IGF-I, IGF-II and IGF-IR expression as molecular markers for egg quality in mullet and grouper

Josette Pesayco Bangcaya Bachelor of Science in Fisheries

School of Life Science Queensland University of Technology Brisbane, Queensland Australia

A dissertation submitted for the degree of Masters in Applied Science (Research) 2004 2

Keywords: Insulin-like growth factor (IGF)-I, IGF-II, IGF-I Receptor, egg quality, vitellogenin, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, mullet, grouper 3

ABSTRACT

Common measures of egg quality have been survival to specific developmental stages, higher hatching rate of fertilized eggs and final production of fry. Determinants of egg quality are variable among and between teleost species and no common unified criteria have been established. Maternally inherited genes influence egg quality and early embryo development is partially programmed by the messenger ribonucleic acid (mRNA). Among the genes, the insulin family is important for growth functions and the presence of their transcripts in the ovary, oocytes and embryos implies their involvement during the reproductive process and their relevance to egg quality. The insulin-like growth factor (IGF) system has three components, the ligands IGF-I and II, the IGFBPs (insulin-like growth factor binding proteins) and the IGF receptors that mediate biological activity of the ligands. Vitellogenin (Vtg) is the major source of nutrients for the developing embryo and elevated levels in female fish plasma signals gonadal development preceding spawning. In oviparous fish where the developing embryo is dependent on the stored food in the yolk, vitellogenin levels in the egg could indicate its capability to support embryonic growth.

This study aimed to develop molecular tools, specifically probes for IGF-I, IGF-II and IGF-IR, for the evaluation of fish egg quality. These probes would be used to determine expression levels of IGF-I, IGF-II and IGF-IR during egg development to assess their potential as molecular indicators for egg quality. In addition, this study also aimed to establish an enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs and determine if differences in Vtg levels could be linked to fertilization and hatching success.

Through reverse-transcription polymerase chain reaction (RT-PCR) putative complementary deoxyribonucleic acid (cDNA) fragments of IGF-I, IGF-II and IGF-IR were cloned and sequenced from mullet (Mugil cephalus) and grouper (Epinephelus coioides). The relative expression ratio of the three genes in the eggs of mullet and grouper were assayed by quantitative PCR (QPCR) and calculated using the Pfaffl 4 method (Pfaffl, 2001). Levels of vitellogenin in different batches of mullet eggs were quantified by ELISA.

Spawned eggs of grouper were grouped into low (<60%) or high (>60%) fertilization rate (FR) and the fertilized eggs that were incubated until hatching were grouped into medium (>90%) or high (>90%) hatching rate (HR). Samples were categorized into sinking eggs, late embryo and hatched larvae. Relative expression ratio of IGF-II was significantly high (P<0.01) compared to IGF-I and IGF-IR in all samples examined. All three genes were strongly expressed in sinking eggs compared to either late embryo or hatched larvae. However, there was no significant interaction effect between the genes and the samples analyzed. Mullet samples all came from a high FR and high HR group and were categorized into sinking, multicell stage, blastula, gastrula, late embryo and hatched larvae. There was a significant interaction effect (P<0.01) between gene and stage, showing that genes are differentially expressed during embryonic development. IGF-II was strongly expressed relative to the other genes in all stages examined and was highest during the gastrula stage.

Vtg levels were examined in mullet oocytes and egg samples that were grouped into 4; oocytes from females that subsequently spawned, had fertilized eggs which hatched (Group A); oocytes from females that did not spawn, therefore no fertilization and no hatching (Group B); eggs that were stripped, artificially fertilized but no hatching (Group C); and eggs that were spawned, assumed to be fertilized but did not hatch (Group D). Group A showed a trend of higher Vtg levels than the other three but this result was not statistically significant. 5

TABLE OF CONTENTS

Page number

Paragraph of key words 2 Abstract 3 List of tables 7 List of figures 8 List of abbreviations 9 Statement of original authorship 11 Acknowledgement list 12 Chapter 1. Introduction 14 Egg quality 16 Insulin-like growth factors 20 Quantitative polymerase chain reaction 25 Enzyme-linked immunosorbent assay (ELISA) 26 Chapter 2. Materials and Methods: Molecular Tools Development 28 Animals and egg samples 28 Spawning and egg collection 29 Gene cloning and sequencing 30 Quantitative polymerase chain reaction assay 37 Vitellogenin purification and ELISA development 40 Chapter 3. Materials and Methods: Molecular Tools Application 44 Quantitative polymerase chain reaction assay on eggs 44 Vitellogenin ELISA 45 Chapter 4. Results 47 Gene cloning 47 Quantitative polymerase chain reaction assay 50 Vitellogenin purification and ELISA in mullet 60 Chapter 5. Discussion and Conclusion 64 Appendix 1. Poster presented to the 6th International Marine Biotechnology Conference in Chiba, Japan (21-27 September 2003) 69

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TABLE OF CONTENTS

Page number

Appendix 2. Spawning protocol for mullet 70 Appendix 3. Pfaffl formula 72 Appendix 4. Mullet and grouper egg samples 73 Bibliography 74 7

LIST OF TABLES Page number

Table 1. Primer pairs used to amplify IGF-I, IGF-II and IGF-IR sequences in the respective mullet and grouper tissues 47 Table 2. Primer pairs designed for QPCR assay 53 Table 3. Master mix of reaction components for QPCR 54 Table 4. Reaction efficiencies and r2 values for QPCR 54 Table 5a. Mean relative expression ratio of target genes in grouper 57 Table 5b. Mean relative expression ratio of target genes in mullet 58 Table 6a. ANOVA of grouper data based on fertilization rate 59 Table 6b. ANOVA of grouper data based on hatching rate 59 Table 7. ANOVA of mullet egg data 60 Table 8. Log means of relative expression ratio in mullet 60 Table 9. Group means of Vtg concentration in mullet sample homogenates 63 Table 10. ANOVA of data of Vtg concentration in mullet egg homogenates 63

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LIST OF FIGURES Page number

Fig 1. Male and female mullet 15 Fig 2. Grouper 16 Fig 3a. Gel photo of amplified mullet IGF-IR cDNA fragments 49 Fig 3b. Gel photo of amplified grouper IGF-IR cDNA fragments 49 Fig 3c. Gel photo of amplified grouper IGF-I cDNA fragment 49 Fig 3d. Gel photo of amplified grouper IGF-II cDNA fragment 49 Fig 4. Alignment of IGF-IR sequence in mullet, grouper and turbot 51 Fig 5. Alignment of grouper IGF-I sequence with other fish species 52 Fig 6. Alignment of grouper IGF-II sequence with other fish species 52 Fig 7. Photomicrograph of grouper egg samples 55 Fig 8. Photomicrograph of mullet egg samples 56 Fig 9. Elution profile of purified Vtg from mullet plasma 62 Fig 10. ELISA standard curve for mullet Vtg 62 9

LIST OF ABBREVIATIONS

AA arachidonic acid AGRF Australian Genome Research Facility ANOVA Analysis of Variance BIARC Bribie Island Aquaculture Research Centre bp base pairs cDNA complementary deoxyribonucleic acid Ct crossing threshold dATP 2’-deoxyadenosine 5-triphosphate dCTP 2’-deoxycytosine 5’-triphosphate dGTP 2’-deoxyguanasine 5’-triphosphate DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate dTTP 2’-deoxythymidine 5’triphosphate EBI European Bioinformatics Institute EDTA ethylenedinitro tetraacetic acid ELISA enzyme-linked immunosorbent assay EPA eicosapentanoic acid FR fertilization rate GnRHa gonadotropin releasing hormone analog HR hatching rate IGF-I insulin-like growth factor I IGF-II insulin-like growth factor II IGF-IR insulin-like growth factor I receptor IPTG isopropyl-beta-D-thiogalactopyranoside kb kilobases kD kilodalton LB Amp Luria-Bertani ampicillin LHRHa luteinizing hormone releasing hormone analog LSD Least Significant Differences mRNA messenger ribonucleic acid 10

NCBI National Center for Biotechnology Information NFC National Fisheries Centre PBS phosphate buffered saline PCR polymerase chain reaction PES polyethersulfone PMSF phenylmethylsulfonyl flouride QPCR quantitative polymerase chain reaction RNA ribonucleic acid RT-PCR reverse-transcriptase polymerase chain reaction SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Tm melting temperature TMB tetramethylbenzidine UDG uracil-DNA glycosylase Vtg vitellogenin X-gal 5-bromo-4-chloro-3-indolyl-ß-D-galactoside

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STATEMENT OF ORIGINAL AUTHORSHIP

The work contained in this dissertation has not been previously submitted for a degree or diploma at any other higher educational institution. To the best of my knowledge and belief, the dissertation contains no material previously published or written by another person except where due reference is made.

Signed :______Date: ______12

ACKNOWLEDGEMENT

Praise and thanks to God Almighty for the guidance and love He has showered me in accomplishing this work, His presence has always been a guiding light throughout this journey.

To Drs. Joebert Toledo, Felix Ayson and Evelyn de Jesus, whose belief in my ability to make it through the unchartered waters gave me confidence to go through with this degree. To John Allwright Fellowship of Australian Centre for International Agricultural Research (ACIAR) for the scholarship award, especially to Sharon Harvey for the quick responses to my queries and, Kaye Toth the International Student Coordinator who assisted me upon my arrival at QUT.

To Dr. Alex Anderson, who made regular visits to Bribie Island to check on my progress, facilitated assistance in all university matters, and provided valuable insights and advice in the conduct of this thesis, I couldn’t wish for a more wonderful principal supervisor; Dr. Abigail Elizur, who is a just a few steps away from my desk guiding me through the research problems, clearing numerous issues in doing this study, pushing me to explore beyond my set narrow research boundary and imparted lots of comforting words during my stay at BIARC; Dr. Neil Richardson who warmly helped my fledgling researcher spirit on the intricacies of assays and never hesitated to answer all my queries; Dr. Mike Rimmer whom without fanfare facilitated financial and logistic support in the conduct of this thesis.

To the BIARC staff especially Leanda, Lana, Gary and Shannon who provided wonderful conversations when all the others have gone, it helped reduced the chill of winter months. To David Mayer, who patiently explained the “mathematics in biology” to my bewildered mind. To NFC staff especially Peter, Anjanette, Julian and Liz who warmly facilitated my stay at Cairns and Ken at Gladstone, who all helped provide valuable samples for my work. To my lab mates, Jason, Luke, Anna, Kim, Liz and Hui Kheng who shared with me the state of the art practices in biotechnology and made the journey towards the goal fun. To Hazra, who became like a second mother to me in Oz, whose hugs equals success in my sequencing and always lent able hands during fish 13 samplings. To Jo, whom I regarded as my “sempai”, as she unselfishly guided a neophyte like me into the mystery of biotechnology as well as the benefits of eating “health foods”.

To Tatay, whose spirit and memory has been with me until the end of this degree; Nanay and my brothers, especially Dongdong whose prayers, confidence and constant communication helped me keep my sanity in the Land Down Under, my niece and nephews whose cute voices always cheer me over the phone. To my dear friends, the salt of my life, especially Salve who was my steadfast e-mail pal as we shared the ups and downs of a research student and beyond; Genghis who showed me that student life in Oz doesn’t just revolve in the lab; Mae, Lorena, and the SEAFDEC dorm people who kept me grounded to the “real” world; Analiza, my invaluable ex-roommate and most trusted financial manager back home; the Glassington family - Bob, Lina, Paul, Lorna and Lani whom without reservations welcomed me into their home and became my second family in Brisbane; Vangie and Ray who helped me tolerate my very first freezing winter months in Oz.

To Gerald, whose love is immeasurable across the miles, made me to focus always on my goals, a wonderful voice to cheer me up at all times and showed so much understanding I’ve never imagined.

My heartfelt gratitude!

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Chapter I. Introduction

Fishes make up more than half of the 48,000 species of living vertebrates. They have been ecological dominants in aquatic habitats through much of the history of complex life. The Food and Agriculture Organization (FAO) report on world capture production of marine fish in 1997 is 86.4 million tons (MT) and 86.0 MT in 2000 and projected to be about 82.8 MT in 2001. After decades of phenomenal growth, it is evident that the marine capture fishery is leveling off and that increased production has, in recent years mainly come from aquaculture. Reports showed that over the last few years, the contribution of capture fishery to food fish supplies have decreased while that of aquaculture has increased (FAO, 2002). In developing Asian countries, it is anticipated that the supply difficulties experienced by capture fisheries will be counterbalanced by increased aquaculture production.

Culture of marine finfish has attracted considerable attention with the promotion of cage culture and improvement of seed production techniques, resulting in an increased number of species for which commercial hatcheries and grow-out operations are available (FAO, 2002). Aquaculture of high value finfish is becoming an industry of increasing importance throughout the Asia-Pacific region including Australia (ACIAR, 1997). Mullet and groupers are among the aquaculture species in this region.

The mullets are a family of near shore, catadromous fishes of considerable economic importance. Grey mullet (Mugil cephalus) is a euryhaline and eurythermal species that has the most cosmopolitan distribution of any of the major food fishes throughout the world (Williams, 2002). They are herbivorous and grow well in polyculture with other aquaculture species contributing to farm production as a secondary crop. They are highly esteemed for the mature ovaries and the dried roe is a gourmet food fetching a very high price (Fig 1). They have also been used as bioremediators in fish farms where they contributed to control of macroalgal biomass in shrimp farm effluent (Erler et al., 2004) and could be an efficient means to improve quality of sediments below intensive net-cage fish farms (Lupatsch et al., 2003). 15

Male mullet with developed testis

Female mullet with roe Fig 1. Male and female mullet (Mugil cephalus)

Groupers belong to the family Serranidae that are among the most highly valued marine finfish for food throughout most of warm and temperate marine regions. They are protogynous hermaphrodites, maturing as females first and may become males later (Tucker, 2003). In Southeast Asia, more than 20 species have been raised commercially by growing out captured wild juveniles and a hatchery technology has been established in some countries (Pomeroy, 2002) but egg survival has been variable (Sugama et al., 2003). Among the cultured groupers, Epinephelus coioides or estuary cod has shown aquaculture potential (Fig. 2).

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(Photo by David Cook, 2004)

Figure 2. Grouper (Epinephelus coioides)

Cultured and wild fish populations are dependent upon the production of good quality eggs. Increasing fish production through aquaculture requires increasing fingerling supply; however high mortality during early life stages remains a significant bottleneck in most of marine finfish production. Hatchery technology for an increasing number of species is continually being developed to meet with the expanding interest in aquaculture and the market demand for diversification, and the production of additional marine species presents an enormous economic opportunity for increasing the supply of high quality, safe and wholesome aquaculture products (Lee and Ostrowski, 2001).

Egg quality Poor egg quality is one of the major constraints in the expansion of aquaculture in both marine and freshwater species, and both cultured and wild fish populations are dependent upon the production of good quality eggs. Determinants of fish egg quality have been extensively studied for decades. The common standard measure of egg quality is survival to specific developmental stages, higher hatching rate of fertilized eggs and/or final production of fry (Kjorsvik et al.1990, Brooks et al., 1997a). 17

Physical factors used as determinants of egg quality includes fertilization rate, chorion appearance, egg shape, buoyancy (for pelagic eggs), pigments, symmetry of early blastomeres and other early stage morphological characters like egg size and number of oil globules. Early stage morphological characteristics like bulged plasma, black spots on egg cortex or wrinkles on egg membrane indicate poor egg quality in turbot and sea bream (reviews by Kjorsvik et al., 1990 & Bromage and Roberts, 1995). High hatchability or higher survival potential of hatched larvae has been indicated by better buoyancy in pelagic eggs (reviewed by Kjorsvik et al., 1990), regular cell division in the first blastomeres (Pavlov and Moksness, 1994; Shields et al., 1997, Moretti et al., 1999) and eggs having more than two oil globules (Bromage and Roberts, 1995). When egg physiology was examined, high quality eggs had significantly higher water uptake than did low quality eggs (Lahnsteiner et al., 1999, 2001, 2002).

Reviews of studies on egg quality determinants have shown that a criterion used in one fish species may not be applicable for other species. Carotenoid pigment associated with bright colors in eggs correlated to hatching rate in salmonid fishes (Craik, 1985) but not in lake trout (Lahnsteiner et al., 1999). Larger egg size was correlated to higher hatchability (Morehead et al., 2001) and larger larvae were found to survive longer without food (Kjorsvik et al., 1990) but under favorable conditions, egg size did not have a direct effect on larval survival (Gisbert and Willot, 2002).

