This article appeared in a journal published by Elsevier
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This article appeared in a journal published by Elsevier
(This is a sample cover image for this issue. The actual cover is not yet available at this time.) This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Aquaculture 368–369 (2012) 61–67 Contents lists available at SciVerse ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online Greening effect on oysters and biological activities of the blue pigments produced by the diatom Haslea karadagensis (Naviculaceae) Romain Gastineau a, Yann Hardivillier a, Vincent Leignel a, Nafissa Tekaya a, Michèle Morançais b, Joël Fleurence b, Nikolai Davidovich c, Boris Jacquette a, Pierre Gaudin b, Claire Hellio d, Nathalie Bourgougnon e, Jean-Luc Mouget a,⁎ a MMS Le Mans, Université du Maine, Avenue Olivier Messiaen, 72085 Le Mans CEDEX 9, France MMS Nantes, Université de Nantes, 2 rue de la Houssinière, Nantes 44322 CEDEX 3, France c Karadag Natural Reserve of the National Academy of Sciences, p/o Kurortnoe, Feodosiya, 98188 Ukraine d Biological Sciences, University of Portsmouth, School of Biological Sciences, King Henry Building, King Henry I Street. Portsmouth PO1 2DY, UK e Université européenne de Bretagne, Laboratoire de Biotechnologie et Chimie Marines (LBCM), Université de Bretagne-Sud, Centre de Recherche Yves Coppens, Campus de Tohannic, 56017 Lorient Cedex, France b a r t i c l e i n f o Article history: Received 5 December 2011 Received in revised form 13 September 2012 Accepted 14 September 2012 Available online 24 September 2012 Keywords: Biological activities Haslea karadagensis Herpesvirus Oyster farming Probiotic Vibrio a b s t r a c t Haslea karadagensis is a recently-described diatom, the second species of blue diatom to be identified, after Haslea ostrearia, the type species of the genus Haslea, which produces marennine, the water soluble pigment involved in the greening of oysters. Haslea karadagensis also produces a blue grey pigment, with different spectral characteristics from marennine itself. This study demonstrates that the pigment from H. karadagensis can colour the gills of two oysters of economic importance, Crassostrea gigas and Ostrea edulis, when they are fed with a suspension of algae, in the same way as the marennine produced by H. ostrearia. Like marennine, the purified pigment produced by H. karadagensis displays several different biological activities. Both the intracellular and extracellular forms of the pigment have been shown to inhibit the growth of marine bacteria (Polaribacter irgensii, Vibrio aestuarianus, Pseudoalteromonas elyakowii) and fungi (Corollospora maritima, Lulworthia sp., and Dendryphiella salina). The pigment also displays antiviral activity against Herpes simplex virus type 1 (HSV-1). In light of these preliminary results, the use of H. karadagensis and putative applications of its pigment in aquaculture, food chemistry and ecophysiological research, are discussed. © 2012 Elsevier B.V. All rights reserved. 1. Introduction The greening of oysters is a natural phenomenon that has long been known (Sprat, 1669) and has been shown to depend on the presence of particular diatoms with blue apices, which produce a pigment able to colour bivalve gills (Gaillon, 1820). Today, greening of oysters at an economic scale is mainly practised on the Atlantic Coast of France, in Marennes-Oléron Bay and Bourgneuf Bay. Subject to market fluctuations, the price of green oysters is about 20% higher than those of other oysters, mainly because of the unpredictability of the greening phenomenon. Greening is carried out in special ponds, usually old salt marshes, which are actively colonised by microalgae, and used to mature and fatten oyster. In some of these oyster ponds, known as ‘claires’, the diatom Haslea ostrearia (Gaillon) Simonsen (1974) can outcompete other microalgae, and has been shown experimentally to be consumed in sufficient quantities to sustain the growth of oysters (Barillé et al., 1994; Cognie, 2001; Piveteau, 1999). The diatom H. ostrearia is known for the water-soluble blue ⁎ Corresponding author. Tel.: +33 2 43 83 32 42; fax: +33 2 43 83 37 95. E-mail address: [email protected] (J.-L. Mouget). 