# Temporal and Spatial Dynamics of Vibrio Harveyi: An Environmental Parameter Correlated Investigation in a 4-Metre-Deep Aquaculture Tank

Alix da Fonseca Ferreira, Roxane Roquigny, Thierry Grard, Cédric Le Bris

► To cite this version:

Alix da Fonseca Ferreira, Roxane Roquigny, Thierry Grard, Cédric Le Bris. Temporal and Spatial Dynamics of Vibrio Harveyi: An Environmental Parameter Correlated Investigation in a 4-Metre-Deep Aquaculture Tank. Microorganisms, 2024, 12 (6), pp.1104. 10.2139/ssrn.4777236. hal-04592601

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https://ulco.hal.science/hal-04592601

Submitted on 29 May 2024

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# Temporal and spatial dynamics of Vibrio harveyi: an environmental parameter correlated investigation in a 4-metre-deep aquaculture tank

A. Da Fonseca Ferreira, R. Roquigny, T. Grard and C. Le Bris

Univ. Littoral Côte d'Opale, UMRt 1158 BioEcoAgro, USC ANSES, INRAe, Univ. Artois, Univ.Lille, Univ. Picardie Jules

Verne, Univ. Liège, Junia, 62200 Boulogne-sur-Mer, France

## Abstract

Nowadays, European sea bass (*Dicentrarchus labrax*) aquaculture knows a significant expansion. Nevertheless, the aquaculture industry is plagued by several microbial diseases, including vibriosis. The spatial and temporal dynamics of pathogenic *Vibrio harveyi* were studied on a European seabass farm in northern France over seven months in 2022. Cylindrical concrete specimens were suspended in a water column and water was pumped from different depths (top: 0.3 m deep, middle: 2.15 m deep, and bottom: 4 m deep), providing insights into biofilm and planktonic *V. harveyi* dynamics. The abundances of *V. harveyi*, in biofilm and free-living forms, were positively correlated. Water parameters were monitored, revealing seasonal fluctuations in temperature, pH, and salinity, with no significant differences observed across the water column. Fish mortality peaks were recorded in July and August, coinciding with elevated temperatures and emphasizing the impact of environmental factors on aquaculture health. Quantification of *V. harveyi* by quantitative PCR revealed no significant differences between water top, middle and bottom layers. *Vibrio harveyi* exhibited seasonality, with peak abundances observed in August, correlating with temperature increases. Principal component analysis identified temperature as a primary driver, but also additional parameters such as salinity and pH. vibriosis outbreaks occurred during the sampling period, providing valuable insights on conditions before, during, and after the outbreak. This study underscores the importance of understanding *V. harveyi* behaviour in aquaculture systems, particularly in the context of global warming, for effective disease management and sustainable practices.

## Keywords

Aquaculture, *Vibrio harveyi*, Biofilms, Planktonic, Environment, Spatio-temporal study

## 1. Introduction

Given the swift expansion of the global population and the sharp decrease in catches from marine fisheries, the advancement of aquaculture has gained substantial attention as a means to guarantee worldwide food supplies (Ahmed et al., 2019). The European seabass (*Dicentrarchus labrax*) stands out as one of the most economically significant fish species harvested in Europe. Over recent decades, the European seabass aquaculture sector has experienced robust growth. Overall production has reached 80,786 tonnes, amounting to a value of € 470 million in 2020 (EUMOFA, 2022). However, like many other species, *D. labrax* is susceptible to various bacterial diseases, including pasteurellosis (*Photobacterium damselae* subsp. *piscicida*), photobacteriosis (*Photobacterium damselae* subsp. *damselae*), tenacibaculosis (*Tenacibaculum* spp.), and vibriosis (*Vibrio* spp.). These diseases pose significant threats to the seabass aquaculture sector (Muniesa et al., 2020). They can not only cause mass mortality events, but also have substantial economic consequences (ANSES, 2019; Novriadi, 2016).

Throughout the whole production chain, vibriosis stands out as the most significant threat to seabass. It is characterised by various symptoms in seabass, including anorexia, erratic swimming behaviour, exophthalmia, darkening of body colour, pale gills, haemorrhage, ulcers, and skin lesions, and can be fatal
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# Temporal and spatial dynamics of Vibrio harveyi: an environmental parameter correlated investigation in a 4-metre-deep aquaculture tank

A. Da Fonseca Ferreira, R. Roquigny, T. Grard and C. Le Bris

Univ. Littoral Côte d'Opale, UMRt 1158 BioEcoAgro, USC ANSES, INRAe, Univ. Artois, Univ.Lille, Univ. Picardie Jules

Verne, Univ. Liège, Junia, 62200 Boulogne-sur-Mer, France

## Abstract

Nowadays, European sea bass (*Dicentrarchus labrax*) aquaculture knows a significant expansion. Nevertheless, the aquaculture industry is plagued by several microbial diseases, including vibriosis. The spatial and temporal dynamics of pathogenic *Vibrio harveyi* were studied on a European seabass farm in northern France over seven months in 2022. Cylindrical concrete specimens were suspended in a water column and water was pumped from different depths (top: 0.3 m deep, middle: 2.15 m deep, and bottom: 4 m deep), providing insights into biofilm and planktonic *V. harveyi* dynamics. The abundances of *V. harveyi*, in biofilm and free-living forms, were positively correlated. Water parameters were monitored, revealing seasonal fluctuations in temperature, pH, and salinity, with no significant differences observed across the water column. Fish mortality peaks were recorded in July and August, coinciding with elevated temperatures and emphasizing the impact of environmental factors on aquaculture health. Quantification of *V. harveyi* by quantitative PCR revealed no significant differences between water top, middle and bottom layers. *Vibrio harveyi* exhibited seasonality, with peak abundances observed in August, correlating with temperature increases. Principal component analysis identified temperature as a primary driver, but also additional parameters such as salinity and pH. vibriosis outbreaks occurred during the sampling period, providing valuable insights on conditions before, during, and after the outbreak. This study underscores the importance of understanding *V. harveyi* behaviour in aquaculture systems, particularly in the context of global warming, for effective disease management and sustainable practices.

## Keywords

Aquaculture, *Vibrio harveyi*, Biofilms, Planktonic, Environment, Spatio-temporal study

## 1. Introduction

Given the swift expansion of the global population and the sharp decrease in catches from marine fisheries, the advancement of aquaculture has gained substantial attention as a means to guarantee worldwide food supplies (Ahmed et al., 2019). The European seabass (*Dicentrarchus labrax*) stands out as one of the most economically significant fish species harvested in Europe. Over recent decades, the European seabass aquaculture sector has experienced robust growth. Overall production has reached 80,786 tonnes, amounting to a value of € 470 million in 2020 (EUMOFA, 2022). However, like many other species, *D. labrax* is susceptible to various bacterial diseases, including pasteurellosis (*Photobacterium damselae* subsp. *piscicida*), photobacteriosis (*Photobacterium damselae* subsp. *damselae*), tenacibaculosis (*Tenacibaculum* spp.), and vibriosis (*Vibrio* spp.). These diseases pose significant threats to the seabass aquaculture sector (Muniesa et al., 2020). They can not only cause mass mortality events, but also have substantial economic consequences (ANSES, 2019; Novriadi, 2016).

Throughout the whole production chain, vibriosis stands out as the most significant threat to seabass. It is characterised by various symptoms in seabass, including anorexia, erratic swimming behaviour, exophthalmia, darkening of body colour, pale gills, haemorrhage, ulcers, and skin lesions, and can be fatal
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(Austin and Zhang, 2006; Korun, 2008; Mohamad et al., 2019). It can be caused by various species of Vibrio, such as Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, Vibrio ordalii, Vibrio parahaemolyticus, and Vibrio vulnificus (Korun, 2008; Muniesa et al., 2020; Novriadi, 2016; Pujalte et al., 2003). Furthermore, the range of Vibrios spp. is expanding, posing an ongoing challenge in open-circuit aquaculture systems supplied with water directly from the open sea (Baker-Austin et al., 2017). Prophylactic measures, such as commercial vaccines, have been extensively developed and successfully implemented to combat Vibrio anguillarum (Dadar et al., 2017). Now that the issue of V. anguillarum is well managed, V. harveyi has recently emerged as a significant concern in seabass aquaculture (Abu Nor et al., 2020; Islam et al., 2022; Vendramin et al., 2001).

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Vibrio harveyi thrives in natural marine ecosystems. Its ability to act as a pathogen in a wide range of cultivated hosts, such as crustaceans, molluscs, and fish is a concern notably for open-circuit aquaculture farms (Austin and Zhang, 2006). Importantly, V. harveyi exhibits various states of existence, including free-swimming and within biofilms attached to both living and non-living surfaces, or engaged in symbiotic and host-pathogen interactions (Montánchez and Kaberdin, 2020). The formation of biofilms on tank walls can significantly heighten the risk of vibriosis outbreaks. Besides, outbreaks of vibriosis in seabass farms exhibit pronounced seasonality, occurring more frequently when water temperatures reach their maximum (Pujalte et al., 2003). Increasing temperatures, particularly with global warming, may boost the abundance and possibly the virulence of viable V. harveyi in the surrounding environment, consequently increasing the presence of V. harveyi and its damages in tanks. Actually, the occurrence of vibriosis episodes remains unpredictable despite high temperatures, as highlighted by Mougin et al. (2021). In their study, conducted in a small tank (75 m³), no instances of vibriosis occurred during the 7-month sampling campaign, despite the clear impact of temperature on V. harveyi concentrations. The study was conducted in a 1.3-metre-deep tank, which is not deep enough to observe the heterogeneity in environmental parameters and their potential impact on V. harveyi and vibriosis development. This underscores the need for further investigations into the relationships between environmental parameters, V. harveyi concentrations dynamics, and vibriosis outbreaks. Additionally, to gain a more comprehensive understanding of these relationships, studies should expand to larger tanks, where environmental heterogeneity is more likely, allowing for a more nuanced examination of repartition within the water column. Importantly, biofilms add an additional layer of complexity to the understanding of vibriosis development. In biofilms, interspecific interactions are facilitated, and previous research conducted by Roquigny et al. (2021) has already highlighted distinctions in microbial communities and their temporal shifts in an aquaculture tank which may interact with V. harveyi. In this context, a first question arises whether the temporal and spatial dynamics of these two forms of V. harveyi — free-swimming and within biofilms — are interconnected, and if one compartment of the tank acts as a reservoir for each form. This uncertainty complicates the understanding of vibriosis onset, introducing additional challenges in managing and mitigating the impact of V. harveyi in open sea environments. In addition to the complexities posed by V. harveyi's presence in various states, vibriosis development is not solely governed by host-pathogen interactions. Rather, it constitutes a multifaceted tripartite relationship, influenced by environmental biotic and abiotic factors (Maulvault et al., 2018). These factors, such as fish density, water temperature, salinity and pH, may collectively play a significant role in shaping the dynamics of vibriosis outbreaks.

