FACTORS INFLUENCING ADULT GONAD PRODUCTION AND LARVAL GROWTH
AND SURVIVAL OF THE PURPLE SEA URCHIN (Strongylocentrotus purpuratus)
by
Md. Abul Kalam Azad
M. Sc. (Fisheries Biology), Bangladesh Agricultural University, Bangladesh, 1997 M. Sc. (Aquaculture), Asian Institute of Technology, Thailand, 2002
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
The Faculty of Graduate Studies
(Animal Science)
THE UNIVERSITY OF BRITISH COLUMBIA
(Vancouver)
August 2011
© Md. Abul Kalam Azad, 2011
Abstract
The continued demand for sea-urchin gonads and overexploitation of natural stocks in many countries have stimulated interest in the aquaculture of various sea-urchin species. The overall aim of this thesis was to develop rearing protocols for larval and adult purple sea urchins
(Strongylocentrotus purpuratus). The specific objectives of this thesis were to determine: (1) the effect of temperature and diet on adult gonad production and (2) the effect of temperature, microalgal diet, ration, and stocking density on embryonic/larval growth and survival. To test these objectives I measured ingestion rate, gravimetric absorption, assimilation efficiency, and various gonad attributes (i.e. wet weight, water content, gonad index, texture, firmness, colour, flavour, and maturity stage) in the experiments using adult sea urchins and developmental progression from egg to prism stage, embryo length, larval length, larval mid-line body length, larval body width, larval post-oral arm length, time to reach metamorphic competency, and survival rate in the embryo/larval experiments. Adult purple sea urchins produced the highest quality gonads at 12°C when fed a prepared diet developed specifically for urchin gonad production by the Norwegian Institute of Fisheries and Aquaculture. Embryos and larvae showed more normal development and had significantly higher percent survival when reared at 11 and
14°C than at 8 and 17°C with best growth and survival occurring at stocking densities ≤100 ind ml-1 of eggs/embryos and ≤1.0 ind ml-1 of larvae. The single-species algal diet of Dunaliella tertiolecta and the bi-species diet of D. tertiolecta and Isochrysis sp. supported the highest larval growth and percent survival of all phytoplankton diets evaluated. Larvae fed an increasing diet ration (i.e. 2,500–10,000 cells ml-1, according to developmental stage) of a mixed-species algal diet (D. tertiolecta and Isochrysis sp.) generally had significantly better growth and survival than any fixed rations (i.e. same ration throughout development) evaluated. Overall, the results from
ii this study suggest that S. purpuratus could be an excellent potential candidate species for aquaculture development.
iii Preface
For this thesis, I contributed to the development of the experimental protocols, conducted the research, performed the statistical analyses, interpreted the data, and prepared the various chapters. I have written all parts of the manuscripts mentioned in this dissertation.
All of my committee members (Dr. Abayomi Alabi, Dr. Colin Brauner, Dr. Marina von
Keyserlingk, Dr. Christopher M. Pearce, and Dr. Robert Scott McKinley) helped to conceive the design of the original experiments. Drs. McKinley and Pearce provided overall supervision and guidance to me and ensured that the experiments were conducted appropriately and that data analyses were correctly performed.
As co-authors, Drs. McKinley and Pearce reviewed and provided critical comments on all of the manuscripts included in this dissertation. A version of Chapter 1 has been published [Azad
A.K., McKinley S. and Pearce C.M. (2010) Factors influencing the growth and survival of larval and juvenile echinoids. Reviews in Aquaculture 2: 121-137]. A version of Chapter 2 has been published [Azad A.K., Pearce C.M. and McKinley R.S. (2011) Effects of diet and temperature on ingestion, absorption, assimilation, gonad yield, and gonad quality of the purple sea urchin
(Strongylocentrotus purpuratus). Aquaculture 317: 187-196]. A version of Chapters 3 and 4 has been published [Azad A.K., Pearce C.M. and McKinley R.S. (2011) Influences of stocking density and temperature on early development and survival of the purple sea urchin,
Strongylocentrotus purpuratus (Stimpson, 1857). Aquaculture Research: in press]. A version of
Chapters 5 and 6 has been accepted for publication [Azad A.K., Pearce C.M. and McKinley R.S.
(2011) Influence of algal species and dietary rations on larval development and survival of the purple sea urchin, Strongylocentrotus purpuratus (Stimpson, 1857). Aquaculture: accepted].
iv Drs. Alabi, Brauner, and Keyserlingk provided critical comments on the manuscript in Chapter 1 as well as all of the chapters in this dissertation.
Dr. Pearce secured introduction and transfer permits from the Pacific Region Introductions and Transfers Committee for the collection of experimental animals and obtained approval of protocols (Animal Use Protocol references : 08-012 and 09-020) for experimental use of animals from the Fisheries and Oceans Canada (DFO) Pacific Region Animal Care Committee (PRACC).
Dr. Pearce provided laboratory space at the Pacific Biological Station and ensured that the experiments were conducted in accordance with the guidelines of the DFO PRACC and the guidelines of the Canadian Council on Animal Care (CCAC).
v Table of contents
Abstract ...... ii
Preface...... iv
Table of contents ...... vі
List of tables...... x
List of figures ...... xii
Acknowledgements ...... xvi
Dedication ...... xviii
CHAPTER 1: Introduction ...... 1 1.1 General introduction...... 1 1.2 General biology and ecology of sea urchins ...... 6 1.3 Aquaculture potential of sea urchins...... 11 1.4 Factors influencing the growth and survival of larval and juvenile echinoids: a review.....13 1.4.1 Larval production ...... 14 1.4.1.1 Food: phytoplankton ...... 14 1.4.1.2 Food: prepared diets...... 22 1.4.1.3 Food: dissolved organic matter ...... 24 1.4.1.4 Stocking density ...... 25 1.4.1.5 Temperature ...... 26 1.4.2 Juvenile production ...... 29 1.4.2.1 Food: algae ...... 29 1.4.2.2 Food: prepared diets...... 35 1.4.2.3 Stocking density ...... 38 1.4.2.4 Temperature ...... 40 1.5 The experimental animal...... 42 1.6 Hypotheses and objectives ...... 45
vi 1.6.1 Experiment 1: Influence of diet and temperature on adult gonad production...... 45 1.6.2 Experiment 2: Influence of stocking density on early development and survival ...... 46 1.6.3 Experiment 3: Influence of temperature on early development and survival ...... 47 1.6.4 Experiment 4: Influence of algal species on larval growth and survival ...... 47 1.6.5 Experiment 5: Influence of dietary rations on larval growth and survival...... 48
CHAPTER 2: Influence of diet and temperature on gonad production of the purple sea urchin...... 50 2.1 Introduction ...... 52 2.2 Materials and methods ...... 54 2.2.1 Experimental urchin collection and maintenance ...... 54 2.2.2 Experimental protocols ...... 55 2.2.3 Statistical analysis ...... 60 2.3 Results ...... 60 2.3.1 Initial sea-urchin attributes...... 60 2.3.2 Ingestion rate...... 64 2.3.3 Gravimetric absorption...... 65 2.3.4 Assimilation efficiency ...... 67 2.3.5 Gonad quantity ...... 67 2.3.5.1 Gonad weight ...... 67 2.3.5.2 Gonad index ...... 68 2.3.6 Gonad quality ...... 69 2.3.6.1 Gonad water ...... 69 2.3.6.2 Gonad firmness, texture, and colour ratings ...... 69 2.3.6.3 Gonad colour (L*, a*, b* values)...... 69 2.4 Discussion ...... 73
CHAPTER 3: Influence of stocking density on early development and survival of the purple sea urchin ...... 80 3.1 Introduction ...... 81 3.2. Materials and methods ...... 82 3.2.1 Broodstock collection and general experimental techniques ...... 82
vii 3.2.2 Experimental protocols ...... 86 3.2.2.1 Influence of stocking density on embryonic development...... 86 3.2.2.2 Influence of stocking density on larval development...... 87 3.2.3 Statistical analysis ...... 88 3.3 Results ...... 88 3.3.1 Influence of stocking density on embryonic development...... 88 3.3.2 Influence of stocking density on larval development...... 90 3.4 Discussion ...... 94
CHAPTER 4: Influence of temperature on early development and survival of the purple sea urchin ...... 98 4.1 Introduction ...... 99 4.2. Materials and methods ...... 100 4.2.1 Broodstock collection and general experimental techniques ...... 100 4.2.2 Experimental protocols ...... 100 4.2.2.1 Influence of temperature on embryonic development...... 100 4.2.2.2 Influence of temperature on larval development...... 101 4.2.3 Statistical analysis ...... 102 4.3 Results ...... 106 4.3.1 Influence of temperature on embryonic development...... 106 4.3.2 Influence of temperature on larval development...... 107 4.4 Discussion ...... 110
CHAPTER 5: Influence of microalgal species on larval development and survival of the purple sea urchin...... 112 5.1 Introduction ...... 113 5.2. Materials and methods ...... 114 5.2.1 Broodstock collection and algal rearing protocols...... 114 5.2.2 Spawning protocols and experimental techniques ...... 115 5.2.3 Statistical analysis ...... 116 5.3 Results ...... 117 5.4 Discussion ...... 121
viii
CHAPTER 6: Influence of dietary rations on larval development and survival of the purple sea urchin ...... 125 6.1 Introduction ...... 126 6.2 Materials and methods ...... 127 6.2.1 Broodstock collection and general experimental techniques ...... 127 6.2.2 Spawning protocols and experimental techniques ...... 127 6.2.3 Statistical analysis ...... 128 6.3 Results ...... 132 6.4 Discussion ...... 134
CHAPTER 7: Conclusions ...... 137
Bibliography ...... 148
Appendix: Conference presentations ...... 181
ix List of tables
Table 1.1 Potential candidate sea-urchin species for aquaculture in various countries of the world………………………………………………………...... 3 Table 1.2 Summary of published rearing diets and conditions used for larval echinoids…………………………………………………………………………..15 Table 1.3 Summary of the published rearing diets and conditions used for juvenile echinoids…………………………………………………………………………..30 Table 2.1 Proximate composition (%) and energy content of experimental diets (based on 100-g wet sample) ……………………………………………………..55 Table 2.2 Criteria for assessment of gonad quality of the sea urchin Strongylocentrotus purpuratus…………………………………………...... 58 Table 2.3 Mean (±SE) initial attributes of experimental sea urchins in different temperature groups and ANOVA results for each attribute comparing among the three temperatures…………………...... 61 Table 2.4 Results of separate two-way ANOVAs on various attributes of the experimental urchins at the end of the experiment. Sources of variation are temperature (T, fixed factor), diet type (D, fixed factor), interaction (T×D), and error. NS= not significant at P>0.05 or more……………………………………………………………………………….62 Table 3.1 Concentration of Dunaliella tertiolecta fed to Strongylocentrotus purpuratus larvae over time. The estimated number of algal cells per larva (taking larval survival into consideration) is given in parentheses………………………………………………………………………..85 Table 3.2 Results of separate one-way ANOVAs or Kruskal-Wallis tests (H values in bold) on various attributes of embryos and larvae of Strongylocentrotus purpuratus. Source of variation is stocking density…………………………………………………………………………….91 Table 3.3 Qualitative and quantitative observations on embryonic development of Strongylocentrotus purpuratus at various stocking densities (mean±SE). Percent survival based on total number of
x eggs/embryos on culture container bottom………………………………………..91 Table 4.1 Results of separate one-way ANOVAs or Kruskal-Wallis tests (H values in bold) on various attributes of embryos and larvae of Strongylocentrotus purpuratus. Source of variation is temperature……...... 103 Table 4.2 Qualitative and quantitative observations on embryonic development of Strongylocentrotus purpuratus at various temperatures (mean±SE). Percent survival based on total number of eggs/embryos on culture container bottom…………………………...... 104 Table 4.3 Influence of temperature on the rate of development (final total length) of larval Strongylocentrotus purpuratus. The Q10 values were calculated over each respective 3°C interval…………………………………….107 Table 5.1 Shape, biovolume, and proximate composition of algae used to feed larval Strongylocentrotus purpuratus, based on previously reported literature values………………………………………………………..115 Table 5.2 Algal diets and concentration on different days during the larval culture period. Cell densities in different algal treatments were standardized to give equal bio-volumes of algae………………………………...118 Table 5.3 Results of separate one-way ANOVAs on various attributes of larvae of Strongylocentrotus purpuratus. Source of variation is algal diet……………………………………………………………………………….118 Table 6.1 Results of separate one-way ANOVAs on various attributes of larvae of Strongylocentrotus purpuratus. Source of variation is algal diet ration………………………………………………………………………...129
xi List of figures
Figure 2.1 Mean ingested (A) wet weight and (B) dry weight in the various diet and temperature treatments at weeks 6 and 12. Error bars are SE and n=6. Letters above bars indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among all 12 treatments within weeks……………………………………………………………………….64 Figure 2.2 Mean (A) gravimetric absorption and (B) assimilation efficiency of ingested diets in the various diet and temperature treatments at weeks 6 and 12. Error bars are SE and n=6. Letters above bars indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among temperature treatments (averaged across diets) within weeks. Numbers (for week 6) and letters (for week 12) beside diet treatments in legends indicate the results of Tukey’s HSD multiple- comparison tests with different letters showing significant (P<0.05) pair-wise differences among diet treatments (averaged across temperatures) within weeks………………………………………………………..66 Figure 2.3 Mean (A) final gonad weight, (B) gonad weight gain, and (C) gonad index in the various diet and temperature treatments. Error bars are SE and n=6. Letters above bars indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among all 12 treatments. Numbers beside diet treatments in legend in C indicate the results of a Turkey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among diet treatments (averaged across temperatures)………………………………..……….68 Figure 2.4 Mean gonad (A) percent water, (B) firmness rating, (C) texture rating, and (D) colour rating in the various diet and temperature treatments. Error bars are SE and n=3 for gonad percent water and
xii n=6 for all other variables. Letters above bars indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among temperature treatments (averaged across diets). Numbers beside diet treatments in legend indicate the results of a Turkey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among diet treatment (averaged across temperatures)……………………………………………………………….70 Figure 2.5 Mean gonad (A) L* value, (B) a* value, (C) b* value, and (D) taste rating in the various diet and temperature treatments. Error bars are SE and n=3 for taste rating and n=6 for all other variables. Letters above bars indicate the results of Tukey’s HSD multiple- comparison tests with different letters showing significant (P<0.05) pair-wise differences among all 12 treatments……………………………………..71 Figure 2.6 Frequency (%) of gonads at different stages of maturity at week 0 and week 12 in the various temperature and diet treatments…………………………...72 Figure 3.1 (A) Egg/embryo sampling areas on the bottom of the culture container on different days (D) of embryonic development. (B)Microphotograph of larva of Strongylocentrotus purpuratus illustrating body dimensions measured: (a) larval total length, (b) mid-line body length (c) body width, and (d) post-oral arm length…………………………………………………………….87 Figure 3.2 Mean (±SE) embryo length (μm) at various stocking densities over time. n=3. Letters above bars indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day………………………………………………………………………89 Figure 3.3 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival at various stocking densities over time. n=4. Letters above bars indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among the
xiii treatments on the final sampling day……………………………………...... 94 Figure 4.1 Mean (±SE) embryo length (μm) at various temperatures over time. n=3. Letters above bars indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day……………………………………………………………………..106 Figure 4.2 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival at various temperatures over time. n=4. Letters above bars indicate the results of Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day………………………………………….109 Figure 5.1 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival on various algal diets over time. n=3. D= Dunaliella tertiolecta, C= Chaetoceros muelleri, I= Isochrysis sp. (Tahitian strain), DI= D. tertiolecta + Isochrysis sp., DC=D. tertiolecta + C. muelleri, DCI=D. tertiolecta + C. muelleri + Isochrysis sp. Letters above bars indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day……………………………………….....120 Figure 6.1 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival on various diet rations over time. n=3. Low ration=1.25 x 103 cells ml-1; Normal ration= 2.5 x 103 cells ml-1; Standardized ration= 2.5 x 103 cells to 10.0 x 103 cells ml-1, with increasing ration according to developmental stage; Medium ration= 5.0 x 103 cells ml-1; and High ration= 10.0 x 103 cells ml-1. Letters above bars indicate the results of Tukey’s multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among the treatments………………...... 132 Figure 6.2 Percent ratio of larval body width and total length over time. Error bars are SE and n=3. Low ration=1.25 x 103 cells ml-1; Normal
xiv ration= 2.5 x 103 cells ml-1; Standardized ration= 2.5 x 103 cells to 10.0 x 103 cells ml-1, with increasing ration according to developmental stage; Medium ration= 5.0 x 103 cells ml-1; and High ration= 10.0 x 103 cells ml-1. Letters above bars indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day……………………………………………..134
xv Acknowledgements
My heartfelt thanks to my committee members [Dr. Abayomi Alabi (Seed Science Ltd.), Dr.
Colin Brauner (Department of Zoology, the University of British Columbia), Dr. Marina von
Keyserlingk (Animal Welfare Program, Faculty of Land and Food Systems, the University of
British Columbia), Dr. Christopher M. Pearce (Fisheries and Oceans Canada), and Dr. Robert
Scott McKinley (Centre for Aquaculture and Environmental Research, Fisheries and Oceans
Canada/the University of British Columbia)] for their continuous consultations, encouragements, collective thoughtfulness, and intellectual lessons throughout the PhD process. Their expertise in biological and hypothesis-driven science was evident in their thoughtful advice that kept me on the right track. I wish to express my sincerest thanks to Dr. Pearce whose scientific prowess and tutelage have dramatically improved my abilities to know the biology and ecology of echinoids as an “echiniculturist”. An excellent advisor, encourager, and mentor, Dr. Pearce guided me through to the completion of this thesis and, for that, I am grateful. The Pacific Biological Station
(PBS) has been a fantastic place to do my PhD research. I immensely benefited from the services of various PBS staff: John Blackburn, Dr. Anya Epelbaum, Sean Williams, Wolfgang Carolsfed,
Doug Brouwer, and Joanne Lessard who helped collect sea urchins and kelp during the study period; and William Bennett who helped make slides for gonad histology. Laurie Keddy deserves special mention for all her help in the algal culture lab, tolerance of bleach fumes, and especially for listening to me about the delicate larvae of sea urchins and their diets for more hours than I can count. Dr. Sten I. Siikavuopio (Norwegian Institute of Fisheries and Aquaculture) provided the prepared diet used to feed the adult sea urchins and Paddy Wong (Paladin International Food
Sales Ltd.) helped to evaluate gonad quality. I am thankful for the many hours of lab assistance contributed by John Blackburn, Dr. Wenshan Liu (Fisheries and Oceans Canada), Robert
xvi Marshall (PhD student, the University of British Columbia) and Janis Webb (MSc student,
University of Victoria). Undergraduate students from Vancouver Island University, including
Kate Rolheiser and Mohammad Bashar, helped count larvae and algal cells during their practicum courses. I am forever indebted to Gordon Miller and George Pattern (PBS Library) for their wisdom on library archives. Prof. C. Kwei Lin (Asian Institute of Technology, Thailand),
Dr. Kathe R. Jensen (University of Copenhagen, Denmark), Erik H.J. Keus (Senior Advisor,
Danida Aquaculture Project, Vietnam), Ms. Rahima Khatun (Program Manager, Danida
Aquaculture Extension Project, Bangladesh), Dr. Mahfujul Haque, and Prof. Mohsin Ali (Faculty of Fisheries, Bangladesh Agricultural University, Bangladesh) deserve special thanks for their encouragement and mental support. I am grateful to my external examiner [Prof. John M.
Lawrence (University of South Florida, Tampa, USA)], as well as internal examiners [Dr. Rachel
Fernandez (Department of Microbiology and Immunology), Dr. Patricia Schulte (Department of
Zoology), and Dr. Daniel M. Weary (Animal Welfare Program)] for their critical review, helpful comments, and valuable advice that greatly improved the contents of this dissertation. I also wish to express gratitude towards my funding sources: the Ontario Student Assistance Program, the Graduate Study Program at the University of British Columbia, as well as Fisheries and
Oceans Canada. Without their assistance, we still would be unaware of the potential of the purple sea urchin, Strongylocentrotus purpuratus, as an aquaculture candidate species.
My father Abul Hassan and maternal uncle Yousuf Ali played inspiring roles for academic ambition and this PhD is really a result of their years of love and encouragement. Special thanks to my wife Shamima (Rina) for her unwavering support and whole-hearted encouragement throughout the journey. My dear daughter and brand-new baby boy also kept me loaded with fun and joy during this journey. I hope this dissertation may provide inspiration for our daughter
Ananna Azad (Anu) and son Abir Azad (Orchid).
xvii
Dedication
To my grandmother “Surupa” who raised me with affection in the absence of my mother “Joygun”– both of them blessing me from paradise.
xviii CHAPTER 1: Introduction1
1.1 General introduction
Sea urchins are one of the most-prized seafood delicacies worldwide and have been long used as a food source by humans, especially in Japan, China, Korea, France, and Chile. Although consumed as a delicacy in many countries for decades, the large international harvesting operations are a relatively recent development over the last 30 years.
Sea-urchin gonads, termed “roe” or “uni” in the commercial trade, are a valuable export commodity on the Japanese sushi market which consumes more than 80% of the world commercial catch (Agatsuma 2010). France is the world’s second largest consumer of sea-urchin roe, consuming approximately 1,000 tonnes per year (Hagen 1996, BIM 2003). In Japan, uni is very popular as a gourmet food. It is marketed in several forms: fresh (Nama uni), frozen (Reito uni), baked and frozen (Kaiyaki uni), steamed (Mushi uni), and salted (Shio uni). Two fermented products, Neri uni-a blended paste and Tsubi uni-a lumpy paste, are also popular (Krause 2003,
Lawrence 2007). Domestic production from Japan has been declining over the years and currently fluctuates between 11,000 and 15,000 tonnes per year (Agatsuma 2010). The country imports about 80–85% of its uni supply. The major suppliers of roe to Japan are, in order of the import market share: Chile (51.7%), USA (21.5%), Canada (8%), North Korea (5%), and South
Korea (4.8%). Significant supplies of live, whole sea urchins are also provided by Russia (10,597 tonnes or 89%), North Korea (735 tonnes or 6%), USA (431 tonnes or 3.6%), and Canada (157 tonnes or 1.3%) (Krause 2003, Agatsuma 2010).
1 A version of this chapter has been published. Azad A.K., McKinley S. and Pearce C.M. 2010. Factors influencing the growth and survival of larval and juvenile echinoids. Reviews in Aquaculture 2: 121-137
1 A major portion of production of sea-urchin roe in the northern hemisphere comes from the red sea-urchin (Strongylocentrotus franciscanus) and green sea-urchin (Strongylocentrotus droebachiensis) fisheries. Red sea urchins (S. franciscanus) are harvested primarily to export processed roe whereas green sea urchins are harvested for live export, mainly to the Japanese market (Perry et al. 2002, Krause 2003). In Chile, sea urchins are viewed as a traditional food, known as “erizo” (Pérez et al. 1995), and the Chilean fishery for Loxechinus albus dominates the world production of sea urchins. In the last few years the Chilean fishery has helped to stabilize overall world production, although fishery landings are already in decline in that country
(Cárcamo et al. 2005).
World production from sea-urchin fisheries peaked in 1995 with a global landing of 120,306 tonnes (Andrew et al. 2002). After a continuous 45 years of increasing world sea-urchin harvest, the total global fisheries has now begun to decrease and in very recent years has dropped 16% in weight (Robinson 2004). The decline in sea-urchin fisheries in major producing countries combined with the strong demand for sea-urchin products have sparked a recent scientific and commercial interest in the aquaculture of a variety of echinoid species (Table 1.1). While the
Japanese have been practising sea-urchin culture or “echiniculture” on a commercial scale for decades (primarily for wild-stock enhancement), it is still in its infancy in most other countries.
Only cultivation processes totally independent of natural stocks – i.e. controlling the complete life cycle of the echinoid – will lower the pressure imposed by fisheries upon natural populations
(Grosjean et al. 1998).
2 Table 1.1 Potential candidate sea-urchin species for aquaculture in various countries of the world.
Urchin species Common name Local name Study location Reference
Anthocidaris crassispina Murasaki uni Japan Kitamura et al. 1993, Rahim et al. 2004,
(Japan) China Liu et al. 2010
Echinus esculentus Edible urchin Scotland Jimmy et al. 2003
Evechinus chloroticus Kina New Zealand Barker and Fell 2004, James et al. 2007
Glyptocidaris crenularis Haidan China Chang and Wang 2004
Loxechinus albus Green urchin Erizo Chile Pérez et al. 1995, Cárcamo et al. 2005
Lytechinus variegatus Green urchin USA, Venezuela Wa tts et al. 1998, George et al. 2004,
Buitrago et al. 2005
Paracentrotus lividus Purple urchin European sea France, Ireland, Byrne 1990, Basuyaux and Blin 1998,
urchin Spain Liu et al. 2007b
Psammechinus miliaris Green urchin Scotland Cook et al. 1998, Kelly 2002
Pseudocentrotus depressus Aka uni Japan Agatsuma et al. 2004
Strongylocentrotus droebachiensis Green urchin Kråkebolle USA, Canada, Pearce et al. 2002a,b,c, 2004,
(Norway) Norway Robinson et al. 2002, Devin et al. 2004,
Kennedy et al. 2007a,b, Siikavuopio et al. 2007
3 Urchin species Common name Local name Study location Reference
Strongylocentrotus franciscanus Red urchin USA, Canada McBride et al. 1998, 2004, Alabi et al. 2001
Strongylocentrotus intermedius Ezo uni (Japan) Japan, China Sakai et al. 2004, Agatsuma et al. 2006,
Xing et al. 2007, Liu et al. 2010
Strongylocentrotus nudus Kita uni Japan, China Agatsuma 1998, Sakai et al. 2004,
(Japan) Agatsuma et al. 2006, Liu et al. 2010
Strongylocentrotus purpuratus Purple urchin USA, Canada Morris and Campbell 1996
Tripneustes gratilla Swaki Australia Dworjanyn et al. 2007, Juinio-Meñez et al. 2008
(Philippines) Philippines
4 As interest in sea-urchin aquaculture increases there is a need for specific information on the rearing protocols for various echinoid species. This includes an examination of the effects of biotic (e.g. diet, diet ration) and abiotic (e.g. temperature, stocking density, ammonia, oxygen) factors on both gonad production of adults and growth and survival of larvae and juveniles. This dissertation examined the effects of various factors (i.e. temperature, diet, ration, stocking density) on gonad production and embryo/larval development and survival of the purple sea urchin, Strongylocentrotus purpuratus. The thesis also suggests future directions for research on echinoid rearing methods that need to be conducted for sustainable sea-urchin aquaculture development.
This document is organized in seven chapters. Chapter 1 is an introductory chapter that consists of the following sections: overview of sea-urchin aquaculture, general biology and ecology of sea urchins, aquaculture potential of echinoids around the world, review on the advances and innovations for larval and juvenile culture of various sea- urchin species (with references to the particular challenges remaining for the researcher and aquaculturist), biological and ecological information about the experimental animal
(S. purpuratus), and the hypotheses and objectives of the thesis. In chapter 2, I report the results of the effects of diet and temperature on yield and quality of gonads of adult S. purpuratus. Chapters 3 and 4 focus on the results of the effects of abiotic factors (i.e. stocking density and temperature) on development and survival of embryonic and larval purple sea urchins. In chapters 5 and 6 I discuss the results of the effects of biotic factors
(i.e. algal diet and diet ration) on growth and survival of larval purple sea urchins. The concluding chapter (chapter 7) focuses on the major findings of the project, strengths and limitations of the thesis, and potential areas of future research.
5 1.2 General biology and ecology of sea urchins
The biology and ecology of various edible sea urchins have recently been reviewed
(Lawrence 2007). Sea urchins are members of a large group of marine invertebrates in the phylum Echinodermata, which also includes sea stars, sand dollars, brittle stars, sea cucumbers, sea lilies, and feather stars. Sea urchins have a pentameric symmetry and a hard, calcareous shell or test. The shell is globular in shape, covered with a thin epithelium and is usually armed with spines. The spines are used for defence and can inflict a painful wound, some species being venomous. Urchins also use these spines – as well as hundreds of tiny, adhesive “tube feet” or podia – for locomotion. The tube feet are also used for holding on to the substratum and trapping drifting macroalgae for food.
Also located over the body surface are pedicellariae, small pincer-like structures mounted on stalks. Muscles at the base of the stalks allow the urchin to move the pedicellariae in various directions in response to a variety of stimuli (Campbell 1973). There are several different forms of pedicellariae, used for a variety of purposes including defence, cleaning the body surface, and holding and breaking up small debris particles (Campbell
1973, 1974). The mouth, located on the underside of the animal, consists of five calcareous plates called “Aristotle’s lantern”, named in honour of the Greek naturalist and philosopher (Kato and Schroeter 1985). Within the Aristotle’s lantern there is a buccal cavity and pharynx. The latter rises up through the lantern and passes into an oesophagus, which joins the tubular digestive tract and empties through the anus located on the top of the test. Five skeins of gonads comprise the most prominent structures in the internal cavity of sea urchins. Between the skeins of gonads are gill-like structures which are part
6 of the water vascular system, important in movement, respiration, and food gathering
(Kato and Schroeter 1985).
