EFFECT OF PROBIOTIC AND SUPPLEMENTED FEED ON GROWTH, SURVIVAL AND DISEASE RESISTANCE OF WHITE SHRIMP LITOPENAEUS VANNAMEI POSTLARVAE

nauplii and supplemented feed produce better survival and growth when compared to that of other concentrations and control group. The specific growth rate was found to be maximum in postlarvae fed with B. megaterium enriched Artemia and supplemented feed when compared to the control group. The biochemical constituents, such as protein, lipid and carbohydrate contents were considerably more in PL fed with both feeds, particularly at 5×10 9 cfu cells. In the challenging study, results showed a higher survival rate in postlarvae fed with enhanced Artemia nauplii and supplemented feed of E2. Therefore, it is evident that 5×10 9 cfu cells of B. megaterium can be considered as an appropriate concentration for the growth and survival of L. vannamei postlarvae.


INTRODUCTION
Globally, aquaculture regarded as the fastest-growing food production technology with high production for several decades (Das et al., 2008;Edwards et al., 2019). In 2016, around 89 per cent of the output originated in Asia, primarily China, which attracts greater attention from global aquaculture production since 62 per cent occurred in China. After China, India takes the second position concerning annual fisheries and aquaculture production. Hence, the country promoting its aquaculture practices for more shellfishes and crustacean's production. In 2017, fish production is expected to reach 12.60 million metric tonnes, with about 65 % coming from the inland sector and about 50% coming from cultural fisheries (FAO, 2018). Aquaculture is a vital source of food, stock augmentation, employment, and profit for millions of people worldwide (Murillo- Gurrea et al., 2001). Many microbiological pathogens, including Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vulnificus, have been weakened in shrimp farming as a result of infectious disease outbreaks (MartinezPorchas and MartinezCordova, 2012). Because of its excellent survival rate, quick development in intensive culture systems, and disease resistance, the Pacific white shrimp Litopenaeus vannamei is widely farmed across the world. With about 3.8 million metric tonnes generated in 2015, intensive culture of Pacific white shrimp Litopenaeus vannamei represents substantial aquaculture operations in terms of production value and a high-value commodity (FAO, 2016). Bioencapsulation is a technology that is currently being explored for the oral administration of carotenoids, chemotherapeutic vaccines, pigments, sterols, vitamins, and vital polyunsaturated fatty acids (PUFAs) via Artemia nauplii. The larval stages of L. vannamei exhibit complicated trophic alterations during development, particularly for penaeid. The phytoplankton-feeding zoea and carnivorous mysis stages come after the non-feeding naupliar stages. The latter stage is particularly reliant on a consistent supply of live food, which is frequently provided in hatcheries as rotifers and Artemia nauplii. Artemia is an important live feed used in the production of shrimp larvae. They are continuous filter feeders that eat suspended particles and are not selective. These qualities allow one to increase their nutritional profile by immersing them in a solution rich in nutrients such as docosahexaenoic acid (Sorgeloos et al., 1986). The main role of aquaculturists is to provide organisms suited for the feed size to the initial feeding stage, as well as an acceptable quantity of feed organisms to ensure greater survival and quicker growth (Arulvasu and Munuswamy, 2009). Disease in shrimp cultivation was induced by a variety of biotic and abiotic causes. Farmers mostly use antibiotics as a preventative or therapeutic measure to combat illness (Hoseinifar et al., 2017). Antibiotics not only disrupt the natural flora of shrimp intestinal tracts but also render diseases resistant to medications; hence, probiotics are the most promising alternative to antibiotics (Tripathi and Giri, 2014). Because of the importance of water quality, nutritional absorption, the immune system, and the survival and growth rates of hosts, probiotics play a key role in aquaculture. Bacillus bacterium strains have produced extremely good outcomes in shrimp farming. This bacterium is a non-infectious Gram-positive sporeforming bacteria that has been utilized to ameliorate the health of shrimp (Keysami et al., 2012). Vibrio harveyi is a scintillating species commonly obtained from marine sources, has been identified as harmful to fish and various crustaceans, most notably Penaeus species. (Lavilla-Pitogo et al., 1990). The quest for sustainable, ecologically friendly aquaculture is driving up research into probiotics for marine creatures. The commercial source of probiotic dietary supplementation provided a superior growth, immuno-physiology of Litopeneaus vannamei than the indigenous source of Bacillus species (Abdollahi-Arpanahi et al., 2018). Several research have been undertaken to investigate the effect of various feeding regimens and additives on shrimp growth performance and immunological state (Bowyer et al., 2019). Since probiotics have been shown to promote host organism growth, nutrition, and survival. The purpose of this study was to see how enriched Artemia nauplii with the probiotic bacteria Bacillus megaterium and supplemented diet affected growth, survival, and disease resistance in postlarvae of white shrimp Litopenaeus vannamei.