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Thus, it is essential to comprehend V. harveyi's spatial and temporal distribution within aquaculture tanks, as well as the environmental conditions that favour its proliferation and persistence. Therefore, through a comprehensive investigation conducted from May to November 2022, encompassing sampling periods before, during, and after episodes of vibriosis outbreaks, we sought to elucidate the spatial persistence of V. harveyi in water and biofilms particularly during "heatwaves" or vibriosis outbreaks. This sampling was done through both biofilm and water sampling, and environmental factors were measured alongside. Besides, the conducted sampling was done at three different depths within the water column in a 4-metre deep tank, to capture potential variations across vertical gradient. This study aimed to identify potential environmental niches within these tanks where V. harveyi may thrive and persist, potentially leading to vibriosis outbreaks. It could lay the groundwork necessary to effectively address its virulence and mitigate the risk of vibriosis in aquaculture settings. Indeed, with escalating concerns over antibiotic resistance and
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(Austin and Zhang, 2006; Korun, 2008; Mohamad et al., 2019). It can be caused by various species of Vibrio, such as Vibrio harveyi, Vibrio alginolyticus, Vibrio anguillarum, Vibrio ordalii, Vibrio parahaemolyticus, and Vibrio vulnificus (Korun, 2008; Muniesa et al., 2020; Novriadi, 2016; Pujalte et al., 2003). Furthermore, the range of Vibrios spp. is expanding, posing an ongoing challenge in open-circuit aquaculture systems supplied with water directly from the open sea (Baker-Austin et al., 2017). Prophylactic measures, such as commercial vaccines, have been extensively developed and successfully implemented to combat Vibrio anguillarum (Dadar et al., 2017). Now that the issue of V. anguillarum is well managed, V. harveyi has recently emerged as a significant concern in seabass aquaculture (Abu Nor et al., 2020; Islam et al., 2022; Vendramin et al., 2001).

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Vibrio harveyi thrives in natural marine ecosystems. Its ability to act as a pathogen in a wide range of cultivated hosts, such as crustaceans, molluscs, and fish is a concern notably for open-circuit aquaculture farms (Austin and Zhang, 2006). Importantly, V. harveyi exhibits various states of existence, including free-swimming and within biofilms attached to both living and non-living surfaces, or engaged in symbiotic and host-pathogen interactions (Montánchez and Kaberdin, 2020). The formation of biofilms on tank walls can significantly heighten the risk of vibriosis outbreaks. Besides, outbreaks of vibriosis in seabass farms exhibit pronounced seasonality, occurring more frequently when water temperatures reach their maximum (Pujalte et al., 2003). Increasing temperatures, particularly with global warming, may boost the abundance and possibly the virulence of viable V. harveyi in the surrounding environment, consequently increasing the presence of V. harveyi and its damages in tanks. Actually, the occurrence of vibriosis episodes remains unpredictable despite high temperatures, as highlighted by Mougin et al. (2021). In their study, conducted in a small tank (75 m³), no instances of vibriosis occurred during the 7-month sampling campaign, despite the clear impact of temperature on V. harveyi concentrations. The study was conducted in a 1.3-metre-deep tank, which is not deep enough to observe the heterogeneity in environmental parameters and their potential impact on V. harveyi and vibriosis development. This underscores the need for further investigations into the relationships between environmental parameters, V. harveyi concentrations dynamics, and vibriosis outbreaks. Additionally, to gain a more comprehensive understanding of these relationships, studies should expand to larger tanks, where environmental heterogeneity is more likely, allowing for a more nuanced examination of repartition within the water column. Importantly, biofilms add an additional layer of complexity to the understanding of vibriosis development. In biofilms, interspecific interactions are facilitated, and previous research conducted by Roquigny et al. (2021) has already highlighted distinctions in microbial communities and their temporal shifts in an aquaculture tank which may interact with V. harveyi. In this context, a first question arises whether the temporal and spatial dynamics of these two forms of V. harveyi — free-swimming and within biofilms — are interconnected, and if one compartment of the tank acts as a reservoir for each form. This uncertainty complicates the understanding of vibriosis onset, introducing additional challenges in managing and mitigating the impact of V. harveyi in open sea environments. In addition to the complexities posed by V. harveyi's presence in various states, vibriosis development is not solely governed by host-pathogen interactions. Rather, it constitutes a multifaceted tripartite relationship, influenced by environmental biotic and abiotic factors (Maulvault et al., 2018). These factors, such as fish density, water temperature, salinity and pH, may collectively play a significant role in shaping the dynamics of vibriosis outbreaks.

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Thus, it is essential to comprehend V. harveyi's spatial and temporal distribution within aquaculture tanks, as well as the environmental conditions that favour its proliferation and persistence. Therefore, through a comprehensive investigation conducted from May to November 2022, encompassing sampling periods before, during, and after episodes of vibriosis outbreaks, we sought to elucidate the spatial persistence of V. harveyi in water and biofilms particularly during "heatwaves" or vibriosis outbreaks. This sampling was done through both biofilm and water sampling, and environmental factors were measured alongside. Besides, the conducted sampling was done at three different depths within the water column in a 4-metre deep tank, to capture potential variations across vertical gradient. This study aimed to identify potential environmental niches within these tanks where V. harveyi may thrive and persist, potentially leading to vibriosis outbreaks. It could lay the groundwork necessary to effectively address its virulence and mitigate the risk of vibriosis in aquaculture settings. Indeed, with escalating concerns over antibiotic resistance and
changing consumer preferences favouring sustainable practices, relying on antibiotic use is not a viable, long-term solution. In this "One Health" context, aquaculture practitioners could solve this problem by devising targeted antivirulence strategies that not only address the immediate challenges posed by *V. harveyi*, but also align with the principles of sustainable and responsible aquaculture. The goal of this research was to contribute to new strategies for effective management and regulation of vibriosis in aquaculture facilities, based on informed preventive measures.

## 2. Material & methods

### 2.1 Sampling site and rearing conditions

From May to November 2022, a sampling campaign was conducted at Aquanord-Ichtus, an aquaculture establishment situated in Gravelines (France), along the coast of the North Sea. This facility is strategically located near a nuclear power plant, which serves as a source of warm water for the flow-through tank systems via its reactor water cooling system. The facility benefits from thermoregulated water throughout the year, except in summer when the facility faces challenges in cooling the water tank more than environmental water.

The cubical aquaculture tank chosen is an outdoor facility with a volumetric capacity of approximately 975 cubic m, a height of 4 m, and a capacity of up to 68,000 sea bass. The average fish stock density within the tank was 32 kg per cubic meter at the beginning of the campaign, and the average individual fish weight was 517 g.

Fish are fed daily and oxygenation is carried out to maintain ideal dissolved oxygen (DO) levels. To overcome the absence of available vaccines against *V. harveyi*, Aquanord-Ichtus has started to create self-vaccines. This approach enables farmers to use oxytetracycline, an antibiotic, only in extreme cases when vibriosis-induced mortalities are exceptionally high and not effectively managed by other zootechnical measures. During vibriosis outbreaks and elevated fish mortality on the farm, the farmers administered oxytetracycline treatment for 7–10 days, ensuring antibiotic coverage persists for about 4 weeks.

### 2.2 Sample collection and environmental parameters

To study the spatial and temporal dynamics of *V. harveyi* in biofilms, cylindrical concrete specimens (5 x 2.5 cm) constructed from the same material as the tank walls, were attached to a rope at three equidistant depths from the surface of the tank (top: 0.3 m deep, middle: 2.15 m deep, and bottom: 4 m deep). At each sampling time, five replicates of concrete were sampled at each depth. To study planktonic *V. harveyi*, alongside the concrete samples, 1 L of water in five replicates was also collected at each depth using a submersible pump. This process yielded 16 independent sampling times, and samples taken from 16 ropes at the wall of the tank. Sampling apparatus was installed when the tank was in an empty state, and the initial sampling was done one day after impoundment of the tank when the fish were added.

Biofilm and planktonic samples were collected every three weeks. Throughout the sampling campaign, environmental seawater temperatures were carefully monitored, based on data provided by the MAREL Carnot buoy consulted from May to November 2022 (MAREL Carnot, 2022). This buoy supplied daily insights into the environmental temperatures of water used to partially supply the tanks. When seawater temperatures exceeded 19°C for two consecutive nights, sampling events were scheduled within a span of 4–5 days to anticipate the onset of vibriosis episodes. Given the sustained temperatures above 20°C from mid-July to the end of August, a weekly sampling regimen was adopted, amounting to 7 samples (Supplementary Table 1). At each sampling time, abiotic parameters such as water temperature, pH, salinity, turbidity, DO, and conductivity were assessed within the tank at each layer, using an HI-9892 multi-parameter probe (Hanna Instruments, Leighton Buzzard, United Kingdom). Throughout the sampling campaign, the aquaculture facility supplied weekly data concerning biomass and mortality rates for the studied rearing tank. A total of 240 water samples and 240 biofilm samples were collected during the sampling campaign.

### 2.3 Bacterial isolation
changing consumer preferences favouring sustainable practices, relying on antibiotic use is not a viable, long-term solution. In this "One Health" context, aquaculture practitioners could solve this problem by devising targeted antivirulence strategies that not only address the immediate challenges posed by *V. harveyi*, but also align with the principles of sustainable and responsible aquaculture. The goal of this research was to contribute to new strategies for effective management and regulation of vibriosis in aquaculture facilities, based on informed preventive measures.

## 2. Material & methods

### 2.1 Sampling site and rearing conditions

From May to November 2022, a sampling campaign was conducted at Aquanord-Ichtus, an aquaculture establishment situated in Gravelines (France), along the coast of the North Sea. This facility is strategically located near a nuclear power plant, which serves as a source of warm water for the flow-through tank systems via its reactor water cooling system. The facility benefits from thermoregulated water throughout the year, except in summer when the facility faces challenges in cooling the water tank more than environmental water.

The cubical aquaculture tank chosen is an outdoor facility with a volumetric capacity of approximately 975 cubic m, a height of 4 m, and a capacity of up to 68,000 sea bass. The average fish stock density within the tank was 32 kg per cubic meter at the beginning of the campaign, and the average individual fish weight was 517 g.

