# Effect of the blue pigment produced by the tropical diatom Haslea nusantara on marine organisms from different trophic levels and its bioactivity

Fiddy S Prasetiya, Sunarto Sunarto, Eri Bachtiar, Mochamad U K Agung, Bram Nathanael, Ardian C Pambudi, Ajeng D Lestari, Sri Astuty, Jean-Luc Mouget

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## To cite this version:

Fiddy S Prasetiya, Sunarto Sunarto, Eri Bachtiar, Mochamad U K Agung, Bram Nathanael, et al.. Effect of the blue pigment produced by the tropical diatom Haslea nusantara on marine organisms from different trophic levels and its bioactivity. Aquaculture Reports, 2020, 17, 10.1016/j.aqrep.2020.100389. hal-04581669

HAL Id: hal-04581669

https://hal.science/hal-04581669

Submitted on 21 May 2024

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# Effect of the blue pigment produced by the tropical diatom *Haslea nusantara* on marine organisms from different trophic levels and its bioactivity

<figure><img src="image_1.png" /></figure>

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Fiddy S. Prasetiya<sup>a,b,1,*</sup>, Sunarto Sunarto<sup>a,1,*</sup>, Eri Bachtiar<sup>a</sup>, Mochamad U.K. Agung<sup>a</sup>, Bram Nathanael<sup>c</sup>, Ardian C. Pambudi<sup>c</sup>, Ajeng D. Lestari<sup>c</sup>, Sri Astuty<sup>a</sup>, Jean-Luc Mouget<sup>d</sup>

## ARTICLE INFO

### Keywords:

* Haslea nusantara

* Haslea ostrearia

* Marennine

* Growth inhibition

* Toxicity

* Antibacterial activity

## ABSTRACT

Marennine is the blue-green pigment produced by the temperate ubiquitous marine diatom *Haslea ostrearia*. This pigment is water-soluble and excreted in seawater during *H. ostrearia* growth and ageing. Previous studies demonstrated the adverse effects of *H. ostrearia* culture supernatant that contains marennine (Blue Water, BW) against marine organisms. In tropical waters in the Southern hemisphere, a new diatom species has recently been discovered, *Haslea nusantara*, which shares a peculiarity with *H. ostrearia* in producing a blue-green, marennine-like pigment. However, little is known about the characteristics of the marennine-like pigment from *H. nusantara* and its bioactivity. The present study aimed to determine spectral characteristics of BW from *H. nusantara* in comparison to BW containing marennine of *H. ostrearia*, using UV-vis spectrophotometry, and to investigate the effects of BWs from these two species on several marine organisms from different trophic levels including autotrophs (*Isochrysis galbana*) and heterotrophs (*Artemia salina*). Additionally, the antibacterial activities of these BWs were also evaluated on two bacterial pathogens that are relevant to aquaculture, *Vibrio harveyi* and *Staphylococcus aureus*. The results show that both BWs from *H. ostrearia* and *H. nusantara* at 0.5 µg mL⁻<fnref n="1" /> significantly reduce the growth of *I. galbana*. At similar range of concentration, toxicity tests reveal that the both BWs from blue *Haslea* species significantly increase mortality rate of *A. salina* to a different extent. Additionally, both BWs from *H. nusantara* and *H. ostrearia* can inhibit the growth of tested bacterial pathogens. This study provides new insight into the different toxicity in the bioactivity of pigments obtained from the two species of blue diatoms from the same genus. Our results also demonstrate the need to determine concentrations that optimize beneficial effects and critical concentrations not to be exceeded before considering the use of marennine as natural antibacterial agents in the field of aquaculture.

## 1. Introduction

In the last decades, increased attention has been paid to the commercial and industrial potential of microalgae. A limited number of species are currently being studied for their ability to synthesize secondary metabolites of interest (pigments, lipids, etc) for biofuel, food or pharmaceutical industry (Falaise et al., 2016; Gastineau et al., 2012b, 2014; Prasetiya et al., 2016; Vo et al., 2011). Due to over-utilization of the terrestrial resources, a constant effort is actually provided for both finding and exploiting microalgae resources and developing their

putative commercial outcomes or industrial applications, e.g., natural drug development from algal compound with antiproliferative activities against tumorous cells and antimicrobial activity (Falaise et al., 2016; Gastineau et al., 2012b, 2012a; Olaizola, 2003). Indeed, microalgae species could be an innovative starting point in drug design. Screening metabolic compounds is essential in targeting chemical compounds that play a role as antibacterial agents. Thus, studies based on bioactive compounds extracted from microalgae could be a sound strategy for disease therapy in aquaculture, and for increasing resistance to pathogen or increasing activity of the immune system in aquatic species

<footnote><sup>a</sup> Marine Science Department, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jl. Raya Bandung Sumedang KM. 21, 45363 Jatinangor, Indonesia</footnote>

<footnote><sup>b</sup> Research Center of Biotechnology and Bioinformatics, Jl. Singaperbangsa No. 2, 40132 Bandung, Indonesia</footnote>

<footnote><sup>c</sup> Study Programme of Marine Science, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jl. Raya Bandung Sumedang KM. 21, 45363 Jatinangor, Indonesia</footnote>

<footnote><sup>d</sup> FR CNRS 3473 IUML, Mer-Molécules-Santé (MMS), Le Mans Université, Avenue O. Messiaen, 72085 Le Mans Cedex 9, France</footnote>

<footnote>* Corresponding authors at: Marine Science Department, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jl. Raya Bandung Sumedang KM. 21, 45363 Jatinangor, Indonesia.
E-mail address: fiddy.semba.prasetiya@unpad.ac.id (F.S. Prasetiya).</footnote>

<footnote n="1">¹ Fiddy S. Prasetiya and Sunarto Sunarto contributed equally to this work.</footnote>
# Effect of the blue pigment produced by the tropical diatom *Haslea nusantara* on marine organisms from different trophic levels and its bioactivity

<figure><img src="image_3.png" /></figure>

Check for
updates

Fiddy S. Prasetiya<sup>a,b,1,*</sup>, Sunarto Sunarto<sup>a,1,*</sup>, Eri Bachtiar<sup>a</sup>, Mochamad U.K. Agung<sup>a</sup>, Bram Nathanael<sup>c</sup>, Ardian C. Pambudi<sup>c</sup>, Ajeng D. Lestari<sup>c</sup>, Sri Astuty<sup>a</sup>, Jean-Luc Mouget<sup>d</sup>

## ARTICLE INFO

### Keywords:

Haslea nusantara

Haslea ostrearia

Marennine

Growth inhibition

Toxicity

Antibacterial activity

## ABSTRACT

Marennine is the blue-green pigment produced by the temperate ubiquitous marine diatom *Haslea ostrearia*. This pigment is water-soluble and excreted in seawater during *H. ostrearia* growth and ageing. Previous studies demonstrated the adverse effects of *H. ostrearia* culture supernatant that contains marennine (Blue Water, BW) against marine organisms. In tropical waters in the Southern hemisphere, a new diatom species has recently been discovered, *Haslea nusantara*, which shares a peculiarity with *H. ostrearia* in producing a blue-green, marennine-like pigment. However, little is known about the characteristics of the marennine-like pigment from *H. nusantara* and its bioactivity. The present study aimed to determine spectral characteristics of BW from *H. nusantara* in comparison to BW containing marennine of *H. ostrearia*, using UV-vis spectrophotometry, and to investigate the effects of BWs from these two species on several marine organisms from different trophic levels including autotrophs (*Isochrysis galbana*) and heterotrophs (*Artemia salina*). Additionally, the antibacterial activities of these BWs were also evaluated on two bacterial pathogens that are relevant to aquaculture, *Vibrio harveyi* and *Staphylococcus aureus*. The results show that both BWs from *H. ostrearia* and *H. nusantara* at 0.5 µg mL⁻<fnref n="1" /> significantly reduce the growth of *I. galbana*. At similar range of concentration, toxicity tests reveal that the both BWs from blue *Haslea* species significantly increase mortality rate of *A. salina* to a different extent. Additionally, both BWs from *H. nusantara* and *H. ostrearia* can inhibit the growth of tested bacterial pathogens. This study provides new insight into the different toxicity in the bioactivity of pigments obtained from the two species of blue diatoms from the same genus. Our results also demonstrate the need to determine concentrations that optimize beneficial effects and critical concentrations not to be exceeded before considering the use of marennine as natural antibacterial agents in the field of aquaculture.

## 1. Introduction

In the last decades, increased attention has been paid to the commercial and industrial potential of microalgae. A limited number of species are currently being studied for their ability to synthesize secondary metabolites of interest (pigments, lipids, etc) for biofuel, food or pharmaceutical industry (Falaise et al., 2016; Gastineau et al., 2012b, 2014; Prasetiya et al., 2016; Vo et al., 2011). Due to over-utilization of the terrestrial resources, a constant effort is actually provided for both finding and exploiting microalgae resources and developing their

putative commercial outcomes or industrial applications, e.g., natural drug development from algal compound with antiproliferative activities against tumorous cells and antimicrobial activity (Falaise et al., 2016; Gastineau et al., 2012b, 2012a; Olaizola, 2003). Indeed, microalgae species could be an innovative starting point in drug design. Screening metabolic compounds is essential in targeting chemical compounds that play a role as antibacterial agents. Thus, studies based on bioactive compounds extracted from microalgae could be a sound strategy for disease therapy in aquaculture, and for increasing resistance to pathogen or increasing activity of the immune system in aquatic species

<footnote><sup>a</sup> Marine Science Department, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jl. Raya Bandung Sumedang KM. 21, 45363 Jatinangor, Indonesia</footnote>

<footnote><sup>b</sup> Research Center of Biotechnology and Bioinformatics, Jl. Singaperbangsa No. 2, 40132 Bandung, Indonesia</footnote>

<footnote><sup>c</sup> Study Programme of Marine Science, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jl. Raya Bandung Sumedang KM. 21, 45363 Jatinangor, Indonesia</footnote>

<footnote><sup>d</sup> FR CNRS 3473 IUML, Mer-Molécules-Santé (MMS), Le Mans Université, Avenue O. Messiaen, 72085 Le Mans Cedex 9, France</footnote>

<footnote>* Corresponding authors at: Marine Science Department, Faculty of Fisheries and Marine Sciences, Universitas Padjadjaran, Jl. Raya Bandung Sumedang KM. 21, 45363 Jatinangor, Indonesia.</footnote>

<footnote>E-mail address: fiddy.semba.prasetiya@unpad.ac.id (F.S. Prasetiya).</footnote>

<footnote n="1">¹ Fiddy S. Prasetiya and Sunarto Sunarto contributed equally to this work.</footnote>
(Gastineau et al., 2018, 2014; Prasetiya et al., 2017a, b; Prasetiya et al., 2019b, 2016).

