Luận án tiến sỹ về di truyền và sinh sản hàu (PhD thesis 2016)

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Genetic and physiological parameters associatedwith oyster reproduction

Vu, Van I

Vu, V. I. (2017). Genetic and physiological parameters associated with oyster reproduction [University ofthe Sunshine Coast, Queensland]. Type: Thesis

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UNIVERSITY OF THE SUNSHINE COAST

Centre of Genetics, Ecology and Physiology, Faculty of Science Health and Education, University of the Sunshine Coast, Locked Bag 4, Maroochydore Dc, QLD 4558, Australia. Tel.:+61 7 5430

2831

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Thesis primary supervisor:

A/Prof. Wayne Knibb

Aquaculture and genetics,

Centre of Genetics, Ecology and Physiology, Faculty of Science Health and Education, University of the Sunshine Coast, Qld

Thesis co-supervisors:

Prof. Abigail Elizur

Aquaculture biotechnology

Centre of Genetics, Ecology and Physiology, Faculty of Science Health and Education, University of the Sunshine Coast, QLD

Dr. Scott Cummins

Molecular and Cellular Biology

Centre of Genetics, Ecology and Physiology, Faculty of Science Health and Education, University of the Sunshine Coast, QLD

Dr. Nguyen Hong Nguyen

Quantitative aquaculture genetics

Centre of Genetics, Ecology and Physiology, Faculty of Science Health and Education, University of the Sunshine Coast, QLD

A/Prof. Wayne O’Connor

Oyster selective breeding and reproduction Port Stephens Fisheries Research Institute, NSW

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Declaration of the origin by author

The work presented in this study does not contain any material which has been previously published or written by any person other than the candidate, except where due and proper reference has been given in the text.

I have stated the contribution of the others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature.

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Statement of Contributions to Jointly Authored Works Contained in the thesis:

1. Vu Van In, Nikoleta Ntalamagka, Wayne O’Connor, Tianfang Wang, Dan Powell, Scott F. Cummins and Abigail Elizur. Reproductive neuropeptides that stimulate spawning in the Sydney rock

oyster, Saccostrea glomerata. Peptide 82 (2016) 109 – 119.Incorporated as Chapter 2.

VVI carried out the experiment design, sampling, in vivo bioassay, data analysis and writing

the draft. NN took part in bioassay, writing the introduction of the draft, and edited the draft. WO took part in experiment design for bioassay carried at Port Stephens NSW. TW took part in the LC-MS/MS. DP took part in identification of GnRH and sampling ganglia. SC took part in experiment design, sampling ganglia, data analysis, ELH identification and edited the draft. AE directed the experiment design, data analysis and edited the draft.

2. Vu Van In, Wayne O’Connor, Michael Dove, Wayne Knibb. 2016. Can genetic diversity be

maintained across multiple mass selection lines of Sydney rock oyster, Saccostrea glomerata despite loss within each? Aquaculture, 454, 210-216.

Incorporated as Chapter 3.

VVI conducted sample collection and analysis, participated in experiment design and drafted the manuscript. WO took part in experiment design and edited the draft. MD took part in

sampling, collecting information of S. glomerata families. WK directed the experiment design,

data analysis and edited the draft.

3. Vu Van In, Wayne O’Connor, Vu Van Sang, Phan Thi Van and Wayne Knibb. Is the Vietnam

aquaculture pacific oyster Crassostrea gigas? and can traditional aquaculture practices retain

genetic variation? A case study on taxonomy and genetic diversity for starting a breeding

program. Aquaculture 473 (2017) 389 - 399.

Incorporated as Chapter 4.

VVI conducted experiment design, data analysis, wrote the draft. WO raised the need to do the research, revised data and the draft PTV took part in experiment in Vietnam. VVS took part in samples collection and data analysis. WK directed the experiment design, data analysis and edited the draft.

4. Vu Van In1,2, Vu Van Sang2, Wayne O’Connor3, Phan Thi Van4, Michael Dove3, Wayne Knibb1 and Nguyen Hong Nguyen1. “Are strain genetic effect and heterosis expression

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altered with culture system and rearing environment in Portuguese oyster,

Crassostrea angulata?”. Aquaculture Research, 2016 1-12. Incorporated as Chapter 5. VVI conducted experiment design, generation of family, data collection and analysis, preparation of the draft. VVS took part in writing draft and data analysis. PTV took part in experiment in Vietnam. WK took part in experiment design and edited the draft. MD took part in cross breeding to produce families and data collection. NHN directed the experiment design, data analysis, edited the draft.

I hereby declare that the content of the above statement is accurate: Signature: Date: February 26, 2016

VU VAN IN

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Published Works by the author incorporated into the thesis

1. Vu Van In, Nikoleta Ntalamagka, Wayne O’Connor, Tianfang Wang, Dan Powell, Scott F.

Cummins and Abigail Elizur. Reproductive neuropeptides that stimulate spawning in the Sydney

rock oyster, Saccostrea glomerata.

Published in Peptides 82 (2016) 109 – 119. Incorporated as Chapter 2.

2. Vu Van In, Wayne O’Connor, Michael Dove, Wayne Knibb, 2016. Can genetic diversity be

maintained across multiple mass selection lines of Sydney rock oyster, Saccostrea glomerata

despite loss within each?

Published in Aquaculture, 454, 210-216. Incorporated as Chapter 3.

3. Vu Van In, Wayne O’Connor, Vu Van Sang, Phan Thi Van and Wayne Knibb. Resolution of the controversial relationship between Pacific and Portuguese oysters internationally and in Vietnam.

Published on Aquaculture, 473 (2017) 389-399 Incorporated as Chapter 4.

4. Vu Van In, Vu Van Sang, Wayne O’Connor, Phan Thi Van, Michael Dove, Wayne Knibb and Nguyen Hong Nguyen. Are strain genetic effect and heterosis expression altered with culture

system and rearing environment in Portuguese oyster, Crassostrea angulata?

Published on Aquaculture Research, 2016, 1-12 Incorporated as Chapter 5.

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Acknowledgments

This thesis could not have been realized without the help of my supervisory board, many colleagues and collaborators which I would like to hereby acknowledge.

First, I would like to express my deepest gratitude and appreciation to my supervisors: A/Prof Wayne Knibb, Prof. Abigail Elizur, Dr. Scott F. Cummins, Dr. Nguyen Hong Nguyen and A/Prof. Wayne O’Connor for their support and guidance, both professionally and personally, and for your patience. I am really happy and proud to be their student. Sincere thanks to A/Prof. Wayne O’Connor, the leader of the ACIAR funded oyster project, for his support of my application for the ACIAR John Allwright Fellowship.

I acknowledge the PSFI, NSW for generously providing samples and access to facilities for spawning and implantation bioassays, special thanks to Steve O’Connor, Michael Dove, Kyle Johnston and Brandt Archer (PSFI, NSW) for their assistance in sampling and bioassays. Many thanks to A/Prof Phan Thi Van, Director of Research Institute for Aquaculture No.1 (RIA1), Vietnam for her support and creating the possibility for oyster genetic breeding trials in Vietnam. Many thanks to the oyster team at the Northern National Broodstock Centre for Mariculture (CatbaMBC), RIA1 for their technical assistance during oyster breeding and grow-out. We gratefully thank Dr Alun Jones (Institute for Molecular Bioscience, the University of Queensland) for advice and assistance with the LC-MS/MS.