Sea urchins are predominantly herbivores (although some species will consume animal material) and in the wild, macroalgae (especially kelps) are the major component of a sea-urchin’s diet (Lawrence 1975). At high densities, sea-urchin overgrazing of macrophytes can lead to the formation of areas devoid of fleshy macroalgae and dominated by encrusting algae, termed coralline flats or barrens (Lang and Mann 1976,
Meidel and Scheibling 1998). Inter-annual events such as El Niño are also known to cause macroalgal dieback, which may lead to lower growth or poor gonad production of urchins due to food limitation (Tegner and Dayton 1991).
Sea urchins are unisexual and broadcast spawners, releasing their sperm and eggs into the water column for fertilisation (Levitan et al. 1992, Levitan 2005, 2006, Rogers-
Bennett 2007). The fertilised eggs hatch into blastulae/gastrulae, which in turn, evolve into a prism stage that is planktonic. With the growth of the skeleton the prism stage develops into the larval form (termed an echinopluteus or pluteus), which has a number of ciliated arms (four, six, or eight depending on the stage of development). Sea-urchin echinoplutei are able to develop for a number of days post-hatch on maternal/egg reserves
(Byrne et al. 2008a,b). Like other planktotrophic invertebrates, once the larval energy reserves are fully consumed, echinoid larvae must secure exogenous food for their development (Emlet 1986, Reitzel et al. 2005). In the natural environment, sea-urchin larvae feed on phytoplankton in the water column.
During their development, larvae go through several developmental changes from the prism-shape stage to the eight-armed echinopluteus stage (Mortensen 1914, 1931,
7 McEdward 1986). When the larvae are ready to metamorphose into the adult form they are said to be competent (Stephens 1972, Strathmann 1987, Kelly et al. 2000). Larvae are considered competent when the juvenile rudiment (i.e. primordial juvenile developing inside the larva) is fully developed and swimming behaviour is directed to the ocean floor in the wild or the walls and/or bottoms of containers in culture (Burke 1980, Fenaux et al.
1994, George et al. 2001, Cárcamo et al. 2005). The rudiment develops on the left side of the larval body, surrounded by an invaginated cavity, which remains open to the exterior at the vestibule (Strathmann 1987). It is the rudiment that gradually develops into the parent-like juvenile echinoid.
Competent larvae are thought to search for habitats for settlement that will increase their chance of survival. Swanson et al. (2004) defined settlement as the attachment of an individual to the substratum after metamorphosis, whereas Rodríguez et al. (1993) described settlement as a process beginning with the onset of a behavioural search for suitable substrata and ending with metamorphosis. In this process of settlement and metamorphosis larvae are considered to have successfully recruited, ending a critical life stage. On the Pacific coast of North America, Strongylocentrotid urchins may settle within 5–9 weeks from fertilization if the conditions are suitable (Strathmann 1978,
Cameron and Schroeter 1980). However, settlement can be delayed if food levels are low, temperature is sub-optimal, or preferred settlement cues are not encountered, and thus may ultimately result in higher larval mortality (Highsmith and Emlet 1986, Lamare and
Barker 1999).
Particular biological, chemical, and/or physical factors associated with substrata in the environment are considered to be the primary stimuli initiating larval settlement and
8 metamorphosis in marine invertebrates (Burke 1980, Pearce and Scheibling 1991, 1994,
Pawlik 1992, Pearce 1997, Martínez and Navarrete 2002, Gordon et al. 2004, Agatsuma et al. 2006). In nature, larvae of some species of sea urchins (particularly
Strongylocentrotid ones) prefer to settle on live coralline algae (Rowley 1990, Pearce and
Scheibling 1990, Kitamura et al. 1993, Lambert and Harris 2000). Field observations showed that newly settled individuals of S. franciscanus and S. purpuratus formed dense populations on rocky areas covered with crustose, coralline red algae (Rowley 1989).
Pearce and Scheibling (1990) observed that the coralline red algae, Lithothamnion glaciale, Phymatolithon laevigatum, Phymatolithon rugulosum, and Corallina officinalis induced >85% of S. droebachiensis larvae to metamorphose in laboratory trials. Swanson et al. (2006) revealed that more than 90% of newly-settled recruits of the sea urchin
Holopneustes purpurascens were located on either the foliose red alga Delasea pulchra or on coralline turf algae, while only 8% of recruits were on the brown alga
Homeostrichus olsenii.
Recently metamorphosed individuals subsist on larval reserves for the first 8–12 days post-metamorphosis until the mouth and gut become fully developed (Gosselin and
Jangoux 1998, Miller and Emlet 1999, Byrne et al. 2008a,b). Post-metamorphic juveniles that have developed a mouth and gut are able to begin grazing the surface they live on, ingesting various components of the microbial film (e.g. benthic diatoms, bacteria, protozoa) and surface epithelial cells of coralline algae (De Ridder and Jangoux 1982). A variety of benthic diatom species, especially Navicula spp., are initial preferred nutrient sources for early post-settled juveniles (Xing et al. 2007, Dworjanyn et al. 2007). At 30–
50 days old, juveniles normally switch to feeding on fleshy macroalgae (Rowley 1990).
9 Juvenile urchins may feed on various genera of foliose macroalgae including (but not limited to) Ulva, Laminaria, Porphyra, Macrocystis, and Nereocystis (Larson et al. 1980,
Kenner 1992, Pearce et al. 2005).
Newly recruited juveniles are cryptic, hiding from potential predators. In the case of
S. franciscanus and S. purpuratus, they may seek shelter under the adult spine canopy
(Nishizaki and Ackerman 2000, Pearse 2006). Young sea urchins emerge from shelter when approximately 4 mm in test diameter (or larger) and forage freely over the rocky sea bottom (Bureau 2000). Predators of North American Strongylocentrotid species include sea otters, sea stars, lobsters, rock crabs, wolf eels, and other fish species
(Himmelman and Steele 1971, Bernstein et al. 1981, Tegner and Levin 1983, Scheibling
1984, Scheibling and Hamm 1991, Hagen and Mann 1992). However, those species commonly found in the intertidal zone, including the purple sea urchin (S. purpuratus), are readily eaten by sea gulls, oystercatchers, and raccoons (Himmelman and Steele 1971,
Parker and Ebert 2003). Although larger adults are generally less susceptible to predation by sea otters than juveniles even the largest urchins are not immune to predation by these voracious marine mammals (Bureau 2000). Time to maturity can range from under a year to many years, depending on species and environmental conditions. Kenner and Lares
(1992) observed that most individuals of purple sea urchins, S. purpuratus, are 16 mm in test diameter or larger and less than 2 years old when reproductively mature.
10 1.3 Aquaculture potential of sea urchins
Harvesting wild sea urchins can be very profitable during the fisheries development stage of a commercially important species. Typically though, a fishery develops faster than the population structure can be adequately assessed resulting in uncertainty and delay in management actions (Perry et al. 2002). This can ultimately lead to overfishing and a collapse of the stocks. For example, unregulated harvesting and poor fisheries management in the Philippines led to the collapse of a multi-million peso (USD $225,000
– $386,000) fishery of the sea urchin Tripneustes gratilla (Talaue-McManus and Kesner
1995, Juinio-Meñez et al. 2008). Chen and Hunter (2003) reported that a multi-million dollar (USD $20.3 million) fishery of the green sea urchin S. droebachiensis in Maine
(USA) has experienced substantial declines in landings, mainly resulting from a large decrease in stock abundance.
Sea urchins, being a relatively sedentary benthic animal, are easily picked by hand at low tide or in the shallow subtidal using simple harvesting equipment. Growth rates are typically too slow for most of the commercial species to allow sufficient replacement of harvested large adults. Indeed, one of the biggest concerns is that animals are collected only for the mature gonad or roe with indivduals having no, or limited, opportunity to spawn before harvest. Japan was the first country to address the over-exploitation issue and some of the earliest (1960s) work directly related to aquaculture occurred in Japan on sea-urchin spawning protocols and fisheries enhancement (Hagen 1996, Agatsuma et al.
2004, McBride 2005). These techniques include habitat enhancement (artificial reefs), natural (algal) and prepared diet development, re-seeding hatchery reared juveniles to
11 suitable habitats, and gonad enhancement of wild-collected adults (Agatsuma and Nakata
2004).
In the west, however, the majority of research on echinoid aquaculture began in the
1980s and 1990s [although one could argue that aquaculture-related research on sea urchins developed as early as the early 1900s from physiologically-based studies in which the feeding habits and digestive processes of echinoderms were investigated
(Robinson 2004)]. As interest in sea-urchin aquaculture developed over the years the majority of research attention has been given to feed development for growth of the adult animal in attempts to accelerate gonad production (e.g. Agatsuma 1998, Pearce et al.
2002a,b,c, 2003, Lawrence and Lawrence 2004, James and Heath 2008). Relatively few studies have investigated larval and juvenile echinoid growth and survival in response to various factors. However, aquaculture techniques for any new candidate urchin species must be refined to suit that particular species and they must be environmentally benign and economically viable. A reliable source of an expected number of quality seed is a pre-requisite for sustainable culture system development. In order to raise large numbers of seed in a commercial context the culture techniques must be refined in terms of diet quality, diet ration, stocking density, and water quality and then shown to be effective once scaled up to commercial-sized batches of seed. The next section of the thesis examines the advances and innovations in the recent past for larval and juvenile culture of sea urchins, with references to the particular challenges remaining for the scientist and echiniculturist.
12 1.4 Factors influencing the growth and survival of larval and juvenile echinoids: a review
This review pertains to all species of echinoids, while acknowledging that not all species may be suitable for commercial-scale aquaculture production. For a discussion on how life-history strategies of sea urchins may be used to assess their suitability as aquaculture candidates, see Lawrence and Bazhin (1998). Most of the species discussed in this section are those that have at least some level of commercial interest for aquaculture, however, there is undoubtedly valuable culture information to be gleaned from research on even non-commercial species. Also, it is important to mention that, with the exception of one study that used commercial-sized (100 L) larval tanks (Buitrago et al. 2005), the larval research cited in the current section has been conducted with small culture volumes (very often 1–5 L). It is unknown if the results from such small volumes can be reliably extrapolated to bigger ones for mass production on a commercial scale, but it is hoped that the small-scale results may provide insight as to what factors might be most important to test on a larger scale. Future research will need to be conducted using mass culture techniques, but such studies are not easy to conduct (due to space and labour requirements) and should be performed in a proper hatchery of at least pilot-scale production size.
13 1.4.1 Larval production
1.4.1.1 Food: phytoplankton
In the natural environment, echinoplutei feed on particles that are suspended in the surrounding seawater (Bullivant 1968, Strathmann 1971, McEdward and Miner 2007).
Particle size, concentration, flavour, and the stage of larval development can all influence larval feeding rates and particle selectivity (Pedrotti 1995). Numerous studies have shown that food has important effects on larval growth, survival, and metamorphosis of benthic marine invertebrates, including echinoids (Walker 1984, Pearce and Scheibling 1991,
Anil and Kurian 1996, Desai and Anil 2004, Schiopu et al. 2006). A large number of phytoplankton species have been used as food for sea urchins under laboratory and hatchery conditions (Table 1.2), but surprisingly little research has compared various microalgal species for their effects on larval echinoid growth and/or survival.
Hart and Scheibling (1988) compared the effects of two algal diets – Dunaliella tertiolecta and Chaetoceros gracilis (both at 1,000 cells ml-1) – on the growth and survival of larval S. droebachiensis. They reported that larvae from cultures fed C. gracilis were generally longer and had greater total arm lengths than those fed D. tertiolecta, although mean survival rates were broadly similar. Kelly et al. (2000) observed, however, that larval Psammechinus miliaris fed D. tertiolecta had improved survivorship at metamorphosis when compared to those fed Pleurocrysis carterae (65.8% versus 48.2%, respectively). Many studies confirm the usefulness of D. tertiolecta for feeding larvae of various echinoid species (Emlet 1986, Sewell et al. 2004, Reitzel et al.
2005, Miner 2007). Hart and Scheibling (1988), however, indicated that generalizations
14 Table 1.2 Summary of published rearing diets and conditions used for larval echinoids.
Urchin species Diet(s) Algal concentration Temperature Larval density Survival Study Location Reference
(cells ml-1) (oC) (inds ml-1) (%)
Anthocidaris Chaetoceros gracilis 8,000–30,000 22–25 0.8 - Japan Kitamura et al. 1993
crassispina
Arbacia punctulata Dunaliella tertiolecta, 350–8,000 23–24 1 - Florida (USA) Metaxas and
Isochrysis galbana, Young 1998a
Thalassiosira weissflogii,
or mixed diet
Echinometra lucunter I. galbana, D. tertiolecta, 2,500–10,000 23–24 1 - Florida (USA) Metaxas and
Thalassiosira weissflogii, Young 1998b
or mixed diet
Echinus esculentus Phaeodactylum tricornutum, 1,000–15,000 17±1 1 46 Scotland (UK) Jimmy et al. 2003
D. tertiolecta, or mixed diet
Evechinus Rhodomonas lens 6,000 14–18 1 - New Zealand Lamare and
chloroticus Barker 1999
E. chloroticus D. tertiolecta 600–6,000 20±1 2 - New Zealand Sewell et al. 2004
15 Urchin species Diet(s) Algal concentration Temperature Larval density Survival Study Location Reference
(cells ml-1) (oC) (inds ml-1) (%)
Glyptocidaris C. gracilis 5,000–40,000 16–17 1 40–65 China Chang and Wang 2004 crenularis
Heliocidaris C. gracilis and R. lens 20,000–50,000 22 - - Australia Hoegh-Guldberg and tuberculata Emlet 1997
Loxechinus albus Chaetoceros calcitrans, 10,000–60,000 16.4 0.7 38–82 Chile Cárcamo et al. 2005
I. galbana,
or Tetraselmis suecica
Lytechinus variegatus R. lens, or D.tertiolecta 8,000 26 0.33 - Florida (USA) McEdward and
Herrera 1999
L. variegatus Chaetoceros muelleri 20,000–60,000 27.5 0.25, 0.5, 1 >65 Venezuela Buitrago et al. 2005
L. variegatus D. tertiolecta 1,000 18–24 1–2 - Florida (USA) George et al. 2000
L. variegatus D. tertiolecta 300–700 22–24 0.5 50 Florida (USA) George et al. 2001
L. variegatus Prepared diet 1,000–4,000 - ~2 80–85 Florida (USA) George et al. 2004
(commercial diet: E-Z larva)
or D. tertiolecta
16 Urchin species Diet(s) Algal concentration Temperature Larval density Survival Study Location Reference
(cells ml-1) (oC) (inds ml-1) (%)
L. variegatus D. tertiolecta 500–5,000 20 ~1 - Nova Scotia Burdett-Coutts and
and I. galbana (Canada) Metaxas 2004
Paracentrotus lividus Phaeodactylum tricornutum 120,000–240,000 - 4 - France Vaїtilingon et al. 2001
P. lividus D. tertiolecta 1,500–7,500 18±2 1.5 68–75 Scotland (UK) Liu et al. 2007a
or prepared diet
Psammechinus D. tertiolecta 1,500–7,500 17 1 67.8–75.6 Scotland (UK) Liu et al. 2007b miliaris or prepared diet
P. miliaris Pleurocrysis elongata, 500–4,000 17 1 48.2–65.8 Scotland (UK) Kelly et al. 2000
Pleurocrysis carterae,
or D. tertiolecta
Pseudocentrotus C. gracilis 8,000–30,000 17–22 0.8 - Japan Kitamura et al. 1993 depressus
Strongylocentrotus D. tertiolecta 10,000 10.8±1.4 ≤ 2 - Nova Scotia Pearce and Scheibling droebachiensis (SD) (Canada) 1990, 1991, 1994
S. droebachiensis Rhodomonas sp. 200–5,000 10.1 0.14 - Washington Bertram and
(USA) Strathmann 1998
17 Urchin species Diet(s) Algal concentration Temperature Larval density Survival Study Location Reference
(cells ml-1) (oC) (inds ml-1) (%)
S. droebachiensis D.tertiolecta 500–5,000 14.2±2.1 0.2 35–46 Nova Scotia Hart and Scheibling
and C.gracilis 3, 6, 9 (Canada) 1988
S. droebachiensis D. tertiolecta 500–5,000 9 ~1 - Nova Scotia Meidel et al. 1999
(Canada)
S. droebachiensis D. tertiolecta 500–5,000 12 ~1 - Nova Scotia Burdett-Coutts and
and I. galbana (Canada) Metaxas 2004
S. franciscanus C. gracilis 18,000 13–15 1 - Oregon (USA) Miller and Emlet 1999
and R. lens 7,000
S. franciscanus D. tertiolecta 500–5,000 17 1 - North Carolina McAlister 2007
Strongylocentrotus C. gracilis 5,000 - 1.5 - Japan Hagen 1996 intermedius
S. intermedius C. gracilis 10,000–70,000 15 1.5–2 - Japan Takahashi et al. 2002
S. intermedius C. gracilis 5,000–20,000 - - - Japan McBride 2005
Strongylocentrotus C. gracilis 10,000–70,000 15 1.5–2 - Japan Takahashi et al. 2002 nudus
18 Urchin species Diet(s) Algal concentration Temperature Larval density Survival Study Location Reference
(cells ml-1) (oC) (inds ml-1) (%)
Strongylocentrotus D. tertiolecta 6,000 ~11–13 ~2 - California Miner 2007 purpuratus (USA)
S. purpuratus C. gracilis 18,000 13–15 1 - Oregon (USA) Miller and Emlet 1999
and R .lens 7,000
S. purpuratus D. tertiolecta 500–5,000 17 1 - North Carolina McAlister 2007
S. purpuratus Rhodomonas sp. 40,000 15 5–10 - California Meyer et al. 2007
(USA)
Tripneustes gratilla Chaetoceros muelleri 3,000–10,000 25 1 - Australia Dworjanyn et al. 2007
T. gratilla C. muelleri 4,000–10,000 25 4 - Australia Dworjanynand
Pirozzi 2008
19 about the effects of food quality on growth are complicated by different algal cell sizes and compositions, and by the unknown nutritional requirements of larvae. Cárcamo et al.
(2005) assessed the effect of single-species and mixed-species microalgal diets (i.e. C. calcitrans, C. calcitrans + I. galbana, and C. calcitrans + I. galbana + Tetraselmis suecica) on larvae of L. albus. Their results suggested that daily larval feeding with a mixed diet of C. calcitrans + I. galbana may produce better results for large-scale production of competent larvae and juveniles than a single-species diet. This prediction is supported by Basch (1996), working with asteroid larvae, who noted that algal mixtures more closely resembled natural planktonic food conditions and generally improved larval development compared with uni-algal diets.
Food ration is another factor that may influence larval echinoid growth and survival.
Meidel et al. (1999) observed that food ration had a strong effect on the rates of larval development, growth, and metamorphosis. They reared larval S. droebachiensis with D. tertiolecta at a “high” (5,000 cells ml-1) and “low” ration (500 cells ml-1) at a larval stocking density of ~1 larva ml-1. Their results showed that the rates of development, growth, and metamorphosis were significantly greater at the “high” ration than at the
“low” one. Sewell et al. (2004) also reported on the effects of food ration on larval development. They tested the developmental plasticity of larvae of Evechinus chloroticus using a “high” or “low” food ration (6,000 or 600 D. tertiolecta cells ml-1, respectively) or with no algal food. Results showed that larvae in the “high” food-ration treatment were largest in all measured dimensions and formed juvenile rudiments within 23 days of fertilization. Larvae fed the “low” ration and those with no algal food were stalled at the four-arm echinopluteus stage and significantly smaller.
20 While low food ration treatments can obviously have negative effects on larval development and growth, high food levels can also have detrimental effects. Kelly et al.
(2000) varied the density of the microalga Pleurocrysis elongata and found that larvae of
P. miliaris showed an extreme reduction in post-oral arm length, and were unable to maintain their position in the water column, on a “high” ration (4,000 cells ml-1 throughout the larval life span). Larvae on an “optimal” ration (1,500–4,000 cells ml-1, according to developmental stage) displayed a more typical morphology; whereas, larvae on a “low” ration (500 cells ml-1) failed to develop to metamorphosis. Jimmy et al. (2003) carried out two trials to assess the potential influence of three microalgal diets (i.e. D. tertiolecta, Phaeodactylum tricornutum, and D. tertiolecta + P. tricornutum) on morphology and survivorship of larval Echinus esculentus. In the first trial, they found that larvae from cultures fed D. tertiolecta or D. tertiolecta + P. tricornutum developed rapidly through metamorphosis. In the second trial, it was observed that a “standard” ration (1,000, 3,000, and 5,000 cells ml-1, according to developmental stage) produced better results – in terms of larval morphology, larval life span, and metamorphosis rate – than a “high” ration (3,000, 9,000, and 15,000 cells ml-1, according to developmental stage). Optimizing larval ration shortened the larval stage from 21–23 days to 16 days, with a survival rate of 46.6%, ten days after metamorphosis (Jimmy et al. 2003).
Studies with echinoid larvae suggest that mixed-species algal diets may give better results than single-species ones. This has also been shown in other benthic marine invertebrates (see review by Marshall et al. 2010 on marine bivalves) and is explained by the fact that larval nutritional requirements are better served by feeding more than one species of phytoplankton. It would be desirable to select single-species or mixed-species
21 diets that have optimal nutritional values for the echinoid species (and developmental stage) of interest in order to produce the highest larval growth and survival rates per unit of culture effort. From a commercial perspective, the species examined must be a good candidate for mass cultivation. The phytoplankton species may have the appropriate biochemical composition, cell size, and/or flavour to optimize larval growth and survival, but if the grower cannot rear the alga at a commercial scale reliably and cost-effectively, then the species will be of little use for commercial production.
Culturing algae under varying conditions (i.e. controlling such factors as irradiance, temperature and nitrogen, silica, and fatty acid levels) can modify their biochemical composition (Dunstan et al. 1993, Thompson et al. 1990, 1992, 1993, Leonardos and
Lucas 2000) and their effects on larval growth and survival (see Marshall et al. 2010 for a review on marine bivalves). Research in this particular area has not been done with echinoids, but could prove beneficial if larval nutritional requirements could be met by feeding only one algal species whose biochemical components had been modified appropriately by controlling the culture conditions. Such a technique would be very useful for a commercial hatchery since it would decrease the cost of maintaining and cultivating different phytoplankton species/strains and also decrease the risk of cross contamination between species.
1.4.1.2 Food: prepared diets
Most research examining larval echinoid feeding has made use of natural diets (i.e. phytoplankton) and only the following three studies have looked at the potential of prepared diets for larval echinoid culture. George et al. (2004) tested the value of a
22 prepared micro-encapsulated diet for larval rearing of L. variegatus. Their results showed that larvae fed the prepared diet were significantly smaller than those fed the phytoplankton D. tertiolecta. Larval survival was 72±6% for individuals fed prepared capsules and 85±4% for those fed D. tertiolecta. To investigate the growth and survival of larvae of P. miliaris on various diets, Liu et al. (2007a) fed larvae a micro- encapsulated formulated feed, D. tertiolecta, and a concentrated algal paste. They found that there was no significant difference in larval survivorship among the three feeding treatments, although faster growth was obtained with D. tertiolecta. In another experiment, they observed similar results for larvae of Paracentrotus lividus (Liu et al.
2007b).
From a commercial aquaculture perspective, prepared feeds will only be useful if they are cost effective and allow for good growth and survival. Echinoculturists, who are accustomed to using phytoplankton for feeding larvae, will only be interested in switching to a prepared feed if it gives as good growth/survival as the natural diet and is as cost-effective. In order to optimize the performance of prepared diets, much more research will be required in order to determine the nutritional requirements of the larvae; requirements that will undoubtedly be dependent on both the species and stage of development. Different types of alternative diets may be assessed for their viability, including yeast-based feeds, lipid emulsion spheres, and grain-based additives such as corn starch and wheat germ.
23 1.4.1.3 Food: dissolved organic matter
Many estuarine and marine invertebrates can obtain sustenance not only from the food they eat, but also through the direct assimilation of dissolved organic compounds in their environments (Stephens 1968, Ferguson 1982). Dissolved organic matter (DOM) represents a complex group of organic solutes including amino acids and sugars which are derived from a variety of biological sources in the marine environment (Hoegh-
Guldberg 1994). The uptake of DOM may have a significant role in the total nutritional requirement of larvae of some species, especially filter-feeders (Stephens1962, Manahan and Crisp 1982, Manahan 1983, Jaeckle and Manahan 1989), and larvae may be able to persist in the face of low particulate food concentrations through DOM uptake (Roditi et al. 2000). A comparative study undertaken by Ferguson (1982) evaluated the overall ecological benefits of free amino acids (FAA) uptake and release in the biology of adults of 21 species of marine invertebrates, including five echinoderms (Astropecten articulatus, Ophiophragmus filograneus, Leptosynapta crassipatina, Lytechinus variegatus, and Mellita quinquiesperforata). The study found that FAA can provide a substantial proportion of invertebrates’ metabolic requirements and play an important part in their well-being by providing much of the organism’s energy requirements.
Manahan et al. (1983) assessed the net rates of removal of amino acids from the water directly by larvae of the purple sea urchin, Strongylocentrotus purpuratus, and concluded that the influx of amino acids has the potential to contribute substantially to the carbon and nitrogen requirements of the larvae. Hoegh-Guldberg (1994) studied the rate of DOM uptake in regards to the energy requirements of larvae of the crown-of-thorns sea star,
Acanthaster planci, and concluded that the extra energy derived from the transport of
24 DOM might be crucial to larval survival, especially in terms of surviving short periods of starvation between food patches. While there is a very good understanding of the types of compounds involved and the rates of transport, the actual contribution of DOM to the nutrition, growth, reproduction, and survival of marine invertebrates in general, and echinoids in particular, remains largely unknown (Wendt and Johnson 2006) and is an area of potential research.
1.4.1.4 Stocking density
The optimum stocking density during larval rearing is crucial, as over-crowding can affect access to food resources (reducing both larval growth and survival rates) and the quality of the rearing water. Studies in marine-invertebrate larval development usually maintain larval rearing densities in the range of 0.2 to 2 larvae ml-1. In some cases, the rearing concentration has been substantially higher, as in the culture of the Babylon snail,
Babylonia spp., in which 8–10 larvae ml-1 has been commonly used (Chaitanawisuti and
Kritsanapuntu 1997, Shieh and Liu 1999). Only one published study has examined the potential effects of stocking density on larval echinoid growth and survival. Buitrago et al. (2005) developed a mass larval-rearing system using static seawater, with an exchange rate of 50% day-1, for culture of larval L. variegatus. They tested three different stocking densities (0.25, 0.50, and 1.00 larva ml-1), while feeding the larvae with C. muelleri at
20,000–60,000 cells ml-1. There were no significant differences among the three stocking densities in terms of survival (all exceeded 65%), larval length, and larval stage index, although larval weight in the low-density treatment was greater than in the high-density treatment. Buitrago et al. (2005) concluded that higher-density (0.50–1.00 larva ml-1)
25 culture had no apparent disadvantages and would reduce the overall cost of seed production.
Obviously, much more research is required to examine the potential effects of larval stocking density on larval growth and survivorship of a variety of echinoid species. Since the effects of stocking density will be greatly dependent on algal ration, the interactive effects of these two factors should be closely examined. Such work will be especially crucial for the development of commercial-scale hatcheries for marketable species.
1.4.1.5 Temperature
Temperature is considered to be one of the most important factors determining larval growth, and changes in temperature can influence both physiological processes and physical structure of larval invertebrates. It is well established that temperature has potential influences on larval development and that optimal performance is obtained within a narrow range of temperatures for a particular invertebrate species (Pechenik
1987, Roller and Stickle 1989, Anil et al. 2001, Ouellet and Chabot 2005, Desai et al.
2006). Below the optimal temperature range, the metabolic activity decreases as does growth and survival. Above the optimal temperature range, larvae have higher metabolic rates resulting in slower growth and lower survival. Despite the known importance of temperature on invertebrate larval development, very little research has examined the effect of this factor on growth and survival of larval echinoids. Hart and Scheibling
(1988) investigated the combined effects of temperature and food ration on larval S. droebachiensis. Larvae were reared at three temperatures (3, 6, 9oC) and provided three rations (500, 1,000, and 5,000 cells ml-1) of a 1:1 mixture of D. tertiolecta and C. gracilis.
26 They found that larvae grew faster at 9oC than at 6oC, while larvae reared at 3oC grew very slowly. Larval length and total arm length were both strongly affected by temperature. The main effects of temperature were always highly significant, while food- ration effects were often not significant. The interaction between these factors was highly significant. Sewell and Young (1999) observed that temperature had an important influence on early development and survivorship in the tropical sea urchin Echinometra lucunter. They examined the effect of four temperature ranges (16.1–35.8oC, 12.9–
23.9oC, 5.5–14.9oC, and 31.0–43.7oC) on egg fertilization rates. The percent fertilization in eggs exceeded 98% at 15.2 to 35.8oC. However, fertilization rate dropped to 87% at
12.9oC and 0% at 5.5–10.8oC and 38.4–40.7oC. In another experiment, they tested temperature (range: 16–36oC) tolerances for early development in E. lucunter. Results showed that embryos developed slowly at 16oC, with only 28% reaching the 2-cell stage after 8 hours of fertilization. At 18–23oC, there was an increase in the rate of development and the majority of embryos had reached the 8-cell stage after 8 hours and had hatched by 24 hours. Sewell and Young (1999) concluded that water temperatures from 27 to 34oC appeared to be optimal for early development of E. lucunter.
