The effect of liquid smoke (Cocos nucifera) in controlling acute hepatopancreatic necrosis disease in Litopenaeus vannamei

Vibrio parahaemolyticus is found in coastal environments throughout the world and is a major pathogen that causes Acute Hepatopancreatic Necrosis Disease (AHPND) with mass mortalities in Pacific whiteleg shrimp (Penaues vannamei). The extract of coconut shell liquid smoke (Cocos nucifera) was tested for antibacterial effect against AHPND in vitro. It was further evaluated for its potential efficacy in preventing AHPND in vannamei shrimp in vivo. The water in this study has been tested and positive for Vibrio parahaemolyticus. Two experimental diets were obtained by adding 0.5% and 1% liquid smoke to a commercial shrimp diet. The dietary supplementation with liquid smoke significantly enhanced shrimp growth performance during 90 days of rearing. A liquid concentration of 0.5% showed a higher growth performance (17.56 gr) than a 1% concentration (14.4 gr). Liquid smoke added to the shrimp fed partially entered the shrimp’s digestive system, and some would dissolve into the water. This water-soluble bioactive is thought to reduce the concentration of V. parahaemolyticus in waters. This was proven by the fact that no shrimp were infected with V. parahaemolyticus during the 90-day rearing.


Introduction
The main obstacles in Indonesia's shrimp farming are preventing and controlling the disease outbreak of shrimp farming [1].Acute Hepatopancreatic Necrosis Disease (AHPND) is one of the deadly diseases for shrimp caused by Vibrio parahaemolyticus infection and has severely damaged the global shrimp industry.This disease outbreak first appeared in China in 2009 and has spread to Vietnam (2011), Malaysia (2011), Thailand (2012), Mexico (2013), Indonesia (2013), and the Philippines (2015) [2] [3], with 100% of mortality within 20 -40 days [4].
The shrimp infected with AHPND display a pale to white and shrunken hepatopancreas, empty digestive tract, yellowish shrimp body color, and soft carapace [5].AHPND has caused a decline in shrimp production up to 60% and US$ 1 billion in annual losses [6].Since vannamei shrimp has no specific immune responses, they mainly rely on their innate and humoral immune system.Since phenol oxidase (PO) is critical for humoral immunity in shrimps against invaders and foreign objects, the study about enhancing PO responses in shrimp has been mostly developed [7].1328 (2024) 012018 IOP Publishing doi:10.1088/1755-1315/1328/1/012018 2 Liquid smoke results from the distillation of steam from burning coconut shells which contains a lot of carbon and other compounds such as hemicellulose and lignin.Liquid smoke contains some phenol group compounds, acid groups, and carbonyl groups which is simultaneously act as antioxidants and antimicrobials disinfectant because it can kill or inhibit the development of bacteria [8].An important component of liquid smoke originating from coconut shells is phenolic compounds, which are strong antioxidants capable of binding free radicals such as reactive oxygen species (ROS) and inhibiting the production of nitric oxide (NO); phenolic compounds also act as antibacterial and antioxidant [9] and play an important role in purification or recovery or renewal of materials including water [10], in color filtration [11], in drinking water treatment [12], and in water sanitation such as in the absorption of heavy metals (Pb, Fe, and Cu), nitrites, and phenol [13].This research aims to determine the effectiveness of liquid smoke from coconut shells in controlling AHPND disease in intensive vannamei shrimp cultivation systems.This was an industrial-scale study, so there was no repetition or control.

