Antimicrobial of nyirih (Xylocarpus granatum) against pathogens of tiger shrimp post-larvae

Leaf extract of X. granatum

Leaves of X. granatum were obtained from a shrimp pond area in the Muara Badak Subdistrict of the Kutai Kartanegara District in East Kalimantan, Indonesia. Leaves were washed and drained until there was no water and, after being dried to around 40% of their original weight, were chopped and wind-dried in a room not exposed to direct sunlight. After about 50 days, the leaves were then macerated with three different solvents namely 80% ethanol, distilled water, and 22‰ seawater for 24 hours. The ratio between leaves and solvents was 300 g of leaves in 2,100 ml solvent. The maceration product was extracted by evaporation, and the extraction products were heated over the bath until the ethanol solvent was evaporated to obtain crude. Extractions with distilled water and seawater were stopped when the solvent reached 10% of the initial volume.

Microbes

Microbes used for the challenge tests were Vibrio harveyi and the fungi Saprolegnia sp., supplied by the Laboratory of Aquatic Microbiology, Faculty of Fisheries and Marine Science, Mulawarman University, Samarinda. Before use, the pathogenicity of V. harveyi was tested by intramuscularly injecting 0.05 ml of the bacteria at a dose of 12.4×10⁴ CFU/ml into five 3-g tiger shrimp. After 5 days, when the shrimp showing signs of redness V. harveyi was then isolated from the hepatopancreas. Furthermore, V. harveyi was isolated and cultured on Thiosulfate Citrate Bile Salt Sucrose Agar medium (Merck KGaA. 1.10263.0500) and incubated for 24 hours at 33°C. Saprolegnia sp. was rejuvenated by culturing it on Potato Dextro Agar medium (Himedia REF M096-500G) incubated at 33°C for 24 hours.

Water and aquarium

Seawater with 22‰ salinity, and 28°C temperature was used as the culture medium for shrimp, and confirmed to be free from pathogens by isolating and identifying Vibrio sp and Saprolegnia sp. The seawater was deposited in a tank for 2 days and then flowed to another tank and aerated. Each aquarium was filled with 5 l sea water and aerated.

Tiger shrimp

The PL-25 tiger shrimp came from a brood stock and controlled hatchery which never applies chemicals or drugs and were confirmed to be Vibrio-free after sampling for isolation and bacterial culture in the medium. Each aquarium was stocked with 25 shrimp.

Treatment

The treatments were leaf extract, extracted with ethanol solvent, distilled water or seawater solvent, and each leaf extract treated to the shrimp had concentrations of 1,000, 800, 600, or 400 ppm (where 1,000 ppm is 1 ml of extract in 1,000 ml of water). Control treatments consisted of a positive control, the antibiotic erythromycin 500 mg/1,000 ml, and a negative control, NaCl 0.85%. Assessments were carried out every 6 hours.

As many as 25 shrimp of PL-25 were soaked in each extract solution at each indicated concentration in each aquarium, and each treatment was replicated three times. The challenge test with each of V. harveyi and Saprolegnia sp. in each aquarium was performed 6 days after extract-soaking by immersing a concentration of 10.6×10⁵ CFU/ml microbes into the shrimp culture medium. The shrimp was reared for 28 days for the challenge test. Clinical symptoms, including changes in activity, appetite, and reflexes, were observed every day. Clinical symptoms were analysed by calculating the percentage of shrimp in an aquarium showing inactive (passive) motion, decreasing appetite, and weakening response of reflex. Observation of pathological anatomy (PA) was performed on dead shrimp and at the end of experiment based on changes in colour, shape, and other abnormalities in the shrimps’ bodies and organs. PA observation included the body condition, carapace, legs, uropod, hepatopancreas, and abdomen (Table 1).

