Seaweed extract of Gracilaria verrucosa as an antibacterial and treatment against Vibrio harveyi infection of Litopenaeus vannamei

The objectives of this study were to investigate the antibacterial activity of G. verrucosa extract in test inhibitory zone with different concentrations (500, 1000, 1500, and 2000 mg/L) and to examine G. verrucosa extract with different dosage (0.5, 1.0, 1.5, 2.0 g/kg) in feed on immune responses (total hemocytes count, phagocytic activity, phenoloxidase activity, respiratory burst) and survival rate in the Litopenaeus vannamei against the pathogenic Vibrio harveyi. Pacific white shrimp with an initial body weight of 5.25 ± 0.55 g was reared in the aquarium (60×30×30 cm3) with a density of 10 shrimp/aquarium. Pacific white shrimp had been fed three times a day as much as 3% in at satiation for 14 days after challenged with V. harveyi. The first results of the inhibitory test showed that all the concentration of G. verrucosa extract was able to inhibit the growth of V. harveyi and the second result showed that the extract of G. verrucosa can increase the immune responses of shrimp. In the result of survival showed that shrimp fed with 0.5, 1.0, 1.5, and 2.0 g/kg has 80, 73, 70, and 70%, respectively. In conclusion, the seaweed extract of G. verrucosa has antibacterial activity and can induce the immune responses and resistance of Pacific white shrimp against V. harveyi infection.


INTRODUCTION
The Pacific white shrimp Litopenaeus vannamei is one of the most important marine aquaculture species in the world. The diseases that most often impact intensive shrimp cultivation are bacterial, viral, and co-infectious diseases (Teixeira-Lopes et al., 2011). Vibriosis is a major disease in Pacific white shrimp culture caused by Vibrio harveyi infection (Widanarni et al., 2012). Shrimp infected by vibriosis experienced symptoms such as browning-skin damage, reddish tail, and swimming legs, necrosis, black lymphoid organ, brown gills, brownish muscle, empty intestine, and weak movement (Cano-Gomez et al., 2009). Antibiotic treatment of bacterial diseases in fish culture has been applied for many years. But antibiotics have been banned because it can cause pathogenic bacteria to be resistant to antibiotics, leaving residues on shrimp & aquatic environment, and harmful to consumers healthy (Zhang et al., 2014).
The natural compound originated from plants have potential in fish farming as an alternative to antibiotic use (Van Hai et al., 2015). The medicinal plant also has an antimicrobial effect on the aquatic organism (Citarasu, 2010). Seaweeds are known to be an important source of secondary metabolites for the pharmaceutical industry in drug development. Seaweed from genus Gracilaria is a potential source for alternative natural medicines for fish because the potential had been reported as an antioxidant, antibacterial, antitumor and many others (Chen et al., 2012;Wongprasert et al., 2014;Vienna et al., 2015). Maftuch et al. (2012) and Vienna et al. (2015) reported that seaweed Gracilaria verrucosa have antibacterial activity and can enhance innate immune of shrimp. Sirirustananun et al. (2011) demonstrated that the supplementation of G. tenuistipitata in shrimp feed at a dose of 0.5-2.0 g/kg for 14 days treatment can increase the immune system of shrimp that infected by WSSV. Jasmanindar et al. (2018a) reported that G. verrucosa extracts increasing the immune system of shrimp and increased survival rate against Vibrio harveyi bacteria, up to 66%. This study aim to investigate the antibacterial activity of G. verrucosa extract and evaluating different doses of G. verrucosa in shrimp feed to increase the immune response of shrimp challenged with V. harveyi.