High fertilization rate, the most common and simple physical determinant for good quality eggs does not always correlate with good survival and development in later embryonic stages (reviews by Kjorsvik et al., 1990; Bromage and Roberts, 1995). A positive correlation with larval viability was found when more than one determinant together with fertilization rate was taken into account such as normal blastomeres at early egg stages (Kjorsvik et al., 2003). High survival was also determined when utilizing more than one factor, such as chorion appearance and regular rounded egg shape (Moretti et al., 1999) and also when several early stage morphological characteristics were considered together such as symmetry, cell size, adhesion, margins and inclusions (Shields et al., 1997). 18

Environmental factors, investigated for affecting egg quality were temperature, photoperiod, salinity, bacterial growth and water pollutants. Maintaining a constant optimum temperature during ovarian development and egg incubation in induced spawners gave higher egg viability compared with subjecting the broodstock to ambient but oftentimes fluctuating temperature in most of the species studied (Kjorsvik et al., 1990; Brooks et al., 1997a; Tveiten et al., 2001). During spawning, conditions with excessive high or low incubation temperature have induced substantial embryo mortality or even zero egg viability (Brooks et al., 1997a; Davies and Bromage, 2002). Photoperiod manipulation was commonly used in hatcheries to delay or advance spawning (Brooks et al., 1997a). This practice has been found to influence egg quality and results showed that delaying spawning by light manipulation have led to increased egg mortality in pink salmon, rainbow trout and Atlantic charr (Kjorsvik et al., 1990) while photoperiodic advancement resulted in poor egg survival in seabass (Brooks et al., 1997a). Studies have shown that salinity has affected the rate of embryo development, bacterial growth increased egg mortality and water pollutants lowered egg hatching rates and was correlated to abnormal egg development (Bromage and Roberts, 1995; Brooks et al., 1997a; Spencer et al., 2002).

Broodstock nutrition affects reproductive performance in terms of fecundity, fertilization and egg quality. Amino acid composition, carbohydrates, total lipid content and vitamin C levels in the broodstock diet has direct correlation with fertilization, successful embryonic development, egg buoyancy, higher hatching rate and egg mortality (reviews by Bromage and Roberts, 1995, Cerda et al., 1995, Brooks et al., 1997a and Kjorsvik et al., 1997; Almansa et al., 1999; Bruce et al., 1999; Santiago and Gonzal, 2000; Izquierdo et al., 2001; Morehead et al., 2001; Furuita et al., 2001). Biochemical components have correlated with egg viability indicators in most fish species (Mukhopadhyay, 2003; Lahnsteiner and Paternello, 2004) and it was shown that lower fertilization and hatching rates were due to dietary effect of essential fatty acid (EFA) deficiency on sperm motility (Vasallo-Agius et al., 2001). Although fertilization rate showed correlation to levels of eicosapentanoic (EPA) and linoleic acid in eggs, no correlation was found with total protein content (Nocillado et al., 2000), while ratio of arachidonic acid (AA) to 19

EPA showed a negative correlation with egg survival (Tveiten et al., 2004). Furthermore, while 14 lipid classes correlated with hatchability in silver and golden perch there was no common one found between them (Anderson and Arthington, 1994). It seemed that to date, no common broodstock nutritional component securing good quality eggs across species has been established.

A range of factors imposed by hatchery practices has been found to influence egg quality such as stress on broodstock brought about by handling, over ripening of eggs and hormone administration in induced spawning. For fish that do not release eggs in captivity, stripping of eggs was necessary and it has been observed that egg viability after ovulation is highly sensitive to time and showed variability among species. Hatching rate was affected by delay or advancement in stripping (Kjorsvik et al., 1990; Shields et al., 2001). Low fertilization, increased occurrence of abnormal larvae and smaller fertilized eggs have been traced to handling stress like anaesthesia and intraperitoneal injection in cod and trout (Bromage and Roberts, 1995; Brooks et al., 1997a; Morgan et al., 1999) but they do not have any effect on fertility and spawning in fathead minnow (Kahl et al., 2001). In induced spawning, hormone administration of lowest dose gave the best egg quality in Dover sole while several low doses gave poor egg quality in mullet, white seabream and ayu (Kjorsvik et al., 1990).

Influence of broodstock age and genetics such as chromosomal arrangements has been linked to egg quality. Cytogenetic studies showed that broodstock subjected to different stresses like pollutants, produced eggs with chromosomal abnormalities that showed high larval mortality (review by Kjorsvik et al., 1990; Gorshkova et al., 2002). As reviewed by Kjorsvik et al. (1990) middle-aged spawners as well as second time ovulaters produced eggs of better quality than first time spawners. It was linked to the capability for lipid synthesis and generative metabolism that changes with fish age. This has been observed for multiple spawning species but not relevant in species that spawn only once in their lifetime.

Other molecular factors that have been studied in relation to egg quality were levels of cathepsin D, an enzyme that mediates the processing of yolk protein (Brooks et al., 20

1997b; Carnevali et al., 1999, 2001), the egg energy content, adenosine triphosphate, which was used as predictor of development and embryo survival in salmonids (Srivastava and Brown, 1991; Wendling et al., 2000) and ovarian pH fluid in lake trout, cyprinid fishes and salmonids (Craik et al., 1985; Lahnsteiner et al., 1999, 2002).

For many species, investigation of egg quality involves comparative tests of several parameters. Physical or visual tools for assessment although simple and convenient to perform, are subjective and may not be reliable. From an economic point of view, it is important to effectively evaluate the quality of hatchery production as early as possible to avoid wasting resources on what may turn out to be poor quality eggs (Planas and Cunha, 1999). Although there maybe species-specific indicators, no universal defining parameter for egg quality has yet been established. It is highly desirable that a single and usable most appropriate egg quality criterion be found, to avoid complications and repetitions especially in large-scale hatcheries. It should be based on a logical and cost- effective parameter that can be validated across species. This would enable hatchery managers to make economically sound decisions as early as possible in the production cycle.

Insulin-like growth factors (IGFs) Studies in the last decade have shown that the vertebrate egg contains a broad representation of different classes of maternal hormones and growth factors that could be vital to egg development and influence egg quality. The maternal ribonucleic acid (RNA) that codes for hormones and growth factors are stored in translationally inactive form until they are activated and turn on protein expression during oocyte maturation, fertilization or early embryonic development before the endocrine glands develop and become functional (Elies et al., 1999).

Growth factors required by the cell for cell-cycle progression (Leroith et al., 1995) include insulin-like growth factors (IGFs), nerve growth factor, epidermal growth factor, platelet derived growth factor, fibroblast growth factor, transforming growth factor, interleukins and hemopoietic growth factors. They represent a diverse group of hormone-like agents that affect a variety of cellular processes including metabolic 21 regulation, cell growth and division, and the maintenance of viability (Sporn and Roberts, 1991) as well as mediate or modify the local actions of classical hormones. Among these polypeptides, it has been established that IGFs mediate the action of growth hormone and in effect stimulate DNA synthesis and cell replication, causing the cell to traverse the successive phases of the cell cycle (Jones and Clemmons, 1995). They are expressed in virtually all cell types where they serve as ubiquitous cellular growth promoters. As multifunctional signaling molecules, they affect cellular proliferation, differentiation, motility, metabolism and survival of many cell types (Alarcon et al., 1998). IGFs also maintain basic cell functions in early development, such as survival, progressively acquiring in late development more specialized roles as well as playing an important role in controlling ovarian development (Maestro et al., 1997; Alarcon et al., 1998; Duan, 1997).

The IGF system consists of insulin, insulin-like growth factor-I and II (IGF-I and IGF- II) that are ligands for their corresponding receptors, insulin receptor (IR), IGF-IR and IGF-IIR, and the IGF binding proteins (IGFBPs). The principal members of the family are insulin, IGF-I and IGF-II. They have similar chemical structures and in-vitro activity but have distinct in-vivo activities (Sporn and Roberts, 1991). Recent studies have suggested an important role for IGFs in fish reproduction where expression in the ovary, oocytes, fish embryos and testicular cells indicates their involvement during the reproductive process (Kagawa and Moriyama, 1995; Funkenstein et al., 1996; Le Gac et al., 1996; de Jesus et al., 2002). Although other growth factors have been suggested to also play a role in fish oocyte growth such as epidermal growth factor and fibroblast growth factor (Tyler et al., 1999) this study will focus on the IGFs.

IGF-I has general growth promoting actions as well as anabolic effects on protein and carbohydrate metabolism in vertebrates (Tveiten et al., 1998; Pozius et al., 2001). IGF- II plays a key role in mammalian growth, influencing fetal cell division and differentiation and possibly metabolic regulation. Most of the biological responses to the IGFs are mediated thru the IGF-IR receptor where both the ligands act through the receptor to stimulate DNA synthesis. IGF-IR mediates several anabolic actions of IGF-I like stimulation of amino acid uptake, proliferation, differentiation and inhibition of 22 protein degradation (review by Florini et al., 1996). It also controls cell proliferation by a number of mechanisms - transformation, inhibition of apoptosis and mitogenesis – that are guided by different regions of the receptor and distinct, albeit potentially overlapping intracellular pathways (Mommsen, 2001). IGF-IIR serves as a cation-independent mannose-6-phosphate receptor that is involved in lysosomal targeting (Duan, 1997) and may function primarily as a degradative pathway to remove IGF-II from the extracellular environment (Jones and Clemmons, 1995). There has been no evidence presented of IGF-IIR in fertilized teleost eggs (Harvey and Kaye, 1991; Perrot et al., 1999) but a ligand-binding assay has detected their presence in fish embryos (Mendez et al., 2001). The actions of IGFBPs can enhance or inhibit the actions of the IGFs (Clemmons, 1998; O’Dell and Day, 1998).

IGF-I is a 70 amino acid peptide hormone with the first fish IGF-I cDNA sequence characterised in Coho salmon. Comparison of the coding sequences of fish and mammalian IGF-I indicates that it has been highly conserved and functional studies showed that the biological potency is remarkably conserved throughout vertebrate evolution (review by Duan, 1997). The major production site of circulating IGF-I in adult fish is the liver while all other tissues produce it locally (Abbot et al., 1992; Kagawa and Moriyama, 1995; Duguay et al., 1996; Schmid et al., 1999; Otteson et al., 2002). IGF-I expression was localized to the outer layer of the zona radiata and peripheral region of the ooplasm at the primary yolk globule stage in the ovary and in granulosa and theca cells in mature oocytes (Kagawa and Moriyama, 1995; Funkenstein et al., 1996).

The alignment of IGF-II sequences reveal that this hormone has also been highly conserved among vertebrate animals with an overall sequence identity of 84% between rainbow trout and human (Duan, 1997). IGF-II transcripts have been detected at all stages of human pre-implantation development including unfertilized oocytes and embryo samples (Lighten et al., 1997). The same results have been found in teleosts (Greene and Chen, 1997; Palamarchuk et al., 2002).

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IGF-IR has a heterotetrameric structure and a tyrosine kinase domain in the cytoplasmic portion of the beta subunit and has considerable structural and functional similarity to the insulin receptor. It encodes one of the longest 5’ untranslated regions (UTR) among the eukaryotic genes (LeRoith et al., 1995). Its presence in teleosts was detected in the ovarian follicular cells at all stages of gonadal development, (Maestro et al., 1997, 1999; Perrot et al., 2000), fertilized eggs, throughout the embryonic development and hatched larvae (Maestro et al., 1997; Elies et al., 1999; Greene and Chen, 1999; Mendez et al., 2001; Maures et al., 2002).

Gene knock-out studies on mice have elucidated the importance of IGFs and their receptors. Embryonic development in mouse lines lacking IGF-I and/or its receptor is impaired and apoptosis occurred in the eye muscles, whiskers and somites of the mouse during embryonic development when IGF-I expression ceased (review by Florini et al., 1996). IGF-II knock-outs resulted in placental and foetal growth retardation (O’Dell and Day, 1998; Regnault et al., 2002). IGF-I and/or IGF-II knock-out mice embryos are viable but showed impaired embryonic development supporting the conclusion that both are required for normal growth (Florini et al., 1996). Knock-out studies on IGF-IR produced mice that are severely growth retarded and die within minutes of birth (Butler and LeRoith, 2001; Kim and Accili, 2002 ).

Studies in teleosts reported four (Greene and Chen, 1997; Schmid et al., 1999) to five (Kermouni et al., 1998) forms of IGF-I transcripts, three IGF-II mRNA transcripts (Schmid et al., 1999) and two IGF-IR mRNA transcripts (Perrot et al., 2000; Maures et al., 2002; Nakao et al., 2002). The several forms may have resulted from gene duplication, allelic differences, population variation, multiple copies or multiple splicing of the gene (Kermouni et al., 1998). A range of techniques had been used to study the presence and levels of IGFs in fish tissues. These include reverse-transcription polymerase chain reaction (RT-PCR), quantitative PCR (QPCR), Western blot, in-situ hybridization, Northern hybridization, ligand binding assays and enzyme immunoreceptor assay (Boge et al., 1994; Drakenberg et al., 1997; Funkenstein et al, 1997; Maestro et al., 1997; Greene and Chen, 1999; Schmid et al., 1999, Aegerter et al., 2003; Armand et al., 2004). 24

In tilapia, which have an asynchronous ovary, expression pattern of IGF-I in gonads was higher than IGF-II (Schmid et al., 1999). A reverse pattern was found in gilthead seabream that have a hermaphroditic gonad where lower levels of IGF-I mRNA compared to IGF-II were observed in the developing gonads but no comparison was made in the testis (Perrot et al., 2000). IGF-II was found to be significantly high in females exhibiting follicular maturational competence (FMC) than those exhibiting medium or low FMC (Bobe et al., 2003a, 2003b). Except for the findings by Ayson et al. (2002) that did not detect IGF-I during embryonic development in rabbit fish, transcripts of IGF-I, IGF-II and IGF-IR were detected in unfertilized eggs, throughout the embryonic stage and hatched larvae and showed variations in levels of expression in zebra fish and rainbow trout (Perrot et al., 1999, Greene and Chen, 1999; Ayaso et al., 2002; Maures et al., 2002). Generally, IGF-I expression level was lower than IGF-II during embryonic development (Greene and Chen, 1999, Ayson et al., 2002), and a study by Maures et al. (2002) showed relatively unchanged levels of both IGF-I and II in all embryonic stages examined. Differential expression patterns of IGF-IR transcripts were observed in the developing eggs with decreasing transcript levels from embryogenesis towards adulthood (Elies et al., 1996; Maures et al., 2002). It was noted that strong hybridization signals of IGF-IR was found in several fast growing areas of the embryo such as the fin buds (Maures et al., 2002). Conflicting findings on expression patterns of the same gene at the same stage of development in different teleosts could probably be attributed to differences in assays used in the studies or reflect inherent physiological differences. With the refinement in techniques and development of new ones, these issues may be resolved. Nevertheless, presence of mRNA transcripts at all stages of pre-implantation development including unfertilized oocytes and during embryonic development suggests that they are maternally inherited (Lighten et al., 1997).

Quantitative polymerase chain reaction (QPCR) Although quantification of mRNA levels does not necessarily directly translate into quantification of biologically active protein (Stankovic and Corfas, 2003), the 25 assessment of gene expression level is an important tool towards the study of its physiological relevance and response to a variety of cues.

Until recently, the quantification of gene expression was based on gel or blot technique. Fluorescence-based real time PCR or QPCR is currently used for the quantification of steady state mRNA levels and is being established as a critical tool for basic research, molecular medicine and biotechnology (Roche, 2002). This technique offers a gel-free detection of mRNA and does away with the use of radioactive substances used in blot assays. With the use of fluorescence-detecting thermocyclers, QPCR allows for the detection of PCR amplification during the early phases of the reaction while traditional PCR detects amplification at the end point of the reaction. The instrument plots the rate of accumulation over the course of an entire PCR and the greater the initial concentration of target sequences in the reaction mixture, the fewer the number of cycles required in attaining a particular yield of amplified products. The threshold line is the level of detection or point at which a reaction reaches a fluorescent intensity above background and the cycle at which the sample reaches this level is called the cycle threshold (Ct) (Applied Biosystems, 1997). The read-out is given as the number of Ct that is proportional to the logarithm of initial amount of target in a sample (Ponchel et al., 2003).

