4) at stage 1; 11.7 ± 5.3 x105 copies/µg total RNA (n=4) at stage 2; 10.6 ± 3.0 x105 copies/µg total RNA (n=4) at stage 3; and to 11.2 ± 2.7 x105 copies/µg total RNA (n=2) at stage 4. During embryogenesis, these levels are low with 0.3 ± 0.1 x105 copies/µg total RNA at the yolk stage (n=7) but rise to 1.1 - 1.4 x105 copies/µg total RNA (n=6-7) from the limb-formation to the hatching- imminent stages, without showing a statistically significant difference (P<0.05). Hatching zoeae show levels of CasBTBDP expression with 0.6 ± 0.1 x105 copies/µg total RNA. 39 Figure 2-6. Expression levels of CasBTBDP by qRT-PCR assays during ovarian and embryo development of C. sapidus. Sample cDNAs (25 ng total RNA eqv.) of ovaries (n=4) and embryos (n= 5-10) at each particular stage. The data are presented as mean ± SE copies/µg total RNA. All data were subjected to a normal distribution using the Shapiro-Wilk test (SigmaPlot). When the data did not show the normal distribution, a nonparametric test (Mann-Whitney Rank Sum test) was employed. Statistical significance was accepted at P < 0.05 and noted with asterisk. N.D. = no statistical difference 2.3.5 CasCHH and CasMIH expression The patterns of CasCHH and CasMIH expression in this study during embryogenesis are similar to the total ecdysteroids and ecdysteroid-responsive factors, especially in CasCHH (Fig. 2-7). At the early stages (yolk and limb- formation), CasCHH is detected with 610 ± 16 (n=8) and 710 ± 24 (n=10) copies/µg total RNA. These levels significantly increase to 1.4 ± 0.4 x104 ** 40 copies/µg total RNA (n=10, P<0.05) at the eye-anlage stage and to 2.6 ± 0.4 x104 copies/µg total RNA (n=9, P<0.05) at the heart-beat stage. CasCHH levels peak at the developing-eye stage (5.0 ± 1.2 x104 copies/µg total RNA, n=6). The levels decline at the hatching-imminent stage: copies/µg total RNA 2.8 ± 0.6 (n=6) and remain low at the zoeal stage: 2.0 ± 0.4 x104 copies/µg total RNA (n=3). Figure 2-7. Expression levels of CasCHH during eyestalk organogenesis in C. sapidus embryos. Each cDNA sample containing 25 ng total RNA eqv. was assayed in duplicate. The expression levels are represented as copies/µg total RNA. The data are presented as mean ± SE (n). All data were subjected to a normality test using the Shapiro-Wilk test (SigmaPlot). When the data did not show the normal distribution, a nonparametric test (Mann-Whitney Rank Sum test) was employed. Statistical significance was accepted at P < 0.05 and noted with letters. CasMIH levels do not fluctuate as we also found in CasCHH (Figs. 2-8). During yolk and eye-anlage stages, the levels of CasMIH are similar, ranging 41 between 4.3 - 7.3 x 103 copies/µg total RNA (n=5-6); however, the levels significantly increase to 2.3 ± 0.6 x104 copies/µg total RNA (n=6, P<0.05) at the heart-beat stage and 2.2 ± 0.6 x104 copies/µg total RNA (n=8) at the developing- eye stage, compared to those measured at earlier developmental stages. Then, the CasMIH levels drop to 1.2 ± 0.3 and 1.1 ± 0.3 x104 copies/µg total RNA (n=5- 6) at the hatching-imminent and zoeal stages, respectively. Figure 2-8. Expression levels of CasMIH during eyestalk organogenesis in C. sapidus embryos. Each cDNA sample containing 25 ng total RNA eqv. was assayed in duplicate. The expression levels are represented as copies/µg total RNA. The data are presented as mean ± SE (n). All data were subjected to a normality test using the Shapiro-Wilk test (SigmaPlot). When the data did not show the normal distribution, a nonparametric test (Mann-Whitney Rank Sum test) was employed. Statistical significance was accepted at P < 0.05 and noted with letters. 2.3.6 Localization of C The presence of CasCHH and CasMIH is noted in embryos at the heart beat stage of the C. sapidus intensity is stronger in the sinus gland than in the cells. Similar to the expression patterns as shown in Fig. show greater staining intensity in the sinus gland than those at the heart stage (Fig. 2-9C). However, there is no difference between the neuropeptides: CasCHH and CasMIH at the same embryonic stages. B) A) asCHH and CasMIH during eyestalk formation embryos (arrow in Figs. 2-9A and B): staining 2-7 and 2-8, embryos at the developing- 42 - eye stage -beat 43 Figure 2-9. Immunolocalization of CasCHH (A) and CasMIH (B) in the embryos at heart-beat and developing-eye stages. The insets show the magnification of the X- organ (XO). C) The analysis of staining intensity and area of CasCHH and MIH of the sinus gland of embryos at heart-beat and developing-eye stages (ImageJ). Statistical significance (P<0.01) between the two stages was noted with ** for CasCHH (closed bar) and CasMIH (open bar). No difference was found between the neuropeptides: CasCHH and CasMIH at the same embryonic stages. The number of adult females are noted in the graph. Arrows indicate axonal tracts; SG= sinus gland. Scale bars are noted in µm. Embryonic stage Heart-beat Developing-eye Re la tiv e s ta in in g in te n s ity a n d a re a 1e+2 1e+3 1e+4 1e+5 1e+6 C) 4 3 3 4 ** ** N.D N.D 44 2.4 Discussion Our study shows the profiling of changes in the titers and transcriptional levels of major molt regulators: ecdysteroids and their receptor (CasEcRs and CasRXRs) and two neuropeptide hormones (CasCHH and CasMIH) during the ovarian development and embryogenesis of C. sapidus. In addition, we isolated the full-length cDNA sequence of C. sapidus BTB domain protein (CasBTBDP), as well as quantified for temporal changes along with these molt regulators. The somatic growth of arthropods requires molting processes, which are coordinated by ecdysteroids and precisely-timed pulsatile release of neurohormones [35,38,60,135]. Ecdysteroids produced and secreted by the Y- organs in crustaceans or prothoracic glands in insects trigger these animals to enter and to prepare for the premolt as well as initiate embryogenesis. Prior to organogenesis of Y-organs or prothoracic glands, ecdysteroids are derived from the mother as conjugated to a large amount of vitellin (VT) [94,156,157]. From the phosphorylated-inactive forms conjugated to VT in the ovary, maternal ecdysteroids are being released via VT utilization at the initiation of embryogenesis [158]. The increase of ecdysteroid levels in the occytes is comparable to the VT contents that is reported in developing ovaries of C. sapidus [89]. Our current results are similar to the findings reported in several crustacean species including C. maenas, M. rosenbergii, Orchestia gammarella and Lysmata seticaudata, where the levels of ecdysteroids are parallele to those of vitellogenesis 45 [95,105,133,159-163]. As stated above, vitellogenin is suggested to carry ecdysteroids to the ovary of crustaceans. By contrast, some insect ovaries synthesize ecdysone [164,165], the function of which is known to stimulate vitellogenesis in fat body and follicle cells of the mosquito, Aedes aegypti [166,167]. The direct involvement of ecdysteroids in vitellogenesis itself has not yet been defined in decapod crustaceans. The maternal ecdysteroids that are accumulated in the oocytes during ovarian development of C. sapidus may trigger the initiation of embryogenesis, as in D. melanogaster [156]. Levels of ecdysteroids change during C. sapidus embryogenesis (Fig. 2- 1), implying their functional importance in embryogenesis. The accumulation of ecdysteroids in the developing ovaries noticeably correlates with ovarian development, specifically with the amount of VT in the ovary [89], suggesting that CasVT serves as a carrier protein, similar to what is reported in insects such as B. mori, Dacus cucurbitae and D. melanogaster [94,156,157]. Hence, the maternal ecdysteroids released from VT through the utilization of CasVT may be responsible for the changes in ecdysteroid levels in early C. sapidus embryogenesis, particularly at the yolk stage, which is similar to what is reported in B. mori and D. melanogaster [94,156]. The levels of ecdysteroids in C. sapidus embryos are akin to those of other crustaceans and insects, ranging between 2-80 pg/embryo, which reflect the size of embryos [94,100,105,161,168-170]. Interestingly in this study, as the C. sapidus embryo begins to develop, ecdysteroid levels are reduced. This finding is similar to the earlier observation in the B. mori embryogenesis when the 46 ecdysteroid levels abruptly decline during cell differentiation [158]. The continuous utilization of VT during embryogenesis indicates the release and use of maternal ecdysteroids. Hence, this condition may result in the reduced levels of total ecdysteroids seen during early embryogenesis. The overall patterns of the changes in embryonic ecdysteroids in later stages are similar to those seen in the juvenile molt [37,119]. Levels of ecdysteroids are low at early embryogenesis, then increased to peak, and returned to the low levels, before hatching of the embryos. The increase in ecdysteroid levels at the limb-formation stage of C. sapidus embryogenesis may reflect the organogenesis and activity of Y-organs. This notion is supported by which levels of cholesterol are gradually reduced during C. sapidus embryogenesis (Prof. R. Lee, personal communication). The presence of Y- organs that were observed at the limb-formation stage of P. serratus embryos coincides with the increase in the total ecdysteroids [100,137]. However, in Machrobrachium rosenbergii, increase in ecdysteroid levels occurs at the eye- anlage stage [105], indicating that the timing of organogenesis of Y-organs may somewhat differ in this species. Taken together, it is suggested that the increase in ecdysteroids during C. sapidus embryogenesis may be primarily contributed to de novo synthesis from the developing Y-organs. Changes in ecdysteroid levels during ovarian development and embryogenesis may often forecast the differences in the amounts of EcRs and its responsive factors. However, ovaries contain levels of CasEcR, CasRXR, and CasBTBDP expression 10-50 times higher than embryos, contrary to the 47 ecdysteroid patterns. Such high expression levels of these genes in ovaries indicate that maternal CasEcR, CasRXR, and CasBTBDP may be immediately translated at early embryogenesis when transcription machinery may not be yet functional [171,172]. Subsequently, these results imply that the ecdysteroid receptor complex (CasEcR-CasRXR) and CasBTBDP may be involved in the early embryogenesis of C. sapidus. Compared to the juvenile molt, the pattern changes of embryonic ecdysteroids, and the receptor complex (CasEcR-CasRXR) we observed are similar to those of the hemolymph and Y-organ, respectively [119]. In embryonic development, levels of both CasEcR and CasRXR are peaked earlier than that of the ecdysteroids. Up-regulation of EcR and RXR by ecdysteroids that occurs in prothoracic gland of M. sexta further modulates this gland’s activity for ecdysteroid synthesis [10,173,174]. Therefore, we infer that ecdysteroids may be responsible for controlling the newly formed Y-organ in the embryos for ecdysteroidogenesis. Our data imply that ecdysteroids and their receptors in C. sapidus embryos may have similar functions in the juvenile molt. BTBDPs, with BTB domain for a protein-protein interaction motif, share a function in protein degradation by ubiquination [175,176]. Particularly, in the embryos of Caenorhabditis elegans and D. melanogaster, ubiquitination through BTBDP is required for degradation of various cell cycle regulators such as CDC25 and cyclin D, and hedgehog morphogen [177]; Zhang, 2006 #494]. Thus, we infer that CasBTBDP may have a similar function in C. sapidus as in C. 48 elegans and D. melanogaster, because its expression levels are maintained high in oocytes and embryos. During early developmental stages (yolk and limb-formation stages), transcriptional levels of CasMIH are higher than those of CasCHH. Our result is somewhat contradictory to earlier reports that MIH levels are much lower than CHH in embryo and adult C. maenas as well as in the eyestalk ganglia of juvenile and adult C. sapidus [23,44,178]. In C. sapidus, CasMIH expression is detected in the ovary and even prior to the eye anlage stage when CasCHH expression is also seen. In C. maenas, the expression of CHH and MIH is seen only at the eye-anlage stage but not in mature oocytes [98]. These observed differences in the timing of occurrence and levels of MIH suggest that the expression pattern of MIH may be species-specific, due to their different life histories and ecological conditions. In particular, C. sapidus inhabits relatively warmer water temperature than C. maenas, which affects the overall duration of embryogenesis, particularly initiation at the yolk stage. Therefore, we suggest that the regulatory mechanism of MIH expression during embryogenesis of C. maenas and C. sapidus may be slightly different. We are currently investigating the presence of ovarian MIH to see if its cDNA is identical to that found in eyestalk ganglia in juveniles and adults of C. sapidus. The overall patterns we observed of CasCHH and CasMIH expression in the eyestalk ganglia mirror the changes of ecdysteroids. Interestingly, the changes in CasCHH and CasMIH expression during embryogenesis seem comparable to and similar to changes seen in the juveniles where the amounts of 49 these neuropeptide expressions are closely related to the measured ecdysteroid titers (unpublished data). With this new data and our findings of the current study, we infer that CasCHH and CasMIH transcription and their hormonal levels may be affected by the presence and amount of ecdysteroids. Our finding suggests that the developing eyestalk during embryogenesis seems to have activity similar to that in the juvenile crabs; therefore, these neuropeptide hormones may function as molt regulator during C. sapidus embryogenesis. Molting results in the increase in the somatic size. Also noteworthy is that there is a difference in the size of decapod crustacean embryos at spawning and just prior to hatching. Similar to the juvenile and adult molt, the hatching process also relies on the uptake of iso-osmotic water that is assisted by the complete discharge of CHH produced by abdominal cells in C. maenas [118] and C. sapidus (unpublished observation). This increase in the size of embryos may be attributed to the first molt that is initiated by maternal ecdysteroids and occurs within the embryonic envelopes without shedding. Zygotic ecdysteroids produced by developing Y-organs, as well as CHH and MIH generated in the developing-eyestalk ganglia, may interact and control both the hatching and molting processes. Although the exact timing of the first molt cycle driven by zygotic ecdysteroids is unknown, we suggest that the first molt cycle can be noted by the presence of CHH expression located in the abdominal segments of C. sapidus (unpublished data) and C. maenas embryos [98], equivalent to gut CHH endocrine cells in adults, occurring only ~ 5-7 days prior to hatching. 