Journal Pre-proof Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus competent larvae

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Journal Pre-proof Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus

competent larvae

Marta Castilla-Gavilán, Meshi Reznicov, Vincent Turpin, Priscilla Decottignies, Bruno Cognie

To cite this version:

Marta Castilla-Gavilán, Meshi Reznicov, Vincent Turpin, Priscilla Decottignies, Bruno Cognie. Jour- nal Pre-proof Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus com- petent larvae. Aquaculture, Elsevier, 2020, 515, pp.734559. �10.1016/j.aquaculture.2019.734559�.

�hal-02378591�

Journal Pre-proof

Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus competent larvae

Marta Castilla-Gavilán, Meshi Reznicov, Vincent Turpin, Priscilla Decottignies, Bruno Cognie

PII: S0044-8486(19)31658-8

DOI:

Reference: AQUA 734559

To appear in:

Received Date: 5 July 2019

Revised Date: 13 September 2019 Accepted Date: 1 October 2019

Please cite this article as: Castilla-Gavilán, M., Reznicov, M., Turpin, V., Decottignies, P., Cognie, B., Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus competent larvae,

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© 2019 Published by Elsevier B.V.

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Sea urchin recruitment: effect of diatom based biofilms on Paracentrotus lividus 1

competent larvae 2

Marta Castilla-Gavilán*

, Meshi Reznicov

, Vincent Turpin, Priscilla Decottignies, 3

Bruno Cognie 4

Université de Nantes, Institut Universitaire Mer et Littoral, EA 2160 Mer Molécules 5

Santé, 2 rue de la Houssinière BP 92208, 44322 Nantes cedex 3 (France) 6

┼

These authors contributed equally 7

*Corresponding author: mcasgavilan@gmail.com 8

Abstract 9

Eight different experimental substrates were tested on Paracentrotus lividus competent 10

larvae in order to evaluate their potential for inducing metamorphosis and enhance 11

survival after recruitment. Two benthic diatoms species, Nitzschia laevis (NL) and 12

Halamphora coffeaeformis (HC), were selected according to their capacity to adhere 13

and to form strong biofilms. They were tested in monocultures and in a mixed biofilm 14

(MIX) that was also tried in combination with Gamma-Aminobutyric Acid, involved in 15

triggering some invertebrate metamorphosis (MIX+GABA). Histamine (HIS) was also 16

used as a treatment according to the high metamorphosis rates that have been recorded 17

for this compound on other sea urchin species. Finally, a natural microphytobenthic 18

biofilm (NATURAL) and oyster shells particles colonized by epiphytic diatoms 19

(SHELL) were sampled from the mud of a refining oyster pond. Batches of 21 days-old 20

larvae were placed on each experimental substrate and their effect was compared to a 21

negative control of filtered sea water (without any treatment; FSW). Metamorphosis rate 22

was daily recorded in each treatment. The sea urchin larvae on substrates NL, 23

NATURAL, GABA+MIX and SHELL showed significantly higher metamorphosis 24

rates than larvae on the other treatments (P < 0.001), reaching more than 90% in 72h.

25

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Survival rate was assessed at 10 days post-metamorphosis in these four treatments. No 26

difference was observed between them in terms of metamorphosis rate or survival rate 27

(more than 60% for the four experimental substrates). Results demonstrate that the 28

transition from planktonic larvae to benthic juvenile could be promoted through diatom- 29

based biofilms. These substrates represent efficient metamorphosis inducers for P.

30

lividus larviculture but we suggest to use preferably N. laevis biofilms in order to 31

promote practical and safe solutions for farmers.

32

Keywords: aquaculture diversification; echinoculture; Nitzschia laevis; metamorphosis 33

rate; post-settlement survival rate 34

Introduction 35

Sea urchin roes are considered as a delicacy and they are among the most valued sea 36

food products. Sea urchin become highly trendy due to their unique taste and the spread 37

of Japanese food around the globe. The leading country in consumption of sea urchins is 38

Japan followed by France, the first market in Europe (Stefánsson et al., 2017). To fulfil 39

this demand, wild populations have been overexploited worldwide leading to a decline 40

since the 90’s (Ceccherelli et al., 2011; Couvray et al., 2015; McBride, 2005). Beyond 41

the economic impact, this depletion has ecological implications as sea urchins have a 42

key role in the infra-littoral rocky shore areas (Giakoumi et al., 2012; Kitching and 43

Thain, 1983; Privitera et al., 2011; Scheibling, 1986).