Relative quantification in QPCR is a technique where the expression of a target gene is measured with respect to a stably expressed reference gene often called housekeeping gene, and the two gene levels are expressed as a ratio (Roche, 2002). This is a faster and less expensive way to analyze expression of multiple genes from a sample of total RNA. In spite of QPCR limitations, as reviewed by Bustin (2002), the technique and results have been validated in most studies (Livak and Schmittgen, 2001; Pfaffl, 2001; Aegerter et al., 2003; Ponchel et al., 2003).

Vitellogenin and ELISA Knowledge of the physiological processes during oogenesis leads to identifying factors in a viable egg. During vitellogonesis, exogenously synthesized proteins generally termed female specific-serum protein (FSSP) are actively incorporated into the growing 26 oocytes in the ovary (Fujita et al., 1998). An extensively studied FSSP is vitellogenin (Vtg), a large and complex phospholipoglycoprotein produced by the liver in large amounts in response to oestrogen that is produced by the ovary following the endocrinal cascade during reproduction. Vtg is taken up by the developing oocytes and enzymatically cleaved into egg yolk proteins and lipids. Together they supply components for growth of the developing embryo (Specker and Sullivan, 1994; Hiramatsu et al., 2002).

Enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blot analysis and Vtg mRNA determination by hybridisation strategies have been used to measure Vtg in fish plasma and liver tissue (Marin and Matozzo, 2004). Different kinds of ELISA have been widely used since it was first developed in 1971 (Specker and Anderson, 1994). Indirect ELISA involves the use of an antigen (Vtg) that is passively adsorbed to a solid surface such as plastic, the addition of a Vtg antibody that can attach to the antigen, followed by the addition of an enzyme labelled reagent that attaches to the antibody and the addition of an enzyme detection system, after which a visual or spectrophotometric reading of the colour reaction is conducted. In employing ELISA, circulating levels of Vtg have been used as a biochemical indicator of maturation in female fish under natural conditions (Blythe et al., 1994; Heppel and Sullivan, 1999; Matsubara et al., 1999; Sherry et al., 1999; Brion et al., 2000; Susca et al., 2001; Hiramatsu et al., 2002; Larsson et al., 2002; Hennies et al., 2003; Kazuto et al., 2002; Koya et al., 2003; Watts et al., 2003).

Vtg ELISA has been established as a plasmatic indicator of the female reproductive state but review of the literature revealed it has not been employed to measure Vtg in fish eggs. The proteolytic breakdown of Vtg upon incorporation in the oocytes produces smaller yolk proteins that showed molecular alterations and seem to differ between species (Matsubara et al., 1999; Tyler et al., 1999) and could probably offer difficulties in establishing a standard assay. The cleaved proteins, like lipovittelin and the ß’- component however have shown to react to Vtg antiserum (Hiramatsu and Hara, 1996) so there is a good probability that a Vtg ELISA on fish eggs can be established. A 27 recent study on the use of ELISA for egg protein to determine fecundity was successfully employed in oysters using a polyclonal antibody (Kang et al., 2003).

It has been observed that several induced spawnings on mullet resulted in failed egg development probably owing to unsuccessful fertilization. It was shown that the number of protein components was higher in floating eggs than sinking eggs where buoyancy often indicates successful fertilization (Carnevali et al., 2001). Because of the importance of sequestered protein for embryo development, determination of protein levels specifically Vtg, could elucidate the failure of fertilization in mullet eggs.

This study proposes the theory that with the use of a reliable technique using mullet and/or grouper as a model species, expression levels of IGFs could be a valid and sensitive molecular maker for egg quality. In addition, the study also aimed to establish enzyme-linked immunoassay (ELISA) for quantifying Vtg in fish eggs to determine if differences in Vtg levels could be linked to fertilization success.

Part of this study was presented to the 6th International Marine Biotechnology Conference in Chiba, Japan on 21-27 September 2003 and was awarded one of the outstanding poster awards from among more than 200 posters presented (Appendix 1). 28

Chapter 2. Materials and Methods: Collection of Samples and Development of Molecular Tools

This chapter discusses cloning of cDNA sequences of IGF-I, IGF-II and IGF-IR in grouper and mullet and the development of a QPCR assay, a molecular tool to quantify gene expression.

To measure Vtg in mullet egg homogenates, purification of mullet Vtg and development of a protocol for a Vtg ELISA was first established. Protein purification and ELISA protocol are discussed in this chapter.

Animals and egg samples Mullet Liver, muscle and gonad tissues were obtained and pooled from three tank-reared and wild caught female mullet (M. cephalus) with >300 g body weight. Tissue collection was done during the spawning season (June-July), ensuring the gonads were vitellogenic or at a maturing stage. The procedure was performed according to Animal Ethics Committee Approval No.: Bribie/032/07/02. Tissues were quickly dissected, cut into small pieces (>0.5 inch thick) and placed in 1.5 mL tube. To prevent RNA degradation, tubes were immediately placed in dry ice and stored at –80oC. When dry ice was not available, tissues were placed in RNA LaterTM (Ambion, USA) and processed for storage at –80oC following the manufacturer’s protocol. RNA Later is an aqueous, non- toxic tissue storage reagent that rapidly permeates the tissue to stabilize and protect cellular RNA in-situ in unfrozen samples. This eliminates the need to immediately process tissue specimens or to freeze samples in liquid nitrogen for later processing. It is claimed to preserve RNA in tissues for up to 1 day at 37oC, 1 week at 25oC and 1 month or more at 4oC and longer at -80oC.

Grouper Tissue samples from wild caught mature female grouper (E. coioides), with body size ranging from 400 to 700 g, were kindly provided by Dr. Mike Rimmer. Muscle, brain 29 and liver tissues were quickly dissected and placed in 1.5 ml Eppendorf (USA) tubes, snapped frozen in liquid nitrogen and stored at -80oC. They were transported in Styrofoam box with dry ice from Northern Fisheries Centre (NFC) to Bribie Island Aquaculture Research Centre (BIARC) and stored at –80oC upon arrival.

Spawning and egg collections Grouper spawners at a ratio of 1:6 male to female were held at the broodfish systems (O’Brien, 2004) at NFC where they spawn naturally. The broodfish system used contained approximately 2.7 tons of seawater at about 2.2 m water depth with a cover that enables photoperiod and temperature manipulation inside the tanks. Natural photoperiod and temperature of 26oC was maintained during spawning. Spawned eggs were taken at different times as well as the newly hatched larvae. Triplicate samples were placed in 1.5 ml tubes with RNA Later. A fourth sample was placed in 10% buffered formalin for microscopic stage identification. Fertilization and hatching rates were noted. Samples were stored at 4oC until sent to BIARC. Upon arrival at BIARC, RNA Later was removed from the samples by syringe, tubes immediately placed in dry ice and stored at –80oC until RNA extraction.

Mullet reared in concrete tanks and/or caught from the wild were induced to spawn at BIARC. All experiments were performed according to Animal Ethics Committee Approval No.: Bribie/032/07/02. Oocytes were sampled by a 5 mm diameter pipette to determine stage of maturation before hormone administration for spawning induction (see Appendix 2). Triplicate samples of spawned eggs were collected at certain stages of development as well as the newly hatched larvae and preserved in RNA Later until processed for RNA extraction. Gladstone Area Water Board hatchery provided a batch of mullet egg samples from their successful spawning employing a similar protocol for spawning induction as in BIARC.

Gene cloning and sequencing To determine the messenger ribonucleic acid (mRNA) sequence of IGF-IR in mullet, and IGF-IR, IGF-I and IGF-II in grouper, total RNA was extracted from mullet and grouper gonad, liver, brain and muscle tissues, reverse-transcribed, cloned and 30 sequenced. Sequence results of the coding region were compared for homology with the data deposited at Gene Bank.

RNA Extraction Total RNA was extracted from the above tissues using a battery powered homogenizer (Astral, USA) in the laboratory area reserved for RNA extraction only, in order to minimize contamination of RNAses and other cloned sequences. Trizol® (Invitrogen, USA) reagent was used for extraction following the manufacturer’s protocol including the additional isolation step for fatty samples. Trizol is a ready-to-use reagent for the total isolation of RNA from cells and tissues. It is a mono-phasic solution of phenol and guanidine isothiocyanate that is an improvement to the single-step RNA isolation developed by Chomczynski and Sacchi in 1987. During sample homogenization or lysis, it maintains the integrity of the RNA while disrupting cells and dissolving cell components.

RNA pellets were resuspended in 22-25 µL autoclaved distilled water by pipetting the pellet up and down and incubated in a dry block heater (Thermoline, Australia) at 55oC for 10 minutes. An aliquot of 3.5 µL and 1 µL of the RNA were used for RNA gel electrophoresis and spectrophotometric reading (UV/VIS Gene Quant, Amersham Biosciences Pty Ltd., Australia) respectively and the rest was stored at –80oC until used in cDNA synthesis. A 1:25 or 1:50 dilution was used to measure RNA concentration using glass capillaries. Concentration was measured at 320 nm wavelength at 0.5 mm path length.

RNA gel electrophoresis Quality and integrity of extracted RNA was confirmed by running RNA samples in 1.2% denaturing formaldehyde agarose gels, following the Qiagen protocol. The 3.5 µL RNA sample aliquot was thawed and mixed with 5x sample loading buffer at a ratio of 4:1. Samples were incubated at 65oC for 5 minutes and chilled in ice until loading time. Samples and RNA marker (0.28-6.58 kb, Promega, USA) were loaded in the gel and subjected to electrophoresis at 60 volts for one hour. Bands were visualized (UVITec illuminator, 365 nm) and photographed (UVITec, Cambridge England). Intensity and 31 band pattern as shown in the gel photo was compared to spectrophotometer readings. Only samples that showed 3 bands (rRNA) that corresponded to 5 kb, 2 kb and between

0.1 to 0.3 kb of the RNA marker (Promega, USA) and showed a valid A260/A280 ratio in the spectrophotometer were used for cDNA synthesis. cDNA synthesis for gene cloning Three µL of total RNA extract were used for 5’ and 3’ RACE using the RACE cDNA Kit following manufacturer’s protocol (Progen, Australia). The first strand reaction product was diluted with 100 uL Tricine EDTA buffer and incubated in a heating block at 72oC for 7 minutes. Samples were aliquoted into tubes and stored at –20oC. These served as cDNA templates for PCR.

Primer Design Primers for the IGF-IR gene were designed based on other fish IGF-IR published sequences. Amino acid and nucleotide sequences from turbot (AJ224993), flounder (AB065098), zebrafish (AF400275), rainbow trout (AAM27467), Atlantic salmon (Q8UWGO) and goldfish (Q9IAA2) were downloaded from the website of National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) and European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk/) and aligned using the Clustal program from EBI. Longest open reading frame available for IGF-IR during the time of the experiment was 1418 amino acids in turbot. A consensus sequence was identified among the fish species and specific forward and reverse nucleotide primer sequences were designed such that the nucleotide base was not less than 18 mer, with an annealing temperature of not less than 50oC, guanine and cytosine (GC) content of not less than 60% and an expected PCR product spanning < 1500 base pairs (bp). Selected primer sequences were checked for self-complementarities, primer dimer formation, self-hybridisation and melting temperature using NarOligo program (Oligo ver. 2 Nar). Complementary primers were designed to have a similar range of annealing temperature. Synthesized primers were purchased from Sigma-Aldrich or Proligo, Australia.

32

IGF-I and IGF-II degenerate and specific primers for mullet were already available in the lab (NCBI Accession numbers: AY 427954 and AY 427955). The degenerate primers were used to amplify the IGF-I and IGF-II fragments in grouper.

Polymerase Chain Reaction (PCR) To amplify synthesized cDNA, PCR was performed. A 12.5 µL total reaction volume for amplification was used, and consisted of the following: 1 µmol of forward and reverse primer, 0.2 mmol dNTPs, 2.5 mM of magnesium chloride (MgCl), 1.25 µL of 10x PCR buffer reaction mix (Invitrogen, USA), 0.3 units of Taq polymerase (Invitrogen, USA or Geneworks, Australia) and 5 µL of the synthesized 5’ or 3’ cDNA for template. Deoxyribonucleotides (dNTP) were prepared from stock solutions of dATP, dTTP, dGTP and dCTP purchased separately.

PCR reactions were carried out in 0.2 mL PCR tubes and thermal cycling was done either in PCR Express or PCR Sprint (Hybaid Limited, Middlesex, UK) that has a 0.2 mL block module. This thermal cycler was designed for fast, accurate, licensed thermal cycling of small number of samples and features high-speed sub-ambient blocks to perform oil-free thermal cycling with excellent dynamic uniformity and precision control of sample temperature. Different primer combinations were first tested using the thermal gradient block. The optimal temperature that produced a positive size band was used to do a PCR for gel excision and cloning. Annealing temperatures used varied from 52.4oC to 64oC, but the basic cycle follows the same format: pre PCR heat step at 94oC for 1-2 minutes followed by 30 cycles of 94oC for 30 seconds for denaturation, annealing temperature (52-64oC) depending on the primer pair used for 30 seconds, extension at 72oC for 1 minute and final extension of 72oC for 10 minutes. All PCR reactions were done in triplicate.

A 1.5% agarose gel was prepared by dissolving 0.75 g agar in 50 ml of 0.5x Tris-Borate EDTA (TBE) buffer. The solution was heated in a microwave until the agarose was melted, allowed to cool to 60oC and 1.0 µL of Ethidium bromide (1 mg/mL) added and the solution was poured into a gel tray with a set comb well. The agar was allowed to solidify at room temperature before the PCR product was loaded in the wells. About 2– 33

5 µL of product mixed with 2 µL of methylene blue or xylene cyanol loading dye were loaded into each well and a DNA ladder (1 Kb, Invitrogen, USA or Gene Ruler 100 bp DNA plus, Geneworks, Australia) was also loaded to serve as standard size gauge. Electrophoresis was done at 140 volts for 25-30 minutes. Band sizes were visualized in the UV light using 320 nm and once the expected band was confirmed, 15 µL of the PCR product was electrophoresed in 1.2% agarose gel at 120 volts for 30 minutes ensuring the buffer in the gel rig was clean. The correct band size was excised using a carbon steel surgical blade sterilized by gamma radiation (Swann-Morton, Sheffield, England).

PCR product gel purification The excised gel band corresponding to the expected amplified cDNA size was purified using Perfect Gel Cleanup Kit (Eppendorf, USA) following the manufacturer’s protocol. On the last part of the protocol, the spin column was placed in a labeled 1.5 mL tube and 30 µL of elution buffer was added. It was centrifuged for 1 minute to elute the bound and purified DNA.

DNA Ligation The purified DNA sample was ligated into pGEMT Easy vector (Promega, USA), following the manufacturer’s protocol. The pGEMT Easy vector has T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the alpha peptide coding region of the enzyme ß-galactosidase whereby insertional inactivation of the alpha peptide allows recombinant clones to be directly identified by color screening on the indicator plates. It also contains multiple restriction sites that would allow for the release of the insert by digestion with a single restriction enzyme. Ligation was carried out at 4oC overnight.

Transformation The ligated plasmids were transformed into XL1-Blue subcloning-Grade Competent Cells (Stratagene, USA) following the manufacturer’s protocol and transformants were plated in Luria Bertani–Ampicillin (LB-Amp) agar plates with X-Gal and IPTG and incubated overnight at 37oC. X-gal is a non-inducing chromogenic substrate for ß- 34 galactosidase that hydrolyzes it to form an intense blue precipitate. X-gal is used in conjunction with IPTG to detect white (recombinants) and to distinguish these from the blue (non-recombinants) colonies. The XL1-Blue competent cell strain allows blue- white color screening for recombinant plasmids and is an excellent host strain for routine cloning applications using plasmid or lambda vectors. To prepare LB Amp agar plates, 250 mL solution comprising of 2.5 g bacto-tryptone, 1.25 g bacto-yeast extract, 1.25 g sodium chloride and 2.5 g of granulated agar was dissolved in water and autoclaved (sterilizing time of 20 min at 121oC). When the solution had cooled down, 6.25 mg of Ampicillin was added and the solution poured into sterile, disposable plates (90 x 14 mm) at about one half the plate’s volume and then allowed to solidify. Plates were stored at 4oC until used.