50 Together with the presence of ecdysteroids and related expression of ecdysteroid-responsive factors, we believe that the process of embryos hatching to zoeae accompanying with the cuticular shedding is the first molt of C. sapidus. Our data demonstrate the presence of ecdysteroids, their receptors, and both CHH and MIH during the ovarian development and embryogenesis in C. sapidus. An inter-relationship between ecdysteroids and the expression of neuropeptides, specifically MIH, still remains to be studied. Since genomic sequences of Cancer pagurus, and Charybdis feriatus MIH contain the binding sites for BR-C, E74A and RXR in the upstream promoter region, we are currently gathering the sequencing information of the upstream promoter elements of C. sapidus [45,47]. 51 3. CHAPTER 3 Ecdysteroids induce the expression of molt-inhibiting hormone of the blue crab, Callinectes sapidus Abstract Arthropod molt is coordinated through the interplay between ecdysteroids and neuropeptide hormones. In crustaceans, changes in the activity of Y-organs during the molt cycle have been regulated by molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Little has been known on the mode of direct effects of ecdysteroids on the levels of MIH and CHH in the eyestalk ganglia during the molt cycle. This study focused on a putative feedback of ecdysteroids on the expression levels of MIH using in vitro incubation study with ecdysteroids and RNAi in the blue crab, Callinectes sapidus. Our results show a tissue-specific expression of ecdysone receptor (EcR) in which the major isoform in eyestalk ganglia is EcR1. The initial elevation of MIH expression at the early premolt stages is replicated by in vitro incubations of ecdysteroids that mimic the intrinsic conditions of D0 stage, in terms of the concentration (75 ng/ml) and the composition (ponasterone A and 20-hydroxyecdysone at a 3:1 ratio). Additionally, multiple injections of EcR1-dsRNA reduce MIH expression by 67%, compared to the controls. Our data provide evidence on a putative feedback mechanism of hormonal regulation in molting, specifically how the molt cycle may be repeated during the life cycle of crustaceans. The elevated concentrations of ecdysteroids at premolt stage may positively act on the expression levels of MIH in the eyestalk ganglia. Subsequently, the increased MIH titers in the 52 hemolymph at postmolt may inhibit the synthesis and release of ecdysteroids by Y-organs, re-setting, succeeding and subsequently, recapitulating the molt cycle. 53 3.1 Introduction Endocrine systems normally have feedback controls to regulate their balance in the organisms. In vertebrates, steroid hormones such as estrogens, glucocorticoids, and androgens regulate their production through negative feedback on neuroendocrine axes [31]. In insect neuropeptides, prothoracicotropic hormone (PTTH) that is produced from the brain and released by corpora cardiaca stimulates the prothoracic gland for ecdysteroidogenesis. PTTH is positively regulated by the ecdysteroids in Manduca sexta [24-27]. The ecdysteroids also generate short-loop feedbacks on prothoracic gland: stimulating at lower levels while inhibiting at the higher titers as reported in M. sexta and Pieris brassicae [10,30-32]. Life stages of arthropods continue through the recapitulated molting process. Molting is complex mechanisms that are hormonally regulated and involves in cell division, synthesis and deposition of new cuticle thereafter shedding of the old shell [36,179,180]. In crustaceans, members of the crustacean hyperglycemic hormone (CHH) family that are originated from the major endocrine tissue, eyestalk ganglia (X-organ-sinus gland) located within the eyestalk, are involved in the regulation of molting: 1) CHH and 2) molt-inhibiting hormone (MIH) [34-36,38,60]. MIH and CHH suppress the synthesis and release of ecdysteroids by Y-organs [178,181]. The hemolymph concentrations of CHH and MIH are closely associated with the levels of ecdysteroids during the molt cycle of the European green crab, Carcinus maenas [48]. 54 Ecdysteroids, arthropods’ molting hormones, are secreted by crustacean Y-organs that are equivalent to insect prothoracic glands. The levels of hemolymphatic ecdysteroids are positively related to molt stages in the following decapod crustaceans including Cancer magister, C. maenas, Callinectes sapidus, Daphnia magna, Hyas araneus, Homarus americanus, Metopograpsus messor, and Procambarus clarkii [3-7,9,59,182]. Y-organs secrete inactive forms of ecdysteroids: ecdysone, and 25-deoxyecdysone (25-dE) [43,49-51] that are subsequently hydroxylated at the peripheral tissues to active forms: 20- hydroxyecdysone (20-HE) and ponasterone A (PoA), respectively [52,57,183,184]. 20-HE is known to be the main active ecdysteroid in insects. However, the hemolymph of a given crustacean species carries more than one active form. In the premolt hemolymph of C. sapidus and C. maenas, PoA is measured as the major type of ecdysteroid and then 20-HE as the second [9,60,183]. Ecdysteroids have been known to affect the activity of Y-organs as well as that of the eyestalks [14,28]. 20-HE influences the Y-organ activity of Orconectes limosus positively or negatively depending on its concentrations [185], while a putative positive feedback of ecdysteroids on eyestalk neuropeptides is suggested in Cancer antennarius [39]. Interestingly, at premolt stages, the concentrations of ecdysteroids are changed, as well as the ratio between the two active forms. At the mid-premolt stage (D2) of C. sapidus, the amounts of PoA to 20-HE are observed at a 3:1 ratio [60]. To date, it has not yet been studied in decapod crustaceans how such inherent changes in ecdysteroid 55 titers and the ratio between PoA and 20-HE may affect the transcription and translation of MIH/CHH in the eyestalk ganglia during the molt cycle. In addition to the presence of two active ecdysteroids in C. sapidus, most of the internal tissues of this species express multiple CasEcRs [119]. Specifically, CasEcR1 distinguishes from CasEcR2, as the putative ligand binding pocket (LBP) of the former contains more hydrophilic amino acids (aa) than those of the latter. Such difference in hydrophobicity may play an important role in a ligand specificity and affinity to a particular type of ecdysteroids. However, it has not yet been examined that the elevated levels of total ecdysteroids or a specific type of ecdysteroid may influence in the expression levels of EcR in relation to the molt regulation of decapod crustaceans. In this study, we aimed to better understand the hormonal regulation of molting that recapitulates throughout the life cycle of arthropods using the hatchery-raised blue crab, C. sapidus with tractable life and molt stage as a model animal. Specifically, to define a putative feedback mechanism, we focused on the interplay between two endocrine systems involving CasMIH from eyestalk ganglia and ecdysteroids from Y-organs. We first examined to test if ecdysteroids influence the expression of CasMIH in eyestalk ganglia using an in vitro incubation. We then also determined the effect of multiple injections of CasEcR-dsRNA on CasMIH expression in the eyestalk ganglia. 56 3.2. Materials and methods 3.2.1 Animals and sample collections Male and female young juvenile C. sapidus (20-40 mm carapace width, CW) obtained from blue crab hatchery, Aquaculture Research Center (ARC, Institute of Marine and Environmental Technology, Baltimore, MD, USA) were reared in the same conditions as stated [119] until they reached the size of 50-90 mm CW (crab stage 13-16 [114]). Molt stages of these animals were determined: intermolt (C4), and premolt (D0-D3-4) stages as stated [114]. After the collection of hemolymph as described [119], the animals were placed on ice for 5-10 min prior to dissection. Eyestalk ganglia and Y-organs were microscopically collected and frozen immediately on dry-ice. The samples were stored at -80oC until further use. 3.2.2 Temporal-spatial distributions of ecdysone receptor (CasEcR) isoforms in eyestalk ganglia and Y-organs at different molt stages Tissues were collected from three animals at the following molt stages: intermolt: C4, early premolt: Do, mid-premolt: D1-D2, and late premolt: D3-4. Tissues were homogenized with 150 µl ice-cold DEPC treated PBS and 100 µl of homogenized samples were subjected to total RNA extraction, as described [119,153]. After treating with DNase I (Fermentas), the total RNAs (1.5 µg) were subjected to the 1st stranded cDNA synthesis using RevertAid Reverse Transcriptase (Fermentas). The cDNA samples were diluted to the final concentration at 12.5 ng total RNA/µl equivalent. Each sample cDNA (12.5 ng 57 total RNA equivalent) was examined using an end-point RT-PCR assay with GoTaq® Green Master Mix (Fermentas) and isoform-specific primers at the following PCR condition: initial denaturation at 94°C for 3 min; 30 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a f inal extension at 72°C for 7 min (Table.3-1). The PCR products were run on a 1.5 % agarose gel and were documented using a Kodak gel-documentation system. The Results shown in Figs. 3-6A and B were the representative pictures of three independent assays. 3.2.3 Effects of in vitro incubations of ecdysteroids on CasMIH expression in eyestalk ganglia The animals (80-90 mm CW) at intermolt were ice-chilled; the eyestalks were dissected under a dissection microscope; and, the retinas were removed carefully and rinsed with ice cold DEPC-treated crustacean saline (pH 7.4). The eyestalks were pre-incubated in DEPC-treated crustacean saline, 0.05% M199 media (incubating media) for 30 min at room temperature for pre-conditioning to an experimental condition. After being rinsed with the incubating media again, the eyestalks were then incubated with the incubating medium containing PoA and 20- 20-HE at a 3:1 ratio. These concentrations (75, 150, and 250 ng/ml) of ecdysteroids for the incubation studies were chosen, based on the levels of intrinsic circulating ecdysteroids at Do, D1, and D2 stages, as reported in C. sapidus [60,119]. The eyestalk ganglia were incubated on a shaker (30 rpm) for 3 hrs at 23-25oC. The incubated eyestalk ganglia were rinsed twice with DEPC- 58 treated crustacean saline, blotted on a Kimwipe to remove the saline before being immediately frozen on dry-ice, and kept at -80oC until further analyses. 3.2.4 Total ecdysteroids in tissues using ecdysteroid-radioimmunoassay (Ecd-RIA) Eyestalk ganglia and Y-organs at various molt stages were homogenized in 100 µl ice-cold DEPC-treated PBS and dissolved in 0.1 M borate buffer (pH 8.0). The homogenized samples were assayed similarly as described [60]. The concentration of ecdysteroids was calculated using AssayZap program (BIOSOFT) and presented in pg/tissue. Protein contents in eyestalk ganglia, Y- organs, and hepatopancreas were estimated using a colorimetric assay, modified Lowry method (Pierce 660). 3.2.5 MIH titers in hemolymph using RIA* A) Iodination Native CasMIH was isolated and quantified as described (Chung and Webster, 2003). 300 pmol of CasMIH was iodinated by following the procedure as described [118,178]. 125ICasMIH was separated on a PD-10 column (GE Healthcare) and diluted in glycerol (final 50%). Then it was stored at 4oC and used within one or two months after iodination. The specific activity of 125ICasMIH ranged from 300 to 500Ci/mmol. * MIH-RIA was performed and analyzed by Dr.Sook Chung 59 B) Hemolymph sample preparation Hemolymphs were sampled from the animals at various molt stages. Hemolymph was directly withdrawn into a marine anticoagulant [186] at a ratio of 1:1 (v/v) via the arthrodial membrane between a chela and the first walking leg. These samples were extracted in isopropanol (final 60%) and centrifuged as described [150]. The supernatants were recovered in Minisorb tubes (Nunc), dried by vacuum centrifugation (Jouan), and assayed in duplicate. Anti-CasMIH was used at a 1:20,000 final concentration as before [23]. Standards of CasMIH RIAs ranged from1.2 X10-12 and 2.5 X 10-11M with ED50 values of 5.7 ± 0.1 ×10- 10M (N = 3) and the detection limit of 1.2 x 10-12M. The RIA data were analyzed as above. 