44

To deal with overexploitation, echinoculture of several species has been developed 45

worldwide (Andrew et al., 2002): for exemple Paracentrotus lividus in Europe, 46

Loxechinus albus in Chile, Strongylocentrotus spp. in China or S. depressus, S.

47

intermedius and S. nudus in Japan. In Europe, certain constraints remain to be solved for

48

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a sustainable and cost-effective industry, notably in the phase of transition between 49

planktonic larvae to a benthic juvenile (Grosjean et al., 1998). Settlement, 50

metamorphosis and post-metamorphosis survival rates are still not high enough to 51

produce juveniles in hatcheries at an industrial-scale (Carbonara et al., 2018; Hannon et 52

al., 2017; Zupo et al., 2018). Researches have been conducted on several sea urchin 53

species to find reliable metamorphosis-inducing-factors for this crucial development 54

stage. Various levels of success have been shown with different species of algae 55

(Carbonara et al., 2018; Castilla-Gavilán et al., 2018b; De la Uz et al., 2013), diatoms 56

and bacterial biofilms (Ab Rahim et al., 2004; Brundu et al., 2016; Dworjanyn and 57

Pirozzi, 2008; Rial et al., 2018; Xing et al., 2007; Zupo et al., 2018), conspecifics 58

(Dworjanyn and Pirozzi, 2008; Gosselin and Jangoux, 1996), and chemical compounds 59

(Carbonara et al., 2018; Pearce and Scheibling, 1990; Swanson et al., 2012). It seems 60

that the most successful metamorphosis-inducing signals are microbial biofilms, 61

whether or not they are linked to the surface of seaweed thalli or inorganic surfaces such 62

as rocks or shells (Hadfield and Paul, 2001). Recent studies have shown the 63

metamorphosis induction effect of benthic diatoms on the culture of P. lividus, 64

obtaining high settlement and survival rates (Rial et al., 2018; Zupo et al., 2018). This 65

zootechnics are also largely used in Japan, China and Chile for sea urchin production in 66

plates covered by natural benthic diatoms biofilms (Ab Rahim et al., 2004; Hagen, 67

1996; McBride, 2005; Rahman et al., 2014; Takahashi et al., 2002; Xing et al., 2007).

68

Moreover, neurotransmitters have been shown to regulate developmental transition in 69

sea urchins, as histamine (Sutherby et al., 2012; Swanson et al., 2012, 2004) and 70

Gamma-Aminobutyric Acid (GABA) (Pearce and Scheibling, 1990; Rahman and 71

Uehara, 2001).

72

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The main objective of the present study was to compare the effect of different inducing 73

cues on the metamorphosis of Paracentrotus lividus and to identify those that could be 74

of easy and cheap application. We tested histamine and GABA, two benthic diatoms 75

(Halamphora coffeaeformis and Nitzschia laevis), a natural benthic biofilm and oyster 76

shells colonized by epiphytic diatoms.

77

Materials and Methods 78

Hatchery of sea urchin larvae 79

Larvae of P. lividus were raised in the Benth’Ostrea Prod aquaculture farm (Bouin, 80

Vendée, France). They were fed on a combined diet consisting of three microalgae 81

species: Isochrysis aff. galbana (clone T-ISO), Rhodomonas sp. and Dunaliella 82

tertiolecta (Castilla-Gavilán et al., 2018a). Larvae were reared in continuous dark at a 83

density of 1 per ml, in 2-m

conical PVC tanks filled with aerated seawater. A complete 84

water exchange and a thorough cleaning of the tanks were carried out every day. Prior 85

to the experiment, pre-competent larvae were transferred to the laboratory and kept at 86

the same density in an aerated 5 L glass reactor balloon, until competence was achieved.

87

Competence was considered when 75% of larvae had a developed rudiment that was 88

equal or larger than the stomach, as proposed by Kelly et al. (2000).

89

Experimental treatments 90

The chosen treatments for P. lividus competent larvae recruitment assay (see next 91

section) were:

92

- Nitzschia laevis biofilm (NL) 93

- Halamphora coffeaeformis biofilm (HC) 94

- Mix biofilm of both N. laevis and H. coffeaeformis (MIX)

95

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- Natural biofilm sampled from refining oyster ponds (NATURAL) 96

- Broken oyster shells 10 g (SHELL) 97

- Histamine 10

M (HIS) 98

- GABA 10

M (GABA) 99

- GABA 10

M + Mix biofilm of both N. laevis and H. coffeaeformis 100

(GABA+MIX) 101

- Control of filtered seawater (FSW) 102

The diatoms N. laevis and H. coffeaeformis were obtained from the Nantes Culture 103

Collection (UTCC58 and NCC39 from the Mer-Molécules-Santé Laboratory, Nantes, 104

France). These species were chosen for their ability to adhere and to form strong 105

biofilms. Prior to experimental assays, growth kinetics of the two diatoms species 106

selected were assessed (lag time, maximal biomass and maximal specific growth rate).

