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Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus competent larvae
Aquaculture 515 (2020) 734559
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Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus competent larvae
T
Marta Castilla-Gavilán∗,1, Meshi Reznicov1, Vincent Turpin, Priscilla Decottignies, Bruno Cognie Université de Nantes, Institut Universitaire Mer et Littoral, EA 2160 Mer Molécules Santé, 2 rue de la Houssinière BP 92208, 44322, Nantes cedex 3, France
A R T I C LE I N FO
A B S T R A C T
Keywords: Aquaculture diversification Echinoculture Nitzschia laevis Metamorphosis rate Post-settlement survival rate
Eight different experimental substrates were tested on Paracentrotus lividus competent larvae in order to evaluate their potential for inducing metamorphosis and enhance survival after recruitment. Two benthic diatoms species, Nitzschia laevis (NL) and Halamphora coffeaeformis (HC), were selected according to their capacity to adhere and to form strong biofilms. They were tested in monocultures and in a mixed biofilm (MIX) that was also tried in combination with Gamma-Aminobutyric Acid, involved in triggering some invertebrate metamorphosis (MIX + GABA). Histamine (HIS) was also used as a treatment according to the high metamorphosis rates that have been recorded for this compound on other sea urchin species. Finally, a natural microphytobenthic biofilm (NATURAL) and oyster shells particles colonized by epiphytic diatoms (SHELL) were sampled from the mud of a refining oyster pond. Batches of 21 days-old larvae were placed on each experimental substrate and their effect was compared to a negative control of filtered sea water (without any treatment; FSW). Metamorphosis rate was daily recorded in each treatment. The sea urchin larvae on substrates NL, NATURAL, GABA + MIX and SHELL showed significantly higher metamorphosis rates than larvae on the other treatments (P < 0.001), reaching more than 90% in 72 h. Survival rate was assessed at 10 days post-metamorphosis in these four treatments. No difference was observed between them in terms of metamorphosis rate or survival rate (more than 60% for the four experimental substrates). Results demonstrate that the transition from planktonic larvae to benthic juvenile could be promoted through diatom-based biofilms. These substrates represent efficient metamorphosis inducers for P. lividus larviculture but we suggest to use preferably N. laevis biofilms in order to promote practical and safe solutions for farmers.
1. Introduction Sea urchin roes are considered as a delicacy and they are among the most valued sea food products. Sea urchin become highly trendy due to their unique taste and the spread of Japanese food around the globe. The leading country in consumption of sea urchins is Japan followed by France, the first market in Europe (Stefánsson et al., 2017). To fulfil this demand, wild populations have been overexploited worldwide leading to a decline since the 90's (Ceccherelli et al., 2011; Couvray et al., 2015; McBride, 2005). Beyond the economic impact, this depletion has ecological implications as sea urchins have a key role in the infra-littoral rocky shore areas (Giakoumi et al., 2012; Kitching and Thain, 1983; Privitera et al., 2011; Scheibling, 1986). To deal with overexploitation, echinoculture of several species has been developed worldwide (Andrew et al., 2002): for exemple Paracentrotus lividus in Europe, Loxechinus albus in Chile, Strongylocentrotus
spp. in China or S. depressus, S. intermedius and S. nudus in Japan. In Europe, certain constraints remain to be solved for a sustainable and cost-effective industry, notably in the phase of transition between planktonic larvae to a benthic juvenile (Grosjean et al., 1998). Settlement, metamorphosis and post-metamorphosis survival rates are still not high enough to produce juveniles in hatcheries at an industrial-scale (Carbonara et al., 2018; Hannon et al., 2017; Zupo et al., 2018). Researches have been conducted on several sea urchin species to find reliable metamorphosis-inducing-factors for this crucial development stage. Various levels of success have been shown with different species of algae (Carbonara et al., 2018; Castilla-Gavilán et al., 2018b; De la Uz et al., 2013), diatoms and bacterial biofilms (Ab Rahim et al., 2004; Brundu et al., 2016; Dworjanyn and Pirozzi, 2008; Rial et al., 2018; Xing et al., 2007; Zupo et al., 2018), conspecifics (Dworjanyn and Pirozzi, 2008; Gosselin and Jangoux, 1996), and chemical compounds (Carbonara et al., 2018; Pearce and Scheibling, 1990; Swanson et al.,
∗
Corresponding author. E-mail addresses: [email protected], [email protected] (M. Castilla-Gavilán). 1 These authors contributed equally. https://doi.org/10.1016/j.aquaculture.2019.734559 Received 5 July 2019; Received in revised form 13 September 2019; Accepted 1 October 2019 Available online 16 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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2012). It seems that the most successful metamorphosis-inducing signals are microbial biofilms, whether or not they are linked to the surface of seaweed thalli or inorganic surfaces such as rocks or shells (Hadfield and Paul, 2001). Recent studies have shown the metamorphosis induction effect of benthic diatoms on the culture of P. lividus, obtaining high settlement and survival rates (Rial et al., 2018; Zupo et al., 2018). This zootechnics are also largely used in Japan, China and Chile for sea urchin production in plates covered by natural benthic diatoms biofilms (Ab Rahim et al., 2004; Hagen, 1996; McBride, 2005; Rahman et al., 2014; Takahashi et al., 2002; Xing et al., 2007). Moreover, neurotransmitters have been shown to regulate developmental transition in sea urchins, as histamine (Sutherby et al., 2012; Swanson et al., 2012, 2004) and Gamma-Aminobutyric Acid (GABA) (Pearce and Scheibling, 1990; Rahman and Uehara, 2001). The main objective of the present study was to compare the effect of different inducing cues on the metamorphosis of Paracentrotus lividus and to identify those that could be of easy and cheap application. We tested histamine and GABA, two benthic diatoms (Halamphora coffeaeformis and Nitzschia laevis), a natural benthic biofilm and oyster shells colonized by epiphytic diatoms.