The dearth of literature on temperature effects on larval urchins is surprising given the amount of biochemical, biological, and environmental research conducted on larval echinoids. From an aquaculture perspective, much more research on temperature effects will be required on commercially-relevant echinoid species in order to optimize larval growth/survivorship and to minimize costs for cooling/heating seawater. Temperature effects will be species dependent and experiments will need to be conducted on all species of commercial interest.
27 1.4.1.6 Salinity
Most of the scientific research on the growth and development of larval and juvenile echinoids has been done with filtered seawater at ambient salinity (generally full-strength or near full-strength seawater). Very little research has examined the potential effect of salinity on echinoid larval development. This may be due to the fact that echinoderms are generally considered to be stenohaline organisms (Roller and Stickle 1993) and that fluctuations in salinity could have adverse effects on larval and juvenile survival and growth (Drouin et al. 1985). The developmental patterns of many invertebrates are known to be influenced by variations in salinity (Roller and Stickle 1989, Pechenik et al.
2000, Anger 2003, Schiopu and George 2004, George and Walker 2007).
Roller and Stickle (1985) examined the developmental status of embryos and larvae of three echinoid species and one asteroid species (S. droebachiensis, S. purpuratus,
Strongylocentrotus pallidus, and Pisaster ochraceus) at various salinities. Results for all species showed that embryos and larvae at lower salinities (20, 22.5, and 25‰) tended to develop more slowly than those at higher salinities (27.5 and 30‰). Similar observations were made by Roller and Stickle (1993) for larvae of L. variegatus when tested at various salinities (10, 15, 20, 25, 27.5, 30, and 35‰). They reported that there was no larval development at salinities less than 27.5‰ and concluded that low salinity prolongs the duration of planktonic existence of echinoderm larvae, thus increasing the probability of larval mortality. Metaxas (1998) examined the effect of six levels (15, 18, 21, 24, 27, and
33 PSU) of salinity on survival and development rate of larvae of the sub-tropical sea urchin E. lucunter. Her results showed that daily and cumulative mortality was
28 significantly lower at 33 PSU than at 21, 24, or 27 PSU over a period of 17 days. At 15
PSU, ~60% of larvae did not develop further than swimming blastulae.
From a commercial-hatchery perspective, the effect of salinity on larval growth and survivorship would only be of concern if the facility was utilizing water that was not full- strength seawater. This may occur if the hatchery is located in an estuary or utilizes saltwater wells. Since echinoids are generally viewed as being stenohaline, the effects of salinity are likely to be less variable across species than the effect of temperature.
1.4.2 Juvenile production
1.4.2.1 Food: algae
A number of studies have been carried out on the feeding preference of sub-adult or adult sea urchins (e.g. Larson et al. 1980, Lemire and Himmelman 1996, Lyons and
Scheibling 2007, Dworjanyn et al. 2007). There is also a relatively large body of literature comparing roe or gonad enhancement of adult sea urchins fed with different formulated diets (e.g. McBride et al. 1998, Pearce et al. 2002a, b, c, 2003, Vidal 2004,
Hammer et al. 2006, Siikavuopio et al. 2007, Hiratsuka and Uehara 2007, Woods et al.
2008), many of which have been assessed in comparison with natural algal diets
(McBride et al. 1997, McLaughlin and Kelly 2001, Pearce et al. 2002a, b, c, 2003,
McBride et al. 2004, Shpigel et al. 2005, Schlosser et al. 2005, Cook and Kelly 2007).
Relatively few studies, however, have investigated juvenile echinoid growth and survival in response to various factors, including diet, and most of these have focused on individuals greater than 5-mm test diameter (Table 1.3).
29 Table 1.3 Summary of published rearing diets and conditions used for juvenile echinoids.
Urchin species Initial Size Diet(s) Temperature Survival Study Location Reference
(mm) (oC) (%)
Paracentrotus 6–20 Palmaria palmata, Laminaria digitata, 18 98.5 France Basuyaux and Blin
lividus pure maize, or mixed diet 1998
Psammechinus 0.89–1.1 Laminaria saccharina, Ulva lactuca, 4–16 55–81 Scotland (UK) Cook et al. 1998
miliaris or prepared diet (Ambient)
P. miliaris 6.5–9.0 L. saccharina, or prepared diet Ambient 100 Scotland (UK) Kelly 2002
Strongylocentrotus 20 Agarum cribrosum, Ascophyllum nodosum, 1.5–3.5 - Maine (USA) Larson et al. 1980
droebachiensis Chondrus crispus, Corallina officinalis, or and
Laminaria longicruris 15–18
S. droebachiensis 2.5 Palmaria palmata, U. lactuca, 5, 10, or 15 - Maine (USA) Devin et al. 2004
or L. saccharina
S. droebachiensis 5–10 L. saccharina and Membranipora Ambient 47–83 New Hampshire Williams and Harris
membranacea or prepared diet (USA) 1998
S. droebachiensis 12 L. saccharina, Macrocystis integrifolia, Ambient - British ColumbiaPearce et al. 2004
Nereocystis luetkeana, or prepared diet (Canada)
30 Urchin species Initial Size Diet(s) Temperature Survival Study Location Reference
(mm) (oC) (%)
S. droebachiensis 2.41 Benthic diatoms (Navicula spp., Amphora 4.7, 9.0, 12.9, 26–100 British Columbia Pearce et al. 2005
spp., and Melosira spp.) and Porphyra sp. 16.0, and 19.7 (Canada)
S. droebachiensis 6–9 Prepared diet or L. longicruris 2.5–14 - New Brunswick Castell et al. 2004
13–16 (Ambient) (Canada)
S. droebachiensis 4.5–13.7 Porphyra purpurea, P. palmata, Ulva 0.9–16 - New Brunswick Daggett et al. 2005
linza, Ulvaria obscura, U. lactuca, (Ambient) (Canada)
L. saccharina, or prepared diet
S. droebachiensis 4–8 Prepared diet or L. longicruris Ambient >95 New Brunswick Kennedy et al. 2005
12–20 (Canada)
S. droebachiensis 13–15 Prepared (pigmented) diet Ambient 94–100 New Brunswick Kennedy et al. 2007a
1–3 or L. longicruris (Canada)
S. droebachiensis 6–9 Prepared diet or L. longicruris 1.7–14 - New Brunswick Kennedy et al. 2007b
13–16 (Ambient) (Canada)
Strongylocentrotus 34.2 Prepared diet 12.5–16.8 - California (USA) McBride et al. 1998 franciscanus (Ambient)
31 Urchin species Initial Size Diet(s) Temperature Survival Study Location Reference
(mm) (oC) (%)
S. franciscanus 10–30 Zostera marina or N. luetkeana 14–16 85 British Columbia Morris and Campbell
(Ambient) (Canada) 1996
Strongylocentrotus - Rhaphoneis surirella, Navicula seminulum, 14–19 33–95 China Xing et al. 2007 intermedius Navicula corymbosa, Navicula parva, (Ambient)
Hantzachia amphioxys var. leptocephala,
Amphora ciffeaeformis, Nitzschia sp.,
or Amphora proteus var. oculata
S. intermedius 0.3–5 Ulvella lens, Ulva pertusa, 18 60–70 Japan Sakai et al. 2004
Laminaria spp., and prepared diet
Strongylocentrotus 0.3–5 Ulvella lens, Ulva pertusa, 18 60–70 Japan Sakai et al. 2004 nudus Laminaria spp., and prepared diet
Tripneustes gratilla - Ecklonia radiata, Sargassum linearifolium, Ambient - Australia Dworjanyn et al.
Dictyota dichotoma, Gracilaria sp., 2007
Lophocladia kuetzingii, U. lactuca,
Zostera capricorni, or prepared diet
32 While early post-metamorphosis is a particularly critical stage in the life-cycle of an echinoid, sparse attention has focused on the effect of diet (or any other factors) on growth and survival of early, post-settled (i.e. <5-mm test diameter) echinoid juveniles.
Many researchers have concluded that metamorphic success and juvenile quality of benthic marine invertebrates may depend on parental investment in the eggs and nutrient acquisition by larvae from the external environment. Vaïtilingon et al. (2001) observed that when competent larvae of P. lividus were starved during the metamorphosis-delay period, most of the energy reserves were used for basic physiological processes and rudiment development. As a consequence, early juveniles showed higher mortality rates than those derived from larvae fed during the same period. In recent studies with T. gratilla, Byrne et al. (2008a, b) showed that growth performances of early juveniles were strongly influenced by maternal egg nutrients (i.e. triglycerides) and nutrient stores sequestered by the larvae.
Xing et al. (2007) assessed the potential value of different species of benthic diatoms as food for early post-settled (size not given) Strongylocentrotus intermedius. In that study, eight cultured diatom species (Amphora coffeaeformis, Amphora proteus var. oculata, Hantzachia amphioxys var. leptocephala, Navicula corymbosa, Navicula parva,
Navicula seminulum, Nitzschia sp., Rhaphoneis surirella) were tested individually, along with treatments of mixed-cultured diatoms, natural assemblages of benthic diatoms, and a control of no food. Results showed that growth and survival of post-settled juveniles were best with N. corymbosa. After 30 days feeding with N. corymbosa the juvenile test diameter and survival were 613±1.14 μm (mean±SE) and 95.83±1.05% (mean±SE), respectively. Devin et al. (2004) tested the effect of algal type on growth and survival of
33 recently-settled (2.5-mm test diameter) S. droebachiensis. The juvenile urchins were fed
Palmaria palmata (red alga), Laminaria saccharina (brown alga), Ulva lactuca (green alga), or a mixed algal diet consisting of P. palmata, U. lactuca, and L. saccharina, ad libitum. The red alga, brown alga, and mixed algae produced greater growth and survival than U. lactuca alone (Devin et al. 2004).
Much more research has examined the effect of diet on growth and survival of larger juveniles (5- to 20-mm test diameter). Algal-diet preference tests were performed by
Larson et al. (1980) for small (test diameter: 20±5 mm) S. droebachiensis. In their study, they selected algal species according to their presence in the local ecosystem and potential economic importance. These included: Agarum cribrosum (brown alga),
Ascophyllum nodosum (brown alga), Laminaria longicruris (brown alga), Chondrus crispus (red alga), and Corallina officinalis (red coralline alga). Their results showed that
L. longicruris was the most preferred alga and that increases in test diameter were greatest when juveniles were feeding on L. longicruris, C. crispus, or C. officinalis.
Differences in test diameter at the end of the experiment among these algal treatments were not significant, but differences among mean monthly increases in test diameter were significant. Mean monthly increases in test diameter were 1.40, 1.29, and 1.19 mm for L. longicruris, C. crispus, and C. officinalis, respectively. Floreto et al. (1996) determined the effects of different seaweed diets (green alga Ulva pertusa, red alga Gloiopeltis furcata, and brown alga Undaria pinnatifida) on the growth and biochemical composition of juvenile T. gratilla and found that highest feed conversion efficiencies were with U. pinnatifida (80.0%) and U. pertusa (76.6%), while the lowest was with G. furcata
(51.5%). The specific growth rates were highest in the juveniles fed U. pinnatifida,
34 followed by G. furcata and U. pertusa. Morris and Campbell (1996) found a higher growth rate for juvenile (10- to 30-mm test diameter) S. franciscanus that were fed with the kelp Nereocystis luetkeana compared with those fed the sea grass Zostera marina.
1.4.2.2 Food: prepared diets
While macro-algae are typically the preferred food for many species of sea urchins
(Lawrence 1975), the use of algae for large-scale, commercial cultivation of echinoids may not be feasible for a variety of reasons: (1) limited natural resources of suitable macroalgal species, (2) restricted harvesting of natural stocks, (3) conflict with other users of the marine environment, (4) commercial expense associated with collecting large quantities of macroalgae, (5) difficulty in storing commercial-scale quantities of algae, and (6) temporal and spatial variation in quantity and/or quality of plants (Lyons and
Scheibling 2007).
In recent years, various alternative feeds have been tested to replace live algae for the somatic growth of juvenile sea urchins. Basuyaux and Blin (1998) used pure maize and different macroalgae (P. palmata, Laminaria digitata) and a mix of maize and algae as food for juvenile (6- to 20-mm test diameter) P. lividus. The best growth rates were obtained with the mix of maize plus P. palmata, although the mortality rates were not significantly different among the diets. Experiments were conducted by Williams and
Harris (1998) to investigate the growth of juvenile (3.5-mm test diameter) S. droebachiensis on a defined prepared diet and a natural diet. The urchins were provided with an ad libitum diet of either L. saccharina, with at least 50% cover of the bryozoan
Membranipora membranacea, or prepared pellets. It was observed that, initially,
35 individuals on the prepared diet had a significantly greater change in size than those on the kelp/bryozoan diet, but overall growth on the natural diet was similar or greater than growth on the prepared feed. Cook et al. (1998) examined the effects of a commercially- manufactured salmon diet and macroalgal diets (U. lactuca and L. saccharina) on growth and survival of juvenile P. miliaris. Their results showed that there was no significant difference in the final test diameter between the urchins receiving the algae and those fed the prepared diet. Cook et al. (1998) observed that the urchins fed on macroalgae had noticeably longer spines, a more flattened test, and lower mortality (18.7%) than those given the salmon feed (45.3%). Pearce et al. (2004) examined the effect of three kelp species (L. saccharina, Macrocystis integrifolia, and N. luetkeana) and a prepared diet on the somatic growth of juvenile (12±0.1 mm) S. droebachiensis. After nine months of feeding, they found that urchins fed the prepared diet were significantly larger and heavier than those fed any of the three kelp species. The authors reported that L. saccharina gave the best results for the kelp species tested, although there were no statistically significant differences among the three kelp treatments. Daggett et al. (2005) observed the effects of different macroalgae [Porphyra purpurea (red alga), P. palmata
(red alga), Ulvaria obscura (green alga), U. lactuca (green alga), Ulva linza (green alga),
L. saccharina (brown alga)] and a prepared diet on the survival and somatic growth of juvenile (4.5- to 13.7-mm test diameter) S. droebachiensis. They also compared the growth rate of hatchery-reared and wild-caught juveniles with different prepared and natural diets and observed, in both experiments, that food type significantly affected somatic growth rate. The survival rates were 95% or greater for all treatments and there were no significant differences in survivorship among dietary treatments. Laminaria
36 saccharina produced significantly slower growth than any other feed treatments, the overall best growth being supported by P. purpurea and the prepared diet. Kennedy et al.
(2005) varied the level of protein (20, 30, 40, and 50% dry mass) in a prepared feed to examine the effect of protein concentration on growth of juvenile (4- to 8-mm test diameter) S. droebachiensis. Their results suggested that sea-urchin juveniles may not require a high concentration of dietary protein for superior growth, as there were no significant differences in growth rate among any of the protein levels tested. Kennedy et al. (2007a) investigated the effects of dietary minerals and pigments in prepared diets on the somatic growth of hatchery-reared and wild juvenile (1- to 15-mm test diameter) S. droebachiensis. They reported that prepared diets with pigment and high mineral concentration can outperform kelp and can be used juvenile production. In another experiment, Kennedy et al. (2007b) observed that juvenile (7.0- to 15.3-mm test diameter) S. droebachiensis fed diets with lower lipid concentration (≤ 3%) had larger test diameters than those fed diets with higher lipid concentrations (≥ 7%). Dworjanyn et al. (2007) investigated the effect of various prepared diets containing different algal species [Ecklonia radiata (brown alga), Sargassum linearifolium (brown alga), and U. lactuca (green alga)] and a control prepared diet without algae on growth of juvenile (5 to
15-g wet weight) T. gratilla. In that study, juvenile T. gratilla fed the diet containing S. linearifolium grew significantly faster than those fed the control diet.
With regard to commercial production of juvenile echinoids, further research is required on the effects of various benthic micro-algal species on early post-settled growth and survivorship (as in Xing et al. 2007). These effects are likely to be species-specific
(and perhaps size-specific) and this work will be required on any echinoid species of
37 commercial interest. Commercial ventures are likely to feed older juveniles with prepared diets (for reasons stated above) and much more work will be required to examine the dietary requirements of various species (as in Kennedy et al. 2005, 2007a, b). These types of studies should refine dietary requirements and optimize juvenile growth and survivorship, while attempting to minimize feed costs. We know of no work that has examined the potential effects of feed ration on juvenile echinoid growth and survivorship. This would be a fruitful avenue of research, especially for commercial-scale production, as profitability will be intimately linked with optimizing growth/survivorship and minimizing feed costs.
1.4.2.3 Stocking density
Juvenile sea urchins have different physical culture requirements relative to swimming larvae or pelagic finfish in that they require a sufficient surface area on which to attach, rather than large water volumes. Typically, they do not use the horizontal bottom of a tank to great extent if vertical surfaces are present (Devin 2002). Cultivated sea urchins tend to position themselves on vertical walls and form dense aggregations, rather than evenly distribute themselves through the available surface area, making cleaning tanks and feeding the animals challenging (Alabi et al. 2001, Devin 2002). Most urchin cultivation systems developed to date make use of relatively shallow containment systems that essentially force the sea urchins to exist on the bottoms of the units. This makes feeding the urchins easier as animals have better access to the food, but makes removal of wastes more difficult (Aas 2004, Daggett et al. 2006). The negative effects of overcrowding on the growth and survival of juveniles have been identified for many other
38 marine invertebrate species (Fréchette and Bacher 1998, Battaglene et al. 1999, Beal and
Kraus 2002, Huchette et al. 2003). However, only one published study has examined the effect of stocking density on growth and survival of juvenile echinoids. Kelly (2002) stocked 550 juvenile (9.0±1.25 mm test diameter, mean±SD) P. miliaris in 250-L tanks and 40 (“high” density) or 20 (“low” density) juveniles in 10-L tanks, each with an independent seawater supply at a flow rate of 1.0–1.5 L min-1. Juvenile urchins were also cultured in hanging baskets (30x18x15 cm with 5-mm mesh) suspended at a depth of approximately 4 m from a long-line system at sea. The stocking number was 40 juveniles basket-1. Kelly (2002) reported that higher stocking densities adversely affected growth, although survival was exceptionally high (only one mortality) in all treatments over the course of the experiment. Comparison of test diameters showed that urchins maintained at the high density (14.1±1.9 mm, mean±SD) were significantly smaller than those at the low density (15.6±1.86 mm, mean±SD).
Juvenile stocking density will have repercussions on the surface area required for cultivation and the number of rearing units and footprint area needed. These ultimately affect the profitability of commercial ventures, and research into the potential effects of juvenile stocking density on growth and survivorship will be much needed to maximize business profitability. Ultimately, these experiments will need to examine the combined effects of stocking density and feed ration, while keeping husbandry and management issues (e.g. feeding, waste management, water quality, harvesting) in mind.
39 1.4.2.4 Temperature
Much of the scientific research on juvenile echinoids has been done with filtered seawater in flow-through systems at ambient temperature and relatively little research has examined the effects of temperature on growth and survival of juvenile echinoids. Miller and Emlet (1999) investigated the effects of temperature (8, 11, and 14oC) on growth and development of juvenile (early post-settled) S. franciscanus and S. purpuratus. They discovered that the juvenile mouth of both species opened and feeding began on day 9 at
14.4–14.7oC and on day 12 at 11oC. Functional pedicellariae occurred in 50% of individuals of S. purpuratus by day 9 at 14.7oC, but not until day 14 at 8oC.Devin et al.
(2004) cultured juvenile (2.5-mm test diameter) S. droebachiensis to test the effects of three temperatures (5, 10, and 15oC) on growth and survival. The authors reported that there were no significant differences in biomass production among the three temperatures, although mortality was lowest at 5oC. Pearce et al. (2005) reared recently settled (<4-mm test diameter) juvenile S. droebachiensis at five temperatures: 4.7±0.8,
9.0±1.1, 12.9±1.1, 16.0±1.5, and 19.7±1.3oC (mean±SD). Urchins were fed natural assemblages of benthic diatoms (predominantly Amphora spp., Melosira spp., and
Navicula spp.) and supplemented with the macroalga Porphyra sp. when they attained 5- mm test diameter. The results showed that the mean test diameter was significantly larger at 9.0 and 12.9oC than at 4.7, 16.0, and 19.7oC and the mean percent survival was significantly greater at 4.7, 9.0, 12.9, and 16.0oC than at 19.7oC and significantly greater at 12.9 and 16.0oC than at 4.7oC. Mean percent survival at the end of the experiment for the various temperature treatments was 76.0±6.0%, 90.0±5.5%, 100.0±0.0%, 98.0±2.0%, and 26.0±11.2% (mean±SE) at 4.7, 9.0, 12.9, 16.0, and 19.7oC, respectively. The
40 researchers concluded that S. droebachiensis should be reared at 9–13oC in order to optimize production for aquaculture.
As with larvae, much more research on temperature effects will be required on commercially-relevant echinoid species in order to optimize juvenile growth/survivorship and minimize costs for cooling/heating seawater. Temperature effects will be species- dependent and experiments will need to be conducted on all species of commercial interest. Temperature will affect water quality (and hence juvenile growth and survivorship), an influence that needs to be examined closely in any studies of the effects of temperature.
41 1.5 The experimental animal
Classification
Kingdom: Animalia
Phylum: Echinodermata
Class: Echinoidea
Order: Echinoida
Family: Strongylocentrodidae
Genus: Strongylocentrotus
Species: S. purpuratus
The purple sea urchin, Strongylocentrotus purpuratus (Stimpson), is common in exposed and semi-protected rocky intertidal and shallow subtidal benthic habitats on the west coast of North America, typically occupying areas that experience strong wave action (Leahy et al. 1978, Mead and Denny 1995, Workman 1999). The species is distributed from Alaska to Cedros Island in Baja California, Mexico (Gonor 1973,
Russell 1987, Ebert et al. 1994, Rogers-Bennett 2007) and occurs over a wide range (5–
20°C) of temperatures (Osovitz and Hofmann, 2005). A laboratory study by
Farmanfarmaian and Giese (1963) reported that S. purpuratus tolerates a temperature range between 5 and 23.5°C.
The major annual spawn of purple sea urchins typically occurs from January to March
(Gonor 1973). Basch and Tegner (2007), however, observed spawning from October through January and their study reported that a prolonged high (>17°C) temperature inhibits the gametogenesis of S. purpuratus. Although S. purpuratus feeds on various species of algae in the natural environment, it clearly prefers giant kelp, Macrocystis
42 integrifolia, and bull kelp, Nereocystis luetkeana, when these are available (Tegner and
Dayton 1981, Springer et al. 2006, Basch and Tegner 2007).
The purple sea urchin is highly valued as a model organism for studies on gene mapping/regulation (Shott et al. 1984, Osovitz and Hofmann 2005, Goel and Mushegian
2006), reproduction (Levitan 2002, 2006), and egg/embryo/larval development
(Hinegardner 1969, Bédard and Brandhorst 1983, Leahy 1986). Echinoids in general are also popular for the curio trade and biological supply warehouses (Kelly et al. 2001,
Livengood and Chapman 2007).
Purple sea urchins are capable of producing marketable gonads (termed “roe” or
“uni” in industry parlance) and are harvested commercially on a limited and experimental basis both in Oregon and California, USA (Richmond et al. 1997, Parker and Ebert
2003). In British Columbia, Canada there was a small test fishery, which occurred between 1989 and 1992, but local depletion led to its closure (Workman 1999). This species is not as popular for harvesters as its close, sympatric relatives the red and green sea urchins (S. franciscanus and S. droebachiensis, respectively), likely because it is substantially more difficult to harvest due to its preference for living in shallow, subtidal locales with high surge and/or turbulence. Purple sea-urchin roe, however, is reportedly very similar in quality to some of the highly desirable, domestic Japanese species and is sought after in Mediterranean countries (Parker and Ebert 2003). In areas along the
Pacific coast of North America where it is fished, populations may be susceptible to overharvesting if the fishery remains unregulated and stocks becomes overexploited
(Olivares-Bañuelos et al. 2008).
43 Canada has extensive stocks of sea urchins on both coasts, although the quality and quantity of gonad or roe can be highly variable when they are harvested from the natural environment. For example, the fishermen of the Bay of Fundy (New Brunswick) fishery noticed that, while the average roe yields of harvested sea urchins ranged between 10 and
15% over the fishing season, there were certain areas and times when roe yields were as high as 30% with excellent colour and overall quality (Pearce and Robinson 2010). The harvesting of sea urchins in cold water in the winter season in the northern hemisphere
(November–January), when the maximum market demands occur in Japan, by fishers is also a painful as well as expensive activity. These issues inevitably led to a solution whereby animals could be collected from the wild during the autumn and reared in captivity to produce large, uniform gonads of high quality before exporting them.
Although, the ultimate goal of sea-urchin aquaculture should be non-destructive to the ecosystem, this kind of research initiative will help to shift the involved people from hunting to farming.
44 1.6 Hypotheses and objectives
The experimental work undertaken for my PhD set out to develop culture methods for the purple sea urchin, S. purpuratus. Specifically, my objectives were to examine the effects of: (1) diet quality (both natural kelps and prepared diet) and temperature on adult gonad production, (2) temperature and stocking density on embryonic and larval development/survival, and (3) micro-algal diet and ration on larval growth/survival.
1.6.1 Experiment 1: Influence of diet and temperature on adult gonad production
Various kelp species, abundant in the habitat of the purple sea urchin and known to be their preferred diet, were chosen as potential food sources. Commercial-scale harvest of wild kelps, however, may conflict with other stakeholders (e.g. fishers and environmentalists who know that kelp beds are important fish/invertebrate spawning grounds) and thus not be sustainable on a commercial-scale basis. Also, previous research has shown that kelps may be inferior to prepared diets for increasing gonad quantity of echinoids. For these reasons, I chose to examine gonad production of the purple sea urchin in response to both a variety of different kelp species as well as a prepared diet.
Temperature is a basic factor that could affect the ingestion rate, absorption rate, assimilation efficiency, and gonad production of candidate urchin species. I tested the following levels of diet and temperature on gonad production of adults in a factorial experiment: four diets [three kelp species (giant kelp, Macrocystis integrifolia; bull kelp,
Nereocystis luetkeana; sugar kelp, Saccharina latissima) and a prepared pellet diet developed by the Norwegian Institute of Fisheries and Aquaculture (NIFA)] and three temperatures (8, 12, and 16°C).
45 HO: There will be no significant affect of diet type on gonad quantity or quality.
HA: Diet type will significantly affect both gonad quantity and quality: the prepared diet will produce significantly larger and better quality gonads than the various kelp species.
HO: There will be no significant affect of temperature on gonad quantity or quality.
HA: Temperature will significantly affect both gonad quantity and quality: higher temperatures will produce significantly larger and better quality gonads than lower temperatures.
HO: There will be no significant interaction affect of diet and temperature on gonad quantity or quality.
HA: The interaction of diet and temperature will significantly affect both gonad quantity and quality.
1.6.2 Experiment 2: Influence of stocking density on early development and survival
Eggs/zygotes of sea urchins are negatively buoyant and settle to the bottom of the rearing unit; hence bottom surface area is a critical factor for embryonic development.
Pelagic, suspension-feeding larvae must swim to acquire food, control their position in the water column, and respond to environmental conditions by making compensatory adjustments in their activities. Larval size and shape are important determinants of larval functional performance and feeding ability. The rearing density is crucial as over- crowding limits swimming area and access to food resources and may lead to reduced water quality; thus over-crowding may impede development/growth and reduce survival.
In separate experiments I evaluated the effects of four stocking densities: 50, 100, 200,
46 and 400 eggs ml-1 on embryonic development and survival and 0.5, 1, 2, and 4 ind ml-1 on larval growth and survival.
HO: There will be no significant affect of stocking density on embryonic/larval development, growth, and survival.
HA: Stocking density will significantly affect embryonic/larval development, growth, and survival: higher stocking densities will significantly reduce embryonic/larval development, growth, and survival.
1.6.3 Experiment 3: Influence of temperature on early development and survival
Although many studies have concluded that environmental temperature is a limiting factor for adult/juvenile echinoid growth and survival, relatively little research has examined temperature influences on sea-urchin larvae. In separate experiments I examined the effiets of four temperatures (8, 11, 14, and 17oC) on embryo and larval development, growth and survival.
HO: There will be no significant affect of temperature on embryonic/larval development, growth, and survival.
HA: Temperature will significantly affect embryonic/larval development, growth, and survival.
1.6.4 Experiment 4: Influence of algal species on larval growth and survival
Different phytoplankton species may have different effects on larval development, growth, and survival. A variety of both uni-algal diets and combinations of various
47 microalgal species were tested to see what diet offers optimal growth and survival. I assessed seven algal diets [Dunaliella tertiolecta, Chaetoceros muelleri, Isochrysis sp.