We gratefully acknowledge financial support from the University of the Sunshine Coast (USC) and Australian Centre for International Agricultural Research (ACIAR) through the John Allwright Fellowship to V-V. I., and through the project “Enhancing bivalve production in northern Vietnam and Australia” (FIS/2010/100), the Australian Seafood CRC and FRDC (Project No.2012/713) for the reproduction part. We highly appreciate the technical assistance from USC staff, Nicole Levi, Emily and IT for their administrative work as well as Genecology group, especially David Bright, Ido Bar, Rob Lamont and Dan Powell for their instruction in lab work and genetic software.

Thanks to my family members and friends for their support and encouraging me during this long but incredible journey.

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Abstract

Genetic programs are an essential component of aquaculture operations and can operate as a management tool to prevent inbreeding as well as facilitate selection for improved traits and increased profitability. A key requirement for the operation of genetic programs is the ability to reproduce select animals in captivity, that is, to have full control of the reproductive process. This study focuses on genetics and reproduction of two important but, before this thesis,

taxonomically confused/ indistinct aquaculture oyster species: Pacific oyster Crassostrea gigas or Portuguese oyster, Crassostrea angulata (this unidentified species is hereafter referred to VNO) and on Sydney rock oyster, Saccostrea glomerata with the aim to develop genetic,

molecular and physiological tools required for an optimal operation of genetic programs for these species. Scientifically, this study intends to provide knowledge about how long term selection affects genetic diversity, about the role of the endocrine system in oyster reproduction and how this information can be used to set up reliable reproduction and genetically sustainable breeding programs.

Research into the above objectives is set out in four data Chapters including: 1) Identification of key genes and peptide hormones associated with reproductive performance of S. glomerata; 2) Analysis of mass selected S. glomerata lines for genetic diversity; 3) Identification of the species identity of oysters cultured in Vietnam and analysis of their genetic diversity and to determine if there is sufficient diversity to initiate a breeding program; 4) Application of the aforementioned discovered genetic and physiological tools to design a trial breeding program for the now identified VNO in Vietnam.

Chapter 1. Literature review overview about the oyster production and reproduction,

genetic and genetic programs as well as about DNA barcoding for resolving close-related oysters.

Chapter 2 was published in Peptides 82 (2016) 109 – 119 and considers the discovery of

genes and neuropeptides that are expressed in the key regulatory tissues of S. glomerata, the

visceral ganglia and gonads (male and female). Special focus was made on the neuropeptides since they encompass a diverse class of chemical messengers that play functional roles in many aspects of an animal’s life, including reproduction. Approximately 28 neuropeptide genes were identified primarily within the visceral ganglia transcriptome that encode precursor proteins containing numerous neuropeptides; 11 were confirmed through mass spectral peptidomics

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analysis of visceral ganglia. These 28 bioactive neuropeptides were synthesized, then tested for their capacity to induce gonad development and spawning. Six of the peptides (ELH, GnRH, APGWamide, buccalin, CCAP and LFRFamide neuropeptides) were found to be potential triggers for spawning in ripe animals. Upon further testing, APGWa and buccalin demonstrated a capacity

to advance gonadal maturation. In summary, our analysis of S. glomerata has found the

neuropeptides that can influence the reproduction cycle of this species, specifically by accelerating gonadal maturation and triggering spawning. Other molluscan neuropeptides identified in this study will enable further research into understanding the neuroendocrinology of oysters, which may have benefits to the cultivation.

Chapter 3 was published in the journal Aquaculture and the paper was entitled “Can

genetic diversity be maintained across multiple mass selection lines of Sydney rock oyster,

Saccostrea glomerata despite loss within each?” Volume 454, 1 March 2016, Pages 210–216.

This is the first assessment of genetic diversity after multiple generations of mass selection for fast growth and disease resistance in Sydney rock oysters using DNA microsatellites. Substantial losses of allelic and haplotypic diversity were evident within each single selected line compared with wild samples. However, considered together, the different selected lines had maintained

levels of diversity not different to that in the wild samples. The S. glomerata data support other

data from shrimp that genetic variation in mass selection program can be kept when multiple independent selection lines are maintained. This Chapter provided important internationally relevant knowledge about the consequences to genetic diversity following generations of mass selection and importantly suggest an approach to restore lost diversity if multiple lines are kept.

Moreover, the seven new microsatellite markers for S. glomerata discovered in this study will

add to the genetic toolbox of resources needed to operate efficient oyster genetic programs.

Chapter 4 was submitted to Aquaculture. It presents evidence, for the first time, to

unequivocally identify and separate between the closely related taxa: The Pacific oyster,

Crassostrea gigas and the Portuguese oyster C. angulata. These two species have been the

subject of taxonomic controversy for some time. The paper is entitled “Is the Vietnam

aquaculture Pacific oyster Crassostrea gigas? and can traditional aquaculture practices retain

genetic variation? A case study on taxonomy and genetic diversity for starting a breeding program”. This study reports for the first time the existence of five nucleotides present in COX1

that show categorical differences between C. angulata and C. gigas, and these diagnostic nucleotides provided evidence that the so-called Pacific oyster in northern Vietnam is C.

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angulata. Further sequence data identified other oyster species in the central/south of Vietnam. Three hatchery oyster lines in northern Vietnam, now identified as C. angulata, were found to

be genetically diverse using mtDNA sequencing and DNA microsatellite analyses, although there is a trend for some genetic loss within each of the three aquaculture lines.

Chapter 5 was published in Aquaculture Research 2016, 1-12. It is a preliminary study to

a future full genetic program, and aims to assess for commercial growth and performance

differences between existing, C. angulata lines in Vietnam based on the outcomes of the

previous chapters. Specifically, an analysis was carried out to estimate the strain additive genetic and heterotic effects on harvest body weight in a 3x3 complete diallel cross involving three populations of oyster from Northern Vietnam. Strip spawning was applied to simultaneously produce full- and half-sib families of nine cross-combinations for performance testing in two locations (assuming two environments), including 1) Catba Islands, Haiphong 2) Vandon Islands, Quangninh province, Vietnam (breeding techniques obtained from Chapter 1) and in two types of culture (single seed and clutch set). A total of 7269 individual oyster were examined over a grow-out period of 270 days. A linear mixed model was used to estimate strain additive genetic and heterotic effects for body weight at harvest. Ranking of strains based on their additive genetic effects did not differ between the testing environments and culture systems. Across the conditions studied, the strain imported from China had the highest additive genetic values (1.66% and 5.86% above the pure strain mean respectively) compared with those of Namdinh and Ria1 strains (0.70% and 0.96% below the pure strain mean respectively for two testing environments and 2.85% and 3.01% below the pure strain mean respectively for two types of culture). The non-additive genetic (heterotic) effect was low and not different from zero for the trait studied across culture systems. There were no differences in levels of heterosis for harvest body weight between the two testing environments or culture systems. Based on the small heterotic and large additive genetic effects among strains used in this study. It can be suggested

that the future breeding program in C. angulata should be based on a wise choice of strain or by

discovering the additive genetic diversity via a selective breeding program.

In summary, the goals of this study were: 1) to provide knowledge about the reproduction of oysters via identification of reproductive neuropeptides; 2) to understand how a long term selection and traditional aquaculture practices can affect genetic diversity; 3) to resolve the

taxonomic relationship of two close-related oyster taxa thought to be aquacultured in Vietnam;

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and 4) to consider how this new information can be used to set up a reliable reproduction and

genetically sustainable breeding program for oyster.