Methods
The study started in June 2022 and lasted three months; it was done at the Aquaculture Teaching Factory, Kampus Politeknik Kelautan dan Perikanan Sidoarjo, located in Paciran Regency, Lamongan.The core research aims to improve the immune system of vannamei shrimp by adding liquid smoke to the feed at different doses.The media (water) used in this study were confirmed to contain Vibrio parahaemolyticus.Thus, feed added with liquid smoke at different concentrations was expected to inhibit mortality in vannamei shrimp.
Our study on the effectiveness of coconut shell liquid smoke as an antibacterial for V. parahameolyticus consisted of two treatments with different doses of liquid smoke mixed into commercial feed for vannamei shrimp infected with AHPND.
Pond A: Treatment of adding coconut shell liquid smoke at a dose of 0.5% Pond B: Treatment of adding coconut shell liquid smoke at a dose of 1% The dosage used came from previous preliminary research through in vitro tests.Five concentrations of liquide smoke were used as inhibitory test (0,5%; 1%; 1.5%; 2%; and 2,5%) of V. parahameolyticus in TCBS agar.1% concentration of liquid smoke showed the highest inhibitory zone followed by 0,5%; 2%; 2,5% and 1,5% of V. parahameolyticus.This resulted were used as in vivo test in this study.
The equipment used to test the effectiveness of coconut shell liquid smoke against Vibrio parahaemolyticus bacteria included a concrete pool with a total area of 81 m 2 , a microbubble aerator with a power capacity of 370 watts, an aeration tube (uniring), an aerator supercharger, plankton net, sample bottles, a microscope, a hand tally counter, digital scales, and anco trays.
The materials included brackish water, distilled water, limestone, Ceraclean fertilizer (an artificial hydrated calcium silicate), chlorine (TCCA), probiotics, F1 fry (PL 30), shrimp feed Number 1, shrimp feed Number 2, NB puper, phosphate block, vitamin C, a DO test kit, an ammonia test kit, a nitrate test kit, a nitrite test kit, phosphate kit, a universal pH, an FE test kit, an AHPND isolate, coconut shell liquid smoke, vitamin B12, and alcohol.

Adding the liquid smoke to the feed
We used two types of commercial feed: feed Number 1 for PL 30 -PL 40 shrimp fry and feed Number 2 for PL 40 -harvested shrimp, which contains 38% protein.We used a spray method for adding liquid smoke solution to shrimp feed.Initially, the feed was weighed according to the daily shrimp feed requirements, then placed on a baking sheet and given different labels according to the concentrations (0.5% and 1%).Liquid smoke solutions with different concentrations (0.5% and 1%) were prepared into spray bottles.The liquid smoke solution was then sprayed on the feed according to the respective doses until the feed looked moist.Then, the feed was air-dried for 30 minutes before being given to the experimental animals (vannamei shrimp).Drying the feed by airing aims to ensure that the contents of the feed are maintained and not damaged.The drying process at high temperatures can cause damage to the active substances contained in a food ingredient.

Preparing the rearing container
We used a concrete tank with a total area of 81 m 2 as the rearing medium.Molasses was added to the pond to remove the remaining mud smell.pH of the pond was improved by applying limestone.Each pond had microbubbles installed on two sides in opposing positions to create a current to rotate the pond water.A blower was installed and distributed at 25 points so that the dissolved oxygen needed by the shrimp could be fulfilled.

Filling the water and growing the plankton
Water was filled to a height of ±80% of the total volume of the rearing container and a salinity of 26 ppt.After filling the water, the microbubble and aeration tube (uniring) were turned on to stir the water and distribute oxygen in the water.The water used as the rearing medium in this study was proven to contain V. Parahemolyticus, and water quality tests were done previously.Plankton was grown with the help of Ceraclean, a commercial product with a composition of 40% silicate and 25% Ca.The dose used was 20 ppm/week.Apart from that, Probiotics (Super Marine), an innovative product of Politeknik Kelautan dan Perikanan Sidoarjo, are also used with a dose of 5 ppm/day.

Maintaining feed
The shrimp was fed three times a day.Feeding at PL 30 -40 was 5% of the total shrimp biomass, PL 41 -50 was 4%, PL 51 -60 was 3%, PL 61 -70 was 2%, and PL 71 -90 was 1% per day in each concrete tank.Feeding times are 07:00 AM, 11:00 AM, 3:00 PM, and 7:00 PM.In DOC 51 -90, feeding was added once at 11:00 PM.We still used commercial feed for PL 30 -35 without adding liquid smoke solution.Feed added with liquid smoke at a dose of 0.5% and 1% was given when the vannamei shrimp were PL 36 until harvested.