Fig. 1. Imaging of variable chlorophyll fluorescence on oyster shells. Scale bar represents intensity in false colour. Outlines of the oyster shells are indicated as a white line. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2. Materials and methods 2.1. Hatchery of sea urchin larvae
All experimental treatments were carried out in four replicates in filtered seawater (5 μm filtered and UV treated). For NL and HC treatments, biofilms were cultured as explained above. For the MIX treatment, biofilms were cultured by inoculating 25 000 cells/ml of each species. Seven days old biofilms were used. One day prior to the recruitment assay, the F/2 media was gently removed and replaced with 100 ml of filtered seawater. For the NATURAL treatment, 3 kg of sediment from an oyster pond of the Benth’Ostrea Prod farm was collected two days prior to the recruitment assay. The natural biofilm was sampled following the protocol described by Eaton and Moss (1966) and homogenized in 400 ml of filtered seawater. Finally, 100 ml were inoculated in four Crystallizing Dishes for 24 h under natural photoperiod at 20 °C. Oyster shells (SHELL treatment) were collected in an oyster pond from the Benth’Ostrea Prod farm. They were broken into small pieces (1.5–5 cm) placed in four Crystallizing Dishes (10 g in each dish) filled with 100 ml filtered seawater. Prior to the recruitment assay, the presence of an active photosynthetic biofilm on the broken shells was checked using a chlorophyll fluorescence imaging system (ImagingPAM M-Series, Maxi version, Waltz GmbH; Fig. 1). HIS and GABA treatments were prepared in 100 ml of filtered seawater at a concentration of 10−6 M and 10−3 M respectively. Concentrations were chosen according to previous works that have been done with other sea urchins species (Rahman and Uehara, 2001; Swanson et al., 2012). For GABA + MIX treatment, a single MIX treatment was reproduced and 10−3 M of GABA was added. A negative control of 100 ml filtered seawater (FSW treatment) was realised in order to estimate the percentage of larvae undergoing spontaneous metamorphosis.
Larvae of P. lividus were raised in the Benth’Ostrea Prod aquaculture farm (Bouin, Vendée, France). They were fed on a combined diet consisting of three microalgae species: Isochrysis aff. galbana (clone T-ISO), Rhodomonas sp. and Dunaliella tertiolecta (Castilla-Gavilán et al., 2018a). Larvae were reared in continuous dark at a density of 1 per ml, in 2-m3 conical PVC tanks filled with aerated seawater. A complete water exchange and a thorough cleaning of the tanks were carried out every day. Prior to the experiment, pre-competent larvae were transferred to the laboratory and kept at the same density in an aerated 5 L glass reactor balloon, until competence was achieved. Competence was considered when 75% of larvae had a developed rudiment that was equal or larger than the stomach, as proposed by Kelly et al. (2000). 2.2. Experimental treatments The chosen treatments for P. lividus competent larvae recruitment assay (see next section) were: -
Nitzschia laevis biofilm (NL) Halamphora coffeaeformis biofilm (HC) Mix biofilm of both N. laevis and H. coffeaeformis (MIX) Natural biofilm sampled from refining oyster ponds (NATURAL) Broken oyster shells 10 g (SHELL) Histamine 10−6 M (HIS) GABA 10-3 M (GABA) GABA 10−3 M + Mix biofilm of both N. laevis and H. coffeaeformis (GABA + MIX) - Control of filtered seawater (FSW)
2.3. Recruitment assay Twenty-one days after fertilisation, when most larvae were competent, 30 larvae were transferred into each experimental treatments. They were kept in the dark at 20 °C (Carbonara et al., 2018). Every 24 h the metamorphosis was recorded under a stereoscope for all treatments and assessed as follows:
The diatoms N. laevis and H. coffeaeformis were obtained from the Nantes Culture Collection (UTCC58 and NCC39 from the MerMolécules-Santé Laboratory, Nantes, France). These species were chosen for their ability to adhere and to form strong biofilms. Prior to experimental assays, growth kinetics of the two diatoms species selected were assessed (lag time, maximal biomass and maximal specific growth rate). They were grown in 80 mm diameter Pyrex® Crystallizing Dishes filled with 100 ml of F/2 media. Each dish was inoculated with 50 000 cells/ml. The diatom biofilms were grown in triplicates under conditions of 15 °C, 14 h:10 h L/D cycle at 120 μmol photons m−2 s−1. Growth curves were assessed by daily cell counting using a haemocytometer.
metamorphosis % =
metamorphosed juveniles x 100 larvae initially dispensed into the dishes
The first four treatments providing a metamorphosis rate of more than 90% were transferred into little tanks (25 × 15 × 10 cm). Each dish was placed on the bottom of a tank filled with aerated filtered seawater changed every 2 days in a 50%. They were kept at 20 °C under 2
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a natural photoperiod in order to assess survival of early juveniles. At 10 days post-metamorphosis (DPM), survival was assessed by counting living juveniles on each treatment under a stereoscope as:
post − settlement survival % living juveniles in the dishes = x 100 post − larvae initially dispensed into the dishes
2.4. Data analysis Growth curves for the diatoms species were established. The collected data from the cell counting was analysed in MATLAB® R2018a software for fitting to the Gompertz model. This model is known to fit well to diatoms growth kinetic analysis (Lépinay et al., 2018; Zwietering et al., 1990):
f (x ) = A exp ⎛−exp ⎛ ⎝ ⎝ ⎜
μ max exp (1) (λ− t) + 1⎞ ⎞ A ⎠⎠ ⎟
where f(x) is expressed in ln (cell ml−1); t is time in days to attain maximum biomass of the culture; μ max stands for maximum specific growth rate per day (ln cell ml−1 d−1); A (ln cell ml−1) represents maximum biomass and λ is the lag time in days. For both diatom species, these three parameters were compared using a t-test. For metamorphosis and survival data, statistical analyses were performed using the SigmaStat® 9.0 software. Differences were tested using a one-way ANOVA test. When normality test failed, a KruskalWallis one-way analysis of variance on ranks was used. StudentNewman-Keuls a posteriori multiple comparisons tests were carried out when significant differences (P < 0.05) were observed.