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Table of Contents

Declaration of the origin by author ... 2

Statement of Contributions to Jointly Authored Works Contained in the thesis: ... 3

Statement of parts of the thesis submitted to qualify for the award of another degree ... 5

Published Works by the author incorporated into the thesis ... 6

1.4 Genetic variation and genetic programs ... 23

1.5 DNA barcoding for resolving close-related oysters ... 26

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2.6 Acknowledgements ... 54

2.7 Supplementary data ... 54

2.8 References ... 54

Chapter 3: Can genetic diversity be maintained across multiple mass selection lines of Sydney rock oyster, Saccostrea glomerata despite loss within each? ... 58

3.2.5 Mitochondrial DNA sequencing and analysis ... 63

3.2.6 Validation and development of microsatellite primers ... 64

3.2.7 Genotyping ... 65

3.2.8 Data analysis and statistical methods ... 66

3.3 Results ... 66

3.3.1 Microsatellite diversity within among lines and wild populations ... 66

3.3.2 mtDNA haplotype diversity among populations ... 68

Chapter 4: Is the Vietnam aquaculture Pacific oyster Crassostrea gigas? and can current aquaculture practices maintain genetic variation among lines? A case study on taxonomy and genetic diversity for a start of breeding program ... 79

4.1 Introduction ... 80

4.2 Materials and methods... 83

4.2.1 Animals ... 83

4.2.2 Sampling ... 83

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4.2.3 DNA extraction ... 84

4.2.4 Mitochondrial DNA sequencing and analysis ... 84

4.2.5 Validation of microsatellite primers ... 85

4.2.6 Genotyping ... 85

4.2.7 Data analysis and statistical methods ... 86

4.3 Results ... 86

4.3.1 ID identity and mtDNA haplotype diversity among lines ... 86

4.3.2 Identification of hatchery oyster ID in Nhatrang, Vietnam ... 90

4.3.3 Genetic diversity among C. angulata lines ... 93

4.3.4 Inbreeding and effective population size assessed by analysis of genotypes using

Chapter 5: Are strain genetic effect and heterosis expression altered with culture system and rearing environment in the Portuguese oyster, Crassostrea angulata? ... 109

5.1 Introduction ... 110

5.2 Materials and methods... 111

5.2.1 Broodstock strains ... 111

5.2.2 Location of experiment ... 113

5.2.3 Family production and rearing ... 114

5.2.4 Grow-out testing environments and harvest measurement ... 115

5.2.5 Statistical analysis ... 115

5.3 Results ... 117

5.3.1 Characteristics of the data ... 117

5.3.2 Effects of culture system (“single” vs. “cultch”) and environment (Catba vs. Vandon) ... 117

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5.3.3 Cross effects ... 118

5.3.4 Strain additive genetic effect ... 119

5.3.5 Heterosis effects ... 120

5.3.6 Reciprocal (maternal) effects ... 123

* = 100*Additive genetic effects / mean of BW. ... 126

**: Pooled all samples from both culture systems for calculation ... 126

5.4 Discussion ... 126

5.5 Acknowledgements ... 129

5.6 Supplementary data ... 129

Chapter 6: Final statement ... 139

6.1 Reproductive neuropeptides of S. glomerata ... 139

6.2 Genetic loss in mass selected S. glomerata ... 140

6.3 Genetic integrity and diversity of aquaculture oyster in Vietnam ... 141

6.4 Evaluate strain genetic effect and heterosis ... 142

6.5 References ... 143

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Lists of Figures

Figure 1.1. Stages of development in bivalves (Helmet al., 2004) ... 22

Figure 1.2. The nervous system of Crassostrea viewed from the right (Fox, 2007) ... 23Figure 2.1. Workflow of the experiments to identify neuropeptides of S. glomerata using

transcriptomics and peptidomics. Position of the visceral ganglia and gonad is shown in schematic [modified from (Paul, 1964)]. ... 35 Figure 2.2. RP-HPLC chromatogram and identification of neuropeptides extracted from visceral

ganglia of female S. glomerata. . ... 43Figure 2.3. Identification and characterization of S. glomerata ELH precursor in comparison with

other oysters.. ... 44

Figure 2.4. Identification and characterization of S. glomerata GnRH precursor in comparison

with other oysters.. ... 45

Figure 2.5. Identification and characterization of S. glomerata APGWa precursor in comparison

with other oysters. ... 46

Figure 2.6. Identification and characterization of S. glomerata buccalin precursor in comparison

with other oysters. ... 47

Figure 2.7. Identification and characterization of other S. glomerata neuropeptides. Schematic

diagrams show the organisation of neuropeptide precursors and multiple sequence alignment of bioactive peptide between mollusc species. ... 48

Figure 2.8 Condition index and fertilization rate of S. glomerata oysters treated with buccalin,

APGWa or neuropeptide-free implant (control) four weeks post-implantation.. ... 50

Figure 3.1. Diagrammatic representation of various S. glomerata lines ... 62

Figure 3.2. Number of alleles and haplotypes among populations. ... 69

Figure 3.3. Genetic distance among individuals of five S. glomerata populations from principal

coordinates analysis (PCoA) using genetic distance matrices. ... Error! Bookmark not defined.

Figure 4.1. Hatchery and sampling sites ... 83 Figure 4.2. Phylogenetic tree using MtDNA COX1 using phylogeny reconstruction analysis with Neighbour-joining statistical method using Bootstrap test with 500 replications, and mode with maximum composite likelihood on Mega6.0.. ... 92 Figure 4.3. Total number of alleles across nine loci in three cultured lines. ... 95

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Figure 4.4. Genetic distance among individuals of three lines from Principal Coordinates Analysis (PCA) using genetic distance matrices. . ... 97

Figure 5.1. Grow-out sites of C. angulata ... 113

Figure 5.2. Mean body weight (g in Y-axis) of crosses in two culture environments (Cat Ba and Van Don). C = China, N = Namdinh and V = Ria1. Cross = male x female ... 119 Figure 5.3. Mean body weight (g in Y-axis) of crosses in two types of culture (“single” and “cultch”). C = China, N = Namdinh and V = Ria1. Cross = male x female ... 119

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List of tables

Table 2.1. Summary of neuropeptides deduced from the S. glomerata transcriptomes. Shading

indicates positive identification. ... 42

Table 2.2. Results of female spawning bioassay using synthetic peptides. Peptide in the same group number were mixed. ... 49

Table 3.1. Number of alleles among loci in selected lines and wild samples ... 67

Table 3.2. Number of full sibs and effective population size among lines and wild samples ... 67

Table 3.3. Number of mtDNA haplotypes in selected lines and wild samples ... 70

Table 4.1. Five exclusive nucleotides in COX1 sequences between C. gigas and C. angulata. Gray shading indicates the exclusive nucleotide. ... 88

Table 4.2. Alignment of oyster sampled from lines of Ria1, Namdinh and China with COX1 references of C. gigas and C. angulata.. ... 89

Table 4.3. Alignment of oyster sampled from Nhatrang with Crassostrea references. . ... 90

Table 4.4. Number of mtDNA haplotypes in hatchery lines ... 93

Table 4.5. Number of DNA microsatellite alleles among loci from Ria1, Namdinh and China lines ... 94