Plasmid culture and DNA purification The positive white colonies were screened by PCR using the universal SP6 and T7 primers. A total amplification reaction volume of 12.5 µL was used per identified colony. The reaction mix consisted of 1 µmol of both SP6 and T7 primers, 0.2 mmol dNTP, 2.5 mmol of magnesium chloride (MgCl), 1.25 µL of 10 x PCR buffer reaction mix (Invitrogen) and 0.3 units of Taq polymerase (Invitrogen, USA or Geneworks, Australia). A pick from the colony was used as template for the PCR by touching a pipette tip in the colony growth, touching it in a fresh agar plate (patching) and then dipping the tip in the 12.5 µL reaction mix in the PCR tube. Template addition was done in the laminar flow to avoid contamination. PCR reaction was carried out at 94oC initial denaturation, 94oC at 30 sec denaturation, 45oC at 45 sec annealing, 72oC at 60 sec extension for 30-35 cycles, with an additional extension of 10 minutes at 72oC.

The PCR products were electrophoresed in a 1.5% agarose gel to confirm which colonies have the correct cDNA insert size. Two isolates from the colonies that have the correct inserts were selected and inoculated in a 2 mL LB media. LB medium was prepared using the components of the LB-Amp minus the agar. Two mL of LB medium were placed in 15 mL Falcon tubes and inoculated with the identified colony. The tubes were incubated at 37oC overnight with constant shaking at 100 rpm. The cultured cells were harvested and the plasmid purified using the Wizard® Plus SV Minipreps DNA 35

Purification Systems (Promega, USA) following the manufacturer’s protocol. Concentration of purified DNA was measured using Gene Quant. To confirm the identity of the clones, purified cDNA clones were digested with the restriction enzyme EcoRI (New England Biolabs, USA), incubated at 37oC overnight and products electrophoresed in 1.5% agarose gel. The digested plasmid that showed the expected size was used as a template for PCR sequencing reaction.

DNA PCR sequencing The sequence reaction mix was as follows: ABI mix dye 3.0 µL, ABI sequencing buffer 1.0 µL, 3.2 pmol M13 primer (either forward or reverse) 1.0 µL, autoclaved distilled water 5.0 µL less the volume of purified DNA. The template was 200-400 ng of purified DNA per reaction mix. PCR conditions were as follows: pre PCR heat step at 94oC for 5 minutes followed by 27 cycles of 94oC for 10 seconds denaturation, annealing temperature of 50oC for 15 seconds, and extension at 60oC for 4 minutes.

The PCR reaction was precipitated using an ethanol and sodium acetate protocol as follows: 1. For each sequencing reaction, a 1.5 ml microcentrifuge tube was prepared containing 2 µL 3M sodium acetate, pH 4.6 and 50 µL of 95% ice cold ethanol. 2. The entire contents of the PCR reaction were pipetted into the tube and vortexed to mix thoroughly. The reaction was left covered at room temperature for 30 minutes and centrifuged for 30 minutes at 13000 rpm. 3. The supernatant was carefully aspirated with a pipette tip and discarded. The pellet which most of the time was barely visible was rinsed with 250 µL of 70% ethanol, vortexed briefly and centrifuged at 13000 rpm for 20 minutes. 4. The supernatant was carefully aspirated and discarded. The pellet was dried at 40oC in a vacuum centrifuge (DNA mini Heto, Medos, Victoria).

The sample was sent by express mail for sequencing at the Australian Genome Research Facility (AGRF) at the University of Queensland, Brisbane. Sequencing was done using Big Dye Terminator Kit ver 3.1. The sequences were sent through the internet set-up connection with the AGRF facility and BIARC. Sequence analysis was done using 36

Sequencher 4.0 (Gene Codes Corporation, USA). This program is a DNA tool written for Microsoft Windows or Mac where users enter or import sequence fragments. Chromatograms were examined for clear and single peaks, nucleotide of the vector sequence trimmed out and the nucleotide sequence examined for homology with reported sequences from other species at NCBI using the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997) specifically BlastX. This tool compares a nucleotide query sequence translated in all reading frames against a protein sequence database.

The different clone sequences were aligned to establish a continuous sequence referred to as contig, using the Sequencher program. This process allowed easier deduction as to where the first nucleotide of the codon starts. Overlapping sequences in the clones were connected to obtain the maximum cDNA sequence. The deduced amino acid sequences were compared for homology with the same gene from other fish species using the ClustalW program at the EBI website.

Quantitative Polymerase Chain Reaction (QPCR) Determination of gene expression levels was conducted by the use of the QPCR assay following a series of steps.

QPCR primer design Using the obtained cDNA sequences of the target genes, primers were designed to amplify the genes of interest. Using the guidelines from different manufacturers (i.e. Applied Biosystems), primers were designed to comply with the following criteria: 1.) Amplify a short segment within the target sequence from 100 to 300 bp, 2.) The segment spans between an intron, 3.) GC content of the segment is 50 to 80%, 4.) Primer pair was specific to the target gene only. In the case of IGFs, the position of the probe was chosen near the 3’ end region where least homology within this family occurs. Runs of identical nucleotides especially four or more Gs, were avoided. No more than two G and/or C in five nucleotides at the 3’ end of the primer and melting temperature (Tm) of each primer 37 was between 60-64oC. Primers were designed using Primer3 program accessed through the website (http://www-genome.wi.mit.edu). The primers identified were rechecked using the NarOligo program and redesigned such that no primer dimer occurs and the annealing temperature ranges from 60-62oC. To further confirm specificity, the designed primers were checked for homology at NCBI using the Blast program to ensure they would only pick up the intended fragment of the target gene. cDNA synthesis for QPCR For QPCR cDNA synthesis, total RNA extract from the eggs and gonads were diluted in water to have a uniform concentration of 1 µg/µL and used as template for first strand cDNA synthesis. DNAse treatment using RQ1 RNAse-free DNAse (Promega, USA) was performed to remove potential contaminating DNA from RNA samples following the manufacturer’s protocol. Starting RNA concentration for cDNA synthesis was tested for 250 ng, 500 and 1 µg. Concentration of the reverse transcriptase enzyme, Superscript III reverse-transcriptase (Invitrogen, USA), was also tested, decreasing the amount from the manufacturer’s recommendation, between 100 and 175 units per 20 µL reactions. From the results, first strand cDNA synthesis was performed using 250 ng RNA and three quarters (175 units) of the recommended enzyme concentration with 200 ng of random primers (Invitrogen, USA) to prime a 20 µL reaction.

QPCR assay optimization All the reagents for the master mix were prepared and aliquoted in the tubes in one designated area of the lab while the template was added in a separate room to avoid cross contamination or to rapidly minimize and isolate potential sources of contamination. QPCR technique is far more stringent than the ordinary PCR, thus a dedicated set of pipettes were used exclusively for the assay. Filter pipette tips were used to avoid carryovers and contaminations and powder-free gloves were used while working.

An assay to determine optimum concentration of MgCl, primers, and Platinum SybrGreen® QPCR SuperMix Uracil-DNA Glycosylase (UDG) (Invitrogen Life Technologies, USA) was conducted using plasmid cDNA as template. The plasmid 38 templates were taken from the clones that were sequenced and determined to contain inserts for which QPCR primers were designed. MgCl2 concentration was tested from 1.5 to 3 mmol/L. Optimum concentration of the primers was tested from 100 to 400 nmol/L. The PCR reaction was carried out in 0.1 mL strip tubes, using rotor gene (Corbett, Australia). 5 µL of cDNA template was used for every 25 µL PCR reaction. PCR conditions were as follows: hold at 50oC for 2 min (UDG incubation), denature at 95oC for 2 min, 40 cycles of denature, annealing and extension at 95oC for 20 sec, 62oC for 20 sec, and 72oC for 20 sec respectively, with the acquisition reading taken at 72oC. Final hold was done at 72oC for 15 minutes. Melting curve analysis was conducted with ramping rate from 72oC to 99oC rising at 1oC at each step, waiting for 5 sec on first step and 5 sec for each step afterwards. This procedure would enable detection of primer dimmers and multiple products in the reaction.

QPCR calculation There are two ways to analyze QPCR data: absolute or relative quantitation. For the purpose of this study, relative quantitation is adequate. With relative quantitation, target genes are referenced to an endogenous gene or a housekeeping gene. The housekeeping genes are necessary for basic cell survival hence their presence in all nucleated cell types (Pfaffl, 2001). The mRNA syntheses of these genes are considered to be stable in various tissues even under experimental conditions. In normalizing the amount of the target gene to the housekeeping gene, differences caused by variations in the initial RNA amount, possible RNA degradation, variations in cDNA synthesis or pipetting errors could be corrected. The final results are normalized to the values in the calibrator or control to generate the relative expression ratio. The calibrator is generally an untreated sample. Results are expressed as the target to reference ratio of each sample, divided by the target to reference ratio of a calibrator.

Beta-actin (ß-actin) was used as the housekeeping gene for this study. Mullet ß-actin cDNA sequence (cloned and sequenced in the BIARC lab by Dr. Kim Guyatt) was used to design primers to amplify 150 bp both from mullet and grouper samples. The determined calibrator for both grouper and mullet assays were the respective gonads taken from the wild-caught fish at pre-spawning stage with oocyte diameter of >450 µm. 39

With the use of calibrator-normalized relative quantification, accuracy of results is influenced by the amplification efficiencies of target and the reference genes determined from the standard curves. Based on the generated standard curves, three methods can be employed for computation: two standard curves, comparative Ct method (Applied Biosystems, 1997) and the Pfaffl method (Pfaffl, 2001).

A standard curve for target genes and the reference gene was constructed using a 10-fold serial dilution of plasmid as templates. Using plasmid templates for construction of standard curves has been recommended since they have optimal reproducibility over a wide range of concentrations (Stankovic and Corfas, 2003). In the conduct of this study, the optimum amount of reagents determined in assays using plasmid templates showed low amplification efficiency and correlation coefficient when used in the actual experimental samples. Therefore, a MgCl2 and primer concentration optimization assay was conducted using the synthesized cDNA of the calibrator in serial dilution as template to establish valid standard curves. The target genes and the reference gene were amplified from the same cDNA source in a separate tube reaction.

Results from the established standard curves showed different reaction efficiencies for the target and the reference genes, thus the Pfaffl method was determined as the most appropriate method to calculate the relative gene expression ratio.

The PCR reaction to amplify target genes and housekeeping gene in the samples was carried out in 0.1 ml tubes strip tubes, using a rotor gene (Corbett, Australia). Four amplification reactions, one each for IGF-I, IGF-II, IGF-IR and β-actin mRNA expression, were carried out in triplicates in different tubes at the same time. A master mix of the reaction component optimized for each gene was aliquoted into 20 uL per reaction tube and 5 uL of cDNA template was added. PCR conditions followed that of QPCR assay optimization (page 38).

Vitellogenin purification and ELISA 40

The development of a mullet Vtg ELISA required purified mullet Vtg and mullet Vtg antibodies. The antibodies were a kind gift from Dr. Akihiko Hara (Hokkaido University, Japan) while the mullet Vtg was purified during this study. The polyclonal Vtg antibodies were raised in rabbits, the antisera purified as an immunoglobulin (IgG) fraction and lyophilized after 40% saturated ammonium sulphate wash. Specificity and sensitivity of the antibody had been evaluated by Oda et al. (2002).

Vtg production in fish was fully under estradiol control. Induction of Vtg in male mullet was performed. Estradiol was dissolved in cocoa butter to a concentration of 5 mg/mL and injected at 5 mg/kg per fish in the male mullets with size ranging from 328 – 384 g. This dose was administered once a week for 3 weeks. On the fourth week, the fish were bled either from the heart or caudal vein. Collection tubes and syringes were rinsed with 1 mmol PMSF with EDTA-NaCl before used (method modified from Yeh et al., 2003). Blood was centrifuged at 1500 g at 4oC for 15 minutes. The supernatant plasma was aliquoted into tubes and stored at –80oC until vitellogenin purification. One fish did not receive any estradiol and served as control. The presence of Vtg in the collected serum was verified by SDS-PAGE in comparison with the control. Samples were run on a 5% gel using the system of Laemmli (1970) in a Bio-Rad Mini Protean II apparatus and using Bio-Rad pre-stained Low Range MW markers. The gels were stained with 0.2% Coomassie Blue in 5:4:1 water:methanol: acetic acid, and destained in the same solvent.

Plasma from estradiol treated fish was used for vitellogenin purification, following a protocol by Heppel and Sullivan (1999). Two mL of plasma in 5% sucrose was applied to a 2 x 24 cm DEAE sepharose CL-6B anion exchanger (Sigma, USA) and connected to the Fast Liquid Purification (FLP) Biologic System (Biorad, USA) placed in a 4oC chamber. The column was equilibrated with 0.07mM NaCl in 25 mmol/L Tris-HCl and chromatography was performed at a gradient flow rate of 0.40 mL/min from 0.07mol/L to 0.5mol/L Tris-HCl. Purified Vtg collected at peak fractions was pooled and the concentration measured following the Folin-Lowry method (Robyt and White, 1987). Confirmation of Vtg was done by observation of a positive reaction in a single radio immuno diffusion (SRID) Plate kit for mullet (Cosmo Bio Co., Ltd, Japan). Purified Vtg was used as the antigen in the indirect ELISA optimization assay. 41

Antigen coating Antigen concentration ranging from 5 to 30 ng/mL in carbonate buffer was used to coat 96 well Nunc plates (Polysorp, Medos, Australia) at 200 µL per well. Blank wells were coated with buffer minus the antigen; the plates were placed in a self-seal plastic bag and incubated at 4oC overnight (12-16 hours). The contents of the well were washed with phosphate buffered saline (PBS) Tween 20 and flicked 3 times to remove unbound antigen.

Specific antibody incubation Lyophilized rabbit antibody raised against mullet vitellogenin was diluted in PBS Tween 20-bovine serum albumin (BSA) to achieve a dilution from 1:2,500 to 1:40,000 and 200 µL were added per well. The plates were incubated for 90 minutes at room temperature (22-23oC) and then washed three times with PBS Tween 20.

Second antibody incubation 200 µL of goat anti-rabbit immuno-λ-globulin, conjugated to horseradish peroxidase (Sigma, USA) as secondary antibody, diluted at 1:5000 or 1:10000, was added to the plate wells. The plates were incubated for 90 minutes at room temperature and excess reagents washed three times with PBS-Tween 20.

Visualization of the reaction For color reaction, 50 and 100 µL of K-Blue TMB Substrate (ELISA Systems, Australia) was added to the wells and incubated for 5 and 10 minutes before 3 mol/L HCl was added to stop the reaction. Plates were read in the plate reader (Dynex Mrx Technologies, USA or Multiskan EX, Thermo Electron Corp, Finland) after 5, 10 and 15 minutes to determine maximum reaction time.

All reactions were done in duplicate. Data was graphed and a linear regression line was generated. A standard curve was constructed and the slope with >0.97 was considered valid. A blank (no antigen) was used to correct for the absorbance readings. Reagent volume and concentration to generate a valid standard curve was as follows: purified 42

Vtg concentration of 50, 40, 30, 20, 10 and 5 ng/mL, primary antibody in 1:40000 dilution, secondary antibody of 1:5000 dilution, 50 uL of K-Blue TMB Substrate in 10 minute reaction development, 50 uL of 3 mol/L HCl, and absorbance measured within 5 minutes of addition of the last reagent.

Egg collection Samples came from tank-reared mullet that were hormonally induced to spawn. Oocytes (diameter > 600 µm) were sampled by gonadal biopsy before hormone administration while egg samples were taken one hour after spawning and stored at -80oC until processed by homogenization. Sampled females were monitored for spawning and hatching. Triplicate samples of oocytes and eggs were collected and later classified into four groups. Group A (2 fish) were oocytes that were spawned, fertilized and hatched, and Group B (7 fish) were oocytes from females that did not spawn and no further egg development and no hatching. Group C (2 fish) were stripped eggs, artificially fertilized but did not show development and hatching, and Group D (2 fish) were eggs that were spawned but did not show development and hatching. To estimate for the mean individual weight, 0.1 g of random samples of oocytes and eggs were taken and the total number in the sample counted. The total weight was divided by the number of eggs counted.

