CONTROL OF GREEN MACROALGAE BLOOMS

Abstract

The control of green macroalgae blooms. More particularly, Ulva algae blooms may be controlled by a living active principle contained in seawater from the Mediterranean Sea. The inventors have observed that seawater from the Mediterranean Sea collected in particular spots (collected at, e.g., latitude 43° 14′N and longitude 5° 21′E, or at latitude 43° 09′N and longitude 5° 36′E) is capable of promoting the death of Ulva lactuca, without the emission of toxic acidic vapors, such as, e.g., H.sub.2S vapors. Altogether, the inventors provide data showing that this seawater comprises an alive microorganism that is responsible for promoting the death of Ulva, in particular of Ulva lactuca. More precisely, the inventors provide experimental data showing that the microorganism that promotes the death of Ulva lactuca, and hence promotes the control of Ulva lactuca blooms, is a virus.

Claims

1.-16. (canceled)

17. A method for controlling and/or preventing blooms of an alga of the genus Ulva in a marine environment in need thereof, comprising the step of contacting said marine environment with seawater collected from the Mediterranean Sea.

18. The method according to claim 17, wherein said alga of the genus Ulva is an alga of the species Ulva lactuca.

19. The method according to claim 17, wherein said seawater is collected at latitude 43° 14′N and longitude 5° 21′E, latitude 43° 09′N and longitude 5° 36′E, latitude 43° 18′N and longitude 5° 17′E, latitude 43° 14′N and longitude 5° 17′E, or at latitude 43° 15′N and longitude 5° 19′E.

20. The method according to claim 17, wherein said seawater is collected at latitude 43° 14′N and longitude 5° 21′E, or at latitude 43° 09′N and longitude 5° 36′E.

21. The method according to claim 17, wherein said seawater is collected from the surface to a depth of at most 30 m.

22. The method according to claim 17, wherein said seawater is conserved at a temperature of from about 4° C. to about 30° C.

23. The method according to claim 17, wherein said seawater comprises an alive microorganism capable of promoting the death of an alga of the genus Ulva.

24. The method according to claim 23, wherein said alive microorganism is a virus.

25. A method for controlling and/or preventing blooms of an alga of the genus Ulva in a marine environment in need thereof, comprising the step of contacting said marine environment with one or more alive microorganism(s) originating from seawater collected in the Mediterranean Sea.

26. The method according to claim 25, wherein said alga of the genus Ulva is an alga of the species Ulva lactuca.

27. The method according to claim 25, wherein said seawater is collected at latitude 43° 14′N and longitude 5° 21′E, latitude 43° 09′N and longitude 5° 36′E, latitude 43° 18′N and longitude 5° 17′E, latitude 43° 14′N and longitude 5° 17′E, or at latitude 43° 15′N and longitude 5° 19′E.

28. The method according to claim 25, wherein said seawater is collected at latitude 43° 14′N and longitude 5° 21′E, or at latitude 43° 09′N and longitude 5° 36′E.

29. The method according to claim 25, wherein said seawater is collected from the surface to a depth of at most 30 m.

30. The method according to claim 25, wherein said seawater is conserved at a temperature of from about 4° C. to about 30° C.

31. The method according to claim 25, wherein said alive microorganism is a virus.

32. The method according to claim 25, wherein said alive microorganism is purified from said seawater.

33. The method according to claim 25, wherein said alive microorganism is filtered from said seawater.

34. The method according to claim 25, wherein the amount of said alive microorganism is ranging from about 105 to about 109 PFU/mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIGS. 1A-1C are photographs of Enteromorpha algae. FIG. 1A: a green tubular alga formerly called Enteromorpha collected in November 2018 after a bloom in the Trieux fjord (TR) in the north coast of Brittany (48° 46′ N, 3° 06′W). FIG. 1B: the tubular form disappears after an incubation for one month at 20° C. and day light exposure with sea water collected in June 2018 in the bay of Marseille (43° 18′ N 5° 16′E or spot X (RS) in FIG. 2). FIG. 1C: the “Enteromorpha” became a typical Ulva lactuca after three months at 20° C. and day light exposure.

