Effect of Methanol Extracts of Arthrospira platensis on Survival and Increased Disease Resistance in Litopenaeus vannamei against Vibriosis

Vibriosis is a common bacterial infection in shrimp that causes mortality in hatcheries and farms. Various steps have been initiated to increase the resistance against bacterial pathogens and decrease the mortality rate through improved culture conditions and feed. Arthrospira platensis (Spirulina), a blue-green alga, is a good source of protein and other nutrients and helps to improve digestion. The effects of the methanol extract of A. platensis on the survival rate and resistance against vibriosis were studied. The minimum inhibitory concentration of the extract for Vibrio species and in vivo antibacterial screening were investigated using Litopenaeus vannamei . Vibrio alginolyticus was inhibited with 2000 µg mL -1 extract and the other two species were inhibited by 1500 µg mL -1 extract. Furthermore, the mortality rate and antioxidant enzyme levels of shrimps injected with pathogens reduced and increased after treatment with the methanol extract, respectively. The survival rate of V. parahaemolyticus and V. harveyi -challenged shrimps were 33.3% and 50%, respectively, after 168 h. The survival rate of V. alginolyticus-infected shrimp reduced (16.6%) 168 h after injection. All surviving shrimp developed resistance to Vibrio pathogens. This study indicated that the bioactive compounds in A. platensis could not only effectively prevent bacterial infection, but also serve as eco-friendly and cost-effective immune stimulants.


INTRODUCTION
White shrimp (Litopenaeus vannamei) is a major shrimp species farmed worldwide for food.These shrimps are susceptible to bacterial and viral diseases leading to huge economic loss. 1 Vibrio species, comprising more than 80% bacterial population in seawater, are gramnegative bacteria causing vibriosis in shrimps. 2 It is a common bacterial infection in L. vannamei, which causes acute hepatopancreatic necrosis syndrome (AHPNS). 3Vibrio harveyi, Vibrio fluvialis, Vibrio parahaemolyticus, Vibrio damsel, Vibrio vulnificus, and Vibrio alginolyticus are the other major Vibrio species causing vibriosis. 4They are opportunistic pathogens and infect shrimps under stressful conditions such as salinity and temperature variations.Additionally, vibriosis occurs due to the combination of physical stress and primary infection by other bacterial or viral pathogens. 1Environmental stress triggers diseases leading to 100% shrimp mortality.Antibiotic treatment to control pathogens has developed antibiotic resistance, 5 leaving antibiotic residues such as parent drug components and metabolites. 6Phytochemical and natural compound use has been advocated to overcome this problem, and there is great interest in the search for novel components to control pathogens.Moreover, owing to inefficient management of the virulent Vibrio strains through antibiotics and the consequent antibiotic residue development, farmers are forced to use probiotics and bioactive compounds. 7Additionally, these compounds will enhance shrimp growth and stimulate the immune system.Thus, the immunostimulatory, antibacterial, and antiviral effects of several algal extracts in shrimp have been studied.
This study uses Arthrospira platensis (Gomont), commonly known as Spirulina, is a bluegreen filamentous alga with numerous therapeutic and nutritional benefits.It is rich in protein, essential fatty acids such as monounsaturated and polyunsaturated fatty acids, amino acids, minerals, fibers, and vitamins and have antimicrobial and antioxidant properties. 8Expensive fishmeal is forcing the farmers to look toward single-cell protein sources such as A. platensis as a promising substitute feed or feed additive for cost-effective shrimp production. 9Several reports have focused on the effect of A. platensis in other shrimps and fishes in improving digestion and increasing immunity against various pathogens. 10Hot water extracts of A. platensis administration in L. vannamei has increased immunity against vibriosis 11 and gene expression upregulation after pH stress. 12However, no reports have focused on the effect of the methanol extract of A. platensis on controlling vibriosis.

Extract preparation
Commercially available A. platensis (200 g, Herbolina, India) was soaked in 2 L methanol and stirred 3 times a day for 7 days.Then, the extract was filtered and the solvent was removed using a rotary evaporator under reduced pressure at 45±5°C until dry residue was obtained.The extract was transferred to airtight glass bottles and stored at 4°C until further use.

Bacterial culture
V. alginolyticus, V. harveyi, and V. parahaemolyticus strains maintained in the laboratory were used for this study.Pure cultures were prepared and inoculated into trypticase soy broth for 24 h.The bacterial pellets obtained using centrifugation at 112 xg for 5 min were resuspended in isotonic sodium chloride solution to obtain different CFU mL -1 according to necessity.

