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Embryonic development, larval morphology and juvenile growth of the sea cucumber 1

Athyonidium chilensis (Semper, 1868) (Holothuroidea: Dendrochirotida) 2

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CHITA GUISADO1 *, ROBERTO MALTRAIN 1, SERGIO A. CARRASCO1 2, DANIELA DÍAZ-5

GUISADO2 3, HERMAN ROJAS 1, and EDUARDO BUSTOS 3 6

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1Facultad de Ciencias del Mar y de Recursos Naturales, Universidad de Valparaíso, Casilla 5080, 9

Viña del Mar, Chile 10

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2Present address: School of Biological Sciences and Coastal Ecology Laboratory, Victoria 12

University of Wellington, Wellington 6140, New Zealand 13

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3Instituto de Ciencia y Tecnología, Universidad Arturo Prat, Ejército 443, Puerto Montt, Chile 15

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*Corresponding author: Facultad de Ciencias del Mar y de Recursos Naturales, Casilla 5080, 17

Reñaca, Viña del Mar, Chile. Phone: (56) (032) 2507862. Fax: (56) (032) 2507859 18

Email: chita.guisado@uv.cl 19

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Running title: Development and growth of Athyonidium chilensis 22

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Abstract 28

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Athyonidium chilensis is the most economically important holothurian in Chile. In spite of the 30

increases in harvest due the high demand from Asian markets, early life stages and juvenile phases 31

of the species have received less attention. The embryonic and larval development of A. chilensis 32

took approximately 5 days and it followed the typical developmental stages described for other sea 33

cucumber species with direct development: the gastrula develops into a vitellaria larva 34

(lecothotrophic stage; 4-5 d pf), which was followed by the pentactula stage (7 d pf). After 35

settlement, individuals were capable of active feeding on micro-algae associated with the sediment. 36

The juveniles (35 d pf) possessed four pairs of ambulacral podia, and after 4 months of cultivation, 37

a substantial number of podia covered their bodies, which were approximately 1.2 mm long. An 38

evident effect of the density during the culture of juvenile was observed, with higher growth rates 39

and survival observed in the lowest density treatment (1 ind/cm2). This study provide the first 40

description of the early life history stages of a sea cucumber in Chilean waters, but also shows that 41

the aquaculture of this species is feasible and could be developed as an alternative to maintain a 42

sustainable harvest and eventually contribute to the restoration of natural populations. 43

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Key words: Athyonidium chilensis, holothuroid, development, juvenile growth, aquaculture 46

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Introduction 54

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Sea cucumbers are a valuable resource for coastal communities in the Indo-Pacific region, where 56

they have been exploited unsustainably during decades. The fishing pressure exerted on the species 57

has increased over the years fuelled by the rising wholesale price of the product, resulting in the 58

depletion of wild stocks worldwide (Purcell, et al. 2002; Hamel, et al. 2003; Ramofafia, et al. 2003). 59

The recent use of sea cucumbers in the pharmaceutical industry as antibacterial agents have also 60

increased the demand of the species (see Al-Haj, et al. 2009), encouraging an improvement of the 61

artificial culture techniques together with the acquisition of an adequate knowledge of critical 62

phases in captivity. However, in the last decades, only a few commercial holothurians have been 63

successfully reared to settlement, including some tropical and temperate species (e.g., Holothuria 64

scabra, H. fuscogilva, Stichopus japonicus, S. mollis) (reviewed by Ramofafia, et al. 2003). 65

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Even though the embryonic and larval development of some species of sea cucumbers has been 67

well documented (e.g., Cameron & Fankboner, 1989; Hamel & Mercier, 1996; Battaglene, et al. 68

2002; Hamel, et al. 2003; Laxminarayana, 2005; Asha & Muthiah, 2002; Asha & Muthiah, 2005), 69

most of the species that have been studied belong to the order Asparochidotida, characterized by an 70

indirect development, i.e., the gastrula develops to an auricularia larva (planktotrophic stage), that 71

undergoes metamorphosis to a dololaria larva (lecothotrophic stage) before settlement as a 72

pentactula (Sewell & McEuen, 2002). However, direct development, i.e., gastrula develops to a 73

vitellaria larva (lecothotrophic stage) before settlement (e.g., with no auricularia stage), is the most 74

common form of development in holoturoids and is dominant in 22 of the 25 families (Smiley, et al. 75