Fish are fed daily and oxygenation is carried out to maintain ideal dissolved oxygen (DO) levels. To overcome the absence of available vaccines against *V. harveyi*, Aquanord-Ichtus has started to create self-vaccines. This approach enables farmers to use oxytetracycline, an antibiotic, only in extreme cases when vibriosis-induced mortalities are exceptionally high and not effectively managed by other zootechnical measures. During vibriosis outbreaks and elevated fish mortality on the farm, the farmers administered oxytetracycline treatment for 7–10 days, ensuring antibiotic coverage persists for about 4 weeks.

### 2.2 Sample collection and environmental parameters

To study the spatial and temporal dynamics of *V. harveyi* in biofilms, cylindrical concrete specimens (5 x 2.5 cm) constructed from the same material as the tank walls, were attached to a rope at three equidistant depths from the surface of the tank (top: 0.3 m deep, middle: 2.15 m deep, and bottom: 4 m deep). At each sampling time, five replicates of concrete were sampled at each depth. To study planktonic *V. harveyi*, alongside the concrete samples, 1 L of water in five replicates was also collected at each depth using a submersible pump. This process yielded 16 independent sampling times, and samples taken from 16 ropes at the wall of the tank. Sampling apparatus was installed when the tank was in an empty state, and the initial sampling was done one day after impoundment of the tank when the fish were added.

Biofilm and planktonic samples were collected every three weeks. Throughout the sampling campaign, environmental seawater temperatures were carefully monitored, based on data provided by the MAREL Carnot buoy consulted from May to November 2022 (MAREL Carnot, 2022). This buoy supplied daily insights into the environmental temperatures of water used to partially supply the tanks. When seawater temperatures exceeded 19°C for two consecutive nights, sampling events were scheduled within a span of 4–5 days to anticipate the onset of vibriosis episodes. Given the sustained temperatures above 20°C from mid-July to the end of August, a weekly sampling regimen was adopted, amounting to 7 samples (Supplementary Table 1). At each sampling time, abiotic parameters such as water temperature, pH, salinity, turbidity, DO, and conductivity were assessed within the tank at each layer, using an HI-9892 multi-parameter probe (Hanna Instruments, Leighton Buzzard, United Kingdom). Throughout the sampling campaign, the aquaculture facility supplied weekly data concerning biomass and mortality rates for the studied rearing tank A total of 240 water samples and 240 biofilm samples were collected during the sampling campaign.

### 2.3 Bacterial isolation
The experimental protocol, established for a previous study in Aquanord-Ichtus (Mougin et al., 2020), was replicated with minor adaptations. Briefly, we gathered five replicates of 1 L seawater samples from aquaculture tanks at different depths and sampling times. These samples were promptly chilled on ice during transportation to the laboratory and processed within a 3-hour window following the sampling. Filtration involved the sequential use of 0.45 µm and 0.22 µm-pore-size nitrocellulose filters (Grosseron SAS, Couëron, France), with the deployment of multiple filters when necessary to prevent clogging due to sample aggregation. Following this, the filters were immersed in a solution consisting of 30 mL of Luria-Bertani broth supplemented with 20 % NaCl (LBS) containing 20 % glycerol and stored at -80°C until further analysis.

Concrete specimens were submerged at various depths: 0.3, 2.15, and 4 m. Post-collection, the specimens were individually collected and preserved in sterile physiological water and put on ice during transportation to the laboratory. They were immediately processed within a 2-hour timeframe upon arrival. The biofilm collection protocol from concrete, following the Mougin et al. (2020) guidelines with slight adaptations, used a systematic swabbing methodology after rinsing the blocks with sterile physiological water, targeting a specific surface area of 2.34 cm². This process involved use of a sterile, stainless steel jig and two consecutive sets of swabs (AB300250, Labomoderne, Gennevilliers, France). Subsequently, the swabs were immersed in LBS enriched with 20 % glycerol and stored at -80°C until further analysis.

To begin the analytical phases, all samples were thawed and subjected to a 3-min vortexing process. After centrifugation at 8,000 x g for 10 min, the resulting cell pellet was resuspended in physiological water. For subsequent steps, the samples were divided into two subsamples, with one of them (1,800 µL) to be used for DNA extraction and *V. harveyi* quantification through real-time PCR, and the other (200 µL) kept for another study.

## 2.4 Quantification of *V. harveyi* using real-time PCR

The 1,800 µL subsamples underwent a centrifugation step at 8,000 x g for 10 min. Subsequently, a DNeasy PowerBiofilm kit (QIAGEN, Hilden, Germany) was used for total DNA extraction from the cell pellet, adhering to the manufacturer's instructions. This method was chosen based on the procedures described by Mougin et al. (2020). This involved the use of *V. harveyi*-specific primers designed to target the *mreb* gene, which is present in *V. harveyi*'s genome as a single copy. The primers were synthesized by TIB MOLBIOL Syntheselabor GmbH (Berlin, Germany). To establish standard curves for quantification, a series of 10-fold dilutions was prepared using pure DNA from the collection strain *V. harveyi* LMG 4044. For each individual assay, calibration curves were generated by plotting the mean threshold cycle (CT) values derived from two replicate series against the logarithm of the respective dilution factors. To mitigate potential interference from real-time PCR inhibitors, each extracted DNA template was analysed after being diluted tenfold. Genome copy equivalent (GE) concentrations were calculated, taking the dilution factor into consideration. All quantitative PCR reactions were conducted using a LightCycler® 480 instrument (Roche Diagnostics, Meylan, France). These assays included a positive control (*V. harveyi* LMG 4044 DNA), a negative control (*V. campbellii* CIP 70.67 DNA), and a no-template control (NTC) devoid of DNA. To confirm the specificity of the fluorescent signals, melting curve analysis was performed. To ensure the reliability of measurements, each extracted DNA template underwent two rounds of analysis.

## 2.5 Statistical analysis

Data normality was assessed using either the Shapiro-Wilk test or the Kruskal-Wallis test, depending on the dataset distribution. Subsequently, the homogeneity of variances was evaluated using Bartlett's test. To determine significant differences among variables across different layers, a one-way analysis of variance (ANOVA) was conducted. Post-hoc comparisons were carried out using the least significant difference (LSD) method with Bonferroni-adjusted *p*-values. In instances where the data did not meet the assumption of normality, the Kruskal-Wallis test was employed as a non-parametric alternative for comparing medians among different groups. Additionally, a principal component analysis (PCA) was performed on environmental parameters (temperature, pH, salinity, DO, and turbidity), *V. harveyi* water
The experimental protocol, established for a previous study in Aquanord-Ichtus (Mougin et al., 2020), was replicated with minor adaptations. Briefly, we gathered five replicates of 1 L seawater samples from aquaculture tanks at different depths and sampling times. These samples were promptly chilled on ice during transportation to the laboratory and processed within a 3-hour window following the sampling. Filtration involved the sequential use of 0.45 µm and 0.22 µm-pore-size nitrocellulose filters (Grosseron SAS, Couëron, France), with the deployment of multiple filters when necessary to prevent clogging due to sample aggregation. Following this, the filters were immersed in a solution consisting of 30 mL of Luria-Bertani broth supplemented with 20 % NaCl (LBS) containing 20 % glycerol and stored at -80°C until further analysis.

Concrete specimens were submerged at various depths: 0.3, 2.15, and 4 m. Post-collection, the specimens were individually collected and preserved in sterile physiological water and put on ice during transportation to the laboratory. They were immediately processed within a 2-hour timeframe upon arrival. The biofilm collection protocol from concrete, following the Mougin et al. (2020) guidelines with slight adaptations, used a systematic swabbing methodology after rinsing the blocks with sterile physiological water, targeting a specific surface area of 2.34 cm². This process involved use of a sterile, stainless steel jig and two consecutive sets of swabs (AB300250, Labomoderne, Gennevilliers, France). Subsequently, the swabs were immersed in LBS enriched with 20 % glycerol and stored at -80°C until further analysis.

To begin the analytical phases, all samples were thawed and subjected to a 3-min vortexing process. After centrifugation at 8,000 x g for 10 min, the resulting cell pellet was resuspended in physiological water. For subsequent steps, the samples were divided into two subsamples, with one of them (1,800 µL) to be used for DNA extraction and *V. harveyi* quantification through real-time PCR, and the other (200 µL) kept for another study.

## 2.4 Quantification of *V. harveyi* using real-time PCR

The 1,800 µL subsamples underwent a centrifugation step at 8,000 x g for 10 min. Subsequently, a DNeasy PowerBiofilm kit (QIAGEN, Hilden, Germany) was used for total DNA extraction from the cell pellet, adhering to the manufacturer's instructions. This method was chosen based on the procedures described by Mougin et al. (2020). This involved the use of *V. harveyi*-specific primers designed to target the *mreb* gene, which is present in *V. harveyi*'s genome as a single copy. The primers were synthesized by TIB MOLBIOL Syntheselabor GmbH (Berlin, Germany). To establish standard curves for quantification, a series of 10-fold dilutions was prepared using pure DNA from the collection strain *V. harveyi* LMG 4044. For each individual assay, calibration curves were generated by plotting the mean threshold cycle (CT) values derived from two replicate series against the logarithm of the respective dilution factors. To mitigate potential interference from real-time PCR inhibitors, each extracted DNA template was analysed after being diluted tenfold. Genome copy equivalent (GE) concentrations were calculated, taking the dilution factor into consideration. All quantitative PCR reactions were conducted using a LightCycler® 480 instrument (Roche Diagnostics, Meylan, France). These assays included a positive control (*V. harveyi* LMG 4044 DNA), a negative control (*V. campbellii* CIP 70.67 DNA), and a no-template control (NTC) devoid of DNA. To confirm the specificity of the fluorescent signals, melting curve analysis was performed. To ensure the reliability of measurements, each extracted DNA template underwent two rounds of analysis.

## 2.5 Statistical analysis

Data normality was assessed using either the Shapiro-Wilk test or the Kruskal-Wallis test, depending on the dataset distribution. Subsequently, the homogeneity of variances was evaluated using Bartlett's test. To determine significant differences among variables across different layers, a one-way analysis of variance (ANOVA) was conducted. Post-hoc comparisons were carried out using the least significant difference (LSD) method with Bonferroni-adjusted *p*-values. In instances where the data did not meet the assumption of normality, the Kruskal-Wallis test was employed as a non-parametric alternative for comparing medians among different groups. Additionally, a principal component analysis (PCA) was performed on environmental parameters (temperature, pH, salinity, DO, and turbidity), *V. harveyi* water
concentration, *V. harveyi* concrete concentration, and fish mortality. Before conducting PCA, the data were standardised to ensure equal weighting of each variable. Relationships among variables were explored using a correlation matrix, employing the Spearman method. Prior to this analysis, pairwise linear relationships were assessed to ensure the absence of linearity. All statistical analyses, including post-hoc procedures, were conducted using R version 4.3.2 and R Studio version 2021.09.0 (R Core Team, 2023; RStudio Team, 2020) with the "agricolae" (De Mendiburu and Yaseen, 2020), "factomineR" (Lê et al., 2008), "factoextra" (Kassambara and Mundt, 2016), and "corrplot" (Wei and Simko, 2021) packages. All graphics were drawn using the "ggplot2" packages (Wickham, 2016).