[0084] FIG. 2 is a scheme showing a statistical analysis of in vitro Breton Ulva lactuca proliferation with distinct samples of seawater of the bay of Marseille. Seawater samples collected in June 2018 in three different spots in the bay of Marseille. X (RN) was 43° 18′N, 5° 16′E; Y (RS) was 43° 15′N, 5° 19′E and Z (PR) was 43° 14′N, 5° 21′E. Seawater samples were divided in three groups of tubes (n=36). Breton Ulva lactuca collected in June 2018 at Brehec (north coast of Brittany 48° 43′N 2° 06′W) were cut in pieces of 1 cm.sup.2 and put in the three groups of tubes corresponding to spots X, Y and Z in the bay of Marseille collected one day before sampling (D1). Ulva lactuca proliferation was carried out at 25° C. with seawater in 50 ml tubes closed with a tape to induce anoxia. Proliferation was observed in 25 tubes/36 (69%) in the X spot that corresponds to open sea, while it was 14 tubes/36 (39%) in the Y spots and only 1 tube/36 (2.7%) in the Z spot, the closest of the shore (see the inserted graph). In tubes where Ulva lactuca could proliferate, confluence was reached after one week and acidity was detected. No acidity was observed in tubes where Ulva lactuca could not grow and Ulva lactuca became white after 5 days. Seawater from the Z spot was kept from D30 to D180 before to be incubated again with Breton Ulva lactuca and proliferation was observed in 12 tubes/36 (33%) for D30 and in 36 tubes/36 (100%) for D180 (see the inserted graph).

[0085] FIGS. 3A-3D are photographs showing the comparison with optical microscopy of Ulva lactuca in three different states. FIG. 3A: Ulva lactuca became white in five days when incubate at 20° C. and day light exposure with seawater from Z spot of the bay of Marseille. FIG. 3B: Optical microscopy (10×) of white Ulva lactuca. Ulva lactuca tissue remains unaffected with a regular organization of Ulva cells. FIG. 3C: Optical microscopy (10×) of healthy Ulva lactuca. FIG. 3D: Optical microscopy (10×) of Ulva lactuca after acidic biodegradation. Ulva lactuca tissue is disrupted with release of chlorophytes that remain green in spite of anoxia. Photographed with a camera Nikon D3100 coupled to a Nikon Eclipse Ti L100 microscope (Nikon, Tokyo, Japan).

[0086] FIGS. 4A-4D are photographs and graph showing the fluorescence microscopy after SYBR staining FIG. 4A-C: sea water from Mediterranean seas inducing bleaching was incubated without Ulva (panel A) and with Ulva (panels B and C). FIG. 4D: shows the virus-like particles amount, expressed as a number of particles/ml. Sea water was filtrated at 0.2 μm.

EXAMPLES

[0087] The present invention is further illustrated by the following examples.

Example: Identification of Seawater Samples that Promote Death of Ulva lactuca

[0088] 1) Materials and Methods

[0089] a) Ulva lactuca Polymorphism

[0090] Green algae were collected in the Trieux fjord in the North coasts of Brittany (48° 46′ N, 3° 06′W). Proliferation was carried out in vitro with seawater samples from the bay of Marseille (Provence, South of France). A green tubular alga formerly called Enteromorpha was collected in November 2018 after a bloom in the Trieux fjord in the north coast of Brittany (48° 46′ N, 3° 06′W). The incubation was carried out for one month at 20° C. and day light exposure with sea water collected in June 2018 in the north bay of Marseille (43° 18′ N 5° 16′E; RN).

[0091] b) Ulva lactuca Proliferation

[0092] Seawater samples were collected in surface in springtime (June 2018, 2019 and 2020) in eight different spots, including three different spots at Marseille (FIG. 2), 2 spots in Provence, and 3 spots in Brittany (see Table 1). Seawater samples were divided in three groups of tubes (n=36). Breton Ulva lactuca collected in June 2018 at Brehec (north coast of Brittany 48° 43′N 2° 6′O) were cut in pieces of 1 cm.sup.2 and put in the three groups of falcon tubes (50 ml) corresponding to spots X, Y and Z in the bay of Marseille collected one day before sampling (D1). Acidity was tested with a Crison pH Meter (Barcelona, Catalonia). The pH meter was calibrated before any measurement. Nitrates was measured with a METRHOM chromato ionic device (Berne, Switzerland) with a Metrosep column A supp 5 150/4 mm with 3.2 mM Na.sub.2CO.sub.3/1 mM NaHCO.sub.3 as eluant. Sea Water was diluted ⅛ and a standard was use to calibrate the amount of nitrates.