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2012.09.016 pigment it produces which accumulates at its apices, and which is known as marennine (or marennin, Lankester, 1886), in reference to the Marennes-Oléron area. When H. ostrearia blooms and releases marennine into the water, the oyster ponds turn dark-green. The oysters, as a result of feeding on H. ostrearia cells, and/or of fixing the pigment released into the water on their gills turn green as well, a phenomenon that has been reproduced experimentally by immersing oysters in a suspension of blue diatoms (the first attempts were made by Lankester, cited in Dyer, 1877; Puységur, 1880; Sauvageau, 1907). The direct role of marennine in oyster greening has also been demonstrated in the laboratory, using either the supernatant (crude extract) of H. ostrearia cultures (Neuville and Daste, 1972; Ranson, 1927), or a solution of purified marennine (Pouvreau, 2006). Marennine can exist in two forms, the intracellular form, which accumulates in the cell apices, and the extracellular form, which is excreted into the medium (Pouvreau et al., 2006a). These two forms have different molecular weights and spectral characteristics, but their structures remain to be defined. Moreover, it has been shown that both forms of marennine can colour oyster gills (Pouvreau, 2006), have antioxidant properties (Pouvreau et al., 2008), biological activities (Gastineau et al., 2012, and could have Author's personal copy 62 R. Gastineau et al. / Aquaculture 368–369 (2012) 61–67 an ecological allelopathic effect on other diatoms in oyster ponds (Pouvreau et al., 2007). In the literature, outside France, naturally-occurring greening of oysters (i.e., not deliberately produced in oyster farms) was first described in Great-Britain (Sprat, 1669), and has also been observed in Denmark (Petersen, 1916), and the United-States (Mitchell and Barney, 1918; Ryder, 1884). For decades, H. ostrearia was believed to be the only diatom able to produce marennine, which was itself considered to be an unusual and indeed unique pigment. The recent discovery and investigation of a pennate diatom with blue-grey apices along the Crimean shores of the Black Sea, which is distinct from H. ostrearia, led to the description of a new species belonging to the genus Haslea, designated Haslea karadagensis (Gastineau et al., in press). Moreover, the pigment synthesised by H. karadagensis, differs from marennine, especially with regard to its in vivo UV– visible spectrophotometry and Raman spectroscopy (Gastineau et al., in press). The first objective of this work was to compare the colouring potential of the pigments produced by the two species of blue diatoms on two different economically important species of oyster, Crassostrea gigas and Ostrea edulis, which were fed on a diet consisting of either of H. ostrearia or H. karadagensis. The second objective was to screen the intracellular and extracellular forms of H. karadagensis pigment for biological activities (antifungal, antibacterial and antiviral properties), with a particular emphasis on its effects on marine micro-organisms. The possible use of this new naturally-occurring substance is discussed, especially as a potential probiotic for use in aquaculture. 2. Material and methods 2.1. Oyster acclimation and growth conditions Two different economically-important species of oysters were used for these experiments, the Pacific oyster C. gigas (Thunberg, 1793), and the flat oyster, O. edulis (Linnaeus, 1758). The samples of C. gigas originated from Saint Germain sur Ay, Normandy, France (49° 14′ 09″ N 1 35′ 32″ W), and those of O. edulis from Saint-Malo, Brittany, France (48° 38′ 53″ N 2° 00′ 27″ W). The oysters were acclimated in 35 × 25 × 20 cm aquaria, containing 5 L of artificial sea water, oxygenated by bubbling air (Rena ® Air 100 air pump). Continuous illumination of 25 μmol photons m −2 s −1 was provided from above by Philips TLD 36 W/965 fluorescent tubes, and measured using a Li-Cor LI-189 quantum meter coupled with a 2π Li-Cor Q21284 quantum sensor. The temperature of the water in the aquaria was 12 ± 1 °C. During a 1 week-acclimation phase, twelve oysters per aquarium were fed with regular inocula of Skeletonema costatum and Entomoneis paludosa cultures. For both species, the first inocula were used at a final concentration of 1.5 · 10 6 cells L −1. Both these microalgae are diatoms commonly encountered in Atlantic oyster ponds (Rincé, 1978). Furthermore, Skeletonema and Entomoneis species have already been used experimentally for oyster feeding (e.g. Knuckey et al., 2002; Barillé et al., 2003). 