Maintaining water quality
We did physical, chemical, and biological water quality measurements.Measurement of water quality and growth parameters are elaborated in Table 1.

Penaeus vannamei
The vannamei shrimp used in this research was shrimp fry PL 30 F1.A total of 50 vannamei shrimp were taken as samples and then weighed for their average weight to determine the initial weight.The average stocking density for each tank was 500 fish/m 2 .A total of 40,000 shrimp were stocked in each rearing pond.The shrimp used in this study was proven negative for AHPND based on clinical observations and disease testing using the PCR method.The shrimp were first acclimatized for 7 days to determine the health used and give them time to adapt to their new environment.
The shrimp were fed by adding fortified feed with liquid smoke solution at different doses (0.5% and 1%) until harvest.To see the level of toxicity of the liquid smoke solution on the shrimp, feeding was carried out by adding liquid smoke solution for 7 days, and the mortality of the shrimp was measured.During the rearing period, the shrimp weight was measured every week by taking samples of the shrimp in a tank and weighing them using analytical scales.Meanwhile, mortality and survival rate observations were carried out every day with anco checks.Additional observations consisted of observing symptoms of disease caused by Vibrio parahaemolyticus bacterial infection until the end of rearing.

Growth parameter
Growth parameters like MBW, ADG, SR, Biomass, population, and size were measured using the formula developed by [14]; the equation is as follows: The average weight of the vannamei shrimp is calculated using the following formula: The daily growth rate of the vannamei shrimp was observed by weighing the shrimp using a digital scale.The data used was shrimp weight data at the beginning and end of the study.The data was analyzed using the formula: Survival Rate (SR) indicates the survival rate of fry stocked in a certain period.

Data analysis
The data processing results were analyzed using a one-way analysis of variance (ANOVA).If significant differences are found in each pool, the analysis continues using Duncan's test with a significance level of 95%.Growth data between the two treatments are analyzed using a T-test.

Shrimp Growth
The average growth of vannamei shrimp in Pond 1 with the addition of liquid smoke to the feed at a dose of (0.5%) during the 90-day rearing was 17.56 ± 0.1 g.The results of the T-test showed a significant difference (p < 0.01) in the average growth of vannamei shrimp in pond B with the addition of liquid smoke to the feed at a dose of (1%), which was 14.4 ± 0.1g.The T-test results also explained that the difference in the final weight value between treatment A (0.5% dose) and treatment B (1% dose) showed a positive number (1.77 g/head), meaning that the average final weight value for treatment A was higher than treatment B, so it can be concluded that treatment A was the best treatment because the average weight of shrimp at the end of rearing was higher than treatment B. The average shrimp weight in Pond A and B is presented in Table 2 and 3.The difference in the increase in the average growth of vannamei shrimp at two different doses of liquid smoke shows that liquid smoke can influence the growth of vannamei shrimp at certain doses.Liquid smoke can be used as an additive to animal feed [15].Liquid smoke contains lactic acid compounds and granules that can optimize the nutrient metabolism process in the digestive tract [16].Liquid smoke has been proven safe and has passed toxicological tests for use as a safe feed additive and alternative to antibiotics in animals [17].The application of liquid smoke as a food additive to animals was first studied by [18], and showed that a 1% dose of liquid smoke could increase the productivity and growth of chickens.Liquid smoke has also been reported to increase growth in tilapia at a dose of 1% [16].Liquid smoke given to livestock also functions as a natural antibiotic [18].This can explain the higher survival rate of vannamei shrimp in treatment B with a dose of 1% (85%) compared to treatment A with a dose of 0.5% (82%).However, there was no significant difference in the survival rate between treatment A and treatment B (P<0.05).The main carbon compounds in liquid smoke are dominated by several phenol derivatives such as phenol 2-methoxy phenol, formic acid anhydride, and 2,6, dimethoxy phenol [19].These compounds are associated with antimicrobial and antioxidant properties.High phenolic compounds provide antioxidant properties and the ability to kill bacteria and fungi [20].The active substances in coconut shell liquid smoke can increase metabolism and protein synthesis and activate digestive enzymes.In fish feed, besides functioning as a flavoring agent, adding coconut shell liquid smoke to the feed functions as a flavoring agent that can directly influence diet, digestive fluid secretion, and total feed consumption.Stimulation of fish digestive secretions, such as saliva, digestive enzymes, bile, and mucus, is considered important from the additives in feed.Interestingly, feed additives can increase growth factors through their ability to increase appetite compared to the previous portion.