(Tahitian strain) and all possible binary and tertiary combinations], along with a control treatment in which they were provided no food.
HO: There will be no significant affect of algal diet on larval development, growth, and survival.
HA: Algal diet will significantly affect larval development, growth, and survival: bi-algal diets will produce significantly better growth and survival than uni-algal diets.
1.6.5 Experiment 5: Influence of dietary rations on larval growth and survival
In nutrient poor conditions, sea-urchin larvae grow long arms that enhance the rate of food-particle capture. While longer arms may provide a feeding benefit, they presumably also come at an energetic cost, resulting in slower growth and longer time to metamorphic competency. In contrast, larvae provided with favourable nutritive conditions maintain short arms and accelerate development to metamorphic competency.
In this experiment, a bi-algal diet (D. tertiolecta and Isochrysis sp. at equal biovolumes) was evaluated using five rations: (1) low ration: 1.25 x 103 cells ml-1; (2) normal ration:
2.5 x 103 cells ml-1; (3) standardized ration: 2.5 x 103 to 10.0 x 103 cells ml-1, with increasing ration according to developmental stage; (4) medium ration: 5.0 x 103 cells ml-
1; and (5) high ration: 10.0 x 103 cells ml-1.
HO: There will be no significant affect of dietary rations on larval development, growth, and survival.
48 HA: Dietary rations will significantly affect larval development, growth, and survival: the standardized ration will produce significantly better growth and survival than the fixed rations.
49 CHAPTER 2: Influence of diet and temperature on gonad production of the purple sea urchin2
In this experiment I examined the combined effects of two factors (diet and temperature) on ingestion rate, absorption, assimilation efficiency, and gonad enhancement of the purple sea urchin, Strongylocentrotus purpuratus, over a 12-week period. Four diets [three kelp species (Macrocystis integrifolia, Nereocystis luetkeana, and Saccharina latissima) and one prepared feed] and three temperatures (8.0±0.4,
12.3±0.3, and 16.2±0.4°C: mean±SD) were tested in a completely-crossed factorial design. Ingestion rate, gravimetric absorption, and assimilation efficiency of urchins fed the various diets were calculated at weeks 6 and 12. In addition, a number of gonad attributes were quantified at the beginning and end of the experiment including wet weight, percent water content, gonad index, texture, firmness, colour, and flavour.
Ingestion rate, gravimetric absorption, and assimilation efficiency were generally significantly affected by both temperature and diet. Wet-weight ingestion rates were significantly higher with the three kelp treatments than with the prepared diet at both weeks 6 and 12. This particular dietary difference was not apparent, however, when ingestion rates were expressed on a dry-weight basis. The highest ingestion rates were recorded for animals held at 12°C at week 6 and at 16°C at week 12. Absorption rates and assimilation efficiencies were significantly higher at 16°C than at 8°C at week 6 and
2 A version of this chapter has been published. Azad A.K., Pearce C.M. and McKinley R.S. 2011. Effects of diet and temperature on ingestion, absorption, assimilation, gonad yield, and gonad quality of the purple sea urchin (Strongylocentrotus purpuratus). Aquaculture 317: 187-196
50 significantly higher at 16°C than at the other two temperatures at week 12. Final gonad weight and gonad index were significantly greater in urchins fed the prepared diet than in those fed any of the three kelp species, with little difference among the kelp treatments.
Final gonad weight was significantly affected by temperature, but this effect was dependent on diet; temperature had little affect on gonad weight (and index) in the three kelp treatments, but 12 and 16°C produced significantly heavier gonads than 8°C for urchins fed the prepared diet. Gonads of urchins fed kelps had more (significantly more in the case of M. integrifolia and N. luetkeana) water than those fed the prepared diet.
Gonad colour, texture, and firmness were generally not significantly affected by temperature, diet, or the interaction of these two factors. Gonad flavour was generally
(sometimes significantly) better in urchins fed the prepared diet than in those fed the three kelp species.
51 2.1 Introduction
Sea-urchin gonads act as the main organ of nutrient storage (Gonor 1973, Siikavuopio et al. 2006) and the amount of nutrient intake, and subsequent gonad growth, depends on food quantity/quality and the rate of consumption, digestion, and absorption (Lawrence
1975, Lares and McClintock 1991, Lawrence et al. 2003). A number of experiments have been carried out on feed intake and assimilation for various sea-urchin species (e.g. Lowe and Lawrence 1976, Frantzis and Grémare 1992, Barker et al. 1998, Klinger et al. 1998,
McBride et al. 1999, Hiratsuka and Uehara 2007). Urchin gonad growth and development may also be controlled by a range of exogenous factors, such as season, photoperiod, and temperature (Spirlet et al. 2000, Garrido and Barber 2001, Kelly 2001, Shpigel et al.
2004, Dumont et al. 2006, Siikavuopio et al. 2006, James et al. 2007). Any or a combination of these exogenous factors may interact with food quantity or quality to affect feed consumption/absorption/assimilation and, ultimately, gonad production and development. For example, McBride et al. (1997) observed that temperature had a significant effect on the feeding rate of Strongylocentrotus franciscanus fed both natural and artificial diets. However, very few studies have examined the direct influence of temperature (or other environmental factors) on sea-urchin gonad production (i.e. quantity or quality) and fewer still have looked at the interactive effects of temperature and diet (see McBride et al. 1997).
A plethora of studies have documented the effects of various macroalgal and prepared diets on gonad growth and quality in numerous sea-urchin species, including Evechinus chloroticus (Barker et al. 1998, James and Heath 2008,Woods et al. 2008), Loxechinus albus (Cárcamo 2004), Lytechinus variegatus (Hammer et al. 2006), Paracentrotus
52 lividus (Spirlet et al. 2001, Shpigel et al. 2005, Cook and Kelly 2007a), Psammechinus miliaris (Cook et al. 1998, McLaughlin and Kelly 2001, Cook and Kelly 2007b), S. droebachiensis (de Jong-Westman et al. 1995; Robinson et al. 2002, Pearce et al.
2002a,b,c, 2004, Siikavuopio et al. 2007), S. franciscanus (McBride et al. 1997, 1998,
1999, 2004), Strongylocentrotus intermedius (Chang et al. 2005, Lawrence et al. 2009), and Strongylocentrotus nudus (Agatsuma 1998, Agatsuma et al. 2005). While initial research was done in the 1970s/1980s to refine techniques and holding facilities for the laboratory culture of adults, embryos, and larvae of Strongylocentrotus purpuratus (i.e.
Leahy et al. 1978, 1981, Leahy 1986) and to look at reproductive periodicity and exogenous cues controlling reproduction and gametogenesis in purple sea urchins
(Boolootian 1963, Gonor 1973, Cochran and Engelmann 1975, Pearse 1981, Pearse et al.
1986, Bay-Schmith and Pearse 1987), no research has examined, from an aquaculture perspective, the effect of temperature and diet on gonad production in this species. The objective of the present study was to examine the interactive effects of temperature and diet on ingestion rate, gravimetric absorption, assimilation efficiency, gonad yield, and gonad quality of the purple sea urchin, S. purpuratus, with an aim towards culturing the species on a commercial basis. I hypothesized that both diet and temperature would significantly affect gonad quantity and quality, with prepared diets and higher temperatures producing significantly larger and better quality gonads than kelp and lower temperatures.
53 2.2 Materials and methods
2.2.1 Experimental urchin collection and maintenance
Adult purple sea urchins were collected by SCUBA divers from the subtidal region at
Sombrio (48°25΄N, 124°3΄W, west Vancouver Island, BC) in mid July 2007. They were transported (~2 h) to the Pacific Biological Station (Nanaimo, BC) in insulated coolers and held in indoor tanks with running, ambient (~9.7°C), sand-filtered seawater prior to experimentation. The animals were fed kelp Nereocystis luetkeana ad libitum twice weekly prior to the experiment. For temperature acclimation, the experimental animals were transferred into three, shallow fibre-glass tanks (Length x Width x Depth: 125 x 80 x 20 cm) and gradually acclimated to the three treatment temperatures (8.0±0.4, 12.3±0.3, and 16.2±0.4°C: mean±SD) by adjusting the seawater by 2°C per 3-d interval until the desired temperature was reached. Specific seawater temperatures were established via appropriate mixing of ambient and chilled (~5°C) or heated (~25°C) seawater in separate plastic header tanks. Twenty four randomly-selected animals from each temperature treatment were placed individually in PVC tubes (Length x Diameter: 15 x 10 cm) with flat PVC bottoms. Urchins were placed randomly in the tubes four weeks prior to beginning the experiment and no food was provided until the experiment started. These tubes were held in the three fibreglass tanks described above. Seawater of specific experimental temperatures flowed (~8 l h-1) into the bottom of each replicate tube and out at the top (i.e. flow-through design with up-welling). Water temperature was automatically recorded in each tank every 30 min during the experiment by temperature loggers (HOBO data logger, Onset Computer Corporation, Pocasset, Massachusetts,
USA). Lighting for the experiment was provided by overhead fluorescent lights which
54 were set for a constant photoperiod of 14 h light:10 h dark. A long-day photoperiod was used in an attempt to prevent gametogenesis, as previous studies suggested that initiation of gametogenesis in some species (e.g. S. droebachiensis) is governed by a short-day photoperiod (Walker et al. 1998, Dumont et al. 2006, Böttger et al. 2006). Urchin survival was 100% at the end of the experiment.
2.2.2 Experimental protocols
The experiment was conducted for 12 weeks (October 12, 2007 to January 4, 2008).
Sea urchins were starved for 4 weeks prior to beginning the experiment to standardise their gonadal and nutritional levels. For each temperature treatment (i.e. 8, 12, and 16°C), six replicate pots were randomly selected for each of four feeding treatments: three kelp species (giant kelp, Macrocystis integrifolia; bull kelp, N. luetkeana; sugar kelp,
Saccharina latissima) and a prepared pellet diet developed by the Norwegian Institute of
Fisheries and Aquaculture (NIFA) (Woods et al. 2008).
Table 2.1 Proximate composition (%) and energy content of experimental diets (based on 100-g wet sample).
Dietary constituents Macrocystis Nereocystis Saccharina Prepared diet
Protein 1.3 2.6 2.2 21.3
Lipid 0.1 0.2 0.2 7.9
Carbohydrate 6.6 4.3 5.4 46.8
Moisture 88.2 89.2 87.8 10.6
Ash 3.8 3.7 4.4 13.4
Energy (Calories) 33 29 32 344
Energy (kJ) 136 123 135 1437
55 The kelp plants were collected locally and frozen in plastic closure bags at approximately -17°C. Only fronds, not stipes, were used for the experiment. The plants were thawed before feeding to the urchins. Proximate and caloric analyses of the experimental diets were performed by CANTEST Ltd. (Burnaby, BC) using standard methods in the Official Methods of Analysis of the Association of Official Analytical
Chemists (2000). These values are given in Table 2.1.
The diets were provided ad libitum twice weekly (Tuesday and Friday). Before each feeding, uneaten food was removed with forceps and faeces were siphoned out from each tube. As the rates of water supply and water siphoning were approximately the same, the animals were disturbed minimally during this cleaning procedure. Ingestion rate, gravimetric absorption, and assimilation efficiency were measured at 6 and 12 weeks during the experiment. The diets were weighed prior to feeding at each sampling period.
Faeces that accumulated at the bottom of the tubes after 48 h of feeding were siphoned into large beakers and uneaten food fragments and spines were carefully removed by hand. Faeces were then collected on filter papers (Whatman GF/C filters) to allow excess seawater to drain off, transferred to pre-weighed ashed crucibles, and wet weighed.
The uneaten food fragments were placed in pre-weighed ashed crucibles, and wet weighed. The food and faecal samples were then dried for ~48 h at 70°C to constant weight and percent dry matter calculated. In addition, the weight of kelps and prepared diet in controls without urchins were determined to correct for any change in the biomass due to factors other than urchin feeding. These controls were also used to estimate the equivalent dry weight of the ingested diets. The ash-free dry weights of the feed and faeces were determined by placing the samples in a muffle furnace at 550°C for 4 h.
56 Ingestion rate was calculated over 48 h using the formula: Ingestion rate (wet or dry) = weight of feed provided (wet or dry) – weight of feed not eaten (wet or dry). Gravimetric absorption was calculated as in Larson et al. (1980): Gravimetric absorption (%) = [(dry weight of feed eaten – dry weight of faeces) / dry weight of feed eaten] × 100.
Assimilation efficiency was determined using the Conover equation (Conover 1966):
Assimilation efficiency U' = [(F' – E') / (1 – E') (F')] x 100 where F' = ash-free dry weight:dry weight ratio of feed (i.e. fraction of organic matter in the ingested food) and E'
= ash-free dry weight:dry weight ratio of faeces (i.e. fraction of organic matter in the faeces).
Gonad quantity (i.e. gonad weight and gonad index) and gonad quality (i.e. gonad water, texture, firmness, and colour) were determined at the beginning and end of the experiment using four randomly-selected animals from each temperature treatment at week 0 (that were not part of the feeding trial) and all of the experimental urchins at week
12. Sea urchins were vigorously shaken to remove external water, weighed, cracked open, and the gonads removed. The gonads were rinsed in seawater and gently shaken using forceps to remove as much water as possible. The gonads were then placed in pre- weighed aluminum pans, weighed, assessed for quality (see below), dried to a constant weight in a 70°C oven for ~48 h, and then re-weighed. Gonad index, gonad weight gain, and gonad percent water were calculated using the following formulae:
Gonad index (%) = (gonad wet weight / whole urchin wet weight) × 100
Gonad weight gain (g) = final gonad weight – initial gonad weight
Gonad water (%) = [(gonad wet weight – gonad dry weight) / gonad wet weight] × 100
57 Gonad colour was quantified using a reflected-light, fibre-optic spectrophotometer
(Minolta Chroma Meter CR-100, Konica Minolta Holdings Inc., Tokyo, Japan) taking three replicate measurements from each gonad of L* (intensity or lightness), a* (hue or redness), and b* (chroma or yellowness) [based on the Commission Internationale de
1’Eclairage (CIE) colour measurement system (see Robinson et al. 2002, for details)].
Table 2.2 Criteria for assessment of gonad quality of the sea urchin Strongylocentrotus purpuratus. Adopted from Pearce et al. (2002a).
Criterion Scale
Gonad firmness
(by eye, rating 1–4) 1 = very firm, 2 = firm, 3 = soft, 4 = very soft
Gonad texture 1 = two distinct gonad segment halves, very smooth
(by eye, rating 1–4) 2 = two distinct gonad segment halves, smooth (distinction and smoothness <1)
3 = distinction of gonad segment halves possible but <2, rough/granular
4 = distinction of gonad segment halves not possible, rough/granular
Colour 1 = bright yellow or orange (equivalent to Grade A in commercial roe industry)
(by eye, rating 1–4) 2 = paler yellow or orange (Grade A or Grade B)
3 = yellow-brown, orange-brown, red-brown, cream (Grade B or Grade C)
4 = other colour (e.g. dark brown, grey) (Grade C)
Gonad taste* 1 = excellent (very sweet)
(rating 1–5) 2 = very good
3 = good
4 = satisfactory (not sweet, not bitter)
5 = poor (bitter and of no commercial value)
. *Gonads were soaked in alum for 30 minutes prior to tasting. Alum [KAl(SO4)2 12 H2O] solution is an ingredient use to maintain the firmness of roe in commercial processing plants (Reynolds and Wilen 2000).
58 The CIE system has been previously used by a number of researchers to assess the gonad colour of other sea-urchin species including S. nudus (Agatsuma 1998), S. droebachiensis
(Robinson et al. 2002, Pearce et al. 2004, Daggett et al. 2006, Dumont et al. 2006), and E. chloroticus (Woods et al. 2008). Gonad firmness, texture, and colour were also subjectively quantified (by one person) using the rating scales developed by Pearce et al.
(2002a) (see Table 2.2). A local sea-urchin processor (Paddy Wong, Paladin International
Food Sales Ltd., Richmond, BC, Canada) rated gonad flavour, based on a scale developed by Pearce et al. (2002a).
In addition, the different stages of gonad maturation at week 0 (starting day) for various temperatures and at week 12 (final sampling day) for various diet and temperatures were identified by Motic Images Advanced 3.2 software (Motic Electric
Group Co., Ltd., Richmond, BC, Canada). About 0.5-1.0 g of randomly sampled (from the centre areas of the gonads) gonad section from four urchins for each temperature group (starting day) and from three urchins for each of the diet at various temperature treatments (final sampling day) were collected. The samples were preserved in
Davidson’s solution and slides (5-μm sections) were prepared using standard histological techniques. Histological sections were classified as six maturity stages (Byrne 1990,
Meidel and Scheibling 1998). The six stages are recovering (Stage I), growing (Stage II), premature (Stage III), mature (Stage IV), partly spawned (Stage V) and spent (Stage VI).
This classification scheme is based on the relative abundance of different cell types (e.g. nutritive phagocytes, ova, or spermatozoa) present in gonads during the maturation process (Dumont et al. 2006, Basch and Tegner 2007). The results of the reproductive condition of the gonads were determined and described qualitatively.
59 2.2.3 Statistical analysis
Statistical analyses were conducted using NCSS 2006 (Number Crunching Statistical
Systems, Kaysville, Utah, USA) using the raw data. The effects of diet and temperature on ingestion, gravimetric absorption, assimilation efficiency, and gonad quantity/quality factors were assessed using general linear model (GLM) two-way analysis of variance
(ANOVA) with diet and temperature as fixed factors. Probability plots were used to confirm that data were normally distributed and Levene’s tests were used to verify homogeneity of variances. Data were transformed appropriately, if required, before analyses to ensure data normality and homogeneity of variances (e.g. gravimetric absorption, assimilation efficiency, and gonad index were arcsine transformed). When a two-way ANOVA with significant interaction term occurred, a one-way ANOVA and
Tukey’s honestly significant difference (HSD) test were utilized to examine all possible pair-wise comparisons among all 12 treatments (P<0.05).
2.3 Results
2.3.1 Initial sea-urchin attributes
There were no differences at the beginning of the experiment among any of the three temperature treatments for any of the measured attributes (i.e. test diameter, urchin wet weight, gonad wet weight, gonad index, gonad percent water, firmness, texture, colour,
L*, a*, and b*) (Table 2.3).
60 Table 2.3 Mean (±SE) initial attributes of experimental sea urchins in different temperature groups and ANOVA results for each attribute comparing among the three temperatures.
Temperature (C°) Test Urchin wet Gonad wet Gonad Gonad Gonad Gonad Colour CIE L* CIE a* CIE b*
(n=5229; ±SD) diameter (mm) weight (g) weight (g) index (%) water (%) firmness texture rating value value value
8.0±0.4 58.1±3.6 86.5±16.7 9.2±2.0 10.7±1.6 77.5±2.0 1.3±0.3 1.8±0.5 2.5±0.3 38.8± 3.2 7.2± 1.2 24.4±2.7
12.3±0.3 58.2±3.9 82.4±9.8 9.8±2.5 11.5±2.4 79.4±0.8 1.8±0.5 2.7±0.5 2.5±0.3 36.5±1.1 6.5±1.0 22.2±3.7
16.2±0.4 58.8±3.2 79.5±6.0 7.6±2.0 9.7±1.3 72.6±3.1 1.5±0.3 1.8±0.3 2.0±0.4 37.2±6.0 7.5±1.3 30.0±6.1
SS (temperature) 0.8 98.8 10.4 5.9 96.6 0.5 2.7 0.7 11.7 1.9 129.6
SS (error) 400.8 4919.8 173.9 175.7 172.7 4.5 8.3 4.0 568.0 47.5 686.9
F=0.01 F=0.09 F=0.27 F=0.15 F=2.52 F=0.50 F=1.45 F=0.75 F=0.09 F=0.19 F=0.84
df=2,9 P=0.99 P=0.91 P=0.77 P=0.86 P=0.14 P=0.62 P=0.28 P=0.49 P=0.91 P=0.83 P=0.45
61 Table 2.4 Results of separate two-way ANOVAs on various attributes of the experimental urchins at the end of the experiment. Sources of variation are temperature (T, fixed factor), diet type (D, fixed factor), interaction (T×D), and error. NS = not significant at P>0.05 or more.
Source df SS F ratio P value df SS F ratio P value df SS F ratio P value
Ingested wet weight (week 6) Ingested wet weight (week 12) Ingested dry weight (week 6)
T 2 250.63 16.05 <0.001 2 1212.31 35.04 <0.001 2 12.32 20.38 <0.001
D 3 1074.23 45.86 <0.001 3 1879.49 36.22 <0.001 3 7.69 8.48 <0.001
T×D 6 186.45 3.98 <0.005 6 801.01 7.72 <0.001 6 8.33 4.59 <0.001
Error 60 468.46 60 1037.82 60 18.14
Ingested dry weight (week 12) Gravimetric absorption (week 6) Gravimetric absorption (week 12)
T 2 26.63 25.97 <0.001 2 0.41 5.40 <0.01 2 0.35 4.97 <0.01
D 3 3.19 2.07 >0.1 NS 3 0.61 5.40 <0.005 3 0.24 2.29 >0.5 NS
T×D 6 11.85 3.85 <0.005 6 0.19 0.89 >0.5 NS 6 0.13 0.63 >0.5 NS
Error 60 30.76 60 2.26 60 2.09
Assimilation efficiency (week 6) Assimilation efficiency (week 12) Final gonad weight (week 12)
T 2 0.26 3.97 <0.05 2 0.25 4.31 <0.05 2 203.63 4.96 <0.01
D 3 0.70 7.08 <0.001 3 0.42 4.80 <0.01 3 1596.77 25.93 <0.001
T×D 6 0.23 1.15 >0.1 NS 6 0.13 0.74 >0.5 NS 6 582.26 4.73 <0.001
Error 60 1.96 60 1.74 60 1231.56
Gonad weight gain Final gonad index (week 12) Percent gonad water (week 12)
T 2 262.69 6.40 <0.01 2 0.004 1.48 >0.1 NS 2 67.67 4.44 <0.05
62 Source df SS F ratio P value df SS F ratio P value df SS F ratio P value
D 3 1596.77 25.93 <0.001 3 0.139 34.96 <0.001 3 119.04 5.20 <0.01
T×D 6 582.26 4.73 <0.001 6 0.012 1.54 >0.1 NS 6 94.00 2.05 >0.05 NS
Error 60 1231.56 60 0.079 24 183.00
Gonad firmness (week 12) Gonad texture (week 12) Gonad colour rating (week 12)
T 2 2.25 1.67 >0.1 NS 2 1.58 1.30 >0.1 NS 2 1.03 1.52 >0.1 NS
D 3 4.11 2.04 >0.1 NS 3 0.26 0.14 >0.5 NS 3 0.17 0.16 >0.5 NS
T×D 6 5.30 1.32 >0.1 NS 6 2.53 0.69 >0.5 NS 6 2.42 1.19 >0.1 NS
Error 60 40.33 60 36.5 60 20.33
CIE Lightness L* value (week 12) CIE Hue or redness a* value (week 12) CIE Chroma or yellowness b* value (week 12)
T 2 235.62 2.35 >0.1 NS 2 5.23 0.47 >0.5 NS 2 158.57 1.79 >0.1 NS
D 3 278.00 1.85 >0.1 NS 3 5.47 0.32 >0.5 NS 3 95.11 0.72 >0.5 NS
T×D 6 949.32 3.16 <0.01 6 49.77 1.48 >0.1 NS 6 604.09 2.28 <0.05
Error 60 3006.10 60 336.81 60 2652.42
Gonad taste (week 12)
T 2 0.89 1.00 >0.1 NS
D 3 9.89 7.42 <0.01
T×D 6 9.11 3.42 <0.05
Error 24 10.67
63 2.3.2 Ingestion rate
Diet, temperature, and the interaction between the two factors all significantly affected wet- weight ingestion rates at both 6 and 12 weeks (Table 2.4). Urchins typically ingested significantly greater amounts of all three species of kelps than the prepared diet at both sampling times (Fig.
2.1A).
A Saccharina Macrocystis Nereocystis Prepared 35
d 30 Week 6 Week 12
25 c d 20 c b b b b c b b b c b c a 15 c c c c c c c a b b b b c 10 a a a 5 a a b b
Ingested wet weight (g) a a 0 8 12 16 8 12 16
B Saccharina Macrocystis Nereocystis Prepared
Week 6 Week 12 d
4 c c c d d d 3 d d a a a a a c c b b d b b b b b c d d c c c a a c c c c 2 d d d b b a a a c c b a b 1 a Ingested dry weight (g) Ingested dry 0 8 12 16 8 12 16 Temperature (°C)
Figure 2.1 Mean ingested (A) wet weight and (B) dry weight in the various diet and temperature treatments at weeks
6 and 12. Error bars are SE and n=6. Letters above bars indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among all 12 treatments within weeks.
64 Wet-weight ingestion rate generally increased with increasing temperature during both sample times, although the trend was not always significant (Fig. 2.1A). Similarly, diet, temperature, and the interaction between the two factors all significantly affected dry-weight ingestion rates at week 6, but the effect of diet was not significant at week 12 (Table 2.4). The decreased ingestion rate on the prepared diets as compared to the kelps, as seen with the wet- weight data, was not apparent with the dry-weight ingestion results and it was difficult to see any obvious trends with respect to diet. As with the wet-weight ingestion data, dry-weight ingestion rate generally increased with increasing temperature at both sample times (Fig. 2.1B)
2.3.3 Gravimetric absorption
Both diet and temperature significantly affected gravimetric absorption at week 6, but only the effect of temperature was significant at week 12. There were no significant interaction effects on gravimetric absorption at either week 6 or 12 (Table 2.4). At week 6, gravimetric absorption was significantly lower on S. latissima than on M. integrifolia or N. luetkeana (with no other significant pair-wise comparisons), but there was no significant dietary effect by week 12. The gravimetric absorption generally increased with increasing temperature at both sample times.
Gravimetric absorption was significantly higher for animals held at 16°C than for those at 8°C at week 6 and significantly higher at 16°C than at both 8 and 12°C at week 12. There was no significant difference in gravimetric absorption between 8 and 12°C at either sample time (Fig
2.2A).
65 A Saccharina (1) Macrocystis (2) Nereocystis (2) Prepared (1,2) Week 6 Week 12 100 A AB B A A B
90
80
70
60
50
40 Gravimetric absorption (%) 8 12 16 8 12 16
B Saccharina (1;b) Macrocystis (2;a,b) Nereocystis (2;b) Prepared (1,2;a)
Week 6 Week 12 100 A AB B AA B
90
80
70
60
50 Assimilation efficiency (%) 8 12 16 8 12 16 Temperature (°C)
Figure 2.2 Mean (A) gravimetric absorption and (B) assimilation efficiency of ingested diets in the various diet and temperature treatments at weeks 6 and 12. Error bars are SE and n=6. Letters above bars indicate the results of
Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among temperature treatments (averaged across diets) within weeks. Numbers (for week 6) and letters (for week 12) beside diet treatments in legends indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among diet treatments (averaged across temperatures) within weeks.
66 2.3.4 Assimilation efficiency
Assimilation efficiency at both weeks 6 and 12 varied significantly for different diets and temperatures, but not with the interaction of these two factors (Table 2.4). At week 6, the assimilation efficiency on S. latissima was significantly lower than that on M. integrifolia or N. luetkeana with no other significant pair-wise comparisons (Fig. 2.2B). At week 12, however, the assimilation efficiency on the prepared diet was significantly lower than on S. latissima or N. luetkeana with no other significant pair-wise comparisons. Assimilation efficiency generally increased with increasing temperature during both sample times (except M. integrifolia at week
12). Assimilation efficiency was significantly higher for the animals held at 16°C than for those at 8°C at week 6 and significantly higher at 16°C than at both 8 and 12°C at week 12. There was no significant difference between 8 and 12°C at either sample time (Fig. 2.2B).
2.3.5 Gonad quantity
2.3.5.1 Gonad weight
Diet, temperature, and the interaction between these two factors all significantly affected final gonad weight (Table 2.4). The prepared diet produced significantly higher gonad weight than all three kelps for the animals held at 12 and 16°C. However when the animals were held at 8°C, there were no significant differences in final gonad weight among any of the diets (Fig. 2.3A).
Similarly, gonad weight gain varied significantly with diet, temperature, and the interaction between these two factors (Table 2.4). As with final gonad weight, the prepared diet produced significantly higher gonad weight gain than the all three kelp diets at 12 and 16°C, but there were no significant differences among any of the diets at 8°C (Fig. 2.3B).
67 A Saccharina Macrocystis B Saccharina Macrocystis Nereocystis Prepared Nereocystis Prepared 35 b 25 b b b 29 20
15 23 a a
a a a a 10 a a a a a a a a 17 a a a a a 5 a 11 0 Gonad weight gain (g) Final gonad weight (g) Final gonad 5 -5 8 12 16 8 12 16
C Saccharina (2,3) Macrocystis (1,2) Nereocystis (3) Prepared (4) 30
25
20
15
Final gonad index index Final gonad 10
5 8 12 16 Temperature (°C)
Figure 2.3 Mean (A) final gonad weight, (B) gonad weight gain, and (C) gonad index in the various diet and temperature treatments. Error bars are SE and n=6. Letters above bars indicate the results of Tukey’s HSD multiple- comparison tests with different letters showing significant (P<0.05) pair-wise differences among all 12 treatments.