2.2. Algal biomass production and experimental diet At the start of the colouring experiments, twelve oysters (randomly chosen) per aquarium were immerged in aquaria containing 1.2 · 10 6 cells L −1 of H. ostrearia and H. karadagensis for 1 week before being observed. Other oysters (twelve per aquarium) continued to be fed with S. costatum and E. paludosa, and served as controls and stock oysters. The following algal strains were provided by the Nantes Culture Collection (University of Nantes, V. Méléder): NCC 18 (E. paludosa), NCC 53 (S. costatum), NCC 148.14c (H. ostrearia) and NCC 313 (H. karadagensis). Prior to experimentation, algae were grown in sterile Erlenmeyer flasks with modified artificial sea water (Mouget et al., 2009), in a controlled-culture room (16 ± 1 °C, 60 μmol photons m −2 s −1, 14 h/10 h L/D cycle, provided by fluorescent Philips TLD 36 W/965 tubes). Prior to use, the algae were maintained in the exponential phase of growth by regular subculturing. 2.3. Extraction and purification of pigments The pigments of H. karadagensis were extracted and purified according to Pouvreau et al. (2006b). The intracellular and extracellular forms were obtained from algal paste and the supernatant, respectively, after gently centrifuging the algal suspensions (10 min at 900 g, with an acceleration/deceleration of 2/2 at 4 °C, using a Sigma 3K15 model from Bioblock Scientific). Extraction was done by crushing the algal biomass in liquid N2 with a 250 mM L −1 NH4HCO3 buffer at pH 8 (Pouvreau et al., 2006b). Purification consisted of a two-step ultrafiltration process using 30 kDa and 3 kDa cut-off membranes, with a 0.23 m 2 Prep/Scale-TFF cartridge from Millipore, and a 313S peristaltic pump from Watson Marlow. It was followed by anion-exchange chromatography, performed with a WatersTM 626 dual piston pump coupled with a WatersTM 486 tuneable absorbance detector, and with the help of DEAE Sepharose Fast Flow resin from Amersham Biosciences. Dialysis was carried out using a spectra/or regenerated cellulose (Spectrum) 3.5 kDa cut-off membrane. Finally, the pigment was freeze-dried on a Heto FD3 apparatus. 2.4. Assessment of antibacterial activities The antibacterial activities of the two forms of the H. karadagensis pigment were studied using three strains of marine bacteria, Polaribacter irgensii (ATCC 700398), Vibrio aestuarianus (ATCC 35048) and Pseudoalteromonas elyakowii (ATCC 700519), which are commonly used in biological assays (Plouguerné et al., 2010), following the protocol from Hellio et al. (2000). Stock cultures of bacteria were maintained in an incubator at 30 °C, regularly diluted with fresh medium, consisting of steam-sterilised sea water mixed with peptone at a final concentration of 5 g L −1. The disc diffusion technique in agar-plated Petri dishes of Hellio et al. (2001) was slightly modified, using sterilised Whatman filter paper discs with a diameter of 6 mm. The purified H. karadagensis pigment (both forms) was diluted in sterile, distilled water at concentrations of 0.01, 0.1, 1, 10, 50 and 100 μg mL −1, and filtered on a 0.22 μm filter. 10 μL aliquots of the solutions were used to load the paper discs, which were dried under sterile conditions at room temperature for 6 h before use. Control discs were inoculated with sterile distilled water. A dilution of the bacterial culture in nutrient broth was made 1 day before the experiments. Aliquots (0.1 mL) of the culture (10 6 CFU mL −1) were used for agar inoculation. After incubating for 2 days at 20 °C, the activity was estimated by measuring the radius of the inhibition zone around the discs. All inhibition assays were done in triplicate. 2.5. Determination of antifungal activities The antifungal activities of the H. karadagensis pigment were studied using three species of marine fungi, Corollospora maritima, Lulworthia sp., and Dendryphiella salina, following Hellio et al. (2000). Stock cultures of fungi were obtained from the University of Portsmouth culture collection and were maintained on maize meal agar (Oxoid) slopes. Pigments were diluted in distilled water, filtered on 0.22 μm filters, and mixed with 6 mL of maize meal agar 12% (Sigma) at pH 6. Controls were made with distilled water. The Petri plates were inoculated with the previous mix (pigments + fungi on maize meal) under sterile conditions to produce an 8 mm agar plug. The inhibitory activity corresponded to a slowdown in mycelium growth, and was measured after 3 weeks of culture at 20 °C by measuring the diameter of the fungal colonies, and comparing it to those of the pigment-free controls. All Author's personal copy R. Gastineau et al. / Aquaculture 368–369 (2012) 61–67 assays were performed in triplicate and results were expressed at MICs values (minimum inhibitory concentration). 