The Total of Vibrio parahaemolyticus in the Water
This study used water previously infected with Vibrio parahaemolyticus bacteria with a concentration of 2.1 x 103 CFU/ml in Pool A and 1.8 x 102CFU/ml in Pool B (Table 4).Vibrio parahaemolyticus in waters is characterized as a green colony bacteria and cannot ferment sucrose.This vibrio causes an infection that enters through wounds in the exoskeleton and spreads through the hemolymph, which will later cause AHPND (Acute Hepatopancreatic Necrosis Disease) in shrimp.
The feed in this study would float in the water column for a while before being eaten by the shrimp.As such, some liquid smoke in the feed will dissolve into the water.Apart from that, feed remains, secretions, and feces from farmed animals also contain bioactive substances originating from liquid smoke, which then dissolve in the water.This was considered an antibacterial agent in the rearing ponds in this study, so it could significantly reduce the concentration of Vibrio parahaemolyticus in the water (Table 4).The bacterial density in this study continued to decrease every week.The results of the analysis of total bacterial density in treatment B with the addition of 1% liquid smoke to the feed showed 0 (zero), meaning no Vibrio parahaemolyticus was found from Week 5 until the end of rearing.
Meanwhile, in treatment A, with the addition of liquid smoke at a dose of 0.5% in the feed, Vibrio parahaemolyticus began to decrease and was not found in Week 12.During the 90-day rearing, no vannamei shrimp were detected infected with AHPND.Liquid smoke has also been reported to inhibit the growth of Escherichia coli, Staphylococcus aureus, Vibrio harveyi, and Vibrio parahaemolyticus bacteria [21].In liquid smoke, phenolic compounds are formed due to the pyrolysis process in wood or coconut shells that contain cellulose, hemicellulose, and lignin.As an antibacterial, this phenolic compound can damage the bacterial cell structure and inhibit cell wall formation, causing lysis of the bacterial cell wall.Apart from that, phenolic compounds can also denature proteins, leading to cell death.Some bacterial cytoplasmic membrane structures contain proteins and fats.Instability in the cell wall and cytoplasmic membrane causes the selective permeability function, active transport function, and control of bacterial cell structure to be disrupted.Disruption of cytoplasmic integrity in bacteria results in the escape of macromolecules and ions from the cell.Bacterial cells lose their shape, and lysis occurs.The use of coconut shell liquid smoke with a concentration of 1% can produce products that have a microbiological safety level in accordance with SNI (Indonesian National Standard) 2009 [22].

Water Quality Parameters of the Rearing Ponds 3.3.1 Physical Parameters
The measurement results (Table 5) show no significant differences between the temperature, transparency, salinity, pH, DO, and oxygen saturation values in the two rearing ponds.The average pond water temperature during rearing was 28.0 -28.8°C; this confirms that the water temperature in this study was still at the optimal value for the growth of vannamei shrimp (28 -32°C) [23].Temperature greatly influences the physiological condition of shrimp.Fluctuations in water temperature can cause shrimp stress, triggering mass deaths of shrimp, especially in environments infected with vibrio [24].
DO represents the concentration of dissolved oxygen in water.DO is needed for respiration, physiological processes, metabolism, and decomposing organic material in ponds.The recommended DO for growing vannamei shrimp is > 4 mg/l [25].Our study shows that the DO values in the two rearing ponds ranged from 7.1 -7.8 mg/l.The high DO levels in this study were due to the application of microbubbles that could break down air bubbles into microparticles to lengthen the presence of oxygen in the water column [26].
The degree of acidity (pH) and salinity in the morning and afternoon of the two rearing ponds was still within the tolerance range for vannamei shrimp, at 8.1 and 20 ppt.The concentration of pH and salinity in the water affects the molting process in vannamei shrimp [27].The transparency of the pond water in both ponds was also very good.The transparency value in this study was 36 cm.The results of water quality measurements can be seen in Table 5.