Research procedures Extraction of seaweed Gracilaria verrucosa
Gracilaria verrucosa seaweed was obtained from cultivation farming area in Muara Gembong, Bekasi, Indonesia. Seaweed cultivated for 1-1.5 months after being planted. Extraction was done by using Zahra et al. (2017) method which had been modified. The seaweed was washed with both seawater and freshwater to remove dispose of salt, microorganisms, and other unwanted materials, and dried under the sun. Afterward, seaweed was finely ground and sieved by mean of a fine sieve (60 mesh size). Extraction was performed by adding ethyl acetate solvent at a ratio of 1:3 (w/v), shaking for 24 hours using thermoshaker 130 rpm at 40°C, precipitating, and finally filtering. The obtained filtrate was then evaporated using a vacuum rotary evaporator at a temperature of 50°C to acquire a crude extract.

Experimental fish
The Pacific white shrimp Litopenaeus vannamei came from a shrimp farm located in Fisheries Academy of Ministry of Marine and Fisheries Affairs Indonesia, Serang Province. Shrimp with an average body weight of 6.07 ± 0.10 g/shrimp were used and acclimatized in fiber tanks for two weeks at 28°C, and shrimp were stocked at a density of 10 shrimp/aquarium.

Bacteria preparation
Vibrio harveyi culture was harvested and recultured in 25 mL of SWC broth consisting 5 g bactopeptone, 1 g yeast extract, 3 mL glycerol, 750 mL seawater, and 250 mL aquades) and incubated in waterbath shaker at a temperature of 37°C for 24 hours. Suspension of bacteria was moved into microtube of 2 mL and serial dilution was performed until the bacterial density of 10 8 CFU/mL was obtained. Approximately 50 µL of bacterial suspension was spread on TCBS agar media at 28°C for 24 hours.
Shrimp feed preparation G. verrucosa extract based on doses all of the treatment previously determined, was dissolved in a 100 mL of distilled water that was previously mixed with egg white as a binder at a concentration of 2%. The obtained solution was then evenly spraying with 1 kg commercial shrimp feed and dried in an oven at 37°C.

Screening phytochemical test
The phytochemical test was performed through the color visualization method by Harborne (2006), which included a test of flavonoid, alkaloid, tannin, saponin, quinone, steroid, and triterpenoid.
Alkaloid test. A total of 0.1 g extract of G. verrucosa has added 1 mL of HCI 2 N and 9 mL of hot distilled water and then heated for 2 minutes. After the cold, the filtrate then shares and is divided into two small tubes. The first tube was added with Bauchardat reagent and the second tube was added by Dragendrauf reagent. Positive results based on the formation of chocolate to blackness in reagent Bauchardat and white deposits in Dragendrauf reagent.
Saponin Test. 0.1 g extract of G. verrucosa added 5 mL of distilled water and then heated for 5 minutes. Then the extract is filtered and the filtrate is shaken. Positive results are indicated by the presence of foam for 10 minutes.
Tanin Test. 0.1 g extract of G. verrucosa added 5 mL of distilled water and then heated for a few minutes. The filtrate is then filtered and added 1% FeCl3. Positive results are indicated by a change in color to dark blue or greenish-black.
Triterpenoid and steroid tests. 0.1 g extract of G. verrucosa added 2 mL ethanol then heated and filtered. The resulting filtrate is evaporated until thick and 1 mL of ether is added, 3 drops of anhydrous acetic acid, and 1 drop of concentrated H2SO4. Triterpenoid positive results are indicated by the presence of red or purple, and a positive result of steroids with the formation of green.
Phenolic and flavonoid tests. As much as 0.1 g extract of G. verrucosa boiled ambon banana are added with 2 mL of methanol and then heated and filtered. The resulting filtrate is divided into two tubes, the first tube is added 10% NaOH and the second tube is added with concentrated H2SO4.
The reddish-orange color shows the presence of phenolic compounds, and the red to brownish color indicates the presence of flavonoid.

Evaluation inhibitory zone test through the agar diffusion method
Inhibitory zone test through the agar diffusion method was performed to observe the inhibition zone around the paper disk. Seaweed extract was made into various concentrations, those were 500, 1000, 1500, and 2000 mg/L and positive control (K+) using 50 mg/L chloramphenicol antibiotic. The extract was dissolved in 10 mL of sterile aquades and dripped 20 μL with a micropipette on a paper disk. The paper disk is placed on the surface of a medium that has been spread by V. harveyi with a concentration of 10 6 CFU/mL and incubated at 28°C. The bacterial inhibition zone is measured based on the diameter of the clear zone formed using caliper with a precision of 0.01 mm.