2.6. Assessment of cytotoxicity and antiviral activities The cytotoxicity and antiviral activity of the pigments were carried out by cell viability (neutral red dye method), as described in Defer et al. (2009). Experiments were conducted using Vero cell line ATCC CCL81 of the African green monkey kidney. Virus stock of Herpes simplex virus type 1, wild strain 17 ACVS and PFAS was obtained from Prof. Billaudel, Laboratoire de Virologie de Nantes (France). The reference drug was the sodium salt of Zovirax IV, 25 mg mL−1, purchased from the Wellcome Foundation Ltd. A series of experiments including assays ranging from extreme concentrations of drugs of 200, 50, 10, 5 and 1 μg mL−1 was done, after diluting the pigments in Eagle's MEM supplemented with 8% FCS. The 96-well plates containing the diluted compound and virus-infected cell suspensions were incubated for 3 days without changing the medium, at 37 °C in 5% CO2. Treated cells and virus controls were run simultaneously. At the end of the experiments, the 50% cytotoxic concentration (CC50) of the test compound was defined as the concentration that reduced the absorbance of virus-infected cells to 50% of that of controls. The 50% antiviral effective concentration (EC50) was expressed as the concentration of the test compound that achieved 50% protection of virus-infected cells from the HSV-induced destruction. 2.7. Microscopy, macro- and microphotography Pictures of the diatoms were taken using a Zeiss Axiostar plus microscope with a Zeiss Acroplan 100×/1.25 oil objective and a Zeiss Axiocam ICc1 camera. Pictures of oysters were taken using a Canon EOS 550 D camera with a Canon Zoom Lens EF 24–105 mm 1:4 objective. 3. Results The protocol used here for the colouration of oysters was inspired by Pouvreau's method (Pouvreau, 2006), in which a marennine concentration of about 2 μg mL −1 and H. ostrearia cultures of 1.2 · 10 6 cells L −1 were used. Compared to the controls (S. costatum + E. paludosa diet, Fig. 2), after 1 week on the experimental diet composed of blue diatoms, the gills and mantle of oysters fed with a suspension either of H. ostrearia (Fig. 1) or of H. karadagensis cells (Fig. 3) had turned green (Figs. 4, 7, and 6, 9, respectively), whereas those of oysters fed with S. costatum and E. paludosa retained their original yellowish colour (Figs. 5, 8). Oysters fed with H. ostrearia had organs strongly coloured green, whereas oysters fed with H. karadagensis exhibited blue-grey coloured organs, which to the naked eye, appeared to be lighter coloured. No oyster mortality occurred on any diet or composition of diatom species. The effects of the pigment produced by H. karadagensis on the three marine bacteria tested are illustrated in Fig. 10. Both forms of the pigment inhibited the growth of V. aestuarianus and P. elyakowii, up to a concentration of 0.1 μg mL −1, in contrast to P. irgensii, which seemed less affected (no inhibition at concentrations lower than to 10 μg mL −1). With regard to their antifungal activity, the minimum inhibitory concentrations (MIC) of the pigment for the growth of the three species of marine fungi tested were in the range 0.1–1 μg mL −1 (Table 1). The cytotoxicity and antiviral activity (anti-HSV-1) of the two forms of the pigments are shown in Table 2. Differences were observed between the two forms. No cytotoxic effect of the extracellular form of the pigment on the Vero cells was observed in the range of the concentrations tested. After 3 days of treatment, no microscopically visible alteration of the normal cell morphology was observed, even at 200 μg mL −1. In addition, a viability assay did not demonstrate any 63 destruction of the cell layer. For an MOI of 0.001 ID50/cells, the extracellular form of the pigment exhibited antiviral activity with an EC50 of 23 μg mL−1. In contrast, the intracellular form displayed lower antiviral activity, with an EC50 of 62 μg mL−1. This form appeared to be more cytotoxic, with a CC50 of 87 μg mL−1. 