Fig. 2. Growth curves of (A) N. laevis and (B) H. coffeaeformis (mean ± sd).
3. Results For diatom biofilms, A and μ max were significantly higher for N. laevis than H. coffeaeformis (P < 0.01; Table 1). Similar λ were obtained for both species. Growth curves represented by the Gompertz model indicated that both species were in the exponential phase when the experiment was carried out (i.e. 7 days old biofilms). Nevertheless, at this moment of the cultures, biomass was significantly higher in N. laevis biofilms than in H. coffeaeformis (P < 0.001; Fig. 2). After 72 h of the experiment, larvae on treatments NL and HC reached more than 91% and 33% of metamorphosis rate respectively (Fig. 3). Both species, on treatment MIX, reached a lower rate (19%). However, when the MIX treatment was associated to the GABA, more than 92% of metamorphosis was observed. The separate treatment of GABA showed a low metamorphosis rate (21%) and no metamorphosed larvae were observed with the HIS treatment. Metamorphosis in the FSW control was less than 1%. With the SHELL and the NATURAL treatment, which were originated from the same environment, metamorphosis reached more than 97%. It is important to note that with the SHELL treatment, larvae did not settled on the shells themselves but on the plates. For easier comparison, we can divide treatments in three groups by their metamorphosis rate results (Fig. 3): group A - NL, NATURAL, GABA + MIX and SHELL, group B - HC, GABA and MIX, and group C -
Fig. 3. Metamorphosis rate (%) of P. lividus larvae after 72 h exposure to the different treatments. NL = N. laevis, NATURAL = natural biofilm, GABA + MIX = GABA + N. laevis + H. coffeaeformis, SHELL = broken oyster shells, HC = H. coffeaeformis, MIX = N. laevis + H. coffeaeformis, HIS = histamine, FSW = filtered seawater. Data are expressed as mean ± confidence interval 95% (n = 4).
HIS and FSW. Treatments in group A showed significantly higher metamorphosis rates than the treatments in group B and C (P < 0.001). Significant differences were also found between treatments in groups B and C (P < 0.05). Between treatments in the same group there was no statistical difference. Larvae with the treatments of the group A were those transferred to the tanks for survival assessment. Survival 10 DPM was higher than 60% for the four treatments. No difference was found between them (Fig. 4).
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 lag time (λ in days) of the two diatoms based biofilms. Data are expressed as mean ± confidence interval.
A μMax λ
N. laevis
H. coffeaeformis
1.3 × 106 ± 0.3 × 106 0.71 ± 0.14 0.04 ± 0.19
0.9 × 106 ± 0.09 × 106 0.39 ± 0.14 0.4 ± 1.69
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also counteract the negative effect that we observed for H. coffeaeformis on N. laevis. Histamine (HIS treatment) did not induced settlement or metamorphosis in any case. This result agree with the study of Carbonara et al. (2018) that tested five different concentrations of histamine on metamorphosis induction of P. lividus and obtained no metamorphosed larvae. A similarity was found on the influence of the SHELL treatment and the sediment from the oyster pond (NATURAL treatment) on the larvae. It can be hypothesized that epibionts population on the oyster shell is similar or very close to the one that colonizes the sediment. The metamorphosis rates obtained with these two treatments were similar to the one obtained with NL treatment. This can be explained by the high relative abundance of Nitzschia spp. on oyster shells and mudflats of this region (Barillé et al., 2017; Méléder et al., 2007). The four treatments (1) N. laevis biofilms, (2) natural biofilms, (3) oyster shells and (4) a combination of GABA + N. laevis + H. coffeaeformis displayed no statistically different survival rates. They represent metamorphosis inducers of high and similar efficiency for P. lividus larviculture. Biofilms coming from oyster ponds (i.e. natural biofilms and oyster shells) could represent a low cost and sustainable source of metamorphosis inducing cue for oyster farmers. However, these substrates can be a contamination vector and their success could be limited by the spatiotemporal variations in the benthic diatoms communities. To overcome these risks and to promote practical and safe solutions for farmers, this study suggests using preferably N. laevis. Its culture can be conducted by farmers all around the year controlling the quality in terms of growth rates and nutritional profile in an industrial production cycle. This can, on the long term, help oyster farmers to diversify through “echinoculture”.
Fig. 4. Survival rate (%) of P. lividus 10 days post-metamorphosis (DPM). NL = N. laevis, NATURAL = natural biofilm, SHELL = broken oyster shells, GABA + MIX = GABA + N. laevis + H. coffeaeformis. Data are expressed as mean ± confidence interval 95% (n = 4).