Evaluation of G. verrucosa extract dose in shrimp feed
A completely randomized design was used in the present research. G. verrucosa extract doses in shrimp feed consisted of six treatments (with three replicates each) as follows: K-(without G. verrucosa extract), K+ (without feed extract + V. harveyi infection), A (0.5 g/kg feed extract + V. harveyi infection), B (1.0 g/kg feed extract + V. harveyi infection), C (1.5 g/kg feed extract + V. harveyi infection), and D (2.0 g/kg feed extract + V. harveyi infection). Shrimp were fed at satiation and feeding treatment fed after the challenge test was 3 times a day for 14 days.

Hemolymph collection
As much as 0.2 mL hemolymph, was collected from the bottom swimming leg of the shrimp using 1 mL syringe (previously filled with 0.3 mL of Na-citrate anticoagulant) and homogenized by a handshake to shape number 8 for 5 minutes. Hemolymph was collected in order to determine parameters such as total haemocyte count (THC), phagocytic activity (PA), phenoloxidase activity (PO), and respiratory burst (RB). Data collection on parameters were done by gathering hemolymph in each replication pre-treatment, first, second and fourth-day after V. harveyi challenge test, and then 14 days post-treatment or challenge test.

Total hemocyte count
Total hemocyte count for each treatment was determined according to the method of Immanuel et al. (2012). An anticoagulant-hemolymph mixture was mixed gently and then a drop of mixture placed and counted with a hemocytometer (Neubauer chamber) using a light microscope at 100 times magnification.

Phagocytic activity (PA)
As much as 0.1 mL shrimp hemolymph was placed into a microplate, mixed with 25 μL of Staphylococcus aureus (107 CFU/mL) bacteria and incubated for 20 minutes. Afterward, 5 μL was dropped on a preparation glass to form preparation pads. The preparations were fixed with absolute methanol for five minutes and stained with Giemsa (10%) for 15 minutes. Phagocytic activity was measured based on the percentage of phagocytic cells that carried out the phagocytic activity.

Phenoloxidase activity (PO)
Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) based on the procedures of Liu and Chen (2004) with slight modification. The optical density of the shrimp's phenoloxidase activity for all test conditions was expressed as dopachrome formation in 50 μL of hemolymph.

Respiratory burst (RB)
Respiratory burst (RB) activity of hemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion production following the method of Cheng et al. (2004) with slight modifications. The optical density of formazan was measured at 630 nm using a 96-well microtiter plate and microplate reader Respiratory burst was expressed as NBT-reduction in 10 μL of hemolymph.

Survival rate
Survival rate (SR) was calculated at the end of the feeding experiment and 14 days post-challenge test. The survival rate was measured by the following formula: Note: SR = survival (%) Nt = the number of shrimp at the end of the rearing No = the number of shrimp at the beginning of rearing

Data analysis
Data analysis was carried out using ANOVA (SPSS 17) methods with 95% (a= 0.05) of confidence level. ANOVA was used to analyze total hemocytes, phagocytic activity, phenoloxidase activity, respiratory burst, and survival rate. An ANOVA test was followed by Duncan's posthoc comparison test if differences were found in software SPSS 22.

