Sonneratia alba Extract Protects the Post Larvae of Tiger Shrimp Penaeus monodon against Vibrio harveyi and Saprolegnia sp

The research aimed to study Sonneratia alba leaves extract to inhibit Vibrio harveyi and Saprolegnia sp., and to evaluate its effectiveness to protect the post larvae of tiger shrimp against the infections. The leaves of S. alba were cleaned, chopped, dried, macerated and extracted in three different solvents namely ethanol 80%, freshwater and saline water. The post larvae 8 then immersed with the extract and infected with Vibrio harveyi and Saprolegnia sp. The clinical signs, pathological anatomy, total vibrio count, prevalence, survival rate, and relative percentage of survival were observed. The results showed that S. alba leaves extract inhibited V. harveyi and Saprolegnia sp. on post larvae of tiger shrimp, improve survival rate and relative percentage survival. The most effective dosage was 1,250 mg/L either for ethanolic, freshwater and saline water solvents.


Introduction
The disease outbreak in tiger shrimp culture in East Kalimantan province, Indonesia is still struggling to be solved. Shrimp mortality occurred in the larvae stage in hatchery to post larvae stage in grow out ponds, and even just before harvesting. The common cause of mortality is Vibrio harveyi and in larvae stadia can also caused by fungi [1]. The main disadvantage in aquaculture is the sudden outbreak of the disease, mainly due to Vibrio spp, which is considered a significant problem for the development of this sector with severe economic losses worldwide [2]. Disease prevention are still not resolved properly due to usage of chemicals and antibiotics. The use of these chemicals are often uncontrolled and leads to bacterial resistance and toxicity in shrimp [3]. Several studies have shown that some plant extracts active against fungi and bacteria both in vitro and in vivo. [3,4,5,6,7,8].
Mangrove grows well in coastal areas of East Kalimantan, Indonesia. Mangrove is widely used for household needs, as well as food, beverages and traditional medicine. Mangrove is a source of several bioactive compounds. Mangrove plants are used as medicines by the community and the extracts of it known to inhibit the growth of pathogenic microorganisms [9,10]. Research in mangrove as a medicine has been widely practiced, as well as mangrove screening as an antibacterial against disease in shrimp [1,4,5,11,12]. *Corresponding author: ginaoesman@gmail.com Mangrove is rich in bioactive compounds [13]. Mangroves can be used as a new bioactive source of potential natural products used to control microbial disorders [14]. The mangrove phytochemical analysis has revealed important chemicals such as saponins, alkaloids, glycosides, tannins, steroids, flavonoids, gums, phytosterols, and reducing sugars [15]. Extracts of methanol bark of S. alba and A. marina fruit showed a 15.00 mm inhibition zone to Salmonella typhi, its acetic acid extract showed a 14.00 mm inhibit zone against Listeria monocytogenes [16]. Extract of S. alba can inhibit V. harveyi and Saprolegnia sp. [17] Mangroves are widely found in Indonesia but the utilization of bioactive materials has not been developed yet [18]. This research aimed to study Sonneratia alba leaf extract to inhibit the growth of V. harveyi and Saprolegnia sp., and to improve the survival of tiger shrimp.

Sonneratia alba Extraction
Leaf of S. alba was collected from the coast in Muara Badak District, Kutai Kartanegara Regency, East Kalimantan. The leaves were cleaned, and drained at room temperature without direct sunlight exposure. After drying, the samples were chopped and macerated with three different solvents, i.e. 80% ethanol, freshwater and saline water at a salinity of 20 ‰ for 24 hours. After maceration, each solvent was evaporated by rotary evaporator until the remaining solvent around 30% of the initial solvent volume. For aqueous extract, the evaporation was carried out until the remaining solvent was around 10% of the initial volume [17].

Microbial Preparation
V. harveyi and Saprolegnia sp. were used for the infected test. They are obtained from the Laboratory of Aquatic Microbiology Faculty of Fisheries and Marine Sciences Mulawarman University. V. harveyi and Saprolegnia sp. were tested for pathogenicity by injecting to ten shrimps with individual body weight of 6 g. The injection was carried out intramuscularly 0.1 mL with at bacterial dilution of 10 3 . Three days post injection, the shrimp showed a reddish on the body. V. harveyi then reisolated from hepatopancreas and reinfected to the shrimp. The procedure was repeated three times. Finally, V. harveyi was isolated and cultured on Thiosulfate Citrate Bile Salt Sucrose agar (TCBSA) medium and incubated for 24 hours at 30 0 C. V. harveyi was cultured in Triptic Soy Broth (TSB) with 2% NaCl. The Saprolegnia sp. Was cultured on Potato Dextro Agar (PDA) medium and incubated at 33 °C for 48 hours.