4. Discussion In this study we demonstrated that the transition from planktonic larvae to benthic juvenile could be promoted through diatom-based biofilms. The higher metamorphosis rate observed in larvae on N. laevis biofilm compared with larvae on H. coffeaeformis biofilm are in agreement with the results obtained by Xing et al. (2007). This study on Strongylocentrotus intermedius showed that, from a variety of eight species of benthic diatoms, Nitzschia sp. induced the highest metamorphosis rate compare to H. coffeaeformis that induced the lowest. Xing et al. (2007) suggested that the observed differences could be explained by the variability in some characteristics of the biofilms, notably the amount of extracellular polymeric substances (EPS). Nitzschia laevis secrets relatively high amount of EPS that allow it to attach strongly to the substrate and create a robust film. On the opposite, H. coffeaeformis secrets moderate amount of EPS making the adhesive strength of the biofilm poorer. Another characteristic that may vary between diatoms species is their ability to produce toxins and repellent metabolites, sometimes as a protection measurement from grazers (Maibam et al., 2014). Therefore, we can also hypothesize that H. coffeaeformis could produce a repellent substance perturbing the cascade of events that induce settlement and metamorphosis. These substances could also explain the significantly lower metamorphosis rate obtained with both N. laevis and H. coffeaeformis (MIX treatment). As in our experiment, Xing et al. (2007) obtained higher metamorphosis rates using monospecific diatom biofilms compare to multispecific ones. Further investigation could be done in order to check if H. coffeaeformis produce antifouling compounds or if a significant chemical compound modification appear when co-cultivated with N. laevis (Paul et al., 2009). Encrusting algae extracts are known to have an inducing metamorphosis effect on invertebrate larvae. This inducer is an oligopeptide that mimics the action of GABA, an inhibitory neurotransmitter (Morse and Morse, 1984; Rowley, 1989). The metamorphosis effect of GABA has been demonstrated on the sea urchin Strongylocentrotus droebachiensis (Pearce and Scheibling, 1990). In the present study this molecule induced a low metamorphosis rate on P. lividus. However, a different behaviour was observed in the larvae exposed to our GABA treatment: they were all found on the bottom of the plate. In the other treatments the metamorphosed larvae were spread in the water column, which is the normal behaviour in P. lividus, metamorphosis occurring before settlement (Fenaux and Pedrotti, 1988). The behaviour pattern observed with GABA could indicate that this neurotransmitter may function in P. lividus as a separate settlement cue and not as a metamorphosis one. Moreover, as we found similar result in the GABA + MIX and NL treatments, we can hypothesize that GABA could
Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements 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 COFUND Biodiversa, ANR-16-EBI13-0008-02). The authors wish to thank Benth’Ostrea Prod for providing the living resources. They are also grateful to V. Méléder and E. Cointet for their assistance during diatombased biofilms culture. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734559. References Ab Rahim, S.A.K., Li, J.-Y., Satuito, C.G., Kitamura, H., 2004. The role of diatom-based film as an inducer of metamorphosis in larvae of two species of sea urchin, Pseudocentrotus depressus and Anthocidaris crassispina. Sessile Org. 21, 7–12. Andrew, N., Agatsuma, Y., Ballesteros, E., Bazhin, A., Creaser, E.,K.A., Barnes, D., Botsford, L., Bradbury, A., Campbell, A., Dixon, J., Einarsson, S., Gerring, P., Hebert, K., Hunter, M., Hur, S., Johnson, C., Juinio-Meñez, M., Kalvass, P., Miller, R., Xiaoqi, Z., 2002. Status and management of world sea urchin fisheries. Oceanogr. Mar. Biol. Annu. Rev. 40, 343–425. Barillé, L., Le Bris, A., Méléder, V., Launeau, P., Robin, M., Louvrou, I., Ribeiro, L., 2017. Photosynthetic epibionts and endobionts of Pacific oyster shells from oyster reefs in rocky versus mudflat shores. PLoS One 12, e0185187. Brundu, G., Monleón, L.V., Vallainc, D., Carboni, S., 2016. Effects of larval diet and metamorphosis cue on survival and growth of sea urchin post-larvae (Paracentrotus lividus; Lamarck, 1816). Aquaculture 465, 265–271.
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Carbonara, S., D'Adamo, R., Novelli, A., Pelosi, S., Fabbrocini, A., 2018. Ground Ulva solution (GUS): a promising metamorphosis cue for Paracentrotus lividus larviculture. Aquaculture 491, 289–294. Castilla-Gavilán, M., Buzin, F., Cognie, B., Dumay, J., Turpin, V., Decottignies, P., 2018a. Optimising microalgae diets in sea urchin Paracentrotus lividus larviculture to promote aquaculture diversification. Aquaculture 490, 251–259. Castilla-Gavilán, M., Turpin, V., Buzin, F., Cognie, B., Decottignies, P., 2018b. Optimizing metamorphosis in Paracentrotus lividus aquaculture using alternative macroalgae species to Corallina sp. Aquacult. Int. 26, 1511–1518. Ceccherelli, G., Pais, A., Pinna, S., Sechi, N., Chessa, L.A., 2011. Human impact on Paracentrotus lividus: the result of harvest restrictions and accessibility of locations. Mar. Biol. 158, 845–852. https://doi.org/10.1007/s00227-010-1611-5. Couvray, S., Miard, T., Bunet, R., Martin, Y., Grillasca, J.-P., Bonnefont, J.