Inhibition of Vibrio parahaemolyticus infection in whiteleg shrimp reared in a biofloc system with different volumes

This study aimed to evaluate the inhibition of V . parahaemolyticus infection in the whiteleg shrimp ( Penaeus vannamei ) reared in different volumes of biofloc system. Post-larval shrimp with an average body weight of 0.28 ± 0.01 g were reared in 15 aquariums with working volume of 33.3 L and stocking density of 3 shrimp per liter. The shrimp were reared for 21 days in a biofloc system (C:N ratio of 10) with biofloc volume of 5, 10, and 15 mL/L, and challenged with V . parahaemolyticus at a density of 10 3 CFU/mL initially. The positive control treatment involved shrimp reared without biofloc and challenged, while the negative control treatment involved shrimp reared without biofloc and without challenged. The shrimp was fed with commercial feed while the protein content of 39-40% four times a day. The results showed that the presumptive Vibrio count (PVC) and the population of V . parahaemolyticus in the biofloc treatments were significantly lower than the positive control (p<0.05). Furthermore, the biofloc treatment with a volume of 15 mL/L demonstrated the best results compared to other treatments in decreasing PVC and V . parahaemolyticus population as evidenced by the immune response, survival rate, and growth performance of whiteleg shrimp.


INTRODUCTION
Penaeus vannamei, commonly known as Pacific white shrimp, represents a key commodity in aquaculture, with global production reaching 5.81 million tons in 2020 (FAO, 2022).Production is projected to increase in 2022, with Indonesia emerging as a major producer (FAO, 2023).Data from the Ministry of Marine Affairs and Fisheries (KKP, 2020) indicates that Indonesia's shrimp production was valued at 860,000 tons, contributing approximately 2 billion USD to global exports in 2019 (FAO, 2022).This represents the highest value in Indonesia's aquaculture sector, followed by species such as Nile tilapia and catfish.The expansion of P. vannamei farming remains promising due to the extensive target markets including Japan, the United States, the United Kingdom, and other shrimp-importing countries (FAO, 2020).
Despite the generally increasing production trend in recent years, white shrimp farming is still constrained by disease outbreaks, particularly bacterial diseases caused by the genus Vibrio, commonly known as vibriosis.Vibrio parahaemolyticus is a significant pathogen affecting white shrimp, causing various abnormalities such as gill necrosis, lethargy, loss of appetite, and in acute cases, up to 100% mortality (Abdel-Latif et al., 2022).Recent years have seen particular global attention on V. parahaemolyticus strains with PirA and PirB toxins, responsible for acute hepatopancreatic necrosis disease (AHPND).The first AHPND outbreak occurred in China (2009), with rapid spread to countries including Vietnam (2010), Malaysia (2011), Thailand (2012), Mexico (2013), the Philippines (2015), and the United States (2016) (Kumar et al., 2021).In Indonesia, V. parahaemolyticus with AHPND (VpAHPND) has been identified in export samples and various regions including Serang, Banten, and Bangkalan, Madura (Han et al., 2020;Saputra et al., 2023;Suryana et al., 2023).
The pathogenicity of V. parahaemolyticus is regulated by quorum sensing (QS) mechanisms (Gode-Potratz & McCarter, 2011), which enable bacteria to communicate via signaling molecules (autoinducers) and synchronize gene expression, including those for virulence factors (Lu et al., 2019;Lin et al., 2022).Through QS, V. parahaemolyticus can produce cytotoxic compounds and molecules facilitating infection, influenced by population density in aquatic environments.One approach to mitigate V. parahaemolyticus infections is by enhancing shrimp immunity and controlling bacterial populations to inhibit QS (Gustilatov et al., 2023).This can be achieved through the application of biofloc technology in shrimp culture.
Biofloc technology utilizes and converts nitrogen from feed waste and metabolic byproducts into heterotrophic microorganism biomass by adding organic carbon sources.This process forms flocs of bacterial biomass and other microorganisms, known as biofloc (Khanjani et al., 2022).Besides improving environmental quality and growth parameters (Kumar et al., 2018), biofloc enhances the immune response of white shrimp (Ekasari et al., 2014;Panigrahi et al., 2018) and serves as a biocontrol for pathogenic bacteria by reducing QS activity and virulence factors (Panigrahi et al., 2018;Aguilera-Rivera et al., 2019;Widanarni et al., 2024).Aguilera-Rivera et al. (2014) reported that biofloc application can prevent outbreaks of opportunistic Vibrio bacteria in aquatic environments.Implementation of biofloc in shrimp farming has shown positive results, including higher survival rates when challenged with V. parahaemolyticus causing AHPND, compared to controls (Hostins et al., 2019;Kumar et al., 2020).
The findings of these studies suggest that biofloc technology can serve as an effective biocontrol agent and enhance shrimp immunity, positioning it as a viable alternative for managing V. parahaemolyticus infections in white shrimp culture.The volume of biofloc, influenced by different C ratios, exhibits varying characteristics and functions (Panigrahi et al., 2018), yet detailed information on the impact of different biofloc volumes on bacterial inhibition and pathogenicity remains scarce.Understanding the optimal protective effects of biofloc at specific volumes can help maintain microbial populations during shrimp farming.This study aims to evaluate the inhibition of V. parahaemolyticus infections in P. vannamei reared in biofloc systems with varying volumes.