Post Larvae Shrimp
Post larvae (PL) 8 was obtained from the hatchery of Windu Permata in Muara Badak of East Kalimantan. Post larvae was antibiotic-free, also free from Vibrio sp. by bacterial testing on TCBSA medium. The shrimp was acclimatized for 1 day in the aquarium at a density of 20 shrimp.

The Treatment
Sterile saline water (free from Vibrio sp. and Saprolegnia sp.) was used in this research. All equipment used in this research were disinfected and dried. The water salinity was 23 ppt, and free of V. harveyi, Aeromonas sp., Pseudomonas sp., and Saprolegnia sp. The aquarium and other facilities were washed with soap and soaked with potassium permanganate (KMNO4). Total volume of water in each aquarium was 5 litres.
The treatments were ethanol, water and sea water extract of S. alba. Each S. alba extract which had concentrations of 750, 1,000, and 1,250 ppm. Control treatments consisted of antibiotic oxytetracycline as a positive control and PBS 0,85% as a negative control. The shrimps were soaked in each aquarium that were filled with each treatment and replicated three times.
The infected test with each of V. harveyi and Saprolegnia sp. was performed after 48 hours of treatment by soaking shrimp in 1 mL/litre with at microbial dilution of 10 6 into each aquarium. The shrimp was reared for 21 days. .

Data Analysis
This study used a complete randomized design with three replications. The data was analyzed using analysis of variance test, then followed by Duncan test if there were significant differences. The clinical symptoms of shrimp were observed and analyzed every day by calculating the percentage of inactive (passive) behavior, decreased appetite, weakened reflexes, reddish and incomplete of body. Anatomical pathology observation, such as damage and changes of the head, legs and tail, was performed on dead shrimp and at the end of the study. The Anatomical pathology were analyzed descriptively. In addition, at the day 14 and 21 of the study, the total bacterial content of V. harveyi of shrimp and water samples were enumerated by total count or total vibrio count (TPC/TVC). The prevalence was obtained from the percentage of shrimp infected with V. harveyi or Saprolegnia sp., that have reddish on the body and positive isolation. The survival rate was calculated by percentage of live shrimp from each treatment, while Relative Percentage of Survival (RPS) was calculated by the formula: RPS=1-[ÿ(prawn treatment mortality) x (control prawn mortality) -1 ]ÿx 100% [1] 3 Result and Discuss

Clinical Symptom
The condition of the shrimp is active and healthy after being treated with the extract of S. alba, however the body of the shrimp turned slightly bluish. This was due to the absorption of extract into the shrimp's body, which was the shrimp response. The shrimp did immune response to the extract that enters its body, it was the chromatophore reaction. The color changing of shrimp indicated the occurrence of body reaction toward the extract treatment, and it was caused by the enlargement of shrimps' cuticle [4,11]. Post larva of shrimp appeared normal, healthy, active and did not cause any mortality and specific clinical symptoms after 48 hours of treatment.
The clinical symptoms were observed at 5 and 10 days after given extract of S. alba. Shrimp shows weakness and appetite decreases after being infected by V. harveyi and Saprolegnia, the clinical symptoms even more severe on negative control, such as weakness, decreased appetite, slow and weak reflexes, and reddish body. The overall clinical symptoms were in Table 1. Sonneratia alba extract was able to suppress V. harveyi and Saprolegnia sp. attack, so the clinical symptoms percentage of S. alba extract was lower than the controls. The extract of mangroves have the potential as antifungal and antibacterial activity, as well as gastric antioxidants, and able to heal wounds [19].