107

They were grown in 80 mm diameter Pyrex

Crystallizing Dishes filled with 100 ml of 108

F/2 media. Each dish was inoculated with 50 000 cells/ml. The diatom biofilms were 109

grown in triplicates under conditions of 15°C, 14h:10h L/D cycle at 120 µmol. photons 110

m

s

. Growth curves were assessed by daily cell counting using a haemocytometer.

111

All experimental treatments were carried out in four replicates in filtered seawater (5 112

µm filtered and UV treated). For NL and HC treatments, biofilms were cultured as 113

explained above. For the MIX treatment, biofilms were cultured by inoculating 25 000 114

cells/ml of each species. Seven days old biofilms were used. One day prior to the 115

recruitment assay, the F/2 media was gently removed and replaced with 100 ml of 116

filtered seawater. For the NATURAL treatment, 3 kg of sediment from an oyster pond 117

of the Benth’Ostrea Prod farm was collected two days prior to the recruitment assay.

118

The natural biofilm was sampled following the protocol described by Eaton and Moss

119

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(1966) and homogenized in 400 ml of filtered seawater. Finally, 100 ml were inoculated 120

in four Crystallizing Dishes for 24 h under natural photoperiod at 20 °C. Oyster shells 121

(SHELL treatment) were collected in an oyster pond from the Benth’Ostrea Prod farm.

122

They were broken into small pieces (1.5-5 cm) placed in four Crystallizing Dishes (10 g 123

in each dish) filled with 100 ml filtered seawater. Prior to the recruitment assay, the 124

presence of an active photosynthetic biofilm on the broken shells was checked using a 125

chlorophyll fluorescence imaging system (Imaging-PAM M-Series, Maxi version, 126

Waltz GmbH; Fig.1). HIS and GABA treatments were prepared in 100 ml of filtered 127

seawater at a concentration of 10

M and 10

M respectively. Concentrations were 128

chosen according to previous works that have been done with other sea urchins species 129

(Rahman and Uehara, 2001; Swanson et al., 2012). For GABA+MIX treatment, a single 130

MIX treatment was reproduced and 10

M of GABA was added. A negative control of 131

100 ml filtered seawater (FSW treatment) was realised in order to estimate the 132

percentage of larvae undergoing spontaneous metamorphosis.

133

Recruitment assay 134

Twenty-one days after fertilisation, when most larvae were competent, 30 larvae were 135

transferred into each experimental treatments. They were kept in the dark at 20°C 136

(Carbonara et al., 2018). Every 24h the metamorphosis was recorded under a 137

stereoscope for all treatments and assessed as follows:

138

metamorphosis % = metamorphosed juveniles

larvae initially dispensed into the dishes x 100

The first four treatments providing a metamorphosis rate of more than 90% were 139

transferred into little tanks (25 x 15 x 10 cm). Each dish was placed on the bottom of a 140

tank filled with aerated filtered seawater changed every 2 days in a 50%. They were

141

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kept at 20°C under a natural photoperiod in order to assess survival of early juveniles.

142

At 10 days post-metamorphosis (DPM), survival was assessed by counting living 143

juveniles on each treatment under a stereoscope as:

144

post − settlement survival % = living juveniles in the dishes

post − larvae initially dispensed into the dishes x 100 Data analysis

145

Growth curves for the diatoms species were established. The collected data from the cell 146

counting was analysed in MATLAB

R2018a software for fitting to the Gompertz 147

model. This model is known to fit well to diatoms growth kinetic analysis (Lépinay et 148

al., 2018; Zwietering et al., 1990):

149

150

where f(x) is expressed in ln (cell ml

); t is time in days to attain maximum biomass of 151

the culture; µ max stands for maximum specific growth rate per day (ln cell ml

d

); A 152

(ln cell ml

) represents maximum biomass and λ is the lag time in days. For both 153

diatom species, these three parameters were compared using a t-test.

154

For metamorphosis and survival data, statistical analyses were performed using the 155

SigmaStat

9.0 software. Differences were tested using a one-way ANOVA test. When 156

normality test failed, a Kruskal-Wallis one-way analysis of variance on ranks was used.