Among microalgae, diatoms, a phytoplankton group with siliceous cell wall structure, attract many attention in the field of biotechnology for various applications such as pharmaceutical, medical, nano-technology as well as aquaculture (Bergé and Barnathan, 2005; Bergé et al., 1999; Brown et al., 1997; Dunstan et al., 1994; Gastineau et al., 2014; Gordon et al., 2009; Lopez et al., 2005; Townley et al., 2008). The blue diatom *Haslea ostrearia* has a peculiarity in producing the blue pigment marennine. This water-soluble pigment has two distinct forms, the intracellular form (IMn) that is accumulated in the apices of the cells, and the extracellular form (EMn) that is released into the medium (Pouvreau et al., 2006). Marennine is responsible for greening oyster, a natural phenomenon famously recognized in the Atlantic Coast of France due to the erratic proliferation of *H. ostrearia* in oyster ponds and the fixation of marennine oyster gills (Sprat, 1669; Gastineau et al., 2018). Indeed, this phenomenon positively contributes to the oyster farmers since the selling price of the green oysters is relatively higher than that of ordinary oysters. Recent studies revealed that new species of blue *Haslea* with marennine-like pigments have been discovered not only in temperate, Northern Hemisphere (Gastineau et al., 2012c, 2014; Gastineau et al., 2016) but also in tropical region of the Southern Hemisphere (Prasetiya et al., 2019a).

Although it has been observed that *H. ostrearia* is not preferred as the main diet in oyster farming due to their size (Prasetiya et al., 2017c) and possibly lower nutritional values when compared to *Skeletonema costatum* (Piveteau, 1999), several studies also showed that marennine, but also marennine-like pigments in both intracellular and extracellular purified forms have several biological activities such as antibacterial activity against important pathogens in aquaculture, antialgae and antioxidant properties (Pouvreau et al., 2007a, 2008; Gastineau et al., 2012b, 2014; Turcotte et al., 2016; Prasetiya et al., 2016). Therefore, these pigments have possibly a good potential for application in aquaculture hatcheries, however, the use of blue waters (BWs, the culture supernatants of blue *Haslea* species) in aquaculture hatcheries seems to be more realistic than using the purified forms, which are costly and require a long process (Pouvreau et al., 2006). However, it has been shown that in certain cases, BW or marennine could also have an adverse effect on the cultured organisms. For instance, diminution of clearance rate and oxygen consumption in mussels and oysters that are found to be species- and size-dependent (Gastineau et al., 2018; Prasetiya et al., 2019b, 2017b). Additionally, recent findings also revealed that marennine not only in its purified form but also as BW can be toxic not only to bivalve larvae but also sea urchins *Sphaerechinus granularis*, barnacle *Chthamalus bisinuatus* as well as winter flounder fish *Pseudopleuronectes americanus* larvae (Falaise et al., 2019a). Hence, further assessment should be conducted to support the application of this pigment in aquaculture practice. Additionally, the searching of prophylactic agent for tropical aquaculture by using a tropical species of blue diatom such *H. nusantara* merits further investigation.

The first objective of this work was thus to determine the spectral characteristics of pigment containing supernatants (BWs) from the two distinct *Haslea* blue species (*H. ostrearia* isolated from the Atlantic Coast of France, and *H. nusantara*, the indigenous species isolated from Indonesia) and to study the effects of their respective BWs on the growth rate of the microalga *Isochrysis galbana*. Secondly, the toxic effect of these BWs on the brine shrimp *Artemia salina* was also assessed. Thirdly, we determine the antibacterial activities of BWs from different *Haslea* species against bacterial pathogens in aquaculture. The prospective utilization of these new substances is discussed, especially as natural antibacterial agents in the fields of aquaculture.

## 2. Materials and methods

### 2.1. Culture conditions

Three marine microalgal strains were used in this work: *Haslea ostrearia* obtained from the Nantes Culture Collection Université de Nantes (NCC-148.7 Nantes, France), *Haslea nusantara* isolated from Semak Daun Island, Indonesia (5°43'49.27" S, 106°34'20.27" E) stored during the experiments in the laboratory of Microbiology and Biotechnology in Universitas Padjadjaran and *Isochrysis galbana* (provided by the Aquaculture Research Station of Jepara). All *Haslea* species were cultured under non-axenic conditions in sterilized 500-mL Erlenmeyer flasks, containing 250 mL of artificial seawater medium (Mouget et al., 2009; Prasetiya et al., 2016) at 16 ± 1 and 26 ± 1 °C for *H. ostrearia* and *H. nusantara* as well as *I. galbana*, respectively. Cultures were grown at an irradiance of 100 µmol photons m⁻² s⁻¹ provided by Philips TLD 36 W/965 fluorescent tubes or a high-intensity discharge lamp (Philips MHN-TD, 250 W). Irradiance was measured with a Li-Cor LI-189 quantum meter and a 2π Li-Cor Q21284 quantum sensor, in a 14:10 h light/dark cycle.

### 2.2. Marennine blue water (BW) preparation

Both *H. ostrearia* and *H. nusantara* were grown on batch mode until pigment concentration in the culture supernatant reached a maximum of 5–10 µg mL⁻¹. At the stationary phase, the biomass was then harvested and filtered through Millipore 0.22 µm syringe filters. This filtered culture supernatant will therefore be referred as Blue Water (BW). Marennine concentration in BW was then estimated on a cell-free culture supernatant (filtered through Millipore 0.2 µm syringe filters) using a spectrophotometer (Cary 100 Bio UV–vis, Agilent Technologies) and the Beer–Lambert's equation (ε677 = 12.13 L g⁻¹ cm⁻¹) as described in Pouvreau et al. (2006) and Prasetiya et al. (2016). BWs from both *H. ostrearia* and *H. nusantara* were stored in the dark at 4 °C before use.

### 2.3. Effect of BW on Isochrysis galbana

Effect of BW on algal growth was carried out at the beginning of the exponential phase in the 24-well microplates Corning Costar Corp., Sigma Aldrich) in triplicate for the concentration 0 (control), 0.5, 1 and 2 µg mL⁻¹. Algae inoculum *I. galbana* was added from the 500 mL Erlenmeyer flask (as explained in the section "culture conditions") to an initial cell density of 5000 cells mL⁻¹. Cell density was measured within 72 h of experiment and cell counts were performed on each sampling day. The samples were gently stirred with the magnetic stirrer in the microplate prior to cell counting to avoid aggregates. Furthermore, cell growth was monitored by measuring cell densities with Neubauer counting chambers under a digital microscope (AmScope Co.) with 40x of magnification. Growth rate (GR) was calculated as described in Prasetiya et al. (2016); GR (day⁻¹) = [ln (initial number cells) - ln (final number cells)]/t. Afterwards, the percentage of growth rate (%GR) was calculated based on the obtained GR value.

### 2.4. Effect of BW on brine shrimp Artemia salina

The eggs of brine shrimp *A. salina* were obtained from the Golden West Artemia Corp. They were hatched in artificial sea water prepared from Instant Ocean 40 g L⁻¹ supplemented with 6 mg L⁻¹ dried yeast and aerated with an aquarium pump. After 48 h of incubation in 25 °C, nauplii were collected with a Pasteur pipette after attracting the organisms to one side of the vessel with a light source. Nauplii were separated from the eggs by pipetting them 2–4 times in small beakers containing sea water prior to toxicity test.

The toxicity test was performed following the protocol from Sorgeloos et al. (1978). Briefly, a suspension of nauplii containing 10
(Gastineau et al., 2018, 2014; Prasetiya et al., 2017a, b; Prasetiya et al., 2019b, 2016).

Among microalgae, diatoms, a phytoplankton group with siliceous cell wall structure, attract many attention in the field of biotechnology for various applications such as pharmaceutical, medical, nano-technology as well as aquaculture (Bergé and Barnathan, 2005; Bergé et al., 1999; Brown et al., 1997; Dunstan et al., 1994; Gastineau et al., 2014; Gordon et al., 2009; Lopez et al., 2005; Townley et al., 2008). The blue diatom *Haslea ostrearia* has a peculiarity in producing the blue pigment marennine. This water-soluble pigment has two distinct forms, the intracellular form (IMn) that is accumulated in the apices of the cells, and the extracellular form (EMn) that is released into the medium (Pouvreau et al., 2006). Marennine is responsible for greening oyster, a natural phenomenon famously recognized in the Atlantic Coast of France due to the erratic proliferation of *H. ostrearia* in oyster ponds and the fixation of marennine oyster gills (Sprat, 1669; Gastineau et al., 2018). Indeed, this phenomenon positively contributes to the oyster farmers since the selling price of the green oysters is relatively higher than that of ordinary oysters. Recent studies revealed that new species of blue *Haslea* with marennine-like pigments have been discovered not only in temperate, Northern Hemisphere (Gastineau et al., 2012c, 2014; Gastineau et al., 2016) but also in tropical region of the Southern Hemisphere (Prasetiya et al., 2019a).

Although it has been observed that *H. ostrearia* is not preferred as the main diet in oyster farming due to their size (Prasetiya et al., 2017c) and possibly lower nutritional values when compared to *Skeletonema costatum* (Piveteau, 1999), several studies also showed that marennine, but also marennine-like pigments in both intracellular and extracellular purified forms have several biological activities such as antibacterial activity against important pathogens in aquaculture, antialgae and antioxidant properties (Pouvreau et al., 2007a, 2008; Gastineau et al., 2012b, 2014; Turcotte et al., 2016; Prasetiya et al., 2016). Therefore, these pigments have possibly a good potential for application in aquaculture hatcheries, however, the use of blue waters (BWs, the culture supernatants of blue *Haslea* species) in aquaculture hatcheries seems to be more realistic than using the purified forms, which are costly and require a long process (Pouvreau et al., 2006). However, it has been shown that in certain cases, BW or marennine could also have an adverse effect on the cultured organisms. For instance, diminution of clearance rate and oxygen consumption in mussels and oysters that are found to be species- and size-dependent (Gastineau et al., 2018; Prasetiya et al., 2019b, 2017b). Additionally, recent findings also revealed that marennine not only in its purified form but also as BW can be toxic not only to bivalve larvae but also sea urchins *Sphaerechinus granularis*, barnacle *Chthamalus bisinuatus* as well as winter flounder fish *Pseudopleuronectes americanus* larvae (Falaise et al., 2019a). Hence, further assessment should be conducted to support the application of this pigment in aquaculture practice. Additionally, the searching of prophylactic agent for tropical aquaculture by using a tropical species of blue diatom such *H. nusantara* merits further investigation.