Development of the protocol for egg homogenization was based on Hiramatsu et al. (2002) and Kang et al. (2003). Egg samples weighing 0.05 to 0.1 g were homogenized in

0.02 M Tris.HCl buffer pH 8.0 with 2% NaCl and 0.1% NaN3 and centrifuged at 12000 x g at 4oC for 20 minutes. Supernatant below the fatty layer was collected, filtered using a 0.22 µm Millex GP PES membrane (Millipore, Ireland) and stored at –20oC until analysis. 43

Chapter 3. Materials and Methods: Application of Molecular Tools

QPCR assay on the eggs Once the amplification efficiency of the target and reference gene from the standard curve was established, QPCR was performed on the egg samples. A master mix of the reaction components specific for each gene was prepared. The reaction mix was placed in a 0.1 mL QPCR tubes (Rotor-Gene, Australia) and 5 µL volume sample template containing 62.5 ng of reverse-transcribed RNA was added. All samples were run in triplicates. PCR conditions were a follows: hold at 50oC for 2 min (UDG incubation), denature at 95oC for 2 min, 40 cycles of denature, annealing and extension of 95oC for 20 sec, 62oC for 20 sec, and 72oC for 20 sec respectively, acquisition reading taken at 72oC to cycling A. Final hold was done at 72oC for 15 minutes. Melting curve analysis was conducted with ramping rate from 72oC to 99oC rising at 1oC at each step, waiting for 5 sec on first step and 5 sec for each step afterwards.

A negative reverse-transcriptase cDNA template for each sample was included to check for any genomic contamination using ß-actin as primer. The presence of an amplification product was further confirmed by gel electrophoresis. All QPCR assays were done within a period of two weeks to ensure that amplification efficiency of the reaction with the given set of reagent components was valid.

Ct values, in any of the triplicate samples that gave a mean standard deviation greater than 0.16 were eliminated. The relative expression ratio was calculated using the Pfaffl method (2001). The Pfaffl mathematical model (see Appendix 3) calculates the relative expression ratio based on the PCR reaction efficiency of the standard curve and the crossing threshold (Ct) of the investigated transcripts when reaction efficiencies of the target and reference gene are not similar. Reaction efficiency of the target and the reference gene was determined by constructing a standard curve using a serial dilution of the template.

44

Statistical Analysis Relative expression ratios of IGF-I, IGF-II and IGF-IR mRNA in sinking eggs, late embryo and hatched eggs of grouper (7 egg batches) was grouped into either low or high fertilization rate (FR) and medium or high hatching rate (HR). Low FR has <60% and high FR has >60% developed eggs over total number of eggs counted while medium HR has <90% and high HR has >90% hatched larvae counted in the sample. Mullet egg samples came from a high FR and high HR group (2 egg batches), except for three sources of unfertilized eggs that came from spawns with no hatching. Relative expression ratio of IGF-I, IGF-II and IGF-IR mRNA were calculated from unfertilized, multicell, blastula, gastrula, during melanocyte formation, late embryo and newly hatched larvae of mullet. Data were log transformed to obtain a normal distribution and analyzed using Analysis of Variance (ANOVA) in Gen Stat version 6.1. Significant differences between the log means were determined by Least Significant Differences (LSD) testing at P=0.05. The results were back transformed to equivalent relative expression ratios using bias correction (Kendall et al., 1983).

Vitellogenin ELISA Vitellogenin in mullet egg extracts was quantified through ELISA. Supernatants were diluted to a concentration of 1:10000 to fit into the validated standard curve of antigen- antibody for ELISA. 200 uL of diluted extracts in carbonate coating buffer was coated to the wells and incubated overnight at 4oC. Plate wells was washed 3x by PBS-Tween 20 and 200 µL of 1:5000 mullet Vtg antibody was added to the wells and incubated at room temperature for 90 minutes and then washed 3x by PBS-Tween 20. Secondary antibody was added to the well (200 µL, 1:5000) and incubated for 90 minutes at room temperature. The plate was washed 3x with PBS-Tween 20 and 50 µL of K-Blue TMB Substrate was added to the wells. After 10 minutes, the color development was stopped with 50 µL of 3mol/L HCl. Optical density of each well was read in the plate reader at 450 nm. A blank well with no antigen was included to correct for absorbance values. Vtg concentration for each egg sample was measured with respect to a standard curve included in every assay. The computed values were corrected with the dilution factor and standardized to 0.1 g of egg weight.

45

Statistical analysis Data was subjected to ANOVA to determine significant effect of Vtg concentration with egg groups. LSD was used to determine differences between group means. 46

Chapter 4. Results

IGF-I, IGF-II and IGF-IR cDNA sequences were isolated in mullet and grouper and their amino acid sequences were deduced. Relative expression levels of the mentioned genes were determined in eggs and hatched larvae in both mullet and grouper

Gene Cloning Using RT-PCR, cDNA sequences of IGF-IR in mullet and IGF-I, IGF-II and IGF-IR in grouper were amplified. Table 1 shows the sequence of primer pairs that were used to amplify the three genes in mullet and grouper, the corresponding tissues that where the source of the RNA template, the PCR primer annealing temperature and the expected DNA product size.

Gel electrophoresis of the amplified cDNA using different primers sets is shown in Figures 3a to 3d. Amplified cDNA fragments for mullet IGF-IR were 485, 818, 1173 and 660 bp and amplification products of grouper IGF-IR fragments were 585, 818, 699 and 1008 bp. IGF-I and IGF-II amplified products in grouper were 585 and 570 bp respectively. Only the expected band size was excised for sequencing, eliminating other non-specific products of different size that at times formed during amplification. Sequencing results that showed overlapping sequences in the clones were aligned to obtain the maximum cDNA sequence. To confirm gene identity, sequences from clones were compared with the deposited sequences at NCBI using BLAST program (Altschul et al., 1997).

The partial deduced amino acid sequence of mullet, grouper and turbot IGF-IR is shown in Figure 4. In teleosts, turbot has the longest deposited IGF-IR sequence at Gene Bank and this was used to provide a better comparison with the sequence results obtained in this study. Comparison of sequence homology between mullet, grouper, turbot and rabbitfish were all done using ClustalW program at EBI website. Sequences corresponding to 711 amino acids (aa) of IGF-IR in mullet were isolated and those correspond to 50% of the coding region in turbot IGF-IR. From grouper, sequences 47 corresponding to 594 aa were isolated and they correspond to 42% of the coding region in turbot. Homology between mullet and grouper was 82%, between mullet and turbot was 72% and, between grouper and turbot was 75%. The transmembrane domain in turbot spans from aa positions 946 to 964 and the tyrosine kinase domain spanned aa positions 1007 to 1134 (Elies et al., 1998). Alignment of grouper and mullet sequence with the identified transmembrane domain in turbot showed 45% homology across the three species and alignment with the identified tyrosine kinase domain showed 87% homology.

Table 1. Primer pair used to amplify gene coding fragments in the respective fish tissue, designated primer name is indicated below the nucleotide sequence Forward Primer Reverse Primer Annealing Expected Tissue temperature product taken oC size (bp) A. mullet IGF-IR gtcgccatccaaactgt gttgtgaagacgccatc 52.4 ~485 Gonad igf1r1034-1039f igf1r1186-1181r tacctgaacgccaacaa ccgttcatgtgtgcgta 53 ~818 Gonad igf1r93-99f igf1r1400-1395r ttygtcttctcgagvac acagtttggatggcgac 55 ~660 Liver igf1r825-833f igf1r6-1r cacaactactgctcyaa acagtttggatggcgac 55 ~1173 Liver igf1r655-661f igf1r6-1r ccsttcachgtbtaccg gcattbccwgcbagvga 55 ~363 Liver igf1r802-808r igf1r941-935r

B. Grouper IGF-IR gtcgccatccaaactgt tggtaaggctgctctgc 52.4 ~818 brain igf1r1034-1039f igf1r1258-1253r ccsttcachgtbtaccg ttgaccacgcccttcgc 55 ~699 brain igf1r802-808f igf1r5-0r gtggtttctcagggaca ccgttcatgtgtgcgga 53 ~1008 brain igf1r51-56f igf1r1400-1395r

C. Grouper IGF-I gcggagacccrwggggatgtctagcg 64 ~585 Liver igf1 -5 to 3fd tacatkckrtarttyckkcccccyghryt igf1 182-174rd

D. Grouper IGF-II catggaracccagmaaagayaacggac 65 ~570 Brain igf2 1-8fd ggmagsstgatsagrggcckgtggwrg igf2 199-190rd

Letters corresponding to the nucleotide: a - adenine; c - cytosine; g - guanine; t - thymine; y - c + t; w - a + t; v - a + c + g; s - c + g; b - c + g + t 48

1 2 3 4 5 6 7 8 9 10

Figure 3a. Gel photo of PCR amplification of mullet IGF-IR, lanes 2 (485 bp), 4 and 5 (818 bp), 7 (1173 bp) indicated with an arrow, 8 (660 bp) and 10 (363 bp); lanes 1, 3, 6 and 9 are DNA markers (0.1-3.0 kb)

11 12 13 14 15 16 17

Fiure 3b. Gel photo of PCR amplification of grouper IGF-IR, lanes 12 (818 bp) designated with arrow, 14 and 15 (699 bp) and 17 (1008 bp); lanes 11, 13 and 16 are DNA markers (0.1-3.0 kb)

18 19 20 21 22 23

Figure 3c. Gel photo of PCR amplification Figure 3d. Gel photo of PCR of grouper IGF-I, lanes 19 and 20 amplification of grouper IGF-II, (585 bp); lane 18 is DNA marker (0.1-3.0 kb) lane 22 and 23 (570 bp); lane 21 is DNA marker (0.1-3.0 kb) 49

The deduced amino acid sequences of IGF-I and IGF-II in grouper brain, grouper liver, mullet and rabbitfish are shown in Figures 5 and 6. IGF-I and IGF-II mRNA sequences from grouper liver have been deposited only this year and comparison with the sequence from grouper brain in this study, showed minor amino acid changes indicated by the boxed letters. In IGF-I, single base differences were located in B, C, A and E domain. Identical sequence of IGF-I in grouper brain and grouper liver was 97% and between grouper and mullet was 93%. Comparison of IGF-I in grouper with rabbitfish showed 94% homology. The known coding sequence of teleost IGF-I including the signal peptide was 186 aa and the length of coding sequence in grouper from this study was also 186 aa. Sequence identity of IGF-II between grouper brain and liver was 97% with three aa differences, two located in the signal peptide region and one in the E region. Sequence homology between grouper, mullet and rabbitfish was 92%. The coding sequence for teleost IGF-II including the signal peptide and the E domain was 215 bp and this study had sequenced 92% of IGF-II in grouper brain.

Quantitative Polymerase Chain Reaction (QPCR) Primers From the determined mRNA sequences, specific primers for IGF-I, IGF-II and IGF-IR in both mullet and grouper were designed to amplify target genes in QPCR assays. The D and E domains that are absent in insulin, were chosen as the regions from which primers for IGF-I and II were designed to ensure only the target sequence were amplified. QPCR primers to amplify IGF-IR were designed in the region towards the 3’ end of the transcript as this has been established to have high variability compared with other gene receptors that also exhibit a tyrosine kinase domain. The primers designed for mullet ß-actin were the same primers used to amplify ß-actin in grouper. Primer pairs designed for QPCR assays are shown in Table 2. 50 mullet ------HNYCS 5 grouper ------turbot RTSPSAPSMPQDVRAYSNSSTQLVVRWSPPVSPNGNQTYYLVRWQQQAEDRELYQHNYCS 660 mullet KELKIPIRVAAIGVGDQE------SEEDQKAEADDASYRKVFE 42 grouper ------turbot KELKIPIRIAAIGVGDQEEDTKPTKPDPEGADKGPCCPCPKSVEDLEAEAADASYRKVFE 720 mullet NFLHNSIFT------LFGVANATNSRRHRLHANSSVIPPLQAG--NGSA--ADLEP 88 grouper ------turbot NFLHNSIFTPRPPDRRRRDLFGFANSTHSRRHRLHTNSSHVPPHQAAGNNSSSTTAEPEQ 780 mullet ADRKFDFMEQSVTERELQISGLKPFTVYRIDIHACNRQVQRCSAAEFVFSRTKPAEKSDD 148 grouper ------PFTVYRIDIHACNRQVQRCSAAEFVFSRTKPAEKADD 37 turbot ADREFEFMEQAVSER-VQIFDLQPFTVYRIDIHACNRQVQRCSAAEFVFSRTKPAEKADD 839 **********************************:** mullet IPGKVTWEGHEDWVFLRWPEPRHPNGLILMYEIKFKLATETEKHECVSGQTYQAQRGVRL 208 grouper IPGPVTWEGHEDWVFLRWPEPPHPNGLILMYEIKFKLAAETEKHECVSGQMYHTQRGVRL 97 turbot IPGQVTWEGHEDWVFLRWPEPPHPNRLVLMYEIKFKLAAETEKHECVSGQTYQNQRGVRL 899 *** ***************** *** *:**********:*********** *: ****** mullet SNLSPGNYSVRVRATSLAGNGSWTQSSDFYVAERDENILYAMIFIPVAIIVLICCLAVML 268 grouper SNLSPGNYSVRVRATSLAGNGSWTHALDLYVAERYENVLYAMIFVPIVIILVICLLVSML 157 turbot SNLSPGHYSVRVRATSLAGNGSWTNAVDLYVAERYENVLYAMIFIPIAIILFICLLVTML 959 ******:*****************:: *:***** **:******:*:.**:.** *. ** mullet VFFNRKRNSDRLGNGVLYASVNPEYFSAAEMYVPDEWEVAREKITLSRELGQGSFGMVYE 328 grouper VVLSRKRNSDRLGNGVLYASVNPEYFSAAEMYVPDEWEVAREKITLSRELGQGSFGMVYE 217 turbot VVLNKKRNSDRLGNGVLYASVNPEYFSAAEMYVPDEWEVAREKIALSRELGQGSFGMVYE 1019 *.:.:***************************************:*************** mullet GVAKGVVKDEPEMRVAIQTVNESASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQP- 387 grouper GLAKGVVKDEPETRVAIKTVNESASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPP 277 turbot GLAKGVVKDEPETRVAIKTVNESASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQP- 1078 *:********** ****:***************************************** mullet TLVIMELMTRGDLKSHLRSLR--KENSTTQVLPPLKKMIQMAGEIADGMAYLNANKFVHR 445 grouper TLVIMELMTRGDLKSYLRSLR--KENATSQVLPPLKKMIQMAGEIADSMSYLNANKFVHR 335 turbot TLVIMELMTRGDLKSYLRSLRPKEQQWSSLSLPPLRKMLQMAGQIADGMAYLNANKFVHR 1138 ***************:***** ::: :: ****:**:****:***.*:********** mullet DLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTMSD 505 grouper DLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTMSD 395 turbot DLAARNCMVADDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTHSD 1198 **********:**********************************************:** mullet VWSFGVVLWEIAT-LAEQPYQGMSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNP 564 grouper VWSFGVVLWEIATLLAEQPYQGMSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNP 455 turbot VWSFGVVLWEIST-LAEQPYQGLSNEQVVRFVMEGGLLEKPQNCPDMLFELMRMCWQFNP 1257 ***********:* ********:*****:*********:**:***************:** mullet KMRPSFLEIISSIKDDLDPPFREMSFFYSEENKPPDTEELDMEVEN-MENIPLDPVSTRQ 623 grouper KMRPSFLEIISSIKDELDPPFREMSFFYSEENKPPDTEELGMEVEN-MENIPLDPASTRQ 514 turbot KMRPAFVEIISSLKDELEPSFKDSSFFYSADNKPVDDPQVHQDKMDSVDDVPLDPPSSTQ 1317 ****:*:*****:**:*:*.*:: ***** :*** * :: : : ::::**** *: * mullet PC-SAVLSPSGCAGGVLPPSTQQLSPMQGPSTPVLLGPMSPSPPGHVASALASPGQTLDK 682 grouper PS-AAAAQQLSPMQGPNTQALDKHSGHVSANGPVYCGPTLMR----CNPSRPSLGQALRT 569 turbot PQQSPVPQQTPPPPSSEAPPAPSLSPSSPSSPCTSTAAMDKQASGASGNGLSGPSHAAGS 1377 * :.. . .: . . . * ...... :: . mullet HSGHVSANGPVVVLRPNFDDMQPYAHMNG------711 grouper CFGQWARG----VLRPNFDEMQPYHAMNG------594 turbot GLG-TSAG---VAMCPSLEELPPYAHMNGGRKNERAMPLLQSSAC 1418 * : . .: *.:::: ** *** Figure 4. Partial deduced amino acid sequence of the mullet and grouper IGF-IR in comparison with turbot (AJ224993). Solid underlined letters identify the transmembrane domain dashed lines show the tyrosine kinase domain. Consensus symbols of: asterisk (*) means that the residues in the column are identical in all sequences in the alignment, colon (:) means that conserved substitutions have been observed, dot (.) means semi-conserved substitutions are observed, dash (-) indicates no corresponding amino acid and was used by the program (EBI) to introduce gaps in order to maximize homology comparison across species.