-L., Coupé, S., 2015. Experimental release of juvenile sea urchins ( Paracentrotus lividus ) in exploited sites along the French mediterranean coast. J. Shellfish Res. 34, 555–563. https://doi.org/10.2983/035.034.0240. De la Uz, S., Carrasco, J.F., Rodríguez, C., Anadon, N., 2013. Metamorphosis, growth and survival of early juveniles of Paracentrotus lividus (Echinodermata: echinoidea): effects of larval diet and settlement inducers. Cah. Biol. Mar. 54, 691–695. Dworjanyn, S.A., Pirozzi, I., 2008. Induction of settlement in the sea urchin Tripneustes gratilla by macroalgae, biofilms and conspecifics: a role for bacteria? Aquaculture 274, 268–274. Eaton, J.W., Moss, B., 1966. The estimation of numbers and pigment content in epipelic algal populations. Limnol. Oceanogr. 11, 584–595. Fenaux, L., Pedrotti, M.L., 1988. Metamorphose des larves d’échinides en pleine eau. Metamorphosis of echinoid larvae in midwater. Mar. Ecol. 9, 93–107. https://doi. org/10.1111/j.1439-0485.1988.tb00201.x. Giakoumi, S., Cebrian, E., Kokkoris, G.D., Ballesteros, E., Sala, E., 2012. Relationships between fish, sea urchins and macroalgae: the structure of shallow rocky sublittoral communities in the Cyclades, Eastern Mediterranean. Estuarine. Coast Shelf Sci. 109, 1–10. Gosselin, P., Jangoux, M., 1996. Induction of metamorphosis in Paracentrotus lividus larvae (echinodermata, echinoidea). Oceanol. Acta 19, 293–296. Grosjean, P., Spirlet, C., Gosselin, P., Vaïtilingon, D., Jangoux, M., 1998. Land-based, closed-cycle echiniculture of Paracentrotus lividus (Lamarck) (Echinoidea: echinodermata): a long-term experiment at a pilot scale. J. Shellfish Res. 17, 1523–1531. Hadfield, M.G., Paul, V.J., 2001. Natural chemical cues for settlement and metamorphosis of marine invertebrate larvae. In: McClintock, J.B., Baker, B.J. (Eds.), Marine Chemical Ecology. CRC Press LLC, pp. 431–461. Hagen, N.T., 1996. Echinoculture: from Fishery Enhancement to Closed Cycle Cultivation. Hannon, C., Officer, R.A., Chamberlain, J., 2017. Evaluation of the efficacy of algal‐conditioned substrates for inducing settlement of Paracentrotus lividus larvae. Aquacult. Res. 48, 1968–1973. Kelly, M.S., Hunter, A.J., Scholfield, C.L., McKenzie, J.D., 2000. Morphology and survivorship of larval Psammechinus miliaris (Gmelin) (Echinodermata: echinoidea) in response to varying food quantity and quality. Aquaculture 183, 223–240. Kitching, J.A., Thain, V.M., 1983. The ecological impact of the sea urchin Paracentrotus lividus (Lamarck) in Lough Ine, Ireland. Philos. Trans. R. Soc. Lond. B 300, 513–552. Lépinay, A., Turpin, V., Mondeguer, F., Grandet-Marchant, Q., Capiaux, H., Baron, R., Lebeau, T., 2018. First insight on interactions between bacteria and the marine diatom Haslea ostrearia: algal growth and metabolomic fingerprinting. Algal Res. 31, 395–405. Maibam, C., Fink, P., Romano, G., Buia, M.C., Gambi, M.C., Scipione, M.B., Patti, F.P., Lorenti, M., Butera, E., Zupo, V., 2014. Relevance of wound-activated compounds produced by diatoms as toxins and infochemicals for benthic invertebrates. Mar. Biol. 161, 1639–1652. McBride, S.C., 2005. sea urchin Aquaculture . Am. Fish. Soc. Symp. 46, 179–208.
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Sea urchin recruitment: Effect of diatom based biofilms on Paracentrotus lividus competent larvae
T
Marta Castilla-Gavilán∗,1, Meshi Reznicov1, Vincent Turpin, Priscilla Decottignies, Bruno Cognie Université de Nantes, Institut Universitaire Mer et Littoral, EA 2160 Mer Molécules Santé, 2 rue de la Houssinière BP 92208, 44322, Nantes cedex 3, France
A R T I C LE I N FO
A B S T R A C T
Keywords: Aquaculture diversification Echinoculture Nitzschia laevis Metamorphosis rate Post-settlement survival rate
Eight different experimental substrates were tested on Paracentrotus lividus competent larvae in order to evaluate their potential for inducing metamorphosis and enhance survival after recruitment. Two benthic diatoms species, Nitzschia laevis (NL) and Halamphora coffeaeformis (HC), were selected according to their capacity to adhere and to form strong biofilms. They were tested in monocultures and in a mixed biofilm (MIX) that was also tried in combination with Gamma-Aminobutyric Acid, involved in triggering some invertebrate metamorphosis (MIX + GABA). Histamine (HIS) was also used as a treatment according to the high metamorphosis rates that have been recorded for this compound on other sea urchin species. Finally, a natural microphytobenthic biofilm (NATURAL) and oyster shells particles colonized by epiphytic diatoms (SHELL) were sampled from the mud of a refining oyster pond. Batches of 21 days-old larvae were placed on each experimental substrate and their effect was compared to a negative control of filtered sea water (without any treatment; FSW). Metamorphosis rate was daily recorded in each treatment. The sea urchin larvae on substrates NL, NATURAL, GABA + MIX and SHELL showed significantly higher metamorphosis rates than larvae on the other treatments (P < 0.001), reaching more than 90% in 72 h. Survival rate was assessed at 10 days post-metamorphosis in these four treatments. No difference was observed between them in terms of metamorphosis rate or survival rate (more than 60% for the four experimental substrates). Results demonstrate that the transition from planktonic larvae to benthic juvenile could be promoted through diatom-based biofilms. These substrates represent efficient metamorphosis inducers for P. lividus larviculture but we suggest to use preferably N. laevis biofilms in order to promote practical and safe solutions for farmers.