1991). 76

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Athyonidium chilensis is one of the most common species of echinoderms along the Chilean coast, 78

with a geographic distribution that extends from Ancón, Perú (11º 44’ 5” S), to Punta Gaviota in 79

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Chiloé Island, Chile (42º 03’ 55” S). This species inhabits intertidal and subtidal zones, where can 80

be found in in rocky pools and crevices, below boulders and also buried in the sand (Deichmann, 81

1941; Pawson, 1964; Pawson, 1969, Fernández, 1998). Although A. chilensis is the most 82

commercially important holothurian in Chile, there is no documented information before 1991 83

(SERNAP, 2003). The average harvest of the species from 1998 to 2008 was 269 ton per year, with 84

a maximum extraction of 1,510 ton in 2000 (Renbo & Yuan, 2004; SERNAP, 2008). The 85

unsustainable exploitation of this resource, added to the growing demand from Asian markets, 86

forebodes a drastic decline of the easily-fished wild stocks of A. chilensis in a near future. 87

Therefore, studies focused on reproduction, development and feeding behavior of the species in 88

captivity are extremely important in order to understand the life history of the species, determine its 89

possible aquaculture potential and also take accurate decisions in terms of stock management. 90

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Despite previous studies have contributed on some aspects of the biology, ecology and culture of A. 92

chilensis (Fernández, 1998; Guisado, et al. 1999; Moreno, 2002; Pérez, 2005; González, 2006; 93

Fernández, 2007; Maltrain, 2007), the early life history of the species has not been completely 94

described and there is a knowledge gap about larval morphology, development, settlement and 95

juvenile growth. In this study we aimed first, to characterize the embryonic and larval development 96

of the sea cucumber A. chilensis under laboratory conditions and second, to evaluate the effects of 97

cultivation density on juvenile growth and survival. 98

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Materials and Methods 101

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Specimen collection and spawning induction 103

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In May 2006, twenty-five adults of A. chilensis were collected at Maitencillo Beach, Chile (32° 38’ 105

S, 71° 26’ W) by SCUBA diving. Individuals were transported to the wet laboratory of Universidad 106

de Valparaíso, where they were placed in five 67-L tanks filled with micro-filtered seawater (5 107

individuals per tank) and kept in a conditioning period of 48 h. Eighteen sea cucumbers with a body 108

length greater than 15 cm (250-400 g) were further selected for spawning induction, placed into 109

three 67-L tanks filled with micro-filtered seawater (6 individuals per tank) and induced to spawn 110

by means of thermal stimulation (Girapsy & Ivy, 2005; Laxminarayana, 2005) and addition of food 111

ad libitum using the micro-alga Chlorella neustonica (Pérez, 2005). 112

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Embryo and larval culture 114

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After spawning, female gametes were deposited in 3-L glass tanks and male gametes were 116

transferred to a 250 ml beaker. In order to carry out fertilization, 2 ml of spermatozoa were added to 117

each 3-L tanks, obtaining a gamete ratio of 1:100 (oocyte - spermatozoa). 118

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Once fertilization was verified, the eggs were transferred into five 67-L tanks (3 eggs ml-1) and each 120

tank was filled with micro-filtered and UV sterilized seawater. Fifty percent of the seawater was 121

renewed on the second day of cultivation. After 3 or 4 days post-fertilization (pf), only larvae 122

swimming in the water column were selected and then transferred into a 1000-L specially 123

conditioned tank (0.5 larva ml-1). The 50 % of the seawater was renewed daily and the temperature 124

was maintained constant (13 ± 0.3ºC). Two air stones positioned at the bottom of the tank provided 125

continuous aeration. Embryonic and larval development was documented by taking random samples 126

(30 larvae) every 15-30 minutes during the first 24 h pf. Hereafter, samples were taken every hour 127

until the early vitellaria stage was observed (4-5 days). Samples were photographed and measured 128

using an optic microscope (4X magnification) equipped with an ocular micrometer and also where 129

analyzed using a scanning electron microscope. After 10 days of cultivation (i.e., when feeding 130

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behavior was observed), the larvae were fed daily with the micro-alga Tetraselmis suecica 131