Results
The bioactive compounds contained in G. verrucosa extracts such as alkaloids, saponins, phenolics, flavonoids, triterpenoids, steroids, and glycosides. Seem liked the result of the research Siregar et al. (2012), seaweed G. verrucosa contained alkaloid compounds, flavonoids, and steroids. The results of the phytochemical analysis of G. verrucosa extract are presented in Table 1.
The results of in vitro showed that G. verrucosa extract is able to inhibit the growth of V. harveyi bacteria and the higher the dose used the greater the inhibit zone (P<0.05). The test results of antibacterial activity G. verrucosa extract presented in Table 2.
The total hemocytes increased the first, second and 14th day after challenge V. harveyi on all of the dose treatment of G. verrucosa extract compared to the control (P<0.05) are presented in Figure 1.
Total hemocytes on all of the extract dose were higher compared with positive control on the first day after challenge (Figure 1). The highest total hemocytes on extract dose B was significantly higher on the first and fourth days after challenge (P<0.05). Total hemocyte on extract dose B was the highest significantly on the second days after challenge (P<0.05) both all of extract dose and control. Total hemocytes on the dose of extract A was the highest significantly of all doses extract and control at the 14 days (P<0.05).
Phenoloxidase activity on all of the dose extract on the first day after challenge increased and significantly different from the positive control (P>0.05) (Figure 2). Phenoloxidase activity on dose extract C was significantly higher on the second day after challenge (P<0.05). Phenoloxidase activity on all of the dose extract was higher than control and dose extract experiment A highest significantly at the 14 days challenge (P<0.05).     The phagocytosis activity on all of the dose extract was higher than positive control at the first, second, fourth and 14 days after challenge ( Figure  3). The phagocytosis activity on dose extracts C and D was higher with positive control on the first day after challenge (P<0.05). Phagocytosis activity on dose extract A was highest at 2 and 4 days after challenge (P<0.05). Phagocytosis activity on dose extract A and B was higher (P<0.05) compared with others at 14 days after challenge.
Respiratory burst activity was increased both dose extract and control (P<0.05) on the first day after challenge (Figure 4). The respiratory burst activity on dose extract A was significantly higher than positive control on the second day after challenge (P<0.05). The respiratory burst activity on dose extract B was higher than other dosage extract and control on the fourth day after challenge (P<0.05). The respiratory burst activity on dose extracts A and B was higher with other dose extracts and control at the 14 days after challenge (P<0.05).
The survival rate on dose extract was compared with the positive control treatment presented in Figure 5. The survival rate was decreased in all of the dose extract and positive control at the first, second and fourth day after challenge (P<0.05). The highest survival rate after challenge V. harveyi on the seventh day is the dose extract A (P<0.05).