## 3. Results

### 3.1 Water quality and fish mortality

The investigation involved a comprehensive examination of water parameters in the top, middle, and bottom layers over the course of May to November 2022. Additionally, fish mortality data were collected and analysed. These data are summarised in Table 1. No significant differences were found across the water column for each sampling time for the recorded parameters, indicating homogeneity of environmental conditions in the tank. Nevertheless, some parameters showed variations throughout the sampling period. For instance, the mean temperature values ranged from a minimum of 16.70°C in November to a maximum of 22.06 °C in August, demonstrating seasonal fluctuation. pH values gradually decreased from an average of 8.07 in May to 6.87 in November. Values also showed increasing acidity with depth in the water column. DO concentrations remained constant. Fish mortality percentages were higher in July and highest in August when peak temperature values were recorded. Fish mortality episodes in May were likely associated with stress induced by transfer of fish into the tank.

### 3.2 Quantification of *V. harveyi*

Abundance of *V. harveyi* was determined using mreb-targeted real-time PCR across the different layers for all samples, as shown in (Fig. 1). The qPCR method has a specified threshold of detection in GE for each sample type: 2.24 GE.mL⁻¹ (0.35 log(GE.mL⁻¹)) for water samples and 501.19 GE.cm⁻² (2.70 log(GE.cm⁻²)) for concrete samples. Despite the potential presence of *V. harveyi*, any concentration below this threshold was considered to be zero in the analysis (Supplementary Table 2). For a given sampling time, no significant differences were found trough the water column for each sample type.

In water samples, the presence of *V. harveyi* started to be detected from the beginning of the sampling campaign in May, persisting consistently thereafter. A significant higher *V. harveyi* concentrations was observed in August compared to other months (p < 0.05). Of note, *V. harveyi* abundance in the bottom layer ranged from 0.52 to 3.36 log(GE.mL⁻¹) (Fig. 1), with a distinct peak in August when the temperature of the water tank was at its highest. Moreover, abundances in the middle and top layers exhibited similar profiles through the months, ranging from 0.60 to 2.43 log(GE.mL⁻¹) and from 0.48 to 2.89 log(GE.mL⁻¹) respectively. Conversely, when oxytetracycline was used during episodes of mortality, a decrease in *V. harveyi* concentrations was observed in water samples, allowing for concentration maintenance below 1.90 log(GE.mL⁻¹), followed by a gradual increase towards the end of the antibiotic's predicted effect.

Many samples were found to be below the qPCR detection threshold for the concrete samples. The estimated concentration of *V. harveyi* in biofilm samples did not exhibit a significant difference over time and through the water column. Vibrio harveyi started to be detected in June and ceased by the end of October for top and middle tank samples. Bottom samples were detected until the end of the sampling campaign. Detected abundances in the bottom, middle and top layers of the tank ranged from 2.88 to 4.51 log(GE.cm⁻²), 3.02 to 4.60 log(GE.cm⁻²), and 2.98 to 4.93 log(GE.cm⁻²), respectively (Fig. 1). A rapid increase in *V. harveyi* abundance was observed during high-temperature periods, particularly from August to September, resulting in two peaks during these months associated with fish mortality in the tank (Table 1). Statistical analysis revealed significant variations in *V. harveyi* concentrations for all layers, with August and September forming a cluster characterised by concentrations significantly higher than those observed for the rest of the months (p < 0.05). Moreover, during these episodes of mortality, oxytetracycline was used and a resulting decrease in *V. harveyi* abundance was observed, enabling concentration maintenance
concentration, *V. harveyi* concrete concentration, and fish mortality. Before conducting PCA, the data were standardised to ensure equal weighting of each variable. Relationships among variables were explored using a correlation matrix, employing the Spearman method. Prior to this analysis, pairwise linear relationships were assessed to ensure the absence of linearity. All statistical analyses, including post-hoc procedures, were conducted using R version 4.3.2 and R Studio version 2021.09.0 (R Core Team, 2023; RStudio Team, 2020) with the "agricolae" (De Mendiburu and Yaseen, 2020), "factomineR" (Lê et al., 2008), "factoextra" (Kassambara and Mundt, 2016), and "corrplot" (Wei and Simko, 2021) packages. All graphics were drawn using the "ggplot2" packages (Wickham, 2016).

## 3. Results

### 3.1 Water quality and fish mortality

The investigation involved a comprehensive examination of water parameters in the top, middle, and bottom layers over the course of May to November 2022. Additionally, fish mortality data were collected and analysed. These data are summarised in Table 1. No significant differences were found across the water column for each sampling time for the recorded parameters, indicating homogeneity of environmental conditions in the tank. Nevertheless, some parameters showed variations throughout the sampling period. For instance, the mean temperature values ranged from a minimum of 16.70°C in November to a maximum of 22.06 °C in August, demonstrating seasonal fluctuation. pH values gradually decreased from an average of 8.07 in May to 6.87 in November. Values also showed increasing acidity with depth in the water column. DO concentrations remained constant. Fish mortality percentages were higher in July and highest in August when peak temperature values were recorded. Fish mortality episodes in May were likely associated with stress induced by transfer of fish into the tank.

### 3.2 Quantification of *V. harveyi*

Abundance of *V. harveyi* was determined using mreb-targeted real-time PCR across the different layers for all samples, as shown in (Fig. 1). The qPCR method has a specified threshold of detection in GE for each sample type: 2.24 GE.mL⁻¹ (0.35 log(GE.mL⁻¹)) for water samples and 501.19 GE.cm⁻² (2.70 log(GE.cm⁻²)) for concrete samples. Despite the potential presence of *V. harveyi*, any concentration below this threshold was considered to be zero in the analysis (Supplementary Table 2). For a given sampling time, no significant differences were found trough the water column for each sample type.

In water samples, the presence of *V. harveyi* started to be detected from the beginning of the sampling campaign in May, persisting consistently thereafter. A significant higher *V. harveyi* concentrations was observed in August compared to other months (p < 0.05). Of note, *V. harveyi* abundance in the bottom layer ranged from 0.52 to 3.36 log(GE.mL⁻¹) (Fig. 1), with a distinct peak in August when the temperature of the water tank was at its highest. Moreover, abundances in the middle and top layers exhibited similar profiles through the months, ranging from 0.60 to 2.43 log(GE.mL⁻¹) and from 0.48 to 2.89 log(GE.mL⁻¹) respectively. Conversely, when oxytetracycline was used during episodes of mortality, a decrease in *V. harveyi* concentrations was observed in water samples, allowing for concentration maintenance below 1.90 log(GE.mL⁻¹), followed by a gradual increase towards the end of the antibiotic's predicted effect.

Many samples were found to be below the qPCR detection threshold for the concrete samples. The estimated concentration of *V. harveyi* in biofilm samples did not exhibit a significant difference over time and through the water column. Vibrio harveyi started to be detected in June and ceased by the end of October for top and middle tank samples. Bottom samples were detected until the end of the sampling campaign. Detected abundances in the bottom, middle and top layers of the tank ranged from 2.88 to 4.51 log(GE.cm⁻²), 3.02 to 4.60 log(GE.cm⁻²), and 2.98 to 4.93 log(GE.cm⁻²), respectively (Fig. 1). A rapid increase in *V. harveyi* abundance was observed during high-temperature periods, particularly from August to September, resulting in two peaks during these months associated with fish mortality in the tank (Table 1). Statistical analysis revealed significant variations in *V. harveyi* concentrations for all layers, with August and September forming a cluster characterised by concentrations significantly higher than those observed for the rest of the months (p < 0.05). Moreover, during these episodes of mortality, oxytetracycline was used and a resulting decrease in *V. harveyi* abundance was observed, enabling concentration maintenance
to remain below 3.97 log(GE.cm⁻²). The abundance of *V. harveyi* gradually rose as the antibiotic's predicted effect started to fade.

## 3.3 Influence of environmental factors

PCA (Fig. 2) was combined with a correlation matrix (Fig. 3) to reveal the interactions of the parameters measured in the aquaculture tank. Both dimensions of the PCA, Dim.1 and Dim.2, captured 64.40% of the data variance, explaining 42.08% and 22.32% of total variance, respectively. Temperature, *V. harveyi* abundance in water ([Vh water]), and *V. harveyi* in biofilm form ([Vh concrete]), emerged as the primary drivers shaping the Dim.1 variance landscape, while Dim.2 was predominantly influenced by DO and turbidity. Strong positive correlations (0.88, *p* < 0.001) were found between *V. harveyi* abundance in water and its abundance in biofilm within the tank, as highlighted in the correlation matrix, indicating that the biofilm results align closely with those observed in the water. A strong positive correlation was observed in the PCA between temperature and both *V. harveyi* abundance in water (0.891, *p* < 0.001) and in biofilm (0.834, *p* < 0.001), but also, to a lesser extent, salinity (0.772, *p* < 0.001). Further confirming and extending these findings, the correlation matrix highlighted significant positive correlations such as temperature with salinity (0.84, *p* < 0.001), *V. harveyi* concentration in water (0.63, *p* < 0.001), concrete (0.55, *p* < 0.001), and fish mortality (0.50, *p* < 0.001). In contrast, pH exhibited a negative correlation with *V. harveyi* abundance in water (-0.43, *p* < 0.001), indicating an inverse relationship. On the other hand, Dim.2 revealed significant correlations with DO (0.798, *p* < 0.001), pH (0.566, *p* < 0.001), fish mortality (0.496, *p* < 0.001), and turbidity (-0.741, *p* < 0.001). Additionally, the correlation matrix revealed strong negative correlations between DO and turbidity (-0.40, *p* = 0.0054), and positive correlation with fish mortality (0.34, *p* = 0.0196).