Numbers beside diet treatments in legend in C indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among diet treatments (averaged across temperatures).
2.3.5.2 Gonad index
Gonad index varied significantly with diet, but not with temperature or the interaction between these two factors (Table 2.4). The prepared diet produced significantly higher indices
68 than all three kelp treatments (Fig. 2.3C). Within the different kelp treatments, urchins fed N. luetkeana had significantly higher gonad indices than those fed M. integrifolia, but there were no other significant pair-wise comparisons (Fig. 2.3C).
2.3.6 Gonad quality
2.3.6.1 Gonad water
Percent gonad water varied significantly with diet and temperature, but not with the interaction between these two factors (Table 2.4). Although there were no significant differences among the kelp treatments, urchins fed the prepared diet had significantly lower percent gonad water than individuals fed M. integrifolia or N. luetkeana (Fig. 2.4A). Urchins held at 16°C had significantly lower percent gonad water than those at 8°C, but there were no other significant pair-wise comparisons among temperatures (Fig. 2.4A).
2.3.6.2 Gonad firmness, texture, and colour ratings
There were no significant effects of diet, temperature, or the interaction between these two factors on gonad firmness, texture, or colour ratings (done by visual observation) (Table 2.4,
Figs. 2.4B,C, D).
2.3.6.3 Gonad colour (L*, a*, b* values)
There were no significant effects of diet or temperature on gonad L* values, but L* was significantly affected by the interaction between these two factors (Table 2.4). A one-way
ANOVA, followed by Tukey’s HD test, showed only three significant pair-wise comparisons among all 12 means: N. luetkeana at 16°C had a significantly lower L* value than S. latissima at
69 16°C and M. integrifolia and N. luetkeana at 12°C (Fig. 2.5A). There were no clear diet or temperature trends in the L* data. Diet, temperature, and the interaction between these two factors did not significantly affect a* values (Table 2.4, Fig. 2.5B).
A B Saccharina (1,2) Macrocystis (2) Saccharina Macrocystis Nereocystis (2) Prepared (1) Nereocystis Prepared 85 B AB A 4 82
79 3
76 2
Gonad water (%) 73 Gonad firmness rating 70 1 8 12 16 8 12 16
D C Saccharina Macrocystis Saccharina Macrocystis 4 Nereocystis Prepared 4 Nereocystis Prepared
3 3 rating by eyes rating by
2 2 Gonad texture rating Gonad texture
1 Gonad colour 1 8 12 16 8 12 16 Temperature (°C) Temperature (°C)
Figure 2.4 Mean gonad (A) percent water, (B) firmness rating, (C) texture rating, and (D) colour rating in the various diet and temperature treatments. Error bars are SE and n=3 for gonad percent water and n=6 for all other variables. Letters above bars indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among temperature treatments (averaged across diets). Numbers beside diet treatments in legend indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among diet treatments (averaged across temperatures).
70 Similar to the L* data, there were no significant effects of diet or temperature on gonad b* values, but b* was significantly affected by the interaction between these two factors (Table 2.4).
However, a one-way ANOVA, followed by Tukey’s HD test, showed only one significant pair- wise comparison among all 12 means: M. integrifolia had a significantly lower b* value at 12 than at 16°C (Fig. 2.5 C).
A Saccharina Macrocystis B Saccharina Macrocystis Nereocystis Prepared Nereocystis Prepared
60 a b b a a 9 b a b b b 54 a b a a 8 * values a b b b L 7 48 b 6 42 a 5 36 CIE Hue a* values 4 CIE Lightness 30 3 8 12 16 8 12 16
D Saccharina Macrocystis C Saccharina Macrocystis Nereocystis Prepared Nereocystis Prepared 40 b 5 a b c b b b 35 b a a a a a 4 c c c c values a b a a a a a a a b a b b b b b b b b* 30 b b b b 3 a b a b 25 2 a
20
Gonad taste rating 1
CIE Chroma 15 0 8 12 16 8 12 16 Temperature (°C) Temperature (°C)
Figure 2.5 Mean gonad (A) L* value, (B) a* value, (C) b* value, and (D) taste rating in the various diet and temperature treatments. Error bars are SE and n=3 for gonad taste rating and n=6 for all other variables. Letters above bars indicate the results of Tukey’s HSD multiple-comparison tests with different letters showing significant
(P<0.05) pair-wise differences among all 12 treatments.
71 2.3.6.4 Gonad taste
Gonad taste rating values were significantly affected by diet and the interaction between the two factors, but not by temperature (Table 2.4). Generally, urchins fed the prepared diet had better tasting gonads (lower rating values) than the three kelp species, although the pair-wise comparisons were not always significant (Fig. 2.5D).
Week 0 Week 12
Recovery Growing Premature Mature Partly spawned 100% 90% 80% 70% 60% 50% 40% 30% 20% 8°C 12°C 16°C C is is s is 8°C 2° ina ed ed ina 1 16°C ared systi p ocyst epar ocystis Pr Prepar cr eocyst Pre Sacchar acr Saccharina Sacchar er M Nereocyst Macro Nereocystis Ma N
Figure 2.6 Frequency (%) of gonads at different stages of maturity at week 0 and week 12 in various temperature and diet treatments.
2.3.6.5 Analysis of gonad histology
The frequencies of the various reproductive stages at weeks 0 and 12 for the various temperature and diet treatments are shown in Fig. 2.6. At the beginning of the experiment, the majority (≥50%) of urchins were in the recovery stage, with the rest in the growing or premature stages. Twelve weeks later, most of the urchins were in the mature stage. A considerable number of urchins held at 12 and 16°C released gametes during the experiment and partly-spawned
72 urchins were observed at the end of the experiment at these two temperatures. No spawning was evident at 8°C.
2.4 Discussion
On a wet weight basis, urchins ingested significantly more kelps than prepared diet. The latter had much higher protein and energy levels than the three kelp species tested (Table 2.1). Lower food quality (e.g. lower energy or protein) may result in larger volumes of kelp plants being ingested by the urchins. Lares and McClintock (1991) reported that when the sea urchin
Eucidaris tribuloides was fed a “low-quality” diet (1% fishmeal) it had significantly higher feeding rates than when it was given a “high-quality” diet (10% fishmeal). They predicted that E. tribuloides compensated for the lower nutritional value of the low-quality diet by increasing the rates of ingestion and absorption. Fernandez and Boudouresque (2000) observed that P. lividus ingested larger volumes of a low-protein-content vegetable diet than a higher-protein-content animal diet. Spirlet et al. (2001) tested four diets – extruded, pellet food based on soya-bean protein; extruded, pellet food based on a mixture of soya-bean and fish protein; dried algae
(Lessonia sp.); and fresh algae (Laminaria sp.) – on gonadal growth of P. lividus. They observed that sea urchins fed the higher-protein prepared diets (both soya-bean and mixture of soya-bean and fish) ingested lower food volumes than individuals given the algal diets, although the differences were not statistically significant. The study also reported that absorption rates were significantly lower on the prepared diets than on the algal diets and also significantly lower on
Lessonia sp. than on Laminaria sp. Higher ingestion rates of natural diets (Laminaria japonica) compared to prepared diets has also been observed by Chang et al. (2005) with S. intermedius.
McBride et al. (2004) found that the rate of food consumption of S. franciscanus is inversely
73 related to the concentration of protein in a prepared diet. Lyons and Scheibling (2007) found that
S. droebachiensis feeding on an invasive alga Codium fragile, a relatively low-energy diet
(8.9±1.8 kJ g-1 dry weight), consistently consumed twice as much (by mass) as those fed the kelp
Laminaria longicruris, a higher-energy diet (12.0±0.8 kJ g-1 dry weight).
Ingestion, absorption, and assimilation are also influenced by factors other than diet, such as temperature and the physiological condition of the experimental animal. In the present study, an increase in temperature led to increased rates of ingestion, absorption, and assimilation, regardless of diet. This relationship between temperature and feeding, absorption, and assimilation rates has also been shown in other echinoid species. Klinger et al. (1986) reported lower ingestion rates and absorption efficiencies for sea urchins (L. variegatus) held at a lower temperature (16°C) than for those held at a higher temperature (23°C). McBride et al. (1997) observed increased feeding rates with an increase in temperature when S. franciscanus was fed either kelp (N. luetkeana) or a prepared diet. Siikavuopio et al. (2006) examined the effects of water temperature (4, 6, 8, 10, 12, and 14°C) and seasons (summer and winter) on food intake of
S. droebachiensis and found that there was a significant and linear increase in feed intake with increasing temperature, both in summer and winter.
For aquaculture purposes, the usefulness of various diets and temperatures for sea-urchin production is ultimately linked to the quantity and quality of the gonads produced on the diets. In the present study, the prepared diet produced significantly higher final gonad weight, gonad weight gain, and final gonad index compared to the other three kelp species tested. Increased gonad production in urchins fed prepared diets versus those given natural algae has been well documented in many echinoid species, including S. franciscanus (McBride et al. 1997), P. miliaris (Cook et al. 1998), P. lividus (Spirlet et al. 2001), and S. droebachiensis (Pearce et al.
2004). Prepared diets typically have much higher protein and energy levels than algae, allowing
74 the urchins to shunt more energy into gonad production. However, Shpigel et al. (2005) investigated the effects of a prepared diet, algal diet, and rotational feeding of these selected diets on the sea urchin P. lividus with the aim of determining which diet regime would produce optimal gonad index and colour. They offered the following diets to adult P. lividus: macroalgae
Ulva lactuca and Gracilaria conferta for 12 weeks; prepared diet for 10 weeks followed by a supply of Ulva and Gracilaria for 2 weeks; prepared diet for 8 weeks followed by Ulva and
Gracilaria for 4 weeks; prepared diet for 6 weeks followed by Ulva and Gracilaria for 6 weeks; prepared diet for 12 weeks; and a combination of Ulva, Gracilaria, and prepared diet for 12 weeks. The study found that the algae alone produced dark-orange coloured gonads but low gonad indices while the prepared diet alone produced good gonad indices but pale colour.
Urchins fed the prepared diet for 8 weeks followed by 4 weeks of algae produced optimal gonad indices and a desired gonad colour.
For those animals fed the prepared diet, final gonad weight and gonad weight gain were significantly higher at 12 and 16°C than at 8°C. Despite the potential importance of temperature on echinoid gonad production, few studies have actually examined the issue. McBride et al.
(1997) compared gonad production of S. franciscanus at 12.9 and 16.1°C, providing either kelp
(N. luetkeana) or a prepared diet. The study found higher gonad indices for the animals held at
16.1°C than those held at 12.9°C, both for animals fed kelp and those given the prepared diet, although there were no significant differences between the temperature treatments. James et al.
(2007) studied the effects of temperature on gonad development in E. chloroticus, comparing constant (~14.7oC) and ambient temperatures in various seasons (austral autumn, winter, spring, and summer). The study reported that gonad index was significantly lower when the temperature was low (in winter: mean of 11.3oC) and significantly higher when the temperature was high (in summer: mean of 18.2oC) as compared to the constant temperature of 14.7oC. James et al. (2007)
75 concluded that the relatively consistent values of gonad index during winter, spring, and summer at constant temperature indicated that temperature had a greater influence on gonad development than any other seasonal effect. However, an increase in temperature (within the animal’s tolerance limits) may only promote gonad growth up to a certain point. For example, Siikavuopio et al. (2006) reared S. droebachiensis at 4, 6, 8, 10, 12, and 14oC and reported that the gonad index value increased to a peak at 10oC, after which there was a trend towards a reduction in gonad index at the highest temperature. In the present study, although both gonad weight and gonad index reached the highest level at the intermediate temperature (12oC), the difference between 12 and 16oC was not significant, suggesting that the limiting upper temperature was not reached in the study.
Although gonad quantity (weight, index) is an important attribute for assessing the market value of an urchin, gonad quality factors – including water content, texture, firmness, colour, and taste – are as important in marketing roe (Pearce et al. 2002a,b, Agatsuma et al. 2005). In the current study, gonads from urchins fed the prepared diet had significantly lower percent water than M. integrifolia and N. luetkeana, although there was no significant difference with S. latissima. While this difference in percent gonad water did not translate into significant differences among the diets in terms of texture or firmness, ratings for these two quality factors were lower (i.e. firmer and better texture) for urchins fed the prepared diet than for those given the kelps at both 12 and 16°C. Similarly, Pearce et al. (2002c) examined the effects of a prepared feed and a diet of kelp (L. longicruris and/or L. digitata) on gonad quality of S. droebachiensis and found that gonads of urchins fed the prepared diet were typically smoother and firmer than those gonads of those fed the kelp diet, although differences between the diets were not significant for these two qualities. In another study, Pearce et al. (2004) reported that higher gonad firmness ratings (i.e. softer gonads) were linked to higher levels of gonad water in S.
76 droebachiensis. In the current study, percent gonad water was significantly lower at 16 than at
8°C, while that at 12°C was intermediate. A similar observation was made by Spirlet et al. (2000) with cultivated P. lividus. These authors reported that percent gonad water was 74.7±1.4,
73.2±0.9, 72.7±1.3, and 65.6±2.6% in urchins held at 12, 16, 20, and 24°C, respectively (i.e. decreasing gonad water with increasing temperature). They reported that percent gonad water values in the 16, 20, and 24°C temperature groups at the end of the experiment were significantly lower than that of the initial values at the beginning of the experiment (i.e. 75.1±2.1%, obtained from urchins held at 12°C), although there were no significant differences between initial and end values at 12°C.
Depending on the local market preference, gonad colour can range from a bright yellow to a dark orange or almost red. Lighter, pale-coloured or dark-brown gonads are not as desirable in the market. In the present study, colour (colour rating, L*, a*, b*,) was not obviously affected by diet or temperature (while there were some significant ANOVA interaction results, there was no clear-cut trend in dietary or temperature affects). The results on redness (a*) and yellowness (b*), obtained by the CIE system, and colour rating by visual judgement were almost the same at the beginning and end of the experiment (Table 2.3 and Fig. 2.5). Woods et al. (2008), working with
E. chloroticus, also reported that the NIFA diet influenced gonad production and taste while not affecting gonad colour. It is known that the pigment carotenoid profile in the diet is the major influencing factor in gonad colouration (Griffiths and Perrott 1976, Agatsuma et al. 2005) and that β-carotene appears to be the particular carotenoid responsible for the yellow-orange colouration in most echinoid species (Griffiths and Perrott 1976, Matsuno and Tsushima 2001,
Robinson et al. 2002, Pearce et al. 2004). Although it is known that β-carotene is the pigment utilized in the NIFA diet (used in the current study), it is unknown what concentration is present
(proprietary information). The level of carotenoid in the NIFA diet (as used at the time of the
77 experiment) may not have been sufficient to significantly affect gonad colouration of S. purpuratus. In a previous study, Robinson et al. (2002) tested AlgroTM – a natural spray-dried preparation of the phytoplankton Dunaliella salina that contains a mixture of carotenoids
[primarily β-carotene (minimum concentration of 2%), but also lesser amounts of α-carotene, cryptoxanthin, lutein, and zeaxanthin] – for gonad colour development of the green sea urchin, S. droebachiensis. In that study a number of concentrations (50, 100, 250, and 500 mg β-carotene kg-1 dry weight of feed) were used in prepared diets and the authors found that 250 mg β-carotene kg-1 dry weight of feed was the most effective concentration for producing suitable gonad colour.
Pearce et al. (2004) compared a prepared diet containing AlgroTM (200 mg of β-carotene kg-1 dry weight of feed) with kelp (L. longicruris and/or L. digitata) for optimizing gonad colour of S. droebachiensis and found better gonad colour in individuals that were fed the prepared diet than in those given kelp. Further work may be required with the NIFA diet to optimize β-carotene levels for generating ideal gonad colouration, but it should be noted that these levels might be species dependent.
In the current study, the NIFA prepared diet gave overall better gonad flavour than the three kelp diets. Morevoer, the NIFA diet produced gonads that ranked as being “very good” (as judged by an urchin processor) in the three tested temperatures. The NIFA diet was graded as giving “good” taste by Woods et al. (2008) in their study on E. chloroticus. These results with a prepared diet are encouraging as previous research on echinoid prepared diets has shown that certain proteins or higher-protein content in prepared diets may be associated with taste bitterness
(Murata et al. 2002, Pearce et al. 2002b, Siikavuopio et al. 2007). The protein concentration of the prepared diet used in the present study is at the optimum level as recommended by de Jong-
Westman et al. (1995). Their study found that a prepared diet with 20% protein was most efficient diet for gonad production of S. droebachiensis. Pearce et al. (2002b) tested three
78 different protein concentrations [19, 24, 29% (percent dry weight)] in prepared diets for gonad production of S. droebachiensis and reported that, although gonad taste was not significantly affected by protein concentration, there was a slight trend of worsening flavour with increasing protein level. However, on the final sampling day, most of the gonads were in mature stages.
McBride et al. (2004) and Dumont et al. (2006) indicated that quality of marketable gonads may be influenced by the reproductive state of gonads of candidate aquaculture species.
Overall, the results of the present study indicated that optimum gonad production and quality in S. purpuratus would be achieved at 12–16°C when feeding a prepared diet (i.e. NIFA diet).
However, commercial gonad production at 16oC not being economically viable that may increase heating cost in comparison to 12oC. Further study, examining various carotenoid concentrations in the NIFA diet or other prepared diets, would be useful for optimizing gonad colour in S. purpuratus. The quantity and quality of the gonads in sea urchins is vital in commercial echinoid farming and is considered critical to the profitability of the aquaculture operation. This study suggests that S. purpuratus could be an excellent potential candidate species for commercial- scale aquaculture as it showed promising gonad yield and quality within 12 weeks of culture.
79 CHAPTER 3: Influence of stocking density on early development and survival of the purple sea urchin3
This study was conducted to determine the optimal stocking density for embryos and larvae of the purple sea urchin, Strongylocentrotus purpuratus. In separate laboratory experiments I evaluated the influence of four initial stocking densities (50, 100, 200, and 400 eggs ml-1 and 0.5,
1, 2, and 4 ind ml-1 for embryos and larvae, respectively) on early development and survival.
Embryo development to prism stage and larval length, mid-line body length, body width, post- oral arm length, time to reach metamorphic competency, and percent survival were assessed during the developmental period. The total length at prism stage was significantly greater in embryos held at 50 and 100 eggs ml-1 than in those held at 200 and 400 eggs ml-1. Larvae grew faster and had significantly higher survival when reared at 0.5 or 1 ind ml-1 then when held at 2 or
4 ind ml-1. Approximately 50% of the larvae held at 0.5 ind ml-1 were competent to metamorphose by day 24 (from prism stage), whereas larvae held at 1 ind ml-1 did not become competent until day 28 and those held at 2 and 4 ind ml-1 failed to develop to metamorphic competency by the termination of the experiment (day 28). Overall survival (from prism stage to metamorphic competency) in the best treatment was 48.9±2.2% (mean±SE) for the 1 ind ml-1 group.
3A version of this chapter has been published. Azad A.K., Pearce C.M. and McKinley R.S. 2011. Influence of stocking density and temperature on early development and survival of the purple sea urchin, Strongylocentrotus purpuratus (Stimpson, 1857). Aquaculture Research: in press
80 3.1 Introduction
The continued demands for sea-urchin gonads or “roe” and over exploitation of natural stocks in many countries have prompted interest in the culture of various sea-urchin species around the world (Hagen 1996, Keesing and Hall 1998, Grosjean et al. 1998, Robinson 2004). Mass seed production is widely recognized as a major bottleneck in the culture of any new candidate aquaculture species and survival and growth rate are benchmark measures of success in the production of young juveniles. With the increasing interest in culturing sea urchins there is a need for information on how various biotic and abiotic factors affect the growth and survival of larval echinoids. Over the past two decades a considerable number of studies has examined the effect of various biotic factors (including diet and ration) on larval development of various sea-urchin species (Hart and Scheibling 1988, Kelly et al. 2000, Jimmy et al. 2003, George et al. 2004,
Cárcamo et al. 2005, Liu et al. 2007a,b), but relatively little attention has focused on the effects of various abiotic factors.
Stocking density is one of the important abiotic factors affecting the early development and survival of marine invertebrates in culture (Ibarra et al.1997, Liu et al. 2006). The optimum rearing density of planktotrophic larvae is crucial as over-crowding may limit both swimming space and access to food resources and can lead to low dissolved oxygen and high nitrogenous waste concentrations – all of which may lead to reduced overall development and survival rates
(Wang and Widdows 1991, Tomasso 1994, Doroudi and Southgate 2000). Studies of marine invertebrate larval development usually maintain larval rearing densities in the range of 0.2 to 5 ind ml-1 (see review by Marshall et al. 2010). In some cases stocking densities have been substantially higher, as in the culture of the Babylon snail, Babylonia spp., (Chaitanawisuti and
Kritsanapuntu 1997, Shieh and Liu 1999) and clam Meretrix meretrix (Liu et al. 2006), where 8–
81 10 ind ml-1 have been commonly used. For echinoid culture, published studies have typically used ≤2 ind ml-1 (see chapter 1). Surprisingly though, only one known published study has examined the potential effects of stocking density on larval sea-urchin growth and survival.
Buitrago et al. (2005) examined three different stocking densities (0.25, 0.50, and 1.00 ind ml-1) of larvae of the sea urchin Lytechinus variegatus and concluded that higher-density culture (i.e.
0.50–1.00 ind ml-1) had no apparent disadvantages and would reduce the overall cost of seed production. While ample studies have examined reproductive periodicity and exogenous cues controlling reproduction and gametogenesis in purple sea urchins (e.g. Boolootian 1963, Gonor
1973, Cochran and Engelmann 1975, Pearse et al. 1986, Bay-Schmith and Pearse 1987), little research has examined the effect of factors such as stocking density on larval development and survivorship. In the present study we examined growth and survival of embryonic and larval
Strongylocentrotus purpuratus in response to a range of stocking densities. It was hypothesized that higher stocking densities will significantly reduce embryonic/larval development, growth, and survival.
3.2. Materials and methods
3.2.1 Broodstock collection and general experimental techniques
Adult purple sea urchins were collected by SCUBA divers from the subtidal region at
Sombrio, west Vancouver Island (48°25΄N, 124°3΄W), BC, Canada. They were transported (~2 hours) to the Pacific Biological Station, Nanaimo, BC in insulated coolers and held in indoor tanks with running, ambient (10.7°C), sand-filtered seawater prior to spawning. The animals were fed ad libitum with Macrocystis integrifolia and Nereocystis luetkeana twice weekly. Before each feeding, uneaten food and faeces were siphoned out from the broodstock maintenance tanks.
82 Spawning induction and larval culture of S. purpuratus were based on methods described by
Leahy (1986), Pearce and Scheibling (1990), and Cárcamo et al. (2005). Individual urchins were induced to spawn by intracoelomic injection with 2.5–3.0 ml of 0.53-M potassium chloride
(KCl). Spawning animals were placed oral side up over 400-ml glass beakers. Females released eggs into 0.2-µm filtered, UV-sterilised seawater (hereafter referred to as UVS seawater) while males released sperm into dry beakers, which were placed on crushed ice. After 25–30 minutes of spawning, eggs of females and sperm of males were pooled separately. The eggs were rinsed three times with UVS seawater to remove any foreign debris. Four to five drops of diluted sperm were added to the eggs and the mixture was stirred gently for approximately five minutes. The inseminated eggs were then rinsed a further three times with UVS seawater to remove excess sperm. The percentages of fertilized eggs were verified by the appearance of the fertilization membrane, and were always >99%. Fertilized eggs were transferred to 20-L white plastic buckets filled with 10 L of UVS seawater. The rearing buckets were placed partially submerged inside fibre-glass seawater tables (Length x Width x Depth: 120 x 90 x 30 cm) with flow-through seawater to the tables to maintain temperature in the buckets at the desired level. Specific seawater temperatures were established via appropriate mixing of ambient and chilled (~5°C) or heated (~25°C) seawater. Water temperature was automatically recorded in each tank every 30 min during the experiments by temperature loggers (HOBO data logger, Onset Computer
Corporation, Pocasset, Massachusetts, USA). Lighting for the experiments was provided by overhead fluorescent lights at a light intensity (at culture bucket level) of 110.3±18.8 lux
(mean±SD, n=10) which were set for a constant photoperiod of 12 h light:12 h dark.
Embryos were reared under static condition until prism-shaped larvae were seen swimming in the water column. On day 4 (post-fertilization), when larvae reached the prism-shape stage and first-feeding point (Miner 2007), they were transferred to clean 20-L white plastic buckets filled
83 with 15 L of UVS seawater. The larvae were fed laboratory-cultured live phytoplankton
Dunaliella tertiolecta (CCMP 1320) daily. The algae were batch cultured in 20-L carboys and harvested at the exponential growth phase for the experiments. Phytoplankton culture methods and nutrients were based on Harrison et al. (1980). Dunaliella tertiolecta – a unicellular flagellate green alga – has been widely used for the culture of larvae of a wide variety of echinoid species including Strongylocentrotus droebachiensis (Hart and Scheibling 1988, Pearce and Scheibling
1990, 1991, 1994), Psammechinus miliaris (Kelly et al. 2000), Echinus esculentus (Jimmy et al.
2003), Lytechinus variegatus (George et al. 2004), and Paracentrotus lividus (Liu et al. 2007a), and has been successfully used to rear echinoid larvae up to metamorphic competency.
Hinegardner (1969) reported that the concentration of algae required for larval echinoids depends on the stage of development, with earlier stages needing much less than older ones. He noted that a daily ration of about 3,000 cells ml-1 (algal species not mentioned) was suitable for young echinoplutei. Jimmy et al. (2003) observed that larvae of E. esculentus grew well when offered a ration of 1,000, 3,000, and 5,000 cells ml-1 (according to developmental stage) of D. tertiolecta every third day of the culture period. In the present study, the ration fed varied according to larval density and developmental stage. To avoid over or under feeding, larvae were checked daily for the presence or absence of algae in the gut, in addition to sampling the culture water for determining the concentration of remaining algal cells. When the algal concentration fed reached more than 4 x 103 cells ml-1 the total amount was provided at two different times per day. Phytoplankton rations during the experimental period are given in Table 3.1. The larval rearing buckets were placed partially submerged inside fibre-glass seawater tables described above. The tables were provided with flow-through water while buckets were kept static.
Constant aeration of the larval cultures was provided through a plastic capillary at the bottom of each bucket. Every second day, the rearing water was completely exchanged for fresh UVS
84 seawater while larval samples were taken every four days. During water changes, larvae were retained on a 60-µm mesh screen submerged in UVS seawater and the rearing buckets bleached and rinsed three times with freshwater as well as UVS seawater.
Table 3.1 Concentration of Dunaliella tertiolecta fed to Strongylocentrotus purpuratus larvae over time.
The estimated number of algal cells per larva (taking larval survival into consideration) is given in parentheses.
Larval density Algal density x 103 ml-1 (x 103 larva-1)
(ind ml-1) Days 0–3 Days 4–7 Days 8–11 Days 12–15 Days 16–19 Days 20–23 Days 24–27
0.5 0.5 0.75 1.0 1.25 1.5 1.75 2.0
(1.0) (1.6) (2.3) (2.9) (4.3) (5.8) (7.7)
1 1.0 1.5 2.0 2.5 3.0 3.5 4.0
(1.0) (1.5) (1.8) (3.0) (4.5) (6.2) (7.3)
2 2.0 3.0 4.0 5.0 6.0 7.0 8.0
(1.0) (1.6) (2.2) (3.5) (4.2) (5.7) (7.3)
4 4.0 6.0 8.0 10.0 9.0 9.0 9.0
(1.0) (1.6) (2.2) (3.8) (3.9) (5.5) (6.8)
Each sampling day the retained larvae were concentrated in 500-ml beakers. After completely mixing the larvae in the beaker, 1-ml (0.5 ml x 2 times) a sample was taken from each replicate bucket and preserved with Lugol’s solution. Larval samples were taken at days 0, 4, 8, 12, 16, 20,
24, and 28 (post-prism stage). Larval cultures were terminated one sampling day after 50% of the larvae in the fastest developing treatment became competent to metamorphose (at day 28 from prism stage). Larvae were considered competent when the rudiment was well developed and
85 swimming was directed to the walls or bottoms of the containers (Burke 1980, Fenaux et al.
1994, George et al. 2001, Cárcamo et al. 2005).
3.2.2 Experimental protocols
3.2.2.1 Influence of stocking density on embryonic development
Adult urchins (3 males and 3 females) were induced to spawn on November 14, 2008. The resulting eggs (diameter: 79.2±0.6 μm, mean±SE, n=21) and fertilized eggs (diameter: 98.2±1.3
μm, mean±SE, n=21) were rinsed with UVS seawater at 11°C. The fertilized eggs were transferred to 20-L plastic buckets with 10 L of UVS seawater (11°C) at four stocking densities
(50, 100, 200, and 400 eggs ml-1 or 940, 1880, 3760, and 7520 eggs cm-2) with three replicates per treatment. The 50 and 100 eggs ml-1 concentrations resulted in a monolayer of eggs on the bottoms of the buckets. The replicate rearing buckets were fully randomized within two fibre- glass seawater tables as described above and the water temperature was 11.1±0.3°C (mean±SD, n=192) during the experimental period.