[0093] c) Optical Microscopy

[0094] Optical microscopy (10×) was performed on healthy Ulva lactuca before confluence, after acidic biodegradation and on white Ulva lactuca after five days with water sample collected in the Z (PR) spot in the bay of Marseille. Photographs were carried out with a camera Nikon D3100 coupled to a Nikon Eclipse Ti L100 microscope (Nikon, Tokyo, Japan).

[0095] d) Diode Array Detection High Performance Liquid Chromatography (DAD HPLC)

[0096] Sea water samples were filtered at 0.2 μm and were analyzed on a Beckman HPLC system gold device with a reverse phase (C8) column using H.sub.2O 0.1% TFA (A) and CH.sub.3CN 0.1% TFA (B). The gradient was from 10% to 50% B in 40 min, and then 10 min at 90% B and 10 min at 10% B. Diode Array Detector Beckman device was coupled after the injector. Flow rate was 0.8 ml/min

[0097] e) Fluorescence Microscopy after SYBR Staining

[0098] Mediterranean seas water without and with Ulva lactuca sample was filtrated through 0.22 μm membrane filter (Millex®; cat. no SLGP033RS) to remove cells and then through 0.02 μm Anodisc filters (Whitman®; cat. no WHA68096002) using a vacuum filtration system to collect viral particles.

[0099] Then filters were stained with SYBR Gold dye (N′,N′-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine) that binds reverently to DNA (Invitrogen®; cat. no S11494) at room temperature for 15 min in the dark, and washed three times with 500 μL of sterile 0.02 μm-filtered mQ water. Stained virus-like particles were observed with an epifluorescence Microscope Leica SP2.

[0100] 2) Results

[0101] a) Breton Ulva lactuca can Grow in Mediterranean Sea and has Different Phenotypes Regarding Salinity

[0102] Ulva lactuca is naturally present in the bay of Marseille (Provence, south of France) and appears each year in winter. Ulva grow rapidly from February to March before disappearing rapidly for springtime. Ulva lactuca blooms, as observed in Brittany, were never reported in the bay of Marseille while this bay has a high concentration in phosphate and nitrogen and shallow beaches. A first hypothesis could be that Breton Ulva lactuca could easily proliferate in Brittany but could not grow in Mediterranean Sea, more particularly, that the nitrate concentration is lower compared to sea water in Brittany. Five spots nearby Marseille were selected and sea water samples were collected and compared to three spots in Brittany (Table 1).

TABLE-US-00001 TABLE 1 Springtime Sea Waters (n = 7) from Brittany and Provence Location TR BR PO RN WF RS PR MU Latitude (N) 48°46′ 48°43′ 47°06′ 43°18′ 43°14′ 43°15′ 43°14′ 43°09′ Longitude 3°06′W 2°56′W 2°07′W 5°16′E 5°17′E 5°19′E 5°21′E 5°36′E Nitrate (μM) 24 + 583 7 + 3 11 + 2 5 + 2 6 + 1 5 + 2 24 + 5 9 + 3 pH 7.9 8.0 8.1 7.9 8.0 8.0 8.0 8.0 Conductimetry 30.9 45.6 48.1 57.4 56.1 57.9 57.8 58.1 (mS) Bleaching* 0 0 0 0 0 8 98 85 (%) *Statistical analysis of in vitro Breton Ulva bleaching with sea water samples collected for springtime. All experiments (n = 8) were carried out with Breton Ulva collected in Trieux fjord (TR) in the north of Brittany in 2018, 2019 and 2020. Ulva were cut in pieces of 1 cm.sup.2 and put in sea water (40 ml) in tubes (n = 25) closed with a tape and let at day light with an average temperature of 25° C.