Experimental animal and Probiotic strain
Litopenaeus vannamei postlarvae were acquired from Royal hatcheries, Chennai, Tamil Nadu, India. The postlarvae were transported in plastic bags containing 5 ppt seawater to the experimental setup kept at our laboratory. Before the trials, postlarvae were cultivated in laboratory tanks filled with aerated seawater at room temperature for 2-3 days. Bacillus megaterium was selected as the probiotic, which was obtained from the Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, Chennai, Tamil Nadu, India. The probiotic strain was grown in Tryptone soy broth (TSB) using a shaking incubator at 30˚C for 24 hours. Using a spectrophotometer, the cell densities of the suspensions were calculated at 600nm.
The present investigation was focused on the effect of Bacillus megaterium enriched to the Artemia nauplii and sprayed on the supplemented feed was fed to the white shrimp Litopenaeus vannamei postlarvae (PL) to obtain high survival and growth. Artemia nauplii enriched with different levels of B. megaterium (5×10 7 , 5×10 9 and 5×10 11 cfu /mL) fed to postlarvae for a period of 30 days. Similarly, supplemented feed with probiotic of different levels fed to PL for a time period of 15 days. Postlarvae fed with 5×10 9 cfu cells (E2) of B. megaterium enriched Artemia nauplii and supplemented feed produce better survival and growth when compared to that of other concentrations and control group. The specific growth rate was found to be maximum in postlarvae fed with B. megaterium enriched Artemia and supplemented feed when compared to the control group. The biochemical constituents, such as protein, lipid and carbohydrate contents were considerably more in PL fed with both feeds, particularly at 5×10 9 cfu cells. In the challenging study, results showed a higher survival rate in postlarvae fed with enhanced Artemia nauplii and supplemented feed of E2. Therefore, it is evident that 5×10 9 cfu cells of B. megaterium can be considered as an appropriate concentration for the growth and survival of L. vannamei postlarvae.

Artemia Cyst Collection and Hatching
Artemia cysts were collected from (100 µm scoop net) Kelambakkam saltpan, Tamil Nadu, India. Cysts were hatched using the standard procedures adopted from previous publications (Sorgeloos et al., 1986). Artemia cyst of one gram was hydrated in freshwater for a period of one hour in a beaker with vigorous aeration. The cysts were collected after one hour and rinsed with tap water and then transferred into the decapsulating solution (4% sodium hypochlorite). After 5 to 10 min, the entire cysts turned to an orange-pink colour indicating decapsulation. The cysts were collected in a 100 µm sieve and rinsed with fresh water to remove all traces of the hypochlorite solution. The decapsulated cysts were transferred to 1litre seawater (salinity 30 ppt) and vigorously aerated for 24 hours. The hatched nauplii were siphoned out by exposing them to light. The nauplii were washed completely and used for the enrichment trials ( Figure 1A -E).