157

Student-Newman-Keuls a posteriori multiple comparisons tests were carried out when 158

significant differences (P < 0.05) were observed.

159

( ) = exp −exp μ max " #(1) (λ − t) + 1&'

-8- Results

160

For diatom biofilms, A and µ max were significantly higher for N. laevis than H.

161

coffeaeformis (P < 0.01; Table 1). Similar λ were obtained for both species.

162

Growth curves represented by the Gompertz model indicated that both species were in 163

the exponential phase when the experiment was carried out (i.e. 7 days old biofilms).

164

Nevertheless, at this moment of the cultures, biomass was significantly higher in N.

165

laevis biofilms than in H. coffeaeformis (P < 0.001; Fig. 2).

166

After 72h of the experiment, larvae on treatments NL and HC reached more than 91%

167

and 33% of metamorphosis rate respectively (Fig. 3). Both species, on treatment MIX, 168

reached a lower rate (19%). However, when the MIX treatment was associated to the 169

GABA, more than 92% of metamorphosis was observed.

170

The separate treatment of GABA showed a low metamorphosis rate (21%) and no 171

metamorphosed larvae were observed with the HIS treatment. Metamorphosis in the 172

FSW control was less than 1%.

173

With the SHELL and the NATURAL treatment, which were originated from the same 174

environment, metamorphosis reached more than 97%. It is important to note that with 175

the SHELL treatment, larvae did not settled on the shells themselves but on the plates.

176

For easier comparison, we can divide treatments in three groups by their metamorphosis 177

rate results (Fig. 3): group A - NL, NATURAL, GABA+MIX and SHELL, group B - 178

HC, GABA and MIX, and group C - HIS and FSW. Treatments in group A showed 179

significantly higher metamorphosis rates than the treatments in group B and C (P <

180

0.001). Significant differences were also found between treatments in groups B and C 181

(P < 0.05). Between treatments in the same group there was no statistical difference.

182

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Larvae with the treatments of the group A were those transferred to the tanks for 183

survival assessment.

184

Survival 10 DPM was higher than 60% for the four treatments. No difference was found 185

between them (Fig. 4).

186

Discussion 187

In this study we demonstrated that the transition from planktonic larvae to benthic 188

juvenile could be promoted through diatom-based biofilms.

189

The higher metamorphosis rate observed in larvae on N. laevis biofilm compared with 190

larvae on H. coffeaeformis biofilm are in agreement with the results obtained by Xing et 191

al. (2007). This study on Strongylocentrotus intermedius showed that, from a variety of 192

eight species of benthic diatoms, Nitzschia sp. induced the highest metamorphosis rate 193

compare to H. coffeaeformis that induced the lowest. Xing et al. (2007) suggested that 194

the observed differences could be explained by the variability in some characteristics of 195

the biofilms, notably the amount of extracellular polymeric substances (EPS). Nitzschia 196

laevis secrets relatively high amount of EPS that allow it to attach strongly to the 197

substrate and create a robust film. On the opposite, H. coffeaeformis secrets moderate 198

amount of EPS making the adhesive strength of the biofilm poorer. Another 199

characteristic that may vary between diatoms species is their ability to produce toxins 200

and repellent metabolites, sometimes as a protection measurement from grazers 201

(Maibam et al., 2014). Therefore, we can also hypothesize that H. coffeaeformis could 202

produce a repellent substance perturbing the cascade of events that induce settlement 203

and metamorphosis.

204

These substances could also explain the significantly lower metamorphosis rate 205

obtained with both N. laevis and H. coffeaeformis (MIX treatment). As in our

206

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experiment, Xing et al. (2007) obtained higher metamorphosis rates using monospecific 207

diatom biofilms compare to multispecific ones. Further investigation could be done in 208

order to check if H. coffeaeformis produce antifouling compounds or if a significant 209

chemical compound modification appear when co-cultivated with N. laevis (Paul et al., 210

2009).

211

Encrusting algae extracts are known to have an inducing metamorphosis effect on 212

invertebrate larvae. This inducer is an oligopeptide that mimics the action of GABA, an 213

inhibitory neurotransmitter (Morse and Morse, 1984; Rowley, 1989). The 214

metamorphosis effect of GABA has been demonstrated on the sea urchin 215

Strongylocentrotus droebachiensis (Pearce and Scheibling, 1990). In the present study 216

this molecule induced a low metamorphosis rate on P. lividus. However, a different 217

behaviour was observed in the larvae exposed to our GABA treatment: they were all 218

found on the bottom of the plate. In the other treatments the metamorphosed larvae were 219

spread in the water column, which is the normal behaviour in P. lividus, metamorphosis 220

occurring before settlement (Fenaux and Pedrotti, 1988). The behaviour pattern 221

observed with GABA could indicate that this neurotransmitter may function in P.