4. Discussion The highest concentrations of pigments used in the present study (50–100 μg mL −1) for the colouration of oysters are in the same range as the very few values available in the literature for marennine produced by H. ostrearia. No data is available about marennine concentrations in oyster ponds during a natural greening event, but in a series of experiments on the outdoor mass production of H. ostrearia, the maximum concentration of marennine measured in 25 m 3 ponds was 2.4 μg mL −1 (Turpin et al., 1999), and 3.4 and 7.1 μg mL -1 for 500 L tanks and 10 m 3 ponds, respectively (Turpin et al., 2001). The concentrations of Haslea cells corresponded to the density observed in cultures in the late exponential growth phase, and this was found to be enough to produce a modification of the gill colour within a week. 4.1. Colouring potential of the marennine-like pigment produced by H. karadagensis Paradoxically, the appearance of a green colouration in oyster gills can reflect two diametrically opposite situations with regard to human use. On the one hand, changes in oyster colour, especially if they turn green, is usually a warning sign of a disease caused by metal pollution (e.g., copper, iron) (Lin and Hsieh, 1999), which can be lethal to oysters (Lee et al., 1996). On the other hand, nowadays mainly in France, the greening of oysters is the consequence of the fixation on the gills of marennine, the pigment produced by H. ostrearia, upon which the bivalves feed in the shallow oyster ponds known as ‘claires’ when this diatom becomes dominant. This well-documented phenomenon is usually required for oyster producers to obtain the red label ‘fine/spéciale de claire verte’ awarded by the French Ministry of Agriculture (AFNOR, 1985), although greening has been found to be rather unpredictable, for reasons as yet unknown. For decades, this unpredictability called for more research on the causal links between the bloom of blue diatoms and the greening of bivalves, and on the factors that control such blooms. It is worth noting that a change in colour is not restricted to oysters, and a greening-like action also occurs in other animals that can be present in oyster ponds (Chaux-Thevenin, 1939), such as in polychaetes, crab, littorina, mussels (Ranson, 1927), sea-anemones (Gaillon, 1820), scallops and cockles (Gastineau, unpublished data). For all these organisms, as for oysters, the specific interactions between microalgal pigments and gills, labial palps, or any coloured organs of the bivalves remain an unexplored field for biochemical and histological investigation. In line with the preliminary experiments dealing with the greening of C. gigas fed with H. ostrearia cultures in the laboratory (Pouvreau, 2006), and regardless of the mechanisms involved in the greening phenomenon, the present study demonstrates for the first time that feeding oysters with the newly-described blue diatom H. karadagensis gives their gills a blue-grey colour, which differs from the fairly intense green obtained with H. ostrearia and marennine. Furthermore, although French oyster production in ponds is now mainly dedicated to C. gigas, both species of oysters used in these experiments demonstrated their capacity to fix marennine-like pigments. Indeed, both Pacific oysters, C. gigas, and flat oysters, O. edulis, were coloured by the pigments produced by the two blue diatom species, revealing possible similarities in their mode of action or their interactions with the physiology of oysters. Author's personal copy 64 R. Gastineau et al. / Aquaculture 368–369 (2012) 61–67 Figs. 1–9. Effect of the algal diet on the colouring of oyster gills. 1: H. ostrearia. 2: S. costatum (up) and E. paludosa (down). 3: H. karadagensis. 4: C. gigas fed with H. ostrearia. 5: C. gigas fed with S. costatum and E. paludosa. 6: C. gigas fed with H. karadagensis. 7: O. edulis fed with H. ostrearia. 8: O.edulis fed with S. costatum and E. paludosa. 9: O. edulis fed with H. karadagensis. Scale bar are 10 μm (1 to 3) and 1 cm (4 to 9). 