51

grouperbrain MSSALSFQWHLCDVFKSAMCCISCSHTLSLLLCVLTLTPTATGAGPETLCGAELVDTLRF 60 grouperliver MSSALSFQWHLCDVFKSAMCCISCSHTLSLLLCVLTLTPTATGAGPETLCGAELVDTLQF 60 mullet MSSAKSFQWHLCDVFKSAMCCISCSHTLSLLLCVLTLTPTATGAGPETLCGAELVDTLQF 60 rabbitfish MSSALSFQWHLCDVFKSAMCCISCSHTLSLLLCILTLTPTATGAGPETLCGAELVDTLQF 60 **** ****************************:************************:* grouperbrain VCGERGFYFSKPTGYGPNARRSRGIVDECCFQSCELRRLEMYCAPAKTSKAARSVRAQRH 120 grouperliver VCGERGFYFSKPTGYGPNVRRSRGIVDECCFQSCELRRLEMNCAPAKTSKAARSVRAQRH 120 mullet VCGDRGFYFSKPTGYGPNARRSRGIVDECCFQSCELRRLEMYCAPAKTNKSVRSVRSQRH 120 rabbitfish VCGERGFYFSKPTGYGPNSRRPRGIVDECCFQSCELRRLEMYCAPAKTSKAARSVRAQRH 120 ***:************** **.******************* ******.*:.****:*** grouperbrain TDMPRAPKVSTAGHKVDKGTERRTAQQPDKTKNKKRPLPGHSHSSFKEVHQKNSSRGNSG 180 grouperliver TDMPRAPKVSTAGHKVDKGTERRTAQQPDKTKNKKRPLPGHSHSSFKEVHQKNSSRGNTG 180 mullet TDMPRTPKVSTAGHKVDKGAERRTAQQPDKTKNKKRPISGHSHSSFKEVHQKNSSRGSTG 180 rabbitfish TDMPRTPKVSAAGQKVDKGTERRTAQQPDKTKSKKRPLSGHSHSSFKEVHQKNSSRGNTG 180 *****:****:**:*****:************.****:.******************.:* grouperbrain GRNYRM 186 grouperliver GRNYRM 186 mullet GTNYRM 186 rabbitfish GRNYRM 186 * **** Figure 5. Partial deduced amino acid sequence of grouper brain IGF-I in comparison with other fish species (NCBI accession number for the following: grouper liver- AAS01183, mullet-AY427954, rabbitfish-AY198184). Boxed letters shows aa difference between liver and brain in grouper.

grouperbrain METQQRYGHHSLCHTCRRTESSRMKKVKMSSSSRALLFALALTLYVVEIASAETLCGGEL 60 grouperliver METPQRYGHHSLCHTCRRTESSRMKVKKMSSSSRALLFALALTLYVVEIASAETLCGGEL 60 mullet METQQRYGHHTLCHTCRRTESSRMKVKKMSSSSRALLFALALTLYVVEMASAETLCGGEL 60 rabbitfish METQQRHGHHSLCHTCRRAESSRMKVRRMSASSRALLFALALTLYVVEIASAETLCGGEL 60 *** **:***:*******:****** :**:*****************:*********** grouperbrain VDALQFVCEDRGFYFSRPTSRGSNRRNQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDV 120 grouperliver VDALQFVCEDRGFYFSRPTSRGSNRRNQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDV 120 mullet VDALQFVCGDRGFYFSRPTSRGNNRRTQSSGIVEECCFRSCNLHLLEQYCAKPAKSERDV 120 rabbitfish VDALQFVCDDRGFYFSRPTSRGNSRRPQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDV 120 ******** *************..** *. ***********:*:**************** grouperbrain SATSLQVIPVMPALKPEVPRKPHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180 grouperliver SATSLQVIPVMPALKPEVPRKPHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180 mullet SATSLQVIPVMPALKQEITRKQHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180 rabbitfish SATSLQVIPVMPAPKPEVSRKPHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180 ************* * *:.** ************************************** grouperbrain KIKAQEQAVHHRPLITLP------198 grouperliver KIKAQEQAVFHRPLISLPSKLPPVLLATDNYVNHK 215 mullet KIKAQEQS------188 rabbitfish KIKAQEQAIFHRPLISLPSKLPPILLTTDNYVSHK 215 *******: Figure 6. Partial deduced amino acid sequence of grouper brain IGF-II in comparison with other fish species (NCBI Accession number for the following: grouper liver- AAS58520, mullet-AY427955, rabbitfish–AY198185). Boxed letters shows aa differencse between liver and brain in grouper.

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Table 2. Primer pairs used to amplify IGF-I, IGF-II, IGF-IR and ß-actin in mullet and grouper for QPCR

Gene Forward Primer Reverse Primer Expected size (bp) ( 5’ to 3’) ( 5’ to 3’) ß-actin ccacgagaccacctacaaca ctctggtggggcaatgat 181 mullet IGF-I agccacaccctctcaactact aagcagcactcgtccacaat 105 IGF-II ctgtgccaaacccgccaagt ctccgcctgcctccgaaact 215 IGF-IR ctccttccacccagcagtta ttggctgaaacatgtcctga 152 grouper IGF-I ctgtgcacctgccaagacta tgtgctgtcctacgctctgt 153 IGF-II aaatagcgtcggcagaga cctctgccacacctcgta 260 IGF-IR ttagcagaacagccttacca acatcctcatcagctcgaat 121

The sequence of the primer pairs yielded a single band when tested in regular PCR assay, confirming that they are not amplifying multiple transcripts. Amplification products of IGF-I, IGF-II and IGF-IR in mullet were 105 bp, 215 bp and 152 bp respectively. Amplified products of grouper IGF-I, IGF-II and IGF-IR were 153 bp, 260 bp and 121 bp respectively. The amplified product for ß-actin was 181 bp in both grouper and mullet. IGF-I, IGF-II and IGF-IR were designated as the target genes and ß-actin was used as the reference gene.

Reaction components Optimization assays for QPCR showed that for each gene, different ratios of the reaction components were required to eliminate formation of non-specific products during amplification. The reaction components for a 25 μL master mix for the different genes were shown in Table 3. These components were used in assays using serial dilutions of the respective mullet and grouper calibrator cDNA for the construction of standard curves. Reaction efficiencies of the target and reference genes based on the standard curves are summarized in Table 4. Reaction efficiencies for mullet IGF-I, IGF-II and IGF-IR were 0.91, 0.94 and 0.99 and the reference gene was 0.99. Except for IGF-IR the efficiencies of the other two genes compared with the reference genes were not the same. In grouper, the reaction efficiency for the reference gene was 0.97 while values for IGF-I, IGF-II and IGF-IR were 0.91, 0.94 and 0.98 respectively.

53

Table 3. Master mix of reaction components for QPCR (per 25 µL reaction) Components Genes amplified ß-actin IGF-I IGF-II IGF-IR (µL) (µL) (µL) (µL) a) mullet RNAse free water 6.5 6.5 5.5 6.5 Platinum SybrGeen QPCR supermix UDG* 12.5 12.5 12.5 12.5 MgCl (50 mM) - - 1.0 - Forward primer (10µmole) 0.5 0.5 0.5 0.5 Reverse primer (10µmole) 0.5 0.5 0.5 0.5 Template 5.0 5.0 5.0 5.0 b) grouper RNAse free water 6.5 7.58 7.58 6.5 Platinum SybrGeen QPCR supermix UDG * 12.5 10.42 10.42 12.5 Forward primer (10µmole) 0.5 1.0 1.0 0.5 Reverse primer (10µmole) 0.5 1.0 1.0 0.5 Template 5 5.0 5.0 5.0 *Platinum® SybrGeen® QPCR supermix UDG (Invitrogen, USA) contains the following: SybrGeen I, 60 u/mL Platinum Taq DNA polymerase, 40 mM Tris-HCl (pH 8.4), 100 mM KCl, mM MgCl2, 400 uM dGTP, 400 uM dATP, 400 uM dCTP, 400 uM dUTP, 40 U/mL UDG, and stabilizers cDNA template contains 62.5 ng starting RNA

Table 4. Reaction efficiency and correlation coefficient (r2) from the constructed standard curves of different genes in mullet and grouper gonad

Gene PCR r2 Efficiency Mullet B-actin 2.08 0.99 IGF-I 2.18 0.91 IGF-II 1.98 0.94 I IGF-IR 1.94 0.99

Grouper B-actin 1.94 0.97 IGF-I 2.13 0.91 IGF-II 1.94 0.94 IGF-IR 1.95 0.98

Samples analyzed 54

Egg stages were classified following Liu and Kelly (1991) from samples collected and preserved in 10% buffered formalin. Egg samples that showed uniform stages were presumed to come from a single female and samples that showed more than one stage of development were eliminated for further analyses (see Appendix 4). This was to ensure that only the expression of a particular stage was being analyzed.

Grouper samples were divided into three groups; sinking eggs, late embryo and hatched larvae. Mullet egg samples were grouped into sinking eggs, multicell, blastula, gastrula, melanocyte formation, late embryo and hatched larvae. Photomicrographs of the different samples analysed for QPCR are shown in Figures 7 and 8.

sinking eggs late embryo

hatched larvae

Figure 7. Photomicrograph of grouper samples

55

sinking eggs multicell stage

blastula gastrula

melanocyte formation late embryo

hatched larvae

Figure 8. Photomicrograph of mullet eggs 56

Data calculation Reaction efficiency between the reference and the target genes were different so the Pfaffl method was used to calculate the relative expression ratio of the three genes. The mean Ct values of the target and reference genes were taken at a set threshold line of 0.0524 for all samples analyzed. The threshold line was set at the point where the fluorescence signal was higher than the level of the background fluorescence and greatest amplification was determined. The final data were expressed as a ratio of the target gene normalized to the reference gene in the eggs relative to the gonads that was designated as the calibrator. The mean relative expression ratio of the three target genes in sinking eggs, late embryo and hatched larvae of grouper were grouped into high or low FR and medium or high HR as shown in Table 5a. The mean relative expression ratio of the three target genes in sinking eggs, hatched larvae and five stages of spawned eggs from mullet are summarized in Table 5b and 5c.

Table5a. Mean relative expression ratio of the three target genes in different grouper samples Mean relative expression ratio Samples IGF-I IGF-II IGF-IR Low FR sinking eggs 68.34 ± 65.6 22.54 ± 9.8 28.07 ± 22.6 late embryo 1.33 ± 1.1 15.3 ± 3.8 2.48 ± 1.4 Hatched larvae 0.66 ± 0.4 2.60 ± 1.4 1.07 ± 1.4

High FR sinking eggs 7.39 ± 0.4 77.75 ± 60.3 7.36 ± 1.0 late embryo 1.04 ± 0.5 22.4 ± 7.9 1.56 ± 1.0 Hatched larvae 0.29 ± 0.1 7.69 ± 2.7 0.63 ± 0.1

Medium HR sinking eggs 69.6 ± 69.9 25.66 ± 8.9 30.31 ± 21.5 late embryo 1.32 ± 1.1 14.31 ± 4.5 2.22 ± 1.6 hatched larvae 0.27 ± 0.2 6.71 ± 2.9 0.39 ± 0.0

High HR sinking eggs 6.07 ± 1.8 76.67 ± 21.5 4.97 ± 0.8 late embryo 0.26 ± 0.0 16.29 ± 0.2 0.77 ± 0.2 hatched larvae 0.73 ± 0.3 5.93 ± 0.7 1.36 ± 0.9 (Low FR = <60% High FR = 68-82%, High HR= 95-99%, Medium HR=85-90%) FR – fertilization rate HR – hatching rate

57

Table 5b. Mean relative expression ratio of the three target genes in mullet Group A samples. Relative expression ratio Samples IGF-I IGF-II IGF-IR

Sinking eggs Multicell 0.37 0.26 4.02 Blastula 0.32 0.14 2.82 Gastrula 0.13 138.10 1.36 Melanocyte formation 1.47 32.58 0.37 Late embryo 3.11 24.73 0.41 Hatched larvae 9.01 12.81 0.51

Table5c. Mean relative expression ratio of the three target genes in mullet Group D samples. Relative expression ratio Samples IGF-I IGF-II IGF-IR

Sinking eggs Multicell 0.25 0.13 4.25 Blastula - 7.83 2.74 Gastrula - 167.16 2.11 Melanocyte formation 0.28 7.43 0.16 Late embryo 4.8 6.57 0.19 Hatched larvae 8.7 15.79 4.14

Statistical Analysis Relative expression ratio of IGF-I, IGF-II and IGF-IR in grouper samples was analyzed by ANOVA using Gen Stat ver. 6. The data were analyzed twice, first based on the fertilization rate and second based on the hatching rate. To obtain normal distribution, raw data were log transformed before analysis. The summary of the ANOVAs based on fertilization rate and hatching rate are shown in Tables 6a and 6b, respectively. Degrees of freedom (d.f.) describes the number of values in the final calculation of an estimate of a statistic that are free to vary, sum of square (s.s.) refers to an interim quantity used in the calculation of an estimate of the population variance, mean of squares (m.s.) refers to an estimate of the population variance based on variability among a given set of measures, variance ratio (v.r.) characterizes the dispersion among the measures in a given population, and F pr shows the statistical significance of the differences obtained among the obtained means of samples from a given population as compared to the table of F values (http://www.animatedsoftware.com). Relative expression levels of IGF-I, 58

IGF-II and IGF-IR in grouper eggs showed no significant interaction effect between the three genes, the samples analyzed (sinking eggs, late embryo and hatched larvae), fertilization or hatching rate. Genes and stage appear as dominant and have a significant main effect (P<0.01) with no evidence of significant interaction. The relative expression ratio of all three genes is significantly higher in sinking eggs than in late embryo or hatched larvae. IGF-II is significantly higher than IGF-I or IGF-IR in all samples examined. There is a suggestive interaction between fertilization rate and samples as well as between gene and samples.

Table 6a. ANOVA of interaction effect between fertilization rate, gene and samples in grouper source of variation d.f. s.s. m.s. v.r. F pr FR 1 3.308 3.308 2.85 0.099 Gene 2 78.465 39.232 33.82 <0.001 Stage 2 68.573 34.286 29.56 <0.001 FR*gene 2 7.720 3.860 3.33 0.046 FR*samples 2 1.951 0.976 0.84 0.439 Gene*samples 4 13.789 3.447 2.97 0.030 FR*gene*samples 4 9.137 2.284 1.97 0.117 Residual 41 47.562 1.160 Total 58 198.052 coefficient of variation = 124.1% d.f. - degrees of freedom; m.s. – mean of squares; s.s. – sum of squares; v.r. – variance ratio; F pr – significance level

Table 6b. ANOVA of interaction effect between hatching rate, gene and samples in grouper

source of variation d.f. s.s. m.s. v.r. F pr HR 1 1.0 1.0 0.62 0.439 Gene 2 48.124 24.062 14.82 <0.001 Samples 2 75.031 37.515 23.10 <0.001 HR*gene 2 0.070 0.035 0.02 0.979 HR*samples 2 2.253 1.127 0.69 0.507 Gene*samples 4 7.146 1.787 1.10 0.374 HR*gene*samples 4 10.528 2.632 1.62 0.194 Residual 31 50.340 1.624 Total 48 184.176 coefficient of variation = 127.9%

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Summary of ANOVA for mullet data is shown in Table 7. Analysis of the relative expression ratio showed a highly significant interaction effect (P<0.01) between gene and stage. Genes were differentially expressed in samples that were analyzed as shown in Table 8. Expression of all the genes was detected in the sinking eggs. Levels of IGF- I decreased from multicell stage to melanocyte formation and increased in late embryo and hatched larvae. IGF-II was significantly higher in sinking eggs than in the multicell stage and the highest level was seen in gastrula stage compared to all other stages and samples analyzed. IGF-IR is highest in sinking eggs and decreased from multicell stage to late embryo. A significant increase was seen in hatched larvae compared to late embryo. Of the three genes, IGF-II was significantly higher in sinking eggs, gastrula, melanocyte formation late embryo and hatched larvae while IGF-IR was the highest in multicell and blastula stage. Table 7. ANOVA of interaction effect between samples and gene expression in mullet eggs source of variation d.f. s.s. m.s. v.r. F pr Gene 2 45.550 22.775 17.58 <0.001 Samples 6 24.985 4.164 3.21 0.024 Gene*samples 11 77.725 7.066 5.45 <0.001 Residual 19 24.611 1.295 Total 38 142.933 coefficient of variation= 151.9%

Table 8. Log means of relative expression ratio in mullet. Means with different superscripts are significantly different (P<0.05). Back transformed data was calculated following Kendall et al. (1983)

Equivalent relative expression Log means ratio (back transformed) IGF-I IGF-II IGF-IR IGF-I IGF-II IGF-IR sinking eggs -0.48abcd 2.71g 1.92efg 0.95 23.14 10.50 multicell -1.58ab -1.35abc 1.39defg 0.28 0.35 5.55 Blastula -1.14abc 0.05abcde 1.02cdefg 0.44 1.45 3.83 Gastrula -2.04a 5.02h 0.53bcdef 0.18 209.22 2.35 melanocyte formation -0.44abcd 2.74fgh -1.41ab 0.89 21.40 0.34 late embryo 1.35defg 2.55fg -1.28abc 5.33 17.70 0.38 hatched larvae 2.18efg 2.65fgh 0.37bcdef 12.22 19.56 2.0

Vitellogenin (Vtg) purification and ELISA in mullet

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Mullet Vtg was purified and Vtg ELISA developed. The developed ELISA protocol was used to determine Vtg concentration in oocytes and egg homogenates of mullet.