3.2.6 Knockdown effects on CasMIH expression in eyestalk and molt interval using double-stranded RNA (dsRNA) CasMIH-, CasEcR1-, and CasRXR-dsRNAs were generated using TranscriptAid T7 High Yield Transcription kit (Fermentas) as described [187]. Prior to the experiments, the hemolymph (100 µl) was withdrawn from the all experimental crabs (60-70 mm CW), mixed in a marine anticoagulant at a 1:1 ratio [186] and kept at -20oC until further assayed for ecdysteroid levels. The knock-down effect with dsRNAs was determined in two ways: 1) the effect within the molt cycle by injecting 5-10 times over 20 days and 2) the effect within and after the molt by injecting ~30 times over 60 days. In both cases, animals received 15 µg CasMIH-dsRNA (in 100ul crustacean saline) or CasEcR1- dsRNAs three times a week as described [187]. The controls received the same 60 volume of crustacean saline. In order to monitor the delivery of dsRNAs into the animals, the crustacean saline was spiked with a 0.01% phenol red as described [187]. At the end of experiments, the animals were at intermolt stage [114] and hemolymph was collected. They were then anesthetized by chilling on ice for 5- 10 min and the tissue samples were collected as stated above. 3.2.7 Expression levels of ecdysone receptor (EcR) and molt-inhibiting hormone (MIH) using qRT-PCR assays The eyestalk ganglia and Y-organs at different molt stages, the eyestalk ganglia from incubation and dsRNA injection experiments were initially homogenized in 150 µl of ice-cold DEPC-treated PBS. Total RNA extraction and cDNA synthesis were carried out as described above. The cDNA samples were diluted to the final concentration at 12.5 ng total RNA/µl equivalent. The eyestalk cDNA samples (25 ng of total RNA equivalent) were assayed in duplicate to estimate the levels of CasMIH and CasEcR1 using Fast SYBR Green Master Mix (Applied Biosystems) and qRT-PCR primers (listed in Table 3-1) on an Applied Biosystems 7500 instrument. The expression of arginine kinase (AK) was determined in the same sample cDNAs as a reference gene for endpoint PCR and qRT-PCR assays. The standards of all transcripts analyzed were generated similarly as described [149,150]. 61 Table 3-1. List of primers that were used for qPCR assays and an end-point RT-PCR assay Primers Sequences (5'-3') CasEcR qF-short (EcR1/EcR2) CCAACAAGTCCAATGTCAGCCGGGG qF-long (EcR1a/EcR2a) CACTCCTAGCATCGTTCAGACT qR1 (EcR1/EcR1a) GGCATCCGAGTCGTCATCACTTATT qR2 (EcR2/EcR2a) CATGTCACTTGTATCTTCACCATCG EcRt-qF GCATTGTGTTTGGAAATACCTTGCC EcRt-qR GCCCTCAATGCATCGAGGTATATTT CasAK qF TTCCTCCACCCTGTCCAA qR GAAGCGGTCACCCTCCTTGA CasMIH qF TCCCTCGCTCACTCCAAATTTTC qR ATTGATAACTCTCGCCGCTGCTT 3.2.8 Statistical analysis All results represent mean ± 1SE (n), in which n is the number of replicates. SigmaPlot (version 12.3) was used to evaluate the statistical significance of the results. The data were subjected to the normality test using the Shapiro-Wilk test (SigmaPlot) prior to all statistical tests. The data that did not show a normal distribution were analyzed using nonparametric ANOVA (Mann-Whitney Rank Sum test). In all cases, statistical significance was accepted at P <0.05 and noted with letters or asterisks. 62 3.3. Results 3.3.1 Levels of molt-inhibiting hormone (CasMIH) expression in eyestalk ganglia and of CasMIH in hemolymph during the molt cycle Levels of CasMIH expression during the molt cycle slightly increase (Fig. 3-1). At intermolt (C4), CasMIH expression levels are measured at 2.4 ± 0.2 x 106 copies/µg total RNA (n=8). These levels are increased to 6.0 ± 1.5 x106 copies/µg total RNA (n=8) at D0 stage (P=0.09). The CasMIH expression reaches 7.1 ± 1.4 x106 copies/µg total RNA (n=22) at D1/D2 stage and declines to 6.1 ± 1.2 x106 copies/µg total RNA (n=7) at D3-4 stages. Figure 3-1. Expression levels of CasMIH in eyestalk ganglia during juvenile molt by qRT-PCR assay. Each cDNA sample containing 25 ng total RNA equivalent was assayed in duplicate. The expression levels are represented as copies/µg total RNA. The data are presented as mean ± SE (n). Statistical significance was accepted at P < 0.05. N.D.= no significant difference P= 0.09 63 The amounts of CasMIH stored in the sinus gland during the molt cycle are presented in Fig. 3-2. At the intermolt and early premolt (D0) stages, CasMIH levels are similar and range from 53.9 ± 5.0 to 38.4 ± 8.3 pmol/sinus gland (n=10-12), respectively. At the mid-premolt stage (D1/D2), CasMIH levels increase significantly to 121.5 ± 13.6 pmol/sinus gland (n=21) and at the late premolt (D3/4) stage, remain high at 105.3 ± 18.5 pmol/sinus gland (n=7), compared to those at preceding molt stages. Figure 3-2. Storage levels of CasMIH in sinus gland (SG) by direct ELISA assay developed from Drexler [44,188]. The concentrations of CasMIH in hemolymphs also change during the molt cycle. CasMIH titers are similar ranging from 14.1 to 15.2 pM at intermolt (n=8) and mid-premolt D2 stages (n=14). At early ecdysis (E0-5%), the CasMIH titers are significantly reduced to 5.8 ± 1.5 pM (n=11), compared to those a a b b 64 measured at intermolt and mid-premolt stages. At post molt (B) stage, the levels of CasMIH are significantly elevated to 36.7 ± 7.0 pM (n= 6) (Fig. 3-3). Figure 3-3. Circulating levels in hemolymph of CasMIH during juvenile molt by RIA assays. The data are presented as mean ± SE (n). Statistical significance was accepted at P < 0.05 and noted with letters. 3.3.2 Endogenous changes in ecdysteroid concentrations in eyestalk ganglia and Y-organs at different molt stage The ecdysteroid contents in eyestalk ganglia and Y-organs vary depending on molt stage (Fig. 3-4). Both tissues have similar levels of ecdysteroids at intermolt stage: 2.3 ± 1.0 pg/µg eyestalk ganglia proteins (n=4) and 9.8 ± 3.4 pg/µg Y-organ proteins (n=6). Ecdysteroid levels increase significantly (P =0.05) by four folds to 9.8 ± 3.5 pg/µg eyestalk ganglia proteins (n=6) at early and mid-premolt (D0/D1) stages, compared to those estimated at intermolt. However, the ecdysteroid levels in Y-organs are increased only to 17.1 ± 3.0 pg/µg Y-organ proteins at early and mid-premolt (D0/D1) stages. These a a b c 65 values do not differ significantly. The hepatopancreas, as a reference tissue has similar levels of ecdysteroids at intermolt: 38.1 ± 5.7 pg/ µg hepatopancreas proteins (n=5) and early and mid-premolt (D0/D1) stages: 36.6 ± 4.3 pg/ µg hepatopancreas proteins (n=7). Figure 3-4. Levels of ecdysteroid presence in the tissues assayed by Ecd-RIA. Eyestalk and Y-organ from intermolt (white bar, n=4-6) and premolt (black bar, n=6) stages of juvenile C. sapidus. Protein estimation was performed by colorimetric assay modified Lowry method (Pierce 660). Statistical significance was accepted at P < 0.05 and noted with asterisk. 3.3.3 Effects of ecdysteroids on CasMIH expression Effects of ecdysteroids on the levels of CasMIH expression in the eyestalk ganglia obtained from the animals at intermolt stage differ by the concentrations of PoA:20-HE at a 3:1 ratio that mimic the concentrations at various molt stages (D0, D1, and D2) (Fig. 3-5). At 75 ng/ml concentration (D0), levels of CasMIH expression are significantly up-regulated to 5.0 ± 1.2 x106 copies/µg total RNA (n=7), compared to those of the control (1.7 ± 0.6 x106 copies/µg total RNA, n= 8). Higher concentrations of ecdysteroids at 150 and 250 ng/ml, levels of * N.D. 66 CasMIH expression tend to decrease to 3.9 ± 1.3 x106 copies/µg total RNA (n= 16) and 0.3 ± 0.1 x106 copies/µg total RNA (n= 5), respectively. However, these reduced CasMIH levels do not differ with the controls. Levels of AK expression measured in the same sample cDNAs remain constant, ranging from 5.1 ± 0.6 x106 copies/µg total RNA (n= 6) with the controls; 4.5 ± 0.5 x106 copies/µg total RNA (n= 8) at 75 ng/ml; 3.9 ± 0.6 x106 copies/µg total RNA (n= 8) at 150 ng/ml; and 5.7 ± 1.7 x106 copies/µg total RNA (n= 8). Figure 3-5. Expression levels of CasMIH in eyestalk ganglia after incubation with different concentrations of active ecdysteroids by qRT-PCR assay. CasMIH expression in different ecdysteroid concentrations that are composed of 3:1 ratio of PoA: 20-HE. The ecdysteroid concentrations of 75, 150, and 250 ng/ml mimic the Do, D1, and D2 stages, respectively. C. sapidus Arginine kinase (CasAK) expression (Gray dot) was quantified from the same cDNA samples. Each cDNA sample containing 25 ng total RNA equivalent was assayed in duplicate. The expression levels are represented as copies/µg total RNA. Statistical significance was accepted at P < 0.05 and noted with letters. ac b ab c 67 3.3.4 Temporal-spatial distributions of ecdysone receptor (CasEcR) isoforms in eyestalk ganglia and Y-organs during the molt cycle Four forms of CasEcRs are differentially expressed in eyestalk ganglia and Y-organs as well as at different molt stages. Overall, the expression of CasEcRs is higher in Y-organs than in the eyestalk ganglia, while the AK expression in these tissues is constant (Figs. 3-6A and B). In the eyestalk ganglia, CasEcR1 serves as the major form, while both CasEcR1 and CasEcR2 seem to be equally distributed in the Y-organs (Figs. 3-6A and B, respectively). During the molt cycle, the patterns of CasEcRs expression are also different in eyestalk ganglia and Y-organs. In eyestalk ganglia, the CasEcR1 and CasEcR2 tend to be gradually increased at premolt stages (D0-D3-4). However, both CasEcR1 and CasEcR2 in the Y-organs seem to be constitutively expressed throughout the molt cycle. CasEcR1a in Y-organs shows a molt-stage dependent expression with greater expression at D1 and D2 stages than at the other stages. Figure 3-6. Spatial and temporal distributions of four CasEcR forms in eyestalk ganglia (A) and Y-organs (B) during the juvenile molt by end-point PCR assay (n=3). CasAK serves as the loading control and obtained from the same cDNA samples. Isoform specific primers are listed in Table 3-1. A) B) 68 3.3.5 Levels of ecdysone receptor (CasEcR) expression in eyestalk ganglia during the molt cycle The expression pattern of CasEcR1, which is the major form in eyestalk ganglia, is similar with Fig. 3-6. Levels of CasEcR1 at the intermolt and mid- premolt (D1) stages remain constant and range from 2.2 - 2.7 x104 copies/µg total RNA (n=6-7). These levels are elevated to 7.0 ± 2.4 x104 copies/µg total RNA (n=8) at mid-premolt (D2) stage and continue to significantly increase (P <0.01) to 1.2 ± 0.2 x105 copies/µg total RNA (n=7) at the late premolt (D3-4) stage, compared to those measured at intermolt (Fig. 3-7). Figure 3-7. Expression levels of CasEcR1 in eyestalk ganglia during juvenile molt by qRT-PCR assay using isoform specific primers (Table 3-1). Each cDNA sample containing 25 ng total RNA equivalent was assayed in duplicate. The expression levels are represented as copies/µg total RNA. The data are presented as mean ± SE (n). Statistical significance was accepted at P < 0.05 and noted with letters. A) a a a ab b 69 3.3.6 Effects of CasMIH- and CasEcR1-dsRNA on CasMIH expression in the eyestalk ganglia and the molt interval A preliminary time-course study with multiple injections of CasMIH-dsRNA significantly reduced levels of CasMIH expression (Suppl. Fig. S3-1). CasMIH- dsRNA injections reduce CasMIH expression by ~50-time: 8.0 ± 3.0 x104 copies/µg total RNA (n= 9), compared to those that received saline (4.0 ± 1.3 x105 copies/µg total RNA, n= 6) and CasRXR-dsRNA (3.4 ± 1.0 x105 copies/µg total RNA, n= 5). Interestingly, CasEcR1-dsRNA significantly reduces the CasMIH levels (1.2 ± 0.4 x105 copies/µg total RNA (n= 9), compared to the controls. However, CasRXR-dsRNA injections have no effect on the levels of CasMIH expression (Fig. 3-8). 70 Figure 3-8. Expression levels of CasMIH in eyestalk ganglia after being administered with dsRNAs by qRT-PCR assay. Each cDNA sample containing 25 ng total RNA equivalent was assayed in duplicate. The expression levels are represented as copies/µg total RNA. The data are presented as mean ± SE (n). Statistical significance was accepted at P < 0.05 and noted with letters. The molt interval of these experimental groups is shown in Fig. 3-9. The animals injected with CasMIH-dsRNA have the molt interval with 14.5 ± 3.0 days (n=4) shorter (P= 0.0737) than the other groups including the control with 20.0 ± 3.2 days (n=4), CasEcR1-dsRNA with 20.8 ± 1.2 days (n=5), and CasRXR- dsRNA with 23.0 ± 3.0 days (n=5). a a c b 71 Figure 3-9. Molt interval after administered with dsRNAs. The data are presented as mean ± SE (n). All data were subjected to a normality test using the Shapiro-Wilk test (SigmaPlot). When the data did not show the normal distribution, a nonparametric test (Mann-Whitney Rank Sum test) was employed. Statistical significance was accepted at P < 0.05. 