In this early development study, samples were taken at days 0, 1, 2, 3, and 4 post- fertilization. Qualitative observations for embryonic development were made by visually each day of the rearing period. Since the eggs were settled on the container bottoms, a1-ml (0.5 ml x 2 times) sample was gently pipetted from each replicate bucket from randomly selected spots within designated areas on the bottom of the rearing buckets (Fig. 3.1A). Live and dead fertilized eggs/embryos, blastulae, and gastrulae were counted in each sample using a Sedgewick-Rafter chamber and a dissecting microscope. Precautionary measures were taken to avoid counting unfertilized and polyspermic eggs. Egg diameter or embryo length (longest dimension) of seven
86 randomly-selected individuals from each replicate bucket of each treatment was measured using
Motic Images Advanced 3.2 software (Motic Electric Group Co., Ltd., Richmond, BC, Canada).
A B
Figure 3.1 (A) Egg/embryo sampling areas on the bottom of the culture container on different days (D) of embryonic development. (B) Microphotograph of larva of Strongylocentrotus purpuratus illustrating body dimensions measured: (a) larval total length, (b) mid-line body length, (c) body width, and (d) post-oral arm length.
3.2.2.2 Influence of stocking density on larval development
Larvae derived from the 100 eggs ml-1 group (total length: 209.3±1.3 μm, mean±SE, n=20) were used for the subsequent larval experiment. Larvae were reared at four stocking densities
(0.5, 1, 2, and 4 ind ml-1) with four replicates per treatment. The replicate rearing buckets were fully randomized within two fibre-glass seawater tables as described above and the water temperature was 11.2±0.2°C (mean±SD, n=1248) during the experimental period. One of the replicates from the 1 ind ml-1 group was lost due to unknown causes after 8 days. Numbers of
87 larvae in each sample were counted using a Sedgewick-Rafter chamber and a dissecting microscope and percent larval survival calculated based on original stocking densities.
Morphological parameters including total length, mid-line body length, body width, and post-oral arm length were measured with Motic Images software (Fig. 3.1B). Larval measurements were made on five randomly-selected individuals from the sample taken from each replicate bucket of each treatment.
3.2.3 Statistical analysis
Statistical analyses were conducted using NCSS 2006 (Number Crunching Statistical
Systems, Kaysville, Utah, USA). The effects of stocking density on various attributes were assessed using one way analysis of variance (ANOVA). Probability plots were used to confirm that data were normally distributed and Levene’s test were used to verify homogeneity of variances. The final day sampling data on morphometric measurements and percent survival were chosen for statistical comparison (Table 3.2). The mean values of the sampled individuals of each bucket were used for the statistical comparison. Tukey’s honestly significant difference (HSD) tests were used to evaluate all pair-wise differences between the means (P<0.05). The nonparametric method Kruskal-Wallis one way ANOVAs were used when normality or equal variance assumption violated and the data could not transformed effectively.
3.3 Results
3.3.1 Influence of stocking density on embryonic development and survival
In general, embryonic developmental and growth rates were slower with increasing stocking density (Table 3.3 and Fig. 3.2). At day 1 (post-fertilization), all embryos in the 50, 100, and 200
88 eggs ml-1 treatments had developed to the motile blastula stage, while most of the individuals stocked at 400 eggs ml-1 were still at the fertilized-egg stage. At day 2, gastrulation began in the
50 and 100 eggs ml-1 treatments, but this was delayed by a day in the 200 and 400 eggs ml-1 densities. At day 3, most of the embryos stocked at 50 eggs ml-1 were near the prism stage while only ~50% of those held at 100 eggs ml-1 reached this point (no prism-stage individuals were evident in the two higher densities at day 3). At day 4, >90% of the embryos held at 50 and 100 eggs ml-1 had reached the prism stage and >50% had developed to the two-arm larval stage whereas only >50% embryos held at 200 eggs ml-1 and 400 eggs ml-1 had reached the prism stage and there were no two-armed larvae. Percent survival (of the embryos on the bottom of the containers) decreased over time (Table 3.3). Embryo length was significantly affected by stocking density at day 4 (Table 3.2) and was significantly greater at stocking densities of 50 and
100 than at 200 and 400 eggs ml-1, with no other significant pair-wise comparisons (Fig. 3.2).
250 50 eggs ml-1 100 eggs ml-1 b -1 -1 m) 200 eggs ml 400 eggs ml b μ 210 a a
170
130 Embryo length ( Embryo length
90 0 1 2 3 4 Days
Figure 3.2 Mean (±SE) embryo length (μm) at various stocking densities over time. n=3. Letters above bars indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day (Table 3.2).
89 3.3.2 Influence of stocking density on larval development and survival
Larval total length, mid-line body length, body width, post-oral arm length, and percent survival were all significantly affected by stocking density (Table 3.2). At day 28, Tukey test results were the same for total length, mid-line body length, body width, and percent survival: values were significantly greater for larvae reared at 0.5 and 1 ind ml-1 than for those held at 2 and 4 ind ml-1, with values for 2 ind ml-1 being significantly greater than those for 4 ind ml-1 (Fig.
3.3A,B,C,E). Results for post-oral arm length were slightly different: larvae reared at 4 ind ml-1 had significantly shorter arm lengths than those held at all other densities, with no other significant pair-wise comparisons (Fig. 3.3D). At 0.5 ind ml-1 50% of the larvae attained metamorphic competency at day 24, while it took 28 days for 50% of the larvae held at 1 ind ml-1 to achieve competency. Larvae grew relatively slower when reared at 2 and 4 ind ml-1 and failed to attain metamorphic competency within 28 days.
90 Table 3.2 Results of separate one-way ANOVAs or Kruskal Wallis tests (H values in bold) on various attributes of embryos and larvae of Strongylocentrotus purpuratus. Source of variation is stocking density. Source df SS F ratio/ P value df SS F ratio/ P value df SS F ratio/ P value
H value H value H value
Embryo final length Larval final total length Larval final mid-line body length
Stocking density 3 1252.2 8.1 <0.05 3 85089.1 145.3 <0.001 3 16611.2 101.0 <0.001
Error 8 412.7 11 2146.7 11 603.3
Larval final body width Larval final post-oral arm length Larval final survival percent
Stocking density 3 12.2 <0.05 3 7681.1 10.2 <0.05 3 1153.0 24.4 <0.001
Error 11 2752.2 11 173.3
Table 3.3 Qualitative and quantitative observations on embryonic development of Strongylocentrotus purpuratus at various stocking densities (mean±SE). Percent survival based on total number of eggs/embryos on culture container bottom. Density (ind ml-1) Observations Day 1 Day 2 Day 3 Day 4
50 Total embryos 248.6±18.9 28.3±3.7 27.7±3.5 22.6±2.2
(940 eggs cm-2) Dead embryos 18.3±1.5 15.0±0.6 18.0±2.9 20.0±2.1
Percent survival 92.6±0.3 45.5±6.1 34.7±7.3 11.7±4.1
Developmental stage All motile blastulae All motile blastulae All near prism shape >90% prism-shaped larvae
Started gastrulation Prominent pigmentation started >50% larvae with 2 arms
Pink colouration of culture
91 Density (ind ml-1) Observations Day 1 Day 2 Day 3 Day 4
100 Total embryos 525.7±13.4 94.3±5 71.6±6.2 67.7±4.1
(1880 eggs cm-2) Dead embryos 50.3±4.3 50.3±1.8 43.3±4.8 53.3±0.3
Percent survival 90.4±0.7 46.4±3.5 39.8±1.5 20.5±5.5
Developmental stage All motile blastulae All motile blastulae 50% larvae near prism shape >90% prism-shaped larvae
Started gastrulation Prominent pigmentation started >50% larvae with 2 arms
Pink colouration of culture
200 Total embryos 1204.0±16.5 239.7±4.3 176.3±3.7 148.0±7.0
(3760 eggs cm-2) Dead embryos 94.6±4.3 109.6±1.5 116.3±12.7 114.3±1.8
Percent survival 92.1±0.5 54.2±1.4 33.8±7.8 22.3±4.5
Developmental stage All motile blastulae All motile blastulae Started gastrulation >50% prism-shaped larvae
Prominent pigmentation started
Pink colouration of culture
400 Total embryos 1906.3±94.4 621.3±11.3 417.0±10.3 312.3±10.2
(7520 eggs cm-2) Dead embryos 143.0±12.5 155.0±10.4 166.7±6.1 181.7±6.0
Percent survival 92.5± 0.3 75.0±1.9 60.0±1.5 41.8±1.4
Developmental stage Most embryos with All motile blastulae Started gastrulation >50% prism-shaped larvae
fertilized membrane Prominent pigmentation started
Few blastulae Pink colouration of culture
92 A 600 0.5 ind ml-1 1 ind ml-1 c 2 ind ml-1 4 ind ml-1 c 520 m) μ
440 b
a 360
Larval total length ( 280
200 0 4 8 12 16 20 24 28
B 350 0.5 ind ml-1 1 ind ml-1 c c 2 ind ml-1 4 ind ml-1
m) 310 μ b 270 a
230
190 Mid-line body length ( Mid-line body
150 4 8 12 16 20 24 28
C 320 c c 0.5 ind ml-1 1 ind ml-1 -1 -1 280 2 ind ml 4 ind ml
) m μ ( 240 b
width 200 y a Bod 160
120 4 8 12 16 20 24 28 Days
93
D b 200 -1 -1 m) 0.5 ind ml 1 ind ml b μ b 2 ind ml-1 -1 170 4 ind ml a 140
110 Post-oral arm length ( 80
50 4 8 12 16 20 24 28 E 100 0.5ind ml-1 1 ind ml-1 2 ind ml-1 4 ind ml-1 80
60 c c b
Larval survival (%) 40 a
20 4 8 12 16 20 24 28 Days
Figure 3.3 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival at various stocking densities over time. n = 4. Letters above bars indicate the results of
Tukey’s HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day (Table 3.2)
3.4 Discussion
Egg density can be measured relative to the volume of water in which the eggs are suspended
(e.g. eggs ml-1) or relative to the surface area of the bottom of the container (e.g. eggs cm-2).
Since the eggs of S. purpuratus are negatively buoyant and settle to the bottom of the rearing unit, bottom surface area is a critical factor for the earliest stages of development. Once embryos
94 become motile and swim up into the water column, the water volume of the container becomes more important. From the present study, it is clear that increasing fertilized-egg concentration beyond a certain point will slow embryonic development and increase egg/embryo mortality.
From the results obtained with the stocking density treatments tested in the present work, we would suggest a maximum stocking density of 1880 eggs cm-2 for the purple sea urchin. It should be noted, however, that increased stocking densities of fertilized eggs and embryos may be achieved by suspending them in the water column through aeration or gentle agitation.
Published work on the influence of stocking density on early development of echinoids is scant, but our results are comparable to those of various studies with other marine invertebrates.
For example, Southgate et al. (1998) examined the effects of stocking density of fertilized eggs of oysters Pinctada maxima (10, 20, 30, 40, and 50 eggs ml-1) and Pinctada margaritifera (10, 20,
30, 50, 100, and 150 eggs ml-1) and observed that overcrowding of fertilized eggs or embryos caused slow development and decreasing survival rates. Although survival rates were poor, there was no significant difference between densities of 20 and 50 eggs ml-1 for P. maxima or between
30 and 100 eggs ml-1 for P. margaritifera. Galley et al. (2010), working with the blue mussel,
Mytilus edulis, reported that culturing fertilized eggs at a lower density (20–200 eggs cm-2) significantly improved the quality of veliger larvae when compared to those cultured at a higher density (400–720 eggs cm-2). Liu et al. (2010) tested eight stocking densities (0.2, 0.5, 1, 2, 5, 10,
20, and 50 ind ml-1) of fertilized eggs of the sea cucumber Apostichopus japonicus and reported that the hatching rate was significantly higher at 0.2–5 ind ml-1 than at 20–50 ind ml-1.
Stocking density also had a significant effect on larval development in S. purpuratus. Our study showed that all larval measurements (except for post-oral arm length) were significantly smaller at densities of 2 and 4 ind ml-1 than at 0.5 and 1 ind ml-1 at the end of the experiment; a similar result was seen with percent survival. Larvae reared at 0.5 and 1 ind ml-1 showed normal
95 development and became competent to metamorphose at days 24 and 28, respectively, whereas larvae reared at 2 and 4 ind ml-1 prolonged their development and failed to attain metamorphic competency by day 28. These results are comparable to other studies that have examined the effect of echinoderm larval stocking densities. Basch (1996), working with the sea star Asterina miniata, noted that larval density had a striking effect on juvenile rudiment development and time to metamorphosis. The study reported that juvenile rudiment diameter was significantly larger in larvae reared at 0.5 ind ml-1 than in larvae held at 1.0 ind ml-1, although there was no significant difference in survival rate between the two treatment groups. Larvae reared at 0.5 ind ml-1 developed to metamorphic competency >1.5 times faster (day 30) than that of siblings held at 1.0 ind ml-1 (day 50). Li and Li (2010) examined the effects of five larval stocking densities (0.05,
0.1, 0.2, 0.4, and 0.8 ind ml-1) on larval development and survival of the sea cucumber A. japonicus. The study observed density-dependent effects, with survival rate being significantly lower for the highest stocking density (0.8 ind ml-1) than for the other treatments. Li and Li
(2010) reported that a larval density between 0.1–0.2 ind ml-1 is best for rearing A. japonicus.
Buitrago et al. (2005) tested three different stocking densities (0.25, 0.50, and 1.00 ind ml-1) for larvae of the sea urchin L. variegatus and reported that there were no significant differences among the three stocking densities in terms of survival (all exceeded 65%), larval length, and larval stage index, although larval weight in the low-density treatment was greater than in the high-density treatment. Buitrago et al. (2005) concluded that a culture density of 0.50–1.00 ind ml-1 had no apparent disadvantages and would reduce the overall cost of seed production. The present study also revealed that a larval density of ≤1.0 ind ml-1 would be most suitable for larval culture of S. purpuratus. Possible explanations for slower development and lower survival of larvae held at higher densities may be competition for food and space or the faster build up of metabolic wastes compared to cultures with animals held at lower densities. In the present study,
96 algal cell concentrations were adjusted according to larval density and developmental stage in an attempt to ensure that larvae in each treatment received suitable rations (Table 3.1). It is unlikely that larvae held at higher densities were underfed. Ration was capped at a certain level (9–10 x
103 cells ml-1) to prevent potential overfeeding effects. With increasing larval density and algal cell concentration, more metabolic waste may accumulate in the rearing water, which can have detrimental effects on larval development and survival. However, in our study, 100% of the culture water was changed every second day and periodical measurements of dissolved oxygen and ammonia concentrations showed that these culture parameters were within suitable levels
(7.4–9.1 mg L-1 and <0.2 mg L-1, respectively) (Tomasso 1994, Saco-Álvarez et al 2010).
Competition for space seems to be the most likely reason for decreased larval performance at higher stocking densities in the current study, although further research would be required to determine the exact contribution of food/space competition and metabolic wastes to decreased larval growth/survival.
In summary, prism stage length was significantly greater in embryos held at 940 and 1880 eggs cm-2 than in those held at 3760 and 7500 eggs cm-2. Larvae grew faster and had significantly higher survival when reared at 0.5 or 1 ind ml-1 then when held at 2 or 4 ind ml-1. Approximately
50% of the larvae held at 0.5 ind ml-1 were competent to metamorphose by day 24 (from prism stage), whereas larvae held at 1 ind ml-1 did not become competent until day 28 and those held at
2 and 4 ind ml-1 failed to develop to metamorphic competency by the termination of the experiment (day 28). Stocking densities of ≤1880 eggs cm-2 and ≤1 larva ml-1 are recommended for commercial hatcheries, although further research at an industrial scale would need to be conducted to ensure that these results translate to larger scales.
97 CHAPTER 4: Influence of temperature on early development and survival of the purple sea urchin4
This study was conducted to determine the optimal temperature for embryos and larvae of the purple sea urchin, Strongylocentrotus purpuratus. In separate laboratory experiments I evaluated the influence of four temperatures (8, 11, 14, and 17oC) on embryonic and larval development, growth, and survival. Embryo development to prism stage and larval length, mid-line body length, body width, post-oral arm length, time to reach metamorphic competency, and percent survival were assessed during the developmental period. Embryos typically had higher percent survival at 11 and 14°C than at 8 and 17°C, while embryo length was significantly smaller in individuals held at 8°C than in those reared at 11, 14, or 17°C. Larvae grew significantly slower at 8°C than at 11, 14, or 17°C (with little difference among the latter three treatments), while survival was significantly reduced at 8 and 17°C compared to 11 and 14°C. Although 50% of the larvae held at 17oC became competent to metamorphose by day 24 (from prism stage), larvae held at 11 and 14oC did not begin to attain metamorphic competency until day 28. Overall survival (from prism stage to metamorphic competency) in the best treatment was 50.0±3.6%
(mean±SE) for the larvae held at 11°C.
4A version of this chapter has been published. Azad A.K., Pearce C.M. and McKinley R.S. 2011. Influence of stocking density and temperature on early development and survival of the purple sea urchin, Strongylocentrotus purpuratus (Stimpson, 1857). Aquaculture Research: in press
98 4.1 Introduction
Water temperature is generally regarded as the dominant factor controlling the development of poikilotherms (Eckert et al.1988). It is well established that temperature can have significant influences on early development of marine invertebrates and that optimal performance is obtained within a relatively narrow range of temperatures for a particular species (Pechenik 1987,
Roller and Stickle 1989, Anil et al. 2001, Ouellet and Chabot 2005, Desai et al. 2006). Despite the known importance of temperature on invertebrate larval development, very few studies have examined the effects of this factor on development and survival of larval echinoids.
Hart and Scheibling (1988) reared larvae of the green sea urchin, Strongylocentrotus droebachiensis, at three temperatures (3, 6, and 9oC) and provided them three rations (500, 1000, and 5000 cells ml-1) of a 1:1 mixture (by cell number) of the phytoplankton Dunaliella tertiolecta and Chaetoceros gracilis. They found that the main effects for temperature were always highly significant and that the larvae grew quicker at 9oC than at 3 and 6oC. Sewell and Young (1999) observed that temperature had an important influence on embryonic development and survivorship in the tropical sea urchin Echinometra lucunter. Their study found that water temperatures from 27 to 34oC appeared to be optimal for early-stage development, although normal growth occurred at temperatures beyond the limits encountered during the breeding season of E. lucunter in any part of its geographical range (Sewell and Young 1999). Aside from these two published studies, no other work has examined the specific effects of temperature on larval echinoid development. Leahy et al. (1978) and Leahy (1986) described general protocols for rearing Strongylocentrotus purpuratus through its entire life cycle under laboratory conditions, but they did not examine the specific effects of stocking density and temperature on larval development. Miller and Emlet (1999) investigated the effects of temperature (8±0.1,
99 11±0.1, and 14.4–14.7oC) on the development of juvenile S. purpuratus and Strongylocentrotus franciscanus. They discovered that the juvenile mouth of both species opened and feeding began on day 9 at 14.4–14.7oC, but not until day 12 at 11±0.1oC. Functional pedicellariae occurred in
50% of individuals of S. purpuratus by day 9 at 14.7oC, but not until day 14 at 8±0.1oC. Apart from this limited work very little is known about the conditions required for larval rearing of purple sea urchins. In the present study we examined survival and growth of embryonic and larval S. purpuratus in response to a range of temperatures. It was hypothesized that temperature will significantly affect embryonic/larval development, growth, and survival.
4.2 Materials and methods
4.2.1 Broodstock collection and general experimental techniques
Broodstock collection, maintenance, algal culture, cell densities, and general experimental techniques were the same as those described in chapter 3 for the stocking density experiment.
4.2.2 Experimental protocols
4.2.2.1 Influence of temperature on embryonic development
Adult urchins (3 males and 3 females) were induced to spawn on March 25, 2009. The resulting eggs (diameter: 80.4±0.6 μm, mean±SE, n=21) and fertilized eggs (diameter: 105.7±0.8
μm, mean±SE, n=21) were rinsed with UVS seawater at 11°C. The fertilized eggs were transferred to 20-L plastic buckets with 10 L of UVS seawater (11°C) and gradually acclimated by adjusting by 2°C per 8 hours (this took a maximum of 24 hours from initial stocking) to four temperatures [8.0±0.2, 11.3±0.2, 13.9±0.4, and 17.1±0.4°C (mean±SD, n=165)] with three replicates per treatment. The stocking density for this experiment was 100 eggs ml-1 (1880 eggs
100 cm-2), which was based on results obtained in the previous stocking density experiment. The replicate rearing buckets were fully randomized within four fibre-glass seawater tables as described above. Sampling, qualitative observations, percent survival calculations, and morphological measurements were done using the same techniques as described for the stocking density (chapter 3) experiment.
4.2.2.2 Influence of temperature on larval development
Larvae derived from the 11oC group (total length: 219.2±1.8 μm, mean±SE, n=20) were used for the subsequent larval experiment. Larvae were reared at four temperatures [8.0±0.3, 11.2±0.3,
14.1±0.4, and 16.8±0.4oC (mean±SD, n=1225)] with four replicates per treatment. The initial larval density was 1 ind ml-1, based on results of the previous stocking density experiment. The larvae were fed laboratory-cultured live phytoplankton Dunaliella tertiolecta (CCMP 1320) daily. The replicate rearing buckets were fully randomized within four fibre-glass seawater tables as described above. To minimize temperature stress, the larvae were acclimated by adjusting seawater temperatures by 1°C per 8 hours (this took a maximum 48 hours from initial stocking).
Percent survival calculations and morphological measurements were done using the same techniques as described in the previous chapter for the stocking density experiment. In addition, to describe the sensitivity of developmental rate (both for post-fertilized pre-feeding larvae and feeding larvae) to changes in temperatures – a temperature quotient Q was determined (Table
4.3). The effect of temperature on physiological processes is traditionally summarized by a Q10 value that gives the relative change in rate of development over a specific 10°C change in temperature (Eckert et al. 1988). The Q10 was calculated by using the van’t Hoff equation:
10/ (T -T ) Q10= (R2/R1) 2 1
Where R2 and R1 are developmental rate at temperatures T2 and T1, respectively.
101 4.2.3 Statistical analysis
Statistical analyses were conducted using NCSS 2006 (Number Crunching Statistical
Systems, Kaysville, Utah, USA) on data from the final sampling day. Mean values of the sampled individuals from each replicate bucket were used for the statistical analyses. The effects of temperature on various embryo/larval attributes were assessed using one-way analyses of variance (ANOVA). Probability plots were used to confirm that data were normally distributed and Levene’s tests were used to verify homogeneity of variances. Tukey’s honestly-significant difference (HSD) tests were used to evaluate all pair-wise differences between the means
(P<0.05). Non-parametric Kruskal-Wallis tests (followed by non-parametric multiple comparisons tests) were used when the data were not normally distributed and the data could not be transformed effectively (see Table 4.1).
102 Table 4.1 Results of separate one-way ANOVAs or Kruskal Wallis tests (H values in bold) on various attributes of embryos and larvae of.Strongylocentrotus purpuratus. Source of variation is temperature.
Source df SS F ratio/ P value df SS F ratio/ P value df SS F ratio/ P value
H value H value H value
Embryo final length Larval final total length Larval final mid-line body length
Temperature 3 4356.6 13.4 <0.001 3 129537.3 148.4 <0.001 3 21155.2 30.7 <0.001
Error 8 868.7 12 3491.7 12 2759.6
Larval final body width Larval final post-oral arm length Larval final percent survival
Temperature 3 57079.1 360.3 <0.001 3 12.3 <0.05 3 1947.2 24.6 <0.001
Error 12 633.8 12 316.7
103 Table 4.2 Qualitative and quantitative observations on embryonic development of Strongylocentrotus purpuratus at various temperatures
(mean±SE). Percent survival based on total number of eggs/embryos on culture container bottom.
Temperature (°C) Observations Day 1 Day 2 Day 3 Day 4
8 Total embryos 523.6±13.9 247.6±7.8 250.6±5.6 175.0±21.4
Dead embryos 37.3±2.2 39.0±2.0 34.7±2.9 34.0±3.6
Percent survival 92.9±0.4 84.3±0.3 86.1±1.2 80.1±2.5
Developmental stage Embryos with fertilized Slowly moving blastulae All motile blastulae Actively swimming
membrane: 482.3±13.0 Size not uniform blastulae
Blastulae: 4.0±1.2 Prominent pigmentation started Started gastrulation
Pink colouration of culture
11 Total embryos 513.3±5.9 222.3±8.5 209.7±4.6 112.7±4.3
Dead embryos 22.7±0.9 25.0±5.0 27.3±3.5 31.0±2.9
Percent survival 95.6±0.1 88.9±1.8 87.0±1.6 72.5±2.3
Developmental stage Embryos with fertilized Actively moving blastulae 50% near prism shape >80% prism-shaped
membrane: 496.3±14.7 Prominent pigmentation larvae
Blastulae: 21.7±5.5 started >50% larvae with 2 arms
Pink colouration of
culture
104 Temperature (°C) Observations Day 1 Day 2 Day 3 Day 4
14 Total embryos 502.7±4.1 202.0±4.9 117.3±5.2 110.3±4.1
Dead embryos 32.0±2.3 26.7±1.8 32.6±2.3 34.0±4.6
Percent survival 93.6±0.5 86.8±1.1 71.9±3.3 69.1±4.3
Developmental stage Embryos with fertilized Started gastrulation Mostly prism-shaped larvae >90% larvae prism -
membrane: 5.0±1.0 Prominent pigmentation 10% larvae with 2 arms shaped
Blastulae: 465.7±6.2 started >50% larvae with 2 arms
Actively motile blastulae Pink colouration of
culture
17 Total embryos 524.6±8.7 106.0±9.8 101.6±13.1 70.3±9.3
Dead embryos 34.7±4.6 31.0±3.5 29.0±5.0 37.3±0.8
Percent survival 93.4 ±0.8 70.1±4.6 68.9±10.1 44.9±7.5
Developmental stage Embryos with fertilized Started gastrulation Mostly prism-shaped larvae >80% larvae with 2 arms
membrane: 9.0±1.2 Prominent pigmentation 50% larvae with 2 arms Body shape exceptionally
Blastulae: 483.3±1.8 started wide
Actively motile blastulae Pink colouration of
culture
105 4.3 Results
4.3.1 Influence of temperature on embryonic development
In general, embryonic developmental and growth rates were accelerated with increasing temperature (Table 4.2, Table 4.3, and Fig. 4.1). At day 1 (post fertilization), most of the embryos held at 14 and 17°C were in the blastula stage, whereas the majority of the individuals at 8 and
11°C were still at the fertilized-egg stage. At day 2, gastrulation had begun in embryos held at 14 and 17°C, but not in individuals held at the two other temperatures.
250 b 8°C 11°C b b
m) 210 14°C 17°C μ a
170
130 Embryo length ( Embryo length
90 01 2 3 4 Days
Figure 4.1 Mean (±SE) embryo length (μm) at various temperatures over time. n=3. Letters above bars indicate the results of a Tukey’s HSD multiple-comparison test with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day (Table 4.1).
At day 3, most of the embryos at 11, 14, and 17°C reached the prism stage and more than
50% of the individuals at 17°C had reached the two-arm stage. At day 4, > 50% of the individuals had reached the two-arm stage in all temperature treatments except the 8°C one, where embryos are just stating gastrulation. Percent survival (of the embryos on the bottom of the containers) generally decreased over time and was lowest at 17°C (Table 4.2). Embryo length was
106 significantly affected by temperature at day 4 (Table 4.1) and was significantly smaller at 8°C than at all other temperatures, with no other significant pair-wise comparisons (Fig. 4.1).
4.3.2 Influence of temperature on larval development and survival
Larval total length, mid-line body length, body width, post-oral arm length, and percent survival were all significantly affected by stocking density (Table 4.1). At day 28, Tukey test results were very similar for total length, mid-line body length, body width, and post-oral arm length: values were significantly smaller for larvae reared at 8°C than for those held at any other temperature with few or no significant pair-wise comparisons among larvae held at 11, 14, and
17°C (Fig. 4.2 A, B, C, D). Results for percent survival were slightly different: there was significantly lower survival at 8 and 17°C than at 11 and 14°C, with no other significant pair- wise comparisons (Fig. 4.2E). At day 24, 50% of the larvae reared at 17°C had attained metamorphic competency, while it took 28 days for 50% of the larvae held at 11 and 14°C to achieve competency. Larvae grew relatively slower when reared at 8°C and failed to attain metamorphic competency within 28 days.
Table 4.3 Influence of temperature on the rate of development (for total length) of larval
Strongylocentrotus purpuratus. The Q10 values were calculated over each respective 3°C interval.