222

lividus as a separate settlement cue and not as a metamorphosis one. Moreover, as we 223

found similar result in the GABA+MIX and NL treatments, we can hypothesize that 224

GABA could also counteract the negative effect that we observed for H. coffeaeformis 225

on N. laevis.

226

Histamine (HIS treatment) did not induced settlement or metamorphosis in any case.

227

This result agree with the study of Carbonara et al. (2018) that tested five different 228

concentrations of histamine on metamorphosis induction of P. lividus and obtained no 229

metamorphosed larvae.

230

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A similarity was found on the influence of the SHELL treatment and the sediment from 231

the oyster pond (NATURAL treatment) on the larvae. It can be hypothesized that 232

epibionts population on the oyster shell is similar or very close to the one that colonizes 233

the sediment. The metamorphosis rates obtained with these two treatments were similar 234

to the one obtained with NL treatment. This can be explained by the high relative 235

abundance of Nitzschia spp. on oyster shells and mudflats of this region (Barillé et al., 236

2017; Méléder et al., 2007).

237

The four treatments (1) N. laevis biofilms, (2) natural biofilms, (3) oyster shells and (4) 238

a combination of GABA+N. laevis+H. coffeaeformis displayed no statistically different 239

survival rates. They represent metamorphosis inducers of high and similar efficiency for 240

P. lividus larviculture. Biofilms coming from oyster ponds (i.e. natural biofilms and 241

oyster shells) could represent a low cost and sustainable source of metamorphosis 242

inducing cue for oyster farmers. However, these substrates can be a contamination 243

vector and their success could be limited by the spatiotemporal variations in the benthic 244

diatoms communities. To overcome these risks and to promote practical and safe 245

solutions for farmers, this study suggests using preferably N. laevis. Its culture can be 246

conducted by farmers all around the year controlling the quality in terms of growth rates 247

and nutritional profile in an industrial production cycle. This can, on the long term, help 248

oyster farmers to diversify through “echinoculture”.

249

Acknowledgements 250

This study was supported by the Erasmus Mundus Joint Master Degree program ACES 251

(Aquaculture, Environment and Society) and by two European projects: TAPAS “Tools 252

for Assessment and Planning of Aquaculture Sustainability” (Horizon 2020 Grant 253

Agreement No 678396) and BIO-Tide “The role of microbial biodiversity in the

254

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functioning of marine tidal flat sediments” (H 2020 ERA-NET Biodiversia, ANR-16- 255

EBI13-0008-02). The authors wish to thank Benth’Ostrea Prod for providing the living 256

resources. They are also grateful to V. Méléder and E. Cointet for their assistance 257

during diatom-based biofilms culture.

258

References 259

Ab Rahim, S.A.K., Li, J.-Y., Satuito, C.G., Kitamura, H., 2004. The role of diatom- 260

based film as an inducer of metamorphosis in larvae of two species of sea 261

urchin, Pseudocentrotus depressus and Anthocidaris crassispina. Sessile 262

Organisms 21, 7–12.

263

Andrew, N., Agatsuma, Y., Ballesteros, E., Bazhin, A., Creaser, E., K. A. Barnes, D., 264

Botsford, L., Bradbury, A., Campbell, A., Dixon, J., Einarsson, S., Gerring, P., 265

Hebert, K., Hunter, M., Hur, S., Johnson, C., Juinio-Meñez, M., Kalvass, P., 266

Miller, R., Xiaoqi, Z., 2002. Status and management of world sea urchin 267

fisheries. Oceanography and Marine Biology: an Annual Review 40, 343–425.

268

Barillé, L., Le Bris, A., Méléder, V., Launeau, P., Robin, M., Louvrou, I., Ribeiro, L., 269

2017. Photosynthetic epibionts and endobionts of Pacific oyster shells from 270

oyster reefs in rocky versus mudflat shores. PloS one 12, e0185187.

271

Brundu, G., Monleón, L.V., Vallainc, D., Carboni, S., 2016. Effects of larval diet and 272

metamorphosis cue on survival and growth of sea urchin post-larvae 273

(Paracentrotus lividus; Lamarck, 1816). Aquaculture 465, 265–271.