# 4. Discussion

## 4.1 Temporal and spatial study of *V. harveyi* in aquaculture

Our investigation examined the temporal and spatial distribution of *V. harveyi* throughout a sampling campaign spanning May to November 2022 on a farm in northern France. This farm has experienced recurring vibriosis episodes attributed to *V. harveyi*. A previous study conducted in a "small tank" (75 m³, 1.3 m deep), revealed the consistent presence and persistence of *V. harveyi* in both planktonic form and biofilms throughout the year (Mougin et al., 2021). This previous study was restricted by the absence of vibriosis outbreaks during the campaign, but also by the small tank size, which prevented in-depth exploration of variations in environmental conditions and their impact on *V. harveyi*. To better understand the dynamics of *V. harveyi* through the water column, we implemented sampling campaign in a 4-metre-deep tank at three different depths. We systematically monitored environmental parameters to find potential explanations for the observed variations. Our study sets itself apart by adopting a comprehensive sampling approach over an extended time frame that included the periods before, during, and after two episodes of vibriosis. Additionally, during our sampling campaign, the two vibriosis outbreaks forced the farmers to use antibiotics. Consequently, this study offers insights into antibiotic effects on *V. harveyi*, considering both planktonic and biofilm forms. This campaign also sheds light on the omnipresence of *V. harveyi* within the aquaculture tank. From the very onset, brought in with the initial filling of the tank with water, *V. harveyi* demonstrated its adaptability, swiftly establishing its presence in the tank environment. *Vibrio harveyi* was present and quantifiable during the whole sampling campaign in planktonic form in the water, and during warmer months in biofilms. Its abundance in biofilms potentially increased due to persistence once established on the tank wall, a finding in line with the previous sampling campaign on the same aquaculture farm in 2018 (Mougin et al., 2021). A distinctive characteristic of *Vibrio*, including *V. harveyi*, is their ability to adapt to changing conditions. This adaptability is underscored by their capacity to transition from free-swimming cells to "swarmer cells" within biofilms (Khan et al., 2020; McCarter, 1999). Furthermore, during the warmer months, it is known that the abundance of other coexisting bacterial families within biofilms such as *Rhodobacteraceae* decreases, providing an environment that enables the *Vibrionaceae* to thrive in the microbial community structure (Bourne et al., 2004; Roquigny et al., 2021)
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to remain below 3.97 log(GE.cm⁻²). The abundance of *V. harveyi* gradually rose as the antibiotic's predicted effect started to fade.

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PCA (Fig. 2) was combined with a correlation matrix (Fig. 3) to reveal the interactions of the parameters measured in the aquaculture tank. Both dimensions of the PCA, Dim.1 and Dim.2, captured 64.40% of the data variance, explaining 42.08% and 22.32% of total variance, respectively. Temperature, *V. harveyi* abundance in water ([Vh water]), and *V. harveyi* in biofilm form ([Vh concrete]), emerged as the primary drivers shaping the Dim.1 variance landscape, while Dim.2 was predominantly influenced by DO and turbidity. Strong positive correlations (0.88, *p* < 0.001) were found between *V. harveyi* abundance in water and its abundance in biofilm within the tank, as highlighted in the correlation matrix, indicating that the biofilm results align closely with those observed in the water. A strong positive correlation was observed in the PCA between temperature and both *V. harveyi* abundance in water (0.891, *p* < 0.001) and in biofilm (0.834, *p* < 0.001), but also, to a lesser extent, salinity (0.772, *p* < 0.001). Further confirming and extending these findings, the correlation matrix highlighted significant positive correlations such as temperature with salinity (0.84, *p* < 0.001), *V. harveyi* concentration in water (0.63, *p* < 0.001), concrete (0.55, *p* < 0.001), and fish mortality (0.50, *p* < 0.001). In contrast, pH exhibited a negative correlation with *V. harveyi* abundance in water (-0.43, *p* < 0.001), indicating an inverse relationship. On the other hand, Dim.2 revealed significant correlations with DO (0.798, *p* < 0.001), pH (0.566, *p* < 0.001), fish mortality (0.496, *p* < 0.001), and turbidity (-0.741, *p* < 0.001). Additionally, the correlation matrix revealed strong negative correlations between DO and turbidity (-0.40, *p* = 0.0054), and positive correlation with fish mortality (0.34, *p* = 0.0196).

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## 4. Discussion

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Our investigation examined the temporal and spatial distribution of *V. harveyi* throughout a sampling campaign spanning May to November 2022 on a farm in northern France. This farm has experienced recurring vibriosis episodes attributed to *V. harveyi*. A previous study conducted in a "small tank" (75 m³, 1.3 m deep), revealed the consistent presence and persistence of *V. harveyi* in both planktonic form and biofilms throughout the year (Mougin et al., 2021). This previous study was restricted by the absence of vibriosis outbreaks during the campaign, but also by the small tank size, which prevented in-depth exploration of variations in environmental conditions and their impact on *V. harveyi*. To better understand the dynamics of *V. harveyi* through the water column, we implemented sampling campaign in a 4-metre-deep tank at three different depths. We systematically monitored environmental parameters to find potential explanations for the observed variations. Our study sets itself apart by adopting a comprehensive sampling approach over an extended time frame that included the periods before, during, and after two episodes of vibriosis. Additionally, during our sampling campaign, the two vibriosis outbreaks forced the farmers to use antibiotics. Consequently, this study offers insights into antibiotic effects on *V. harveyi*, considering both planktonic and biofilm forms. This campaign also sheds light on the omnipresence of *V. harveyi* within the aquaculture tank. From the very onset, brought in with the initial filling of the tank with water, *V. harveyi* demonstrated its adaptability, swiftly establishing its presence in the tank environment. *Vibrio harveyi* was present and quantifiable during the whole sampling campaign in planktonic form in the water, and during warmer months in biofilms. Its abundance in biofilms potentially increased due to persistence once established on the tank wall, a finding in line with the previous sampling campaign on the same aquaculture farm in 2018 (Mougin et al., 2021). A distinctive characteristic of *Vibrio*, including *V. harveyi*, is their ability to adapt to changing conditions. This adaptability is underscored by their capacity to transition from free-swimming cells to "swarmer cells" within biofilms (Khan et al., 2020; McCarter, 1999). Furthermore, during the warmer months, it is known that the abundance of other coexisting bacterial families within biofilms such as *Rhodobacteraceae* decreases, providing an environment that enables the *Vibrionaceae* to thrive in the microbial community structure (Bourne et al., 2004; Roquigny et al., 2021)
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In comparison to Mougin et al. (2021) our study observed higher concentrations of *V. harveyi* in our water tank. They found, actually, a maximum concentration of 9.51×10¹ GE.mL⁻¹ in rearing tanks, while a maximum concentration of 2.54 x 10³ GE.mL⁻¹ was found in our water tank. This contrast could be due to differences in environmental conditions, but also due to different tank sizes and fish. In our study, which aimed to investigate the water column, all measured abiotic parameters in the water column exhibited clear stability, a characteristic likely shaped by the conditions of fish farming. These conditions, which ensure efficient water circulation, additionally play a role in homogenising tank parameters overall. The concentrations of *V. harveyi* reflected the same stability through the water column. Similar observations regarding *Vibrio* spp. concentrations in the water column were found in a natural oyster farming site (Cruz et al., 2020). The only significant difference observed by Cruz et al. (2020) appeared for some oysters collected close to the seafloor where sediment accumulation was high. Even though our tank water appeared to be homogeneous, organic matter accumulated at the floor of the tank. This phenomenon is caused by fewer fish activities at the bottom, resulting in less associated movement. It can also be attributed to the sedimentation and accumulation of nutrient-rich sediments consisting of food scraps and fish faeces. Moreover, sediments are known to serve as a source of nutrients for *V. harveyi*, which not only sediment themselves in the tank, but are also able to absorb dissolved organic matter (Neogi et al., 2018). Sediments also provide an additional surface for colonisation, protecting the bacteria from biotic and abiotic stresses, and enabling their persistence in the tank (Pianetti et al., 2004; Vezzulli et al., 2002). This hypothesis regarding sediment dynamics and *V. harveyi* colonization could be tested by conducting further sampling after emptying the tank, which would provide insights into the role of sediments in *V. harveyi* dynamics in aquaculture settings.

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## 4.3 Environmental factors influencing *V. harveyi* distribution

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Even though *V. harveyi* concentrations were spatially homogeneous throughout the water column, temporal variations were observed through the 7-month sampling campaign. Relationships between bacterial concentrations and measured environmental parameters were investigated through PCA (Fig. 2) and a correlation matrix (Fig. 3). Interestingly, temperature emerged as the foremost factor influencing the abundance of *V. harveyi*, as abundance was consistently noted to be more favourable as temperatures rose above 20 °C. Given that *V. harveyi* was introduced into the tank water from the sea, its persistence within the tank is facilitated by favourable tank conditions, enabling it to form biofilms on the tank walls and promoting its growth. Additionally, extensive studies have demonstrated the crucial influence of temperature on the presence of *V. harveyi* in natural aquatic systems, displaying a clear seasonal cycle, particularly during the summer when temperatures exceed 25 °C (Chimetto et al., 2008; Zhou et al., 2012). This seasonality was also observed in our study, as *V. harveyi* exhibited higher abundance during the warmest months, when temperatures reached up to 22 °C. However, it is important to note that vibriosis does not necessarily occur when temperature reach 22 °C, suggesting that additional factors may influence its occurrence. Additionally, the correlation matrix (Fig. 3) suggests that salinity and pH, which are predicted to change with overall ocean temperatures (Gulev et al., 2021), also positively affected the presence of *V. harveyi* in the aquaculture tank. These findings are consistent with prior researches on the effects of environmental parameters on *Vibrio* species in natural and in aquaculture settings, where a positive correlation between salinity and temperature with the frequency of detection of *Vibrio* species has been reported (Abioye et al., 2021; Scro et al., 2022). DO concentration is reported to have a negative correlation with temperature, given that increased water temperatures lead to decreased DO levels (Pfeffer et al., 2003). However, in our case, DO levels were managed continuously by the farmers, which explains the absence of any observable effect on the abundance of *V. harveyi*. A second negative correlation was observed with pH values. Although *V. harveyi* is known to tolerate wide ranges of pH levels (i.e., 5.5 to 9.0), the correlation matrix in our study shows that pH is negatively correlated with both *V. harveyi* abundance in water and salinity (Prayitno and Latchford, 1995). This parameter cannot be fully controlled in aquaculture, adding another challenge to vibriosis management in tanks. In addition, global warming and ocean acidification may alter the microbial balance, favouring the presence of *Vibrio*, while diminishing populations of other bacterial genera (Montánchez and Kaberdin, 2020). All these
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In comparison to Mougin et al. (2021) our study observed higher concentrations of *V. harveyi* in our water tank. They found, actually, a maximum concentration of 9.51×10¹ GE.mL⁻¹ in rearing tanks, while a maximum concentration of 2.54 x 10³ GE.mL⁻¹ was found in our water tank. This contrast could be due to differences in environmental conditions, but also due to different tank sizes and fish. In our study, which aimed to investigate the water column, all measured abiotic parameters in the water column exhibited clear stability, a characteristic likely shaped by the conditions of fish farming. These conditions, which ensure efficient water circulation, additionally play a role in homogenising tank parameters overall. The concentrations of *V. harveyi* reflected the same stability through the water column. Similar observations regarding *Vibrio* spp. concentrations in the water column were found in a natural oyster farming site (Cruz et al., 2020). The only significant difference observed by Cruz et al. (2020) appeared for some oysters collected close to the seafloor where sediment accumulation was high. Even though our tank water appeared to be homogeneous, organic matter accumulated at the floor of the tank. This phenomenon is caused by fewer fish activities at the bottom, resulting in less associated movement. It can also be attributed to the sedimentation and accumulation of nutrient-rich sediments consisting of food scraps and fish faeces. Moreover, sediments are known to serve as a source of nutrients for *V. harveyi*, which not only sediment themselves in the tank, but are also able to absorb dissolved organic matter (Neogi et al., 2018). Sediments also provide an additional surface for colonisation, protecting the bacteria from biotic and abiotic stresses, and enabling their persistence in the tank (Pianetti et al., 2004; Vezzulli et al., 2002). This hypothesis regarding sediment dynamics and *V. harveyi* colonization could be tested by conducting further sampling after emptying the tank, which would provide insights into the role of sediments in *V. harveyi* dynamics in aquaculture settings.