Table 4.6. Number of full-sibs and effective population size among lines... 96

Table 4.7. Pairwise Population Fst value ... 96

Table 5.1. Descriptive statistics for harvest total weight of a 3 x 3 diallel cross population of C. angulata oyster in two different culture environments and methods (g) ... 117

Table 5.2. Statistical significance of fixed effects on harvest total weight of oyster ... 118

Table 5.3. Strain additive genetic effects on body weight (BW) at the two culture environments (CatBa vs. Van Don) ... 120

Table 5.4. Strain additive genetic effects on body weight (BW) by two types of culture (“Single”

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Table 5.8. Maternal effects on body weight (BW) by two types of culture (“Single” vs. “Cultch”)

... 125

Supplementary files

Supplementary file S1. Amino acid sequences of Saccostrea glomerata and other molluscan

neuropeptides used for schematic diagrams and alignments……….……..……. 56

Supplementary file S2. Allelic and haplotypic data of Sydney rock oyster, Saccostrea glomerata….…..… 78

data……….……….……….…....…107

Chapter 1: Literature Review

1.1 Introduction

Genetic programs are an essential component of aquaculture operations being a management tool not only to prevent inbreeding but also to allow selection for improved traits and increase profitability. A key requirement for the operation of genetic programs is the ability to reproduce select animals in captivity, that is, to have full control of the reproductive process. This study will focus on genetics and reproduction of two important aquaculture species: Pacific

oyster, Crassostrea gigas / Portuguese oyster, C. angulata (VNO) and Sydney rock oyster, Saccostrea glomerata with its aim to develop genetic, molecular and physiological tools required for an optimal operation of genetic programs and apply those to the VNO and S. glomerata.

Scientifically, it will provide knowledge about how long term selection affects genetic diversity, about the endocrine system controlling reproduction in oysters and how this information can be used to permit reliable reproduction and set up sustainable genetic improvement programs. To

achieve the above objectives, four experiments were implemented including: 1) Analysis of selected S. glomerata for genetic diversity; 2) Identification of oyster populations cultured in Vietnam and analysis of their genetic diversity; 3) Application of the genetic and physiological tools to design a trial breeding program for the VNO in Vietnam; 4) Identification of key genes and peptide hormones associated with reproductive performance of S. glomerata.

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1.2 Oyster production

In 2010, world oyster production reached approximately 5 million tonnes (FAO, 2014). In Australia, it is one of the oldest aquaculture species with a history of 120 years (Nell, 2005), mainly present in South Australia, New South Wales, Tasmania and to a lesser extent in Queensland and Western Australia (Maguire and Nell, 2005) with two main species: Sydney rock

oyster, S. glomerata and Pacific Oysters, C. gigas.

C. gigas, native to Japan, was introduced to mainland Australia in the late 1940s and to

Tasmania in the early 1950s; it then quickly established wild populations in Australia (English,

2000, Ward et al., 2000). C. gigas was introduced to France in the early 1970s because of the mass mortality of C. angulata (Boudry, 2008). Curently, it is now being cultured in 27 countries and is the most highly produced mollusc species in the world (Boudry, 2008) as C. gigas has

relatively high disease resistance and fast growth compared to other oyster species. The average

time for S. glomerata to grow to a marketable size (40-60g) is about 3.5 years , whereas it only takes about 17-18 months for C. gigas to reach market size in Tasmania (Maguire and Nell, 2005)

and as short as 12 months in NSW.

In Vietnam, before 2008, mollusc production was dominated by the aquaculture of hard

clams (Meretrix meretrix and M. lyrata) with seed collected from the wild; other mollusc species

accounted for only a small proportion of the total Vietnamese production (RIA1 annual report, 2012). In 2012, the total mollusc production was appropximately 190,000 t/annum (FIS/2010/100, 2013). The so-called “Pacific Oyster” (VNO) was introduced into Vietnam for both hatchery and grow-out production in 2008 via the support of an ACIAR project (Building bivalve mollusc hatchery production capacity project in Vietnam and Australia, FIS/2005/114). The growth of VNO production in Vietnam was very impressive, rising from no production in 2006-07 to 7000 t by 2010-11 , worth A$7.0-9.8 million annually (FIS/2010/100, 2013). Currently, VNO production consists of approximately 200 small farms, operating 2,200 rafts in northern Vietnam (FIS/2010/100, 2013). One of the major constraints in VNO production in Vietnam is lack of reliable seed supply, poor quality of spat and quality assurance to meet the growing demands of farmers and producers (FIS/2010/100, 2013).

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1.3 Reproduction

One of the key requirements for the operation of genetic programs is the ability to reproduce selected animals in captivity, that is, to have full control of the reproductive process. However, the molecular and biochemical cues associated with oyster reproduction are poorly understood. Therefore, identification of genes and peptide hormones that regulate oyster reproduction will help establish knowledge for a better understanding of this process. Through transcriptome analysis, I aimed to identify genes and gene products that can trigger reproduction and investigate how this knowledge could be applied to optimize the design for a genetic breeding programs.

Oysters obtain food by filtering and extracting algae, bacteria and nutrients from the

surrounding water. Most oyster species, including S. glomerata and C. gigas/VNO, change sex

during their life. The first spawning is usually as a male and subsequent spawning as a female

while some remain as hermaphrodites. In adult S. glomerata populations, the percentage of

males and females can differ greatly, for example 33% and 67% (Dove and O'Connor, 2012).

During spawning, adult S. glomerata females can spawn up to 20 million eggs (O'Connor et al., 2008b), while a large C. gigas may release up to 100 million eggs per spawn and males release

hundreds of millions of sperm into the water when the tides and currents are optimal for the widest distribution. Fertilization takes place in the water column and embryo development continues for up to 3-4 weeks as the larval stages of the oyster swim and grow, ultimately settling on a suitable hard surface, then never leaving their settled position.

The development of oysters has been described in terms of five developmental phases, where phase I is the ripening period, phase II is fully ripe, phase III is post spawning, phase IV is regression with the presence of phagocytes, and phase V is a regressive phase where gonial cells are indeterminate (Dinamani, 1974). The stages of development of bivalves are shown in Figure

1.1. The development of embryos to D-veliger larvae of S. glomerata is saffected by temperature,

salinity and the interaction of these factors. Salinity of 35 ppt and 26°C are optimal for embryonic development whereas D-veliger larvae grow best at 28°C and at maximum salinity level of 34 ppt. The highest growth rate of spat was observed at a salinity of 35 ppt and a temperature of 30°C (Dove and O'Connor, 2007).

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The development of embryos and larvae for both C. glomerata, C. gigas are well-known

and recorded in manuals for hatchery production (Dove and O'Connor, 2012). However, the information on regulation of oyster reproduction or sex changes is very limited. In molluscs, neuropeptides and peptide hormones are known to have multiple functions from physiology, behaviour and reproduction. Therefore, the identification and investigation of functions of neuropeptides involved in oyster reproductive regulation will greatly support reliable hatchery production.

Figure 1.1. Stages of development in bivalves (Helm et al., 2004)

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In bivalve molluscs, neurohormones are generated in ganglia of the central nervous system that consists a large number of neurosecretory cells. There are two main parts: the cerebral ganglia and the visceral ganglia (Figure 1.2) where the visceral ganglia are much larger than the cerebral ganglia. Several vertebrate-like hormone immunoreactivities have been found in the mussel ganglia: insulin-like, α-MSH-like, CCK-like, somatostatin-like, FMRFa-like, substance P-like, and neuropeptide F-like. However, their involvement in the reproductive process of molluscs is still unknown (Pazos and Mathieu, 1999). The role of the various other peptides reported in the endocrine system for the regulation of gonad development, spawning behaviour and sex changes is unclear and needs to be examined.