72 3.4 Discussion Endocrine systems usually involve feedback mechanisms to maintain physiological homeostasis. The present study describes the interplays between the levels of ecdysteroids and MIH in the blue crab, C. sapidus. Our results demonstrate that eyestalk ganglia and Y-organs, although containing all four CasEcR expression, elevate the expression of a tissue-dependent major form during the molt cycle. Effect of ecdysteroids on the levels of CasMIH expression in the eyestalk ganglia has been investigated by in vitro incubation studies with different concentrations and ratios of ecdysteroids. In addition, the effects of ecdysteroids acting through EcR were examined by in vivo RNAi studies. Molting is one of the most fundamental physiological processes in the animals belonging to the phylum Arthropoda and recapitulates throughout their life cycle. Crustacean molting and the molt cycle are coordinated and controlled by the levels of hormones produced by two endocrine systems: eyestalk ganglia and Y-organs. It has been suggested in a simple way that animals at the intermolt stage carry low levels of ecdysteroids due to high levels of MIH that suppresses the activity of Y-organs. The suppression of MIH on Y-organs is noted in a reduction of protein synthesis and cholesterol uptake in these organs, resulting in the reduction of ecdysteroid synthesis and secretion [39,181]. The MIH binding affinity to the Y-organ membranes prepared from different molt stage as well as the levels of its second messenger, cGMP do not change significantly [178]. On the other hand, the maximal number of binding sites increase significantly at post molt (B) stage [178]. These data indicate that the 73 inhibitory effects of MIH on the Y-organ may not result from the binding capacity or affinity of receptors. Interestingly, it is reported at the premolt stage that the phosphodiesterase (PDE) in Y-organs increase and affect the levels of cAMP and cGMP in P. clarkii, thus alleviating the MIH/CHH actions [20]. However, the exact mechanism by which MIH regulates Y-organ activity through the molt cycle, remain still unknown. The levels of CasMIH expression in the eyestalk ganglia vary and increase at early premolt stage. This result is rather different from those reported in C. maenas in which the expression levels remain constant [178]. In addition, the levels of CasMIH in the hemolymph and sinus gland appear to be higher at premolt than those of C. maenas [48] and CHH in C. sapidus [153]. Our results indicate that the transcription of CasMIH may be under the regulation of ecdysteroids as the upstream promoter region of CasMIH gene may contain EcR binding site. Indeed, it is noted that the sequence of genomic amplification of CasMIH upstream promotor region (GenBank accession no KJ813010, Suppl Fig. S3-2) includes the binding site of RXR, and broad complex , similar as reported in C. pagurus [45] and Charybdis feriatus [47]. If CasMIH expression is under the control of ecdysteroids, the eyestalk ganglia should respond to ecdysteroids in the hemolymph and show the increase in its binding capacity to ecdysteroids at premolt stage, compared to the intermolt. To answer this question, the ecdysteroid contents in the eyestalk ganglia and Y-organs that were obtained from the animals at different molt stages were measured. The results show that there are different levels of 74 ecdysteroid concentrations between these tissues. The Y-organs contain higher concentrations of ecdysteroids than the eyestalk ganglia at both molt stages. However, the eyestalk ganglia at premolt seem to be able to concentrate or accumulate ecdysteroids by increasing four folds, whereas Y-organs show only ~1.7-fold increase. This indicates that the eyestalk ganglia are more sensitive to the hemolymphatic ecdysteroids than the Y-organs and may elevate their binding capacity to ecdysteroids. The exact type of ecdysteroids in the tissues has not been characterized. However, we infer that PoA and/or 20-HE may be the ones present in these tissues. Levels of hemolymphatic ecdysteroids change during the molt cycle, along with increase in the total concentrations as well as a particular active ecdysteroid [60]. The intrinsic concentrations of ecdysteroids mimicking the levels of early premolt stage with PoA:20-HE at a 3:1 ratio increase CasMIH expression in the eyestalk ganglia that were obtained from the animals at intermolt stage. Our results are pertinent to an earlier finding in C. antennarius that ecdysteroids stimulate neuropeptide hormone production [28]. Interestingly, ecdysteroid concentration similar to that of early premolt stage is able to stimulate the CasMIH expression, but not with the highest concentrations (250ng/ml). This implicates that there may be difference in a molt-dependent binding capacity of the eyestalk ganglia to ecdysteroids. At premolt, the increase in the total concentration of ecdysteroids is due to the elevated levels of PoA, yielding the ratio at 3:1 between PoA and 20-HE. Such changes in total levels and PoA may indicate the relative changes in 75 EcR/RXR expression. The signal transduction of ecdysteroids requires a heterodimer complex of EcR and its binding partner RXR. Interestingly, our results show that the distribution of CasEcR isoforms differs by tissues and molt stages. In eyestalk ganglia, CasEcR1 expression contributes around 70% towards total CasEcR expression. Structural property of the putative ligand binding pocket of CasEcR1 seems to be less hydrophobic than that of CasEcR2; therefore, CasEcR1 may prefer to bind to 20-HE rather than PoA. However, levels of CasMIH expression in the eyestalk ganglia incubated with of a 1:3 ratio of PoA to 20-HE do not differ from the controls (Suppl. Fig. S3-3). On the other hand, incubation of these two active ecdysteroids at a 3:1 ratio shows that only the ecdysteroids that mimicked intrinsic condition of early premolt (75 ng/ml) significantly and specifically increase the levels of CasMIH expression, while it has no effect on the levels of CasAK expression. Thus, it appears that the initial elevation of ecdysteroids, particularly PoA, may be important, specifically in regulation of CasMIH expression. As our aim was to examine the initial stimulatory effect of ecdysteroids on CasMIH expression, we carried out 3 hr incubation studies. Levels of CasEcR in the eyestalk ganglia do not change compared to those measured in the control group. EcR is in general known to be early-ecdysteroid responsive gene. However, there may be a critical and limited window of its expression after the exposure to ecdysteroids. Indeed, up-regulation of EcR expression in limb buds of U. pugilator occurs only at 1.5 hr incubation with 20-HE but not at 2 or 2.5 hr incubation [189]. Thus, it is implied that up-regulation of CasEcR might have 76 taken placed earlier, which was then subsequently translated for transducing the signal and trans-activating CasMIH expression as shown in Fig. 3-5. However, further studies are required particularly for establishing a relationship between the levels of transcription and translation of EcR in crustaceans using a time- course incubation study. RNAi technique, specifically dsRNA, has been successfully employed in decapod crustaceans for functional study of a gene of interest [187,190]. Prior to carrying out the current experiment, injections of 5 and 10 µg CasMIH-dsRNA were tested in order to find an optimal dose that yielded the maximal reduction of the target gene expression. Our preliminary data obtained from three injections at a two-day interval of 10 µg CasMIH-dsRNA showed ~ 90% reduction in CasMIH expression. Our study with 30-time injections to the animals normally exhibiting molt interval (~20 days) shows that molting is slightly induced after CasMIH-dsRNA injections, having the shorter molt interval (14. 5 ± 0.6 days, P = 0.0727), compared to other groups. Our finding is pertinent to an earlier finding in the much smaller sized crayfish, C. quadricarinatus where multiple injections of CqMIH-dsRNA induce molting [190]. CHH neuropeptides specifically produced in crustacean eyestalk ganglia tend to accumulate in the sinus gland, as similar to adipokinetic hormone in corpora cardiaca in insects [191]. The progress of life cycle accentuates their accumulation, suggesting that the amounts of MIH/CHH translation are far greater than what is actually required by the animal. Although CasMIH levels are reduced >90% by multiple injections of CasMIH-dsRNA, it does not yield 77 dramatic molt inductions. Perhaps the prolonged reduction of CasMIH levels over 60 days may induce the release of CasMIH stored in the sinus gland, although the newly translated materials in the neurosecretory neuronal cells are preferentially released first from the neurosecretory terminals in crustaceans and mammals [192-194]. Interestingly, a long-term administration of CasMIH-dsRNA seems to be lethal as half of the animals died during the mid-premolt (D1 and D2) stage. This might have occurred as off-target effects of CasMIH-dsRNA [195-197]. However, it is suggested that the lack of CasMIH may impair the Y-organ activity in terms of the ecdysteroid levels as well as the specific type of an active ecdysteroid. The eyestalk ablated C. sapidus at premolt stage carry 20-HE as the major ecdysteroid in their hemolymph, in contrast to the intact animals that use PoA as the major form [60]. Although it is not clarified in this current study, it will be intriguing to examine in the future, if indeed multiple injections of CasMIH-dsRNA may replicate a similar effect of the eyestalk ablation. We have also observed that CasCHH-dsRNA administrations produced a high mortality of the animals, particularly those that were undergoing the ecdysis process. These animals were unable to shed the old exoskeleton. As a result, they were usually trapped in the old skeleton. Crustacean ecdysis relies on the hormonal regulation of a large uptake of iso-osmotic water [118]. The expression and release of gut CHH from the endocrine cells present in the fore- and hindgut are required for the completion of successful ecdysis [118,198]. It has been often observed in C. maenas that untimely discharged CHH from these 78 endocrine cells causes the death of animals during the ecdysis, exhibiting by the typical symptom of swelling of the body trapped in the old exoskeleton. What we have observed is similar to what was reported in C. maenas [118]. And, we consider that CasCHH-dsRNA may affect the expression of the gut CHH expression, possibly its release, which results in a high mortality of these animals during ecdysis. The same amounts of multiple injections of CasEcR1- dsRNA indeed reduce CasMIH expression, strongly suggest that transcription of CasMIH may be under the control of ecdysteroids. However, CasEcR1-dsRNA injections seem to be less effective in knock-down of CasEcR1 expression and produce much less reduction, compared to those determined with CasMIH-dsRNA injections. The levels of knock-down of a target gene by RNAi vary, depending on the levels of its expression as well as the function or property of that particular gene product [199-201]. We suggest, however, that this result may be due to the low translation rate of CasEcR or a slow turnover of CasEcR in the eyestalk ganglia. Indeed, translation of crustacean EcR including CasEcR appears to be tightly regulated. The 5´untranslated region (5´UTR) of CasEcR cDNA contains upstream open reading frame as well as internal-ribosome entry site [119]. This indicates that translation of the CasEcR mRNAs may require a particular physiological condition(s). Reduction of CasMIH expression upon CasEcR1- dsRNA injection supports the fact that levels of CasMIH expression respond to the presence of ecdysteroids. 30-time injections of CasEcR1-dsRNA, but not of 79 CasRXR-dsRNA reduce by 70% of CasMIH expression. EcR binds to ecdysteroids, while its partner, RXR, facilitates the liganded EcR to bind to the ecdysteroid-responsive elements. As alluded to earlier, the upstream promoter regions of MIH genes of C. pagurus [45], C. feriatus [47], and C. sapidus (GenBank accession no. KJ813010, Suppl. Fig. S3-2) contain the binding sites for ecdysteroid-responsive factors. Hence, it is suggested that reduced levels (~67%) of CasEcR might have generated the downstream effect on decreased levels (three folds) of CasMIH expression. With the data obtained from this study including the direct effects of ecdysteroids on the CasMIH expression, together with the previous findings [14,28,39,45], a regulatory mechanism for molt control in crustaceans is proposed using C. sapidus as a model (Fig. 3-10). 