[0103] The bay of Marseille is at 20 km of the mouth of the Rhone River and North West winds (Mistral and Tramontane) that are dominant blow regularly from Rhone River to Marseille. Table 1 shows that pH and conductimetry (related mainly to salinity) are lower in RN due probably to the influence of Rhone river. Table 1 shows that the concentration in nitrates in open coastal sea water is equivalent in Brittany (BR and PO) and in Provence (RN, WF, RS). However, nitrate concentration can be much higher in Brittany fjord (TR) or in calanque (MU) and marina (PR) in Provence.

[0104] As shown above, Breton Ulva lactuca can grow rapidly with sea waters from Marseille (FIG. 1). Ulva lactuca polymorphism was tested with a green tubular alga formerly called Enteromorpha (FIG. 1A) collected in the Trieux fjord nearby the city of Paimpol (North Brittany) that became a typical Ulva lactuca after three months at 20° C.±10° C. and day light exposure (FIG. 1C). This experiment illustrates the importance of salinity in the polymorphism of Ulva lactuca as previously described (Rybak, Ecological Indicators, 2018, 85, 253-261).

[0105] b) Breton Ulva lactuca Proliferation is Different Regarding the Location and the Timing of the Water Sampling in the Bay of Marseille

[0106] Natural biodegradation on beaches occurs when Ulva lactuca reach confluence inducing anoxia characterized by production of H.sub.2S. For this biodegradation, Ulva can become white due to dehydration. However, this is a different phenomenon that we observed with Breton Ulva lactuca in sea water collected at Marseille. Breton Ulva lactuca were turning white (bleaching) rapidly sometime in one day without dehydration. To simulate this natural process proliferation of Ulva lactuca was carried out with sea water in 50 ml tubes closed with a tape to induce anoxia.

[0107] A statistical analysis was carried out with seawater samples collected in three different spots in Brittany and five spots in Provence, including Marseille bay (Table 1 and FIG. 2). Seawater samples were divided in eight groups of tubes (n=36). Breton Ulva lactuca were cut in pieces of 1 cm.sup.2 and put in the eight groups of tubes corresponding to spots X (RN), Y (RS) and Z (PR) in the bay of Marseille, PR and MU in Provence, TR and BR (North Brittany) and PO (South Brittany), collected one day before sampling (D1).

[0108] No bleaching was observed with sea water collected in Brittany for springtime when Ulva proliferation is the highest. Regarding the five different spots in Provence, the number of tubes where proliferation could happen was not the same. Proliferation was observed in 25 tubes/36 (69%) for the X spot that corresponds to open sea, while it was 14 tubes/36 for the Y spots and only 1 tube/36 for the Z spot, the closest of the shore. In tubes were Ulva lactuca could not grow, Ulva lactuca were becoming white under day light at 20° C. in five days with no acidity detected, as shown in FIG. 3A. This Ulva lactuca white phenotype was not similar to white dehydrated Ulva lactuca as observed in Brittany, when Ulva lactuca are staying on the shore in low tides. For tubes where Ulva lactuca could proliferate, confluence was reached after one week and acidity was observed according to the regular process of Ulva lactuca biodegradation (Dominguez and Loret, Mar Drugs. 2019 Jun. 14; 17(6). Pii: E357). As observed in natural conditions Ulva lactuca remained green under biodegradation. Sea water from Z spot (FIG. 2) were kept from D30 to D180 before to be incubated again with Breton Ulva lactuca and the proliferation were observed in 12 tubes/36 for D30 and 36 tubes/36 for D180 (FIG. 2). The active principle that promote the death of Breton Ulva lactuca cells is not a pollutant that would have produced the same effect from D1 to D180. Other Breton algae (mainly brown) were not affected with seawaters from Marseille bay (data not shown).