Figure 1.2. The nervous system of Crassostrea viewed from the right (Fox, 2007)

1.4 Genetic variation and genetic programs

One of the problems in oyster industries is degraded stock due to inbreeding (Knibb et al., 2014b). For sustainable culture, a selective breeding program which delivers genetic

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improvement for specific traits, yet prevents inbreeding, is indispensable. Maintaining a wide range of genotypes could give a hatchery population more flexibility of response to constantly changing environments (Boudry, 2008, Taris et al., 2006). Genetic diversity is the main resource for a stock improvement program, however it may be eroded due to domestication selection, or poor husbandry practices in hatcheries where often a very limited number of broodstock individuals are kept resulting in high inbreeding and deterioration of quality seed stock (Boudry, 2008, Nguyen, 2009, Taris et al., 2006). Loss of genetic variation and inbreeding may then lead to poor future genetic gains in a breeding program (In et al., 2016a) . Inbreeding depression may reduce fitness (ability to survive and reproduce) and production performance of the animals. Therefore, maintenance of additive genetic variance is important as it provides bbetter selection repsonse over generations of selective breeding.

Growth rate and disease resistance can be improved through a genetic program as heritable variation exists for these traits (Boudry et al., 1997). Mass selection and family based selection are two kind of approaches in selective breeding. Mass selection is suitable only when a single trait is chosen for improvement, such as fast growth; meanwhile other traits especially with low or moderate heritability such as shell shape may be better improved through family

selection (Ward et al., 2000). To date, breeding programs for S. glomerata and C. gigas have been

set up in many countries including Australia, New Zealand, Korean, China and elsewhere in the world to improve growth rate and disease resistance (Appleyard and Ward, 2006, Hwang et al., 2013, Kaspar et al., 2013, Li et al., 2013, O'Connor and Dove, 2006). The main traits of interest for a genetic program have been fast growth. Selection for fast growth has been successful for

several oyster species including Crassostrea virginica, Ostrea edulis and Ostrea chilensis (Dove

and O'Connor, 2009, Nell et al., 1999, O'Connor and Dove, 2006).

Specifically for C. gigas, selection for fast growth was initated in Tasmania, Australia in

1996 (Appleyard and Ward, 2006) and subsequently selective breeding developed fast growth and disease resistant stocks. However, a high level of inbreeding due to mating of closely related individuals could result in depression of performance characteristics (Evans et al., 2004b). This resulted in a decline in response on first or second generation (Evans et al., 2004b, Toro and Newkirk, 1990). In order to balance maximum selection intensity and the need to retain an adequate number of broodstocks, inbreeding accumulation of no more than 1% is considered acceptable as it may not adversely affect performance gains too much (Falconer and Mackey,

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1996, Frankel and Soule, 1981). Specifically for S. glomerata, the third generation stock from

selection for high growth was 74% heavier (50g greater) than controls which saves up to 11

months (28 for selected vs. 38 months for non-selected oysters) of the time otherwise taken to

reach the market size (Nell, 2005, Nell, 2006).

However other traits such as disease resistance, shape and uniformity are increasingly

becoming important (Appleyard and Ward, 2006, Kube et al., 2013). C. gigas mortality syndrome

(POMS) occurred and caused mass mortality in Europe, New Zealand, Korea and Australia during

summer times raising the need to develop POMS resistant stocks for the C. gigas industry (Hwang

et al., 2013, Kube et al., 2013). In Australia, the resistance to POMS has been added in the

breeding objective for C. gigas in order to produce resistant stock for growers (Kube et al., 2013). In S. glomerata, Winter mortality syndrome (Bonamia roughleyi) and QX disease (Marteilia sydneyi) has caused more than 40% reduction in S. glomerata production in Australia (Heasman et al., 2000, Nell, 2003). QX may kill 80% or more of the infected S. glomerata population (Nell

and Perkins, 2006, Simonian et al., 2009). However, the mortality of both diseases was reduced by selective breeding conducted by the NSW DPI (Simonian et al., 2009). After two generations

of selection, the new strains resistant to QX (Marteilia sydneyi) and winter mortality diseases had

29% less mortality from QX disease at Lime Kiln Bar, Georges River (Nell, 2003). Selective breeding was also developed for fast growth and MSX disease resistance in lines of American

Oyster (Crassostrea virginica) in the United States in order to avoid high mortality rates from

MSX disease (Standish et al., 1993).

Understanding the level of genetic variation for traits of commercial importance and their genetic correlations is important in selective breeding programs as it shapes how to do selection (what selection indices to use, what weights to put on particular traits). This involves the estimation of genetic parameters such as heritabilities (h2) and genetic correlations. High heritabilities for a trait of interest show that a large proportion of the phenotypic variation is due to genetics (Falconer, 1981, Newkirk et al., 1977). Heritability values of 0.20 or larger indicate that genetic improvement can easily be achieved through a selective breeding program (Newkirk et al., 1977). Correlation implies that the alteration of one trait will cause correlated changes in other traits (Falconer, 1981). The changes can be either positive or negative. The magnitude of changes will be affected by degree of correlation between the traits involved (Toro and Newkirk, 1990).

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In summary, for both C. gigas and S. glomerata, there are a limited number of genetic

tools available to calibrate and manage diveristy in hatchery stocks. There is also limited knowledge of the molecular and biochemical cues associated with reproduction. The genetic

diversity studies will determine if current breeding practices for C. gigas and S. glomerata are

sustainable in the long term. The discovery of bioactive compounds associated with reproduction will support more reliable reproduction, which in turn will support the genetic breeding programs.

1.5 DNA barcoding for resolving close-related oysters

In Vietnam, an industry based on a newly introduced oyster, called “Pacific oyster” (VNO)

started in 2008, however, it has not been clear whether the VNO is C. gigas. This is because they were imported from a population in Taiwan which is known as a pure stock of C. angulata

(Boudry et al., 1997). In addition, Vietnamese farmers also imported oyster spat from South of

China which are assumed C. angulata but they may consist of C. gigas or hybrid stocks. Unfortunately, C. angulata and C. gigas are the genetically very close taxa which are difficult to

identify using morphology and physiology criteria (Huvet et al., 2000b). In the past, they were classified as two different species by Thunberg in 1793 and Lanmarck in 1819 as they apparently

were distributed in two separate areas: C. angulata in Europe and C. gigas in Asia (Lapegue et

al., 2004). However, the two taxa were then considered as a single species due to high genetic similarity between the taxa based on morphologic e.g. Boudry et al. (1998) comparision, experimental hybridization (Huvet et al., 2001, Huvet et al., 2002) and allozyme studies . In the past, mitochondrial DNA (mtDNA) studies were used for clarification of taxonomic status and analysis of stock structure in some commercially important oysters (Klinbunga et al., 2003). Boudry et al. (1998) and Huvet et al. (2000b) suggested mtDNA show clear differences between

C. gigas and C. angulata and thus mtDNA sequencing may be an effective tool for identification

of oyster populations in Vietnam.

Specifically for differentiation of C.gigas and C.angulata, allozyme and molecular DNA

markers such as microsatellites and mitochondrial DNA (mtDNA) have been used. mtDNA is sometimes considered as preferable to nuclear DNA for phylogenetic studies due to its uniparental inheritance, high evolutionary rate, lack of introns, large copy numbers in every cell and limited recombination (Radulovici et al., 2010).