Discussion
G. verrucosa can be used as an immunostimulant because it contained polysaccharide compounds (Vienna et al., 2015). G. verrucosa extraction resulted in contained sulfate and galactose compounds (Jasmanindar et al., 2018b). The bioactive compound affected the immune system in the shrimp. The bioactive compounds in G. verrucosa extract through the mechanism of molecular interaction with receptor surfaces in shrimps an important role in the immune system (Zahra et al., 2017).
Hemocyte is an important role in both cellular and humoral immune responses in immune defenses of crustaceans . The results of the first, second, fourth and 14 days after challenge showed that total hemocytes of G. verrucosa extract were higher than the positive control. That was thought to be due to the effect of G. verrucosa extract as immunostimulant during maintenance to improve the Pacific white shrimp immune system. Changes in the increase and decrease in the amount of hemocyte are the health status and stress indicator on the shrimp. This increase in total hemocytes indicates that the increased defense reaction in the shrimp body due to the presence of foreign particles entering the body of the shrimp, that V. harveyi bacteria. Antigen entering the body of the shrimp will be recognized by the hemocyte cell receptor to produce cellular responses such as phagocytosis (Lin et al., 2013). An increasing number of hemocytes is a response to resistance to a pathogen invasion caused by an increase in the number of pathogens in the host.
Phenoloxidase activity is an ability or activity of the body's defense of Pacific white shrimp in recognized foreign objects entering (Costa et al., 2009). Phenoloxidase activity after the first-day challenge experienced an increase in treatment and control. Total hemocyte is closely related to the activation of proPO to produce phenoloxidase activity. Phenoloxidase is an enzyme that plays a role in the melanization process. This enzyme is produced through a proPO (prophenoloxidase) system that can be activated by immunostimulant. Activation of the prophenoloxidase system results in the production of melanin, a dark brown pigment responsible for several processes, including to inactivate foreign particles, and to protect its spread on the host, and to repair cuticle damage. The proPO system plays an important role in the introduction of foreign bodies including phagocytosis, melanization, cytotoxic reactant production, particle encapsulation, and the formation of nodules and capsules (Amparyup et al., 2013).
The phagocytosis activity continued to increase at the first, second, fourth and 14 days after challenge. During the process of phagocytosis, the foreign particles or bacteria will be recognized by the receptors on the cell surface, then deleting by cells that rearrange the cytoskeleton for the formation of phagosomes. The previous study has suggested an increase in phagocytosis activity in shrimp also occurs after the challenge test as a shrimp defense mechanism (Febriani et al., 2013). Wongprasert et al. (2014) that polysaccharides of seaweed can stimulate non-specific immune systems, in this case, phagocytosis and respiratory burst activity through the interaction mechanisms of polysaccharide molecules with receptormediated surfaces.
Phagocytosis activity was associated with an increased respiratory burst. Respiratory burst activity is a mechanism of particle removal by phagocytic hemocyte cells involving the release of degradative enzymes into the phagosome (an oxygen-dependent killing mechanism) and generating reactive oxygen intermediates (Sirirustananun et al., 2011). Respiratory burst of the dose extract has increased compared to the initial measurement at first and second day after challenge. The respiratory burst was a decrease between treatment and control on the fourth day after challenge.
Extract of G. verrucosa has antibacterial activity derived from flavonoid & saponin compounds as well as sulfated galactan compounds as immunostimulants in white shrimp. Mechanisms to stimulate the immune system in shrimp through sulfated galactan compounds. Sulfated galactan compound in G. verrucosa extract is a type of carbohydrate element that is lipopolysaccharide. The introduction of pathogens through molecular patterns by an identifiable protein called pattern recognition protein (PRRs). The protein can recognize carbohydrates from bacterial cell walls or microorganisms such as lipopolysaccharide (LPS) or peptidoglycan (Cochet & Peri, 2017). Receptors in shrimp that are pattern recognition receptors (PRRs) have an important role in the immune system of shrimp consisting of lipopolysaccharide and β-1.3glucan binding protein (LGBP) and toll receptors. The introduction of pathogens is triggered by PRRs activation via the serine protease pathway which is then broken down into proPO to produce phenoloxidase (Li & Xiang, 2013). Sulfated galactan in G. verrucosa extract binds to LGBP on the hemocytic membrane. Sulfated galactan capable of stimulating the immune system in shrimp is assumed through the mediation or interaction of sulfated galactans with receptors on the surface of the hemocytes (Wongprasert et al., 2013). The presence of a bond between sulfated galactan and receptor activates signal signaling to increase the proliferation of hemocytes and stimulates immune system activity.
The best survival rate that G. verrucosa dose extract has a higher compared with positive control. This suggests that G. verrucosa extract is capable of controlling V. harveyi infections that are supported through the enhancement of noncellular and humoral specific immune systems. The best survival is the treatment of the dose extract A (0.5 g/kg of feed). Previous research has shown that G. verrucosa extract is to maintain the survival of white shrimp against diseases caused by Vibrio bacterial infection (Kanjana et al., 2011;Sirirustananun et al., 2011;Jasmanindar et al., 2018). Seaweed Gracilaria genus is a potential source for alternative natural medicines for the fish cause this species had been reported had potential as anti-oxidant, anti-bacterial, anti-tumor, anti-viral, antibacterial activity and enhances innate immune of shrimp (Maftuch et al., 2016;Saraswaty et al., 2015).

CONCLUSION
The effective dose extract G. verrucosa which gives the best results in enhancing the immune response and also the white shrimp resistance to V. harveyi infection is a dose of 0.5 g/kg of feed. The application of G. verrucosa extract in white shrimp feed has antibacterial properties and can increase immune responses and resistance to vibriosis disease caused by V. harveyi infection.