80 Figure 3-10. Proposed model of molting control in crustaceans. (1) Neuropeptides (MIH and CHH) secreted from the eyestalk ganglia suppress the ecdysteroidogenesis in Y-organs. Autocrine feedbacks of ecdysteroids on the Y-organs may be generated from different ecdysteroid levels: (2) ecdysteroid concentrations at early premolt stage generate positive feedback while the levels at the premolt stages (3) inhibit the ecdysteroidogenesis in the Y-organs. This study provides supporting evidence that premolt ecdysteroids have a stimulatory effect on eyestalk ganglia for neuropeptide production, particularly at transcriptional level (4), generating the long-loop feedback on Y-organs. Based on the endogenous changes of CasMIH expression and storage in this study, it suggests that ecdysteroids might have positive effect on translational and storage processes (5). On the contrary, ecdysteroids might suppress the secretion of neuropeptides as reported in Cancer antennarius [28](6). Overall, this define model of molting control suggests that the interactions of ecdysteroids and neuropeptides coordinate the molting into the repetitive cycle. (Modified from Hopkins [14]) 1 2 3 4 5 6 81 At the early premolt, elevated levels of ecdysteroids may stimulate the MIH expression in eyestalk ganglia which is confirmed by an in vitro incubation of ecdysteroids as well as CasEcR1-dsRNA injections. The exact cue for MIH release has not yet been identified in crustaceans. However, the levels of hemolymphatic ecdysteroids may negatively affect the release of MIH. The low levels of CasMIH at D2 stage are measured at the peak concentrations of hemolymphatic ecdysteroids, as an earlier report in C. antennarius [39]. The highest levels of CasMIH in the hemolymph are found at post-molt stage during which animals have the lowest levels of hemolymphatic ecdysteroids (Fig. 3-3). Such high levels of CasMIH preceding intermolt stage inhibit the synthesis and release ecdysteroids by the Y-organs, which may result in re-setting the molt cycle. This overall proposed model for molt control describes the two feedback mechanisms involving MIH in the eyestalk ganglia and ecdysteroids produced by Y-organs. Firstly, the long-loop feedback includes the neuroendocrine axis between the eyestalk ganglia and Y-organs in that MIH inhibits the activity of the Y-organs at intermolt and post molt stage. At premolt stage, elevated levels of ecdysteroids stimulate the synthesis of the MIH and may suppress its release, while ecdysteroids and their signaling pathway through EcR may have the short- loop feedback for stimulating and inhibiting the ecdysteroidogenesis. However, further studies for examining these following topics are required: 1) the levels of CasMIH synthesis in X-organs at different molt stages in response to the varying concentrations of ecdysteroids; 2) the binding affinity of a specific EcR on each 82 active ecdysteroid type; and, 3) consequent effects on MIH expression in the eyestalk, and the direct effects of PoA and 20-HE on Y-organs. 83 3.5 Supplement figures Figures S3-1. Expression levels of CasMIH from a time course study of CasMIH- dsRNA injections in eyestalk ganglia by qRT-PCR assay. Each cDNA sample containing 25 ng total RNA equivalent was assayed in duplicate. The expression levels are represented as copies/µg total RNA. The data are presented as mean ± SE (n). Statistical significance was accepted at P < 0.05 and noted with letters. Figure S3-2. Genomic arrangement of sapidus using GenomeWalker (Clontech). The upstream promoter region was extended 2,509 nt from the start codon. The binding site for RXR (RXRE) is predicted on the region between -2,131 to found at the proximal region and clustered in tandem repeats using web prediction of regulatory elements MIH gene (Accession no. KJ813010) in -2,107 nt, while the binding site for broad complex (BR - program (http://consite.genereg.net/). 84 C. -CE) is based 85 Figure S3-3. Expression levels of CasMIH in eyestalk ganglia after incubation with different ratios of active ecdysteroids by qRT-PCR assay. Two different ratios of PoA:20-HE were determined: 3:1 ratio ( black bar) and 1:3 ratio (Gray bar). Each cDNA sample containing 25 ng total RNA equivalent was assayed in duplicate. The expression levels are represented as copies/µg total RNA. Statistical significance was accepted at P < 0.05 and noted with asterisk a b ab * 86 CHAPTER 4 General conclusions and future studies This study supported the hypothesis that ecdysteroids stimulate CasMIH expression as evidenced by the effect of ecdysteroids on neuropeptide production in eyestalk ganglia. Ecdysteroid levels as well as expression levels of CasMIH and CasEcR were examined in two different molt cycles: embryonic and juvenile molts. In addition, both in vitro and in vivo experiments were conducted to verify the functional roles of ecdysteroids and the ecdysteroid receptor. The endogenous changes in ecdysteroid levels as well as expressions of neuropeptides and ecdysteroid-responsive factors during embryogenesis and juvenile molt are similar. These results suggest that embryogenesis includes a molting process that occurs inside the embryonic envelope prior to hatching. During ovarian development, levels of maternal mRNAs encoding for ecdysteroid-responsive factors (CasEcR, CasRXR, and CasBTBDP) are very high, but the levels abruptly decrease when embryonic development is initiated. This suggests that these maternal factors are immediately utilized and involved in the initiation of embryogenesis when the transcription machinery has not yet functioned. During embryogenesis, the changing of ecdysteroid levels was similar to that found in the juvenile molt. This result indicates that the embryonic ecdysteroids are produced by the newly developed Y-organs. Interestingly, expression levels of CasEcR and CasRXR peak before ecdysteroids. This 87 lagged peak of ecdysteroids is also found in the Y-organs of juvenile molt when expression levels of all CasEcR forms are peaked at the D1 stage, while the ecdysteroid peak occurs at the D2 stage. This pattern is reported as a common response for negative feedback of ecdysteroidogenesis in both insects and crustaceans. Therefore, CasEcR and CasRXR may be involved in the regulation of ecdysteroidogenesis in Y-organs. In addition, temporal changes of ecdysteroid-responsive factors during embryogenesis may be derived mainly from the expressions in embryonic Y-organs. There appears to be a relationship between CasMIH and CasCHH expressions and the changes of ecdysteroid levels during embryogenesis. Similarly, during the last juvenile molt cycle, CasMIH levels at premolt were higher than those at intermolt. The similar patterns of temporal expressions obtained from two different molt cycles in different life stages indicate that in every molt cycle, the interactions between ecdysteroids and neuropeptides are repeated. Since the embryonic molt is considered the first cycle of molting in the C. sapidus lifecycle, interactions between ecdysteroids and neuropeptide hormones are roughly determined from the timing of their expressions. As the ecdysteroid system is initiated before neuropeptides during embryogenesis, it is implied that ecdysteroids may stimulate neuropeptide expressions. However, much is still unknown about the translation and storage activities of CasMIH in X-organs and sinus glands, respectively. The present findings suggest that high levels of CasMIH expression and storage during the late premolt may be utilized in postmolt and the consecutive intermolt as the secreted 88 levels at the postmolt stage are higher than other stages. However, all the activities of CasMIH in transcription, translation, and storage are not completed: the expression and storage levels were only examined during intermolt and premolt stages while the secreted levels were covered in one molt cycle. In order to fully understand CasMIH activities during the molt cycle, further studies in the following topics are required: 1) how much CasMIH is expressed and stored during the ecdysis and postmolt stages compared to the intermolt and premolt stages; and 2) how much CasMIH is translated in the X-organ in one molt cycle. An analysis of the effects of ecdysteroids on CasMIH expression were assessed via in vitro incubation with active ecdysteroids composed of PoA and 20-HE at concentrations and ratios that mimicked the premolt stages of C. sapidus. Only the 3:1 ratio of PoA:20-HE at concentrations that simulate early premolt and mid premolt (Do and D1) stages induce CasMIH expression. This result suggests that PoA has a stimulatory effect on CasMIH expression whereas 20-HE may interfere with PoA. It is implicated that PoA may be the key factor in regulation of CasMIH expression. This incubation experiment was performed based on the endogenous ratios of ecdysteroids during the mid premolt (D2) stage. The endogenous ratio of PoA to 20-HE during intermolt, and early premolt (Do and D1) stages are still unknown. Therefore, the intrinsic ratios of PoA:20-HE in these stages need to be evaluated. Thru results in this area, further incubation studies might be attempted to define the effects of PoA and 20-HE on CasMIH expression. 89 In the eyestalk ganglia, CasEcR1 is the major form and has LBP that is more hydrophilic than that of CasEcR2. Considered to be an ecdysteroid property, the hydrophilic LBP of CasEcR1 is predicted to bind with 20-HE in a manner stronger than PoA. The decreased stimulatory effects of ecdysteroids on CasMIH expression in higher concentrations of ecdysteroids may result from the increasing amounts of 20-HE in the ecdysteroid mixture that interferes with PoA binding with CasEcR1. This suggestion requires further study to demonstrate the binding affinity of CasEcR1 and/or CasEcR2 to PoA and 20-HE. Unlike the effects on eyestalk ganglia, it is still unknown which type of active ecdysteroids regulate the feedback on ecdysteroidogenesis in Y-organs. Due to the challenges inherent in distinguishing ecdysteroids in the incubating media and newly secreted ones, experiments that focus on the Y-organ incubation are necessary. By obtaining these results, the regulation process of ecdysteroidogenesis in Y-organs would be better understood. Based on the knowledge that ecdysteroids transduce signals through CasEcR, a master transcription factor in the ecdysteroid system, transcription of the downstream-responsive genes will be affected by CasEcR depletion. In vivo experiments demonstrated that knockdown of CasEcR1 lead to a reduction in CasMIH expression. This indirect effect of reducing CasEcR1 on CasMIH expression indicates that CasEcR1 may be involved in CasMIH transcription. In eyestalk, the expression profile of CasEcR2 is also similar to that of CasEcR1, and this putative isoform is predicted to better bind with PoA than CasEcR1. Therefore, it would be interesting to know whether CasEcR2 is 90 involved in CasMIH expression. Since the expression levels of CasEcR2 are much lower than CasEcR1, it is difficult to measure thru a SYBR green qRT-PCR assay. This suggests a need to find other methods that are able to detect very low transcript amounts. Therefore, changes of CasEcR2 expression in incubation studies and knockdown experiments could be analyzed and considered in CasMIH expression. Moreover, translation of crustacean EcR including CasEcR appears to be tightly regulated as its 5´untranslated region (5´UTR) contains an upstream open reading frame as well as internal-ribosome entry site [119]. This indicates that translation of CasEcR mRNAs may require a particular physiological condition. Protein levels of CasEcR during the molting and experiment need to be considered. When the investigation focused on the genomic arrangement of MIH genes in crustaceans, it was discovered that an upstream promoter of MIH contained the binding site for RXR and broad complex (Suppl. Fig. S3-2). Thus, there is great evidence to support the hypothesis that ecdysteroids are acting through their responsive factors (EcR, RXR, or BTBDP) to trans-activate the MIH expression. Trans-activation of ecdysteroids on MIH expression could be determined through binding experiments in which the ecdysteroid-responsive elements of the MIH gene are tested for their ability to bind with different ecdysteroid-responsive factors. By performing these experiments, the following questions can be answered: 1) Which forms of EcR have effects on MIH expression at the 91 transcription level? and, 2) What are the pathways through which ecdysteroids trans-activate MIH expression. Is it acting through an EcR-RXR complex or other ecdysteroid-responsive factors such as a broad complex or BTBDP? All together, this investigation provided lines of evidence that demonstrate the effects of ecdysteroids on eyestalk ganglia. Through this expansion of knowledge, the regulation of molting in crustaceans can be re-defined (Fig. 3-10). Premolt ecdysteroids consisting of PoA:20-HE at a 3:1 ratio stimulates MIH expression in eyestalk ganglia. This increase of MIH production during premolt stages leads to the accumulation of MIH in the sinus gland as herein reported. This storage hormone could be further utilized in later stages. It seems possible that premolt ecdysteroids also have a positive effect on MIH at the translational and storage levels. On the contrary, secretion of MIH into the hemolymph may be inhibited by ecdysteroids as reported previously in C. antennarius [28]. Considering all possible responses in MIH together, large amounts of MIH would be stored in the sinus gland and then are utilized during postmolt and in the consecutive intermolt as this study has shown. A high secretion of repository MIH into the hemolymph at postmolt will inhibit ecdysteroidogenesis in Y-organs, resulting in re-setting of the molt cycle. This model proposes that long-loop feedback of ecdysteroids generates negative feedback for ecdysteroidogenesis in the Y-organ through the neuroendocrine axis in which ecdysteroids at premolt stages stimulate the synthesis of neuropeptide hormones. In addition, temporal expression of 92 CasEcR in the Y-organs suggests that ecdysteroids may have short-loop feedback that stimulates and inhibits ecdysteroidogenesis. In summary, the major findings of this research study were: • C. sapidus experiences one molt cycle during embryogenesis, and this molt is considered the first cycle in its lifecycle. • Maternal ecdysteroids as well as CasEcR, CasRXR, and CasBTBDP are deposited in oocytes during ovarian development. The presence of these maternal factors indicates that ecdysteroids may be required for initiating embryogenesis. • In both embryonic and juvenile molts, the levels of ecdysteroids and the expression levels of neuropeptide hormones (CasMIH and CasCHH) were correlated. Higher levels of CasMIH expression were observed when ecdysteroid titers increase. • In vitro incubation studies revealed that only the combinations of PoA:20-HE ratios that mimic premolt ecdysteroids stimulate CasMIH expression in eyestalk ganglia. • In vivo studies indicated that the stimulatory effect of ecdysteroids on CasMIH expression is acting through CasEcR. This study still requires further research to detail the stimulatory effect of ecdysteroids on the neuropeptide axis. The following lists present remaining questions that need to be answered. 