Temperature (°C) Developmental rate (Q10)
Post-fertilized larvae (day 4) Feeding larvae (day 28)
8 - -
11 3.3 28.4
14 1.1 1.2
17 1.5 1.3
107 A 600 c 8°C 11°C b b c
m) 520 14°C 17°C μ 440
360 a
280 Larval total length (
200 0 4 8 12 16 20 24 28
B 350 8°C 11°C b b
m) b μ 310 14°C 17°C
270 a 230
190 Mid-line body length ( Mid-line body
150 4 8 12 16 20 24 28
C 335 b 8°C 11°C b b 290 14°C 17°C m) μ 245
200 a Body width ( 155
110 4 8 1216202428 Days
108
D 230 c b 8°C 11°C c
m) 195 14°C 17°C b μ a 160
125
90 Post-oral arm length (
55 4 8 12 16 20 24 28 E 100 8°C 11°C 14°C 17°C 80
60 b b
Larval survival (%) 40 a a
20 4 8 12 16 20 24 28 Days
Figure 4.2 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival at various temperatures over time. n = 4. Letters above bars indicate the results of Tukey’s
HSD multiple-comparison tests with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day (Table 4.1).
The Q10 values at day 4 (post-fertilization) for the pre-feeding larvae and at day 28 for the feeding larvae indicate that the acceleration in developmental was higher during the both pre- feeding and feeding larval stages at 11°C than the other temperature groups (Table 4.3).
109 4.4 Discussion
Temperature is considered to be one of the most important factors determining early development and studies on various echinoid species have suggested that the optimal temperature for fertilization and normal development of embryos is species-specific and occurs within the range of temperatures found during the breeding season in the natural environment (Stephens
1972, Mita et al. 1984, Fujisawa 1989, Fujisawa and Shigei 1990). In the present study, embryos developed faster with increasing tested temperature with embryo length at day 4 being significantly smaller in individuals held at 8°C than in those reared at 11, 14, or 17°C.
While the upper tolerance limit for normal embryonic development remains undetermined, it is clear that 8°C (and probably below) would not be optimal for early culture of S. purpuratus.
This finding is consistent with earlier work by Farmanfarmaian and Giese (1963) who examined embryonic development of S. purpuratus and reported that there was no fertilization membrane formation when mixing eggs and sperm at 5 or 30°C. These authors also reported that, at 25°C, fertilization membranes appeared, but divisions of the fertilized eggs were abnormal and no further development occurred; development was normal between 13 and 20°C. Hinegardner
(1969) noted that S. purpuratus larvae require a temperature of 15°C or lower for normal development, while Leahy (1986) used 16°C and Miner (2007) used 11–14°C for laboratory culture of embryos of purple sea urchins. In the present study, water temperatures of 11 and 14°C appeared to be most suitable for embryonic development of S. purpuratus.
Larval growth was strongly affected by temperature, being significantly reduced at 8°C, compared to 11, 14, or 17°C. Survival was also influenced by temperature, with percent survival being significantly lower at 8 and 17°C than at 11 and 14°C. While survival was reduced at 17°C, larvae reared at this temperature developed faster (achieving metamorphic competency by day
110 24) than those reared at 11 and 14°C (competence reached on day 28). The results on duration for attaining metamorphic competency are consistent with Miller and Emlet’s (1999) observation that larvae of S. purpuratus reared at ambient seawater temperature (13–15°C) became competent to metamorphose in 29 days. In the present study, larval development at 8°C was extremely slow, a result supported by Strathmann (1978) who observed relatively long larval developmental periods with various Strongylocentroid sea urchins at low temperatures. However, the Q10 values of the present study indicated that the acceleration in developmental rate during the feeding larval stages was more temperature-sensitive than during the pre-feeding embryonic stages (Table 4.3).
The Q10 value for feeding larvae between 8 and 11°C was extremely high, which indicated that larval developmental rate at 8°C was very slow in comparison to that at 11°C. This slow growth rate at 8°C may be due to poor metabolic and physical responses of the larvae at prolonged low temperatures. This result is similar to the findings of McEdward’s (1985) studies on sand dollars
Dendraster excentricus, who tested developmental rates at 12, 17 and 22°C temperatures and found the Q10 values of 2.4 and 3.6 for pre-feeding and feeding larvae, respectively.
In summary, embryos typically had higher percent survival at 11 and 14°C than at 8 and
17°C, while embryo length was significantly smaller in individuals held at 8°C than in those reared at 11, 14, or 17°C. Larvae grew significantly slower at 8°C than at 11, 14, or 17°C (with little difference among the latter three treatments), while survival was significantly reduced at 8 and 17°C compared to 11 and 14°C. Although 50% of the larvae held at 17oC became competent to metamorphose by day 24 (from prism stage), larvae held at 11 and 14oC did not begin to attain metamorphic competency until day 28. Taking all of these results into consideration, a temperature of 11°C is recommended for commercial hatcheries, although further research at an industrial scale would need to be conducted to ensure that these results translate to larger scales.
111 CHAPTER 5: Influence of microalgal species on larval development and survival of the purple sea urchin5
This study was conducted to determine the optimal microalgal diet for larvae of the purple sea urchin, Strongylocentrotus purpuratus. Seven algal diets [i.e. Dunaliella tertiolecta, Chaetoceros muelleri, Isochrysis sp. (Tahitian strain), and all possible binary and tertiary combinations of these species] were assessed, along with a control treatment of no food. Larval total length, mid- line body length, body width, post-oral arm length, time to reach metamorphic competency, and survival rate were assessed during the experimental period (28 days). Larvae reared with the bi- algal diet of D. tertiolecta/Isochrysis sp. and the uni-algal diet of D. tertiolecta provided significantly better growth than all other dietary treatments. Larvae were successfully raised to metamorphic competency with both the binary combination of D. tertiolecta/Isochrysis sp. and the single species D. tertiolecta within 28 days of culture, whereas larvae in all the other dietary treatments failed to reach competence within 28 days. The survival (from prism stage to metamorphic competency) for the best treatments was 44.4±2.9 and 42.2±1.1% (mean±SE) for the binary diet of D. tertiolecta/Isochrysis sp. and single algal diet of D. tertiolecta, respectively.
5A version of this chapter has been accepted for publication. Azad A.K., Pearce C.M. and McKinley R.S. 2011.
Influence of microalgal species and dietary rations on larval development and survival of the purple sea urchin,
Strongylocentrotus purpuratus (Stimpson, 1857). Aquaculture: accepted
112 5.1 Introduction
In the natural environment, like other planktotrophic larvae of marine invertebrates, sea- urchin larvae rely largely on phytoplankton for food (Bullivant 1968, Strathmann 1971, Bertram and Strathmann 1998, McEdward and Miner 2007). A number of phytoplankton species have been used to feed larval sea urchins under laboratory and hatchery conditions, but very few studies have examined the specific effects of different microalgal species or dietary rations on larval growth and survival (see chapter 1). Hinegardner (1969) tested the effect of a variety of microalgal species for growth and survival of larval Arbacia punctulata and Lytechinus pictus and found that three particular species – Dunaliella tertiolecta, Rhodomonas sp., and
Pyramimonas sp. – best supported successful development to metamorphosis. Many studies confirm the usefulness of D. tertiolecta for feeding larvae of various species of ehinoids including Strongylocentrotus droebachiensis (Hart and Scheibling 1988, Pearce and Scheibling
1990, 1991, 1994), Psammechinus miliaris (Kelly et al. 2000, Liu et al. 2007a), Evechinus chloroticus (Sewell et al. 2004), Lytechinus variegatus (George et al. 2004), and Paracentrotus lividus (Liu et al. 2007b). Gonzalez et al. (1987), however, reported that D. tertiolecta alone does not provide optimal nutrition for larval Loxechinus albus and needs to be supplemented with
Isochrysis aff. galbana.
It is desirable to select single-species or mixed-species of phytoplankton that have optimal nutritional value for the cultured species of interest in order to produce the highest larval growth and survival rates per unit of culture effort. From a commercial perspective, the phytoplanckton species examined must be a good candidate for mass cultivation (i.e. easily and cheaply produced). In the present study, a variety of both uni-algal diets and combinations of various microalgal species were tested to see what diet offers optimal growth and survival. I examined
113 the effect of single algal species diets [i.e. Dunaliella tertiolecta (CCMP 1320), Chaetoceros muelleri (CCMP 1316), and Tahitian strain of Isochrysis sp. (CCMP 1324)] and all possible binary and tertiary combinations of these species on larval growth and survival of S. purpuratus.
These three algal species were selected as they differ in size and nutritional quality (see Table
5.1), are widely used in the aquaculture industry, and easily cultured. It was hypothesized that bi- algal diets will produce significantly better growth and survival than uni-algal diets because the former may provide a more comprehensive or balanced nutrient profile than the latter.
5.2. Materials and methods
5.2.1 Broodstock collection and algal rearing protocols
Adult purple sea urchins were collected by SCUBA divers from the subtidal at Bamfield, west Vancouver Island (48°50΄N 125°9΄W), BC, Canada. They were transported (~3.5 hours) to the Pacific Biological Station, Nanaimo, BC, in insulated coolers and held in indoor tanks with running, ambient (~10.7°C), sand-filtered seawater. The animals were fed kelp N. luetkeana ad libitum twice weekly. Before each feeding time the uneaten food and faeces were siphoned out from the broodstock maintenance tanks. Spawning induction and embryo rearing were carried out using the same techniques as described in chapter 3 for the stocking density experiment.
The experimental larvae were reared in 20-L plastic buckets with 15-L UVS seawater. The larvae were fed laboratory cultured live algal diets daily. The microalgae were batch cultured in
20-L carboys or 200-L columns with cultures being harvested at the exponential growth phase for the experiments. The methods and nutrients used for algal culture were based on Harrison et al.
(1980). A modification of Harrison’s algal medium was used; this change was a partial substitution of organic phosphates with inorganic phosphates. The sizes of algal cells were
114 determined using a compound microscope with image analysis software (Motic Images, Motic
Group Co., Ltd., Richmond, BC, Canada) and cell concentrations were determined with a haemocytometer. The algal species were mixed according to equal bio-volume per species
(Hillebrand et al. 1999, Liu et al. 2009) and algal ration was determined on the basis of previous studies (see chapter 1) and the recent work examining the effects of stocking density. Although the size and biochemical composition of a particular algal species may vary according to culture condition and developmental phase (Harrison et al. 1990, Thompson et al. 1992, Dunstan et al.
1993, Leonardos and Lucas 2000) the estimated published quantitative and qualitative data of algal species fed in the present study are given in Table 5.1.
Table 5.1 Shape, biovolume, and proximate composition of algae used to feed larval Strongylocentrotus purpuratus, based on previously reported literature values*.
Species Shape* Bio-volume Ratio to D Proximate composition (%)
3 (μm ) Protein Carbohydrate Lipid
Dunaliella tertiolecta (D) Spheroid 250.5 1:1 32 34 7
Chaetoceros muelleri (C) Cylinder 71.4 3.5:1 34.1 15.6 11.1
Isochrysis sp. (I) Cylinder 61.7 4:1 41 18 23
*Adapted from Pillsbury 1985, Volkman et al. 1989, Brown 1991, *Hillebrand et al. 1999, Rivero-Rodríguez et al.
2007, Liu et al. 2009
5.2.2 Spawning protocols and experimental techniques
Adult urchins (3 males and 3 females) were induced to spawn on July 12, 2009. The resulting eggs (diameter: 87.7±1.1 μm, mean±SE, n=21) and fertilized eggs (diameter: 115.8±0.7 μm, mean±SE, n=21) were rinsed with 11°C UVS seawater. The fertilized eggs were transferred into
115 six 20-L plastic buckets containing 10 L of UVS seawater. The stocking density was approximately 100 eggs ml-1, which was found to be optimum in previous experiment (chapter
3). The rearing buckets were kept static and placed partially submerged inside two fibre-glass seawater tables (described above) with flow-through seawater at 11.3±0.1°C (mean±SD, n=192).
On day 4, prism-shaped larvae were transferred to clean 20-L plastic buckets containing 15 L of
UVS seawater and fed one of seven algal diets [i.e. D. tertiolecta (D), C. muelleri (C), Isochrysis sp. (Tahitian strain: TISO) (I) and all possible binary and tertiary combinations (DC, DI, CI,
DCI)] daily. In addition, there was a no-fed control treatment to examine how long larvae could survive on maternal/egg reserves and or dissolved organics in the water. Feeding rations varied according to larval densities and developmental stages and are given in Table 5.2.
The larvae were 228.1±2.2 μm (mean±SE, n=21) in total length at the beginning of the experiment. Initial larval stocking density was 1 ind ml-1 and this was not adjusted over time.
Three replicate buckets were randomly selected for each treatment. These buckets were placed
(fully randomized), partially submerged, inside three fibre-glass seawater tables described above.
The tables were provided with flow-through water at 11.2±0.2°C (mean±SD, n=1248) while buckets were kept static (constant aeration was provided through a plastic capillary at the bottom of each bucket). Sampling, percent survival calculations and morphological measurements were done using the same techniques as described in chapter 3 for the stocking density experiment.
5.2.3 Statistical analysis
Statistical analyses were conducted using NCSS 2006 (Number Crunching Statistical
Systems, Kaysville, Utah, USA). Mean values of the sampled individuals from each replicate bucket were used for the statistical analyses. The effects of algal diets and diet rations on various
116 body dimensions and survivorships were assessed using one way analysis of variance (ANOVA).
Probability plots were used to confirm that data were normally distributed and Levene’s test were used to verify homogeneity of variances. The final sampling day data on morphometric measurements and survivorships were chosen for statistical comparison and no data from the no fed treatment (Table 5.3). Tukey’s honestly significant difference (HSD) tests were used to evaluate all pair-wise differences between the means (P<0.05).
5.3 Results
Larval total length, mid-line body length, body width, post-oral arm length, and percent survival at the end of the experiment were all significantly affected by diet type (Table 5.3). The larval total length, mid-line body length, body width, and post-oral arm length with all dimensions being significantly larger in the DI and D treatments than in all other dietary treatments (Figs. 5.2A, B, C, D). There were no significant differences between D and DI treatments and few or no significant differences between C, I, DC, CI, and DCI treatments for any of the measured dimensions. Larvae fed the DI diet displayed more typical echinoid development (in all body dimensions) than larvae fed any of the other diets with >50% of DI-fed larvae progressing to the competency stage within 28 days (from prism stage). Although development was slightly slower for larvae fed the D diet than those on the DI diet, 50% of the larvae in the former treatment attained competency by the end of the experiment (day 28).
117 Table 5.2 Algal diets and concentration on different days during the larval culture period. Cell densities in different algal treatments were standardized to give equal bio-volumes of algae (sea Table 5.1)
Algal diet Algal density (x 103 ml-1)
Day (0-3) Day (4-7) Day (8-11) Day (12-15) Day (16-19) Day (20-23) Day (24-27)
Dunaliella tertiolecta (D) 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Chaetoceros muelleri (C) 3.50 5.25 7.00 8.75 8.75* 8.75* 8.75*
Isochrysis sp. (I) 4.00 6.00 8.00 10.00 12.00 14.00 16.00
D + C (DC) 0.50+1.75 0.75+2.63 1.00+3.50 1.25+4.38 1.50+5.25 1.75+6.10 2.00+7.00
D + I (DI) 0.50+2.00 0.75+3.00 1.00+4.00 1.25+5.00 1.5+6.00 1.75+7.00 2.00+8.00
C + I (CI) 1.75+2.00 2.63+3.00 3.50+4.00 4.40+5.00 5.25+6.00 6.10+7.00 7.00+8.00
D + C + I (DCI) 0.33+1.17+1.33 0.50+1.75+2.00 0.67+2.33+2.67 0.83+2.92+3.33 1.00+3.50+4.00 1.17+4.08+4.67 1.33+4.67+5.33
* Ration did not increase beyond this point as a considerable number of uningested cells were found on the bottoms of the culture vessels in this treatment.
Table 5.3 Results of separate one-way ANOVAs on various attributes of larvae of Strongylocentrotus purpuratus. Source of variation is algal diet.
Source df SS F ratio P value df SS F ratio P value df SS F ratio P value
Larval total length Larval mid-line body length Larval body width
Diets 6 49885.2 40.6 <0.001 6 45058.0 221.7 <0.001 6 69218.5 119.7 <0.001
Error 14 2870.1 14 474.2 14 1349.5
Larval post-oral arm length Percent survival
Diets 6 19414.1 152.3 <0.001 6 501.6 3.7 <0.05
Error 14 297.4 14 318.5
118
A 600 D C I DI DC CI DCI Unfed b b m)
μ 500 a a a aa 400
300
200
100 Larval total length ( 0 0 4 8 12 16 20 24 28 Days
400 B D C I DI DC CI DCI Unfed b 350 b m)
μ 300 a a a 250 a a
200
150
100
Mid-line body length ( Mid-line body 50
0 4 8 12 16 20 24 28 Days
C 350 D C I DI DC CI DCI Unfed c c 300
250 a m) b b μ b 200 a a 150
100 Body width (
50
0 4 8 12 16 20 24 28 Days
119
D 250 I DI DC CI DC Unfed D C I c c
m) 200 μ b b b 150 b a
100
50 Post-oral arm length (
0 4 8 12 16 20 24 28 Days
E 100 D C I DI DC CI DCI Unfed 90 80 70 60 a 50 a b b b a a b b b 40 Survival (%) a 30 20 10 0 4 8 12 16 20 24 28 Days
Figure 5.1 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival on various algal diets over time. n = 3. D = Dunaliella tertiolecta, C = Chaetoceros muelleri, I = Isochrysis sp. (Tahitian strain), DI = D. tertiolecta + Isochrysis sp., DC = D. tertiolecta + C. muelleri,
DCI = D. tertiolecta + C. muelleri + Isochrysis sp. Letters above bars indicate the results of Tukey’s HSD multiple- comparison tests with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day (Table 5.3).
120 For all other diets, larval development was relatively slower and individuals failed to attain metamorphic competency within 28 days. Larvae in the no-fed control showed longer post-oral arm length than individuals in any other treatment on day 12, but were stunted in all other dimensions (Figs. 5.2A, B, C, D). At day 12 unfed larvae became much narrower, lost pigmentation, and most were very transparent.
The mixed algal diet DI (44.4±2.9%, mean±SE) and single algal diet D (42.2±1.1%) were similar in surviatl rate, and were significantly higher than all other treatments (Fig. 5.2E). The single algal diet C had significantly lower percent survival (27.8±1.1%) than DI and D, with no other differences noted between the remaining treatments. Many of the unfed control larvae died by day 12 with only a negligible amount surviving beyond 16 days.
5.4 Discussion
Larval development of S. purpuratus (for all body dimensions measured) was significantly influenced by the types of algal species provided. Of the seven tested phytoplankton diets, larval growth and survival were best with DI and D. Larvae fed the single algal species diet of D. tertiolecta (D) had similar growth and survival to those given the mixed algal species diet of D. tertiolecta and Isochrysis sp. (DI). McEdward (1984) and Fenaux et al. (1985) reported that the morphometric changes during larval development have important functional consequences, as form or shape is related to feeding capability and metabolic demand in larval echinoids. Many authors have reported that D. tertiolecta are easily ingested by echinoderm larvae (i.e. has an appropriate cell size) and that it quickly breaks down in their stomachs and is capable of producing healthy larvae (Strathmann 1971, Cameron and Hinegardner 1974, Basch 1996).
Hinegardner (1969) tested the effect of 14 phytoplankton species (i.e. Amphidinium operculatum,
121 Coccolithus huxleyi, Cryptomonas, Cyclotella nana, Cylindrotheca closterium, D. tertiolecta,
Eutreptiella, Isochrysis galbana, Melosira nummuloides, Monochrysis lutheri, Nitzschia brevirostris, Phaeodactylum tricornutum, Pyramimonas, Rhodomonas) on the development of echinoid larvae and reported that the larvae of A. punctulata grew well (through to metamorphosis) on D. tertiolecta. Kelly et al. (2000) compared the effects of two algal diets – D. tertiolecta and Pleurochrysis carterae – on growth and survival of larval P. miliaris. They reported that D. tertiolecta produced more morphologically typical larvae and gave better results in terms of survival at metamorphosis (65.8%) than P. carterae (48.2%). George et al. (2004) examined the efficacy of a prepared micro-encapsulated diet compared to an algal diet of D. tertiolecta for larval rearing of L. variegatus and found that larvae fed the prepared diet were significantly smaller than those fed D. tertiolecta. Larval survival was also higher for those fed
D. tertiolecta (85±4%) than those individuals given the prepared feed (72±6%). Liu et al. (2007a) offered a micro-encapsulated formulated feed, an algal diet (D. tertiolecta), and a concentrated algal paste (shellfish diet 1800: a mixture of Isochrysis sp. Pavlova sp., Tetraselmis sp., and
Thalassiosira weissflogii) to P. miliaris larvae. They observed faster growth for individuals fed
D. tertiolecta than those given the other two diets, although there was no significant difference in survival among the three feed treatments. Liu et al. (2007b) also reported similar results for P. lividus and the authors concluded that larvae of both species showed a tendency of food selection towards D. tertiolecta.
Some studies with echinoderm larvae suggest that mixed-species algal diets may give better results than single-algal diets. This has also been observed in other benthic marine invertebrates
(see review by Marshall et al. 2010 on marine bivalves for example) and has been attributed to the hypothesis that a mixed-algal diet should provide a more balanced nutrient profile than a single-algal one. In the current study, larvae fed the mixed-algal species diet DI grew the fastest,
122 had the highest percent survival, and attained metamorphic competency the earliest of all the tested diets, although there were no significant differences between DI and the single-algal species diet D for any of the measured variables. Basch (1996), working with larvae of the sea star Asterina miniata, noted that algal mixtures more closely resembled natural food conditions and generally improved larval development compared with single-algal diets. Gonzalez et al.
(1987) reported that larvae of L. albus showed more typical growth when provided a mixed algal- species diet of D. tertiolecta and Isochrysis aff. galbana than when given a single-algal diet of D. tertiolecta. Cárcamo et al. (2005) assessed the effect of single-species and mixed-species microalgal diets (i.e. Chaetoceros calcitrans, C. calcitrans + I. galbana, and C. calcitrans + I. galbana + Tetraselmis suecica) on larval L. albus. Their results suggested that daily larval feeding with a mixed diet of C. calcitrans + I. galbana may give better results for large-scale production of competent larvae than a single-species diet.
While some species of algae may be insufficient to provide maximal growth and survival when fed to echinoid larvae alone [e.g. Isochrysis sp. (Pechenik 1987, Schiopu et al. 2006)], this appears not to be the case with D. tertiolecta fed to S. purpuratus and other echinoid species.
Interestingly, this algal species has lower lipid levels than other commonly cultured phytoplankton species such as C. muelleri and Isochrysis sp. (Table 5.1) and is deficient in the long-chain polyunsaturated fatty acids (PUFAs) 20:5n–3 (eicosapentaenoic acid) and 22:6n–3
(docosahexaenoic acid) (Volkman et al.1989), which have been shown to be essential for optimal larval growth of various bivalve species (see review by Marshal et al. 2010). Schiopu et al.
(2006), however, reported that larvae of the sand dollar Dendraster excentricus demonstrated the ability to elongate and de-saturate shorter chain (18 carbon) PUFAs to longer chain (20 carbon) ones while Liu et al. (2007b) reported that larvae of P. lividus performed well on diets with 32% protein and 7% lipid content, both studies indicating that larvae of at least certain species of
123 echinoids may have relatively low lipid requirments. Despite the important relationships between the nutritional requirements of larval sea urchins and their growth and survival, little has been done to determine what levels of various dietary components (e.g. protein, lipid, carbohydrate, micro-nutrients) need to be supplied to echinoid larvae to obtain optimum performance. Further study of the nutritional requirements of sea-urchin larvae and algal biochemical composition need to be conducted before causative relationships can be established.
In summary, larvae reared with the bi-algal diet of D. tertiolecta/Isochrysis sp. and the uni- algal diet of D. tertiolecta provided significantly better growth than all other dietary treatments.
Larvae were successfully raised to metamorphic competency with both the binary combination of
D. tertiolecta/Isochrysis sp. and the single species D. tertiolecta within 28 days of culture, whereas larvae in all the other dietary treatments failed to reach competence within 28 days.
Since there were no significant differences between the single algal diet of D. tertiolecta and the bi-algal diet of D. tertiolecta/Isochrysis sp., and it would be easier for industry to rear only one species as opposed to two, I would recommend that D. tertiolecta alone be used in commercial hatcheries, although further research at an industrial scale would need to be conducted to ensure that these results translate to larger scales.
124 CHAPTER 6: Influence of dietary rations on larval development and survival of the purple sea urchin6
This study was conducted to determine the optimal dietary ration for larvae of the purple sea urchin, Strongylocentrotus purpuratus. In this experiment, a bi-algal diet (Dunaliella tertiolecta and Isochrysis sp. at equal biovolumes) was evaluated using five rations: (1) low ration: 1.25 x
103 cells ml-1; (2) normal ration: 2.5 x 103 cells ml-1; (3) standardized ration: 2.5 x 103 cells to
10.0 x 103 cells ml-1, with increasing ration according to developmental stage; (4) medium ration:
5.0 x 103 cells ml-1; and (5) high ration: 10.0 x 103 cells ml-1. Larval total length, mid-line body length, body width, post-oral arm length, time to reach metamorphic competency, and survival rate were assessed during the experimental period (28 days). Typical echinoid larval development in all body dimensions was only achieved for larvae offered the standardized ration. Larvae reared on this ration grew significantly faster that those fed any of the fixed rations; larvae fed the former ration attained metamorphic competency within 28 days whereas larvae fed on the fixed rations (lower or higher concentrations) failed to attain competency by the end of the experiment
(28 days). In general, in comparison to larvae fed the standardized ration, larvae on the low ration showed longer post-oral arm length and narrower body width whereas larvae on the high ration showed shorter post-oral arm length and body width in relation to their total length. Overall survival (from prism stage to metamorphic competency) was 53.3±1.9% (mean±SE) for the best treatment of standardized ration.
6 A version of this chapter has been accepted for publication. Azad A.K., Pearce C.M. and McKinley R.S. 2011.
Influence of microalgal species and dietary rations on larval development and survival of the purple sea urchin,
Strongylocentrotus purpuratus (Stimpson, 1857). Aquaculture: accepted
125 6.1 Introduction
Diet ration had an important influence on larval development and metamorphosis of
Strongylocentrotus droebachiensis (Meidel et al. 1999). Sewell et al. (2004) tested the developmental plasticity of larvae of Evechinus chloroticus using a “high” or “low” food ration
(6,000 or 600 Dunaliella tertiolecta cells ml-1, respectively) or with no algal food and reported that larvae in the “high” food-ration treatment were largest in all measured dimensions and formed juvenile rudiments within 23 days of fertilization, whereas larvae fed the “low” ration and those with no algal food were stalled at the four-arm echinopluteus stage.
Hinegardner (1969) reported that the concentration of algae needed for feeding larval echinoids depends on the stage of development, with younger stages requiring much less food than older ones. He noted that a daily ration of about 3000 cells ml-1 (species not mentioned) was suitable for young echinoplutei. Kelly et al. (2000) found that increasing the algal ration according to developmental stages of larvae of Psammechinus miliaris gave better results than the fixed ration. Later studies have also reported beneficial effects of varying ration according to developmental stage. For example, Jimmy et al. (2003) observed that larvae of Echinus esculentus grew well when provided Dunaliella tertiolecta at rations of 1000, 3000, and 5000 cells ml-1 every third day, according to developmental stage. In addition to larval stage, however, algal particle size, concentration, and flavour can all influence larval feeding rates and particle selectivity (Hart and Scheibling 1988, Pedrotti 1995).
In the present study I examined a mixed algal species diet of D. tertiolecta (CCMP 1320) and
Tahitian strain of Isochrysis sp. (TISO: CCMP 1324) (the optimal diet determined in chapter 5) with varying rations. The objective of this study was to determine the optimal ration that will support the highest survival and growth of purple sea-urchin larvae for successful hatchery
126 production. It was hypothesized that diet ration will be optimised at some level, with significantly lower/higher rations negatively impacting larval growth and survival.
6.2 Materials and methods
6.2.1 Broodstock collection and general experimental techniques
Broodstock collection, broodstock maintenance, and algal culture were done using the same techniques as described in chapter 5 for the algal species diet experiment.
6.2.2 Spawning protocols and experimental techniques
Adult sea urchins (3 males and 3 females) were induced to spawn on December 10, 2009. The resulting eggs (diameter: 86.4 ± 0.5μm, mean±SE, n=21) and fertilized eggs (diameter: 116.4 ±
0.6μm, mean±SE, n=21) were rinsed with UVS seawater at 11°C temperature. All rearing protocols for fertilized eggs/embryos and resulting larvae (total length: 196.8 ± 2.2μm, mean±SE, n=21) as well as survival determination and morphological parameters measured were as in chapter 3. In addition, one index of larval shape was calculated, i.e. the ratio of body width to larval total length.
In this experiment a mixed algal-species D. tertiolecta and TISO was evaluated with varying rations. The diet rations for the five treatments were (a) low ration (LR): 1.25 x 103 cells ml-1
(e.g. D. tertiolecta at 0.25 x 103 cells ml-1 and TISO at 1.0 x 103 cells ml-1 [based on equal bio- volumes of the two species(see chapter 5)], (b) normal ration (NR): 2.5 x 103 cells ml-1, (c) standardized ration (SR): 2.5 x 103 cells to10.0 x 103 cells ml-1 (increasing ration according to developmental stages and stocking density; e.g. DI in chapter 5) (d) medium ration (MR): 5.0 x
103 cells ml-1 and (e) high ration (HR): 10.0 x 103 cells ml-1. All treatments were carried out in
127 triplicate and the replicates were fully randomized among the sea water tables. The initial larval density was 1 ind ml-1, based on results of the previous stocking density (chapter 3) experiment.