[0109] c) Comparison with Optical Microscopy of Ulva lactuca in Three Different States Shows that Tissue is not Disrupted in White Ulva lactuca

[0110] What happens in FIG. 3A showing white Ulva lactuca was studied at a tissue level with optical microscopy. FIG. 3B shows that the white tissue of Ulva lactuca remains unaffected with a regular organization of Ulva lactuca cells comparable to healthy Ulva lactuca, which have a thallus composed of tight cells with chlorophytes present in cytoplasm giving a green color to cells (FIG. 3C). The white color in FIG. 3B indicates that cells are dead but this death is not due to a macro predator or environmental conditions that would have disrupted the tissue organization of the alga tissue as shown in FIG. 3C. This is not a sporulation that could provide a white color. The main explanation emerging from these preliminary experiments is that a microorganism specific of Ulva lactuca control Ulva lactuca blooms in the Mediterranean Sea. Only a microorganism attack, in particular a viral attack, could explain this rapid death of Ulva lactuca cells without tissue damage. Furthermore, this hypothesis was confirmed. Indeed, when sea water is filtrated at 0.2 μm, Ulva lactuca can still turn white showing that the bleaching activity is not due to planktons, amoeba or bacteria that have a size superior to 0.2 μm.

[0111] d) Diode Array Detection Coupled to High Performance Liquid Chromatography (DAD HPLC)

[0112] Mediterranean sea water inducing bleaching was filtrated at 0.2 μm and then analyzed with a DAD HPLC that makes possible to have a UV spectral analyses of each entities eluting at different times from a hydrophobic C8 column with an acetonitrile gradient. Most of the peaks eluting between 5 to 45 min are characterized by a UV spectral signature with a maximum absorption at 243 nm and correspond to organic macromolecules call colloids. The 3D view of the DAD HPLC run shows that colloids are the major components of sea water filtrated at 0.2 μm. Three peaks have a different UV spectral signature. The peak indicated with a red arrow at 3.5 min might correspond to the presence of viral particles and is characterized by a first max. abs. at 266 nm due to nucleic acids and aromatic amino acids. The two other peaks correspond to free nucleic acids at 6 min and free proteins at 45 min characterized respectively by a max. abs. at 260 and 280 nm. When Breton Ulva lactuca are added to Mediterranean seas water for five days and when bleaching occurs, the peak corresponding to virus increases significantly with a maximum absorbance at 266 nm ranging from 7 to 32 mAU. Interestingly, this peak compatible with viral particles increases 78%, while the colloid peaks decrease (due probably to Ulva eating).

[0113] e) Virus-Like Particle Stain and Fluorescence Microscopy

[0114] Mediterranean sea water without and with Ulva lactuca was filtrated at 0.2 μm and then stained with an aromatic compounds called SYBR Gold dye (for N′,N′-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine) that binds preferentially to DNA. This dye is widely used in virology to stain and visualize virus like particles (VLPs) present in seawater and other aquatic samples. There are hundreds of published reports using this methodology to count and detected viruses in biological samples (Shibata et al., Aquat Microb Ecol. 2006, 43, 223-231). FIG. 4A-C shows that fluorescence microscopy after SYBR staining reveals a high viral production when Ulva lactuca is added to sea water. This high viral production is already significant when Ulva lactuca are still green. However, when Ulva lactuca become white the viral abundance reaches 6.5×10.sup.8 viruses/ml, which is an atypical viral high concentration (FIG. 4D). This experiment suggests that viruses are actively produced and released with higher rates when Ulva lactuca become bleached.