Chemical Parameters
Chemical parameters, such as ammonia, nitrite, nitrate, TOM, and alkalinity, and biological parameters, such as plankton and bacteria, were measured weekly.Measurement in Pool A showed that the average value of ammonia in this study was 1.46 mg/L, nitrate 1.85 mg/L, nitrite 6.38 mg/L, alkalinity 134.5, and TOM was 227.8.Meanwhile, measurement in Pool B showed that the average value of ammonia was 1.73 mg/L, nitrate was 1.87 mg/L, nitrite was 6.38 mg/L, alkalinity was 134.5, and TOM was 227.8.
The measurement results showed very high values above the threshold (<0.1 mg/L) for ammonia, nitrite, and nitrate [25].The high ammonia, nitrite, and nitrate content in water can inhibit shrimp growth rates by up to 50% and kill several important organisms [28].The high content of ammonia, nitrite, and nitrate in this study did not affect the growth or survival rate of the cultivated shrimp due to the addition of oxygen and high levels of dissolved oxygen in the water column [26].Dissolved oxygen is an important factor in the conversion of ammonia to nitrate.The transformation of nitrite to nitrate requires more oxygen than ammonia to nitrite.Ammonia and other nitrogen compounds (nitrite and nitrate) originate from the nitrogen cycle regulated by biological activity.The first stage is fixation, namely the conversion of dinitrogen gas (N2) into nitrogen that can be used directly by phytoplankton.

Biological Parameter
The total density of phytoplankton in Pond A was 262 ind/mL, and Pond B was 225 ind/mL.There were no significant differences (P<0.05) in the chemical and biological water quality parameter values in Pond A and B. The results of water quality measurements showed that the water quality parameter values in the two ponds were optimal for rearing vannamei shrimp.In this research, water quality management was carried out routinely to maintain the media (water) environment optimally to increase the growth of vannamei shrimp and reduce mortality rates.

Maintaining Water Quality
One of the factors determining the success of shrimp cultivation is the management of water quality as a medium for rearing shrimp.Several things were done to manage water quality in this study, including giving probiotics every day at a dose of 2 ppm.Probiotics are live microorganisms that are deliberately introduced into ponds or in feed to provide beneficial effects on shrimp health [29].A transparency value of 30 -40 cm in aquaculture waters is caused by the growth of plankton, a condition very much needed in super-intensive shrimp cultivation because it leads to shady pond conditions, suppresses the growth of algae and moss at the bottom of the pond, and prevents the presence of phytoplankton, which can IOP Publishing doi:10.1088/1755-1315/1328/1/01201810 absorb ammonia, nitrate, and nitrite; all are dangerous for shrimp growth.Circulation was managed by reducing the water by 20 cm and replacing it with new water from a drilled well.To binding dead plankton so as not to pollute the water quality, captan were applied regularly and spread every morning and increasing or producing calcium minerals in farm water [30].

Conclusion
Adding liquid smoke at a dose of 0.5% to the feed was able to significantly increase the average growth rate of vannamei shrimp during the 90-day rearing; however, the survival rate for vannamei shrimp at a dose of 0.5% was lower than at a dose of 1%.Some liquid smoke added to the feed will enter the shrimp's digestive system, and some will dissolve into the water.This water-soluble bioactive is thought to be able to reduce the concentration of V. parahaemolyticus in the water.No vannamei shrimp were infected by V. parahaemolyticus bacteria during the 90-day rearing.

Table 1 .
Water quality parameter

Table 2 .
The Average Shrimp Weight in Pond A (Dose 0.5%)

Table 3 .
The Average Shrimp Weight in Pond B (Dose 1%)

Table 5 .
Water quality measurement results