(MLB1094; 1,200 cells ml-1), and after the day 42, 0.5 g of Algamac 2000 TM were also provided 132

once a week. Once settlement was observed, 145 g of fine sand (> 0.5 mm) were added to the tank 133

in order to increase survival (C. Guisado, unpublished data 2006). Size was daily determined by 134

taking measures of the maximum length (ML in mm, defined as the distance between the mouth and 135

the anus) (Hamel & Mercier, 1996; Hamel, et al. 1999) of 30 individuals during the first 8 months 136

of cultivation. Survival was monthly evaluated using 100 cm2 PVC quadrants with six replicates in 137

each measurement. 138

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Effects of cultivation density on juvenile growth and survival of Athyonidium chilensis 140

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Three experimental densities were tested in this study. Juveniles obtained in laboratory (75 days pf; 142

0.9 ± 0.1 mm) were placed in 300-ml glass tanks at densities of 1, 3 and 5 individuals per cm2 (i.e., 143

64, 191 and 318 individuals per tank, respectively), with each density being conducted in triplicate. 144

Fine sand was used as substrate based on preliminary experiments (3 g per tank; C. Guisado, 145

unpublished data 2006). Seawater was renewed daily and the temperature was maintained constant 146

(13 ± 0.3 °C). Juveniles were fed daily with the micro-alga T. suecica (1,200 cells/ml) and the 147

supplement Algamac 2000 TM was added once a week. At the beginning and at the end of the 148

experimental period, the ML of 20 randomly selected juveniles per treatment was measured and the 149

absolute growth rate (AGR) was calculated as follow, AGR = MLt-ML0 t-1, where: MLt= final 150

maximum length (μm), ML0 = initial maximum length (μm) and t = experimental period (days). In 151

order to evaluate survival, the total number of individuals in each tank was visually quantified twice 152

a month. 153

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One- way analyses of variance (ANOVA) were used to test for differences in AGR among the 155

experimental densities examined. Differences in survival of juvenile over time and among different 156

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experimental densities were tested using a two-way ANOVA. In both cases, significant differences 157

were further examined using multiple pairwise comparison tests (Tukey). Percentage variables were 158

arcsine transformed. Normality and homocedasticity requirements were tested using Lilliefors and 159

Levene tests, respectively. When assumptions were not met, the analyses were performed in rank-160

transformed data (Conover1980). 161

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Results 164

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Characterization of developmental stages of Athyonidium chilensis 166

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Spawning and embryonic development 168

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The chronology of development for A. chilensis, with the corresponding size of the embryos and the 170

photographic illustrations of the stages of development, are presented in Table 1 and Figures 1 and 171

2. After spawning induction, only 7 of the 18 individuals spawned (6 males and 1 female). Once 172

fertilization was completed, the estimated quantity of viable eggs was ~ 6 x 105. The eggs were 173

spherical and could be easily seen (359.81 ± 16.13 μm [mean diameter ± S.D]). The fertilization 174

envelope (fe) was observed after 10 min post-fertilization (pf) (Fig. 1A). 175

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The first cleavage occurred between 1 to 3 h pf. The second (Fig. 1B), third and fourth cleavages 177

took place between 3 to 6 h pf. During these stages blastomeres (b) of equal size and spherical 178

shape were observed, with a clear separation between them. The animal and vegetal poles continued 179

dividing until the 32-cell stage. At this stage the fertilization envelope was still observed (Fig 1C). 180

After 24-25 h pf, a process of embryo compaction, a considerable reduction in the space between 181

the blastomeres and the presence of the blastocele (bc) was observed, being the precursor of the 182

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blastula stage (Fig. 1D). Rotating movements and constant migration throughout the water column 183

were observed in the embryos, and after 48 h pf the blastula began to elongate and transform into a 184

gastrula (Fig. 1E). The blastopore (bp) and blastopore scams (bs) were clearly visible in the aboral 185

region (Fig. 2A, B) and a small lateral depression at the equatorial region of the embryo’s body 186

began to form, indicating the position of the future vestibule (v) (Fig. 2C). After 4 to 5 d pf the 187

embryo began its transformation into a vitellaria larva (Fig. 1F). 188

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Larval development 190

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The vitellaria larva of A. chilensis (433 ± 36.96 μm [mean diameter ± S.D]) was characterized as a 192

free swimming and lecithotrophic larva. The vestibule (v) was clearly visible and the five primary 193

buccal tentacles (pt) were observed inside it (Fig. 1F). After 7 days pf these tentacles began to 194

protrude from the vestibule, process that was the precursor of the pentactula stage (Fig. 1G). The 195

pentactula was characterized by a progressive decrease in the swimming capacity due to the loss of 196

the body cilia. Also, the buccal tentacles (bt) were completely protruded, showing an important role 197

on feeding and locomotion. The anal pore was also observed in the aboral region of the body. At 21 198

days pf the late pentactula possesses, in addition to the buccal tentacles, a pair of ambulacral podia 199