Antibacterial activity
The minimum inhibitory concentration (MIC) was detected in microplates using broth dilution method. 13Briefly, 2500, 2000, 1500, and 1000 µg mL -1 extract were mixed with 1x10 8 CFU mL -1 bacterial culture prepared using broth dilution method in trypticase soy broth using 0.5 MacFarland standard.Gentamycin (50 µg mL -1 ) was used as a control.The lowest extract concentration that completely inhibited visible bacterial growth was considered the MIC.For determining the minimum bactericidal concentration (MBC), 20 µL test broth was re-inoculated into trypticase soy agar and incubated at 37°C for 24 h.The minimum concentration that reduced the colony count to 99.9% was considered as the MBC.

Experimental animal collection and maintenance
L. vannamei were collected from a local hatchery (Best Aqua Star Shrimp Hatcheries, Villupuram, India) and maintained in 1000 L fiberglass tanks containing seawater with airlift biological filters at 27-30°C and 20±1 ppt salinity.For each experimental trial, 12 shrimp were transferred into a 30L capacity tank (plastic) with proper aeration, and 10% water volume was exchanged daily.The animals were initially fed commercially available feeds, and the extracts were administered along with the feed during the experiments.The salinity was measured at regular intervals using a refractometer (Weswox hr-05, India).All applicable international, national, and institutional guidelines for animal care and use were followed.

In vivo antibacterial activity
A. platensis extract (20 mg) was dissolved in 10 mL Tris-EDTA buffer (pH 7.4) for preliminary in vivo antibacterial activity assay. 14Equal volume (10 µL) extract and V. alginolyticus, V. harveyi, and V. parahaemolyticus suspensions were mixed separately and incubated at 37°C for 3 h.The test mixture diluted in normal saline (200 µL) was intramuscularly injected in 6-8 g healthy animals at the sixth abdominal segment.Control shrimps were administered normal saline and the general health and bacterial infection status of all animals were monitored for 168 h.The survival rates of pathogen-challenged and control shrimp were calculated using the following formula:

Challenge test
The shrimps were fed with commercially available diets (Bay White -Vannamei Shrimp Feed, India) for four weeks and further fed 2500 µg g -1 methanol extract of A. platensis.The shrimp were challenged by injecting 1x10 8 CFU ml -1 of the three Vibrio species separately into the ventral sinus of the cephalothorax.The survival rate was recorded every 24 h for168 h.The control shrimp were fed the extracts and injected with 1 mL normal saline.

Antioxidant response determination
The shrimp were dissected, and 100 mg tissue was separated and crushed using a homogenizer in tubes containing 1 mL 50 mM phosphate buffer (pH 7.0).The homogenate was centrifuged at 10,000 rpm for 10 min at 4°C.The supernatant was removed and used for the catalase and superoxide dismutase (SOD) activity assays.Catalase activity was calculated using hydrogen peroxide reduction kinetics with 0.04 nm cm -1 extinction coefficient at 240 nm using a UV-vis spectrophotometer. 13Specific activity was calculated as unit per mg protein (CAT U mg "1 protein).SOD activity was calculated using nitro blue tetrazolium (NBT) with riboflavin. 15The specific activity was calculated as unit per mg protein using absorbance values.
RT-PCR was performed using a thermal cycler (QIAGEN Rotor-Gene Q, Software 2.3.1.49).The amplification was performed at 95°C for 3 min for initial denaturation, followed by 3s denaturation at 95°C and annealing for 30s at 60°C.Fluorescent signals based on SYBR green were sensed separately at each elongation step, and the signal was measured as positive after the threshold cycle (Ct) was set to less than 2.