1. Introduction Sea urchin roes are considered as a delicacy and they are among the most valued sea food products. Sea urchin become highly trendy due to their unique taste and the spread of Japanese food around the globe. The leading country in consumption of sea urchins is Japan followed by France, the first market in Europe (Stefánsson et al., 2017). To fulfil this demand, wild populations have been overexploited worldwide leading to a decline since the 90's (Ceccherelli et al., 2011; Couvray et al., 2015; McBride, 2005). Beyond the economic impact, this depletion has ecological implications as sea urchins have a key role in the infra-littoral rocky shore areas (Giakoumi et al., 2012; Kitching and Thain, 1983; Privitera et al., 2011; Scheibling, 1986). To deal with overexploitation, echinoculture of several species has been developed worldwide (Andrew et al., 2002): for exemple Paracentrotus lividus in Europe, Loxechinus albus in Chile, Strongylocentrotus
spp. in China or S. depressus, S. intermedius and S. nudus in Japan. In Europe, certain constraints remain to be solved for a sustainable and cost-effective industry, notably in the phase of transition between planktonic larvae to a benthic juvenile (Grosjean et al., 1998). Settlement, metamorphosis and post-metamorphosis survival rates are still not high enough to produce juveniles in hatcheries at an industrial-scale (Carbonara et al., 2018; Hannon et al., 2017; Zupo et al., 2018). Researches have been conducted on several sea urchin species to find reliable metamorphosis-inducing-factors for this crucial development stage. Various levels of success have been shown with different species of algae (Carbonara et al., 2018; Castilla-Gavilán et al., 2018b; De la Uz et al., 2013), diatoms and bacterial biofilms (Ab Rahim et al., 2004; Brundu et al., 2016; Dworjanyn and Pirozzi, 2008; Rial et al., 2018; Xing et al., 2007; Zupo et al., 2018), conspecifics (Dworjanyn and Pirozzi, 2008; Gosselin and Jangoux, 1996), and chemical compounds (Carbonara et al., 2018; Pearce and Scheibling, 1990; Swanson et al.,
∗
Corresponding author. E-mail addresses: [email protected], [email protected] (M. Castilla-Gavilán). 1 These authors contributed equally. https://doi.org/10.1016/j.aquaculture.2019.734559 Received 5 July 2019; Received in revised form 13 September 2019; Accepted 1 October 2019 Available online 16 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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2012). It seems that the most successful metamorphosis-inducing signals are microbial biofilms, whether or not they are linked to the surface of seaweed thalli or inorganic surfaces such as rocks or shells (Hadfield and Paul, 2001). Recent studies have shown the metamorphosis induction effect of benthic diatoms on the culture of P. lividus, obtaining high settlement and survival rates (Rial et al., 2018; Zupo et al., 2018). This zootechnics are also largely used in Japan, China and Chile for sea urchin production in plates covered by natural benthic diatoms biofilms (Ab Rahim et al., 2004; Hagen, 1996; McBride, 2005; Rahman et al., 2014; Takahashi et al., 2002; Xing et al., 2007). Moreover, neurotransmitters have been shown to regulate developmental transition in sea urchins, as histamine (Sutherby et al., 2012; Swanson et al., 2012, 2004) and Gamma-Aminobutyric Acid (GABA) (Pearce and Scheibling, 1990; Rahman and Uehara, 2001). The main objective of the present study was to compare the effect of different inducing cues on the metamorphosis of Paracentrotus lividus and to identify those that could be of easy and cheap application. We tested histamine and GABA, two benthic diatoms (Halamphora coffeaeformis and Nitzschia laevis), a natural benthic biofilm and oyster shells colonized by epiphytic diatoms.
Fig. 1. Imaging of variable chlorophyll fluorescence on oyster shells. Scale bar represents intensity in false colour. Outlines of the oyster shells are indicated as a white line. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2. Materials and methods 2.1. Hatchery of sea urchin larvae
All experimental treatments were carried out in four replicates in filtered seawater (5 μm filtered and UV treated). For NL and HC treatments, biofilms were cultured as explained above. For the MIX treatment, biofilms were cultured by inoculating 25 000 cells/ml of each species. Seven days old biofilms were used. One day prior to the recruitment assay, the F/2 media was gently removed and replaced with 100 ml of filtered seawater. For the NATURAL treatment, 3 kg of sediment from an oyster pond of the Benth’Ostrea Prod farm was collected two days prior to the recruitment assay. The natural biofilm was sampled following the protocol described by Eaton and Moss (1966) and homogenized in 400 ml of filtered seawater. Finally, 100 ml were inoculated in four Crystallizing Dishes for 24 h under natural photoperiod at 20 °C. Oyster shells (SHELL treatment) were collected in an oyster pond from the Benth’Ostrea Prod farm. They were broken into small pieces (1.5–5 cm) placed in four Crystallizing Dishes (10 g in each dish) filled with 100 ml filtered seawater. Prior to the recruitment assay, the presence of an active photosynthetic biofilm on the broken shells was checked using a chlorophyll fluorescence imaging system (ImagingPAM M-Series, Maxi version, Waltz GmbH; Fig. 1). HIS and GABA treatments were prepared in 100 ml of filtered seawater at a concentration of 10−6 M and 10−3 M respectively. Concentrations were chosen according to previous works that have been done with other sea urchins species (Rahman and Uehara, 2001; Swanson et al., 2012). For GABA + MIX treatment, a single MIX treatment was reproduced and 10−3 M of GABA was added. A negative control of 100 ml filtered seawater (FSW treatment) was realised in order to estimate the percentage of larvae undergoing spontaneous metamorphosis.
Larvae of P. lividus were raised in the Benth’Ostrea Prod aquaculture farm (Bouin, Vendée, France). They were fed on a combined diet consisting of three microalgae species: Isochrysis aff. galbana (clone T-ISO), Rhodomonas sp. and Dunaliella tertiolecta (Castilla-Gavilán et al., 2018a). Larvae were reared in continuous dark at a density of 1 per ml, in 2-m3 conical PVC tanks filled with aerated seawater. A complete water exchange and a thorough cleaning of the tanks were carried out every day. Prior to the experiment, pre-competent larvae were transferred to the laboratory and kept at the same density in an aerated 5 L glass reactor balloon, until competence was achieved. Competence was considered when 75% of larvae had a developed rudiment that was equal or larger than the stomach, as proposed by Kelly et al. (2000). 2.2. Experimental treatments The chosen treatments for P. lividus competent larvae recruitment assay (see next section) were: -
Nitzschia laevis biofilm (NL) Halamphora coffeaeformis biofilm (HC) Mix biofilm of both N. laevis and H. coffeaeformis (MIX) Natural biofilm sampled from refining oyster ponds (NATURAL) Broken oyster shells 10 g (SHELL) Histamine 10−6 M (HIS) GABA 10-3 M (GABA) GABA 10−3 M + Mix biofilm of both N. laevis and H. coffeaeformis (GABA + MIX) - Control of filtered seawater (FSW)
2.3. Recruitment assay Twenty-one days after fertilisation, when most larvae were competent, 30 larvae were transferred into each experimental treatments. They were kept in the dark at 20 °C (Carbonara et al., 2018). Every 24 h the metamorphosis was recorded under a stereoscope for all treatments and assessed as follows:
The diatoms N. laevis and H. coffeaeformis were obtained from the Nantes Culture Collection (UTCC58 and NCC39 from the MerMolécules-Santé Laboratory, Nantes, France). These species were chosen for their ability to adhere and to form strong biofilms. Prior to experimental assays, growth kinetics of the two diatoms species selected were assessed (lag time, maximal biomass and maximal specific growth rate). They were grown in 80 mm diameter Pyrex® Crystallizing Dishes filled with 100 ml of F/2 media. Each dish was inoculated with 50 000 cells/ml. The diatom biofilms were grown in triplicates under conditions of 15 °C, 14 h:10 h L/D cycle at 120 μmol photons m−2 s−1. Growth curves were assessed by daily cell counting using a haemocytometer.