(ap) (Fig. 2 D, E, F). After the settlement of the pentactula, individuals were capable of active 200

feeding on micro-algae associated with the sediment, using their buccal tentacles to collect and 201

ingest food. 202

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Juvenile growth and survival 204

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Thirty-five days pf the fourth pair of ambulacral podia appeared, and juvenile with green 206

pigmentation towards the posterior portion of the body were observed (Fig. 1H). During this period, 207

a survival of ~18 % from the total viable larvae was recorded (Fig. 3A). After 4 months, a 208

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substantial number of completely developed podia and a notable pigmentation on the body wall 209

were observed, and juveniles (1,200 µm ML), began to show some common adult behaviors such as 210

burying and anal pumping movements. During this period, a survival of 1.6 % from the total viable 211

larvae was recorded (Fig 3A). After 8 months of cultivation the survival decreased until 0.24 %, and 212

a maximum size of 1,900 µm was recorded (Fig. 1I, 3A, 3B). 213

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Effects of cultivation density on growth and survival of juvenile Athyonidium chilensis 215

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After 3 months, the AGR (µm d-1) of juvenile A. chilensis varied significantly among the three 217

cultivation densities evaluated (one-way ANOVA, F2,177=4.947, P=0.008). The maximum AGR 218

was recorded in the treatment with the lowest density (8.78 ± 4.17 µm d-1 [mean ± S.D]; 1 ind cm2), 219

which was not significantly different from the 7.12 ± 3.57 µm d-1 obtained in the treatment with 3 220

ind cm2 (Tukey, P=0.052); however, differed significantly from the 6.64 ± 4.61 µm d-1 obtained 221

with 5 ind cm2 (Tukey, P=0.008) (Fig. 4). 222

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The survival of juvenile A. chilensis decreased significantly in all density treatments over the 75 224

days of experiments until a mean value of 39.7 % ± 6.7 % [mean ± S.D] (two-way ANOVA, F5,36= 225

50.204; P < 0.001). Significant differences were detected among treatments (two-way ANOVA, 226

F2,36= 5.101; P = 0.011; Fig. 5), with no interactive effect between time and treatments (two-way 227

ANOVA, interaction time x treatment: F10,36= 1.131; P = 0.368). Overall, the survival of juveniles 228

in the treatment with 1 ind cm2 was not significantly different to the survival in the treatment with 3 229

ind cm2 (Tukey, P=0.378); however, the former differed significantly with the lower values 230

obtained in the treatment with 5 ind cm2 (Tukey, P = 0.008) (Fig. 5). 231

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Discussion 235

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Liberation of gametes in A. chilensis was only achieved after stimulation via an increase in the 237

water temperature and the addition of food, a technique previously used to induce spawning in a 238

range of tropical sea cucumbers, including H. scabra, Actinopyga mauritiana (Battaglene, 1999) 239

and H. fuscogilva (Battaglene, et al. 2002; Ramofafia, et al. 2003). Spawning occurred during the 240

night, and invariably, males spawned before the female. This same reproductive behaviour was 241

previously described by Costelloe (1988) in Aslia lefevrei and by Renbo & Yuan (2004) in 242

Apostichopus japonicus. Indeed, some authors have suggested that sperm, or something associated 243

with it, is a proximal signal for synchronizing female to spawn (see Bataglene, et al. 2002). 244

Therefore, in order to successfully carry out fertilization in laboratory is essential to maintain a total 245

control over the broodstocks, because spontaneous spawning could cause polyspermia, a common 246

problem with in vitro fertilizations and a lethal condition in marine invertebrates. 247

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The average diameter of newly released A. chilensis eggs was 359.81 ± 16.13 μm. Larger sizes have 249

been reported by Costelloe (1988) in the dendrochirote A. lefevrei (400-650 μm) and, Hamel & 250

Mercier (1996) found that the mean diameter of the eggs in Cucumaria frondosa was more than 251

twice the size registered in our study (i.e., 900 μm). By contrast, in species that present indirect 252

development eggs are smaller, as has been recorded for H. scabra (156.4 μm), H. fuscogilva (151.7 253