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Previous studies using protein allozyme markers supported the hypothesis that the two species should be classified as the same species (Boudry et al., 1998). Similarly, nuclear markers did not help clarify the two taxa Huvet et al. (2000a) and Reece et al. (2008). However, mtDNA has revealed possible genetic differences between them and proposed mtDNA can be used to distinguish these taxa (Boudry et al., 1998, Huvet et al., 2000b). The differentiation between the taxa tended to increase considering mtDNA sequences rather than nuclear sequences (Huvet et al., 2000a). This can be explained by the fact that mtDNA is often more polymorphic than nuclear DNA since the former has a relatively faster mutation rate, which results in greater variation between species. Indeed recently, mtDNA sequences were used to consider relationships among

six Asian oysters including C gigas and C. angulata (Wu and Yu, 2009).

Among genes of the mtDNA chromosome, the mitochondrial cytochrome C oxidase subunit I (COX1) gene is one of the most commonly used markers in molecular studies and barcoding as it provides strong phylogenetic differentiation(Hebert et al., 2003). It was widely

used as a DNA barcode for attempted phylogenetic separation of C. angulata and C. gigas. Other

mtDNA genes: rrnL and MNR, 16S rDNA, 12S rDNA (David and Savini, 2011, Lam and Morton, 2006, Masaoka and Kobayashi, 2005, Stepien et al., 2001) and nuclear genes: ITS1, ITS2 18S and 28S rDNA (David and Savini, 2011, Larsen et al., 2005) were also used to analyze the phylogeny

of bivalves but they are less diverse than COX1 (Boudry et al., 2003, Radulovici et al., 2010,

Stepien et al., 2001). Sequence comparison of the Internal transcribed spacer (ITS) region is another tool for taxonomy and molecular phylogeny. Although ITS has high degree of variation

among some species of Ostreidae, it could not separate the closely related species such as C. gigas and C. angulata (Wang and Guo, 2008).

Boudry et al. (1998) used mtDNA-RFLP for analysis of COX1 of C. gigas and C. angulata with TaqI, MseI, Sau3AI, HhaI to reveal 6 mitotypes composed of A (ccab), B(cdab), C(dcad), D(dcab), E(dcbd), and J(acab) showing 76% of C. gigas contain C mitotypes, but 88% of C. angulata hold A mitotypes. COX1 was also considered an effective tool to identify the European flat oyster Ostrea edulis, the native O. angasi in Oyster Harbour, Western Australia (Morton et al., 2003), C. iredalei and Saccostrea cucullata in Thailand (Klinbunga et al., 2003), Malaysian Crassostrea oyster species including Crassostrea iredalei, Crassostrea belcheri and Crassostrea madrasensis (Mustaffa et al., 2010) and to discover new oyster species, such as Crassostrea hongkongensis (Lam and Morton, 2003).

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Therefore, the principal aim of this study/thesis was to establish foundational knowledge to develop an optimal genetic program for oysters in Australia and Vietnam. The study involved four major experiments: 1) Identify genes and peptide hormones that regulate oyster reproduction, 2) Develop the molecular tools and genetic markers to analyse

genetic diversity for selected S. glomerata and hatchery VNO stocks, 3) Investigate genetic integrity of VNO to understand whether they are C. gigas or C. angulata and whether they

have adequate genetic variation to start a breeding program. The fourth experiment was to evaluate strain genetic effects and heterotic expression of selected VNO in various culture systems and different rearing environments.

Specifically, the thesis includes the six chapters that have been prepared in the format of four papers as the followings:

1) Chapter 1: Literature Review

2) Chapter 2: Vu Van In, Nikoleta Ntalamagka, Wayne O’Connor, Tianfang Wang, Dan Powell, Scott

F. Cummins and Abigail Elizur. Reproductive neuropeptides that stimulate spawning in the Sydney

rock oyster, Saccostrea glomerata. Peptide .

3) Chapter 3: Vu Van In, Wayne O’Connor, Michael Dove, Wayne Knibb. 2016. Can genetic diversity

be maintained across multiple mass selection lines of Sydney rock oyster, Saccostrea glomerata

despite loss within each? Aquaculture, 454, 210-216.

4) Chapter 4: Vu Van In, Phan Thi Van, Vu Van Sang and Wayne Knibb. Is the Vietnam aquaculture

pacific oyster Crassostrea gigas? And can aquaculture practice remain gentic variation among? A case study on taxonomy and genetic diversity for a start of breeding program. To be submitted

to Aquaculture.

5) Chapter 5: Vu Van In, Phan Thi Van, Nguyen Thi Thu Hien, Wayne Knibb and Nguyen Hong

Nguyen. Are strain genetic effect and heterosis expression altered with culture system and

rearing environment in Portuguese oyster, Crassostrea angulata?. Aquaculture Research, 2016

1-12.

6) Chapter 6: Final statement

1.6 References

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BOUDRY, P., HEURTEBISE, S., COLLET, B., CORNETTE, F. & GÉRARD, A. 1998. Differentiation between

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BOUDRY, P., HEURTEBISE, S. & LAPÈGUE, S. 2003. Mitochondrial and nuclear DNA sequence

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ENGLISH, L. J., MAGUIRE, G. B., WARD, R. D. 2000. Genetic variation of wild and hatchery populations

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EVANS, F., MATSON, S., BRAKE, J. & LANGDON, C. 2004. The effects of inbreeding on performance

traits of adult Pacific oysters, Crassostrea gigas. Aquaculture, 230, 89-98.

FALCONER, D. S. 1981. Introduction to quantitative genetics, 2nd edition. Logman Group Ltd. New York.

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FOX, R. 2007. Invertebrate Anatomy Online, Crassostrea virginica, American Oyster. The American Oyster, Crassostrea virginica [Online]. [Accessed 2007].

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HUVET, A., BALABAUD, K., BIERNE, N. & BOUDRY, P. 2001. Microsatellite Analysis of 6-Hour-Old Embryos Reveals No Preferential Intraspecific Fertilization Between Cupped Oysters

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hybrids enough to conclude that the two oysters Crassostrea gigas and Crassostrea angulata

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HUVET, A., LAPEGUE, S., MAGOULAS, A. & BOUDRY, P. 2000b. Mitochondrial and nuclear DNA

phylogeography of Crassostrea angulata, the Portuguese Oyster endangered in Europe.

Conservation Genetics, 1, 251-262.

HWANG, J. Y., PARK, J. J., ARZUL, I. & PARK, M. A. 2013. Ostreid herpesvirus infection in farmed Pacific

Oyster larvae, Crassostrea gigas in Korea. 5th International Oyster Symposium (IOS5), World Oyster Society. Ho Chi Minh city, December 2013.

IN, V.-V., O'CONNOR, W., DOVE, M. & KNIBB, W. 2016. Can genetic diversity be maintained across

multiple mass selection lines of Sydney rock oyster, Saccostrea glomerata despite loss within

each? Aquaculture, 454, 210-216.

KASPAR, H. F., JANKE, A. R., CAMARA, M. D., KING, N., YEN, S. & RAGG, N. L. C. 2013. 5th International Oyster Symposium (IOS5), World Oyster Society. Ho Chi Minh city, December 2013.