Time and place of study
This study was conducted from June to August 2022 at the Aquatic Organism Health Laboratory, Department of Aquaculture, Faculty of Fisheries and Marine Sciences, IPB University.

Experimental design
This research used a completely randomized design (CRD) consisting of five treatments and three replications (Table 1).

Study procedure Preparation of containers and rearing media
The containers used for the adaptation process and initial maintenance of white shrimp seedlings are 200×100×40 cm 3 fiber tanks.Subsequently, the containers used for maintenance during the treatment period are 60×30×30 cm 3 glass aquariums, with a total of 15 units.The aquariums are thoroughly cleaned and filled with freshwater, then disinfected using chlorine at a dose of 30 µl/L for 24 hours.The aquariums are then rinsed with freshwater and dried.After that, the aquariums are filled with seawater with a salinity of 25 g/L up to a water volume of 33.3 L, and aeration is installed.

Biofloc preparation
The preparation of biofloc suspension for white shrimp maintenance comes from biofloc culture conducted in white shrimp cultivation tanks using molasses as the organic carbon source.During maintenance, molasses is directly added to the shrimp maintenance aquariums once a day, two hours after the morning feeding, with an estimated C/N ratio of 10 (Ekasari et al., 2010).The amount of carbon added to support the floc formation process by heterotrophic bacteria in each treatment follows the carbon requirement calculation scheme by De Schryver et al. (2008).

Preparation of test bacteria
V. parahaemolyticus used as the challenge test bacteria in this study were made resistant to the antibiotic rifampicin at 50 μg/mL (1 g rifampicin, 95 mL absolute ethanol, and 5 mL distilled water) as a marker on agar media.The bacteria were then cultured on Sea Water Complete (SWC) or Thiosulfate-Citrate-Bile-Salt Sucrose (TCBS) agar.Colonies that had grown on the agar media after 24 hours were then taken using an inoculation loop and inoculated into 15 mL of liquid SWC media.The culture was then incubated using a shaker at 160 rpm for 18 hours at 28°C until a bacterial density of 10 8 CFU/mL was obtained.The bacterial density was then diluted to a concentration of 10 3 CFU/mL in the shrimp maintenance media.

White shrimp maintenance
White shrimp were stocked into the aquariums at a density of 3 individuals per liter (100 individuals per aquarium) and maintained for 21 days.During the maintenance period, the shrimp were fed commercial feed with a protein content of 39-40%, four times a day (07:00, 12:00, 17:00, and 22:00 WIB) at a feeding rate of 10%.The challenge test using V. parahaemolyticus was conducted using an immersion method at the start of the treatment.The challenge test used a V. parahaemolyticus concentration of 10 3 CFU/ mL, obtained from the preliminary LC50 (lethal concentration 50%) test results (Torpee et al., 2021), as shown in Table 1.
Weekly counts of the bacterial populations of Vibrio sp. and V. parahaemolyticus Rf R in the rearing water and shrimp gut were performed.Water quality parameters, including dissolved oxygen (DO), pH, and salinity, were measured daily, while total ammonia nitrogen (TAN), nitrite, and nitrate were measured weekly at the Aquaculture Environment Laboratory, Department of Aquaculture, IPB University.