The first objective of this work was thus to determine the spectral characteristics of pigment containing supernatants (BWs) from the two distinct *Haslea* blue species (*H. ostrearia* isolated from the Atlantic Coast of France, and *H. nusantara*, the indigenous species isolated from Indonesia) and to study the effects of their respective BWs on the growth rate of the microalga *Isochrysis galbana*. Secondly, the toxic effect of these BWs on the brine shrimp *Artemia salina* was also assessed. Thirdly, we determine the antibacterial activities of BWs from different *Haslea* species against bacterial pathogens in aquaculture. The prospective utilization of these new substances is discussed, especially as natural antibacterial agents in the fields of aquaculture.

# 2. Materials and methods

## 2.1. Culture conditions

Three marine microalgal strains were used in this work: *Haslea ostrearia* obtained from the Nantes Culture Collection Université de Nantes (NCC-148.7 Nantes, France), *Haslea nusantara* isolated from Semak Daun Island, Indonesia (5°43'49.27" S, 106°34'20.27" E) stored during the experiments in the laboratory of Microbiology and Biotechnology in Universitas Padjadjaran and *Isochrysis galbana* (provided by the Aquaculture Research Station of Jepara). All *Haslea* species were cultured under non-axenic conditions in sterilized 500-mL Erlenmeyer flasks, containing 250 mL of artificial seawater medium (Mouget et al., 2009; Prasetiya et al., 2016) at 16 ± 1 and 26 ± 1 °C for *H. ostrearia* and *H. nusantara* as well as *I. galbana*, respectively. Cultures were grown at an irradiance of 100 µmol photons m⁻² s⁻¹ provided by Philips TLD 36 W/965 fluorescent tubes or a high-intensity discharge lamp (Philips MHN-TD, 250 W). Irradiance was measured with a Li-Cor LI-189 quantum meter and a 2π Li-Cor Q21284 quantum sensor, in a 14:10 h light/dark cycle.

## 2.2. Marennine blue water (BW) preparation

Both *H. ostrearia* and *H. nusantara* were grown on batch mode until pigment concentration in the culture supernatant reached a maximum of 5–10 µg mL⁻¹. At the stationary phase, the biomass was then harvested and filtered through Millipore 0.22 µm syringe filters. This filtered culture supernatant will therefore be referred as Blue Water (BW). Marennine concentration in BW was then estimated on a cell-free culture supernatant (filtered through Millipore 0.2 µm syringe filters) using a spectrophotometer (Cary 100 Bio UV–vis, Agilent Technologies) and the Beer-Lambert's equation (ε677 = 12.13 L g⁻¹ cm⁻¹) as described in Pouvreau et al. (2006) and Prasetiya et al. (2016). BWs from both *H. ostrearia* and *H. nusantara* were stored in the dark at 4 °C before use.

## 2.3. Effect of BW on Isochrysis galbana

Effect of BW on algal growth was carried out at the beginning of the exponential phase in the 24-well microplates Corning Costar Corp., Sigma Aldrich) in triplicate for the concentration 0 (control), 0.5, 1 and 2 µg mL⁻¹. Algae inoculum *I. galbana* was added from the 500 mL Erlenmeyer flask (as explained in the section "culture conditions") to an initial cell density of 5000 cells mL⁻¹. Cell density was measured within 72 h of experiment and cell counts were performed on each sampling day. The samples were gently stirred with the magnetic stirrer in the microplate prior to cell counting to avoid aggregates. Furthermore, cell growth was monitored by measuring cell densities with Neubauer counting chambers under a digital microscope (AmScope Co.) with 40x of magnification. Growth rate (GR) was calculated as described in Prasetiya et al. (2016); GR (day⁻¹) = [ln (initial number cells) - ln (final number cells)]/t. Afterwards, the percentage of growth rate (%GR) was calculated based on the obtained GR value.

## 2.4. Effect of BW on brine shrimp Artemia salina

The eggs of brine shrimp *A. salina* were obtained from the Golden West Artemia Corp. They were hatched in artificial sea water prepared from Instant Ocean 40 g L⁻¹ supplemented with 6 mg L⁻¹ dried yeast and aerated with an aquarium pump. After 48 h of incubation in 25 °C, nauplii were collected with a Pasteur pipette after attracting the organisms to one side of the vessel with a light source. Nauplii were separated from the eggs by pipetting them 2–4 times in small beakers containing sea water prior to toxicity test.

The toxicity test was performed following the protocol from Sorgeloos et al. (1978). Briefly, a suspension of nauplii containing 10
individuals was added to each well in a 24-well microplate (Corning Costar Corp., Sigma Aldrich) that corresponded to 0, 0.5, 1 and 2 µg mL⁻¹ of marennine. Experiments were run in triplicate with plates incubated at 25 °C for 24 h during 72 h. The plates were then verified under a binocular microscope (AmScope, × 12.5) and the numbers of non-motile (dead) nauplii in each well were counted to calculate the median lethal concentration (LC₅₀).

## 2.5. Assessment of antibacterial activities

The antibacterial activities of BW from both *Haslea* species were studied using two different strains of marine bacteria, the Gram-negative Vibrio harveyi, and the Gram-positive Staphylococcus aureus, which are pathogens often found among Indonesian aquaculture facilities and frequently used in biological assays, following the protocol from Hellio et al. (2000). Briefly, stock cultures of bacteria were maintained in an incubator at 30 °C, frequently diluted with fresh medium, containing steam-sterilised sea water mixed with peptone at a final concentration of 5 g L⁻¹. The disc diffusion method from Hellio et al. (2001) in agar-plated Petri dishes was slightly modified, using sterile Whatman filter paper discs with a diameter of 6 mm (Whatman International Ltd.). The BW (both from *H. nusantara* and *H. ostrearia*) was diluted in sterile sea water at concentrations of 0, 5, 6, 7, 8, 9 and 10 µg mL⁻¹, and filtered on a 0.22 µm filter in order to avoid the presence of cells of *Haslea* or dead frustules that may alter the experiment. A total of 100 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. Other control discs were inoculated with sterile distilled water to eliminate possible salt influence, while control positive discs resulted from the use of the antibiotic chloramphenicol (30 µg mL⁻¹, Sigma Aldrich). A dilution of the bacterial culture in nutrient broth was made a day before the experiments started. Aliquots (0.1 mL) of the culture (10⁶ CFU mL⁻¹) were used for agar inoculation. After two days of incubation at 30 °C, the antibacterial activity was estimated by measuring the radius of the inhibition zone around the discs. All inhibition assays were performed in triplicate.

## 2.6. Data analysis

Statistical analysis was performed using Graphpad Prism 5 for Mac OS X software. All data were tested for assumptions of normality and homoscedasticity by the Bartlett's test. The fourth root square transformation was performed when required. The percent growth rate of *I. galbana* percent growth rate for each concentration and endpoints, as well as the inhibition zone, were analyzed using one-way ANOVA, followed by Tukey's post-hoc test. Significance of differences were defined at *p* < 0.05. The LC₅₀ values for dead nauplii were then calculated by using Graphpad Prism 5 for Mac OS X software.

# 3. Results

## 3.1. Marennine and Marennine-like pigment from *Haslea ostrearia* and *H. Nusantara*

Spectral characteristics of both types of pigment solutions were assessed using UV-vis spectrophotometer. Extracellular marennine-like pigment of *H. nusantara* in BW solutions displayed similar signatures to that of *H. ostrearia*. This is indicated by two peaks, one in the UV region and the other in the red part of the spectrum (Fig. 1). The maximum absorbance of BWs for *H. nusantara* and *H. ostrearia* at neutral pH in the visible part of the spectra was observed at 663 and 666 nm, respectively (Fig. 1).

## 3.2. Effect of BW on percent growth rate of Isochrysis galbana

In this study, the effect of BWs from *H. nusantara* and *H. ostrearia* on

<figure><img src="image_2.png" /><figcaption>Fig. 1. Spectral characteristics of *Haslea nusantara* and *Haslea ostrearia* extra-cellular pigment solutions (BWs) from 300 to 700 nm at pH 7.</figcaption></figure>

the percent growth rate of *I. galbana* was tested. The percent growth rate of *I. galbana* was affected by both pigment solutions (Fig. 2). ANOVA analysis showed that both BWs from *H. nusantara* and *H. ostrearia* significantly reduced the percent growth rate of *I. galbana* (Fig. 2A, F₃,₁₁ = 66.49, *P* value = 0.001 and Fig. 2B, F₃,₁₁ = 64.06, *P* value = 0.001, respectively).

## 3.3. Effect of BW on *Artemia salina*

In this study, mortality of *A. salina* due to exposure to BW containing marennine-like pigments was indicated by no motile activity and change in body color from white to transparent (Fig. 3). After a 24 h exposure to BW from *H. nusantara* and *H. ostrearia*, the percentage of mortality of *A. salina* was assessed. The median lethal effect concentration (LC₅₀) of BW from *H. nusantara* on *A. salina* for 24, 48 and 72 h was 0.707, 1.086 and 0.504 µg mL⁻¹, respectively (Fig. 4A). On the other hand, LC₅₀ value of BW from *H. ostrearia* was 0.701, 2.614 and 0.552 µg mL⁻¹ for 24, 48 and 72 h of exposure, respectively (Fig. 4B). There was no significant differences between LC₅₀ of both pigments for 24 h of exposure on *A. salina* (*p*-value > 0.05). However, it was found that BW from *H. nusantara* significantly affect percent mortality of *A. salina* after 48 h of exposure on this pigment (*p*-value < 0.05). This effect was found to be larger than what was observed in *H. ostrearia*.