μm) and Actinopyga mauritiana (109.9 μm) by Ramofafia et al. (2003). These differences in egg 254

size between species could be due to their different reproductive strategies, as species that present 255

direct development only presents a lecitotrophic stage, in which survival and growth rely on 256

endogenous reserves. 257

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Athionidium chilensis, as other dendrochirotes species (e.g., C. frondosa; Hamel & Mercier 1996) 259

presents direct development, during which the gastrula develops into a lecitotrophic larva (i.e., 260

vitellaria). The mean size recorded in this study for the vitellaria larvae was 433 ± 36.96 μm (mean 261

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± S.D), while Hamel & Mercier (1996) recorded in C. frondosa a much larger size (1.55 ± 0.3 mm). 262

Apparently, the different larval stages of A. chilensis do not exhibit a significant increase in the 263

maximum length during the larval development, a condition previously observed in C. frondosa as 264

well (Hamel & Mercier, 1996). 265

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According to several studies, the embryonic and larval development of dendrochirotes is considered 267

brief in comparison with species that present indirect development. For example, in C. frondosa the 268

pentactula stage was recorded between 11-11.5 days at 12 °C (Hamel & Mercier, 1996), in A. 269

chilensis between 3-4 days at 14-16 ºC (Pérez 2005) and according to this study between 6-7 days 270

at 13 ± 0.3 ºC. By contrast, in aspidochirotes the final larval stage is reached later, for example, in 271

A. japonicus it was recorded between 13-17 days at 21.5-23 ºC (Rembo & Yuan, 2004), in 272

Isostichopus fuscus between 22-27 days at 27-29 ºC (Hamel, et al. 2003) and in H. scabra between 273

13-15 days at 25-27 ºC (Ramofafia, et al. 2003). These results suggest that environmental factors 274

such as temperature may play an important role during the developmental stages of several 275

holothurian species and can be especially important as a technique to manage the extension of the 276

larval stage in laboratory reared species (Hamel & Mercier, 1996; Ramofafia et al. 2003). 277

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Juvenile A. chilensis obtained after 35 days pf were capable of actively use their crown of tentacles 279

for feeding, and their four pairs of ambulacral podia for locomotion and attachment to the substrate. 280

During the first 8 months of cultivation in laboratory, juvenile A. chilensis reached an average 281

length of 1,900 µm, contrasting with the grater sizes recorded by Hamel & Mercier (1996) and 282

Costelloe (1988) during the first year of cultivation in the dendrochirotes C. frondosa and A. 283

lefevrei (10 mm and 3.4 cm, respectively). However, the slow growth rates observed in A. chilensis 284

were more notable when compared with aspidochirotes species. For example, I. fuscus reached a 285

length of 3.5 cm after only 72 days pf (Hamel, et al. 2003), and a similar size was recorded in H. 286

spinifera after 4 months of cultivation (4.8 cm; Asha & Muthiah, 2005). These differences in 287

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juvenile growth between the orders Aspidochirotida and Dendrochirotida could be due to their 288

completely different biological and ecological characteristics, since aspidochirotes move actively 289

and experience daily cycles of burying, while dendrochirotes are usually buried or covered by 290

substrate (e.g., sand or algal detritus) displaying less mobility and a passive foraging behavior 291

(Giese, et al. 1991). 292

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As in others sea cucumber species obtained in land-based nursery systems, A. chilensis showed low 294

survival during the first 30 days of cultivation. High mortalities were also recorded during the first 295

20 days post-metamorphosis in C. frondosa by Hamel et al. (2003) and during hatch in H. scabra 296

(Ramofafia, et al. 2003). Few procedures have been proposed in order to avoid high mortalities in 297

early stages of holothurians during cultivation, but one of the most important is the maintenance of 298

an appropriate density of individuals during culture. Past experience has shown that a high density 299

of individuals increases mortality and reduces the availability of space and food, causing 300

malnutrition, slow growth and high variability of size (Xiyin, et al. 2004; Agudo, 2006). Our results 301

suggested that at low density (1 ind/cm2) juveniles grew faster and survive better than conspecifics 302

maintained at high densities (5 ind/cm2), agreeing with the findings of Battaglene et al. (1999) and 303