274

Carbonara, S., D’Adamo, R., Novelli, A., Pelosi, S., Fabbrocini, A., 2018. Ground Ulva 275

solution (GUS): A promising metamorphosis cue for Paracentrotus lividus 276

larviculture. Aquaculture 491, 289–294.

277

Castilla-Gavilán, M., Buzin, F., Cognie, B., Dumay, J., Turpin, V., Decottignies, P., 278

2018a. Optimising microalgae diets in sea urchin Paracentrotus lividus 279

larviculture to promote aquaculture diversification. Aquaculture 490, 251–259.

280

Castilla-Gavilán, M., Turpin, V., Buzin, F., Cognie, B., Decottignies, P., 2018b.

281

Optimizing metamorphosis in Paracentrotus lividus aquaculture using 282

alternative macroalgae species to Corallina sp. Aquaculture International 26, 283

1511–1518.

284

Ceccherelli, G., Pais, A., Pinna, S., Sechi, N., Chessa, L.A., 2011. Human impact on 285

Paracentrotus lividus: The result of harvest restrictions and accessibility of 286

locations. Marine Biology 158, 845–852. https://doi.org/10.1007/s00227-010- 287

1611-5 288

Couvray, S., Miard, T., Bunet, R., Martin, Y., Grillasca, J.-P., Bonnefont, J.-L., Coupé, 289

S., 2015. Experimental Release of Juvenile Sea Urchins ( Paracentrotus lividus ) 290

in Exploited Sites along the French Mediterranean Coast. Journal of Shellfish 291

Research 34, 555–563. https://doi.org/10.2983/035.034.0240 292

De la Uz, S., Carrasco, J.F., Rodríguez, C., Anadon, N., 2013. Metamorphosis, growth 293

and survival of early juveniles of Paracentrotus lividus (Echinodermata:

294

-13-

Echinoidea): Effects of larval diet and settlement inducers. Cahiers de Biologie 295

Marine 54, 691–695.

296

Dworjanyn, S.A., Pirozzi, I., 2008. Induction of settlement in the sea urchin Tripneustes 297

gratilla by macroalgae, biofilms and conspecifics: A role for bacteria?

298

Aquaculture 274, 268–274.

299

Eaton, J.W., Moss, B., 1966. The estimation of numbers and pigment content in epipelic 300

algal populations. Limnology and Oceanography 11, 584–595.

301

Fenaux, L., Pedrotti, M.L., 1988. Metamorphose des larves d’échinides en pleine eau.

302

Metamorphosis of echinoid larvae in midwater. Marine Ecology 9, 93–107.

303

https://doi.org/10.1111/j.1439-0485.1988.tb00201.x 304

Giakoumi, S., Cebrian, E., Kokkoris, G.D., Ballesteros, E., Sala, E., 2012. Relationships 305

between fish, sea urchins and macroalgae: The structure of shallow rocky 306

sublittoral communities in the Cyclades, Eastern Mediterranean. Estuarine, 307

coastal and shelf science 109, 1–10.

308

Gosselin, P., Jangoux, M., 1996. Induction of metamorphosis in Paracentrotus lividus 309

larvae (Echinodermata, Echinoidea). Oceanol. Acta 19, 293–296.

310

Grosjean, P., Spirlet, C., Gosselin, P., Vaïtilingon, D., Jangoux, M., 1998. Land-based, 311

closed-cycle echiniculture of Paracentrotus lividus (Lamarck) (Echinoidea:

312

Echinodermata): a long-term experiment at a pilot scale. J. Shellfish Res. 17, 313

1523–1531.

314

Hadfield, M.G., Paul, V.J., 2001. Natural chemical cues for settlement and 315

metamorphosis of marine invertebrate larvae, in: McClintock, J.B., Baker, B.J.

316

(Eds.), Marine Chemical Ecology. CRC Press LLC, pp. 431–461.

317

Hagen, N.T., 1996. Echinoculture: from fishery enhancement to closed cycle 318

cultivation.

319

Hannon, C., Officer, R.A., Chamberlain, J., 2017. Evaluation of the efficacy of algal ‐ 320

conditioned substrates for inducing settlement of Paracentrotus lividus larvae.

321

Aquaculture Research 48, 1968–1973.

322

Kelly, M.S., Hunter, A.J., Scholfield, C.L., McKenzie, J.D., 2000. Morphology and 323

survivorship of larval Psammechinus miliaris (Gmelin) (Echinodermata:

324

Echinoidea) in response to varying food quantity and quality. Aquaculture 183, 325

223–240.