## 3.4. Antibacterial activity of BW from *Haslea nusantara* and *H. Ostrearia*

In this study, the effects of the pigment solutions (BWs) produced by *H. nusantara* and *H. ostrearia* on the two marine bacteria tested are illustrated in Fig. 5. Both BWs inhibited the growth of *V. harveyi* and *S. aureus*, up to a concentration of 10 µg mL⁻¹ (Fig. 5). It was shown that BW from *H. nusantara* at concentration 9 µg mL⁻¹ had the highest radius of inhibition (2.19 mm) against *V. harveyi* (Fig. 5A). Whereas the highest inhibition of BW *H. nusantara* against *S. aureus* was found at concentration 7 µg mL⁻¹ (Fig. 5B).

As for *H. ostrearia*, different concentrations of BW significantly affected the radius of inhibition for *V. harveyi* tested (Fig. 5C, *p* < 0.05). Marennine from *H. ostrearia* at concentration 10 µg mL⁻¹ had the highest radius of inhibition (1.1 mm) for *V. harveyi* compared to others. ANOVA revealed that BW of *H. ostrearia* significantly inhibited the growth of *S. aureus* (Fig. 5D, *p* < 0.05). Tukey post-hoc test showed
individuals was added to each well in a 24-well microplate (Corning Costar Corp., Sigma Aldrich) that corresponded to 0, 0.5, 1 and 2 µg mL⁻¹ of marennine. Experiments were run in triplicate with plates incubated at 25 °C for 24 h during 72 h. The plates were then verified under a binocular microscope (AmScope, × 12.5) and the numbers of non-motile (dead) nauplii in each well were counted to calculate the median lethal concentration (LC₅₀).

## 2.5. Assessment of antibacterial activities

The antibacterial activities of BW from both *Haslea* species were studied using two different strains of marine bacteria, the Gram-negative Vibrio harveyi, and the Gram-positive Staphylococcus aureus, which are pathogens often found among Indonesian aquaculture facilities and frequently used in biological assays, following the protocol from Hellio et al. (2000). Briefly, stock cultures of bacteria were maintained in an incubator at 30 °C, frequently diluted with fresh medium, containing steam-sterilised sea water mixed with peptone at a final concentration of 5 g L⁻¹. The disc diffusion method from Hellio et al. (2001) in agar-plated Petri dishes was slightly modified, using sterile Whatman filter paper discs with a diameter of 6 mm (Whatman International Ltd.). The BW (both from *H. nusantara* and *H. ostrearia*) was diluted in sterile sea water at concentrations of 0, 5, 6, 7, 8, 9 and 10 µg mL⁻¹, and filtered on a 0.22 µm filter in order to avoid the presence of cells of *Haslea* or dead frustules that may alter the experiment. A total of 100 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. Other control discs were inoculated with sterile distilled water to eliminate possible salt influence, while control positive discs resulted from the use of the antibiotic chloramphenicol (30 µg mL⁻¹, Sigma Aldrich). A dilution of the bacterial culture in nutrient broth was made a day before the experiments started. Aliquots (0.1 mL) of the culture (10⁶ CFU mL⁻¹) were used for agar inoculation. After two days of incubation at 30 °C, the antibacterial activity was estimated by measuring the radius of the inhibition zone around the discs. All inhibition assays were performed in triplicate.

## 2.6. Data analysis

Statistical analysis was performed using Graphpad Prism 5 for Mac OS X software. All data were tested for assumptions of normality and homoscedasticity by the Bartlett's test. The fourth root square transformation was performed when required. The percent growth rate of *I. galbana* percent growth rate for each concentration and endpoints, as well as the inhibition zone, were analyzed using one-way ANOVA, followed by Tukey's post-hoc test. Significance of differences were defined at *p* < 0.05. The LC₅₀ values for dead nauplii were then calculated by using Graphpad Prism 5 for Mac OS X software.

# 3. Results

## 3.1. Marennine and Marennine-like pigment from *Haslea ostrearia* and *H. Nusantara*

Spectral characteristics of both types of pigment solutions were assessed using UV-vis spectrophotometer. Extracellular marennine-like pigment of *H. nusantara* in BW solutions displayed similar signatures to that of *H. ostrearia*. This is indicated by two peaks, one in the UV region and the other in the red part of the spectrum (Fig. 1). The maximum absorbance of BWs for *H. nusantara* and *H. ostrearia* at neutral pH in the visible part of the spectra was observed at 663 and 666 nm, respectively (Fig. 1).

## 3.2. Effect of BW on percent growth rate of Isochrysis galbana

In this study, the effect of BWs from *H. nusantara* and *H. ostrearia* on

<figure><img src="image_4.png" /><figcaption>Fig. 1. Spectral characteristics of *Haslea nusantara* and *Haslea ostrearia* extra-cellular pigment solutions (BWs) from 300 to 700 nm at pH 7.</figcaption></figure>

the percent growth rate of *I. galbana* was tested. The percent growth rate of *I. galbana* was affected by both pigment solutions (Fig. 2). ANOVA analysis showed that both BWs from *H. nusantara* and *H. ostrearia* significantly reduced the percent growth rate of *I. galbana* (Fig. 2A, F₃,₁₁ = 66.49, *P* value = 0.001 and Fig. 2B, F₃,₁₁ = 64.06, *P* value = 0.001, respectively).

## 3.3. Effect of BW on *Artemia salina*

In this study, mortality of *A. salina* due to exposure to BW containing marennine-like pigments was indicated by no motile activity and change in body color from white to transparent (Fig. 3). After a 24 h exposure to BW from *H. nusantara* and *H. ostrearia*, the percentage of mortality of *A. salina* was assessed. The median lethal effect concentration (LC₅₀) of BW from *H. nusantara* on *A. salina* for 24, 48 and 72 h was 0.707, 1.086 and 0.504 µg mL⁻¹, respectively (Fig. 4A). On the other hand, LC₅₀ value of BW from *H. ostrearia* was 0.701, 2.614 and 0.552 µg mL⁻¹ for 24, 48 and 72 h of exposure, respectively (Fig. 4B). There was no significant differences between LC₅₀ of both pigments for 24 h of exposure on *A. salina* (*p*-value > 0.05). However, it was found that BW from *H. nusantara* significantly affect percent mortality of *A. salina* after 48 h of exposure on this pigment (*p*-value < 0.05). This effect was found to be larger than what was observed in *H. ostrearia*.

## 3.4. Antibacterial activity of BW from *Haslea nusantara* and *H. Ostrearia*

In this study, the effects of the pigment solutions (BWs) produced by *H. nusantara* and *H. ostrearia* on the two marine bacteria tested are illustrated in Fig. 5. Both BWs inhibited the growth of *V. harveyi* and *S. aureus*, up to a concentration of 10 µg mL⁻¹ (Fig. 5). It was shown that BW from *H. nusantara* at concentration 9 µg mL⁻¹ had the highest radius of inhibition (2.19 mm) against *V. harveyi* (Fig. 5A). Whereas the highest inhibition of BW *H. nusantara* against *S. aureus* was found at concentration 7 µg mL⁻¹ (Fig. 5B).

As for *H. ostrearia*, different concentrations of BW significantly affected the radius of inhibition for *V. harveyi* tested (Fig. 5C, *p* < 0.05). Marennine from *H. ostrearia* at concentration 10 µg mL⁻¹ had the highest radius of inhibition (1.1 mm) for *V. harveyi* compared to others. ANOVA revealed that BW of *H. ostrearia* significantly inhibited the growth of *S. aureus* (Fig. 5D, *p* < 0.05). Tukey post-hoc test showed
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Percent growth rate (%) of *I. galbana* exposed to different concentration of pigment solutions (BWs) from *Haslea nusantara* (A) and *Haslea ostrearia* (B). Values are mean ± standard error (SE) of mean based on three replicates per concentration. Error bars with different lower case letters are significantly different.</figcaption></figure>

that the highest radius of inhibition was found at concentration 9 µg mL⁻¹ (1.75 mm). Nevertheless, the radius of inhibition formed by chloramphenicol 30 µg mL⁻¹ was significantly higher than that of BWs from both *H. nusantara* and *H. ostrearia* for *V. harveyi* and *S. aureus* (*p* < 0.05).

# 4. Discussion

## 4.1. Blue pigments from *Haslea nusantara* and *H. Ostrearia*

In the present study, two types of extracellular pigment solutions from the two distinct blue *Haslea* species were characterized. The UV-vis spectrophotometry revealed that at pH 7, the extracellular form of the pigment produced by *H. nusantara* displays a maximum absorbance in the red region with little difference when compared to marennine (663 vs. 666 nm, respectively). This confirms that both pigments

belong to the same chemical family. Furthermore, the peaks of maximum absorbance in the red region measured in this study for BWs from *H. ostrearia* and *H. nusantara* are comparable to those observed in previous works (Pouvreau et al., 2007b, 2006; Prasetiya et al., 2019a).

## 4.2. Effect of BW on Isochrysis galbana

In this study, two types of filtered culture supernatants (BWs) from the two distinct *Haslea* blue species were tested against *I. galbana*, microalgae commonly used in aquaculture as natural feed. Both types of pigment solutions appeared to inhibit the growth of *I. galbana*. Indeed, previous studies revealed that marennine produced by *H. ostrearia* can inhibit the growth of other microalgal species, not only as purified marennine solutions (Pouvreau et al., 2007b), but also in culture supernatants (Neuville and Daste 1978; Robert and Turpin 1993; Windarto et al. 2014). Furthermore, Prasetiya et al. (2016) revealed that the

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Effect of pigment solutions (BWs) (2 µg mL⁻¹) on nauplii of *A. salina* during the toxicity experiment. The red and black circles indicate dead and alive nauplii, respectively. Scale bar is 2000 µm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Percent growth rate (%G) of *I. galbana* exposed to different concentration of pigment solutions (BWs) from *Haslea nusantara* (A) and *Haslea ostrearia* (B). Values are mean ± standard error (SE) of mean based on three replicates per concentration. Error bars with different lower case letters are significantly different.</figcaption></figure>

that the highest radius of inhibition was found at concentration 9 µg mL⁻¹ (1.75 mm). Nevertheless, the radius of inhibition formed by chloramphenicol 30 µg mL⁻¹ was significantly higher than that of BWs from both *H. nusantara* and *H. ostrearia* for *V. harveyi* and *S. aureus* (*p* < 0.05).