Collected plasma isolated from blood of male mullets that were injected with estradiol to induce Vtg production, was pooled. Plasma of treated male males showed a very intense band of molecular weight approximately 220 kD after SDS-PAGE that was absent in the control confirming the presence of Vtg (Figure 9). Purification of Vtg was carried out by anion exchange chromatography and the elution profile of the plasma is shown in Fig. 10. The purified Vtg collected at peak fractions had a concentration of 275µg/mL. Purified samples were aliquoted into 1 mL tubes and stored at 80oC until use.

Antigen-antibody dilution was optimized in an indirect ELISA. Figure 11 shows the standard curve constructed using optimum antigen-antibody dilution. The established standard curve has a quantification range of 5 to 50 ng/mL Vtg.

For the ELISA of mullet oocytes and spawned eggs, all samples came from tank-reared mullet that were hormonally induced to spawn and were monitored. Group A and B were samples obtained by gonadal biopsy, while Group C and D eggs collected after spawning, incubated in the hatching tank and were monitored for hatching. Mean Vtg level of homogenates from Group A oocytes (7.84 mg/g sample) was higher compared to 3.62, 5.44 and 1.82 mg/g sample in Group B, C and D respectively (Table 9). Statistical analysis of group means showed no significant difference between the four groups, as shown on Table 10. 61

1 2 3 4 5 6 7 8 9

Figure 9. Vitellogenin (shown by the arrow) in plasma of mullet after induction with estradiol. Lane 5: MW markers; Lanes 1 and 6 - untreated males showing the absence of Vtg; lanes 2, 3, 4 and 9 - males treated with estradiol showing an intense Vtg band; lane 8 – untreated female showing a minor Vtg band. 62

Figure 10. Elution profile of mullet plasma during purification, purified Vtg were collected at peak fractions 22 to 24

1.20 y = 0.0191x + 0.0595 R2 = 0.9792 1.00

0.80

0.60

0.40

absorbance (600 nm) 0.20

0.00 0 102030405060 protein concentration (ng/ml)

Figure 11. Standard curve for developed mullet ELISA

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Table 9. Group means of Vtg concentration in mullet sample homogenates

Samples Vtg level μg/mg sample (mean) Oocytes, subsequently spawned and hatched (Group A, n=2) 7.840 Oocytes, female not spawned (Group B, n=7) 3.616 Eggs, stripped, artificially fertilized, no hatching (Group C, n=2) 5.440 Eggs, females subsequently spawned , no hatching (Group D, n=2) 1.825

Table 10. ANOVA of Vtg concentration in mullet egg homogenates source of variation d.f. s.s. m.s. v.r. F pr Group 3 43.180 14.393 2.94 0.092 Residual 9 44.117 4.902 Total 12 87.297 coefficient of variation= 51.8 %

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Chapter 5. Discussion and Conclusion

Gene cloning and QPCR

This study cloned and sequenced partial IGF-IR in mullet and IGF-I, IGF-II and IGF-IR in grouper. The relative expression levels of IGF-I, IGF-II and IGF-IR were determined and analyzed during embryonic development in mullet and grouper.

A high sequence homology between the putative coding regions of the IGFs exists across fish species and the degenerate primers designed according to the regions of high homology were successful in isolating IGF-I, IGF-II and IGF-IR in both grouper and mullet. Only one form of IGF-IR fragment was isolated in mullet and grouper, consistent with the findings of one distinct type of IGF-IR in turbot, trout (Elies et al., 1996) and barramundi (Drakenberg et al., 1997). This pattern differs from the situation in salmon (Chan et al., 1997), rainbow trout (Greene and Chen, 1999), zebrafish (Maures et al., 2002), Japanese flounder (Nakao et al., 2002) and gilthead seabream (Perrot et al., 2000) where two IGF-IR forms were isolated. It still remains to be determined if this pattern reflects a genuine differences between species or whether a second IGF-IR is yet to be identified in the species that so far revealed only one form. A single form of both IGF-I and IGF-II was cloned from grouper while several studies on teleosts reported multiple forms. Five forms were reported for IGF-I in goldfish (Kermouni et al., 1998), four in rainbow trout (Greene and Chen, 1997) and tilapia (Schmid et al., 1999), and two in gilthead seabream (Perrot et al., 2000). Only one form of IGF-II was reported in daddy sculpin (Loffing-Cueni et al., 1999), rainbow trout (Greene and Chen, 1997), rabbitfish (Ayson et al., 2002) and chum salmon (Palamarchuk et al., 2002) while three forms have been isolated in tilapia (Schmid et al., 1999). The occurrence of multiple forms of IGFs in different species was either due to alternative RNA splicing of sequence domains (Duguay et al., 1992) or as products of different or distinct genes (Greene and Chen, 1999; Nakao et al., 2002). The different forms that exhibited temporal expression patterns probably made distinct functional contributions to growth regulation and development (Maures et al., 2002). Comparison of IGF-I and IGF-II sequences in grouper brain from this study and grouper liver submitted to Gene Bank by a group of F. 65

Shi from Guangdong, China (NCBI Accession nos. AAS01183 and AAS58520) showed a sequence identity of 97% due to minor amino acid changes. These differences could be attributed to different tissue source, variations in locality or simply PCR sequencing error. It is likely to be due to different forms as the different forms of IGF-I in goldfish ovary showed 28 aa mismatches in the coding sequence between them (Kermouni et al., 1998) while IGF-I forms in rainbow trout showed different size of cDNA bands in the agarose gel.

With the development of real-time chemistry in QPCR, quantification of gene expression has proven to be relatively easy to perform, produces high throughput with high sensitivity and reliable specificity (Bustin, 2002). From the obtained IGF-I, IGF-II and IGF-IR cDNA sequences, primers were designed to amplify these genes for QPCR assays in sinking eggs, during embryonic development and in hatched larvae from grouper and mullet. ß-actin was present and expressed at similar levels in each of the samples analyzed and was chosen as the housekeeping gene for the QPCR assay. The use of ß-actin as a housekeeping gene has been verified in other studies on teleosts (Dyer et al., 2001; Caipang et al., 2003; Caelers et al., 2004; Lam et al., 2004; Raine et al., 2004). The relative expression ratio of IGF-I, IGF-II and IGF-IR was calculated using the Pfaffl method (Pfaffl, 2001).

Expression levels of the growth factors IGF-I, IGF-II and IGF-IR have been determined to play an important physiological role in animal growth, especially during embryonic development (Jones and Clemmons, 1995; Florini et al., 1996; Accili et al., 1998; review by Le Roith et al., 2001). Studies in teleosts have confirmed their conserved role during evolution (reviews by Peter and Marchant, 1995, Duan, 1997, and Moriyama et al., 2000). The presence of IGF-I, IGF-II and IGF-IR mRNA transcripts in oocytes and embryos, stages when the endocrine organs are not yet functional, indicates these growth factors are maternally inherited and points to their functional role in early development (Greene and Chen, 1997; Perrot et al., 1999).

The relative expression ratios of IGF-I, IGF-II and IGF-IR were determined in three groups of grouper samples: sinking eggs, late embryo and hatched larvae. Expression 66 levels of the three genes were significantly high in sinking eggs compared to late embryo or hatched larvae. Sinking eggs were presumed to be unfertilized and detection of IGF-I, IGF-II and IGF-IR transcripts in these samples suggested they were maternally inherited (Greene and Chen, 1997, 1999; Perrot et al., 1999). The significantly high level of IGF-II expression compared to IGF-I or IGF-IR in the three samples analyzed supported the concept that IGF-II represents the fetal growth factor (Humbel, 1990).

To relate the expression levels of IGF-I, IGF-II and IGF-IR to egg quality using fertilization and hatching rate as physical determinants, grouper samples were classified into low or high FR and medium or high HR. Statistical analysis of the relative expression ratio in grouper samples showed no significant interaction effect among the genes, samples and fertilization or hatching rate. In rainbow trout, IGF-I and IGF-II mRNA in oocytes showed significantly high expression levels that were correlated to high embryonic survival (Aegerter et al, 2003). Maternally inherited mRNA is selectively degraded during embryonic development concurrent with an increase in newly transcribed mRNA from the embryonic genome (Telford et al., 1990). Consideration of the temporal expressions of the target genes could determine at what stage of development - a sensitive correlation to egg quality can be established.

In mullet, the samples analyzed were sinking eggs, multicell stage, blastula, gastrula, melanocyte formation, late embryo and hatched larvae. These all came from high FR and high HR batches. IGF-II transcript levels were significantly higher than those of IGF-I or IGF-IR in sinking eggs, melanocyte formation, late embryo, hatched larvae and highest expression was seen in the gastrula stage. Previous findings in rainbow trout and rabbit fish also showed that IGF-II was strongly expressed relative to IGF-I during embryonic development (Greene and Chen, 1999; Ayson et al, 2002). The highest level of IGF-II in gastrula may signify the importance of its role at this stage of embryo development, as this is the stage when the axial relationship of the mature fish body plan is being established. A negative correlation pattern between IGF-I and IGF-IR expression level was observed, where IGF-I transcript levels increased from multicell stage to hatched larvae while IGF-IR transcript levels decreased from multicell stage to late embryo. The levels of IGF receptors have been shown to be down-regulated by 67 increasing concentrations of exogenous IGFs (Florini et al., 1996). The increasing level of IGF-I transcripts was also seen during the embryonic development in gilthead seabream while the level of IGF-IR remained unchanged (Perrot et al., 1999). This pattern was in contrast to that observed in zebrafish embryos where IGF-I levels were relatively unchanged while the two forms of IGF-IR showed different temporal expression patterns (Maures et al., 2002).

The identification of a strong relative expression level of IGF-II compared to IGF-I and IGF-IR makes it a potential marker in further studies during embryonic development in fish. Examination of the IGF-II expression levels in oocytes or during the gastrula stage in a larger number of samples exhibiting different fertilization and hatching performance could determine if indeed IGF-II expression levels provides a reliable criterion in distinguishing between good and poor quality eggs. Furthermore, the sensitivity of the established QPCR assay to detect even low mRNA expression levels could be used in other studies that need to determine gene expression levels such when confirming micro- array results.

Vtg ELISA To determine whether Vtg could be used as a maker for egg quality in mullet, this study purified Vtg and developed an ELISA to measure Vtg concentration in mullet sample homogenates.

High production of Vtg in male mullet was induced by estradiol administration. This technique had been conducted successfully with other male teleosts as Vtg production in liver is under estradiol regulation (Matsubara et al., 1994; Chang et al., 1996; Roubal et al., 1997; Mosconi et al., 1998; Heppel et al., 1999; Heppel and Sullivan, 1999; Sherry et al., 1999; Scholz et al., 2004). The successful purification of Vtg from the collected plasma using anion exchange chromatography followed an established protocol that was used in other teleost species (Parks et al., 1999; Heppel and Sullivan, 1999). The ELISA results demonstrated that oocytes from a female that subsequently had successful hatching had a higher amount of Vtg relative to oocytes and spawned eggs from females that did not show embryonic development and hatching. The high level of Vtg in 68 oocytes that were viable indicates the importance of this protein for successful embryonic development. ELISA was also used to measure egg protein quantity in oyster eggs and showed that it was sensitive enough to measure fecundity of female oysters collected even during the non-reproductive season (Kang et al., 2003), however to date no other publications are available to address possible correlation of Vtg levels to egg quality.

Overall, this study has developed tools to address a commercially important question – can we identify molecular markers to assess egg quality? While the results are still preliminary, it has been identified that both IGF-II transcript levels and Vtg levels in the oocytes are worth pursuing in further studies. The molecular tools for detecting IGF-I, IGF-II and IGF-IR transcripts can be used for other applications in research into fish biology, and the the production of Vtg as a response to endocrine disrupting compounds in waterways (Sherry et al., 1999; Marin and Matozzo, 2004) could be measured with the developed ELISA.

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Appendix 1

Poster presented to the 6th International Marine Biotechnology Conference 21-27 September 2003, Chiba, Japan

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Appendix 2 Spawning protocol for mullet

1. The administration of reproductive hormones has been used to induce oocyte maturation and spawning of mullet in captivity. The following are the hormone implants used during this study: a) Luteinizing hormone releasing hormone analog (LHRHa) in cholesterol-cellulose delivery system - LHRHa pellets were prepared using the following formulation: 1 mg LHRH (des-gly14,D trp 6, Pep Tech Animal Health DF D27, Australia) dissolved in 0.4 ml 80% ethanol. Solution was mixed thoroughly with 680 mg cholesterol and 120 mg cellulose and pressed in molder to form pellets. Computed final concentration of LHRHa in the pellet was 1.25 ug/mg of mix. b) LHRHa in saline solution - LHRHa (des-gly14,D trp 6); 1 mg was dissolved in saline solution to a final concentration of 200 ug/ml. c) Gonadotropin-releasing hormone (GnRH) in ethylene and vinyl acetate (EVAC) – GnRHa pellets at a concentration of 40 ug/pellet were a kind gift of Dr. Hanna Rosenfeld (Israel Oceanographic and Limnological Research Station, Eilat, Israel). d) Hypophysation – collected pituitaries from mature wild caught female were grounded to a 1 ml of saline solution just before administration at the rate of 1 pituitary for < 1 kg of spawner and 2 pituitaries to >1 kg female spawner.

2. Oocytes from female mullet with body weight of >800 g were sampled by the use of a 5 mm diameter pipelle. Females with mean oocyte diameter of > 500 uM were selected for induced spawning. Mature males with running milt were selected by gently squeezing the abdomen. Identified spawners were transferred to spawning tanks at the ratio of 3:1 (9 males, 3 females) male to female ratio.

3. Females were injected with either LHRHa pellet at a concentration of 50 ug/kg fish on the first day and a second dose of LHRH in saline solution at 50 ug/kg fish on the next day if germinal vesicle breakdown was not observed in sampled oocytes. For females receiving GnRHa in EVAC, one pellet per fish was administered once. Females 71 receiving pituitary extracts were given a dose of LHRH in saline solution (50 ug/kg fish) on the following day.