Enrichment Procedure
Enrichment of the second instar nauplii was carried out by the standard procedure of Sorgeloos and Kulasekarapandian (1984). First instar Artemia nauplii appeared after 24 h incubation. Cell suspensions of B.megaterium were enriched after 12 h incubation. The second instar stage Artemia nauplii were segregated from the container through a 120 μm sieve and moved to a glass beaker at a mass of 200,000 nauplii / L of seawater. The three different concentrations of Bacillus megaterium, 5 ×10 7 cfu / mL (Experiment 1), 5×10 9 cfu / mL (Experiment 2) and 5 ×10 11 cfu / mL (Experiment 3). After 6 hours of enrichment period, the Artemia nauplii were picked and rinsed with seawater. Unenriched Artemia nauplii served as control. The Artemia nauplii were reaped from the enrichment glass containers. They were washed rigorously with fresh water and stored for further use. Bacillus strain present in the gut of transparent nauplii was observed under a light microscope ( Figure 1F).

Experimental setup
Experiments were accomplished by dividing the Litopenaeus vannamei postlarvae into four groups such as Control, Experiment 1(E1), Experiment 2 (E2) and Experiment 3 (E3). A total count of 180 postlarvae individuals with a weight of 0.58 ± 0.27 mg on average were randomly divided into 12 tanks with 15 individuals per tank placed. Three replicate groups of postlarvae were fed with unenriched and enriched Artemia nauplii.

Preparation of the experimental diet
Pellet feed obtained from Central Institute of Brackishwater Aquaculture, Indian Council of Agricultural Research, Chennai, Tamil Nadu, India. After 24 h culture, the probiotic bacteria were collected by centrifuge at 10,000 rpm for 15 minutes. The cells collected were washed and re-suspended in phosphate-buffered saline (PBS pH 7.4). The prepared suspension containing probiotic bacteria sprayed uniformly over the pellet. Then feed was dried and stored at 4°C. Experimental diets E1, E2 and E3 supplemented with Bacillus megaterium were 5 ×10 7 cfu, 5×10 9 cfu and 5 ×10 11 cfu cells and control (without probiotic) were prepared once every 15 days.

Experimental Design and Feeding Schedule
After completion of live feed enrichment studies, same postlarvae were used for the supplemented feed experiment. Experiments were accomplished by divided the animals into four groups such as Control, E1, E2 and E3. A total of 40 postlarvae individuals with a weight of 0.17 ± 0.005 on average were randomly divided into 8 tanks with 5 individuals per tank were placed. Feed was given at a ratio of 50% of the postlarvae body weight. The daily ration was split into three equal parts and was fed at 10:00 h, 14:00 h and 18:00 h. Excess feed was removed daily. Residuals removal and seawater exchange was done every day.

Growth parameters
Growth of Litopenaeus vannamei postlarvae fed with enriched Artemia and probiotic supplemented feed of different concentrations of Bacillus megaterium was intended using the formula given by Nimrat et al. (2011). The shrimps' postlarvae of each tank were weighed individually and measured to calculate the growth parameters.

Biochemical analysis
The biochemical constituents, comprising protein, lipid, and carbohydrate, were assessed according to protocol. The estimation of protein, lipid and carbohydrates from all experimental groups was quantified using the methods of Bradford (1976), Barnes and Blackstock (1973) and Roe (1955).

Challenge study
Postlarvae were exposed to a virulent strain of Vibrio harveyi (Sritunyalucksana et al., 2005) for 10 days. Ten PL were added to aerated seawater that contained 1.4 × 10 7 cfu/mL of V. harveyi. During the first 24 hours, there was no water exchange. Post larvae were fed as normal during the challenge experiment. After 10 days, the number of survivals were counted. After the 15-day feeding experiments, three shrimp per tank were challenged. The data is displayed as a relative survival percentage (rps).

Statistical Analysis
One-way analysis of variance was used to conduct statistical analyses of the data (ANOVA). Statistical analyses of the data were carried out, using a one-way analysis of variance (ANOVA). Differences were deemed to be significant when p < 0.05. Mean ± standard deviation of the data is displayed.

RESULTS
The present experiment aimed to analyse the growth, survival and disease resistance of Litopenaeus vannamei postlarvae fed with different concentrations of probiotic-enriched Artemia nauplii and supplemented feed.