Dead shrimp were recorded each day in order to calculate shrimp survival rate. In addition, the total content of V. harveyi proliferating in each shrimp was calculated using total plate count (TPC) method. The shrimp bacteria were isolated and cultured on Thiosulfate Citrate Bile Salt Sucrose Agar medium, after incubation for 24 hours at 33°C the number of growing V. harveyi colonies was counted. The relative percentage of survival (RPS) was also applied to know the effectiveness of leaf extract X. granatum in preventing shrimp from V. harveyi infection using the following formula:

RPS = (1- (mortality of treatment shrimp) × (mortality of control shrimp)^-1) × 100%

Effect of extract treatment

The body colour of shrimp changed darker and shifted back to normal colour again, after 24 to 36 hours of extract soaking. The change of colour indicated the intrusion of extract into the body fluids of the shrimp as osmoregulatory processes try to balance the osmotic pressure of body fluids with its surrounding environment. The extract-penetrating body fluids will stimulate the immunity mechanism of shrimp. Discolouration in the shrimp is a sign that foreign substances are penetrating the body fluids, influencing the chromatophore, which is part of a shrimp’s immunity system^1,4. Colour change in crustaceans can be stimulated by many factors as a behavioural form for adaptation and protection^10–12.

Effects of challenge

At 2 days after the challenge test with V. harveyi, the activities and appetites of the shrimp were decreasing. Clinical symptoms on day 14 were increasingly apparent in negative controls. Clinical symptoms of the shrimp incubated with seawater extract were more apparent than other treatments, especially at concentrations of 400 ppm and 600 ppm. Those incubated with ethanol and distilled water extract appeared to be better than the positive control. Ethanol extract 800 ppm and 1,000 ppm and distilled water extract 1,000 ppm resulted in a better average activity of shrimp. Better appetite was also shown by shrimp in the culture medium with ethanol and distilled water extract 1,000 ppm, and the reflex response of shrimp was found better in the ethanol extract 1,000 ppm. However, all clinical symptoms in the shrimp were showing improvement until day 28, except for negative controls.

Clinical symptoms on shrimp challenged with Saprolegnia sp. began to appear on day 6, and shrimp appeared healthier than shrimp challenged with V. harveyi. In the negative control, legs were visibly dirty, a symptom that did not appear in treatments challenged with V. harveyi. Clinical symptoms on shrimp on days 14 and 28 in all extract treatments appeared better than antibiotic-added positive controls. Clinical symptoms that appeared on shrimp for all treatments are presented in Table 2. Shrimp infected with of V. harveyi exhibited symptoms of decreasing activity, weakening reflex responses and loss of appetite^1,13. Raw data behind each table is available on OSF¹⁴.

Pathological effects of challenge

Better anatomical pathology was shown by shrimp in all extract treatments when compared to negative controls. Some individual shrimp in negative controls challenged with V. harveyi experienced anatomical pathology issues, such as: incomplete organs; reddish carapace; broken rostrum; reddish and broken legs; swollen and gripped uropod; brownish, reduced, and disturbed hepatopancreas; and brownish, hard abdomen. Shrimp in negative controls challenged with Saprolegnia sp., showed a lighter anatomical pathology than those challenged with V. harveyi. The anatomical pathology of shrimp was indicated with reddish appearance in the body, legs, and uropod or with incomplete carapace, legs, and uropod. The hepatopancreas softened, and both it and the stomach changed brownish. Vibrio sp. causes gills to turn dull pale and reddish yellow and carapace dark reddish, pleopods and uropod to break, and hepatopancreas slightly reddish to dark red¹⁵. Vibriosis brings reddish black colour on shrimp, red spots on legs and uropod, haemorrhage in the body, and deformity and moulting failure¹³.

Based on clinical symptoms and anatomical pathology, shrimp culture in a medium with extract treatments showed a better physiological condition and resistance against for V. harveyi and Saprolegnia sp. infection, compared to negative controls. The positive control gave almost the same result as the ethanol and distilled water extract treatments. This evidence indicated that X. granatum leaf extract is supposed to be effective at preventing tiger shrimp from both V. harveyi and Saprolegnia sp. infection. Vibrio infection may occur in all phases of shrimp development, from egg to broodstock¹⁶. Vibriosis, especially caused by the luminous Vibrio, often brings about serious losses in shrimp hatcheries¹⁷. Vibrio species are abundant in a seawater environment, and some opportunistic pathogenic strains are associated with the immunity of cultured shrimp in unfavourable environmental conditions¹⁸.