KLINBUNGA, S., KHAMNAMTONG, N., TASSANAKAJON, A., PUANGLARP, N., JARAYABHAND, P. & YOOSUKH, W. 2003. Molecular Genetic Identification Tools for Three Commercially Cultured

Oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand.

Marine Biotechnology, 5, 27-36.

KNIBB, W., WHATMORE, P., LAMONT, R., QUINN, J., POWELL, D., ELIZUR, A., ANDERSON, T., REMILTON, C. & NGUYEN, N. H. 2014. Can genetic diversity be maintained in long term mass selected populations without pedigree information? A case study using banana shrimp

Fenneropenaeus merguiensis. Aquaculture, 428–429, 71-78.

KUBE, P., HICK, P., CUNNINGHAM, M., KIRKLAND, P., ELLIOTT, N., O’CONNOR, W. & DOVE, M. C. 2013. Current progressin genetic selection for resistance to acific Oyster mortality syndrome

caused by OsHV-1 microvariant (μ-var) in Australia. 5th International Oyster Symposium (IOS5), World Oyster Society. Ho Chi Minh city, December 2013.

LAM, K. & MORTON, B. 2003. Mitochondrial DNA and morphological identification of a new species

of Crassostrea (Bivalvia: Ostreidae) cultured for centuries in the Pearl River Delta, Hong Kong,

China. Aquaculture, 228, 1-13.

LAM, K. & MORTON, B. 2006. Morphological and mitochondrial-DNA analysis of the Indo-West Pacific

rock oysters (Ostreidae: Saccostrea species). Journal of molluscan studies, 72, 235-245.

LAPEGUE, S., BATISTA, F., HEURTEBISE, S., YU, Z. & BOUDRY, P. 2004. Evidence for the presence of

the Portuguese oyster, Crassostrea angulata in northern China. Journal of Shellfish Research,

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MAGUIRE, G. B. & NELL, J. A. 2005. History, status and future of oyster culture in Australia. The 1stInternational Oyster Symposium Proceedings, Oyster Research Institute News No.19.

MASAOKA, T. & KOBAYASHI, T. 2005. Species identification of Pinctada imbricata using intergenic

spacer of nuclear ribosomal RNA genes and mitochondrial 16S ribosomal RNA gene regions.

Fisheries Science, 71, 837-846.

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highlights taxonomic ambiguities of Malaysian Crassostrea (Sacco, 1897) oyster species. The 7th IMT-GT UNINET and the 3rd Joint International PSU-UNS Conferences, Proceedings, 7-8 October 2010, Prince of Songkla University, Hat Yai, Songkhla, Thailand / Prince of Songkla

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Chapter 2:

Reproductive neuropeptides that stimulate spawning in the Sydney

rock oyster Saccostrea glomerata

Vu Van Ina,b, Nikoleta Ntalamagkaa, Wayne O’Connora,c, Tianfang Wanga, Dan Powella, Scott F. Cumminsa and Abigail Elizura,d

Published on Peptide 82 (2016) 109 – 119

a University of the Sunshine Coast, Maroochydore, Queensland 4558, Australia

b Northern National Broodstock Center for Mariculture, RIA1, Catba Islands, Haiphong, Vietnam

c Industry and Investment NSW, Department of Primary Industries, Port Stephens Fisheries Institute, Taylors Beach, NSW, 2316, Australia

d Corresponding author at: Genecology Research Center, Faculty of Science Health and Education, University of the Sunshine Coast, Locked Bag 4, Maroochydore Dc, QLD 4558, Australia. Tel.:+61 7 5430 2831. E-mail address:

Abstract

The Sydney rock oyster, Saccostrea glomerata, is a socio-economically important species

in Australian waters. Little is known at the molecular level about this species in terms of reproduction. To address this gap, we have performed a combination of high throughput transcriptomic and peptidomic analysis, to identify genes and neuropeptides that are expressed

in the key regulatory tissues of S. glomerata, the visceral ganglia and gonads (male and female).

Special focus was made on the neuropeptides since they encompass a diverse class of chemical messengers that play functional roles in many aspects of an animal’s life, including reproduction. Approximately 28 neuropeptide genes were identified, primarily within the visceral ganglia that encode precursor proteins containing numerous neuropeptides; some were confirmed through mass spectral peptidomics analysis of visceral ganglia. Of those, 28 bioactive neuropeptides were synthesized, and then tested for their capacity to induce gonad development and spawning. ELH, GnRH, APGWamide, buccalin, CCAP and LFRFamide were found to trigger spawning in ripe animals. Upon further testing, APGWa and buccalin demonstrated a capacity to also advance

conditioning and gonadal maturation. In summary, our analysis of S. glomerata has identified

neuropeptides that can influence the reproduction cycle of this species, specifically by accelerating gonadal maturation and triggering spawning. Other molluscan neuropeptides

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identified in this study will enable further research into understanding the neuroendocrinology of oysters, which may benefit their cultivation.

Keywords: Molluscs, Egg-laying hormone, Gonadotropin-releasing hormone, Mass

spectrometry, Neuropeptides, Saccostrea glomerata, Reproduction.

2.1 Background

The Sydney rock oyster, Saccostrea glomerata, is one of the most ecologically and commercially important species of the oyster family (Ostreidae) in Australian waters. In the wild,

it dominates sheltered shorelines of intertidal and immediate subtidal regions along the Eastern Australian coast. It also forms the basis of an extensive oyster industry in South-East Queensland

and New South Wales. S. glomerata production is one of the oldest aquaculture industries in

Australia and its current production has reached 7,793,390 dozen of oysters (at farm gate), valued at around $34.7 million for 2014/2015 (Trenaman et al., 2015).

Critical to the production and marketing of S. glomerata is their physical and reproductive condition. S. glomerata is a protandric species, where the gonad condition cycles are broadly

understood, and to some extent can be manipulated through gross environmental changes (O'Connor et al., 2008a, O'Connor et al., 2008b). However, our understanding of the molecular and biochemical processes underpinning changes in gonadal condition, as well as our capacity to

monitor these changes, is limited. Thus, our ability to manipulate maturation and spawning in S. glomerata is also limited. The identification of neuropeptides that may regulate S. glomerata

reproduction provides an important research area which should contribute to our understanding

of S. glomerata biology as well as help to facilitate hatchery production (Dove and O'Connor,

2009, Nell, 2006).

Neuropeptides are produced and released by neurons through a regulated secretory pathway (Burbach, 2011). They represent a highly diverse and multifunctional group of signalling molecules that include hormones, neurotransmitters and neuromodulators (Conzelmann et al., 2013, Stewart et al., 2014). Their roles in the molluscan physiology, behaviour and reproduction are well established (Fricker, 2012, Morishita et al., 2010), and includes APGWamide, gonadotropin releasing hormone (GnRH), and egg-laying hormone (ELH), which have each been

investigated through in vitro or in vivo studies. These neuropeptides are generated from

precursor sequences (Cummins et al., 2011, Morishita et al., 2010, Nuurai et al., 2010).