Growth and survival of shrimp post larvae
After the 30-day experiment, the growth parameters of shrimp PL in different concentrations of probiotic and control, such as initial length & weight, final length & weight, specific growth rate and survival rates are shown in Table 1. The initial average body length and total body mass of the PL was 5.47mm and 0.58 mg respectively. Initial weight and length did not differ much between experiments (p>0.05). Also, the final lengths of the shrimp PL were 20.98, 23.68, 25.22 and 21.14 mm respectively. The final weights of the shrimps PL were 19.0, 24.8, 27.0 and 22.1mg in control, E1, E2 and E3 respectively. Statistical analysis revealed that shrimps fed with E2-enriched Artemia nauplii grew noticeably more than the control group (p<0.05) at the conclusion of the trial. Additionally, significant (p<0.05) differences between the E2 group and control were noted as SGR percentage variances. There were no discernible variations in SGR between the E3 group and control (p>0.05).The survival rates were found at 71.11, 84.44, 93.33 and 80.00 % in control, E1, E2 and E3 respectively. However, the survival rate of the shrimp PL fed with the experimental group was significantly different (p<0.05) from that of the control group. Similarly, after 15-days completion all the growth indicators measured in the experiment were significantly different (p<0.05). The effects of probiotic B.megaterium sprayed pellet feed in L.vannamei postlarvae are presented in Table  2. The present investigation is the consecutive process of the previous experiment.
Since the same PL utilized in this experiment, the initial average length and weight showed a substantial variation between the groups (p<0.05). The final length of shrimp PL were 31.32, 33.01, 37.30 and 32.00 mm in control, E1, E2 and E3. The data were statistically significant (p<0.05) between experimental groups at various doses compared to the control group. Similarly, weight gain in L. vannamei post larvae fed with probiotic supplemented feed varied significantly (p<0.05) compared to the post larvae fed with pellet feed. The post larvae of L.vannamei fed with probiotic supplemented feed attained maximum weight in E2 followed by E3 and E1 were 48.10, 32.41 and 31.52 mg respectively, whereas control showed comparatively less weight of 25.11 mg. The highest specific growth rate found in E2 group followed by E1 and E3 were 3.31, 1.82 and 1.70 % respectively. In comparison to the control and other groups, the E2 probiotic supplemented group demonstrated a significant difference (p< 0.05). Compared to the group provided control feed and the probiotic-supplemented feed had a greater survival rate. In probiotic supplemented feed survival rate were ranged from 95.00, 100.00 and 90.00% in E1, E2 and E3 respectively. The highest survival rate of 100% was recorded in shrimp's PL fed with probiotic supplemented feed while the control feed exhibited the survival rate of 80%.

Biochemical parameters of shrimp PL
The results obtained with biochemical constituents such as protein, lipid and carbohydrate in Litopenaeus vannamei postlarvae enriched Artemia nauplii with various concentration of probiotics are given in Figure 2A. Protein content in postlarvae of E1 (5 × 10 7 cfu), E2 (5 × 10 9 cfu) and E3 (5 × 10 11 cfu) of probiotic were observed to be 57.40 mg/g, 63.40 mg/g and 56.30 mg/g respectively. The results showed a moderate difference in enriched groups compared to that of control (55.40 mg/g). The total lipid content of post larvae in experimental groups such as E1, E2 and E3 were observed to be 9.20, 11.50 and 8.09 mg/g respectively. However, lipid content was moderately increased in the E2 group than that of the control and other experimental group. The carbohydrate content in postlarvae fed with enriched Artemia in E1, E2 and E3 concentrations was recorded as 12.78, 16.32 and 12mg/g. However, the carbohydrate content was less in the unenriched group. Maximum carbohydrate content was recorded in E2 (5 × 10 9 cfu) of enriched Artemia fed to postlarvae followed by the other concentration, such as E1 (5 × 10 7 cfu) and E3 (5 × 10 11 cfu) compared to unenriched Artemia fed postlarvae group. Among all groups, the E2 is statistically significant group p<0.05. Artemia nauplii enriched with 5 × 10 11 cfu cells. B-Control: pellet feed without probiotic, E1: probiotic supplemented feed of 5 × 10 7 cfu, E2: probiotic supplemented feed of 5 × 10 9 cfu and E3: probiotic supplemented feed of 5 × 10 11 cfu cells.
After 15 days of feeding experiments, the results obtained with biochemical constituents of Litopenaeus vannamei postlarvae fed with probiotic supplemented feed are shown in Figure 2B. Protein content in L. vannamei PL of E1, E2 and E3 of probiotic supplemented feed were observed as 63.21, 69.46 and 62.12 mg respectively. Protein content among the probiotic supplemented feed groups showed moderate difference compared to that of control. The total lipid content of L. vannamei in experimental groups such as E1, E2 and E3 were observed as 10.13, 12.35 and 9.03 mg respectively. The variation present in the experimental groups is statistically significant (p < 0.05). However, lipid content was moderately increased in the E2 group compared to that of control and other experimental groups. The carbohydrate content of L. vannamei PL fed with probiotic supplemented feed sprayed with probiotic of different concentration of E1, E2 and E3 was recorded as 13.81, 17.84 and 13.41 mg respectively. However, the carbohydrate content in control was 12.50 mg compared to that of probiotic supplemented feed. In the biochemical analysis, the protein content in the probiotic supplemented feed is statistically significant when compared to the other two biochemical content of the same experiment. The changes in the biochemical levels in postlarvae fed with enriched Artemia and probiotic-supplemented feed were moderately increased in the E2 group compared to that of control group and other experimental group.