Floc volume observation
Floc volume was measured after settling in a cone, and floc particle density was observed using the method of Sumitro et al. (2022).Approximately 1000 mL water sample was settled for 60 minutes in a 1000 mL capacity cone tube.If there was a significant increase in floc volume in the treatment using biofloc, dilution was performed by replacing 50% of the maintenance water to keep the floc volume stable.

Observation parameter Total Vibrio count and total V. parahaemolyticus in water and shrimp bodies
On the 7 th , 14 th , and 21 st days of maintenance, total Vibrio count and V. parahaemolyticus Rf R were measured in the maintenance media and the bodies of white shrimp following the method of Madigan et al. (2003).Total bacteria were counted using the total plate count method on TCBS media, while total V. parahaemolyticus was counted on TCBS media supplemented with 50 µg/mL rifampicin.The maintenance water and homogenized shrimp bodies were serially diluted and 50 µL was spread on each type of media.The media with bacterial cultures were then incubated for 24 hours at 28°C-29°C.The bacteria that grew were then counted to determine the total bacterial count.

Immune response
The measurement of shrimp immune response refers to Hamsah et al. (2019) and Widanarni et al. (2020).Total hemocyte count (THC) was conducted by placing shrimp samples in a mortar containing an anticoagulant (3.8% Na-citrate) with a ratio of 1:3 (shrimp weight: anticoagulant).The shrimp were then ground, and the body fluid was extracted with a micropipette, dropped onto a hemocytometer, and observed under a microscope at 100× magnification.Phenoloxidase (PO) activity was measured by the formation of dopachrome produced by L-DOPA, with optical density (OD) measured using a spectrophotometer at a wavelength of 492 nm (Sutthangkul et al., 2017).Respiratory burst (RB) activity was measured according to the method of Hampton et al. (2020), with RB expressed as NBT reduction per 10 μl hemolymph.Immune response sampling was conducted on days 7, 14, and 21.

Growth performance
The shrimp's weight was measured at the beginning and end of the rearing period.Sampling was conducted before feeding when the shrimp's intestines were empty.Specific growth rate (SGR) and feed conversion ratio (FCR) were calculated

Statistical analysis
The obtained data were tabulated using Microsoft Excel 2013.Analysis of growth performance, immune response, total bacteria, and total V. parahaemolyticus was performed using analysis of variance (ANOVA) with SPSS version 20.If significant differences were found, further tests were conducted using Tukey's test with a 95% confidence interval.

Floc volume range
The floc volume in each treatment was measured daily and maintained for 21 days during the maintenance period.Throughout the maintenance period, the floc volume remained within the range corresponding to each treatment.In treatment B5, the floc volume ranged from 4.6 to 4.9 mL/L; treatment B10 had a floc volume ranging from 9.4 to 9.8 mL/L; and treatment B15 had a floc volume range of 14.3 to 14.5 mL/L.The weekly range of floc volume values is presented in Table 2.