326

Kitching, J.A., Thain, V.M., 1983. The ecological impact of the sea urchin 327

Paracentrotus lividus (Lamarck) in Lough Ine, Ireland. Phil. Trans. R. Soc.

328

Lond. B 300, 513–552.

329

Lépinay, A., Turpin, V., Mondeguer, F., Grandet-Marchant, Q., Capiaux, H., Baron, R., 330

Lebeau, T., 2018. First insight on interactions between bacteria and the marine 331

diatom Haslea ostrearia: Algal growth and metabolomic fingerprinting. Algal 332

research 31, 395–405.

333

Maibam, C., Fink, P., Romano, G., Buia, M.C., Gambi, M.C., Scipione, M.B., Patti, 334

F.P., Lorenti, M., Butera, E., Zupo, V., 2014. Relevance of wound-activated 335

compounds produced by diatoms as toxins and infochemicals for benthic 336

invertebrates. Marine biology 161, 1639–1652.

337

-14-

McBride, S.C., 2005. Sea Urchin Aquaculture   : American Fisheries Society Symposium 338

46, 179–208.

339

Méléder, V., Rincé, Y., Barillé, L., Gaudin, P., Rosa, P., 2007. Spatiotemporal changes 340

in microphytobenthos assemblages in a macrotidal flat (Bourgneuf Bay,France).

341

Journal of Phycology 43, 1177–1190. https://doi.org/10.1111/j.1529- 342

8817.2007.00423.x 343

Morse, A.N.C., Morse, D.E., 1984. Recruitment and metamorphosis of Haliotis larvae 344

induced by molecules uniquely available at the surfaces of crustose red algae.

345

Journal of Experimental Marine Biology and Ecology 75, 191–215.

346

https://doi.org/10.1016/0022-0981(84)90166-7 347

Paul, C., Barofsky, A., Vidoudez, C., Pohnert, G., 2009. Diatom exudates influence 348

metabolism and cell growth of co-cultured diatom species. Marine Ecology 349

Progress Series 389, 61–70. https://doi.org/10.3354/meps08162 350

Pearce, C.M., Scheibling, R.E., 1990. Induction of metamorphosis of larvae of the green 351

sea-urchin, Strongylocentrotus droebachiensis by coralline red algae. Biol. Bull.

352

http://dx.doi.org/10.2307/1542322 353

Privitera, D., Noli, M., Falugi, C., Chiantore, M., 2011. Benthic assemblages and 354

temperature effects on Paracentrotus lividus and Arbacia lixula larvae and 355

settlement. Journal of Experimental Marine Biology and Ecology 407, 6–11.

356

Rahman, M.A., Arshad, A., Yusoff, F.M., 2014. Sea urchins (echinodermata:

357

Echinoidea): their biology, culture and bioactive compounds. Presented at the 358

Proceedings of the International Conference on Agricultural, Ecological and 359

Medical Sciences (AEMS’14), pp. 39–48.

360

Rahman, M.A., Uehara, T., 2001. Induction of metamorphosis and substratum 361

preference in four sympatric and closely related species of sea urchins (Genus 362

Echinometra) in Okinawa. Zoological Studies 40, 29–43.

363

Rial, D., Rial, P., Casal, A., Costoya, N., Costas, D., 2018. Induction of settlement , 364

growth and survival of juveniles of Paracentrotus lividus. Aquaculture 483, 16–

365

20. https://doi.org/10.1016/j.aquaculture.2017.10.005 366

Rowley, R.J., 1989. Settlement and recruitment of sea urchins (Strongylocentrotus spp.) 367

in a sea-urchin barren ground and a kelp bed: are populations regulated by 368

settlement or post-settlement processes? Mar. Biol. 100, 485–494.

369

https://doi.org/10.1007/BF00394825 370

Scheibling, R., 1986. Increased macroalgal abundance following mass mortalities of sea 371

urchins (Strongylocentrotus droebachiensis) along the Atlantic coast of Nova 372

Scotia. Oecologia 68, 186–198.

373

Stefánsson, G., Kristinsson, H., Ziemer, N., Hannon, C., James, P., 2017. Markets for 374

sea urchins: a review of global supply and markets. Matís report, Reykjavik, 375

Iceland.

376

Sutherby, J., Giardini, J.-L., Nguyen, J., Wessel, G., Leguia, M., Heyland, A., 2012.

377

Histamine is a modulator of metamorphic competence in Strongylocentrotus 378

purpuratus (Echinodermata: Echinoidea). BMC developmental biology 12, 14.