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In the oysters, it has been demonstrated that the tetrapeptide APGWa plays a role in the

reproduction of Crassostrea gigas female oysters, where it can induce in vitro adductor muscle

contraction followed by oocyte release (Bernay et al., 2006). GnRH is a well-known reproductive regulator in vertebrates, but has also been found in the CNS of bivalve molluscs (Bigot et al., 2012, Nakamura et al., 2007. GnRH-like peptides have been identified in the visceral, cerebral

and pedal ganglia of scallops Patinopecten yessoensis, the oyster: C. gigas and P. fucata (Treen,

2012, Pazos and Mathieu, 1999, Stewart et al., 2014). In support of their role in reproduction,

there exists a highly expressed orthologue of the GnRH receptor in mature gonads of C. gigas (Morishita et al., 2010). Also, in vitro trials in C. gigas and the mussel Mytilus edulis show that

GnRH stimulates proliferation of gonadal cells (Pazos and Mathieu, 1999). Finally, the ELH is a

well-known spawning inducer in the aquatic snails Aplysia and Lymnaea (Conn and Kaczmarek, 1989, Strumwasser et al., 1987) and its gene sequence has been identified in C. gigas and P. fucata (Stewart et al., 2014). Numerous other molluscan neuropeptides exist, although their role

in reproduction is less well known.

The main objective of the present study was to identify neuropeptide genes that may be

important in regulating S. glomerata reproduction. We focused on the neural and gonad tissues through transcriptome and peptidomic analysis, and then performed in vivo maturation and

spawning bioassays to elucidate potential reproduction-associated roles.

2.2 Materials and methods 2.2.1 Experimental Animals

Animals for tissue collection for RNA

Wild live adult S. glomerata were obtained from Port Stephens Fisheries Research

Institute, New South Wales (PSFI). The stage of gonadal development of each individual was determined (stages I-V) as described by Dinamani (1974). Gonad and visceral ganglia tissues were isolated from each individual within three out of five gonadal development stages: 1) stage I - Ripening; 2) stage II - Fully ripe and 3) Stage III - Post spawning (20-25 oysters/each stage, N=70) in July, 2012. Tissues from males and females were kept separately for gonad and ganglia at -80oC until used for total RNA and peptide extraction.

Animals for physiological bioassays

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Live adult S. glomerata from wild and hatchery lines were obtained from either local retail

outlets on the Sunshine coast, QLD (for bioassays carried out at University of the Sunshine Coast, USC) or from PSFI (for those carried out at PSFI). Animals were acclimatized in culture tanks for at least 24 h, fed with algae before used for the experiments.

2.2.2 Transcriptomics and peptidomics

The overall procedure applied in this study is shown in Figure 2.1.

Figure 2.1. Workflow of the experiments to identify neuropeptides of S. glomerata using

transcriptomics and peptidomics. Position of the visceral ganglia and gonad is shown in schematic [modified from (Paul, 1964)].

Detecting peptides in each fractionList of posible peptide precursors

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RNA extraction and transcriptome sequencing

Total RNA was isolated using TRIsureTM Reagent (Bioline USA Inc.) following the manufacturer’s specifications. The quality and concentration of the total RNA were checked by gel electrophoresis and spectrophotometry (Nanodrop 2000, Thermo Scientific, USA). Total RNA of each sex was pooled separately from all developmental stages (stages I-V). Twenty

micrograms of total RNA of each tissue were freeze dried and sent to BGI for de novo

transcriptome sequencing. BGI conducted RNA-Seq with 2 lanes of 100 paired-end sequencing

on the illumine HiSeq2000, assembly and functional annotation. De novo assemblies were

performed by SOAPdenovo software using trimmed reads from the Illumina sequencing. The assembler was run with the parameter sets as following: seqType, fq; minimum kmer coverage = 4; minimum contig length of 100 bp; group pair distance = 250.

Bioinformatics analyses

To identify target sequences, gender-specific transcriptomes for the gonadal and visceral

ganglia of S. glomerata were imported into the CLC Main Workbench (v7.0.2; CLC-bio, Denmark).

Previously identified molluscan neuropeptide sequences were then queried (tBLASTn) against

the transcriptomes. To complement this, Open Reading Frames (ORF) were retrieved from the S. glomerata databases and screened for signal sequences using SignalP 4.0 Server

( The presence of recurrent KK; KR; RK cleavage sites was identified using NeuroPred (Web-based software on Multiple sequence alignments were done by MEGA software version 6.06 (Tamura et al., 2013). Derived and amino acid sequences were aligned, guided by chain cleavage sites and conserved cysteines (Brunak et al., 1991) Domain graph 2.0 and Miktex-2.9 were used to build the peptide schematics and sequence alignments, respectively. Data of other species used for alignment of amino acid sequences and schematic diagrams was obtained from the supplementary list of neuropeptides provided by Stewart et al. (2014). Web-based Clustal Omega (

was used to estimate percentage of identity between S. glomerata peptide amino acid sequences

and other mollusc species.

Reverse phase-high performance liquid chromatography (RP-HPLC)

The collected ganglia tissues were homogenized on ice in a solution of 0.1% Trifluro-acetic acid (TFA – Solution A), with subsequent sonication consisting of three times 30 s pulses separated by 20 seconds. The homogenized tissues were then centrifuged at 16,000 rpm for 20

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minutes at 4°C and the supernatants were collected. This process was repeated with the pellet leftover. The extracted peptide mixture was analysed by RP-HPLC (PerkinElmar series 200 pump/autosampler, Flexar PDA detector and Chromera v3.2 software). The total collected peptides from the extractions were loaded on the HPLC. Samples were separated and eluted with a protocol of 100% to 40% solution A at a flow rate of 1 mL/min over 20 min for the synthetic peptides and 60 min for the extracted peptide mixture. Eluted compounds were detected at wavelengths of 210 nm and 280 nm. Mobile phases used were solution A (0.1% TFA) and solution B (0.1% TFA in acetonitrile). A total of 12 fractions were collected in 5 min intervals for further analysis by mass spectroscopy (MS). Control synthetic peptides were tested in RP-HPLC and observed to elute at 42.5% acetonitrile for GLDRYSFMGGI-NH2; 43.5% acetonitrile for GMPMLRL-NH2; 42% acetonitrile for MRYFL-NH2; and 58.5% acetonitrile for RPGW-NH2. Five-minute fractions were lyophilised and resuspended in 1% formic acid for MS analysis.

Mass spectrometry analysis (LC-MS/MS analysis) and protein identification

Resuspended HPLC fractions were analyzed by LC-MS/MS on a Shimadzu Prominance Nano HPLC (Japan) coupled to a Triple T of 5600 mass spectrometers (ABSCIEX, Canada) equipped with a nano electrospray ion source. The protocol has been detailed elsewhere [25]. Briefly, approximately 6 µL of each extract was injected and de-salted on the trap column before entering a nano HPLC column (Agilent Technologies, Australia) for mass spectrometry analysis. The mass spectrometer acquired 500 ms full scan TOF-MS data followed by 20 by 50 ms full scan product ion data. Full scan TOFMS data was acquired over the mass range 350-1800 and for product ion MS/MS 100-1800. Ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion. The data were acquired and processed using Analyst TF 1.5.1 software (ABSCIEX, Canada).

Fragmentation data was analyzed by PEAKS v6.0 (BSI, Canada) software. Sequences of peptides were determined by comparing the fragmentation patterns with those predicted from

the S. glomerata transcriptomes. Search parameters were as follows: no enzyme was used;

variable modifications included methionine oxidation, conversion of glutamine/glutamate to pyroglutamic acid, deamidation of asparagine and peptide amidation. Precursor mass error tolerance was set to 0.1 Da and a fragment ion mass error tolerance was set to 0.1 Da. de novo sequencing, database search and characterising unspecific post-translational modifications (PTMs) were used to maximise the identifications; false discovery rate (FDR) was set to ≤ 1%, and

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