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Even though *V. harveyi* concentrations were spatially homogeneous throughout the water column, temporal variations were observed through the 7-month sampling campaign. Relationships between bacterial concentrations and measured environmental parameters were investigated through PCA (Fig. 2) and a correlation matrix (Fig. 3). Interestingly, temperature emerged as the foremost factor influencing the abundance of *V. harveyi*, as abundance was consistently noted to be more favourable as temperatures rose above 20 °C. Given that *V. harveyi* was introduced into the tank water from the sea, its persistence within the tank is facilitated by favourable tank conditions, enabling it to form biofilms on the tank walls and promoting its growth. Additionally, extensive studies have demonstrated the crucial influence of temperature on the presence of *V. harveyi* in natural aquatic systems, displaying a clear seasonal cycle, particularly during the summer when temperatures exceed 25 °C (Chimetto et al., 2008; Zhou et al., 2012). This seasonality was also observed in our study, as *V. harveyi* exhibited higher abundance during the warmest months, when temperatures reached up to 22 °C. However, it is important to note that vibriosis does not necessarily occur when temperature reach 22 °C, suggesting that additional factors may influence its occurrence. Additionally, the correlation matrix (Fig. 3) suggests that salinity and pH, which are predicted to change with overall ocean temperatures (Gulev et al., 2021), also positively affected the presence of *V. harveyi* in the aquaculture tank. These findings are consistent with prior researches on the effects of environmental parameters on *Vibrio* species in natural and in aquaculture settings, where a positive correlation between salinity and temperature with the frequency of detection of *Vibrio* species has been reported (Abioye et al., 2021; Scro et al., 2022). DO concentration is reported to have a negative correlation with temperature, given that increased water temperatures lead to decreased DO levels (Pfeffer et al., 2003). However, in our case, DO levels were managed continuously by the farmers, which explains the absence of any observable effect on the abundance of *V. harveyi*. A second negative correlation was observed with pH values. Although *V. harveyi* is known to tolerate wide ranges of pH levels (i.e., 5.5 to 9.0), the correlation matrix in our study shows that pH is negatively correlated with both *V. harveyi* abundance in water and salinity (Prayitno and Latchford, 1995). This parameter cannot be fully controlled in aquaculture, adding another challenge to vibriosis management in tanks. In addition, global warming and ocean acidification may alter the microbial balance, favouring the presence of *Vibrio*, while diminishing populations of other bacterial genera (Montánchez and Kaberdin, 2020). All these
340 observations highlight the complexity to identify the multiparameter ideal Vibrio environment; varying
341 based on geographic location and environmental conditions (Trinanes and Martinez-Urtaza, 2021).

342 **4.4 Challenges and considerations in managing vibriosis outbreaks**

343 The repeated occurrences and unpredictability of vibriosis outbreaks associated with *V. harveyi* present a
344 significant challenge to the seabass aquaculture sector, as highlighted by Vendramin et al. (2001) and
345 Firmino et al. (2019). The issue becomes particularly problematic in open-system aquaculture, since the
346 prevalence of this pathogen in fish closely aligns with its abundance in the surrounding environment (Kim
347 and Lee, 2017), but also because water quality cannot be fully controlled. Moreover, in the context of
348 climate change, there has been a temperature increase of about 0.88 °C in sea and ocean surface
349 temperatures over the last century (Gulev et al., 2021). This temperature rise is known to affect the
350 distribution and abundance of marine organisms (IPCC, 2022), including *Vibrio*, which has seen its
351 abundance increase sharply in the North Sea over the period 1958–2011 (Vezzulli et al., 2002). According
352 to the Intergovernmental Panel on Climate Change, the ocean is projected to continue warming, leading
353 to long-term impacts on ocean ecosystems and human societies (IPCC, 2022). Open aquaculture systems
354 may therefore need to address an increase in vibriosis episodes. This heightened risk could require
355 increased antibiotic use, posing risks to fish health, environmental integrity, and consumer safety. In
356 response to these challenges, it is imperative to develop specific and effective solutions. Antibiotics, as
357 suggested by our findings, fall short as a sustainable solution, since despite their use by farmers, the
358 concentrations of *V. harveyi* in water and biofilms quickly rebound. Within a three-month period, farmers
359 had to resort to antibiotics twice to avoid massive fish losses (Fig. 2). Given the escalating importance of
360 addressing the *Vibrio* challenge in response to global warming, it is crucial to adopt a comprehensive
361 approach to understand the virulence mechanisms used by *V. harveyi*. As an opportunistic pathogen, *V.
362 harveyi* thrives in warmer temperatures, requiring proactive measures in response to changing climate
363 conditions that could now favour its growth in previously cooler waters. Addressing the core issue involves
364 reducing bacterial virulence through antivirulence therapies (Clatworthy et al., 2007; D’Angelo et al.,
365 2018). Environmental effects impact not only to behaviour of the bacterium, but also to its area of
366 distribution, which is bound to evolve. For some geographic areas, including the one in the present study
367 and more generally temperate waters, such changes may pose challenges regarding *Vibrio* expansion
368 (Trinanes and Martinez-Urtaza, 2021). Moreover, they may even compromise host immunity, thereby
369 enhancing the bacterium’s potential to increase mortality. Understanding these multifaceted interactions
370 becomes a keystone in formulating nuanced and effective strategies aimed at mitigating the impact of
371 potential vibriosis outbreaks.

372 **5. Conclusion**

373 In conclusion, this study highlighted the challenges of managing *V. harveyi* outbreaks in aquaculture
374 systems. The persistence of *V. harveyi* pose major challenges, particularly during warmer months. The
375 high adaptability of *Vibrio* and particularly *V. harveyi*, implies its considerable permissiveness, rendering
376 the task of attempting to control the entire environment to prevent vibriosis highly challenging, especially
377 in the context of global warming and ocean acidification (Montánchez and Kaberdin, 2020). Additionally,
378 significant correlations were found between *V. harveyi* concentrations and various environmental
379 parameters, highlighting the complex dynamics influencing its distribution. While temperature emerged
380 as a primary influencer, the absence of vibriosis outbreaks every summer suggests the influence of
381 additional parameters such as salinity and pH. Furthermore, in the context of escalating *Vibrio* abundance
382 linked to rising global ocean temperatures, concerns arise regarding the heightened pathogenic potential
383 of certain *Vibrio* strains, potentially leading to an increased incidence of diseases in the marine
384 environment. This poses a real challenge for open aquaculture systems which are specifically impacted by
385 these temperature increases as they are unable to regulate water temperature during the hottest months.
386 In navigating these intricate ecological challenges, the integration of a “One Health” framework becomes
387 imperative, emphasising the interconnectedness of animal, environmental, and human health (McEwen
388 and Collignon, 2018; Zinsstag et al., 2011). Understanding the virulence mechanisms of *V. harveyi* is crucial
389 for developing effective countermeasures and adaptive strategies that align with changing ecological
390 systems. This emphasizes the need to explore its virulence genes and their responses to environmental
340
341

observations highlight the complexity to identify the multiparameter ideal Vibrio environment; varying based on geographic location and environmental conditions (Trinanes and Martinez-Urtaza, 2021).

342

## 4.4 Challenges and considerations in managing vibriosis outbreaks

343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371

The repeated occurrences and unpredictability of vibriosis outbreaks associated with *V. harveyi* present a significant challenge to the seabass aquaculture sector, as highlighted by Vendramin et al. (2001) and Firmino et al. (2019). The issue becomes particularly problematic in open-system aquaculture, since the prevalence of this pathogen in fish closely aligns with its abundance in the surrounding environment (Kim and Lee, 2017), but also because water quality cannot be fully controlled. Moreover, in the context of climate change, there has been a temperature increase of about 0.88 °C in sea and ocean surface temperatures over the last century (Gulev et al., 2021). This temperature rise is known to affect the distribution and abundance of marine organisms (IPCC, 2022), including *Vibrio*, which has seen its abundance increase sharply in the North Sea over the period 1958–2011 (Vezzulli et al., 2002). According to the Intergovernmental Panel on Climate Change, the ocean is projected to continue warming, leading to long-term impacts on ocean ecosystems and human societies (IPCC, 2022). Open aquaculture systems may therefore need to address an increase in vibriosis episodes. This heightened risk could require increased antibiotic use, posing risks to fish health, environmental integrity, and consumer safety. In response to these challenges, it is imperative to develop specific and effective solutions. Antibiotics, as suggested by our findings, fall short as a sustainable solution, since despite their use by farmers, the concentrations of *V. harveyi* in water and biofilms quickly rebound. Within a three-month period, farmers had to resort to antibiotics twice to avoid massive fish losses (Fig. 2). Given the escalating importance of addressing the *Vibrio* challenge in response to global warming, it is crucial to adopt a comprehensive approach to understand the virulence mechanisms used by *V. harveyi*. As an opportunistic pathogen, *V. harveyi* thrives in warmer temperatures, requiring proactive measures in response to changing climate conditions that could now favour its growth in previously cooler waters. Addressing the core issue involves reducing bacterial virulence through antivirulence therapies (Clatworthy et al., 2007; D'Angelo et al., 2018). Environmental effects impact not only to behaviour of the bacterium, but also to its area of distribution, which is bound to evolve. For some geographic areas, including the one in the present study and more generally temperate waters, such changes may pose challenges regarding *Vibrio* expansion (Trinanes and Martinez-Urtaza, 2021). Moreover, they may even compromise host immunity, thereby enhancing the bacterium's potential to increase mortality. Understanding these multifaceted interactions becomes a keystone in formulating nuanced and effective strategies aimed at mitigating the impact of potential vibriosis outbreaks.