379

Swanson, R.L., Byrne, M., Prowse, T.A.A., Mos, B., Dworjanyn, S.A., Steinberg, P.D., 380

2012. Dissolved histamine: a potential habitat marker promoting settlement and 381

metamorphosis in sea urchin larvae. Mar. Biol. 159, 915–925.

382

-15-

Swanson, R.L., Williamson, J.E., de Nys, R., Kumar, N., Bucknall, M.P., Steinberg, 383

P.D., 2004. Induction of Settlement of Larvae of the Sea Urchin Holopneustes 384

purpurascens by Histamine From a Host Alga. Biol Bull 206, 161–172.

385

Takahashi, Y., Itoh, K., Ishii, M., Suzuki, M., Itabashi, Y., 2002. Induction of larval 386

settlement and metamorphosis of the sea urchin Strongylocentrotus intermedius 387

by glycoglycerolipids from the green alga Ulvella lens. Marine Biology 140, 388

763–771.

389

Xing, R., Wang, C., Cao, X., Chang, Y., 2007. The potential value of different species 390

of benthic diatoms as food for newly metamorphosed sea urchin 391

Strongylocentrotus intermedius. Aquaculture 263, 142–149.

392

Zupo, V., Glaviano, F., Caramiello, D., Mutalipassi, M., 2018. Effect of five benthic 393

diatoms on the survival and development of Paracentrotus lividus post-larvae in 394

the laboratory. Aquaculture 495, 13–20.

395

https://doi.org/10.1016/j.aquaculture.2018.05.028 396

Zwietering, M., Jongenburger, I., Rombouts, F., Van’t Riet, K., 1990. Modeling of the 397

bacterial growth curve. Applied and environmental microbiology 56, 1875–

398

1881.

399

400

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402

Fig. 1 Imaging of variable chlorophyll fluorescence on oyster shells. Scale bar 403

represents intensity in false colour. Outlines of the oyster shells are indicated as a white 404

line.

405

Fig. 2 Growth curves of (A) N. laevis and (B) H. coffeaeformis (mean ± sd).

406

Fig. 3 Metamorphosis rate (%) of P. lividus larvae after 72h exposure to the different 407

treatments. NL = N. laevis, NATURAL = natural biofilm, GABA+MIX = GABA+N.

408

laevis+H. coffeaeformis, SHELL = broken oyster shells, HC = H. coffeaeformis, MIX = 409

N. laevis+H. coffeaeformis, HIS = histamine, FSW = filtered seawater. Data are 410

expressed as mean ± confidence interval 95% (n=4).

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Fig. 4 Survival rate (%) of P. lividus 10 days post-metamorphosis (DPM). NL = N.

412

laevis, NATURAL = natural biofilm, SHELL = broken oyster shells, GABA+MIX = 413

GABA+N. laevis+H. coffeaeformis. Data are expressed as mean ± confidence interval 414

95% (n=4).

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Table 1 Maximum biomass (A in ln cell ml^-1), maximum specific growth rate per day (µMax in ln cell ml^-1 d^-1) and

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lag time (λ in days) of the two diatoms based biofilms. Data are expressed as mean ± confidence interval.

418

N. laevis H. coffeaeformis A 1.3 x 10

± 0.3 x 10

0.9 x 10

± 0.09 x 10

µMax 0.71 ± 0.14 0.39 ± 0.14

λ 0.04 ± 0.19 0.4 ± 1.69

419

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Highlights

• Eight different substrates have been tested on Paracentrotus lividus larvae in order to study their metamorphosis and post-settlement survival and to choose those substrates of easy and cheap application for echinoculture

• Organic substrates as Nitzschia laevis biofilm and natural biofilms sampled from oyster ponds are appropriate substrates for recruitment, enhancing metamorphosis and survival rate

• We preferably suggest the use of N. laevis as its culture allow farmers to implement

quality controls

Dear editors,

This work is original and is not under consideration by any other journal. All individuals listed as authors are qualified as author, have approved the submitted version and have permission to reproduce any previously published material.

This study was supported by the Erasmus Mundus Joint Master Degree program ACES (Aquaculture, Environment and Society) and by two European projects: TAPAS “Tools for Assessment and Planning of Aquaculture Sustainability” (Horizon 2020 Grant Agreement No 678396) and BIO-Tide “The role of microbial biodiversity in the functioning of marine tidal flat sediments” (H 2020 ERA-NET Biodiversia, ANR-16-EBI13-0008-02).

The authors declare that they have no conflict of interest.

This article does not contain any studies with human participants performed by any of the

authors. All applicable international, and/or institutional guidelines for the care and use of

animals were followed.