# 4. Discussion

## 4.1. Blue pigments from *Haslea nusantara* and *H. Ostrearia*

In the present study, two types of extracellular pigment solutions from the two distinct blue *Haslea* species were characterized. The UV-vis spectrophotometry revealed that at pH 7, the extracellular form of the pigment produced by *H. nusantara* displays a maximum absorbance in the red region with little difference when compared to marennine (663 vs. 666 nm, respectively). This confirms that both pigments

belong to the same chemical family. Furthermore, the peaks of maximum absorbance in the red region measured in this study for BWs from *H. ostrearia* and *H. nusantara* are comparable to those observed in previous works (Pouvreau et al., 2007b, 2006; Prasetiya et al., 2019a).

## 4.2. Effect of BW on Isochrysis galbana

In this study, two types of filtered culture supernatants (BWs) from the two distinct *Haslea* blue species were tested against *I. galbana*, microalgae commonly used in aquaculture as natural feed. Both types of pigment solutions appeared to inhibit the growth of *I. galbana*. Indeed, previous studies revealed that marennine produced by *H. ostrearia* can inhibit the growth of other microalgal species, not only as purified marennine solutions (Pouvreau et al., 2007b), but also in culture supernatants (Neuville and Daste 1978; Robert and Turpin 1993; Windarto et al. 2014). Furthermore, Prasetiya et al. (2016) revealed that the

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Effect of pigment solutions (BWs) (2 µg mL⁻¹) on nauplii of *A. salina* during the toxicity experiment. The red and black circles indicate dead and alive nauplii, respectively. Scale bar is 2000 µm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).</figcaption></figure>
## Haslea nusantara

<figure><img src="image_5.png" /><figcaption>Fig. 4. Percentage of dead *Artemia salina* at different time of exposure (24, 48 and 72 h, indicated by circle, square and triangle, respectively) to the pigment solutions (BWs) from *Haslea nusantara* (A) and *Haslea ostrearia* (B) in each concentration.</figcaption></figure>

growth of *Tisochrysis lutea* was inhibited by the presence of BW from *H. ostrearia*. It can thus be hypothesized that BW from the tropical *Haslea* species also has the ability to inhibit the growth of other species of microalgae with almost similar magnitude to that of marennine from the temperate blue diatom *H. ostrearia*.

### 4.3. Effect of BW on Artemia salina

To our knowledge, the effect of BWs from both blue *Haslea* species had never been tested on the brine shrimp *A. salina*. In this study, we observed that exposure to the tested BW solutions could cause unfavourable effects against *A. salina*, which depend on the exposure dose. Concentrations from 0.5 µg mL⁻¹ significantly increased the percentage of mortality of *A. salina* neonate. Significantly higher mortality was observed for *A. salina* exposed to 2 µg mL⁻¹ for 72 h for both BW solutions from *H. nusantara* and *H. ostrearia*. This confirms previous studies that evidenced several adverse effects due to marennine, including mortality using different animal models. For instance, a recent study

## Haslea ostrearia

<figure><img src="image_6.png" /></figure>

from Falaise et al. (2019) demonstrated that significant mortality rates were observed in veliger larvae of blue mussel *Mytilus edulis* exposed to 5 µg mL⁻¹ of BW from *H. ostrearia*. Other study from Turcotte et al. (2016) showed that exposure of veliger larvae of blue mussel to lower dose (1 µg mL⁻¹) of BW from *H. ostrearia* for 20 d cause 100 % mortality of larvae. Furthermore, the oxygen consumption and clearance rates of adult *M. edulis* were significantly reduced due to BW exposure of 3 µg mL⁻¹ (Prasetiya et al., 2019b). Thus, deleterious effects of BWs were species specific and could hardly be generalized.

### 4.4. Antibacterial activity of Haslea nusantara and H. Ostrearia

Among marine bacteria tested, both BWs from *H. nusantara* and *H. ostrearia* were able to inhibit the growth of *V. harveyi* and *S. aureus*. *V. harveyi* and other *Vibrio* species are pathogenic bacteria, commonly found in aquaculture systems (Franco et al., 2017; Ke et al., 2017; Muroga, 2001). These bacteria are also able to infect a large variety of hosts, such as shrimp, fish and commercially important molluscs
## Haslea nusantara

<figure><img src="image_7.png" /><figcaption>Fig. 4. Percentage of dead *Artemia salina* at different time of exposure (24, 48 and 72 h, indicated by circle, square and triangle, respectively) to the pigment solutions (BWs) from *Haslea nusantara* (A) and *Haslea ostrearia* (B) in each concentration.</figcaption></figure>

## Haslea ostrearia

<figure><img src="image_8.png" /></figure>

growth of *Tisochrysis lutea* was inhibited by the presence of BW from *H. ostrearia*. It can thus be hypothesized that BW from the tropical *Haslea* species also has the ability to inhibit the growth of other species of microalgae with almost similar magnitude to that of marennine from the temperate blue diatom *H. ostrearia*.

### 4.3. Effect of BW on Artemia salina

To our knowledge, the effect of BWs from both blue *Haslea* species had never been tested on the brine shrimp *A. salina*. In this study, we observed that exposure to the tested BW solutions could cause unfavourable effects against *A. salina*, which depend on the exposure dose. Concentrations from 0.5 µg mL⁻¹ significantly increased the percentage of mortality of *A. salina* neonate. Significantly higher mortality was observed for *A. salina* exposed to 2 µg mL⁻¹ for 72 h for both BW solutions from *H. nusantara* and *H. ostrearia*. This confirms previous studies that evidenced several adverse effects due to marennine, including mortality using different animal models. For instance, a recent study

from Falaise et al. (2019) demonstrated that significant mortality rates were observed in veliger larvae of blue mussel *Mytilus edulis* exposed to 5 µg mL⁻¹ of BW from *H. ostrearia*. Other study from Turcotte et al. (2016) showed that exposure of veliger larvae of blue mussel to lower dose (1 µg mL⁻¹) of BW from *H. ostrearia* for 20 d cause 100 % mortality of larvae. Furthermore, the oxygen consumption and clearance rates of adult *M. edulis* were significantly reduced due to BW exposure of 3 µg mL⁻¹ (Prasetiya et al., 2019b). Thus, deleterious effects of BWs were species specific and could hardly be generalized.

### 4.4. Antibacterial activity of Haslea nusantara and H. Ostrearia

Among marine bacteria tested, both BWs from *H. nusantara* and *H. ostrearia* were able to inhibit the growth of *V. harveyi* and *S. aureus*. *V. harveyi* and other *Vibrio* species are pathogenic bacteria, commonly found in aquaculture systems (Franco et al., 2017; Ke et al., 2017; Muroga, 2001). These bacteria are also able to infect a large variety of hosts, such as shrimp, fish and commercially important molluscs
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Antibacterial activities of the pigment solutions (BWs) produced by *Haslea nusantara* and *Haslea ostrearia* against *Vibrio harveyi* (above) and *Staphylococcus aureus* (below). Values are means ± SE (n = 3), and are expressed in mm.</figcaption></figure>

(Falaise et al., 2019b; Santhyia et al., 2015; Sarjito et al., 2018; Saulnier et al., 2010). Indeed, *V. harveyi* is of particular concern to the penaeid industry as this species was involved in most devastating mortality events observed the South East of Asia in the last decade (Rungrassamee et al., 2016; Sarjito et al., 2018; Stalin and Srinivasan, 2016).

The present work is the first demonstration of the capacity of the BW from the tropical *H. nusantara* to inhibit the growth of the aquaculture relevant *V. harveyi*. This result is reinforced by the comparable inhibition on the growth of *V. harveyi* produced by BW from the temperate *H. ostrearia* evidenced in our study, which is comparable to a previous result by Falaise et al. (2019b) at similar range of concentration (10 µg mL⁻¹).

The inhibition on the growth of *V. harveyi* and *S. aureus* by BWs from both *Haslea* species could suggest that BW has a broad spectrum of antibacterial activity (Gram positive and negative bacteria). Nevertheless, our results also show that the growth inhibition of marine bacteria tested are variable. Indeed, previous study from Falaise et al. (2016) demonstrated that variability of BW in inhibiting the growth of bacteria is probably due to linkability of marennine to cell surface or membrane receptors and trigger stress responses or even apoptotic signals by inducing changes in cell metabolism. This is supported by variability of BW effects on the growth inhibition of the three different species of *Vibrio* tested (V. aestuarianus, V. coralliilyticus and V. tubia-shii), which could reveal differences in membrane receptor affinities and in mechanisms of action.

## 4.5. Marennine and marennine-like pigments as potential antibacterial agents

Like the purified forms of marennine and marennine-like pigments, the culture supernatant of the blue *Haslea* species (*H. nusantara*, *H. ostrearia*, *H. karadagensis*, etc.), or so called BW solutions, exhibit antibacterial activity by inhibiting the growth of marine bacteria (Falaise et al., 2016, 2019b, this study). However, it could be questioned whether the observed effect of inhibition from this BW solutions were caused by the pigments or by other molecules that could be present in the non-axenic *Haslea* culture medium (e.g., exopolysaccharides of *Haslea* or epibiont bacteria). Although the present work was not aimed to answer this question (aquaculture-oriented application, see below), previous studies conducted using the purified form of extracellular marennine (EMn) against *Vibrio* species confirmed that the purified pigment is responsible for the antibacterial activity (Gastineau et al., 2013, 2012). Additionally, recent study from Falaise et al. (2019a) has

demonstrated the biological activities of both marennine solutions (BW and EMn) on various marine organism, and the observed effects were comparable between the different solutions tested. Moreover, from the feasibility point of view, the utilization of BW solutions as natural antibacterial agents in aquaculture is by far more realistic compared to that of any purified form that requires expensive and longer processing procedures (Pouvreau et al., 2006).

In line with the previous antibacterial activity evidenced in vitro, the inhibition of the growth of the marine bacterium *V. harveyi* demonstrated by the marennine-like pigment produced by *H. nusantara* is also prospective in the field of aquaculture. Indeed, this pathogenic bacterium has been reported to cause serious infections in shrimp farming systems worldwide that can lead to a 100 % mortality rate (De Schryver et al., 2014; Franco et al., 2017; Kumara and Hettiarachchi, 2016). Despite the mechanisms of action responsible for the antibacterial activity of marennine remain little explored, recent study have revealed that marennine interacts with the outer membrane of Gram-negative bacteria (Tardy-Laporte et al., 2013). To our knowledge, the in vivo interaction between marennine and *Vibrio* bacteria remains unclear, whether the pigment has a direct effect on *Vibrio* growth or if it could decrease its pathogenicity by fixing on cell membranes, both of which could explain the better survival of bivalve larvae exposed to marennine and challenged with *V. splendidus* as shown in Turcotte et al. (2016). This suggests that marennine antibacterial mechanisms will have to be better understood prior to validate the upscaling of any application of *Haslea* and marennine as antibacterial agents in aquaculture.