Total Vibrio count and total V. parahaemolyticus Rf R in water and shrimp body
Total Vibrio count and V. parahaemolyticus were observed through the maintenance media and the shrimp bodies.The observation of total presumptive Vibrio count (TPC) of Vibrio sp. in the maintenance media showed that the total Vibrio count for treatments B5, B10, and B15 was higher on day 7 compared to treatments KN and KP.However, on days 14 and 21, the total Vibrio count tended to decrease (Table 3).In treatment B5, the count was higher on day 7 at 5.62 log CFU/mL, but decreased on days 14 and 21 to 3.92 log CFU/mL and 3.55 log CFU/mL, respectively.
Similarly, treatments B10 and B15 had the highest total Vibrio count on day 7 and the lowest on day 21.The total Vibrio count in the shrimp bodies showed similar values to those in the maintenance media.Treatments B5, B10, and B15 had the highest count on day 7 at 5.98 ± 0.57 log CFU/mL and the lowest count on day 21 at 3.75 ± 0.42 log CFU/mL.The complete data for total Vibrio count in the water and white shrimp bodies are presented in Table 3.
The observation of total V. parahaemolyticus Rf R bacteria in the maintenance water showed a range of 3.18-4.80log CFU/mL on day 7, while no V. parahaemolyticus growth was detected on days 14 and 21.However, in the positive control (KP) treatment, bacteria were found on days 14 and 21 with total counts of 3.27 ± 0.20 log CFU/ mL and 1.83 ± 0.42 log CFU/mL, respectively.The observation of total V. parahaemolyticus Rf R bacteria in white shrimp bodies showed higher counts in the KP treatment, ranging from 2.77 to 3.78 log CFU/mL.In the biofloc treatments (B5, B10, and B15), the highest total V. parahaemolyticus counts were observed on day 7, with a tendency to decrease on days 14 and 21, with the lowest count in treatment B10 at 1.72 ± 0.28 log CFU/mL (Table 4).

White shrimp immune response
The results of the immune response observations in white shrimp, including total hemocyte count (THC), phagocytic activity (PA), phenoloxidase (PO) activity, and respiratory burst (RB) activity, are presented in Table 5.On day 7, THC values showed a significant difference (P<0.05) in treatments B5, B10, and B15 compared to KN and KP treatments.The highest THC value was found in treatment B15, with a value of 7.70 ± 0.35 10 6 cells/mL, while the lowest value was in KP treatment, with a value of 3.27 ± 0.31 10 6 cells/mL.Phagocytic activity in the treatments with increased biofloc volume (B5, B10, and B15) on days 7, 14, and 21 of rearing showed significant differences (P<0.05)compared to KP treatment but did not differ significantly from KN treatment.
The highest phagocytic activity (PA) was observed on day 21 in the biofloc treatment (B15), with a value of 54.67 ± 2.08%, while the lowest value on day 21 was in the positive control treatment (KP), with a value of 31.67 ± 2.08%.Observations of phenoloxidase (PO) activity in all treatments on day 0 showed a value of 0.24 ± 0.01 (100 µL).PO values with increased biofloc volume (B5, B10, and B15) showed significantly different results from KP treatment but did not differ significantly from KN treatment.The highest PO activity during 21 days of rearing was observed on day 21 in the B15 treatment, with a value of 0.30 ± 0.01 (100 µL), and the lowest PO activity was observed on day 14 in the KP treatment, with a PO value of 0.12 ± 0.02 (100 µL).
The observation of respiratory burst (RB) on day 0 in all treatments showed a value of 0.15 ± 0.01 (10 µL).On days 7, 14, and 21, the increased biofloc volume (B5, B10, and B15) significantly increased RB values.A significant effect was observed on day 21, where the increased biofloc volume (B15) challenged with V. parahaemolyticus 10 3 CFU/mL showed higher RB activity, with a value of 0.5 ± 0.00 (10 µL), and was significantly different (P<0.05) from both the positive control (KP) and negative control (KN) treatments.However, there were no significant differences between treatments B5 and B10.