372

## 5. Conclusion

373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390

In conclusion, this study highlighted the challenges of managing *V. harveyi* outbreaks in aquaculture systems. The persistence of *V. harveyi* pose major challenges, particularly during warmer months. The high adaptability of *Vibrio* and particularly *V. harveyi*, implies its considerable permissiveness, rendering the task of attempting to control the entire environment to prevent vibriosis highly challenging, especially in the context of global warming and ocean acidification (Montánchez and Kaberdin, 2020). Additionally, significant correlations were found between *V. harveyi* concentrations and various environmental parameters, highlighting the complex dynamics influencing its distribution. While temperature emerged as a primary influencer, the absence of vibriosis outbreaks every summer suggests the influence of additional parameters such as salinity and pH. Furthermore, in the context of escalating *Vibrio* abundance linked to rising global ocean temperatures, concerns arise regarding the heightened pathogenic potential of certain *Vibrio* strains, potentially leading to an increased incidence of diseases in the marine environment. This poses a real challenge for open aquaculture systems which are specifically impacted by these temperature increases as they are unable to regulate water temperature during the hottest months. In navigating these intricate ecological challenges, the integration of a “One Health” framework becomes imperative, emphasising the interconnectedness of animal, environmental, and human health (McEwen and Collignon, 2018; Zinsstag et al., 2011). Understanding the virulence mechanisms of *V. harveyi* is crucial for developing effective countermeasures and adaptive strategies that align with changing ecological systems. This emphasizes the need to explore its virulence genes and their responses to environmental
# 6. Acknowledgements

# 7. References
391 factors like temperature, which are expected to evolve in the future. These insights are crucial for guiding
392 future research and developing strategies for disease management and sustainable aquaculture practices.

393 6. Acknowledgements

394 The authors would like to thank Aquanord-Ichtus and especially Guillaume Tielie, Anabelle Duhamel, and
395 Céline Doyen. The authors also thank Lorédane Clause for technical support. Alix Da Fonseca Ferreira
396 would like to thank the PMCO council and Hauts-de-France regional council for their financial support of
397 her PhD studies. This work was funded by the French government, Ifremer, and the Hauts-de-France
398 region in the framework of the CPER 2021-2027 IDEAL project, and by IFSEA Graduate School (which
399 originates from the French National Research Agency under the Investments for the Future programme
400 ANR-21-EXES-00 11).

401 7. References

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448 Gulev, S.K., Thorne, P.W., Ahn, J., Dentener, F.J., Domingues, C.M., Gerland, S., Gong, D., Kaufman, D.S., Nnamchi, H.C., Quaas, J., 2021. Changing state of the climate system. Climate change 287-422.

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456 Kassambara, A., Mundt, F., 2016. factoextra: Extract and Visualize the Results of Multivariate Data Analyses.

458 Khan, F., Tabassum, N., Anand, R., Kim, Y.-M., 2020. Motility of *Vibrio* spp.: regulation and controlling strategies. Appl Microbiol Biotechnol 104, 8187-8208. https://doi.org/10.1007/s00253-020-10794-7

461 Kim, J.Y., Lee, J.-L., 2017. Correlation of Total Bacterial and *Vibrio* spp. Populations between Fish and Water in the Aquaculture System. Frontiers in Marine Science 4.

463 Korun, J., 2008. Marine *Vibrios* associated with diseased sea bass (*Dicentrarchus labrax*) in Turkey. Journal of Fisheriesciences.com. https://doi.org/10.3153/jfscom.2008007

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487 Mougin, J., Roquigny, R., Flahaut, C., Bonnin-Jusserand, M., Grard, T., Le Bris, C., 2021. Abundance and spatial patterns over time of Vibrionaceae and *Vibrio harveyi* in water and biofilm from a seabass aquaculture facility. Aquaculture 542, 736862. https://doi.org/10.1016/j.aquaculture.2021.736862

491 Mougin, J., Roquigny, R., Travers, M.-A., Grard, T., Bonnin-Jusserand, M., Le Bris, C., 2020. Development of a mreB-targeted real-time PCR method for the quantitative detection of *Vibrio harveyi* in
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442 De Mendiburu, F., Yaseen, M., 2020. agricolae: Statistical Procedures for Agricultural Research.

443 EUMOFA, 2022. The EU fish market: 2022 edition. Publications Office, LU.

444 Firmino, J., Furones, M.D., Andree, K.B., Sarasquete, C., Ortiz-Delgado, J.B., Asencio-Alcudia, G., Gisbert, E., 2019. Contrasting outcomes of *Vibrio harveyi* pathogenicity in gilthead seabream, *Sparus aurata* and European seabass, *Dicentrachus labrax*. Aquaculture 511, 734210. https://doi.org/10.1016/j.aquaculture.2019.734210

448 Gulev, S.K., Thorne, P.W., Ahn, J., Dentener, F.J., Domingues, C.M., Gerland, S., Gong, D., Kaufman, D.S., Nnamchi, H.C., Quaas, J., 2021. Changing state of the climate system. Climate change 287-422.

450 IPCC, 2022. Climate Change 2022: Impacts, Adaptation and Vulnerability. Contribution of Working Group II to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change. IPCC Sixth Assessment Report.

453 Islam, S.I., Mou, M.J., Sanjida, S., Tariq, M., Nasir, S., Mahfuj, S., 2022. Designing a novel mRNA vaccine against *Vibrio harveyi* infection in fish: an immunoinformatics approach. Genomics Inform 20, e11. https://doi.org/10.5808/gi.21065

456 Kassambara, A., Mundt, F., 2016. factoextra: Extract and Visualize the Results of Multivariate Data Analyses.

458 Khan, F., Tabassum, N., Anand, R., Kim, Y.-M., 2020. Motility of *Vibrio* spp.: regulation and controlling strategies. Appl Microbiol Biotechnol 104, 8187-8208. https://doi.org/10.1007/s00253-020-10794-7

461 Kim, J.Y., Lee, J.-L., 2017. Correlation of Total Bacterial and *Vibrio* spp. Populations between Fish and Water in the Aquaculture System. Frontiers in Marine Science 4.

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471 Maulvault, A.L., Camacho, C., Barbosa, V., Alves, R., Anacleto, P., Fogaça, F., Kwadijk, C., Kotterman, M., Cunha, S.C., Fernandes, J.O., Rasmussen, R.R., Sloth, J.J., Aznar-Alemany, Ò., Eljarrat, E., Barceló, D., Marques, A., 2018. Assessing the effects of seawater temperature and pH on the bioaccumulation of emerging chemical contaminants in marine bivalves. Environmental Research 161, 236-247. https://doi.org/10.1016/j.envres.2017.11.017

476 McCarter, L., 1999. The multiple identities of *Vibrio parahaemolyticus*. J Mol Microbiol Biotechnol 1, 51-57.

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480 Mohamad, N., Amal, M.N.A., Saad, M.Z., Yasin, I.S.M., Zulkiply, N.A., Mustafa, M., Nasruddin, N.S., 2019. Virulence-associated genes and antibiotic resistance patterns of *Vibrio* spp. isolated from cultured marine fishes in Malaysia. BMC veterinary research 15, 176. https://doi.org/10.1186/s12917-019-1907-8

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Figure 1: Biofilm concentrations of *V. harveyi* in log genome copy equivalents (GE).cm⁻² and *V. harveyi* concentrations in log GE.mL⁻¹ according to tank depth and time. The coloured lines correspond to the averages of the 5 replicates, which are represented by the shapes. Antibiotic treatments with oxytetracycline are indicated in dark green, and their period of effect is estimated in light green. The dashed line marks the qPCR detection threshold for *V. harveyi* quantification, delineating the limit above which presence is reliably detected.

Figure 2: Circle of correlation of the PCA for parameters including *V. harveyi* in water ([Vh water]), *V. harveyi* in biofilms ([Vh concrete]), dissolved oxygen, temperature, salinity, turbidity, pH, and fish mortality (Mortality). The contribution of each variable indicates its importance in explaining the overall variance in the dataset.

Figure 3: Correlation matrix of tank environmental parameters including *V. harveyi* concentrations in water ([Vh Water]), *V. harveyi* in biofilms ([Vh Concrete]), dissolved oxygen, temperature, salinity, turbidity, pH, and fish mortality (Mortality). The size of the circles corresponds to the strength of correlation, with larger circles indicating stronger correlations and small ones indicating weak correlations. Only statistically significant correlations (*p* < 0.05) between the parameters are shown.

Table 1: Mean values ± standard deviations of parameters recorded from May to November 2022 in aquaculture facilities, categorised by the top, middle (mid), and bottom (bot) layers of the rearing tank. The 'n' signifies the number of sampling times averaged. For each sampling time, the average was calculated from 10 probe measurements.

