Acute Hepatopancreatic Necrosis Disease (AHPPND) control in Whiteleg shrimp ( Litopenaeus vannamei Boone ) used inulin source from Dahlia uber ( Dahlia variabilis Willd.)

. The effect of Inulin derived from Dahlia tubers as a functional diet on the growth of white legs shrimp was studied to see its impact on AHPND infection. The research was conducted in a fiber tank filled with 500 L of seawater sterilized with chlorine and stocked with 100 PL30 shrimps/container. Shrimp were treated with feed containing Inulin extract from dahlia tubers at a dose of 2.5%, 5.0%, and 5.0% commercial Inulin and compared with a control. Each treatment was given 2 replications. Shrimp were then challenged with Vibrio parahaemolyticus strain AHPND at a density of 10 6 CFU/mL. The live shrimp were then processed histopathologically to view the microanatomical gut structure (midgut). The results showed that the mean survival of the shrimp in the control and 2.5% Inulin treatment groups was 97.3 ± 1.15% and 97.3 ± 2.3%, respectively, and the 5% Inulin treatment and commercial Inulin were each 96.9 ± 2.7%. Histopathological observations on Inulin treatment of 2.5% dahlia tuber extract and commercial Inulin showed that shrimp intestine had more lactic acid bacteria abundance than control and 5% Inulin extract. In conclusion, shrimp treated with inulin can reduce the risk of shrimp being infected with AHPND.


Introduction
The acute hepatopancreatic necrosis disease (AHPND) outbreak is a frightening threat to shrimp farmers today.Mortality caused by AHPND can even reach 100%, so it is categorized as an extraordinary epidemic.AHPN disease resulted in the shrimp's death, which occurred at the beginning of rearing from the second week after stocking.Because of the death that occurs at an early age, the disease was initially known as Early Mortality Syndrome (EMS) [12].The results showed that shrimp infected with AHPND were characterized by the dominance of Vibrio bacteria in the intestines of shrimp.Compared to healthy shrimp, the intestinal community was more varied (seeds were highly associated with an increased risk of outbreaks).This is because the seeds that are stocked are already infected with the V. parahaemolyticus bacteria that causes AHPND [14] and immediately multiply in ponds and induce disease attacks.Bacteria can live in water, soil, or other carriers, which will then find the right medium for their growth [5].
Bacteria that are included in the feed cannot reach the hepatopancreas, due to the mechanical sieve gastric (gastric sieve) with a porous size of 0.2 -0.7 microns, and only small particles measuring 0.1 microns can pass through the filter and enter and accumulate into the stomach [9].Therefore, Vibrio bacteria cannot pass into the hepatopancreas but fill the intestines and produce toxins that allow entry into the hepatopancreas, damaging organic structures [5].
Subsequent research found that VP-AHPND expressing a toxin encoded by the plasmid Pirvp was homologous to Pir (a plasmid like that of Photorhabdus, which infects insects), a binary toxin.The binary toxin is formed from two subunits, namely PirAvp and PirBvp.Unlike other binary poison pears, PirB (PirBvp, a protein with a molecular weight of 50.1 kDa) V. parahaemolyticus triggers AHPND with the pathology of acute hepatopancreatic tubular damage, while PirAvp (a protein with a molecular weight of 12.7 kDa) only causes changes small histologically [3].Unlike other bacteria, which are generally opportunistic pathogens, V. parahaemolyticus AHPND is unique because colonization in the intestines of shrimp will secrete PirABvp toxin to the hepatopancreas organ, which is the target organ [2].
Prebiotics can be defined as a substrate or food ingredient that cannot be digested but selectively fermented by some colonic microflora, stimulating the growth and activity of bacteria that are beneficial to health [10].Compounds belonging to the prebiotic group include Inulin, fructooligosaccharides (FOS), isomalto oligosaccharides, Lactosucrose, lactulose, pyro1dextrins, soy oligosaccharides, trans1 galactooligosaccharides, xylo1oligosaccharides.However, in 2007, only two species met the prebiotic criteria, namely Inulin and trans-galactoligosaccharides (TOS) [11].
Inulin has been widely used as food ingredients for human health because it can prevent constipation, colitis, and cancer, just like soluble dietary fiber, which cannot be digested by shrimp digestive enzymes but can be fermented by microflora in the large intestine.Once consumed, inulin compounds reach the large intestine in a state of unchanged structure.However, Inulin is immediately fermented by the microflora in the large intestine.Inulin is fermented into short-chain fatty acids, and lactic acid is produced from several specific microflora.Such conditions lower the degree of acidity (pH) in the large intestine.Lactic acid in the large intestine stimulates intestinal peristalsis.
Sources of natural Inulin are very diverse, ranging from grass families to tubers.The chemical compound inulin found in plants is stored as a carbohydrate reserve, especially in plants of the Compositae and Graminae families.Until now, only a few plants have been reported to have high inulin content, such as Brambang utan (Pancratium zeylanicum) of 3.65 mg/gram, Brojol onions (Crimum asiaticum) of 1.32 mg/gram and reeds (Imperata cylindrica) of 1.27 mg/sample.Several other types of plants can be a source of Inulin, the parts that can be utilized are fruit, stems and tubers, with inulin content (% fresh weight) are Banana Fruit 0.3 -0.7, Wheat Cereal 0.5 -1 Onion Bulbs 2 -6, Scallion Bulbs 3 -10, Garlic Bulbs 9 -16 [8].
Applying Inulin to shrimp can increase shrimp's immune response and defense against WSSV [15].Treatment of larvae with inulin-enriched artemia nauplii resulted in significantly higher survival at PL than without inulin treatment [4].The results of this study indicate that the administration of Inulin can improve life in the seed stage, which may be due to the increased responsiveness of the larvae.
The aim of this activity was to observe the effect of inulin administration on the intestinal structure of shrimp, and the resistance of shrimp to infection with the bacterium Vibrio parahaemolyticus.