## 5. Conclusions

This study has successfully documented for the first time, that the newly-discribed diatom from the tropic *H. nusantara* produces a marennine-like pigment that has almost similar traits to that of marennine pigment from *H. ostrearia*. At certain concentrations, the culture supernatants (BWs) of both *Haslea* species have the ability to inhibit the growth of *I. galbana* and display some toxicity against *A. salina*. Both BW solutions also display antibacterial activity against Gram-positive and -negative bacteria relevant to aquaculture. These results reveal that marennine-like pigments are bio-active compounds, which allow us to hypothesise that they could be used as a natural antibacterial agents in aquaculture. However, it remains to study beforehand the chronic exposure to low concentrations of BW solutions. When the *H. nusantara* culture supernatant is considered to be applied in the aquaculture field as a preventive or curative antipathogen, effective dose should be considered in order to avoid the possible adverse effects.

### CRediT authorship contribution statement

**Fiddy S. Prasetiya:** Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing, Project administration. **Sunarto Sunarto:** Conceptualization, Methodology, Validation, Formal analysis, Investigation, Project administration, Funding acquisition, Supervision. **Eri Bachtiar:** Conceptulization, Methodology, Funding acquisition, Supervision. **Mochamad U.K. Agung:** Resources, Supervision. **Bram Nathanael:** Validation, Formal analysis, Investigation. **Ardian C. Pambudi:** Validation, Formal analysis, Investigation. **Ajeng D. Lestari:** Validation, Formal analysis, Investigation. **Sri Astuty:** Resources, Supervision. **Jean-Luc Mouget:** Resources, Writing - review & editing, Project administration, Funding acquisition.

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Antibacterial activities of the pigment solutions (BWs) produced by *Haslea nusantara* and *Haslea ostrearia* against *Vibrio harveyi* (above) and *Staphylococcus aureus* (below). Values are means ± SE (n = 3), and are expressed in mm.</figcaption></figure>

(Falaise et al., 2019b; Santhyia et al., 2015; Sarjito et al., 2018; Saulnier et al., 2010). Indeed, *V. harveyi* is of particular concern to the penaeid industry as this species was involved in most devastating mortality events observed the South East of Asia in the last decade (Rungrassamee et al., 2016; Sarjito et al., 2018; Stalin and Srinivasan, 2016).

The present work is the first demonstration of the capacity of the BW from the tropical *H. nusantara* to inhibit the growth of the aquaculture relevant *V. harveyi*. This result is reinforced by the comparable inhibition on the growth of *V. harveyi* produced by BW from the temperate *H. ostrearia* evidenced in our study, which is comparable to a previous result by Falaise et al. (2019b) at similar range of concentration (10 µg mL⁻¹).

The inhibition on the growth of *V. harveyi* and *S. aureus* by BWs from both *Haslea* species could suggest that BW has a broad spectrum of antibacterial activity (Gram positive and negative bacteria). Nevertheless, our results also show that the growth inhibition of marine bacteria tested are variable. Indeed, previous study from Falaise et al. (2016) demonstrated that variability of BW in inhibiting the growth of bacteria is probably due to linkability of marennine to cell surface or membrane receptors and trigger stress responses or even apoptotic signals by inducing changes in cell metabolism. This is supported by variability of BW effects on the growth inhibition of the three different species of *Vibrio* tested (V. aestuarianus, V. coralliilyticus and V. tubiashii), which could reveal differences in membrane receptor affinities and in mechanisms of action.

### 4.5. Marennine and marennine-like pigments as potential antibacterial agents

Like the purified forms of marennine and marennine-like pigments, the culture supernatant of the blue *Haslea* species (*H. nusantara*, *H. ostrearia*, *H. karadagensis*, etc.), or so called BW solutions, exhibit antibacterial activity by inhibiting the growth of marine bacteria (Falaise et al., 2016, 2019b, this study). However, it could be questioned whether the observed effect of inhibition from this BW solutions were caused by the pigments or by other molecules that could be present in the non-axenic *Haslea* culture medium (e.g., exopolysaccharides of *Haslea* or epibiont bacteria). Although the present work was not aimed to answer this question (aquaculture-oriented application, see below), previous studies conducted using the purified form of extracellular marennine (EMn) against *Vibrio* species confirmed that the purified pigment is responsible for the antibacterial activity (Gastineau et al., 2013, 2012). Additionally, recent study from Falaise et al. (2019a) has

demonstrated the biological activities of both marennine solutions (BW and EMn) on various marine organism, and the observed effects were comparable between the different solutions tested. Moreover, from the feasibility point of view, the utilization of BW solutions as natural antibacterial agents in aquaculture is by far more realistic compared to that of any purified form that requires expensive and longer processing procedures (Pouvreau et al., 2006).

In line with the previous antibacterial activity evidenced in vitro, the inhibition of the growth of the marine bacterium *V. harveyi* demonstrated by the marennine-like pigment produced by *H. nusantara* is also prospective in the field of aquaculture. Indeed, this pathogenic bacterium has been reported to cause serious infections in shrimp farming systems worldwide that can lead to a 100 % mortality rate (De Schryver et al., 2014; Franco et al., 2017; Kumara and Hettiarachchi, 2016). Despite the mechanisms of action responsible for the antibacterial activity of marennine remain little explored, recent study have revealed that marennine interacts with the outer membrane of Gram-negative bacteria (Tardy-Laporte et al., 2013). To our knowledge, the in vivo interaction between marennine and *Vibrio* bacteria remains unclear, whether the pigment has a direct effect on *Vibrio* growth or if it could decrease its pathogenicity by fixing on cell membranes, both of which could explain the better survival of bivalve larvae exposed to marennine and challenged with *V. splendidus* as shown in Turcotte et al. (2016). This suggests that marennine antibacterial mechanisms will have to be better understood prior to validate the upscaling of any application of *Haslea* and marennine as antibacterial agents in aquaculture.

### 5. Conclusions

This study has successfully documented for the first time, that the newly-discribed diatom from the tropic *H. nusantara* produces a marennine-like pigment that has almost similar traits to that of marennine pigment from *H. ostrearia*. At certain concentrations, the culture supernatants (BWs) of both *Haslea* species have the ability to inhibit the growth of *I. galbana* and display some toxicity against *A. salina*. Both BW solutions also display antibacterial activity against Gram-positive and -negative bacteria relevant to aquaculture. These results reveal that marennine-like pigments are bio-active compounds, which allow us to hypothesise that they could be used as a natural antibacterial agents in aquaculture. However, it remains to study beforehand the chronic exposure to low concentrations of BW solutions. When the *H. nusantara* culture supernatant is considered to be applied in the aquaculture field as a preventive or curative antipathogen, effective dose should be considered in order to avoid the possible adverse effects.

#### CRediT authorship contribution statement

**Fiddy S. Prasetiya:** Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing, Project administration. **Sunarto Sunarto:** Conceptualization, Methodology, Validation, Formal analysis, Investigation, Project administration, Funding acquisition, Supervision. **Eri Bachtiar:** Conceptulization, Methodology, Funding acquisition, Supervision. **Mochamad U.K. Agung:** Resources, Supervision. **Bram Nathanael:** Validation, Formal analysis, Investigation. **Ardian C. Pambudi:** Validation, Formal analysis, Investigation. **Ajeng D. Lestari:** Validation, Formal analysis, Investigation. **Sri Astuty:** Resources, Supervision. **Jean-Luc Mouget:** Resources, Writing - review & editing, Project administration, Funding acquisition.

#### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

## Acknowledgements

The authors wish to thank to the Directorate of Research and Community Service (DRPM) Universitas Padjadjaran and the Ministry of Research and Higher Education (KEMENRISTEKDIKTI) of Indonesian Government for their support, and the close collaboration between Universitas Padjadjaran (UP) and Le Mans Université (LM). This study was funded by Hibah Internal Unpad scheme of Lecturer Competency Unpad (HIU-RKDU, grant number: 2122/UN6.M/LT/2019) granted to SS, FSP and EB. The authors also acknowledge the Horizon 2020 Research and Innovation Programme GHANA (The Genus Haslea, New marine resources for blue biotechnology and Aquaculture, grant agreement No [734708/GHANA/H2020-MSCA-RISE-2016]) (JLM).

## Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aqrep.2020.100389.

## References

Bergé, J., Barnathan, G., 2005. Fatty acids from lipids of marine organisms: molecular biodiversity, roles as biomarkers, biologically active compounds, and economical aspects. Adv. Biochem. Eng. Biotechnol. 49–125.

Bergé, J.P., Bourgougnon, N., Alban, S., Pojer, F., Billaudel, S., Chermann, J.C., Robert, J.M., Franz, G., 1999. Antiviral and anticoagulant activities of a water-soluble fraction of the marine diatom Haslea ostrearia. Planta Med. 65, 604–609. https://doi.org/10.1055/s-1999-14032.

Brown, M.R., Jeffrey, S.W., Volkman, J.K., Dunstan, Ga., 1997. Nutritional properties of microalgae for mariculture. Aquaculture 151, 315–331. https://doi.org/10.1016/S0044-8486(96)01501-3.

De Schryver, P., Defoirdt, T., Sorgeloos, P., 2014. Early Mortality Syndrome Outbreaks: A Microbial Management Issue in Shrimp Farming? PLoS Pathog. 10, 10–11. https://doi.org/10.1371/journal.ppat.1003919.

Dunstan, Ga., Volkman, J.K., Barrett, S.M., Leroi, J.M., Jeffrey, S.W., 1994. Essential polyunsaturated fatty acids from 14 species of diatom (Bacillariophyceae). Phytochemistry 35, 155–161. https://doi.org/10.1016/S0031-9422(00)90525-9.

Falaise, C., François, C., Travers, M.A., Morga, B., Haure, J., Tremblay, R., Turcotte, F., Pasetto, P., Gastineau, R., Hardivillier, Y., Leignel, V., Mouget, J.L., 2016. Antimicrobial compounds from eukaryotic microalgae against human pathogens and diseases in aquaculture. Mar. Drugs 14, 1–27. https://doi.org/10.3390/ md14090159.