93 • The endogenous ratios of PoA and 20-HE at other molt stages such as intermolt (C4) and premolt (D0 and D1) stages: different forms of hemolymphatic ecdysteroids from these stages could be isolated and assayed by HPLC-RIA as described [60]. By having this result, the response of eyestalk ganglia on these ecdysteroid conditions could be tested and compared with the recent finding. • The expression of CasMIH including transcription, translation, and storage at ecdysis and postmolt stages. By obtaining these results, effects of ecdysteroids on CasMIH at different levels could be determined. • Effects of different ecdysteroid ratios and levels on the sinus gland in eyestalk ganglia for CasMIH secretion. As proposed, the inhibitory effect of ecdysteroids on the CasMIH secretion could be determined. This result would determine whether ecdysteroids have an effect on CasMIH at the secretion levels. • Expression of CasEcR at protein level in eyestalk ganglia and Y-organ during molting cycle and in experimental conditions. Since the 5’UTR of CasEcR mRNA contains uORF and IRES, the translation of CasEcR seems to be tightly regulated and depends on specific conditions. • Localization of CasEcR isoform in eyestalk ganglia and Y-organ. Antibodies that recognize CasEcR1/CasEcR1a and CasEcR2/CasEcR2a are required to detect the localization of CasEcRs in different molt stages and in experimental conditions. This result will provide evidence to 94 support the presence of specific isoforms that transduce ecdysteroids in different tissues and conditions. • Binding ability of different CasEcRs with 20-HE and PoA as the putative CasEcR1/CasEcR1a and CasEcR2/CasEcR2a are predicted to bind with 20-HE and PoA, respectively. By obtaining this knowledge, the induction of CasMIH expression in eyestalk ganglia will be determined in a CasEcR form where the PoA exerts its signal. In addition, as CasEcR expression in Y-organ is molt-stage dependent, the binding result would also provide information about the possible ecdysteroid that regulates ecdysteroidogenesis in Y-organs. • Binding of ecdysteroid-responsive factors (CasEcR, CasRXR, and CasBTBDP) on the upstream promoter of CasMIH. Binding of these factors on regulatory elements can be detected by Pull-down assay or Gel Shift assay (EMSA). This result will provide strong evidence supporting the hypothesis that ecdysteroids stimulate CasMIH expression at a transcriptional level. 95 5. REFERENCES [1] Spindler, K.-D., Przibilla, S. and Spindler-Barth, M. (2001) Molting hormones of arthropods: molecular mechanisms. Zoology 103, 189–201. [2] Spindler, K.D., Honl, C., Tremmel, C., Braun, S., Ruff, H. and Spindler- Barth, M. (2009) Ecdysteroid hormone action. Cell Mol Life Sci 66, 3837- 3850. [3] Anger, K. and Spindler, K.-D. (1987) Energetics, moult cycle and ecdysteroid titers in spider crab (Hyas araneus) larvae starved after the Do threshold. Mar Biol 94, 367-375. [4] Suganthi, A.S. and Anikumar, G. (1999) Moult-related fluctuation in ecdysteroid titre and spermatogenesis in the crab, Metapograpsus messor (Brachyura: Decapoda). Zool Stud 38, 314-321. [5] Nakatsuji, T. and Sonobe, H. (2004) Regulation of ecdysteroid secretion from the Y-organ by molt-inhibiting hormone in the American crayfish, Procambarus clarkii. Gen Comp Endocrinol 135, 358-364. [6] Thomton, J.D., Tamone, S.L. and Atkinson, S. (2006) Circulating ecdysteroid concentrations in Alaskan dungeness crab (Cancer magister). J Crust Biol 26, 176-181. [7] Martin-Creuzburg, D., Westerlund, S.A. and Hoffmann, K.H. (2007) Ecdysteroid levels in Daphnia magna during a molt cycle: determination by radioimmunoassay (RIA) and liquid chromatography-mass spectrometry (LC-MS). Gen Comp Endocrinol 151, 66-71. [8] Huang, X., Warren, J.T. and Gilbert, L.I. (2008) New players in the regulation of ecdysone biosynthesis. J Gen Genom 35, 1-10. [9] Styrishave, B., Lund, T. and Anderson, O. (2008) Ecdysteroids in female shore crabs Carcinus maenas during the moulting cycle and oocyte development. . J Mar Biol Asso UK 88, 575-581 96 [10] Gilbert, L.I., Rybczynski, R. and Warren, J.T. (2002) Control and biochemical nature of the ecdysteroidogenic pathway. Annu Rev Entomol 47, 883-916. [11] McBrayer, Z., Ono, H., Shimell, M., Parvy, J.P., Beckstead, R.B., Warren, J.T., Thummel, C.S., Dauphin-Villemant, C., Gilbert, L.I. and O'Connor, M.B. (2007) Prothoracicotropic hormone regulates developmental timing and body size in Drosophila. Dev Cell 13, 857-71. [12] Rewitz, K.F., Yamanaka, N., Gilbert, L.I. and O'Connor, M.B. (2009) The insect neuropeptide PTTH activates receptor tyrosine kinase torso to initiate metamorphosis. Science 326, 1403-5. [13] Lachaise, F., Meister, M.F., Hetru, C. and Lafont, R. (1986) Studies on the biosynthesis of ecdysone by the Y-organs of Carcinus maenas. Mol Cell Endocrinol 45, 253-261. [14] Hopkins, P.M. (1992) Hormonal control of the molt cycle in the fiddler crab Uca pugilator. Amer Zool 32, 450-458. [15] Webster, J.L., Polgar, W.E., Brandt, S.R., Berzetei-Gurske, I.P. and Toll, L. (1993) Comparison of kappa 2-opioid receptors in guinea pig brain and guinea pig ileum membranes. Eur J Pharmacol 231, 251-258. [16] Lachaise, F., Somme, G., Carpentier, G., Granjeon, E., Webster, S. and Baghdassarian, D. (1996) A transaldolase : An enzyme implicated in crab steroidogenesis. Endocrine 5, 23-32. [17] Han, D.W. and Watson, R.D. (2005) Trimeric G proteins in crustacean (Callinectes sapidus) Y-organs: occurrence and functional link to protein synthesis. J Exp Zool A Comp Exp Biol 303, 441-447. [18] Han, D.W., Patel, N. and Douglas Watson, R. (2006) Regulation of protein synthesis in Y-organs of the blue crab (Callinectes sapidus): involvement of cyclic AMP. J Exp Zool A Comp Exp Biol 305, 328-434. 97 [19] Lee, S.G. and Mykles, D.L. (2006) Proteomics and signal transduction in the crustacean molting gland. Integr Comp Biol 46, 965-977. [20] Nakatsuji, T., Han, D.W., Jablonsky, M.J., Harville, S.R., Muccio, D.D. and Watson, R.D. (2006) Expression of crustacean (Callinectes sapidus) molt- inhibiting hormone in Escherichia coli: characterization of the recombinant peptide and assessment of its effects on cellular signaling pathways in Y- organs. Mol Cell Endocrinol 253, 96-104. [21] Nakatsuji, T., Sonobe, H. and Watson, R.D. (2006) Molt-inhibiting hormone-mediated regulation of ecdysteroid synthesis in Y-organs of the crayfish (Procambarus clarkii): involvement of cyclic GMP and cyclic nucleotide phosphodiesterase. Mol Cell Endocrinol 253, 76-82. [22] Lee, S.G., Bader, B.D., Chang, E.S. and Mykles, D.L. (2007) Effects of elevated ecdysteroid on tissue expression of three guanylyl cyclases in the tropical land crab Gecarcinus lateralis: possible roles of neuropeptide signaling in the molting gland. J Exp Biol 210, 3245-3254. [23] Zmora, N., Trant, J., Zohar, Y. and Chung, J.S. (2009) Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement. Saline Systems 5, 7. [24] Watson, R.D., Whisenton, L.R., Bollenbacher, W.E. and Granger, N.A. (1986) Interendocrine regulation of the corpora allata and prothoracic glands of Manduca sexta. Insect Biochem 16, 149–155. [25] Whisenton, L.R., Granger, N.A. and Bollenbacher, W.E. (1987) A kinetics analysis of brain-mediated 20-hydroxyecdysone stimulation of the corpora allata of Manduca sexta. Mol. Cell. Endocrinol. 54, 171-178. [26] Whisenton, L.R., Watson, R.D., Granger, N.A. and Bollenbacher, W.E. (1987) Regulation of juvenile hormone biosynthesis by 20- hydroxyecdysone during the fourth larval instar of the tobacco hornworm, Manduca sexta. Gen. Comp. Endocrinol. 66, 62-70. 98 [27] Takaki, K. and Sakurai, S. (2003) Regulation of prothoracic gland ecdysteroidogenic activity leading to pupal metamorphosis. Insect Biochem Mol Biol 33, 1189-1199. [28] Mattson, M.P. and Spaziani, E. (1986) Evidence for ecdysteroid feedback on release of Molt-inhibiting hormone from crab eysestalk ganglia. Biol Bull 171, 264-273. [29] Rybczynski, R. and Gilbert, L.I. (2003) Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca(2+)- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis. Mol Cell Endocrinol 205, 159- 168. [30] Beydon, P. and Lafont, R. (1983) Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae. J. Insect. Physiol. 29, 529–533. [31] Bidmon, H.-J., Stumpf, W.E. and Granger, N.A. (1992) Eedysteroid receptors in the neuroendoerine-endocrine axis of a moth Experientia 48, 42-47. [32] Sakurai, S. and Williams, C.M. (1989) Short-loop negative and positive feedback on ecdysone secretion by prothoracic gland in the tobacco hornworm, Manduca sexta. Gen. Comp. Endocrinol. 75, 204-216. [33] Zeleny, C. (1905) Compensatory regulation. J. Exp. Zool, 21-102. [34] Chan, S.M., Gu, P.L., Chu, K.H. and Tobe, S.S. (2003) Crustacean neuropeptide genes of the CHH/MIH/GIH family: implications from molecular studies. Gen Comp Endocrinol 134, 214-219. [35] Chang, E.S., Chang, S.A. and Mulder, E.P. (2001) Hormones in the lives of crustaceans: an overview. American Zoologist, 1090-1097. [36] Chang, E.S. and Mykles, D.L. (2011) Regulation of crustacean molting: a review and our perspectives. Gen. Comp. Endocrinol. 172, 323-330. 99 [37] Chung, J.S., Zmora, N., Katayama, H. and Tsutsui, N. (2010) Crustacean hyperglycemic hormone (CHH) neuropeptidesfamily: Functions, titer, and binding to target tissues. Gen. Comp. Endocrinol. 166, 447-454. [38] Webster, S.G., Keller, R. and Dircksen, H. (2012) The CHH-superfamily of multifunctional peptide hormones controlling crustacean metabolism, osmoregulation, moulting, and reproduction. Gen. Comp. Endocrinol. 175, 217-233. [39] Mattson, M.P. and Spaziani, E. (1986) Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: involvement of cyclic AMP-mediated protein synthesis. Gen Comp Endocrinol 63, 414- 423. [40] Mattson, M.P. and Spaziani, E. (1985) Cyclic AMP mediates the negative regulation of Y-organ ecdysteroid production. Mol Cell Endocrinol 42, 185- 189. [41] Covi, J.A., Chang, E.S. and Mykles, D.L. (2009) Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland. Comp Biochem Physiol A Mol Integr Physiol 152, 470-7. [42] Nakatsuji, T., Lee, C.Y. and Watson, R.D. (2009) Crustacean molt- inhibiting hormone: structure, function, and cellular mode of action. Comp Biochem Physiol A Mol Integr Physiol 152, 139-148. [43] Spaziani, E. and Wang, W.L. (1993) Biosynthesis of ecdysteroid hormones by crustacean Y-organs: conversion of cholesterol to 7- dehydrocholesterol is suppressed by a steroid 5 alpha-reductase inhibitor. Mol. Cell. Endocrinol. 95, 111-114. [44] Zmora, N., Sagi, A., Zohar, Y. and Chung, J.S. (2009) Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 2: novel specific binding sites in hepatopancreas and cAMP as a second messenger. Saline Systems 5, 6. 100 [45] Lu, W., Wainwright, G., Webster, S.G., Rees, H.H. and Turner, P.C. (2000) Clustering of mandibular organ-inhibiting hormone and moult- inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression. Gene 253, 197-207. [46] Okumura, T., Ohira, T., Katayama, H. and Nagasawa, H. (2005) In vivo effects of a recombinant molt-inhibiting hormone on molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. Zoolog. Sci. 22, 317-320. [47] Chan, S.M., Chen, X.G. and Gu, P.L. (1998) PCR cloning and expression of the molt-inhibiting hormone gene for the crab (Charybdis feriatus). Gene 224, 23-33. [48] Chung, J.S. and Webster, S.G. (2005) Dynamics of in vivo release of molt- inhibiting hormone and crustacean hyperglycemic hormone in the shore crab, Carcinus maenas. Endocrinology 146, 5545-5551. [49] Lachaise, A., LeRoux, A., Hubert, M. and Lafont, R. (1993) The molting gland of crustaceans: localization, activity,and endocrine control (a review). J. Crust. Biol. 13, 198–234. [50] Lachaise, F., Carpenter, G., Somme, G., Colandeau, J. and Beydon, P. (1989) Ecdysteroid synthesis by crab Y-organs. J Exp Zool 252, 283-292. [51] Rees, H.H. (1995) Ecdysteroid biosynthesis and inactivation in relation to function. Eur. J.Entomol. 92, 9–39. [52] Mykles, D.L. (2011) Ecdysteroid metabolism in crustaceans. J Steroid Biochem Mol Biol 127, 196-203. [53] Chang, E.S. and O'Connor, J.D. (1977) Secretion of alpha-ecdysone by crab Y-organs in vitro. Proc Natl Acad Sci USA 74, 615-618. [54] Spaziani, E., Rees, H.H., Wang, W.L. and Watson, R.D. (1989) Evidence that Y-organs of the crab Cancer antennarius secrete 3-dehydroecdysone. Mol Cell Endocrinol 66, 17-25. 