[0115] 3) Discussion

[0116] The average nitrate concentration in sea water worldwide and in Mediterranean seas is about 1 μM. If nitrate concentration was the reason for the absence of Ulva lactuca proliferation at Marseille, one could have expected nitrate concentration up to 100 μM on Brittany north coast where green tides are the most important in Western Europe particularly for springtime, but such is not the case excepted in river or fjord (Table 1). Nitrate concentrations are variable regarding seasons. In Brittany north coast there is an average of 5 μM at the marine station of Roscoff that went to almost 10 μM in winter to 1 μM in summer in 2018 and 2019 (Service d'Observation en Milieu Littoral (SOMLIT), INSU-CNRS, Roscoff and Marseille” http://somlit-db.epoc.u-bordeauxl.fr/bdd.php). Other parameters such as pH and conductimetry are also variable regarding season at Roscoff (see http://somlit-db.epoc.u-bordeauxl.fr/bdd.php). Data at BR in Briton North coast (Table 1) are in the range of the nitrate concentration observed at Roscoff and a same variability regarding season is observed at Marseille (http://somlit-db.epoc.u-bordeauxl.fr/bdd.php). This variability regarding seasons was also observed in Galicia at the west of Spain (Villares et al., Bol. Inst. Esp. Oceanogr. 1999, 15, 337-341). It is also important to point out that the origin of Ulva green tides does not necessarily come from Brittany coasts. Ulva proliferations are observed in the middle of North Atlantic and Ulva drift to Brittany due to dominant western winds in North Atlantic. Chlorophyll anomalies appear to be more and more frequent in North Atlantic and the main cause of green tides could be due mainly to the global warming. A continuous survey of nitrate concentrations was not performed because the purpose was to compare with the same analytical method and only for springtime if nitrate concentration could be much lower in Marseille compare to Breton north coast to explain the absence of Ulva proliferation. This was found not to be the case and a very interesting survey carried out in Marseille bay in 2007 and 2008 by IFREMER shows that nitrate concentration can be as high in open sea nearby Marseille that it is in North Brittany coast with nitrate concentration superior to 8 μM measured three times in June 2008 (Young et al., PLoS One. 2016, 11(5):e0155152). Furthermore, the chlorophyll activity appears to be abnormally low (0.2 μg/ml) regarding nutriments concentration and can grow up to 1 μg/ml just for very short period that might be explain by viral lyses controlling proliferation (Young et al., PLoS One. 2016, 11(5):e0155152).

[0117] Viruses are well known to participate in the control of microalgae bloom but this has so far not been demonstrated for macroalgae. Virus control of microalgae blooms were recently observed in the USA with the two microalgae Aureococcus anophagefferens inducing harmful bloom algae on the east coast (Moniruzzaman et al., Front Microbiol. 2018, 9,752-758) or Tetraselmis in Hawaii (Schvarcz and Steward, Virology 2018, 518,423-433). In the two cases, it was due to viruses recently discovered called giant viruses. Giant viruses were first discovered in amoebae (La Scola et al., Science 2003, 299, 2033-2038). It is interesting to note that moving amoebae were detected in the microscope in FIG. 3B. While most viruses known since one century had size <400 nm, with for instance 160 nm for HIV or 20 nm for the smallest (Parvoviridae infecting pigs), giant viruses have size up to 1 μm. Since then, giant viruses have been discovered all over the world infecting many species, particularly marine species (Abergel et al., FEMS Microbiol Rev 2015, 39, 779-796).

[0118] Ulva lactuca blooms will remain a source of troubles that could grow with the global warming. However, there is a natural law hypothesis called “Kill the winner” that may interrupt this Ulva lactuca success story. When there is proliferation of a species, a predator of this species appears to control this proliferation. Among the most powerful natural predators, the biggest is not necessarily the most efficient. The apparition of a predator specific of Ulva lactuca may be a consequence of the high concentration of predators in the Mediterranean Sea, such as viruses, marine bacteria and amoebae. Viruses are the most abundant biological entities in seawaters that can be found even in the bathypelagic (1,000 to 2,000 m) zone and the Mediterranean Sea appears to have the highest concentration mainly in the epipelagic (5 m) zone. If prokaryotes and unicellular algae appear to be the main viral hosts, only 9% of sequences obtained from the viral fraction had an identifiable viral origin and no research was carried out with sequences specific of giant viruses. The predator dynamics can be different regarding temperatures, which could explain why Ulva lactuca disappear in the bay of Marseille for springtime when temperature reach 15° C.

[0119] The experiments described for this invention demonstrate that it is possible to control Breton Ulva lactuca proliferation with water samples from Marseille Bay. This control is made by a microscopic living active principle and its concentration is not the same regarding different spots in the bay of Marseille. Of importance, the sample collections in the spring of 3 consecutive years (2018, 2019, 2020) at the same spot (PR) in the bay of Marseille were all able to achieve Ulva lactuca bleaching, indicating that the microorganism, in particular the virus, was persistently retrieved in this marine environment.