RESULTS AND DISCUSSION
The methanolic extract of A. platensis effectively inhibited the growth of all the three Vibrio species (Table 2).The MIC for V. alginolyticus was 2000 µg mL -1 , whereas 1500 µg mL -1 extract inhibited V. harveyi and V. parahaemolyticus.These two strains were the most susceptible to the extracts.Gentamycin was used as a positive control which inhibited all the Vibrio species at very low concentrations (50 µg mL -1 ).The MBC of the extract was higher than the MIC, as the MBC for V. alginolyticus, V. harveyi,and V. parahaemolyticus were 2500, 2000, and 2000 µg mL -1 .The antimicrobial activity could be due to the presence of bioactive compounds such as alkaloids, flavonoid glycosides, phenols, and saponins in A. platensis. 19The mechanism of antimicrobial activity of these compounds is still unclear and may be genotoxic or due to the interaction with the outer membrane of the bacteria. 20o control acute hepato-pancreatic necrosis disease (AHPND), new approaches for using bioactive compounds from algal sources need to be considered to reduce production loss. 21he in vitro antibacterial activity of various A. platensis extracts, particularly on Vibrio strains, has been reported previously. 22The protective effect of methanol extract of A. platensis against vibriosis in L. vannamei was studied in vivo.The control shrimp inoculated with Vibrio strains were severely infected within 72 h.Vibrio alginolyticusand V. parahaemolyticus-infected shrimp died within 120 h, whereas V. harveyi-infected shrimp survived slightly longer (Figure 1).V. harveyi is a luminescent bacterium that accounts for the total mortality of shrimp in aquaculture farms. 23njecting the Vibrio culture mixed with methanol extract into the test animals increased survival rate and reduced shrimp mortality.The survival rate of shrimp exposed to V. alginolyticus was 16%, whereas that of shrimp exposed to V. parahaemolyticus and V. harveyi increased (25%).
The mortality rate was higher with V.alginolyticus because of the virulence of the strain.The survival rate of shrimp not infected with Vibrio, but administered normal saline was 100%.These results clearly show that the methanol extract of A. platensis considerably protected L. vannamei against vibriosis.This result was substantiated by the observation that dry powder 24 and hot water extract 25 of A. platensis increased resistance against V. alginolyticus through increased phagocytic and lysozyme activities for pathogen clearance from shrimp.Based on the inhibitory effect of methanol extract on Vibrio species, the incorporation of extracts along with feed for Vibrio-challenged shrimp was studied (Figure 2).The shrimp challenged with V. alginolyticus had a reduced mortality rate (16.7%) than the mortality rate 48 h after injecting 4x10 6 CFU mL -1 V. alginolyticus (76.6%). 26Further, the survival rate reduced to 16.6% after 168 h.The survival rate of V. parahaemolyticusand V. harveyi-challenged shrimp were 33.3% and 50%, respectively, after 168 h.The survival rate of control shrimp administered saline and methanol extracts was 100%.The methanol extract of A. platensis considerably influences pathogen multiplication regulation.Further studies should determine the optimal dosage and administration.The effect of metabolites on inhibitory activity on pathogen depends on the dose, extract concentration, administration route, and exposure time. 27This study clarified that extract along with feed controlled infections and increased shrimp immunity.An extract concentration below the MIC may not be effective in developing disease resistance against Vibrio.To determine the efficiency of the extract in increasing immunity and disease resistance, the soluble protein content and antioxidant enzymes were measured in shrimp after the experimental period (Table 3).
The soluble protein content in Vibrioinfected shrimp was lower than that in control shrimp.The soluble protein content in V. alginolyticus-infected shrimp on normal feed and methanol extract along with feed were 14.31 and 28.68 mg mL -1 , respectively.Moreover, the soluble protein content in V. harveyi-infected shrimp on normal feed and methanol extract along with feed were 12.52 and 24.84 mg mL -1 , respectively.Similarly, the soluble protein content in V. parahaemolyticus-infected shrimp on normal feed and methanol extract along with feed were 13.57 and 26.56 mg mL -1 , respectively.Previous studies have reported that blue-green algae activate both humoral and cellular immune system to reduce environmental stress and infectious agents in the freshwater prawn Macrobrachium rosenbergii. 28ot water extracts of A. platensis induced and activated the antioxidant enzymes in L. vannamei. 25OD is an essential antioxidant enzyme that plays vital role in the defense mechanism of shrimp immune system in scavenging superoxide anion that damages the host tissues. 12The SOD activity increases in infected shrimps as a defense against the pathogens and also shrimps fed with herbal extract-supplemented feed for resistance against the infected pathogens. 29In this study,  16.6%) after challenging with Vibrio alginolyticus and with and fed with methanol extract along with feed (b).L. vannamei survived (50%) after challenging with V. harveyi and fed with methanol extract along with feed (c) L. vannamei fed with 2.5 mg concentration of extract along with feed, challenged with Vibrio parahaemolyticus and survived (33.3%) along with control (d) shrimps fed with methanol extract along with feed the SOD activity in infected untreated shrimps were lower (1.78-1.98U mg -1 protein) than that in the control (2.05 U mg -1 protein) and extractfed shrimps.The SOD levels in V. alginolyticus-, V. harveyi-, and V. parahaemolyticus-infected shrimp fed with extracts were 3.02, 2.35, and 2.63 U mg -1 protein, respectively.SOD increased to lessen the self-damage caused by superoxide anion damage in extract-fed shrimp.It may be due to the presence of antioxidants, such as C-phycocyanin, beta carotene minerals, protein, carbohydrates vitamins and lipids, in A. platensis. 30eta carotene and C-phycocyanin scavenge free radicals such as alkoxyl, peroxyl, and hydroxyl radicals. 31The increased SOD and other antioxidant molecules in the shrimp might have increased H 2 O 2 concentration, which in turn increases catalase production to detoxify it.Catalase activity in extract-treated infected shrimp was higher than that in untreated challenged shrimp.Therefore, the immunostimulatory effects and antioxidant properties of various phytochemical components present in A. platensis should be evaluated.Furthermore, this study showed that the extracts stimulated SOD and catalase activities, thereby increasing the innate immunity in L. venammei.Similarly, A. platensis extracts and feeding microalgae stimulated phagocytosis in other shrimp species, such as Penaeus merguieneis and fish Cyprinus carpio. 32he shrimp survived for 168 h and were subjected to RT-PCR to detect Vibrio infection.Real-time PCR is used for the molecular detection of target DNA under automated conditions by fluorescence changes at low cost and in modest steps, avoiding difficult electrophoretic methods.Currently, it is widely used to detect pathogens at earlier stages.V. alginolyticus was not detected in the tissue samples of challenged shrimp that survived after 168 h (Figure 3a).The gene encoding the beta subunit of DNA gyrase is gyrB, which has a unique sequence for V. alginolyticus that is recognized as a molecular marker. 33The survival rate of V. alginolyticus-infected shrimp was minimal than that of other strain-infected shrimp.The V. alginolyticus-infected shrimp are more susceptible to other pathogens and prone to decreased immunity, ultimately leading to death.However, the survival time increased, and the surviving shrimp were not infected when compared with that in the control.The survival rate of L. vannamei on methanol extract-incorporated feed after challenge with V. harveyi was 50%.None of the surviving shrimp showed signs of vibriosis in RT-PCR analysis (Figure 3b-d).
V. harveyi is considered one of the most common opportunistic pathogens in L. vannamei and other shrimp farms and hatcheries and infects shrimp under environmental stress, thus increasing the mortality rate. 34The secretion of extracellular products such as enzymes (proteases and lipases), toxins (hemolysins), lipopolysaccharides, and bacteriocin helps in the survival of these bacteria in the host. 35V. harveyi causes luminous vibriosis and a 382 bp toxR gene fragment is widely used for the rapid and specific identification of this pathogen.The survival rate of V. harveyi-infected shrimp increased after treatment.V. parahaemolyticus produces a thermolabile hemolysin responsible for its pathogenicity via tlh gene, which is a basic molecular marker for the species.The survival rate of L. vannamei fed with 2500 µg extract along with feed and challenged with V. parahaemolyticus was 33.3% and the shrimps were subjected to RT-PCR analysis for pathogen infection.All surviving shrimps were negative for Vibrio infection.V. parahaemolyticus is a major pathogenic strain for L. vannamei and causes Early Mortality Syndrome (EMS), with 100% mortality within 20 days of stocking.It is present in the water and digestive tract of shrimp and enters the host through gills, feed, and injuries.Shrimps are susceptible to infection due to stress or less immune response. 36revious invivo studies have showed that ethanol extracts of seaweed Gracilaria fisheri decreased V. harveyi and V. parahaemolyticus colonization and protected against mortality. 37This study shows that, to some extent, A. platensis extract helps to overcome stress and increase immunity against the pathogens.