White shrimp growth performance
The growth performance of white shrimp reared in a biofloc system with different volumes is presented in Table 6.Based on Table 6, it is evident that the B10 treatment provided better feed conversion ratio (FCR) values, which were significantly different from the positive control (KP), B5, and B15 treatments according to statistical analysis.Additionally, the B10 treatment yielded higher values in final weight and specific growth rate parameters, although these values were not significantly different from the B5 and B15 treatments.The highest weight obtained during the rearing period was 0.99 ± 0.08 g, the highest specific growth rate was 6.42 ± 0.28%, and the lowest feed conversion ratio was 1.45 ± 0.08.
The total feed consumption during the 21day rearing period ranged from 83.35 to 102.65 g.The highest survival rate during the rearing period was in the range of 84.23-89.96%,which was significantly different from the positive control (KP) but not significantly different from the negative control (KN), B5, B10, and B15 treatments.The results of water quality measurements during the rearing of white shrimp are presented in Table 7. Overall, the values of TAN (total ammonia nitrogen) and NO2 -(Nitrite) in the biofloc treatments were lower compared to the control treatments, while the NO3 -(Nitrate) values in the biofloc treatments were higher than those in the control treatments.The lowest DO (Dissolved Oxygen) value was 5.0 mg/L, which is within the normal range, while the highest value was 6.10 mg/L.The pH values (7.67-8.00),temperature (26.2-29.3°C),and salinity (31-34 g/L) were all within the normal range for white shrimp rearing.In general, the water quality conditions during the white shrimp rearing were optimal and in accordance with SNI 8008:2014 BSN (2014).

Discussion
Vibriosis disease, caused by bacteria from the Vibrio genus, particularly Vibrio parahaemolyticus, is one of the most serious and prevalent diseases affecting shrimp aquaculture (Valente & Wan, 2021).V. parahaemolyticus is an opportunistic pathogen whose high population in aquatic environments plays a role in regulating virulence factors and contributing to bacterial defense mechanisms as well as host infection activities, such as enhanced attachment and penetration, interbacterial interactions, and environmental stress responses (Wang et al., 2013).Typically, bacterial infections are managed using antibiotics, but their usage has been restricted.An environmentally friendly alternative to control V. parahaemolyticus infections involves inhibiting bacterial growth to reduce virulence and enhance shrimp immune responses, thereby increasing resistance to bacterial infections (Zhao et al., 2014), such as through the application of biofloc technology (Gustilatov et al., 2022).
This study demonstrates that the presence of biofloc at all tested volumes (5 mL/L, 10 mL/L, and 15 mL/L) reduces the density of Vibrio spp.and V. parahaemolyticus, both in the culture medium and on the shrimp bodies.According to Gustilatov et al. (2023), biofloc acts as a biocontrol agent and can inhibit the growth and pathogenicity of Vibrio bacteria due to extracellular components that disrupt bacterial QS activity.Biofloc has also been shown to produce compounds such as bromophenol, carotenoids, poly-beta-hydroxybutyrate, and various hydrolytic enzymes (Fatimah et al., 2019).Gustilatov et al. (2022) reported that biofloc can reduce V. parahaemolyticus density in vitro and inhibit biofilm formation.
Biofilm formation, mediated by QS (Liu et al., 2018), allows bacteria to efficiently utilize nutrients, increase resistance to antimicrobial agents, stress, and enhance bacterial virulence (Packiavathy et al., 2013).Additionally, biofloc can play an antagonistic role by competing for nutrients, energy, and sites, thereby suppressing the growth of pathogenic bacteria (Ferreira et al., 2020).The immune parameter profiles measured in this study align with previous findings indicating the positive effects of biofloc on aquaculture organism immunity, as reported by Tepaamorndech et al. (2020), Panigrahi et al. (2018), andEkasari et al. (2014).Biofloc, consisting of bacteria with lipopolysaccharides or peptidoglycans, fungi with β-glucans, and other microbes, can stimulate hemocytes and other immune parameters through pathogen-associated molecular patterns (Kim et al., 2014).
In this study, the total hemocyte count (THC) and phagocytic activity (PA) of shrimp reared in biofloc systems showed significant differences compared to controls, confirming the immunostimulatory effects of biofloc and corroborating previous research by Ferreira et al. (2015).The best results were observed in the 15 mL/L biofloc volume treatment compared to other volumes.Hemocytes are involved in several pathogen resistance activities, including phagocytosis, encapsulation, foreign particle aggregation, and prophenoloxidase (proPO) system functions (Sahoo et al., 2008).The humoral immune response in shrimp, indicated by phenoloxidase (PO) activity a precursor to melanin formation that inactivates and prevents pathogen spread was enhanced in biofloc treatments (Amparyup et al., 2013).
A similar trend was observed in pathogen destruction activity through reactive oxygen intermediates (ROI) represented by reactive blue (RB) parameters (Duan et al., 2015).Consistent with the increase in hemocytes, the activities of phenoloxidase (PO) and reactive blue (RB) in the 15 mL/L biofloc treatment were also significantly higher compared to the 5 mL/L and 10 mL/L treatments.This indicates that a 15 mL/L biofloc treatment can provide better protection against Vibrio parahaemolyticus infections.Based on the obtained results and corroborated by several previous studies, biofloc is capable of protecting shrimp from pathogen infections, partly through the enhancement of immune responses.The reduction in bacterial virulence impacts its ability to infect shrimp.
In addition to weakening the bacteria's ability to infect shrimp, biofloc also improves the shrimp's immune response to V. parahaemolyticus infections, enabling the immune system to better control the bacterial infection due to the influence of biofloc (Ekasari et al., 2014).The positive role of biofloc is also evident in shrimp growth performance.Higher growth rates in biofloc treatments are attributed to the comprehensive nutrient composition of biofloc, including proteins, carbon, ash, fatty acids, minerals, and other nutrients, which can serve as a natural feed consistently available in the culture medium (Toledo et al., 2016).Additionally, biofloc can enhance digestive enzyme activity, leading to more efficient feed utilization for growth (Wang et al., 2015).This effect is indicated by higher length-to-weight ratios (SGR) and lower feed conversion ratios (FCR) in biofloc treatments compared to controls in this study.
Furthermore, the survival rate of shrimp challenged with Vibrio parahaemolyticus was higher in biofloc treatments compared to positive controls.This improvement is due to reduced V. parahaemolyticus virulence and enhanced immune responses in shrimp.The survival rate in the 15 mL/L biofloc treatment was 89.90 ± 0.00%, which was not significantly different from the 10 mL/L (84.23 ± 3.31%) and 5 mL/L (84.23 ± 3.66%) treatments, but significantly higher than the positive control (61.32 ± 13.59%).These findings are consistent with reports by Sajali et al. (2019) and Hostins et al. (2019), which observed that biofloc treatment enhances the survival of white shrimp challenged with V. parahaemolyticus.Biofloc also positively impacts water quality by reducing ammonia and nitrite concentrations, which can be toxic to shrimp, through nitrogen assimilation by heterotrophic bacteria (Robles-Porchas et al., 2020).