Falaise, C., Cormier, P., Tremblay, R., Audet, C., Deschênes, J.S., Turcotte, F., François, C., Seger, A., Hallegraeff, G., Lindquist, N., Sirjacobs, D., Gobert, S., Lejeune, P., Demoulin, V., Mouget, J.L., 2019a. Harmful or harmless: biological effects of mare- nnine on marine organisms. Aquat. Toxicol. 209, 13–25. https://doi.org/10.1016/j. aquatox.2019.01.016.

Falaise, C., James, A., Travers, M.-A., Zanella, M., Badawi, M., Mouget, J.-L., 2019b. Complex relationships between the blue pigment marennine and marine Bacteria of the genus Vibrio. Mar. Drugs 17, 160. https://doi.org/10.3390/ md17030160.

Franco, R., Martín, L., Arenal, A., Santiesteban, D., Sotolongo, J., Cabrera, H., Mejías, J., Rodríguez, G., Moreno, A.G., Pimentel, E., Castillo, N.M., 2017. Evaluation of two probiotics used during farm production of white shrimp Litopenaeus vannamei (Crustacea: decapoda). Aquac. Res. 48, 1936–1950. https://doi.org/10.1111/are. 13031.

Gastineau, R., Hardivillier, Y., Leignel, V., Tekaya, N., Morançais, M., Fleurence, J., Davidovich, N., Jacquette, B., Gaudin, P., Hellio, C., Bourgougnon, N., Mouget, J.L., 2012a. Greening effect on oysters and biological activities of the blue pigments produced by the diatom Haslea karadagensis (Naviculaceae). Aqua. 368–369, 61–67. https://doi.org/10.1016/j.aquaculture.2012.09.016.

Gastineau, R., Pouvreau, J.B., Hellio, C., Morançais, M., Fleurence, J., Gaudin, P., Bourgougnon, N., Mouget, J.L., 2012b. Biological activities of purified marennine, the blue pigment responsible for the greening of oysters. J. Agric. Food Chem. 60, 3599–3605. https://doi.org/10.1021/jf205004x.

Gastineau, R., Davidovich, N.A., Bardeau, J.-F., Caruso, A., Leignel, V., Hardivillier, Y., Jacquette, B., Davidovich, O., Rincé, Y., Gaudin, P., Cox, E., Mouget, J.L., 2012c. *Haslea karadagensis* (Bacillariophyta): a second blue diatom, recorded from the Black Sea and producing a novel blue pigment. Eur. J. Phycol. 47, 469–479.

Gastineau, R., Turcotte, F., Pouvreau, J.-B., Morançais, M., Fleurence, J., Windarto, E., Prasetiya, F.S., Arsad, S., Jaouen, P., Babin, M., Coiffard, L., Couteau, C., Bardeau, J.-F., Jacquette, B., Leignel, V., Hardivillier, Y., Marcotte, I., Bourgougnon, N., Tremblay, R., Deschênes, J.-S., Badawy, H., Pasetto, P., Davidovich, N., Hansen, G., Dittmer, J., Mouget, J.-L., 2014. Marennine, promising blue pigments from a wide- spread Haslea diatom species complex. Mar. Drugs 12, 3161–3189. https://doi.org/ 10.3390/ md12063161.

Gastineau, R., Hansen, G., Davidovich, N.A., Davidovich, O., Bardeau, J.-F., Kaczmarska, I., Ehrman, J.M., Leignel, V., Hardivillier, Y., Jacquette, B., Poulin, M., Morançais, M.,

Fleurence, J., Mouget, J.-L., 2016. A new blue-pigmented hasleoid diatom, *Haslea provincialis*, from the Mediterranean Sea. Eur. J. Phycol. 51, 156–170. https://doi.org/10.1080/09670262.2015.1110861.

Gastineau, R., Prasetiya, F.S., Falaise, C., Cognie, B., Decottignies, P., Morançais, M., Méléder, V., Davidovich, N., Turcotte, F., Tremblay, R., Pasetto, P., Dittmer, J., Bardeau, J.-F., Pouvreau, J.-B., Mouget, J.-L., 2018. Marennine-like pigments: Blue diatom or Green oyster cult? In: In: La Barre, S., Bates, S.S. (Eds.), Blue Biotechnology: Production and Use of Marine Molecules, vol 1 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. https://doi.org/10.1002/9783527801718.529–551.

Gordon, R., Losic, D., Tiffany, M.A.S., Nagy, S.S., Sterrenburg, Fa S., 2009. The GlaS. Menagerie: diatoms for novel applications in nanotechnology. Trends Biotechnol. 27, 116–127. https://doi.org/10.1016/j.tibtech.2008.11.003.

Ke, H.-M., Prachumwat, A., Yu, C.-P., Yang, Y.-T., Promsri, S., Liu, K.-F., Lo, C.-F., Lu, M.-Y.J., Lai, M.-C., Tsai, I.J., Li, W.-H., 2017. Comparative genomics of Vibrio campbellii strains and core species of the Vibrio harveyi clade. Sci. Rep. 7, 41394. https://doi.org/10.1038/srep41394.

Kumara, K.R.P.S., Hettiarachchi, M., 2016. Regular monitoring and controlling &i&Vibrio &/i&- a critical bio-security measure for sri lankan shrimp (&i&Penaeus monodon&/i &) hacheries. Fish Pathol. 51, S46–S53. https://doi.org/10.3147/jsfp.51.S46.

Lopez, P.J., Desclés, J., Allen, A.E., Bowler, C., 2005. Prospects in diatom research. Curr. Opin. Biotechnol. 16, 180–186. https://doi.org/10.1016/j.copbio.2005.02.002.

Mouget, J.-L., Gastineau, R., Davidovich, O., Gaudin, P., Davidovich, N.A., 2009. Light is a key factor in triggering sexual reproduction in the pennate diatom Haslea ostrearia. FEMS Microbiol. Ecol. 69, 194–201. https://doi.org/10.1111/j.1574-6941.2009.00700.x.

Muroga, K., 2001. Viral and bacterial diseases of marine fish and shellfish in Japanese hacheries. Aquaculture 202, 23–44. https://doi.org/10.1016/S0044-8486(01) 00597-X.

Olaizola, M., 2003. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomol. Eng. 20, 459–466. https://doi.org/10.1016/S1389-0344(03)00076-5.

Piveteau, F., 1999. Étude des arômes de l'huîte creuse Crassostrea gigas : conséquences d'une affinité à l'aide des microalgues Skeletonostea costatum et Haslea ostrearia = Aroma of oyster Crassostrea gigas: effect of supplementation with the microalga Skeletonostea costatum (Doctoral dissertation). Nantes. .

Pouvreau, J.B., Morançais, M., Massé, G., Rosa, P., Robert, J.M., Fleurence, J., Pondaven, P., 2006. Purification of the blue-green pigment "marennine" from the marine ty-choplasic diatom Haslea ostrearia (Gaillon/Bory) Simonsen. J. Appl. Phycol. 18, 769–781. https://doi.org/10.1007/s10811-006-9088-9.

Pouvreau, J.B., Morançais, M., Fleurence, J., Pondaven, P., 2007a. Method for the quantification of the blue-green pigment "marennine" synthesized by the marine diatom Haslea ostrearia (Gaillon/Bory) Simonsen using HPLC gel-filtration and photodiode-array detection. J. Appl. Phycol. 19, 263–270. https://doi.org/10.1007/ s10811-006-9133-8.

Pouvreau, J.B., Housson, E., le Tallec, L., Morançais, M., Rincé, Y., Fleurence, J., Pondaven, P., 2007b. Growth inhibition of several marine diatom species induced by the shading effect and allelopathic activity of marennine, a blue-green polyphenolic pigment of the diatom Hasleostrearia (Gaillon/Bory) Simonsen. J. Exp. Mar. Biol. Ecol. 352, 212–225.

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influence the work reported in this paper.

## Acknowledgements

The authors wish to thank to the Directorate of Research and Community Service (DRPM) Universitas Padjadjaran and the Ministry of Research and Higher Education (KEMENRISTEKDIKTI) of Indonesian Government for their support, and the close collaboration between Universitas Padjadjaran (UP) and Le Mans Université (LM). This study was funded by Hibah Internal Unpad scheme of Lecturer Competency Unpad (HIU-RKDU, grant number: 2122/UN6.M/LT/2019) granted to SS, FSP and EB. The authors also acknowledge the Horizon 2020 Research and Innovation Programme GHANA (The Genus Haslea, New marine resources for blue biotechnology and Aquaculture, grant agreement No [734708/GHANA/H2020-MSCA-RISE-2016]) (JLM).

## Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aqrep.2020.100389.

## References

Bergé, J., Barnathan, G., 2005. Fatty acids from lipids of marine organisms: molecular biodiversity, roles as biomarkers, biologically active compounds, and economical aspects. Adv. Biochem. Eng. Biotechnol. 49–125.

Bergé, J.P., Bourgougnon, N., Alban, S., Pojer, F., Billaudel, S., Chermann, J.C., Robert, J.M., Franz, G., 1999. Antiviral and anticoagulant activities of a water-soluble fraction of the marine diatom Haslea ostrearia. Planta Med. 65, 604–609. https://doi.org/10.1055/s-1999-14032.

Brown, M.R., Jeffrey, S.W., Volkman, J.K., Dunstan, Ga., 1997. Nutritional properties of microalgae for mariculture. Aquaculture 151, 315–331. https://doi.org/10.1016/S0044-8486(96)01501-3.

De Schryver, P., Defoirdt, T., Sorgeloos, P., 2014. Early Mortality Syndrome Outbreaks: A Microbial Management Issue in Shrimp Farming? PLoS Pathog. 10, 10–11. https://doi.org/10.1371/journal.ppat.1003919.

Dunstan, Ga., Volkman, J.K., Barrett, S.M., Leroi, J.M., Jeffrey, S.W., 1994. Essential polyunsaturated fatty acids from 14 species of diatom (Bacillariophyceae). Phytochemistry 35, 155–161. https://doi.org/10.1016/S0031-9422(00)90525-9.