Number of infectious colonies

The colonial density of V. harveyi (TVC) in shrimp soaked with leaf extract on the last day of the experiment ranged from 14.67×10³ to 22.67×10³ CFU/ml. This bacterial count indicated that leaf extract for all solvents at concentrations ranging from 400 to 1,000 ppm could inhibit the proliferation of V. harveyi better than negative controls with TVC of 25.67×10³ CFU/ml. The TVC value in the positive controls included in the value range of leaf extract treatments, which was 16.00×10³ CFU/ml. The strongest inhibition of extract against V. harveyi, consecutively, was distilled water extract at 1,000 ppm, distilled water extract at 800 ppm, ethanol extract of 800 ppm, and ethanol extract of 1,000 ppm, as presented in Table 3. The bacterial density of V. harveyi in water, sediment, and shrimp samples varied between 6.0×10³ to 88×10³ CFU/ml,1,200×10³ to 10,400×10³ CFU/g, and 5.0×10³ to 73 ×10³ CFU/ml, respectively¹⁹. Bacterial density of V. harveyi in hepatopancreas of 1.5-month-old tiger shrimp, 14 days after the challenge test with 10⁵ CFU/ml, was about 14.67 ×10³ CFU/ml.

The above facts indicated that X. granatum leaves extracted with distilled water and ethanol solvents, at concentrations between 800 to 1,000 ppm, were potentially antibacterial and able to inhibit V. harveyi growth better than antibiotics. All Vibrio isolates were found to be resistant to ampicillin, gentamycin, oxytetracyclin, chloramphenicol, trimethoprim, and kanamicin—the antibiotics commonly used in aquaculture²⁰. Crude ethanolic extract of X. granatum, in vitro at 400 ppm, could inhibit the growth of Staphylococcus epidermis, Staphylococcus aureus, Shigella boydii, Proteus spp., Escherichia coli, and Streptococcus pyogenes²¹.

Survival rates

The survival rate of shrimp incubated with extracts of X. granatum was better than the negative control, ranging from 53.33% to 78.67% following to V. harveyi infection, and 54.67% to 76.00% following to Saprolegnia sp. infection. The survival rates in the positive control was recorded as 78.67% and 77.33%, and in the negative control were 21.33%–29.33% (Table 4). The RPS of shrimp soaked with extract, in the negative controls, and in the positive controls ranged from 40.61% to 72.89%, from 35.84% to 66.12%, and from 67.97% to 72.98%, respectively. The highest RPS against V. harveyi infection was 72.89% obtained in treatments of ethanol extract at 1,000 ppm, distilled water extract of 1,000 ppm, and positive control, followed consecutively, by distilled water extract of 800 ppm 71.14%, ethanol extract of 800 ppm 69.39%, and seawater extract of 1,000 ppm 67.81%. The highest RPS against Saprolegnia sp. infection was in ethanol extract of 1,000 ppm (66.12%), followed by ethanol extract of 800 ppm (66.01%), distilled water extract of 1,000 ppm (65,90%), and seawater extract (64.27%) (Table 5).

The above results indicated that X. granatum extract had the potential to protect shrimp from both V. harveyi and Saprolegnia sp. infection, and, thus, may be applied to improve the survival rate of shrimp in captivity. Vibrio attacks may cause shrimp death from the larva to adult stage if reared in ponds²². The causative vibriosis generally is V. harveyi leading to mortality range of around 40–65%, while the causative agent for the failure of larval development in hatcheries is commonly Saprolegnia sp.

X. granatum has been utilized by the local community for food, animal feed, food preservatives, and traditional medicine. Mangroves are the best choice to isolate natural or bioactive products to challenge against bacteria and fungi²³. Substances extracted from mangrove may function as herbal remedies to treat various biological dysfunctions with minimal side effects but with maximum healing potential²⁴. Mangroves provide rich secondary metabolites, such as alkaloids, flavonoids, phenolics, steroids, and terpenoids. Natural phenols, alkaloids, and flavonoids have antioxidant, antibacterial, anti-tumour, and anti-viral properties^25–27. Flavonoids are synthesized by plants to respond to microbial infections, and, in vitro, these metabolites are effective antimicrobial substances against microorganisms extensively²⁸. Flavonoid and phenolic compounds from natural sources are known to be associated with a variety of biological activities, such as antioxidant properties, anti-inflammatory actions, and anticancer activities²³.