Xilin (2004), whose observed that the highest growth and survival occurred in the low density 304

treatments (i.e., 1.7 ind/cm2 and 1 ind/cm2 for H. scabra and A. japonicus, respectively). 305

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In summary, Athyonidium chilensis as well as other holothurian species, undergoes critical phases 307

in captivity, such as fertilization, larval development and settlement. Nevertheless, this species 308

showed a rapid embryonic and larval development. Juvenile growth and survival was affected by 309

the cultivation density, factor that could indirectly mediate the food availability on cultured 310

conditions. This study provide the first data on embryonic development, larval morphology and 311

juvenile growth of A. chilensis, showing that this sea cucumber can be successfully reared in land-312

based nursery systems and that the culture of this species is feasible and can potentially be 313

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developed as an alternative to maintain sustainable harvest and eventually contribute to the 314

restoration of natural populations. Further research should target to improve the spawning induction 315

and principally, the feeding and growth of juveniles in captivity. 316

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Acknowledgements 319

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We would like to thank L. Amaro and M. Torres for their help during field collections and 321

laboratory experiments. The comments of anonymous reviewers that improved initial versions of 322

the manuscript are also appreciated. This research was supported by funds of the project FONDEF 323

from CONICYT: DO3I 1072. 324

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435

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439

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cucumber, Apostichopus japonicus. In: A. Lovatelli, C. Conand, S. Purcell, S. Uthicke, J. F. Hamel 441

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Technical Paper n°. 463. Rome, Italy, pp. 273-276. 443

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Figure captions 462

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Fig.1. Optic microscopy of the embryonic and larval development of A. chilensis. (A) Fertilized 464

oocyte with fertilization envelope (fe) completely elevated. Scale bar = 120 μm. (B) 4-cell stage, 465

where (b) blastomere. Scale bar = 180 μm. (C) 32-cell stage. Scale bar = 150 μm (D) Blastula, 466

where (bc) blastocoele. Scale bar = 100 μm (E) Gastrula, where (v) vestibule. Scale bar = 100 μm 467

(F) Vitellaria, where (pt) primary tentacles. Scale bar = 150 μm (G) Pentactula, where (bt) buccal 468

tentacles. Scale bar = 200 μm. (H) Juvenile 35 d pf, where (a) anus, (bt) buccal tentacles, (ap) 469

ambulacral podia. Scale bar = 230 μm. (I) Juvenile 120 d pf. Scale bar = 275 μm. 470

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Fig.2. Electronic microscopy of the larval development of A. chilensis. (A) Gastrula with blastopore 472

(bp) in the aboral region. (B) Blastopore scams (bs). (C) Gastrula with the future vestibule (v) in 473

formation. (D and E) Late pentactula, where (bt) buccal tentacles, (ap) ambulacral podia. (F) 474

Buccal tentacles in detail. Scale bar (A, C, D, E) = 100 μm. Scale bar (B) = 10μm. 475

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Fig. 3. (A) Survival and (B) Size of A. chilensis during the first eight months of cultivation. 477

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Fig.4. Absolute growth rate (AGR) of juvenile A. chilensis reared in laboratory under three 479

experimental densities (1, 3 and 5 individuals per cm2). Letters indicate significant differences in 480

growth rate. Vertical bars represent S.Es. 481

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Fig.5. Survival of juvenile A. chilensis reared in laboratory under three experimental densities (1, 3 483

and 5 individuals per cm2). Letters and asterisks indicate significant differences in growth rate. 484

Vertical bars represent S.Es. 485

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Table 1. Chronology of development and approximate sizes (mean ± S.D) of A. chilensis under 487

laboratory conditions (13 ± 0.3 ºC). Time of development is presented in hours (h) and days (d). 488

489

Stage Time (h, d) Size (μm)

Fertilized oocyte 0 min 359.81 ± 16.13

2-cell 1-3 h 341.67 ± 14.43

4-cell 3-5 h 366.67 ± 72.17

8-cell 5-6 h 358.33 ± 14.43

16-cell 6-8 h 400.00 ± 25.00

32-cell 8-9 h 441.67 ± 14.43

Blastula 24-25 h 325.00 ± 25.00

Gastrula 48-49 h 483.33 ± 38.19

Vitellaria 4-5 d 433.00 ± 36.96

Pentactula 7 d 629.00 ± 35.61

Late pentactula 21 d 689.00 ± 64.11

Juvenile 35 d 683.71 ± 11.11

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