metamorphosis % =
metamorphosed juveniles x 100 larvae initially dispensed into the dishes
The first four treatments providing a metamorphosis rate of more than 90% were transferred into little tanks (25 × 15 × 10 cm). Each dish was placed on the bottom of a tank filled with aerated filtered seawater changed every 2 days in a 50%. They were kept at 20 °C under 2
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a natural photoperiod in order to assess survival of early juveniles. At 10 days post-metamorphosis (DPM), survival was assessed by counting living juveniles on each treatment under a stereoscope as:
post − settlement survival % living juveniles in the dishes = x 100 post − larvae initially dispensed into the dishes
2.4. Data analysis Growth curves for the diatoms species were established. The collected data from the cell counting was analysed in MATLAB® R2018a software for fitting to the Gompertz model. This model is known to fit well to diatoms growth kinetic analysis (Lépinay et al., 2018; Zwietering et al., 1990):
f (x ) = A exp ⎛−exp ⎛ ⎝ ⎝ ⎜
μ max exp (1) (λ− t) + 1⎞ ⎞ A ⎠⎠ ⎟
where f(x) is expressed in ln (cell ml−1); t is time in days to attain maximum biomass of the culture; μ max stands for maximum specific growth rate per day (ln cell ml−1 d−1); A (ln cell ml−1) represents maximum biomass and λ is the lag time in days. For both diatom species, these three parameters were compared using a t-test. For metamorphosis and survival data, statistical analyses were performed using the SigmaStat® 9.0 software. Differences were tested using a one-way ANOVA test. When normality test failed, a KruskalWallis one-way analysis of variance on ranks was used. StudentNewman-Keuls a posteriori multiple comparisons tests were carried out when significant differences (P < 0.05) were observed.
Fig. 2. Growth curves of (A) N. laevis and (B) H. coffeaeformis (mean ± sd).
3. Results For diatom biofilms, A and μ max were significantly higher for N. laevis than H. coffeaeformis (P < 0.01; Table 1). Similar λ were obtained for both species. Growth curves represented by the Gompertz model indicated that both species were in the exponential phase when the experiment was carried out (i.e. 7 days old biofilms). Nevertheless, at this moment of the cultures, biomass was significantly higher in N. laevis biofilms than in H. coffeaeformis (P < 0.001; Fig. 2). After 72 h of the experiment, larvae on treatments NL and HC reached more than 91% and 33% of metamorphosis rate respectively (Fig. 3). Both species, on treatment MIX, reached a lower rate (19%). However, when the MIX treatment was associated to the GABA, more than 92% of metamorphosis was observed. The separate treatment of GABA showed a low metamorphosis rate (21%) and no metamorphosed larvae were observed with the HIS treatment. Metamorphosis in the FSW control was less than 1%. With the SHELL and the NATURAL treatment, which were originated from the same environment, metamorphosis reached more than 97%. It is important to note that with the SHELL treatment, larvae did not settled on the shells themselves but on the plates. For easier comparison, we can divide treatments in three groups by their metamorphosis rate results (Fig. 3): group A - NL, NATURAL, GABA + MIX and SHELL, group B - HC, GABA and MIX, and group C -
Fig. 3. Metamorphosis rate (%) of P. lividus larvae after 72 h exposure to the different treatments. NL = N. laevis, NATURAL = natural biofilm, GABA + MIX = GABA + N. laevis + H. coffeaeformis, SHELL = broken oyster shells, HC = H. coffeaeformis, MIX = N. laevis + H. coffeaeformis, HIS = histamine, FSW = filtered seawater. Data are expressed as mean ± confidence interval 95% (n = 4).
HIS and FSW. Treatments in group A showed significantly higher metamorphosis rates than the treatments in group B and C (P < 0.001). Significant differences were also found between treatments in groups B and C (P < 0.05). Between treatments in the same group there was no statistical difference. Larvae with the treatments of the group A were those transferred to the tanks for survival assessment. Survival 10 DPM was higher than 60% for the four treatments. No difference was found between them (Fig. 4).
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 lag time (λ in days) of the two diatoms based biofilms. Data are expressed as mean ± confidence interval.