Falaise, C., François, C., Travers, M.A., Morga, B., Haure, J., Tremblay, R., Turcotte, F., Pasetto, P., Gastineau, R., Hardivillier, Y., Leignel, V., Mouget, J.L., 2016. Antimicrobial compounds from eukaryotic microalgae against human pathogens and diseases in aquaculture. Mar. Drugs 14, 1–27. https://doi.org/10.3390/ md14090159.

Falaise, C., Cormier, P., Tremblay, R., Audet, C., Deschênes, J.S., Turcotte, F., François, C., Seger, A., Hallegraeff, G., Lindquist, N., Sirjacobs, D., Gobert, S., Lejeune, P., Demoulin, V., Mouget, J.L., 2019a. Harmful or harmless: biological effects of mare- nnine on marine organisms. Aquat. Toxicol. 209, 13–25. https://doi.org/10.1016/j. aquatox.2019.01.016.

Falaise, C., James, A., Travers, M.-A., Zanella, M., Badawi, M., Mouget, J.-L., 2019b. Complex relationships between the blue pigment marennine and marine Bacteria of the genus Vibrio. Mar. Drugs 17, 160. https://doi.org/10.3390/ md17030160.

Franco, R., Martín, L., Arenal, A., Santiesteban, D., Sotolongo, J., Cabrera, H., Mejías, J., Rodríguez, G., Moreno, A.G., Pimentel, E., Castillo, N.M., 2017. Evaluation of two probiotics used during farm production of white shrimp Litopenaeus vannamei (Crustacea: decapoda). Aquac. Res. 48, 1936–1950. https://doi.org/10.1111/are. 13031.

Gastineau, R., Hardivillier, Y., Leignel, V., Tekaya, N., Morançais, M., Fleurence, J., Davidovich, N., Jacquette, B., Gaudin, P., Hellio, C., Bourgougnon, N., Mouget, J.L., 2012a. Greening effect on oysters and biological activities of the blue pigments produced by the diatom Haslea karadagensis (Naviculaceae). Aqua. 368–369, 61–67. https://doi.org/10.1016/j.aquaculture.2012.09.016.

Gastineau, R., Pouvreau, J.B., Hellio, C., Morançais, M., Fleurence, J., Gaudin, P., Bourgougnon, N., Mouget, J.L., 2012b. Biological activities of purified marennine, the blue pigment responsible for the greening of oysters. J. Agric. Food Chem. 60, 3599–3605. https://doi.org/10.1021/jf205004x.

Gastineau, R., Davidovich, N.A., Bardeau, J.-F., Caruso, A., Leignel, V., Hardivillier, Y., Jacquette, B., Davidovich, O., Rincé, Y., Gaudin, P., Cox, E., Mouget, J.L., 2012c. *Haslea karadagensis* (Bacillariophyta): a second blue diatom, recorded from the Black Sea and producing a novel blue pigment. Eur. J. Phycol. 47, 469–479.

Gastineau, R., Turcotte, F., Pouvreau, J.-B., Morançais, M., Fleurence, J., Windarto, E., Prasetiya, F.S., Arsad, S., Jaouen, P., Babin, M., Coiffard, L., Couteau, C., Bardeau, J.-F., Jacquette, B., Leignel, V., Hardivillier, Y., Marcotte, I., Bourgougnon, N., Tremblay, R., Deschênes, J.-S., Badawy, H., Pasetto, P., Davidovich, N., Hansen, G., Dittmer, J., Mouget, J.-L., 2014. Marennine, promising blue pigments from a wide- spread Haslea diatom species complex. Mar. Drugs 12, 3161–3189. https://doi.org/ 10.3390/ md12063161.

Gastineau, R., Hansen, G., Davidovich, N.A., Davidovich, O., Bardeau, J.-F., Kaczmarska, I., Ehrman, J.M., Leignel, V., Hardivillier, Y., Jacquette, B., Poulin, M., Morançais, M.,

Fleurence, J., Mouget, J.-L., 2016. A new blue-pigmented hasleoid diatom, *Haslea provincialis*, from the Mediterranean Sea. Eur. J. Phycol. 51, 156–170. https://doi.org/10.1080/09670262.2015.1110861.

Gastineau, R., Prasetiya, F.S., Falaise, C., Cognie, B., Decottignies, P., Morançais, M., Méléder, V., Davidovich, N., Turcotte, F., Tremblay, R., Pasetto, P., Dittmer, J., Bardeau, J.-F., Pouvreau, J.-B., Mouget, J.-L., 2018. Marennine-like pigments: Blue diatom or Green oyster cult? In: In: La Barre, S., Bates, S.S. (Eds.), Blue Biotechnology: Production and Use of Marine Molecules, vol 1 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. https://doi.org/10.1002/9783527801718.529–551.

Gordon, R., Losic, D., Tiffany, M.A.S., Nagy, S.S., Sterrenburg, Fa S., 2009. The GlaS. Menagerie: diatoms for novel applications in nanotechnology. Trends Biotechnol. 27, 116–127. https://doi.org/10.1016/j.tibtech.2008.11.003.

Ke, H.-M., Prachumwat, A., Yu, C.-P., Yang, Y.-T., Promsri, S., Liu, K.-F., Lo, C.-F., Lu, M.-Y.J., Lai, M.-C., Tsai, I.J., Li, W.-H., 2017. Comparative genomics of Vibrio campbellii strains and core species of the Vibrio harveyi clade. Sci. Rep. 7, 41394. https://doi.org/10.1038/srep41394.

Kumara, K.R.P.S., Hettiarachchi, M., 2016. Regular monitoring and controlling &i&Vibrio &/i&- a critical bio-security measure for sri lankan shrimp (&i&Penaeus monodon&/i &) hacheries. Fish Pathol. 51, S46–S53. https://doi.org/10.3147/jsfp.51.S46.

Lopez, P.J., Desclés, J., Allen, A.E., Bowler, C., 2005. Prospects in diatom research. Curr. Opin. Biotechnol. 16, 180–186. https://doi.org/10.1016/j.copbio.2005.02.002.

Mouget, J.-L., Gastineau, R., Davidovich, O., Gaudin, P., Davidovich, N.A., 2009. Light is a key factor in triggering sexual reproduction in the pennate diatom Haslea ostrearia. FEMS Microbiol. Ecol. 69, 194–201. https://doi.org/10.1111/j.1574-6941.2009.00700.x.

Muroga, K., 2001. Viral and bacterial diseases of marine fish and shellfish in Japanese hacheries. Aquaculture 202, 23–44. https://doi.org/10.1016/S0044-8486(01) 00597-X.

Olaizola, M., 2003. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomol. Eng. 20, 459–466. https://doi.org/10.1016/S1389- 0344(03)00076-5.

Piveteau, F., 1999. Étude des arômes de l'huîte creuse Crassostrea gigas : conséquences d'une affinité à l'aide des microalgues Skeletonema costatum et Haslea ostrearia = Aroma of oyster Crassostrea gigas: effect of supplementation with the microalga Skeletonema costatum (Doctoral dissertation). Nantes. .

Pouvreau, J.B., Morançais, M., Massé, G., Rosa, P., Robert, J.M., Fleurence, J., Pondaven, P., 2006. Purification of the blue-green pigment "marennine" from the marine ty-choplasic diatom Haslea ostrearia (Gaillon/Bory) Simonsen. J. Appl. Phycol. 18, 769–781. https://doi.org/10.1007/s10811-006-9088-9.

Pouvreau, J.B., Morançais, M., Fleurence, J., Pondaven, P., 2007a. Method for the quantification of the blue-green pigment "marennine" synthesized by the marine diatom Haslea ostrearia (Gaillon/Bory) Simonsen using HPLC gel-filtration and photodiode-array detection. J. Appl. Phycol. 19, 263–270. https://doi.org/10.1007/ s10811-006-9133-8.

Pouvreau, J.B., Housson, E., le Tallec, L., Morançais, M., Rincé, Y., Fleurence, J., Pondaven, P., 2007b. Growth inhibition of several marine diatom species induced by the shading effect and allelopathic activity of marennine, a blue-green polyphenolic pigment of the diatom Hasleostrearia (Gaillon/Bory) Simonsen. J. Exp. Mar. Biol. Ecol. 352, 212–225.

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Saulnier, D., de Decker, S., Haffner, P., Cobret, L., Robert, M., Garcia, C., 2010. A large-scale epidemiological study to identify bacteria pathogenic to Pacific Oyster Crassostrea gigas and correlation between virulence and metalloprotease-like activity. Microb. Ecol. 59, 787-798. https://doi.org/10.1007/s00248-009-9620-y.

Sorgeloos, P., Remiche-Van Der Wielen, C., Persoone, G., 1978. The use of Artemia nauplii for toxicity tests-A critical analysis. Ecotoxicol. Environ. Saf. 2, 249-255. https://doi.org/10.1016/S0147-6513(78)80003-7.

Sprat, T., 1669. The history of the generation and ordering of green oysters, commonly called Colchester oysters. Hist. R. Soc. 307-319.

Stalin, N., Srinivasan, P., 2016. Molecular characterization of antibiotic resistant Vibrio harveyi isolated from shrimp aquaculture environment in the south east coast of India. Microb. Pathog. 97, 110-118. https://doi.org/10.1016/j.micpath.2016.05.021.

Tardy-Laporte, C., Arnold, A.A., Genard, B., Gastineau, R., Morançais, M., Mouget, J.L., Tremblay, R., Marcotte, I., 2013. A 2H solid-state NMR study of the effect of anti-microbial agents on intact Escherichia coli without mutating. Biochim. Biophys. Acta Biomembr. 1828, 614-622. https://doi.org/10.1016/j.bbamem.2012.09.011.

Townley, H.E., Parker, A.R., White-Cooper, H., 2008. Exploitation of diatom frustules for nanotechnology: tethering active biomolecules. Adv. Funct. Mater. 18, 369-374. https://doi.org/10.1002/adfm.200700609.

Turcotte, F., Mouget, J.-L., Genard, B., Lemarchand, K., Deschênes, J.-S., Tremblay, R., 2016. Prophylactic effect of *Haslea ostrearia* culture supernatant containing the pigment marennine to stabilize bivalve hatchery production. Aquat. Living Resour. 29, 401. https://doi.org/10.1051/alr/2016032.

Vo, T.-S., Ngo, D.-H., Ta, Q.Van, Kim, S.-K., 2011. Marine organisms as a therapeutic source against herpes simplex virus infection. Eur. J. Pharm. Sci. 44, 11-20. https://doi.org/10.1016/j.ejps.2011.07.005.