Anatomical Pathology
Better anatomical pathology was shown by shrimp in all extract treatment when compared to controls. The tails and legs of some shrimps that treated with 750 ppm water extract of S. alba became reddish, as well as the tails of some shrimp with 750 ppm ethanol extract and saline water of S. Alba. The Anatomical pathology of shrimp on the negative control showed symptoms of vibrio attack, which was the tail, legs, gills and body redness. Some shrimp were deformed and failed moulting, this condition caused mortality. The anatomical pathology of shrimp on negative control that was infected with Saprolegnia showed dirty feet and body, while the shrimp were treated the extract showed normal reaction.
Vibrio sp. caused dirty gills, changing the color to yellow or pale red, dark and in the carapace, abdominal, and tail, also dark slightly redness hepatopancreas [20]. The shrimp were attacked by vibrio showed changing appearance symptoms like changing color to reddish black, red patches on the legs and tail, hemorrhage on the body, deformity and molting failure [11].

Total Vibrio Count
V. harveyi was isolated from shrimp and water media. The Total Vibrio Count (TVC) showed that the lowest measurements was in 1,250 ppm ethanol extract of S. alba treatment, followed by the treatment of 1,250 ppm saline water extract of S. alba and control positive, then 1,000 ppm ethanol extract of S. alba (Fig. 1). The ethanol, freshwater and saline water extracts of S. alba were able to suppress the attack of V. harveyi and Saprolegnia sp. in shrimp.
The ability of S. alba extract as an antimicrobial was comparable to antibiotics. The methanol extract of S. alba bark showed a 15 mm inhibition zone against Salmonella typhi. Acetone extract of S. alba leaves showed a 14 mm inhibition zone against Listeria monocytogenes [16]. The inhibition zone of S. alba ethanol extract 1,000 ppm to V. harveyi is 12.67 mm, and Saprolegnia is 12.00 mm, and Minimal inhibitory concentration (MIC) of S. alba to V. harveyi is 3.91μgmL -1 , and MIC against Saprolegnia sp. is 7.81 μgmL -1 [1]. MIC of mangrove plants against pathogenic bacteria ranged from 20-640 mg/mL [21]. Methanol extracts of Sonneratia caseolaris was effective on all gram negative bacteria [9].

Prevalence
The prevalence infected of the V. harveyi and Saprolegnia sp. in shrimps that were treated with the extract S. alba leaves, showed that bioactive of S. alba could reduce the prevalence of microbial infected (Fig. 2). The average prevalence attack of V. harveyi and Saprolegnia sp. on extract treatment was 12.67-56.67 %, and 2.33-47.33%. The average prevalence attack of V. harveyi and Saprolegnia sp. on the negative control was 75.00-81.33 %, and 69.33-72.33%. Sonneratia caseolaris bark has the potential as a stable antimicrobial and antioxidant and can be used as an antimicrobial and natural antioxidant agent in the clinical, pharmaceutical and food processing industries [22]. Mangrove plants can be utilized as a source of natural antifungal drugs [23]. Several researchers around the world have found evidence that mangrove extract has great potential against pathogenic microbes. Mangrove extract using ethanol solvent, freshwater and saline water inhibit the growth of various microbes by in vitro test [17]. Antimicrobial compounds of the plant have therapeutic potential because natural ingredients are functionally work without side effects which often associated with synthetic antimicrobials [24].

Relative Percentage of Survival
The ability of S. alba extracts 1,250 ppm to protect the post larvae of tiger shrimp against V. harveyi attacks compared to antibiotic were not different. The second best treatment is 1,000 ppm in ethanol and 1,000 ppm in saline water. The ability of S. alba extracts protects against Saprolegnia sp. the best is also on saline water solvent of S. alba 1,250 ppm leaf which is different from antibiotic treatment but not different from freshwater and ethanol solvent treatment. The second best is 1,250 ppm in ethanol, followed by 1,250 ppm in water (Fig. 4). Sonneratia alba leaf extract has the ability to inhibit V. harveyi and Saprolegnia sp. infection and improve survival of post larvae of tiger shrimp. The ability to inhibit these microbes can be seen from the results of TVC test, which showed the content of TVC on shrimp that was treated with extracts have low values, especially on ethanol extract and sea water extract. S. alba leaf extract is able to protect against microbial attacks.

Conclusion
Sonneratia alba leaf extract is potentially to be used as an antimicrobial. These plants are able to inhibit V. harveyi and Saprolegnia sp., reduce prevalence attack of microbial, improve survival rate and relative percentage of survival post larvae of tiger shrimp.