Materials and tools
Dahlia tubers were obtained from farmers, washed, and peeled, then chopped thinly.After mixing the chopped tuber in a 1:1 ratio with water, a kitchen blender was used to turn the mixture into sludge.The sludge was heated to 80°C by using a heater and stirrer for 30 minutes, after which it was filtered through a filter of mesh 60.The filtrate was then stored in the refrigerator for 3 days.The white precipitate was collected and dried using an oven (60°C) for 48 hours, followed by flouring using a kitchen blender and stored in airtight bottles as research material.Commercial inulin was purchased from the online shop.Vanname post larva 30 (PL-30) was obtained from commercial hatcheries in Jepara Regency.12 units of 750-liter conical glass fiber containers filled with 500 liters of sterile seawater were used as research containers.Each container was filled with 100 shrimp seeds.The dose of inulin used is 0% (Inu-1), 2.5% (Inu-2), 5% (Inu-3), 5% commercial Inulin (Inu-4).Inulin according to the dosage is mixed with pellets using progol as an adhesive.Shrimp rearing was carried out for 45 days.

Measurements
Measurement of shrimp growth, and shrimp mortality, is done every week.Challenge test for infection with Vibrio parahaemolyticus AHPND bacteria with a density level of 106 /mL, and observed the condition of the shrimp at 3, 6, 12, 24, 36, 46, 72, and 96 hours after treatment with Inulin.At the end of the study, 5 prawns were dissected, and their intestines were homogenized and implanted in MRS media to see lactic acid bacteria growing in the intestines.Five prawns were taken for Davidson fixation and then histology was carried out using the histology method, Hematoxylene Eosin staining to see the microanatomical structure of the shrimp intestine in the midgut section.

Observation
Bacteriological observations were made at the end of the test by observing the abundance of Lactobacillus bacteria in the shrimp intestines.Histological, looking at the condition of the intestine at the end of the test, by observing the attachment of bacteria to the intestinal pili.Shrimp growth and Survival Rate are carried out every two weeks.Observation of water quality is carried out daily and weekly, growth measurements are carried out weekly.

Analysis
Test total bacteria and lactic acid bacteria in the intestine, survival, growth, challenge test performed Anova analysis followed by Duncan's test to see the difference in treatment.

Vanname shrimp survival and growth
The survival rate of shrimp in the Inulin treatment from 2.5% tubers and the control showed high survival, higher than the Inulin treatment from 5% tubers and 5% commercial inulin, which is more than 96%.Statistical test results showed that the treatment with 2.5% Inulin and the control was not significantly different, while the 5.0% Inulin and 5.0% commercial Inulin were significantly different from the control and 2.5% Inulin (Table 2).In general, the survival rate for all treatments was still high, indicating good rearing conditions, and the shrimp had a high survival rate.Remarks: Inu-1 = 0 % inulin; Inu-2 = 2,5% inulin tuber; Inu-3 = inulin 5% tuber; Inu-5 = Inulin commercial 5%; ns=not significant at p<5%

Total bacteria and Lactic Acid Bacteria (LAB) in the shrimp intestine
Observations on the content of Lactic Acid Bacteria (LAB) and shrimp varied between treatments.Observation of BAL using MRS media and 1% CaCO3 was added to the media.If there is growth of LAB, then there is a reaction of lactic acid produced by LAB with CaCO3 to form calcium lactate which dissolves in the MRS medium, resulting in a clear/transparent color (Figure 1).The observation results showed that treatment with Commercial Inulin showed the highest LAB content, namely 5.2 x 10 2 CFU/mL, followed by 2.5% Inulin treatment of 3.2 x 10 2 CFU/mL.In the 5% Inulin treatment, the control was 0.6 x 102 CFU/mL, and the smallest was the 5% Inulin treatment containing 0.1 x 10 2 CFU/mL.