4.2. Biological activities of the H. karadagensis pigment Both the intracellular and extracellular forms of the pigment produced by H. karadagensis exhibited antibacterial and antifungal activities at concentrations > 0.1 μg mL −1. In the absence of values for marennine concentrations in oyster ponds in the literature, these concentrations appear to be realistic, compared to the maximum concentrations of marennine measured in ponds and tanks Author's personal copy R. Gastineau et al. / Aquaculture 368–369 (2012) 61–67 65 Table 2 Evaluation of anti-HSV activity on vero cell line of Haslea karadagensis pigments using neutralised red dye method. Compound tested CC50 μg mL−1 EC50 μg mL−1 Zovirax Inta Exta >200.0 87.0 >200.0 0.2 62.0 23.0 The 50% cytotoxic concentration (CC50) is the concentration that reduced the absorbance of mock-infected cells to 50% of that of controls. The 50% antiviral effective concentration (EC50) is the concentration that achieved 50% protection of virus-infected cells from the HSV-induced destruction. a Int for intracellular form, Ext for extracellular form. Fig. 10. Antibacterial activities of the intracellular (I) and extracellular (E) forms of the pigment produced by Haslea karadagensis. Estimation was made by measuring the radius of the inhibition halo. Values are means ± S.E. (n = 3), and are expressed in mm. used for the outdoor mass production of H. ostrearia (2–7 μg mL −1, Turpin et al., 1999, 2001). For most of the micro-organisms and concentrations tested, the intracellular form seemed more active, possibly due to its higher cytotoxicity (see below). Among marine bacteria tested, the growth of which was inhibited by the H. karadagensis pigment, V. aestuarianus is especially relevant for aquaculture. Vibrio species and isolates are bacteria commonly encountered in aquaculture systems, and are able to infect a wide variety of hosts (Vandenberghe et al., 2003). They are responsible for diseases in different organisms, such as pleuronectiform (Planas et al., 2005) and anguilliform fish (Esteve-Gassent et al., 2004), shrimp (e.g., Sudheesh and Xu, 2001), holothurians (Gu et al., 2011; Zhao et al., 2011) or molluscs with a high economic value, such as abalone (Cheng et al., 2004). The Vibrio species V. aestuarianus and V. splendidus are of particular concern to the oyster industry as they may have been involved in most of the severe summer mortality Table 1 Evaluation of the minimum inhibitory concentration (MIC) of Haslea karadagensis pigments for marine fungi. Inta Exta Corollospora maritima Lulworthia sp. Dendryphiella salina 0.1 1.0 1.0 1.0 0.1 1.0 Results are minimum inhibitory concentrations (MICs) expressed in μg mL−1. a Int for intracellular form, Ext for extracellular form. events observed in the last decade (Garnier et al., 2008; Lago et al., 2009). The inhibition of the growth of V. aestuarianus produced by the H. karadagensis pigment confirms a previous rather preliminary observation involving intracellular marennine and V. anguillarum (Pouvreau, 2006), and is particularly encouraging given the current aquacultural need to control mortality events caused by these pathogenic bacteria. In line with this anti-Vibrio activity evidenced in vitro, the inhibition of the growth of the marine bacterium P. elyakowii demonstrated by both forms of the marennine-like pigment produced by H. karadagensis is also of potential interest for aquaculture. Indeed this bacterium has been implicated in damages caused to seaweed production (Narita et al., 2001), and a related species, P. peptidolytica, has been shown to damage mussel threads (Venkateswaran and Dohmoto, 2000). Both forms of the H. karadagensis pigment also inhibit fungal growth, a result possibly relevant for aquaculture, as some fungi are pathogens of bivalves, e.g. Ostracoblabe implexa, known to be an important causal agent of disease or mortality in oysters (Bower et al., 1994), especially in the Black Sea (Pirkova and Demenko, 2008). Moreover, marine fungi, such as those used in this study, are often involved in the phenomenon of biofouling. They can adhere to immersed structures, such as oyster shells, trays and cages, leading to additional costs in terms of time and money as a result of the need for cleaning (Sala and Lucchetti, 2008). Marine fungi can also cause corrosion of immersed surfaces, like bacteria (Little et al., 1999). Given the allelopathic activity of marennine observed by Pouvreau et al. (2007) against different microalgae encountered in oyster ponds, some of them being fouling diatoms (Nitzschia closterium, Entomoneis pseudoduplex, Haslea crucigera), and despite the limited number of target species used for the present study, possible antifouling effect of marennine-like pigments deserves further studies. For instance, antifouling experiments against a largest range of bacteria, macroalge and fungi should be performed, extending to macroalgal spores and barnacle larvae fixation. Indeed, the search for natural antifouling substances produced by algae has been mainly conducted on brown or red macroalgae (e.g. Bazes et al., 2009; Bianco et al., 2009; Silkina et al., 2009), but some compounds isolated from microalgae also have the potential for being environmentally friendly antifouling agents (Bhadury and Wright, 2004). 