4. Fifty percent of the males in the spawning tank were injected with 10 ug/kg of LHRHa in saline solution on the same day that the females receive their first hormone implants. 72

Appendix 3 Pfaffl formula

∆CP (control-sample) R= Etarget target ∆CP (control-sample) Ereference ref

where: R = relative expression ratio CP = crossing point (or Ct/crossing threshold using the Corbett thermal cycler) ∆CPtarget (control-sample) Etarget = efficiency for gene of interest to the power of delta CP target

∆CPref (control-sample) Ereference = efficiency for calibrator gene to the power of delta CP calibrator

∆ CP ref = mean CP of reference gene in calibrator – mean CP of gene of interest in calibrator

∆ CP target = mean CP of target gene in sample – mean CP of target gene in sample

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Appendix 4 Egg stages collected from grouper and mullet spawns a) Grouper Batch Stages 1 2 3 4 5 6 7 Sinking eggs √ √ √ √ √ √ 2-cell √ 16 cell √ Blastula √ Early germ ring √ √ Gastrula √ Early embryo √ Somite √ √ Late embryo √ √ √ √ √ √ Newly hatched larvae √ √ √ √ √ √ b) Mullet Batch Stages 1 2 Sinking eggs √ √ Multicell √ √ Blastula √ √ Early germ ring √ Gastrula √ √ Early embryo √ Somite √ Melanocyte formation √ √ Late embryo √ √ Newly hatched larvae √ √

√ - sample available } – two stages in one sample words highlighted in red represents the samples analyzed for QPCR

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Appendix 5 Primer sequences designed for IGF-IR Amino acids in red font, sequence name indicated below it, forward arrow indicates forward primer, reverse arrow indicates reverse primer mullet ------HNYCS 5 grouper ------turbot RTSPSAPSMPQDVRAYSNSSTQLVVRWSPPVSPNGNQTYYLVRWQQQAEDRELYQHNYCS 660 igf1r655-661f mullet KELKIPIRVAAIGVGDQE------SEEDQKAEADDASYRKVFE 42 grouper ------turbot KELKIPIRIAAIGVGDQEEDTKPTKPDPEGADKGPCCPCPKSVEDLEAEAADASYRKVFE 720 mullet NFLHNSIFT------LFGVANATNSRRHRLHANSSVIPPLQAG--NGSA--ADLEP 88 grouper ------turbot NFLHNSIFTPRPPDRRRRDLFGFANSTHSRRHRLHTNSSHVPPHQAAGNNSSSTTAEPEQ 780 mullet ADRKFDFMEQSVTERELQISGLKPFTVYRIDIHACNRQVQRCSAAEFVFSRTKPAEKSDD 148 grouper ------PFTVYRIDIHACNRQVQRCSAAEFVFSRTKPAEKADD 37 turbot ADREFEFMEQAVSER-VQIFDLQPFTVYRIDIHACNRQVQRCSAAEFVFSRTKPAEKADD 839 igf1r802-808f igf1r825-833f

mullet IPGKVTWEGHEDWVFLRWPEPRHPNGLILMYEIKFKLATETEKHECVSGQTYQAQRGVRL 208 grouper IPGPVTWEGHEDWVFLRWPEPPHPNGLILMYEIKFKLAAETEKHECVSGQMYHTQRGVRL 97 turbot IPGQVTWEGHEDWVFLRWPEPPHPNRLVLMYEIKFKLAAETEKHECVSGQTYQNQRGVRL 899 mullet SNLSPGNYSVRVRATSLAGNGSWTQSSDFYVAERDENILYAMIFIPVAIIVLICCLAVML 268 grouper SNLSPGNYSVRVRATSLAGNGSWTHALDLYVAERYENVLYAMIFVPIVIILVICLLVSML 157 turbot SNLSPGHYSVRVRATSLAGNGSWTNAVDLYVAERYENVLYAMIFIPIAIILFICLLVTML 959 mullet VFFNRKRNSDRLGNGVLYASVNPEYFSAAEMYVPDEWEVAREKITLSRELGQGSFGMVYE 328 grouper VVLSRKRNSDRLGNGVLYASVNPEYFSAAEMYVPDEWEVAREKITLSRELGQGSFGMVYE 217 turbot VVLNKKRNSDRLGNGVLYASVNPEYFSAAEMYVPDEWEVAREKIALSRELGQGSFGMVYE 1019 mullet GVAKGVVKDEPEMRVAIQTVNESASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQP- 387 grouper GLAKGVVKDEPETRVAIKTVNESASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQPP 277 turbot GLAKGVVKDEPETRVAIKTVNESASMRERIEFLNEASVMKEFNCHHVVRLLGVVSQGQP- 1078 igf1r5-0R igf1r 1034-1039f igf1r51-56F igf1r6-1R

mullet TLVIMELMTRGDLKSHLRSLR--KENSTTQVLPPLKKMIQMAGEIADGMAYLNANKFVHR 445 grouper TLVIMELMTRGDLKSYLRSLR--KENATSQVLPPLKKMIQMAGEIADSMSYLNANKFVHR 335 turbot TLVIMELMTRGDLKSYLRSLRPKEQQWSSLSLPPLRKMLQMAGQIADGMAYLNANKFVHR 1138 igf1r93-99f mullet DLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTMSD 505 grouper DLAARNCMVAEDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTMSD 395 turbot DLAARNCMVADDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMSPESLKDGVFTTHSD 1198 igf1r 1186-1181r mullet VWSFGVVLWEIAT-LAEQPYQGMSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNP 564 grouper VWSFGVVLWEIATLLAEQPYQGMSNEQVLRFVMEGGLLDKPDNCPDMLFELMRMCWQYNP 455 turbot VWSFGVVLWEIST-LAEQPYQGLSNEQVVRFVMEGGLLEKPQNCPDMLFELMRMCWQFNP 1257 igf1r 1258-1253R mullet KMRPSFLEIISSIKDDLDPPFREMSFFYSEENKPPDTEELDMEVEN-MENIPLDPVSTRQ 623 grouper KMRPSFLEIISSIKDELDPPFREMSFFYSEENKPPDTEELGMEVEN-MENIPLDPASTRQ 514 turbot KMRPAFVEIISSLKDELEPSFKDSSFFYSADNKPVDDPQVHQDKMDSVDDVPLDPPSSTQ 1317 mullet PC-SAVLSPSGCAGGVLPPSTQQLSPMQGPSTPVLLGPMSPSPPGHVASALASPGQTLDK 682 grouper PS-AAAAQQLSPMQGPNTQALDKHSGHVSANGPVYCGPTLMR----CNPSRPSLGQALRT 569 turbot PQQSPVPQQTPPPPSSEAPPAPSLSPSSPSSPCTSTAAMDKQASGASGNGLSGPSHAAGS 1377 mullet HSGHVSANGPVVVLRPNFDDMQPYAHMNG------711 grouper CFGQWARG----VLRPNFDEMQPYHAMNG------594 turbot GLG-TSAG---VAMCPSLEELPPYAHMNGGRKNERAMPLLQSSAC 1418 igf1r 1400-1395r

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Appendix 6 Primer sequences designed for IGF-I Amino acids in red font, sequence name indicated below it, forward arrow indicates forward primer, reverse arrow indicates reverse primer

grouperbrain MSSALSFQWHLCDVFKSAMCCISCSHTLSLLLCVLTLTPTATGAGPETLCGAELVDTLRF 60 grouperliver MSSALSFQWHLCDVFKSAMCCISCSHTLSLLLCVLTLTPTATGAGPETLCGAELVDTLQF 60 mullet MSSAKSFQWHLCDVFKSAMCCISCSHTLSLLLCVLTLTPTATGAGPETLCGAELVDTLQF 60 rabbitfish gcggagacccgtggggMSSALSFQWHLCDVFKSAMCCISCSHTLSLLLCILTLTPTATGAGPETLCGAELVDTLQF 60 igf1 -5 to 3f grouperbrain VCGERGFYFSKPTGYGPNARRSRGIVDECCFQSCELRRLEMYCAPAKTSKAARSVRAQRH 120 grouperliver VCGERGFYFSKPTGYGPNVRRSRGIVDECCFQSCELRRLEMNCAPAKTSKAARSVRAQRH 120 mullet VCGDRGFYFSKPTGYGPNARRSRGIVDECCFQSCELRRLEMYCAPAKTNKSVRSVRSQRH 120 rabbitfish VCGERGFYFSKPTGYGPNSRRPRGIVDECCFQSCELRRLEMYCAPAKTSKAARSVRAQRH 120 grouperbrain TDMPRAPKVSTAGHKVDKGTERRTAQQPDKTKNKKRPLPGHSHSSFKEVHQKNSSRGNSG 180 grouperliver TDMPRAPKVSTAGHKVDKGTERRTAQQPDKTKNKKRPLPGHSHSSFKEVHQKNSSRGNTG 180 mullet TDMPRTPKVSTAGHKVDKGAERRTAQQPDKTKNKKRPISGHSHSSFKEVHQKNSSRGSTG 180 rabbitfish TDMPRTPKVSAAGQKVDKGTERRTAQQPDKTKSKKRPLSGHSHSSFKEVHQKNSSRGNTG 180 igf1 182-174 rd grouperbrain GRNYRM 186 grouperliver GRNYRM 186 mullet GTNYRM 186 rabbitfish GRNYRM 186

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Appendix 7

Primer sequences designed for IGF-II Amino acids in red font, sequence name indicated below it, forward arrow indicates forward primer, reverse arrow indicates reverse primer

grouperbrain METQQRYGHHSLCHTCRRTESSRMKKVKMSSSSRALLFALALTLYVVEIASAETLCGGEL 60 grouperliver METPQRYGHHSLCHTCRRTESSRMKVKKMSSSSRALLFALALTLYVVEIASAETLCGGEL 60 mullet METQQRYGHHTLCHTCRRTESSRMKVKKMSSSSRALLFALALTLYVVEMASAETLCGGEL 60 rabbitfish METQQRHGHHSLCHTCRRAESSRMKVRRMSASSRALLFALALTLYVVEIASAETLCGGEL 60 igf2 1-8f

grouperbrain VDALQFVCEDRGFYFSRPTSRGSNRRNQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDV 120 grouperliver VDALQFVCEDRGFYFSRPTSRGSNRRNQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDV 120 mullet VDALQFVCGDRGFYFSRPTSRGNNRRTQSSGIVEECCFRSCNLHLLEQYCAKPAKSERDV 120 rabbitfish VDALQFVCDDRGFYFSRPTSRGNSRRPQNRGIVEECCFRSCDLNLLEQYCAKPAKSERDV 120

grouperbrain SATSLQVIPVMPALKPEVPRKPHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180 grouperliver SATSLQVIPVMPALKPEVPRKPHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180 mullet SATSLQVIPVMPALKQEITRKQHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180 rabbitfish SATSLQVIPVMPAPKPEVSRKPHVTVKYSKYEVWQRKAAQRLRRGVPAILRAKKFRRQAE 180

grouperbrain KIKAQEQAVHHRPLITLP------198 grouperliver KIKAQEQAVFHRPLISLPSKLPPVLLATDNYVNHK 215 mullet KIKAQEQS------188 rabbitfish KIKAQEQAIFHRPLISLPSKLPPILLTTDNYVSHK 215 igf2 199-190r

77

Appendix 8. Reprinted Genbank entry for E. coioides IGF-I cDNA sequence.

1: AY776159. Reports Epinephelus coioi...[gi:54399797] Links

LOCUS AY776159 558 bp mRNA linear VRT 26-OCT-2004 DEFINITION Epinephelus coioides insulin-like growth factor I (IGF-I) mRNA, partial cds. ACCESSION AY776159 VERSION AY776159.1 GI:54399797 KEYWORDS . SOURCE Epinephelus coioides (orange-spotted grouper) ORGANISM Epinephelus coioides Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Actinopterygii; Neopterygii; Teleostei; Euteleostei; Neoteleostei; Acanthomorpha; Acanthopterygii; Percomorpha; Perciformes; Percoidei; Serranidae; Epinephelinae; Epinephelus. REFERENCE 1 (bases 1 to 558) AUTHORS Bangcaya,J., Nocillado,J., Anderson,A., Richardson,N., Rimmer,M., Thaggard,H. and Elizur,A. TITLE IGF-II mRNA as a possible molecular marker for egg quality in grouper and mullet JOURNAL Unpublished REFERENCE 2 (bases 1 to 558) AUTHORS Bangcaya,J., Nocillado,J., Anderson,A. and Elizur,A. TITLE Direct Submission JOURNAL Submitted (11-OCT-2004) School of Life Science, Queensland University of Technology, George St., Brisbane, Queensland 4001, Australia FEATURES Location/Qualifiers source 1..558 /organism="Epinephelus coioides" /mol_type="mRNA" /db_xref="taxon:94232" /tissue_type="brain" /country="Australia" gene <1..>558 /gene="IGF-I" CDS <1..>558 /gene="IGF-I" /codon_start=1 /product="insulin-like growth factor I" /protein_id="AAV34198.1" /db_xref="GI:54399798" /translation="MSSALSFQWHLCDVFKSAMCCISCSHTLSLLLCVLTLTPTATGA GPETLCGAELVDTLQFVCGERGFYFSKPTGYGPNARRSRGIVDECCFQSCELRRLEMY CAPAKTSKAARSVRAQRHTDMPRAPKVSTAGHKVDKGTERRTAQQPDKTKNKKRPLPG HSHSSFKEVHQKNSSRGNTGGRNYRM" ORIGIN 1 atgtctagcg ctctttcctt tcagtggcat ttatgtgatg tcttcaagag tgcgatgtgc 61 tgtatctcct gtagccacac cctctcacta ctgctgtgcg tcctcaccct gactccgacg 121 gcaacagggg cgggcccaga gaccctgtgc ggggcggagc tggtcgacac gctgcagttt 181 gtgtgtggag agagaggctt ttatttcagt aaaccaacag gctatggccc caatgcacgg 241 cggtcacgtg gcattgtgga cgaatgctgc ttccaaagct gtgagctgcg gcgcctggag 301 atgtactgtg cacctgccaa gactagcaag gctgctcgct ctgtgcgtgc acagcgccac 361 acagacatgc cgagagcacc caaggttagt accgcagggc acaaagtgga caaaggcaca 421 gagcgtagga cagcacagca gccagacaag acaaaaaaca agaagagacc tttacctgga 481 catagtcatt catccttcaa ggaagtgcat cagaaaaact caagtcgagg caacacgggg 541 ggccgaaact accgaatg //

78

Appendix 9. Reprinted Genbank entry for E. coioides IGF-II cDNA sequence.

1: AY776158. Reports Epinephelus coioi...[gi:54399795] Links

LOCUS AY776158 534 bp mRNA linear VRT 26-OCT-2004 DEFINITION Epinephelus coioides insulin-like growth factor II (IGF-II) mRNA, partial cds. ACCESSION AY776158 VERSION AY776158.1 GI:54399795 KEYWORDS . SOURCE Epinephelus coioides (orange-spotted grouper) ORGANISM Epinephelus coioides Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Actinopterygii; Neopterygii; Teleostei; Euteleostei; Neoteleostei; Acanthomorpha; Acanthopterygii; Percomorpha; Perciformes; Percoidei; Serranidae; Epinephelinae; Epinephelus. REFERENCE 1 (bases 1 to 534) AUTHORS Bangcaya,J., Nocillado,J., Anderson,A. and Elizur,A. TITLE IGF-II mRNA as a possible molecular marker for egg quality in grouper and mullet JOURNAL Unpublished REFERENCE 2 (bases 1 to 534) AUTHORS Bangcaya,J., Nocillado,J., Anderson,A. and Elizur,A. TITLE Direct Submission JOURNAL Submitted (10-OCT-2004) School of Life Science, Queensland University of Technology, George St., Brisbane, Queensland 4001, Australia FEATURES Location/Qualifiers source 1..534 /organism="Epinephelus coioides" /mol_type="mRNA" /db_xref="taxon:94232" /tissue_type="brain" /country="Australia" gene <1..>534 /gene="IGF-II" CDS <1..>534 /gene="IGF-II" /codon_start=1 /product="insulin-like growth factor II" /protein_id="AAV34197.1" /db_xref="GI:54399796" /translation="METQQRYGHHSLCHTCRRTESSRMKVKKMSSSSRALLFALALTL YVVEIASAETLCGGELVDALQFVCEDRGFYFSRPTSRGSNRRNQNRGIVEECCFRSCD LNLLEQYCAKPAKSERDVSATSLQVIPVMPALKPEVPRKPHVTVKYSKYEVWFRRQAE KIKAQEQAVHHRPLITLP" ORIGIN 1 atggagaccc agcaaagata cggacaccac tcactttgcc acacctgccg gagaacggag 61 agcagcagaa tgaaggtcaa gaagatgtcc tcgtccagtc gcgcgctgct gtttgcactg 121 gccctaacgc tctacgttgt ggaaatagcg tcggcagaga cgctgtgtgg gggagagctg 181 gtggatgcgc tgcagttcgt ctgtgaagac agaggcttct atttcagtag gccaaccagc 241 aggggtagca accggcgcaa ccagaaccgt gggatcgtag aggagtgttg tttccgtagc 301 tgtgacctca acctgctgga gcagtactgt gccaaacccg ccaagtccga aagggacgtg 361 tcggccacct ctctgcaggt catacccgtg atgcccgcac taaaaccgga agtcccgagg 421 aagccgcatg tgaccgtgaa gtattccaaa tacgaggtgt ggtttcggag gcaggcggag 481 aagatcaaag cccaggagca ggcagtccac caccggcccc tcatcaccct tcca //

79

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