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th rowspan="2"></th><th colspan="7">Sampling month</th></tr><tr><th>May<br>(n = 2)</th><th>June<br>(n = 2)</th><th>July<br>(n = 3)</th><th>August<br>(n = 4)</th><th>September<br>(n = 2)</th><th>October<br>(n = 2)</th><th>November<br>(n = 1)</th></tr></thead><tbody><tr><td rowspan="3">Temperature (C°)</td><td rowspan="3">pH</td><td rowspan="3">Salinity (psu)</td><td>Bot</td><td>Mid</td><td>Top</td><td>18.29 ± 0.77</td><td>19.06 ± 0.30</td><td>20.89 ± 0.28</td><td>22.06 ± 0.58</td><td>20.69 ± 0.03</td><td>17.82 ± 0.36</td><td>16.71 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>18.29 ± 0.74</td><td>19.06 ± 0.30</td><td>20.89 ± 0.28</td><td>22.05 ± 0.57</td><td>20.71 ± 0.05</td><td>17.82 ± 0.36</td><td>16.70 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>18.29 ± 0.74</td><td>19.06 ± 0.30</td><td>20.89 ± 0.28</td><td>22.06 ± 0.57</td><td>20.71 ± 0.04</td><td>17.82 ± 0.36</td><td>16.70 ± 0</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td rowspan="3">Salinity (psu)</td><td>Bot</td><td>Mid</td><td>Top</td><td>8.07 ± 0.71</td><td>7.53 ± 0.16</td><td>7.37 ± 0.22</td><td>7.29 ± 0.04</td><td>7.39 ± 0.06</td><td>7.17 ± 0.19</td><td>7.06 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>8.06 ± 0.70</td><td>7.55 ± 0.06</td><td>7.31 ± 0.24</td><td>7.23 ± 0.07</td><td>7.33 ± 0.03</td><td>7.12 ± 0.17</td><td>7.03 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>8.06 ± 0.70</td><td>7.55 ± 0.06</td><td>7.31 ± 0.24</td><td>7.23 ± 0.07</td><td>7.33 ± 0.03</td><td>7.12 ± 0.17</td><td>7.03 ± 0</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td rowspan="3">Salinity (psu)</td><td>Bot</td><td>Mid</td><td>Top</td><td>33.83 ± 0.68</td><td>34.75 ± 0.16</td><td>35.14 ± 0.16</td><td>35.07 ± 0.16</td><td>34.97 ± 0.11</td><td>34.09 ± 0.05</td><td>33.08 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>33.40 ± 1.34</td><td>34.74 ± 0.18</td><td>35.17 ± 0.18</td><td>35.16 ± 0.07</td><td>34.91 ± 0.05</td><td>34.00 ± 0.18</td><td>31.99 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>33.45 ± 1.34</td><td>34.63 ± 0.27</td><td>35.12 ± 0.15</td><td>35.18 ± 0.10</td><td>34.83 ± 0.20</td><td>33.81 ± 0.30</td><td>32.67 ± 0</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td rowspan="3">Salinity (psu)</td><td>Bot</td><td>Mid</td><td>Top</td><td>5.27 ± 0.15</td><td>4.13 ± 0.04</td><td>3.20 ± 0.01</td><td>4.70 ± 0.21</td><td>5.46 ± 0.02</td><td>3.47 ± 0.06</td><td>6.47 ± 0.15</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>5.45 ± 0.03</td><td>5.19 ± 0.02</td><td>3.39 ± 0.01</td><td>3.63 ± 0.02</td><td>5.83 ± 0.09</td><td>3.46 ± 0.04</td><td>3.64 ± 0.26</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>5.74 ± 0.03</td><td>5.49 ± 0.03</td><td>3.44 ± 0.02</td><td>4.04 ± 0.03</td><td>7.30 ± 0.43</td><td>3.82 ± 0.05</td><td>3.65 ± 0.11</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td rowspan="3">Salinity (psu)</td><td>Bot</td><td>Mid</td><td>Top</td><td>4.51 ± 1.02</td><td>5.02 ± 0.07</td><td>4.90 ± 1.44</td><td>2.84 ± 0.82</td><td>6.89 ± 0.52</td><td>7.15 ± 4.95</td><td>3.30 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>4.26 ± 1.32</td><td>3.40 ± 1.45</td><td>4.17 ± 0.75</td><td>3.86 ± 0.83</td><td>4.64 ± 1.65</td><td>5.56 ± 2.36</td><td>6.67 ± 0</td></tr><tr><td>Top</td><td>Mid</td><td>Top</td><td>3.99 ± 0.47</td><td>3.99 ± 0.77</td><td>6.41 ± 2.73</td><td>3.83 ± 0.80</td><td>6.39 ± 3.13</td><td>5.35 ± 1.77</td><td>5.29 ± 0</td></tr></tbody></table>
Figure 1: Biofilm concentrations of *V. harveyi* in log genome copy equivalents (GE).cm⁻² and *V. harveyi* concentrations in log GE.mL⁻¹ according to tank depth and time. The coloured lines correspond to the averages of the 5 replicates, which are represented by the shapes. Antibiotic treatments with oxytetracycline are indicated in dark green, and their period of effect is estimated in light green. The dashed line marks the qPCR detection threshold for *V. harveyi* quantification, delineating the limit above which presence is reliably detected.

Figure 2: Circle of correlation of the PCA for parameters including *V. harveyi* in water ([Vh water]), *V. harveyi* in biofilms ([Vh concrete]), dissolved oxygen, temperature, salinity, turbidity, pH, and fish mortality (Mortality). The contribution of each variable indicates its importance in explaining the overall variance in the dataset.

Figure 3: Correlation matrix of tank environmental parameters including *V. harveyi* concentrations in water ([Vh Water]), *V. harveyi* in biofilms ([Vh Concrete]), dissolved oxygen, temperature, salinity, turbidity, pH, and fish mortality (Mortality). The size of the circles corresponds to the strength of correlation, with larger circles indicating stronger correlations and small ones indicating weak correlations. Only statistically significant correlations (*p* < 0.05) between the parameters are shown.

Table 1: Mean values ± standard deviations of parameters recorded from May to November 2022 in aquaculture facilities, categorised by the top, middle (mid), and bottom (bot) layers of the rearing tank. The 'n' signifies the number of sampling times averaged. For each sampling time, the average was calculated from 10 probe measurements.

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th rowspan="2"></th><th rowspan="2"></th><th colspan="7">Sampling month</th></tr><tr><th>May<br>(n = 2)</th><th>June<br>(n = 2)</th><th>July<br>(n = 3)</th><th>August<br>(n = 4)</th><th>September<br>(n = 2)</th><th>October<br>(n = 2)</th><th>November<br>(n = 1)</th></tr></thead><tbody><tr><td rowspan="3">Temperature (C°)</td><td rowspan="3">pH</td><td rowspan="3">Salinity (psu)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td>Bot</td><td>Top</td><td>18.29 ± 0.77</td><td>19.06 ± 0.30</td><td>20.89 ± 0.28</td><td>22.06 ± 0.58</td><td>20.69 ± 0.03</td><td>17.82 ± 0.36</td><td>16.71 ± 0</td></tr><tr><td>Mid</td><td>Top</td><td>18.29 ± 0.74</td><td>19.06 ± 0.30</td><td>20.89 ± 0.28</td><td>22.05 ± 0.57</td><td>20.71 ± 0.05</td><td>17.82 ± 0.36</td><td>16.70 ± 0</td></tr><tr><td>Bot</td><td>Top</td><td>18.29 ± 0.74</td><td>19.06 ± 0.30</td><td>20.89 ± 0.28</td><td>22.06 ± 0.57</td><td>20.71 ± 0.04</td><td>17.82 ± 0.36</td><td>16.70 ± 0</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">pH</td><td rowspan="3">Salinity (psu)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td>Bot</td><td>Top</td><td>8.07 ± 0.71</td><td>7.53 ± 0.16</td><td>7.37 ± 0.22</td><td>7.29 ± 0.04</td><td>7.39 ± 0.06</td><td>7.17 ± 0.19</td><td>7.06 ± 0</td></tr><tr><td>Mid</td><td>Top</td><td>8.06 ± 0.70</td><td>7.55 ± 0.06</td><td>7.31 ± 0.24</td><td>7.23 ± 0.07</td><td>7.33 ± 0.03</td><td>7.12 ± 0.17</td><td>7.03 ± 0</td></tr><tr><td>Bot</td><td>Top</td><td>7.95 ± 0.72</td><td>7.51 ± 0.02</td><td>7.25 ± 0.23</td><td>7.18 ± 0.07</td><td>7.25 ± 0.08</td><td>7.02 ± 0.16</td><td>6.87 ± 0</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">pH</td><td rowspan="3">Salinity (psu)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td>Bot</td><td>Top</td><td>33.83 ± 0.68</td><td>34.75 ± 0.16</td><td>35.14 ± 0.16</td><td>35.07 ± 0.16</td><td>34.97 ± 0.11</td><td>34.09 ± 0.05</td><td>33.08 ± 0</td></tr><tr><td>Mid</td><td>Top</td><td>33.40 ± 1.34</td><td>34.74 ± 0.18</td><td>35.17 ± 0.18</td><td>35.16 ± 0.07</td><td>34.91 ± 0.05</td><td>34.00 ± 0.18</td><td>31.99 ± 0</td></tr><tr><td>Bot</td><td>Top</td><td>33.45 ± 1.34</td><td>34.63 ± 0.27</td><td>35.12 ± 0.15</td><td>35.18 ± 0.10</td><td>34.83 ± 0.20</td><td>33.81 ± 0.30</td><td>32.67 ± 0</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">pH</td><td rowspan="3">Salinity (psu)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td>Bot</td><td>Top</td><td>5.27 ± 0.15</td><td>4.13 ± 0.04</td><td>3.20 ± 0.01</td><td>4.70 ± 0.21</td><td>5.46 ± 0.02</td><td>3.47 ± 0.06</td><td>6.47 ± 0.15</td></tr><tr><td>Mid</td><td>Top</td><td>5.45 ± 0.03</td><td>5.19 ± 0.02</td><td>3.39 ± 0.01</td><td>3.63 ± 0.02</td><td>5.83 ± 0.09</td><td>3.46 ± 0.04</td><td>3.64 ± 0.26</td></tr><tr><td>Bot</td><td>Top</td><td>5.74 ± 0.03</td><td>5.49 ± 0.03</td><td>3.44 ± 0.02</td><td>4.04 ± 0.03</td><td>7.30 ± 0.43</td><td>3.82 ± 0.05</td><td>3.65 ± 0.11</td></tr><tr><td rowspan="3">Turbidity (FNU)</td><td rowspan="3">pH</td><td rowspan="3">Salinity (psu)</td><td rowspan="3">Dissolved oxygen<br>(mg.L⁻¹)</td><td>Bot</td><td>Top</td><td>4.51 ± 1.02</td><td>5.02 ± 0.07</td><td>4.90 ± 1.44</td><td>2.84 ± 0.82</td><td>6.89 ± 0.52</td><td>7.15 ± 4.95</td><td>3.30 ± 0</td></tr><tr><td>Mid</td><td>Top</td><td>4.26 ± 1.32</td><td>3.40 ± 1.45</td><td>4.17 ± 0.75</td><td>3.86 ± 0.83</td><td>4.64 ± 1.65</td><td>5.56 ± 2.36</td><td>6.67 ± 0</td></tr><tr><td>Bot</td><td>Top</td><td>3.99 ± 0.47</td><td>3.99 ± 0.77</td><td>6.41 ± 2.73</td><td>3.83 ± 0.80</td><td>6.39 ± 3.13</td><td>5.35 ± 1.77</td><td>5.29 ± 0</td></tr></tbody></table>
<table><thead><tr><th>Fish Mortality (%)</th><th>1.26</th><th>0.98</th><th>1.31</th><th>4.57</th><th>0.90</th><th>0.06</th><th>0.01</th></tr></thead></table>
<table><thead><tr><th>Fish Mortality (%)</th><th>1.26</th><th>0.98</th><th>1.31</th><th>4.57</th><th>0.90</th><th>0.06</th><th>0.01</th></tr></thead></table>