4.3. Antiviral activity of the pigment produced by H. karadagensis First observations of an antiviral effect using a water soluble extract of H. ostrearia cultures were made by Bergé et al. (1999). This antiviral activity has been recently confirmed using purified marennine (Gastineau et al., 2012). The present study demonstrates that the purified pigment of H. karadagensis also displays antiviral activity, thus confirming that marennine-like molecules are a potential source of natural active compounds. When considering aquaculture, the search for natural antiviral products is a current challenge. Indeed, one of the major threats associated with oyster farming was identified more than 30 years ago to be a specific virus known as oyster herpes-type virus (OsHV) (Farley, 1978; Farley et al., 1972). These Author's personal copy 66 R. Gastineau et al. / Aquaculture 368–369 (2012) 61–67 virus belong to the recently defined order of Herpesvirales (Davison, 2010; Davison et al., 2005). They have long been known to be responsible for many oyster mortality episodes worldwide (Hine et al., 1992; Nicolas et al., 1992). In particular, one strain, OsHV-1, has been associated with most of the severe summer mortality events reported by most French oyster production sites (Arzul and Renault, 2002; Arzul et al., 2001; Renault et al., 1994; Schikorski et al., 2011; Segarra et al., 2010). However, one criticism that could be made is that the antiviral evaluation consisted of testing algal pigments on mammalian fibroblastic cells (Vero cells) infected by HSV-1, a model currently used to screen antiviral molecules derived from marine organisms (Bergé et al., 1999; Maier et al., 2001; Yasin et al., 2000). The choice of a heterologous model was unavoidable due to the lack of bivalve cell lines (Azumi et al., 1990; Lee and Maruyama, 1998; Li and Traxler, 1972; Prescott et al., 1966; Tamamura et al., 1993). Another concern could be that the intracellular form of the pigment produced by H. karadagensis has been shown to be cytotoxic towards Vero cells, which questions the possible toxicity of marenninelike pigments. If the dose makes the poison, we must bear in mind that green oysters have been eaten for centuries, and as far as we are aware, have never been of any particular health and safety concern for gourmets, nor has the marennine produced by H. ostrearia ever been implicated in any oyster pathology. Nevertheless, the results and limitations of the present investigations highlight the fact that more research is needed to establish the relative cytotoxicity of the marennine-like pigments, a molecular approach being currently designed. In conclusion, the newly-described diatom H. karadagensis produces a marennine-like pigment that has been shown to fix to the gills and to colour two economically-important species of oysters, C. gigas and O. edulis. Like marennine, this pigment has been shown to display antibacterial, antifungal, and antiviral activities. These results demonstrate that marennine-like pigments are bio-active compounds, which allow us to hypothesise that they could be used as a prophylactic treatment based on a microalgal diet for bivalves, a strategy which has already received some attention in the last two decades (Naviner et al., 1999). Nevertheless, it remains to explore beforehand the possible physiological interactions of these blue pigments with the cells, tissues and organs of oysters. This also does not preclude the need for investigation of the safety and organoleptic qualities of the coloured oysters, as well as their acceptability to consumers, two of the main conditions necessary for probiotics or probiotic-sources to be viable in aquaculture (Verschuere et al., 2000; Kesarcodi-Watson et al., 2008). 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