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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: https://doi.org/10.1016/j.aquaculture.2019.734559 Reference: AQUA 734559

To appear in: Aquaculture

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, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734559.

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© 2019 Published by Elsevier B.V. 1 Sea urchin recruitment: effect of diatom based biofilms on Paracentrotus lividus

2 competent larvae

3 Marta Castilla-Gavilán*┼, Meshi Reznicov ┼, Vincent Turpin, Priscilla Decottignies,

4 Bruno Cognie

5 Université de Nantes, Institut Universitaire Mer et Littoral, EA 2160 Mer Molécules 6 Santé, 2 rue de la Houssinière BP 92208, 44322 Nantes cedex 3 (France) 7 ┼ These authors contributed equally 8 *Corresponding author: [email protected]

9 Abstract

10 Eight different experimental substrates were tested on Paracentrotus lividus competent

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

32 promote practical and safe solutions for farmers.

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

34 rate; post-settlement survival rate

35 Introduction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

68 1996; McBride, 2005; Rahman et al., 2014; Takahashi et al., 2002; Xing et al., 2007).

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

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

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

72 Uehara, 2001).

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

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

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

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

77 shells colonized by epiphytic diatoms.

78 Materials and Methods

79 Hatchery of sea urchin larvae

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

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

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

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

84 density of 1 per ml, in 2-m3 conical PVC tanks filled with aerated seawater. A complete

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

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

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

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

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

90 Experimental treatments

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

92 section) were:

93 - Nitzschia laevis biofilm (NL)

94 - Halamphora coffeaeformis biofilm (HC)

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

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

97 - Broken oyster shells 10 g (SHELL)

98 - Histamine 10 -6 M (HIS)

99 - GABA 10 -3 M (GABA)

100 - GABA 10 -3 M + Mix biofilm of both N. laevis and H. coffeaeformis

101 (GABA+MIX)

102 - Control of filtered seawater (FSW)

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

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

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

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

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

108 They were grown in 80 mm diameter Pyrex ® Crystallizing Dishes filled with 100 ml of

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

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

111 m-2 s-1. Growth curves were assessed by daily cell counting using a haemocytometer.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

128 seawater at a concentration of 10 -6 M and 10 -3 M respectively. Concentrations were

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

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

131 MIX treatment was reproduced and 10 -3 M of GABA was added. A negative control of

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

133 percentage of larvae undergoing spontaneous metamorphosis.

134 Recruitment assay

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

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

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

138 stereoscope for all treatments and assessed as follows:

metamorphosed juveniles metamorphosis % = x 100 larvae initially dispensed into the dishes

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

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

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

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

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

144 juveniles on each treatment under a stereoscope as:

living juveniles in the dishes post − settlement survival % = x 100 post − larvae initially dispensed into the dishes

145 Data analysis

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

147 counting was analysed in MATLAB ® R2018a software for fitting to the Gompertz

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

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

μ max (1) () = exp −exp (λ − t) + 1 150

151 where f(x) is expressed in ln (cell ml -1); t is time in days to attain maximum biomass of

152 the culture; µ max stands for maximum specific growth rate per day (ln cell ml -1 d-1); A

153 (ln cell ml -1) represents maximum biomass and λ is the lag time in days. For both

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

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

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

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

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

159 significant differences ( P < 0.05) were observed.

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160 Results

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

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

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

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

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

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

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

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

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

170 GABA, more than 92% of metamorphosis was observed.

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

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

173 FSW control was less than 1%.

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

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

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

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

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

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

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

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

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

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

184 survival assessment.

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

186 between them (Fig. 4).

187 Discussion

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

189 juvenile could be promoted through diatom-based biofilms.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

204 and metamorphosis.

205 These substances could also explain the significantly lower metamorphosis rate

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

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

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

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

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

211 2009).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

226 on N. laevis.

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

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

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

230 metamorphosed larvae.

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

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

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

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

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

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

237 2017; Méléder et al., 2007).

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

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

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

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

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

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

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

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

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

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

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

249 oyster farmers to diversify through “echinoculture”.

250 Acknowledgements

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

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

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

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

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

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

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

258 during diatom-based biofilms culture.

259 References

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401

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402 List of figures

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

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

405 line.

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

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

408 treatments. NL = N. laevi s, NATURAL = natural biofilm, GABA+MIX = GABA+ N.

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

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

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

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

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

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

415 95% (n=4).

416

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417 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 418 lag time ( λ in days) of the two diatoms based biofilms. Data are expressed as mean ± confidence interval.

N. laevis H. coffeaeformis

A 1.3 x 10 6 ± 0.3 x 10 6 0.9 x 10 6 ± 0.09 x 10 6

µMax 0.71 ± 0.14 0.39 ± 0.14

λ 0.04 ± 0.19 0.4 ± 1.69

419

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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 were followed.