101 [55] Christiaens, O., Iga, M., Velarde, R.A., Rouge, P. and Smagghe, G. (2010) Halloween genes and nuclear receptors in ecdysteroid biosynthesis and signalling in the pea aphid. Insect Mol Biol 19 Suppl 2, 187-200. [56] Chan, S.M., Rankin, S.M. and Keeley, L. (1988) Characterization of the molt stages in Penaeus vannamei: setogenesis and hemolymph levels of total protein, ecdysteroids, and glucose. Bio. Bull., 185-192. [57] Snyder, M.J. and Chang, E.S. (1991) Metabolism and excretion of injected [3H]-ecdysone by female labsters, Homarus americanus. Biol Bull 180, 475-484. [58] Snyder, M.J. and Chang, E.S. (1991) Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. II. excretion of metabolites. Gen Comp Endocrinol 83, 118-131. [59] Snyder, M.J. and Chang, E.S. (1991) Ecdysteroids in relation to the molt cycle of the American lobster, Homarus americanus. I. Hemolymph titers and metabolites. Gen Comp Endocrinol 81, 133-145. [60] Chung, J.S. (2010) Hemolymph ecdysteroids during the last three molt cycles of the blue crab, Callinectes sapidus: quantitative and qualitative analyses and regulation. Arch. Insect. Biochem. Physiol. 73, 1-13. [61] Escriva, H., Delaunay, F. and Laudet, V. (2000) Ligand binding and nuclear receptor evolution. Bioessays 22, 717-727. [62] Thornton, J.W. (2003) Nonmammalian nuclear receptors: evolution and endocrine disruption. Pure Appl Chem 75, 1827-1839. [63] Krust, A., Green, S., Argos, P., Kumar, V., Walter, P., Bornert, J.M. and Chambon, P. (1986) The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J 5, 891-897. 102 [64] Hu, X., Cherbas, L. and Cherbas, P. (2003) Transcription activation by the ecdysone receptor (EcR/USP): identification of activation functions. Mol Endocrinol 17, 716-731. [65] Hewitt, S.C., Harrell, J.C. and Korach, K.S. (2005) Lessons in estrogen biology from knockout and transgenic animals. Ann Rev Physiol 67, 285- 308. [66] King-Jones, K. and Thummel, C.S. (2005) Nuclear receptors--a perspective from Drosophila. Nat Rev Gene 6, 311-323. [67] Robinson-Rechavi, M., Escriva Garcia, H. and Laudet, V. (2003) The nuclear receptor superfamily. J Cell Sci 116, 585-586. [68] Ashburner, M. (1972) Puffing patterns in Drosophila melanogaster and related species. Results Probl Cell Differ 4, 101-51. [69] Ashburner, M. (1973) Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration. Dev Biol 35, 47-61. [70] DiBello, P.R., Withers, D.A., Bayer, C.A., Fristrom, J.W. and Guild, G.M. (1991) The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics 129, 385-97. [71] Andres, A.J. and Thummel, C.S. (1992) Hormones, puffs and flies: the molecular control of metamorphosis by ecdysone. Trends Genet 8, 132-8. [72] Jindra, M. (1994) Gene regulation by steroid hormones: vertebrates and insects. . Eur. J. Entomol. 91, 163–187. [73] Fletcher, J.C. and Thummel, C.S. (1995) The ecdysone-inducible Broad- complex and E74 early genes interact to regulate target gene transcription and Drosophila metamorphosis. Genetics 141, 1025-35. 103 [74] Riddiford, L.M., Hiruma, K., Zhou, X. and Nelson, C.A. (2003) Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol 33, 1327-38. [75] Kim, H.W., Lee, S.G. and Mykles, D.L. (2005) Ecdysteroid-responsive genes, RXR and E75, in the tropical land crab, Gecarcinus lateralis: differential tissue expression of multiple RXR isoforms generated at three alternative splicing sites in the hinge and ligand-binding domains. Mol Cell Endocrinol 242, 80-95. [76] Guay, P.S. and Guild, G.M. (1991) The ecdysone-induced puffing cascade in Drosophila salivary glands: a Broad-Complex early gene regulates intermolt and late gene transcription. Genetics 129, 169-75. [77] Burtis, K.C., Thummel, C.S., Jones, C.W., Karim, F.D. and Hogness, D.S. (1990) The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins. Cell 61, 85-99. [78] Fletcher, J.C., D'Avino, P.P. and Thummel, C.S. (1997) A steroid-triggered switch in E74 transcription factor isoforms regulates the timing of secondary-response gene expression. Proc Natl Acad Sci U S A 94, 4582- 6. [79] Fletcher, J.C., Burtis, K.C., Hogness, D.S. and Thummel, C.S. (1995) The Drosophila E74 gene is required for metamorphosis and plays a role in the polytene chromosome puffing response to ecdysone. Development 121, 1455-65. [80] Fletcher, J.C. and Thummel, C.S. (1995) The Drosophila E74 gene is required for the proper stage- and tissue-specific transcription of ecdysone-regulated genes at the onset of metamorphosis. Development 121, 1411-21. [81] Feigl, G., Gram, M. and Pongs, O. (1989) A member of the steroid hormone receptor gene family is expressed in the 20-OH-ecdysone inducible puff 75B in Drosophila melanogaster. Nucleic Acids Res 17, 7167-78. 104 [82] Segraves, W.A. and Hogness, D.S. (1990) The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev 4, 204-19. [83] Karim, F.D. and Thummel, C.S. (1992) Temporal coordination of regulatory gene expression by the steroid hormone ecdysone. Embo J 11, 4083-93. [84] Williams, A.B. (1974) The swimming crabs of the genus Callinectes (Decapoda: Portunidae). Fish. Bull. 72, 685-798. [85] Hines, A., Jivoff, P.R., Bushmann, P.J., Montfrans, J., Reed, S.A., Wolcott, D.J. and Wolcot, T.G. (2003) Evidence for sperm limitation in the blue crab Callinectes sapidus. . Bull. Mar. Sci. 72, 287-310. [86] Adiyodi, K.G. and Adiyodi, R.G. (1974) Comparative physiology of reproduction in arthropods. Adv. Comp. Physiol. Biochem 5, 37-107. [87] Meusy, J.J. (1980) Vitellogenin, the extraovarian precursor of the protein yolk in Crustacea : A review. Reprod. Nutr. Develop. 20, 1-20. [88] Brown, M. (1986) Expression of the Genes Coding for Vitellogenin (Yolk Protein). Ann. Rev. Entomol. 31, 507-531. [89] Zmora, N., Trant, J., Chan, S.M. and Chung, J.S. (2007) Vitellogenin and its messenger RNA during ovarian development in the female blue crab, Callinectes sapidus: gene expression, synthesis, transport, and cleavage. Biol. Reprod. 77, 138-146. [90] Tsukimura, B. (2001) Crustacean Vitellogenesis: Its Role in Oocyte Development. Amer Zool 41, 465–476. [91] Cakouros, D., Daish, T., Martin, D., Baehrecke, E.H. and Kumar, S. (2002) Ecdysone-induced expression of the caspase DRONC during hormone- dependent programmed cell death in Drosophila is regulated by Broad- Complex. J. Cell. Biol. 157, 985-995. 105 [92] Kozlova, T. and Thummel, C.S. (2003) Essential roles for ecdysone signaling during Drosophila mid-embryonic development. Science 301, 1911-1914. [93] Makka, T., Seino, A., Tomita, S., Fujiwara, H. and Sonobe, H. (2002) A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori. Arch Insect Biochem Physiol 51, 111-20. [94] Sonobe, H. and Yamada, R. (2004) Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions. Zoolog Sci 21, 503-16. [95] Subramoniam, T., Tirumalai, R., Gunamalai, V. and Hoffman, K.H. (1999) Embryonic ecdysteroids in a mole crab, Emerita asiatica (Milne-Edwards). J. Biosci. 24, 91-96. [96] White, K.P., Hurban, P., Watanabe, T. and Hogness, D.S. (1997) Coordination of Drosophila metamorphosis by two ecdysone-induced nuclear receptors. Science 276, 114-117. [97] Churchill, E.P. (1917) Life history of the blue crab., 95-125. [98] Chung, J.S. and Webster, S.G. (2004) Expression and release patterns of neuropeptides during embryonic development and hatching of the green shore crab, Carcinus maenas. Development 131, 4751-4761. [99] Helluy, S.M. and Beltz, B.S. (1991) Embryonic Development of the American Lobster (Homarus americanus): Quantitative Staging and Characterization of an Embryonic Molt Cycle Biol Bull 180, 355-371. [100] Spindler, K.D., Van Wormhoudt, A., Sellos, D. and Spindler-Barth, M. (1987) Ecdysteroid levels during embryogenesis in the shrimp, Palaemon serratus (Crustacea Decapoda): quantitative and qualitative changes. Gen. Comp. Endocrinol. 66, 116-122. 106 [101] Walker, A., Ando, S., Smith, G.D. and Lee, R.F. (2006) The utilization of lipovitellin during blue crab (Callinectes sapidus) embryogenesis. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 143, 201-208. [102] Tilburg, C., Kernehan, C., Andon, A. and Epifanio, C. (2008) Modeling estuarine ingress of blue crab megalopae: Effects of temporal patterns in larval release. J. Mar. Res. 66, 391-412. [103] Garcia, F., Cunningham, M.L., Garda, H. and Heras, H. (2008) Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae). Comp Biochem Physiol B Biochem Mol Biol 151, 317-22. [104] Sibert, V., Ouellet, P. and Brêthes, J.-C. (2004) Changes in yolk total proteins and lipid components and embryonic growth rates during lobster (Homarus americanus) egg development under a simulated seasonal temperature cycle. Mar. Biol. 144, 1075-1086. [105] Wilder, M.N., Okumura, T., Aida, K. and Hanyu, I. (1990) Ecdysteroid fluctuations during embryogenesis in the giant freshwater prawn, Macrobrachium rosenbergii. Gen. Comp. Endocrinol. 80, 93-100. [106] Costlow, J.D., Bookhout, C.G. and Bulletin, B. (1959) The Larval Development of Callinectes sapidus Rathbun Reared in the Laboratory. Bio. Bull. 116, 373-396. [107] Liu, B., McConnell, L.L. and Torrents, A. (2002) Herbicide and insecticide loadings from the Susquehanna River to the northern Chesapeake Bay. J Agric Food Chem 50, 4385-4392. [108] Kuang, Z., McConnell, L.L., Torrents, A., Meritt, D. and Tobash, S. (2003) Atmospheric deposition of pesticides to an agricultural watershed of the Chesapeake Bay. J Environ Qual 32, 1611-1622. [109] Huang, D.J., Wang, S.Y. and Chen, H.C. (2004) Effects of the endocrine disrupter chemicals chlordane and lindane on the male green neon shrimp (Neocaridina denticulata). Chemosphere 57, 1621-1627. 107 [110] Palma, P., Palma, V.L., Fernandes, R.M., Soares, A.M. and Barbosa, I.R. (2009) Endosulfan sulphate interferes with reproduction, embryonic development and sex differentiation in Daphnia magna. Ecotoxicol Environ Saf 72, 344-350. [111] Palma, P., Palma, V.L., Matos, C., Fernandes, R.M., Bohn, A., Soares, A.M. and Barbosa, I.R. (2009) Effects of atrazine and endosulfan sulphate on the ecdysteroid system of Daphnia magna. Chemosphere 74, 676-681. [112] Bhavan, P.S. and Geraldine, P. (2001) Biochemical stress responses in tissues of the prawn Macrobrachium malcolmsonii on exposure to endosulfan. Pest Biochem Physiol 70, 27–41. [113] Bucholz, F. (1982) Drach's molt staging system adapted for euphausiids. Mar Biol 66, 301-305. [114] Drach, P., Tchernigovtzeff, C. and (1967) Sur la me´thode de de´termination des stades d’intermue et son application ge´ne´rale aux Crustace´s. Vie et milieu serie A. Biol. 18, 595–610. [115] Chang, E.S. (1995) Physiological and biochemical changes during the molt cycle in decapod crustacean: an overview. J Exp Mar Biol Ecol 193, 1-14. [116] Priya, T.A., Li, F., Zhang, J., Wang, B., Zhao, C. and Xiang, J. (2009) Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis. Comp Biochem Physiol B Biochem Mol Biol 153, 121-129. [117] Mangum, C. (1992) Physiological Aspects of Molting in the blue crab Callinectes sapidus. Amer Zool 32, 459-469. [118] Chung, J.S., Dircksen, H. and Webster, S.G. (1999) A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas. Proc Natl Acad Sci U S A 96, 13103-7. 108 [119] Techa, S. and Chung, J.S. (2013) Ecdysone and retinoid-X receptors of the blue crab, Callinectes sapidus: Cloning and their expression patterns in eyestalks and Y-organs during the molt cycle. Gene 527, 139-153. [120] Lackner, D.H. and Bahler, J. (2008) Translational control of gene expression from transcripts to transcriptomes. Int Rev Cell Mol Biol 271, 199-251. [121] Gray, N.K. and Wickens, M. (1998) Control of translation initiation in animals. Ann Rev Cell Dev Biol 14, 399-458. [122] Lopez de Quinto, S., Lafuente, E. and Martinez-Salas, E. (2001) IRES interaction with translation initiation factors: functional characterization of novel RNA contacts with eIF3, eIF4B, and eIF4GII. RNA 7, 1213-1226. [123] Sonenberg, N. (2006) Protein Synthesis in Eukaryotes: IRES‐mediated Internal Initiation. eLS. [124] Cherbas, P., Cherbas, L., Lee, S.S. and Nakanishi, K. (1988) 26- [125I]iodoponasterone A is a potent ecdysone and a sensitive radioligand for ecdysone receptors. Proc Natl Acad Sci U S A 85, 2096-100. [125] Carmichael, J.A., Lawrence, M.C., Graham, L.D., Pilling, P.A., Epa, V.C., Noyce, L., Lovrecz, G., Winkler, D.A., Pawlak-Skrzecz, A., Eaton, R.E., Hannan, G.N. and Hill, R.J. (2005) The X-ray structure of a hemipteran ecdysone receptor ligand-binding domain: comparison with a lepidopteran ecdysone receptor ligand-binding domain and implications for insecticide design. J Biol Chem 280, 22258-69. [126] Hopkins, P.M., Durica, D. and Washington, T. (2008) RXR isoforms and endogenous retinoids in the fiddler crab, Uca pugilator. Comp Biochem Physiol A Mol Int Physiol 151, 602-614. [127] Wu, X., Hopkins, P.M., Palli, S.R. and Durica, D.S. (2004) Crustacean retinoid-X receptor isoforms: distinctive DNA binding and receptor- receptor interaction with a cognate ecdysteroid receptor. Mol Cell Endocrinol 218, 21-38. 109 [128] Durica, D.S., Wu, X., Anilkumar, G., Hopkins, P.M. and Chung, A.C. (2002) Characterization of crab EcR and RXR homologs and expression during limb regeneration and oocyte maturation. Mol Cell Endocrinol 189, 59-76. [129] Davidson, E.H., Rast, J.P., Oliveri, P., Ransick, A., Calestani, C., Yuh, C.H., Minokawa, T., Amore, G., Hinman, V., Arenas-Mena, C., Otim, O., Brown, C.T., Livi, C.B., Lee, P.Y., Revilla, R., Rust, A.G., Pan, Z., Schilstra, M.J., Clarke, P.J., Arnone, M.I., Rowen, L., Cameron, R.A., McClay, D.R., Hood, L. and Bolouri, H. (2002) A genomic regulatory network for development. Science 295, 1669-1678. [130] Mueller, N.S. (1963) An experimental analysis of molting in embryos of Melanoplus differentialis. Dev. Biol. 8, 222–240. [131] Chaix, J.C. and De Reggi, M. (1982) Ecdysteroid levels during ovarian development and embryogenesis in the spider crab Acanthonyx lunulatus. Gen Comp Endocrinol 47, 7-14. [132] Chang, E.S., Bruce, M.J. and Tamone, S.L. (1993) Regulation of crustacean molting: a multi-hormonal system. American Zoologist, 324- 329. [133] Subramoniam, T. (2000) Crustacean ecdysteriods in reproduction and embryogenesis. Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 125, 135-156. [134] Gunamalai, V., Kirubagaran, R. and Subramoniam, T. (2004) Hormonal coordination of molting and female reproduction by ecdysteroids in the mole crab Emerita asiatica (Milne Edwards). Gen. Comp. Endocrinol. 138, 128-138. [135] Charmantier, G. and Charmantier-Daures, M. (1998) Endocrine and neuroendocrine regulations in embryos and larvae of crustaceans. Invertebr. Reprod. Dev. 33, 273-287. [136] Lachaise, F. and Hoffmann, J.A. (1982) Ecdysteroids and embryonic 110 development in the shore crab, Carcinus maenas. Hoppe. Seylers. Z. Physiol. Chem. 363, 1059-1067. [137] McCarthy, J.F. and Skinner, D.M. (1979) Changes in ecdysteroids during embryogenesis of the blue crab, Callinectes sapidus rathbun. Dev Biol 69, 627-33. [138] Konopova, B. and Jindra, M. (2008) Broad-Complex acts downstream of Met in juvenile hormone signaling to coordinate primitive holometabolan metamorphosis. Development 135, 559-568. [139] Emery, I.F., Bedian, V. and Guild, G.M. (1994) Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis. Development 120, 3275-87. [140] Tzolovsky, G., Deng, W.M., Schlitt, T. and Bownes, M. (1999) The function of the broad-complex during Drosophila melanogaster oogenesis. Genetics 153, 1371-1383. [141] Parthasarathy, R., Tan, A., Bai, H. and Palli, S.R. (2008) Transcription factor broad suppresses precocious development of adult structures during larval-pupal metamorphosis in the red flour beetle, Tribolium castaneum. Mech. Dev. 125, 299-313. [142] Spokony, R.F. and Restifo, L.L. (2007) Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis. Dev. Genes. Evol. 217, 499-513. [143] Spokony, R.F. and Restifo, L.L. (2009) Broad Complex isoforms have unique distributions during central nervous system metamorphosis in Drosophila melanogaster. J. Comp. Neurol. 517, 15-36. [144] Buaklin, A., Klinbunga, S. and Mensveta, P. (2011) Identification and expression analysis of the Broad-Complex core protein isoform 6 (BR-C Z6) gene in the giant tiger shrimp Penaeus monodon (Penaeidae: Decapoda). Genet. Mol. Res. 10, 2290-2306. 111 [145] Nagle, L., Place, A.R., Schott, E.J., Jagus, R., Messick, G. and Pitula, J.S. (2009) Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus. Dis. Aquat. Org. 84, 79e87. [146] Bowers, H.A., Messick, G.A., Hanif, A., Jagus, R., Carrion, L., Zmora, O. and Schott, E.J. (2010) Physicochemical properties of double-stranded RNA used to discover a reo-like virus from blue crab Callinectes sapidus. Disease of Aquatic Organisms 93, 17–29. [147] Lee, K.J., Watson, R.D. and Roer, R.D. (1998) Molt-inhibiting hormone mRNA levels and ecdysteroid titer during a molt cycle of the blue crab, Callinectes sapidus. Biochem Biophys Res Commun 249, 624-627. [148] Chung, J.S., Wilcockson, D.C., Zmora, N., Zohar, Y., Dircksen, H. and Webster, S.G. (2006) Identification and developmental expression of mRNAs encoding crustacean cardioactive peptide (CCAP) in decapod crustaceans. J Exp Biol 209, 3862-3872. [149] Chung, J.S. and Zmora, N. (2008) Functional studies of crustacean hyperglycemic hormones (CHHs) of the blue crab, Callinectes sapidus - the expression and release of CHH in eyestalk and pericardial organ in response to environmental stress. Febs J 275, 693-704. [150] Chung, J.S., Katayama, H. and Dircksen, H. (2012) New functions of arthropod bursicon: inducing deposition and thickening of new cuticle and hemocyte granulation in the blue crab, Callinectes sapidus. PLoS One 7, e46299. [151] Chung, J.S., Bembe, S., Tamone, S., Andrews, E. and Thomas, H. (2009) Molecular cloning of the crustacean hyperglycemic hormone (CHH) precursor from the X-organ and the identification of the neuropeptide from sinus gland of the Alaskan Tanner crab, Chionoecetes bairdi. Gen Comp Endocrinol 162, 129-133. [152] Chung, J.S., Manor, R. and Sagi, A. (2011) Cloning of an insulin-like androgenic gland factor (IAG) from the blue crab, Callinectes sapidus: implications for eyestalk regulation of IAG expression. Gen. Comp. Endocrinol. 173, 4-10. 112 [153] Chung, J.S. (2008) A trehalose 6-phosphate synthase gene of the hemocytes of the blue crab, Callinectes sapidus: cloning, the expression, its enzyme activity and relationship to hemolymph trehalose levels. Saline Systems 4, 18. [154] Huang, H.Y., Chien, C.H., Jen, K.H. and Huang, H.D. (2006) RegRNA: an integrated web server for identifying regulatory RNA motifs and elements. Nucleic Acids Res 34, 429-434. [155] Huang, H.Y., Chien, C.H., Jen, K.H. and Huang, H.D. (2006) RegRNA: an integrated web server for identifying regulatory RNA motifs and elements. Nucleic. Acids. Res. 34, W429-34. [156] Bownes, M., Shirras, A., Blair, M., Collins, J. and Coulson, A. (1988) Evidence that insect embryogenesis is regulated by ecdysteroids released from yolk proteins. Proc Natl Acad Sci U S A 85, 1554-7. [157] Sanders, D.A. (1990) Occurence of free ecdysteroids and yolk protein ecdysteroid conjugates in the embryo of the melon fly, Dacus cucurbitae Coquillet. University Microfilms International. [158] Sonobe, H. and Ito, Y. (2009) Phosphoconjugation and dephosphorylation reactions of steroid hormone in insects. Mol. Cell. Endocrinol. 307, 25-35. [159] Blanchet, M.F., Charniaux-Cotton, H., Croisille, Y., Junera, H., Martin, M., Meusy, J.J. and Zerbib, C. (1980) Hormonal control of vitellogenin synthesis in the Amphipoda Crustacea Orchestia gammarella (Pallas). Reprod Nutr Dev 20, 539-544. [160] Young, N.J., Webster, S.G. and Rees, H.H. (1993) Ovarian and hemolymph ecdysteroid titers during vitellogenesis in Macrobrachium rosenbergii. Gen Comp Endocrinol 90, 183-191. [161] Lanot, R., Thiebold, J., Costet-Corio, M.F., Benveniste, P. and Hoffmann, J.A. (1988) Further experimental evidence for the involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta, Orthoptera). Dev. Biol. 126, 212-214. 113 [162] Lachaise, F., Goudeau, M., Hetru, C., Kappler, C. and Hoffmann, J.A. (1981) Ecdysteroids and ovarian development in the shore crab, Carcinus maenas. Hoppe-Seyler's Z Physiol Chem 362, 521-529. [163] Lachaise, F. and Hoffmann, J.A. (1977) Ecdysone et development ovarien chez un Decapode, Carcinus maenas. C R Acad Sci (Paris) 285, 701-704. [164] Lagueux, M., Harry, P. and Hoffmann, J.A. (1981) Ecdysteroids are bound to vitellin in newly laid eggs of locusta. Mol Cell Endo 24, 325-338. [165] Hagedorn, H.H., O'Connor, J.D., Fuchs, M.S., Sage, B., Schlaeger, D.A. and Bohm, M.K. (1975) The ovary as a source of alpha-ecdysone in an adult mosquito. Proc Natl Acad Sci U S A 72, 3255-9. [166] Martin, D., Wang, S.F. and Raikhel, A.S. (2001) The vitellogenin gene of the mosquito Aedes aegypti is a direct target of ecdysteroid receptor. Mol Cell Endocrinol 173, 75-86. [167] Chen, L., Zhu, J., Sun, G. and Raikhel, A.S. (2004) The early gene Broad is involved in the ecdysteroid hierarchy governing vitellogenesis of the mosquito Aedes aegypti. J Mol Endocrinol 33, 743-761. [168] Tawfik, A.I., Vedrova, A. and Sehnal, F. (1999) Ecdysteroids during ovarian development and embryogenesis in solitary and gregarious Schistocerca gregaria. Arch. Insect. Biochem. Physiol. 41, 134-143. [169] Tawfik, A.I., Tanaka, Y. and Tanaka, S. (2002) Possible involvement of ecdysteroids in embryonic diapause of Locusta migratoria. J. Insect. Physiol. 48, 743-749. [170] Minelli, A., Brena, C., Deflorian, G., Maruzzo, D. and Fusco, G. (2006) From embryo to adult--beyond the conventional periodization of arthropod development. Dev. Genes. Evol. 216, 373-383. [171] Lin, R.-J. (2010) Maternal mRNA and the PolyA Tail in Oocytes. Nat. Educat. 3, 47. 114 [172] Oh, B., Hwang, S., McLaughlin, J., Solter, D. and Knowles, B.B. (2000) Timely translation during the mouse oocyte-to-embryo transition. Development 127, 3795-3803. [173] Gilbert, L.I., Song, Q. and Rybczynski, R. (1997) Control of ecdysteroidogenesis: activation and inhibition of prothoracic gland activity. Invert Neurosci 3, 205-216. [174] Song, Q. and Gilbert, L.I. (1998) Alterations in ultraspiracle (USP) content and phosphorylation state accompany feedback regulation of ecdysone synthesis in the insect prothoracic gland. Insect Biochem Mol Biol 28, 849- 860. [175] Perez-Torrado, R., Yamada, D. and Defossez, P.-A. (2006) Born to bind: the BTB protein–protein interaction domain. BioEssays 28, 1194–1202. [176] Stogios, P.J., Downs, G.S., Jauhal, J.J., Nandra, S.K. and Prive, G.G. (2005) Sequence and structural analysis of BTB domain proteins. Genome Biol 6, R82. [177] Pintard, L., Willis, J.H., Willems, A., Johnson, J.L., Srayko, M., Kurz, T., Glaser, S., Mains, P.E., Tyers, M., Bowerman, B. and Peter, M. (2003) The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425, 311-316. [178] Chung, J.S. and Webster, S.G. (2003) Moult cycle-related changes in biological activity of moult-inhibiting hormone (MIH) and crustacean hyperglycaemic hormone (CHH) in the crab, Carcinus maenas. From target to transcript. Eur J Biochem 270, 3280-3288. [179] Travis, D.F. (1955) The molting cycle of the spiny lobster, Panulirus argus latreille. iii. physiological changes which occur in the blood and urine during the normal molting cycle Bio. Bull. 109, 484-503. [180] Truman, J.W. (1996) Ecdysis control sheds another layer. Science 271, 40-41. 115 [181] Webster, S.G. (1986) Neurohormonal control of ecdysteroid biosynthesis by Carcinus maenas Y-organs in vitro, and preliminary characterization of the putative molt-inhibiting hormone (MIH). Gen Comp Endocrinol 61, 237- 247. [182] Tamone, S.L. and Chang, E.S. (1993) Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro. Gen Comp Endocrinol 89, 425-32. [183] Lachaise, F. and Lafont, R. (1984) Ecdysteroid metabolism in a crab: Carcinus maenas L. Steroids 43, 243-259. [184] Wang, S.F., Ayer, S., Segraves, W.A., Williams, D.R. and Raikhel, A.S. (2000) Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors. Mol Cell Biol 20, 3870-9. [185] Dell, S., Sedlmeier, D., Bocking, D. and Dauphin-Villemant, C. (1999) Ecdysteroid biosynthesis in crayfish Y-organs: feedback regulation by circulating ecdysteroids. Arch Insect Biochem Physiol 41, 148-155. [186] Söderhäll, K. and Smith, V.J. (1983) Separation of the haemocyte populations of Carcinus maenas and other marine decapods and prophenoloxidase distribution. Dev Comp Immunol 7, 229-239. [187] Zmora, N. and Chung, J.S. (2014) A Novel Hormone Is Required for the Development of Reproductive Phenotypes in Adult Female Crabs. Endocrinology 155, 230-239. [188] Drexler, A.L., Harris, C.C., dela Pena, M.G., Asuncion-Uchi, M., Chung, S., Webster, S. and Fuse, M. (2007) Molecular characterization and cell- specific expression of an ion transport peptide in the tobacco hornworm, Manduca sexta. Cell. Tissue. Res. 329, 391-408. [189] Durica, D.S., Kupfer, D., Najar, F., Lai, H., Tang, Y., Griffin, K., Hopkins, P.M. and Roe, B. (2006) EST library sequencing of genes expressed during early limb regeneration in the fiddler crab and transcriptional responses to ecdysteroid exposure in limb bud explants. Integr Comp Biol 46, 948-964. 116 [190] Pamuru, R.R., Rosen, O., Manor, R., Chung, J.S., Zmora, N., Glazer, L., Aflalo, E.D., Weil, S., Tamone, S.L. and Sagi, A. (2012) Stimulation of molt by RNA interference of the molt-inhibiting hormone in the crayfish Cherax quadricarinatus. Gen Comp Endocrinol 178, 227-36. [191] Van Der Horst, D.J. (2003) Insect adipokinetic hormones: release and integration of flight energy metabolism. Comp Biochem Physiol B 136, 217-226. [192] Stachura, M.E., Tyler, J.M. and Kent, P.G. (1989) Pituitary immediate release pools of growth hormone and prolactin are preferentially refilled by new rather than stored hormone. Endocrinology 125, 444-449. [193] Stuenkel, E., Gillary, E. and Cooke, I. (1991) Autoradiographic evidence that transport of newly synthesized neuropeptides is directed to release sites in the X-organ--sinus gland of Cardisoma carnifex. Cell. Tissue. Res. 264, 253-262. [194] Ollivaux, C. and Soyez, D. (2000) Dynamics of biosynthesis and release of crustacean hyperglycemic hormone isoforms in the X-organ-sinus gland complex of the crayfish Orconectes limosus. Eur. J. Biochem. 267, 5106- 5114. [195] Oates, A.C., Bruce, A.E. and Ho, R.K. (2000) Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo. Dev. Biol. 224, 20-28. [196] Kulkarni, M.M., Booker, M., Silver, S.J., Friedman, A., Hong, P., Perrimon, N. and Mathey-Prevot, B. (2006) Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. Nat. Methods 3, 833-838. [197] Seinen, E., Burgerhof, J.G., Jansen, R.C. and Sibon, O.C. (2011) RNAi- induced off-target effects in Drosophila melanogaster: frequencies and solutions. Brief. Funct. Genomics. 10, 206-214. 117 [198] Webster, S.G., Dircksen, H. and Chung, J.S. (2000) Endocrine cells in the gut of the shore crab Carcinus maenas immunoreactive to crustacean hyperglycaemic hormone and its precursor-related peptide. Cell. Tissue. Res. 300, 193-205. [199] Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E. and Prydz, H. (2002) Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30, 1757-1766. [200] Kretschmer-Kazemi Far, R. and Sczakiel, G. (2003) The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res. 31, 4417- 4424. [201] Luo, K.Q. and Chang, D.C. (2004) The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem. Biophys. Res. Commun. 318, 303-310.