CONCLUSION
Vibriosis, the most common infection caused by V. alginolyticus, V. harveyi, and V. parahaemolyticus, is a serious threat to the aquaculture industry.Introducing novel bioactive compounds against bacterial pathogens through feed will diminish the bacterial community and improve shrimp immunity.Incorporating A.

Figure 1 .Figure 2 .
Figure 1.In-vivo assessment of shrimp survival rate (%) after challenging with Vibrio species along with Spirulina extract for preliminary study (each treatment is done with twelve shrimps)

Figure 3 .
Figure 3. Real time PCR detection of pathogens in L. vannamei after feeding with Spirulina extract.(a) L. vannamei survived (16.6%) after challenging with Vibrio alginolyticus and with and fed with methanol extract along with feed (b).L. vannamei survived (50%) after challenging with V. harveyi and fed with methanol extract along with feed (c) L. vannamei fed with 2.5 mg concentration of extract along with feed, challenged with Vibrio parahaemolyticus and survived (33.3%) along with control (d) shrimps fed with methanol extract along with feed

Table 1 .
Primers used in the study

Table 2 .
Minimum inhibitory concentration and minimum bactericidal concentration of methanol extract of Spirulina on Vibrio species

Table 3 .
Antioxidant response of infected L. vannamei fed normal feed and with Spirulina extract incorporated feed