CONCLUSION
Biofloc can inhibit Vibrio parahaemolyticus, enhance immune responses, improve growth performance, and increase the resistance of Penaeus vannamei, with the best results observed at a volume application of 15 mL/L.
using the equation provided by Liu et al. (2019): Note: Wt = Average shrimp weight at the end of the rearing period (g) W0 = Average shrimp weight at the beginning of the rearing period (g) Ba = Final shrimp biomass (g) B0 = Initial shrimp biomass (g) Bm = Biomass of dead shrimp (g) t = Duration of rearing (days) FC = Total feed consumption (g)

Table 1 .
Experimental design for white shrimp maintenance in a biofloc system with different volumes for controlling V. parahaemolyticus infection.

Table 2 .
Range of floc volume in white shrimp maintenance media during the maintenance period.

Table 3 .
Total Vibrio count in water and body of white shrimp maintained in biofloc system with different volumes and challenged with V. parahaemolyticus.
Note: Different superscript letters in the same column indicate significantly different results (Tukey p<0.05).

Table 4 .
Total V. parahaemolyticus Rf R in water and body of white shrimp maintained in biofloc system with different volumes and challenged with V. parahaemolyticus.Different superscript letters in the same column indicate significantly different results (Tukey p<0.05).

Table 5 .
Immune response of white shrimp reared in biofloc systems with different volumes and challenged with V. parahaemolyticus Different superscript letters in the same column indicate significantly different results (Tukey p<0.05).

Table 6 .
Growth performance of white shrimp reared in biofloc systems with different volumes and challenged with V. parahaemolyticus.

Table 7 .
Water quality of the maintenance media for white shrimp maintained in the biofloc system with different volumes and tested against V. parahaemolyticus.