6.2.3 Statistical analysis
Statistical analyses were conducted using NCSS 2006 (Number Crunching Statistical
Systems, Kaysville, Utah, USA) on data from the final sampling day. Mean values of the sampled larvae from each replicate bucket were used for the statistical analyses (Table 6.1).The effects of diet rations on various body dimensions and survivorships were assessed using one way analysis of variance (ANOVA). Probability plots were used to confirm that data were normally distributed and Levene’s test were used to verify homogeneity of variances. Tukey’s honestly significant difference (HSD) tests were used to evaluate all pair-wise differences between the means (P<0.05).
128 Table 6.1 Results of separate one-way ANOVAs on various attributes of larvae of Strongylocentrotus purpuratus. Source of variation is algal diet ration.
Source df SS F ratio P value df SS F ratio P value df SS F ratio P value
Larval final total length (TL) Larval final mid-line body length Larval final body width
Rations 4 41335.2 35.6 <0.001 4 8425.3 11.5 <0.001 4 51455.2 52.5 <0.001
Error 10 2901.4 10 1836.4 10 2448.6
Larval final post-oral arm length Larval body width: TL (%) Final larval percent survival
Rations 4 4561.7 13.7 <0.001 4 961.5 30.9 <0.001 4 2854.8 24.1 <0.001
Error 10 830.9 10 77.8 10 296.3
129
600 A c Low ration Normal ration Standardized ration 500 b m) Medium ration High ration μ a a 400 a
300
200 Larval total length ( 100
0 0 4 8 12 16 20 24 28 Days
350 B Low ration Normal ration Standardized ration c 300 b Medium ration High ration b b m) μ 250 a
200
150
100 Mid-line body length ( Mid-line body 50
0 4 8 12 16 20 24 28 Days
130 350 C Low ration Nornal ration Standardized ration e 300 Medium ration High ration d 250 m) c μ 200 b
150 a
Body width ( 100
50
0 4 8 12 16 20 24 28 Days
D 250 Low ration Normal ration Standardized ration Medium ration High ration c b m) 200 a c a μ b b a 150
100
Post-oral arm length ( 50
0 4 8 12 16 20 24 28 Days
131
E 100 Low ration Normal ration Standardized ration 90 Medium ration High ration 80 70
60 b b b 50 40
Survival (%) Survival (%) a 30 a 20 10 0 4 8 12 16 20 24 28 Days
Figure 6.1 Mean (±SE) (A) larval total length, (B) mid-line body length, (C) body width, (D) post-oral arm length, and (E) percent survival on various diet rations over time. n = 3. Low ration=1.25 x 103 cells ml-1; Normal ration=
2.5 x 103 cells ml-1; Standardized ration= 2.5 x 103 cells to 10.0 x 103 cells ml-1, with increasing ration according to developmental stage; Medium ration= 5.0 x 103 cells ml-1; and High ration= 10.0 x 103 cells ml-1. Letters above bars indicate the results of Tukey’s multiple-comparison tests with different letters showing significant (P<0.05) pair- wise differences among the treatments (Table 6.1).
6.3 Results
Larval total length, mid-line body length, body width, post-oral arm length, post-oral arm length/total length ratio, body width/total length ratio, and percent survival at the end of the experiment were all significantly affected by diet ration (Table 6.1). The total length of SR larvae was significantly greater than all other treatment groups (Fig. 6.1A). Larvae in the MR treatment had significantly greater total length than those in the LR, NR, and HR groups with no significant difference among the latter three treatments. Similarly, mid-line body length for larvae in the SR treatment was significantly greater than for those in all other treatments (Fig. 6.1B).
132 Larvae in the HR group had significantly shorter mid-line body length than those in all other treatments, with no significant differences among LR, NR, and MR treatments. All pair-wise comparisons for body width were significant with the largest dimensions occurring in the SR and
MR treatments (Fig. 6.1C). Post-oral arm length was significantly greater for SR larvae than LR,
NR, and HR individuals with only one other significant pair-wise comparison (LR vs MR) (Fig.
6.1D). The body width: larval total length ratio was significantly smaller for LR followed by NR larvae. All pair-wise comparisons for the body width: total length ratio was significantly higher in the SR larvae than in the LR, NR, and HR treatments with LR and NR ratios being significantly smaller than MR and HR ones (Fig.6.2). Larvae (50%) in the SR treatment reached the competent stage within 28 days of culture (post-prism stage), whereas larvae in all other treatments failed to attain metamorphic competency by the end of the experiment (day 28).
The SR treatment had significantly higher percent survival (53.3±1.9%, mean±SE) at day 28 than the LR (17.8±1.1) and NR (25.6±2.2%) treatments at the end of the experiment (Fig. 6.1E).
There were no significant differences in percent survival among the SR, MR (47.8±1.1%), and
HR (45.6±6.2%) treatments on the final sampling day of the experiment.
133
70 Low ration Normal ration Standardized ration c 60 d Medium ration High ration d c 50 b
40 a
30
20
10 Body width: Total length (%) (%) Total length Body width:
0 4 8 12 16 20 24 28 Days
Figure 6.2 Percent ratio of larval body width and total length over time. Error bars are SE and n=3. Low ration=1.25 x 103 cells ml-1; Normal ration= 2.5 x 103 cells ml-1; Standardized ration= 2.5 x 103 cells to 10.0 x 103 cells ml-1, with increasing ration according to developmental stage; Medium ration= 5.0 x 103 cells ml-1; and High ration= 10.0 x 103 cells ml-1.Letters above bars indicate the results of a Tukey’s HSD multiple comparison test with different letters showing significant (P<0.05) pair-wise differences among the treatments on the final sampling day (Table 6.1).
6.4 Discussion
The present study provides evidence that algal ration can have a marked effect on larval development and survival. Larvae fed the SR diet (i.e.increasing ration according to larval developmental stage and density) showed typical echinoid larval development in all measured body dimensions and progressed to metamorphic competency with the highest percent survival of all ration treatments tested. Similar results were reported by Kelly et al. (2000) for larvae of P. miliaris fed the microalga P. elongata. In that study, larvae on an “optimal” ration (1,500–4,000 cells ml-1, according to developmental stage) displayed a more typical morphology, whereas larvae on a “low” ration (500 cells ml-1) failed to develop to metamorphosis. In a study by Jimmy
134 et al. (2003), on larval E. esculentus, it was observed that a “standard” ration (1,000, 3,000, and
5,000 cells ml-1, according to developmental stage) of D. tertiolecta produced better results – in terms of larval morphology and metamorphic rate – than a “high” ration (3,000, 9,000, and
15,000 cells ml-1, according to developmental stage). They reported that optimizing algal ration also shortened the larval stage from 21–23 days to 16 days, with a survival rate of 46.6%, ten days after metamorphosis.
In the present study, larvae demonstrated considerable plasticity in morphology in response to various diet rations. Larvae fed the LR and NR rations exhibited significantly lower body width: total length ratios than individuals in any other ration treatment, indicating that the body had become elongated in relation to the body width. Many studies have reported that, in low-food conditions, echinoid larvae increase their arm length to increase the number of feeding cilia, leading to a delay in broadening the body width and development of the echinus rudiment
(Boidron-Metairon 1988, Strathmann et al. 1992, Fenaux et al. 1994, Kelly et al. 2000, Sewell et al. 2004). While low-food rations can obviously have negative effects on larval development and survival, high food levels may also have detrimental effects. In the present study, reduced total length, mid-line body length, body width, post-oral arm length, and percent survival (especially during the initial culture period) was evidenced in larvae from the HR group when compared with those from the SR treatment. These results are consistent with Kelly et al. (2000) who reported that larvae of P. miliaris showed an extreme reduction in post-oral arm length, and were unable to maintain their position in the water column, on a “high” ration (4,000 cells ml-1 throughout the larval life span) of the microalga P. elongata. It is apparent that larval echinoid growth, development, and survival will be maximized at a certain ration level, with reduced growth/survival occurring at rations above and below this critical concentration. This optimum ration level will be dependent on both species and developmental stage. Research to date on
135 echinoid larvae suggests that increasing rations with larval developmental stage may lead to overall better growth and survival than fixed rations.
In summary, typical echinoid larval development in all body dimensions was only achieved for larvae offered the standardized ration. Larvae reared on this ration grew significantly faster that those fed any of the fixed rations; larvae fed the former ration attained metamorphic competency within 28 days whereas larvae fed on the fixed rations (lower or higher concentrations) failed to attain competency by the end of the experiment (28 days). Increasing ration with larval development stage is recommended over fixed rations for commercial hatcheries, although further research at an industrial scale would need to be conducted to ensure that these results translate to larger scales.
136 CHAPTER 7: Conclusions
For my Ph.D. research I have investigated various aspects of culturing the purple sea urchin,
S. purpuratus. The major objectives of this study were to examine the effects of various biological (e.g. stocking density, food, food ration) and physical (e.g. temperature) factors on gonad production of adult animals and development, growth, and survival of embryos and larvae.
The rest of this chapter will briefly summarize my findings, provide recommendations to industry on how to best culture this species, and suggest potential future areas for research on echinoid culture.
The gonad experiment in Chapter 2 examined the combined effect of diet (three kelp species and a prepared diet) and temperature (8, 12, 16°C) on ingestion rate, absorption efficiency, assimilation rate, and gonad quantity/quality of adult urchins. At two different sampling times
(weeks 6 and 12) wet-weight ingestion rates were significantly higher with the three kelp species than with the prepared diet. The highest ingestion rates were recorded for animals held at 12 and
16°C at weeks 6 and 12, respectively. The prepared diet produced significantly higher gonad weight and gonad index than any of the three kelp species, with no significant differences among the kelp treatments. Final gonad weight was significantly affected by temperature, but this effect was dependent on diet; temperature had little affect on gonad weight and index in the three kelp treatments, but 12 and 16°C produced significantly heavier gonads than 8°C for urchins fed the prepared diet. Temperature and diet did not significantly affect gonad colour, texture, or firmness, but the prepared diet generally imparted better flavour to the roe than the three kelp species.
These findings agree with my alternative hypothesis for this research that diet type would significantly affect both gonad quantity and quality, with the prepared diet producing significantly larger and better quality gonads than the various kelp species.
137 In recent years, the effects of macroalgae and prepared diets on gonad yield and quality have been examined in various sea-urchin species, including E. chloroticus (Barker et al. 1998, Woods et al. 2008), L. albus (Cárcamo 2004), L. variegatus (Hammer et al. 2006), P. lividus (Spirlet et al. 2001, Shpigel et al. 2005, Cook and Kelly 2007a), P. miliaris (Cook et al. 1998, McLaughlin and Kelly 2001, Cook and Kelly 2007b), S. droebachiensis (de Jong-Westman et al. 1995,
Robinson et al. 2002, Pearce et al. 2002a, 2003, 2004, Siikavuopio et al. 2007), S. franciscanus
(McBride et al. 1997, 2004), S. intermedius (Chang et al. 2005, Lawrence et al. 2009), and S. nudus (Agatsuma 1998, Agatsuma et al. 2005). All of these studies reported that sea urchins perform well with either natural or prepared diets, but that they typically produce higher gonad yields with prepared diets compared to natural macroalgae. Similar conclusions were drawn in the present study.
The prepared diet produced by the Norwegian Institute of Fisheries and Aquaculture (NIFA) worked very well and produced larger and better tasting gonads than the three kelp species tested.
Prepared diets have a number of advantages over macroalgae: (1) they are manufactured and do not rely on harvesting large quantities of marine plants, (2) they can be more easily stored long- term than algae, and (3) they have a known nutrient profile which does not vary seasonally or geographically (as macroalgae do) which can be easily manipulated for further improvements in gonad quality. For these reasons, I would recommend prepared diets over kelp for commercial gonad enhancement of the purple sea urchin. This information will be beneficial to entrepreneurs starting either full life-cycle culture or gonad enhancement of purple sea urchins. Commercial gonad enhancement may primarily rely on adult urchins harvested from the wild. Adult urchins could be collected from the wild and reared in captivity for 10–12 weeks to produce large, uniform gonads of high quality before exporting them.
138 Further results from Chapter 2 revealed that S. purpuratus fed a prepared (NIFA) diet produced significantly heavier gonads at 12 and 16°C than at 8°C. Although both gonad weight and gonad index reached the highest level at the intermediate temperature (12oC), the difference between 12 and 16oC was not significant, suggesting that the limiting upper temperature was not reached in the study. These findings agree with my alternative hypothesis that temperature would significantly affect gonad quantity, with higher temperatures producing significantly larger gonads than lower temperatures. Temperature effects are species specific and previous studies have documented different optimal temperatures for different urchin species. For instance,
Siikavuopio et al. (2006) reared S. droebachiensis at 4, 6, 8, 10, 12, and 14oC and reported that the gonad index value increased to a peak at 10oC, after which there was a trend towards a reduction in gonad index at the highest temperature. The choice of temperature for commercial- scale operations, however, will depend not only on the preferences of the given species but also on the temperature of their incoming seawater and the cost of heating/cooling. This will be geographically and seasonally dependent. Operations may choose to run at cooler temperatures without incurring the cost of heating the water, knowing that it may take longer to produce suitable gonads. There may be a trade off between the cost of heating/cooling and the time it will require for gonad enhancement. If heating/cooling cost is not an issue, I would recommend that the industry in British Columbia uses 12°C, the lowest temperature that produced heaviest and highest quality gonads.
However, for sustainable aquaculture development of any candidate species, a reliable source of high-quality seed is necessary. Information on optimum culture parameters (both abiotic and biotic) for producing high-quality juveniles with the highest growth and survival rates per unit of culture effort is critical for the species of interest. Determining the optimal rearing conditions for embryos and larvae will allow more efficient production of competent larvae while decreasing
139 the length of the culture period as well as food and labour costs. In Chapter 3 I examined the effect of stocking density on development, growth, and survival of embryos (50, 100, 200, and
400 eggs ml-1) and larvae (0.5, 1, 2, and 4 ind ml-1) of the purple sea urchin, S. purpuratus.
Prism-stage individuals that had been stocked initially at 50 and 100 eggs ml-1 were significantly longer than those held initially at 200 and 400 eggs ml-1. Larvae had significantly higher survival and grew faster when held at 0.5 or 1 ind ml-1 then when reared at 2 or 4 ind ml-1. Approximately
50% of the larvae held at 0.5 ind ml-1 were competent to metamorphose by day 24 (from prism stage), whereas larvae held at 1 ind ml-1 did not become competent until day 28 and those held at
2 and 4 ind ml-1 failed to develop to metamorphic competency by the termination of the experiment. These findings agree with my alternative hypothesis for this research that higher stocking densities would significantly reduce embryonic/larval development, growth, and survival rates. They are also in agreement with the one published study directly examining the effect of echinoid larval stocking density on growth and survival; Buitrago et al. (2005) having reported that a stocking density of 0.5 to 1.0 ind ml-1 would be most suitable for seed production of L. variegatus. For commercial production I would recommend an egg/embryo density of ≤100 inds ml-1 and a larval density of ≤1 ind ml-1, however, further research at an industrial scale would need to be conducted to ensure that these results translate to larger volumes.
In Chapter 4 I examined the effect of temperature (8, 11, 14, 17°C) on development, growth, and survival of embryos and larvae of the purple sea urchin, S. purpuratus. The results revealed that embryos typically had higher percent survival at 11 and 14°C than at 8 and 17°C, while embryo length was significantly larger in individuals held at 11, 14, or 17°C than in those reared at 8°C. Larvae grew significantly slower at 8°C than at 11, 14, or 17°C (with little difference among the latter three treatments), while survival was significantly reduced at 8 and 17°C compared to 11 and 14°C. Although 50% of the larvae held at 17oC became competent to
140 metamorphose by day 24 (from prism stage), larvae held at 11 and 14oC did not begin to attain metamorphic competency until day 28. These findings agree with my alternative hypothesis for this research that temperature would significantly affect embryonic/larval development, growth, and survival. I would recommend that commerical hatcheries use temperatures between 11 and
14°C for mass production of competent purple urchin larvae. However, further research at an industrial scale would need to be conducted to ensure that these results translate to larger volumes.
Chapters 5 and 6 examined the effects of various biotic factors (i.e. diet type and ration) on growth and survival of larval purple urchins. Many studies have reported that D. tertiolecta alone is a suitable feed for rearing larvae of a variety of sea-urchin species including S. droebachiensis
(Hart and Scheibling 1988, Pearce and Scheibling 1990, 1991, 1994), P. miliaris (Kelly et al.
2000, Liu et al. 2007a), E. chloroticus (Sewell et al. 2004), L. variegatus (George et al. 2004), and P. lividus (Liu et al. 2007b), but little or no work has examined the direct effect of diet on echinoid larval growth or survival. A single algal species, however, may not provide balanced nutritional requirements for sea-urchin larvae and it may need to be supplemented with other phytoplankton species (see Gonzalez et al. 1987). In Chapter 5 I examined the effect of seven algal diets [i.e. Dunaliella tertiolecta, Chaetoceros muelleri, Isochrysis sp. (Tahitian strain), and all possible binary and tertiary combinations of these species], along with a control treatment of no food, on larval growth and survival of S. purpuratus. Larvae reared with the uni-algal diet of
D. tertiolecta or the bi-algal diet of D. tertiolecta/Isochrysis sp. provided significantly better larval growth than all other dietary treatments tested. Larvae were successfully raised to metamorphic competency with both the binary combination of D. tertiolecta/Isochrysis sp. and the single species D. tertiolecta within 28 days of culture, whereas larvae in all the other dietary treatments failed to reach competence within 28 days. In previous studies many researchers have
141 successfully reared echinoid larvae with Chaetoceros spp. (see review by Azad et al. 2010), but the present study revealed that C. muelleri is not suitable for S. purpuratus. These findings do not agree with my alternative hypothesis for this research that bi-algal diets will produce significantly better growth and survival than uni-algal diets.
This information will help hatchery operators to select only a single algal diet of D. tertiolecta or a mixed algal diet of D. tertiolecta and Isochrysis sp., microalgal species that are widely used and easily produced, for production of purple urchin larvae. Since there were no significant differences between the single algal diet of D. tertiolecta and the bi-algal diet of D. tertiolecta/Isochrysis sp., and it would be easier for industry to rear only one species as opposed to two, I would recommend that D. tertiolecta alone be used in commercial hatcheries, although further research at an industrial scale would need to be conducted to ensure that these results translate to larger volumes.
In Chapter 6 I used a bi-algal diet (D. tertiolecta and Isochrysis sp. at equal biovolumes) to evaluate the effect of five rations [(1) low ration: 1.25 x 103 cells ml-1; (2) normal ration: 2.5 x
103 cells ml-1; (3) standardized ration: 2.5 x 103 cells to 10.0 x 103 cells ml-1, with increasing ration according to developmental stage; (4) medium ration: 5.0 x 103 cells ml-1; and (5) high ration: 10.0 x 103 cells ml-1] on growth and survival of larval S. purpuratus. Results showed that larvae fed an increasing diet ration (i.e. 2.5 x 103 cells to 10.0 x 103 cells ml-1, according to developmental stage) generally had significantly better growth and survival than any fixed rations
(i.e. same ration throughout development) evaluated. These findings agree with my alternative hypothesis for this research that diet ration would be optimised at some level, with lower/higher rations significantly negatively impacting larval growth and survival. These findings are also in agreement with various other studies that have reported negative effects of low food rations
(Boidron-Metairon 1988, Strathmann et al. 1992, Fenaux et al. 1994, Kelly et al. 2000, Sewell et
142 al. 2004) and detrimental effects of high rations (Kelly et al. 2000, Jimmy et al. 2003) on echinoid larval development and survival. My results are also similar to those of Hinegardner
(1969) and Basch (1996) who reported that the optimal concentration of algal cells may depend on larval developmental stage and stocking density and also to Kelly et al. (2000) who found that a gradual increase in ration over time, up to a certain level, may accelerate larval development over fixed rations. Increasing ration with larval development stage is recommended over fixed rations for commercial hatcheries, although further research at an industrial scale would need to be conducted to ensure that these results translate to larger volumes.
The purple sea urchin, S. purpuratus, is widely used as a model organism for ecological, bio- medical, and genetic research (Boolootian 1963, Gonor 1973, Leahy et al. 1981, Shott et al. 1984,
Pearse et al. 1986, Osovitz and Hofmann 2005, Goel and Mushegian 2006). Work by Leahy and colleagues (Leahy et al. 1978, 1981, Leahy 1986) and the present study show that the purple urchin is easily cultured and would make an ideal aquaculture candidate (i.e. responds well to both natural and prepared diets in captivity, can spawned year round, and has marketable roe).
The whole life cycle of this animal depends essentially on primary productivity (i.e. macroalgae and microalgae) and the culture of this species should be environmentally friendly in terms of feed requirements, as large quantities of animal protein are not necessary. Sea urchins echinoid may be excellent potential candidate aquaculture species for integrated multi-trophic aquaculture
(IMTA) systems as they can feed on the heavier settleable solids (i.e. faecal material and uneaten feed) coming out of the cultured fish system (Kelly et al.1998, Cook and Kelly 2007a.), thus decreasing the organic load to the benthos. Research will be required to examine the effect of various urchin stocking densities, various urchin holding systems, and the effect urchins have on the level of organics reaching the seafloor.
143 In the present study, I examined gonad production of adults fed various species of kelps and a prepared diet under laboratory conditions. Future research is required to examine the effects of other species of macroalgae or locally-available terrestrial plants and the effects of combining macroalgae and prepared diets (as in Spirlet et al. 2001, Shpigel et al. 2005, Cook and Kelly
2007b) on gonad production, both in the laboratory and in the field under pilot-scale conditions.
Although previous research on gonad quality has suggested that β-carotene is responsible for the attractive yellow-orange colouration of gonads of some echinoid species (Griffiths and Perrott
1976, Matsuno and Tsushima 2001, Robinson et al. 2002, Pearce et al. 2004, Agatsuma et al.
2005), the present study failed to achieve an improvement in colour with the NIFA prepared diet which used proprietary levels of β-carotene. Further research with prepared diets would need to examine the effect of various concentrations of β-carotene on gonad colour and flavour in this
(and other) echinoid species. Indeed, further feed development studies – examining various sources and levels of protein, lipid, carbohydrate, and minerals (as in Kennedy et al. 2005,
2007a,b) – would be invaluable in developing suitable prepared diets in terms of gonad production and quality. This avenue of research is required for every echinoid species being considered for culture. These studies should also take into consideration feed cost and ease of production.
Very little work has focused on the effect of prepared diets on gonad flavour and this needs to be examined much more closely in future gonad enhancement research. There is no point in producing large, nicely-coloured roe that tastes awful – the market will not accept it. Assessing flavour is not easy though and researchers will most likely need to work closely with professional taste panels or commercial urchin processors to get useful information about the flavour of urchin roe.
144 In my study, temperature did not affect any of the gonad quality factors measured, but I believe that it would be useful to examine gonad index over time at more refined gradations of temperature (e.g. 2°C intervals between 10 and 20°C) to determine the least amount of time needed to produce marketable gonads at various temperatures. Very little research has examined the effect of temperature on echinoid gonad production and this type of research will be required for any echinoid species of commercial interest.
Research on the effects of temperature and stocking density on the early development of echinoids is scant (see review by Azad et al. 2010). Only one earlier study (Farmanfarmaian and
Giese 1963) has examined the effects of temperature on embryonic development of purple sea urchins. While the current study examined the effect of temperature on early development of purple urchins in small-scale laboratory culture units, future research is required to determine if the results are applicable to larger-scale (pilot-scale or commercial-scale) production of pre- feeding prism shaped larvae and feeding echinoplutei. As purple sea urchins occur over a wide latitudinal range and are exposed to a large range of temperatures in their distribution, further research might examine if there are population differences in embryo/larval temperature responses of this species. The examination of temperature effects on embryonic/larval development and survival is virtually non-existent for all echinoid species being considered for culture and would be a fruitful area of research.
The current study found that a stocking density of ≤ 1 ind ml-1 gave the best larval growth and survival. Although many studies on echinoid development have typically used a larval stocking density of 1–2 inds ml-1, surprisingly little research has focused on the direct effects of larval density, or the interaction effects of larval density and diet concentration, on larval growth and survival (review by Azad et al. 2010). Further research in this area on all echinoid species of interest is required. The combined effect of flow rate and stocking density may also be a useful
145 area for research; larvae may be able to be held at higher stocking densities if held in a flow- through system as opposed to a static one (as was used in the current study). Small-scale laboratory research will need to be followed up by commercial-scale trials to confirm that small- scale results are transferable to industry. Possible interactive effects between larval stocking density and ration should also be explored.
The current study identified that D. tertiolecta or a combination of D. tertiolecta and
Isochrysis sp. are suitable phytoplankton diets for rearing purple urchin larvae, but this work only examined three algal species and their binary and tertiary combinations. Far more research needs to be conducted with purple urchins and all echinoid species of interest to examine the effects of other phytoplankton species and combinations of species, with the proviso that the selected algal species should be good candidates for mass cultivation under commercial-scale conditions (i.e. easily and cheaply produced). The present study did not examine the potential use of prepared or artificial diets for feeding larvae and this is a particular avenue of study which requires much more research (see George et al. 2004, Liu et al. 2007a,b). Potential alternative diets to be tested might include dried heterotrophically-grown algae, algal pastes, yeast-based feeds, lipid emulsion spheres, and grain-based additives such as corn starch and wheat germ. Ultimately, research on prepared diets for larvae would examine protein, lipid, carbohydrate, and mineral requirements of this life stage. This type of research will be pertinent for any echinoid species of commercial interest.
For my project, I investigated the effects of various factors on larval growth and survival up to metamorphic competency. Further research, examining the effects of various factors on early post-metamorphic growth and survival, will be required for this species and other echinoids (as in
Xing et al. 2007, Dworjanyn et al. 2007). There is a dearth of information on the life stage from post-metamorphosis to 5-mm test diameter (see review by Azad et al. 2010).
146 My research was conducted with relatively small culture volumes (15 L) in well-controlled laboratory conditions. The majority of studies on echinoid larvae have also been conducted in small culture volumes (very often 1–5 L). Only one publiched study, by Buitrago et al. (2005), used commercial-sized (100 L) tanks for rearing larval sea urchins (L. variegatus). An obvious weakness to my research is that it is unknown whether the results from my small-scale studies can be extrapolated to commercial-scale production. Future research will need to be conducted using mass production techniques and should be performed in a proper hatchery, of at least pilot- scale production size, in order to determine if the small-scale results obtained here are transferrebale to larger scales.
In conclusion, there is a general trend (see Robinson 2004) that most aquaculture operations for marine species do not start until the wild stocks have been reduced to a point where the earnings and lifestyle of the fishers and related industry people are negatively affected. As the wild fisheries of sea urchins have been depleted around the globe and demand for roe or “uni” remains high, the possibility for commercial sea-urchin aquaculture is bright. Given the steady increase in interest in echinoid aquaculture, more studies on quality gonad production for adults and mass production technology of juveniles will be required. By systematically examining a number of biotic and abiotic variables in the rearing of purple sea urchins (adults, embryos, and larvae), I was able to determine that S. purpuratus should make an ideal candidate for aquaculture. While my study focused on S. purpuratus, I believe that this information would benefit the study of biological and environmental criteria of other closely related echinoid species.
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180 Appendix: Conference presentations
My scientific activities undertaken during my Ph. D. have been diverse and I have participated in a number of aquaculture and fisheries resource development and management conferences in different parts of the world. The following is a list of the conferences in which I have participated and presented papers during my Ph.D. study:
Azad A.K., Jensen K.R and Lin C.K. 2008. Coastal aquaculture development in Bangladesh:
unsustainable and sustainable experiences. International Institute of Fisheries Economics and
Trade (IIFET) conference (July 22-25, 2008), Nha Trang University, Nha Trang, Vietnam.
This article was published in Environmental Management 44 (2009): 800-809 as well as in
conference (IIFET 2008) proceedings.
Azad A.K., McKinley R.S. and Pearce C.M. 2008. Ingestion, absorption and gonad yield and
quality of purple sea urchins (Strongylocentrotus purpuratus): influence of temperature and
diet. Aquaculture Pacific Exchange conference (September 25-26, 2008), Campbell River,
BC, Canada.
Azad A.K., McKinley R.S. and Pearce C.M. 2009. Larval development and survivorship of
purple sea urchins (Strongylocentrotus purpuratus): influence of stocking density.
Aquaculture Association of Canada (AAC) conference (May 10-13, 2009), Nanaimo, BC,
Canada.
Azad A.K., McKinley R.S. and Pearce C.M. 2010. Factors influencing larval development and
survivorship of laboratory-reared purple sea urchins (Strongylocentrotus purpuratus):
implications for aquaculture. Australasian Aquaculture conference (May 23-26, 2010),
Hobart, Australia.
181