Challenge study
After 30 days, the 10-day challenge test against V. harveyi was undertaken ( Figure  3A). The relative percent of survival (RPS) in the E2 group considerably outperformed the control (p < 0.05). No significant difference in survival rates was observed among control, E1 and E3 groups when PL were challenged with V. harveyi (p > 0.05). The survival rate of shrimp's PL against V.harveyi from 10 hours challenging study was conducted after 15 days ( Figure 3B). The probioticsupplemented feed showed a higher level of survival (p>0.05) than the group fed the control feed after 10 hours of the experimental challenge with V. harveyi. No significant difference in survival rates was observed between E1and E3 groups.   Venkat et al., 2004).

DISCUSSION
The current study demonstrates that shrimp PL fed with B. megaterium enriched feed saw increased levels of growth and survival, with improved growth performances seen compared to controls. Similar outcomes were shown in shrimp fed diets containing B. subtilis L10 and G1, which had greater survival rates of 100 and 95.5%, respectively, than the control, which had a survival rate of 86.5% (Zokaeifar et al., 2012). In the current study, post larvae survival significantly increased, which may be attributable to Bacillus' capacity to outcompete other hazardous bacteria. In this study, E1, E2, and E3 supplemented groups outperformed controls in terms of growth and survival rate. Similar outcomes were attained when Tilapia Oreochromis niloticus was supplemented with the probiotic Bacillus coagulans (Wang et al., 2008). The stress tolerance, survival rate, immunological responses, and disease resistance of aquatic animals were all improved by the addition of Bacillus probiotics. Additionally, B. licheniformis supplementation increased resistance to V. parahaemolyticus infection (Gao et al., 2018). (Gao et al., 2018). Similar results obtained when V. harveyi injected to the post larvae L.vannamei fed with probiotic supplemented feed showed a significantly higher level (P<0.05) of survival rate than control group. Higher survival of shrimp fed probiotic-supplemented feed may be attributable to the host's immunological response to the probiotics. In our study, bioencapsulation and supplementation of a B.megaterium was shown to have positive effects on growth indices of L.vannamei PL. Among the experimental groups, PL fed with probiotic in E2 had improved weight, SGR and survival. These results are consistent with the specific B. megaterium concentration in shrimp PL, which led to a better survival rate following V. harveyi challenge.

CONCLUSION
The potential probiotic Bacillus megaterium enriched Artemia nauplii and supplemented diet enhance the growth, biochemical composition and disease resistance of L.vannamei postlarvae. Therefore, our study suggests that Bacillus megaterium can be utilised as a feed enhancer for enriching both live and supplemented diets in order to increase white shrimp, Litopeneaus vannamei postlarvae growth and production.