Challenge test with VP-AHPND bacteria
The challenge test was carried out by means of shrimp treated with Inulin after the test period and was then challenged with VP-AHPND bacteria at a dose of 10 6 CFU/mL.The test results showed that the control shrimp experienced 80% mortality at 48 hours after exposure, while the shrimp treated with either 2.5% Inulin tuber, 5% Inulin tuber, or 5% Commercial Inulin did not experience death.The results of the challenge test showed that Inulin was able to increase the content of lactic acid bacteria in the shrimp intestine, thereby inhibiting the growth of V. parahaemolyticus AHPND bacteria in the intestine, thereby preventing bacterial colonization and production of PyrAB toxin and preventing shrimp death.Remarks: Inu-1 = 0 % inulin; Inu-2 = 2,5% inulin tuber; Inu-3 = inulin tuber 5%; Inu-5 = Inulin commercial 5%

Histological observation of microanatomical structures
An overview of the microanatomical structure of the shrimp intestine in both the untreated and inulin-treated groups is presented in Figure 2, the microanatomical structure of the intestine, and Figure 3.The structure of the hepatopancreas.Histopathological change in hepatopancreas could be a sign specific to AHPND.AHPND is characterized by massive sloughing of tubule epithelial cells of the hepatopancreas (HP) [16].Three stages (initial, acute, and terminal) have been commonly reported as part of the pathogenic course of AHPND [14,17].
During the acute phase, the reduction of vacuole and elongation of tubule epithelial cells followed by massive sloughing of epithelial cells inside the tubular lumen could be a sign of the acute stage [14,17,18].In this research, a sloughing of the hepatopancreatic tubule is observed.According to histopathological studies, the hepatopancreas of shrimp treated with Inulin seems normal, while that of control shrimp has slough tubule epithelial cells.Shrimp received Inulin also show normal midgut epithelial, possibly due to the vibrio population retarded by lactobacillus in the midgut.Based on data in this research, lactobacillus content in the shrimp intestine is more abundant compared to the control.

Water quality
The water quality during the research showed that the water quality parameters were still in the feasibility category for shrimp farming.The water temperature is still above 26°C, the average oxygen level is above 6.7 mg/L, the total ammonia nitrogen is below 1 mg/L, and the average salinity is more than 27 ppt.The condition of the research water quality is not a problem for the life of the reared shrimp.Water quality observation data is presented in Table 4.The water temperature has dropped to 22°C, but in general, the temperature is still above 26°C.Vanname shrimp are relatively resistant to low temperatures, unlike tiger prawns, which are sensitive to low temperatures.Oxygen is also still very feasible to show normal shrimp metabolism, which is more than 6.2 mg/L.The minimum safe limit for oxygen levels for normal shrimp growth is 4 mg/L.
Water quality seems to have no effect by inulin treatment.Almost no difference between water quality parameters among treatments compared to the control.All observed parameters are considered optimal for shrimp growth.
Inulin has the opportunity to be a prebiotic material candidate that can be used to increase the responsiveness of shrimp to bacterial infections that cause AHPND.The results of this study indicate that shrimp treated with Inulin is likely without AHPND attack, possibly due to abundance of Lactobacillus in shrimp intestine.There is a negative correlation between V. parahaemolyticus bacteria and relatively varied gutdwelling bacteria.The results of this study follow the results of previous studies found that several types of intestinal bacteria, such as Streptomyces, Roseobacter, and Ruegeria show a negative relationship with Vibrio, so they have the potential as probiotics that enhance shrimp health [1,4].Considering that until now, AHPND has become a frightening disease for farmers, Inulin is applied at the beginning of rearing by providing inulin-enriched feed.Giving Inulin will enrich the shrimp gut microbiome and stimulate the formation of a BAL-type bacterial population so that it becomes a competition for Vibrio bacteria, especially Vibrio parahaemolyticus AHPND.Inulin also increases vanname shrimp phenoloxidase activity and reduces the risk of lethal WSSV attacks [7].Antibiotic administration has not had satisfactory results for the control of AHPND.It will even trigger the formation of antibioticresistant bacteria.However, for use on a production scale, more intensive studies are needed on a mass scale to see the efficiency of Inulin.

Conclusion
The study results can be concluded that Inulin derived from Dahlia tubers, which are given at the beginning of vannamei shrimp farming, can reduce the risk of shrimp being infected with AHPND.Shrimp that received inulin treatment showed healthier hepatopancreas than the control.

Fig. 1 .
Fig. 1.Vibrio bacteria in shrimp gut treated with inulin tuber of 2.5%, 5.0% and 5% commercial Inulin from feed grown on TCBS media (left).The yellow colonies are V. alginolyticus, the green ones are V. parahaemolyticus bacteria.B. Lactic acid Bacteria (LAB grown on MRS media.Colony with clear zone is LAB.

Table 1 .
Survival rate, final shrimp weight, final shrimp length, and weight gain on inulin testing.

Table 2 .
Total bacteria and lactic acid bacteria in the intestines of treated shrimp

Table 3 .
Observation of shrimp mortality after challenge test with Vibrio parahaemolyticus AHPND

Table 4 .
Water quality during research