A μMax λ
N. laevis
H. coffeaeformis
1.3 × 106 ± 0.3 × 106 0.71 ± 0.14 0.04 ± 0.19
0.9 × 106 ± 0.09 × 106 0.39 ± 0.14 0.4 ± 1.69
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also counteract the negative effect that we observed for H. coffeaeformis on N. laevis. Histamine (HIS treatment) did not induced settlement or metamorphosis in any case. This result agree with the study of Carbonara et al. (2018) that tested five different concentrations of histamine on metamorphosis induction of P. lividus and obtained no metamorphosed larvae. A similarity was found on the influence of the SHELL treatment and the sediment from the oyster pond (NATURAL treatment) on the larvae. It can be hypothesized that epibionts population on the oyster shell is similar or very close to the one that colonizes the sediment. The metamorphosis rates obtained with these two treatments were similar to the one obtained with NL treatment. This can be explained by the high relative abundance of Nitzschia spp. on oyster shells and mudflats of this region (Barillé et al., 2017; Méléder et al., 2007). The four treatments (1) N. laevis biofilms, (2) natural biofilms, (3) oyster shells and (4) a combination of GABA + N. laevis + H. coffeaeformis displayed no statistically different survival rates. They represent metamorphosis inducers of high and similar efficiency for P. lividus larviculture. Biofilms coming from oyster ponds (i.e. natural biofilms and oyster shells) could represent a low cost and sustainable source of metamorphosis inducing cue for oyster farmers. However, these substrates can be a contamination vector and their success could be limited by the spatiotemporal variations in the benthic diatoms communities. To overcome these risks and to promote practical and safe solutions for farmers, this study suggests using preferably N. laevis. Its culture can be conducted by farmers all around the year controlling the quality in terms of growth rates and nutritional profile in an industrial production cycle. This can, on the long term, help oyster farmers to diversify through “echinoculture”.
Fig. 4. Survival rate (%) of P. lividus 10 days post-metamorphosis (DPM). NL = N. laevis, NATURAL = natural biofilm, SHELL = broken oyster shells, GABA + MIX = GABA + N. laevis + H. coffeaeformis. Data are expressed as mean ± confidence interval 95% (n = 4).
4. Discussion In this study we demonstrated that the transition from planktonic larvae to benthic juvenile could be promoted through diatom-based biofilms. The higher metamorphosis rate observed in larvae on N. laevis biofilm compared with larvae on H. coffeaeformis biofilm are in agreement with the results obtained by Xing et al. (2007). This study on Strongylocentrotus intermedius showed that, from a variety of eight species of benthic diatoms, Nitzschia sp. induced the highest metamorphosis rate compare to H. coffeaeformis that induced the lowest. Xing et al. (2007) suggested that the observed differences could be explained by the variability in some characteristics of the biofilms, notably the amount of extracellular polymeric substances (EPS). Nitzschia laevis secrets relatively high amount of EPS that allow it to attach strongly to the substrate and create a robust film. On the opposite, H. coffeaeformis secrets moderate amount of EPS making the adhesive strength of the biofilm poorer. Another characteristic that may vary between diatoms species is their ability to produce toxins and repellent metabolites, sometimes as a protection measurement from grazers (Maibam et al., 2014). Therefore, we can also hypothesize that H. coffeaeformis could produce a repellent substance perturbing the cascade of events that induce settlement and metamorphosis. These substances could also explain the significantly lower metamorphosis rate obtained with both N. laevis and H. coffeaeformis (MIX treatment). As in our experiment, Xing et al. (2007) obtained higher metamorphosis rates using monospecific diatom biofilms compare to multispecific ones. Further investigation could be done in order to check if H. coffeaeformis produce antifouling compounds or if a significant chemical compound modification appear when co-cultivated with N. laevis (Paul et al., 2009). Encrusting algae extracts are known to have an inducing metamorphosis effect on invertebrate larvae. This inducer is an oligopeptide that mimics the action of GABA, an inhibitory neurotransmitter (Morse and Morse, 1984; Rowley, 1989). The metamorphosis effect of GABA has been demonstrated on the sea urchin Strongylocentrotus droebachiensis (Pearce and Scheibling, 1990). In the present study this molecule induced a low metamorphosis rate on P. lividus. However, a different behaviour was observed in the larvae exposed to our GABA treatment: they were all found on the bottom of the plate. In the other treatments the metamorphosed larvae were spread in the water column, which is the normal behaviour in P. lividus, metamorphosis occurring before settlement (Fenaux and Pedrotti, 1988). The behaviour pattern observed with GABA could indicate that this neurotransmitter may function in P. lividus as a separate settlement cue and not as a metamorphosis one. Moreover, as we found similar result in the GABA + MIX and NL treatments, we can hypothesize that GABA could
Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements 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 COFUND Biodiversa, ANR-16-EBI13-0008-02). The authors wish to thank Benth’Ostrea Prod for providing the living resources. They are also grateful to V. Méléder and E. Cointet for their assistance during diatombased biofilms culture. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734559. References Ab Rahim, S.A.K., Li, J.-Y., Satuito, C.G., Kitamura, H., 2004. The role of diatom-based film as an inducer of metamorphosis in larvae of two species of sea urchin, Pseudocentrotus depressus and Anthocidaris crassispina. Sessile Org. 21, 7–12. Andrew, N., Agatsuma, Y., Ballesteros, E., Bazhin, A., Creaser, E.,K.A., Barnes, D., Botsford, L., Bradbury, A., Campbell, A., Dixon, J., Einarsson, S., Gerring, P., Hebert, K., Hunter, M., Hur, S., Johnson, C., Juinio-Meñez, M., Kalvass, P., Miller, R., Xiaoqi, Z., 2002. Status and management of world sea urchin fisheries. Oceanogr. Mar. Biol. Annu. Rev. 40, 343–425. Barillé, L., Le Bris, A., Méléder, V., Launeau, P., Robin, M., Louvrou, I., Ribeiro, L., 2017. Photosynthetic epibionts and endobionts of Pacific oyster shells from oyster reefs in rocky versus mudflat shores. PLoS One 12, e0185187. Brundu, G., Monleón, L.V., Vallainc, D., Carboni, S., 2016. Effects of larval diet and metamorphosis cue on survival and growth of sea urchin post-larvae (Paracentrotus lividus; Lamarck, 1816). Aquaculture 465, 265–271.
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