Effect of Asiatic mangrove plant ( Rhizophora mucronata ) extract on the growth and virulence of Vibrio harveyi causing bioluminescence disease in Penaeus monodon larviculture

Aim of study: Vibrio harveyi bacteria are affecting shrimps during grow-out practices. The application of chemicals to control V. harveyi has resulted in antibiotic‐resistance among bacteria. An extract of the leaves of Rhizophora mucronata was tested to control the growth and virulences of V. harveyi. Area of study: This study was conducted in the Crustacean Culture Division of ICAR-CIBA, Chennai city, Tamil Nadu State, India. Material and methods: R. mucronata plants were collected from the Pitchavaram area, and the leaves contents extracted. The resultant extract was prepared and tested against the growth of V. harveyi and its virulence factors. The various functional compounds of R. mucronata were screened and volatile compounds were analyzed. Main results: When R. mucronata extract was treated against V. harveyi (350 µg/mL) an inhibitory zone of 14 ± 0.1 mm was observed. At 300 µg/mL, the extract was found to be active in decreasing the luciferase to a maximum of 76 counts per second in 30 days and a similar level of bioluminescence was reduced in 15 days. During, shrimp larviculture a reduction in the cumulative percent of mortality 15.70% ( p <0.033) was observed when treated with the extract of R. mucronata. Research highlights: When extract (200 μg/mL) of R. mucronata was tested against V. harveyi during Penaeus monodon larviculture, the V. harveyi counts decreased ( p <0.049). Volatile compounds viz, tetramethyl-6,7,8,8a-tetrahydro-5H-naphthalene-1-one (38.63%), squalene (31.19%), α-amyrin, (7.07%) and β-amyrin (8.75%) were detected. It would be desirable to use crude extracts of R. mucronata during shrimp culture to control V. harveyi .


Introduction
Vibrio is one of the virulent bacterial pathogens, responsible for causing large-scale mortality during the developmental stages of penaeid shrimps. Bioluminescence disease-causing Vibrio harveyi bacteria and its related species can affect almost all types of organisms (Defoirdt et al., 2007)  of antimicrobials in the aquaculture system has resulted in bacteria developing resistance to antibiotics (Igbinosa et al., 2017). Edible shrimps are also perhaps a source in spreading multiple antibiotic resistances (MAR) among the bacterial pathogens to the consumers (Beshiru et al., 2020). Copious technologies have been developed to control bioluminescent V. harveyi causing disease in the aquaculture system. "Green water culture system" is a technique used in aquaculture, wherein the shrimps or fish are co-cultured with abundant microalgae such as Chlorella. However, the micro algae production essential for aquaculture is yet to be quantified (Amir Neori, 2011). Probiotic bacteria have been used for monitoring bacterial infections and to reduce organic matter in shrimp grow-out practices, but more quantity of cells are necessary, which is cost-effective (Defoirdt et al., 2008). There is an urgent need to search for other methods to control disease-causing vibrios, which is an imperative challenge for sustainable aquaculture development. It is possible that instead of chemicals, alternative bio-agents, extracted from marine plants/mangroves could be used to control resistant marine vibrios. Rhizophora mucronata, commonly known as Asiatic mangrove or 'red mangrove' has been reported to have healing properties against nausea, haemorrhage, and angina (Manilal et al., 2015). Among the mangrove plants, R. mucronata, and Avicennia marina are known to have anti-hyperglycemic, anti-radical and anti-diabetic properties (Sur et al., 2015;Aljaghthmi et al., 2017) in humans.
The preliminary phytochemical analysis of R. mucronata revealed phenols, alkaloids, terpenoids, carbohydrates, saponins, and tannins (Chitra et al., 2019). R. mucronata extracts at 100 µg/mL hold more potent antioxidant activity than those of R. apiculata and R. annamalayana, as evident by the presence of phenolic N-H and OH components found in mangroves leaves extract (Arulkumar et al., 2020). The fruit extract of R. stylosa was found to control vibriosis in larvae of the mangrove crab, Scylla serrata (Burhanuddin et al., 2019). R. mucronata extract was screened for antagonism against multi-drug resistant V. harveyi and V. campbellii (Baskaran & Mohan, 2012). The crude ethanol extract of R. mucronata could protect tiger shrimp from V. harveyi infection (Saptiani et al., 2019). R. mucronata could be used as a bioindicator for monitoring, lead (Pb) pollution in the " Muthupet mangrove ecosystem " of Tamil Nadu, India (Ganeshkumar et al., 2019). The methanolic fraction of R. mucronata extract offers a substantial decrease in diabetes and metabolic impairment in rats (Adhikari et al., 2016). R. apiculata extract improved the immune system in clownfish against infection by V. alginolyticus (Dhayanithi et al., 2015). Therefore, an attempt was made, in the present study, to evaluate in-vitro antagonistic effect of crude extracts of R. mucronata on the growth and virulence factors of V. harveyi for protection against bioluminescence disease in Penaeus monodon larviculture.

A sampling of mangrove leaves and preparation of crude extracts
Leaves of R. mucronata (Fig. S1 [suppl]) were collected from a mangrove forest at Pichavaram in Tamil Nadu, India (Lat 11º27'N; Lon 79º47'E). The leaves were washed in a 10 mg/L solution of KMnO 4 for 10 min to eliminate epiphytes, sand, and other extraneous matters. The leaves were cleaned in freshwater and shade-dried at room temperature for 12 hrs. Later the leaves were pulverized by a sterile pestle and mortar and stored at -20ºC for further use. The 2.0g powder was treated with 100 mL of ethyl acetate and then extracted by the Soxhlet apparatus. The extracts were then neutralized to pH 7.0 by using 0.1 N NaOH and filtered through Whatman filter paper No.1. The extracts were later dried at 42ºC in a hot air oven for 6 hrs. For cold extraction, 1.0 g of powder was mixed with 10 mL of ethyl acetate and thereafter placed in a shaker incubator at 37°C at 50 rpm for 96 hrs. The extract was then filtered through Whatman filter paper No.1 and rotary evaporated (30°C) under vacuum and stored at 4°C. The pH was neutralized as stated earlier. The resultant extract was liquefied with 5mg/mL of 30% (v/v) dimethyl sulfoxide (DMSO) and used for testing antagonism against luminescent V. harveyi .

Antibacterial assay
Antibacterial activity of the extracts was carried out by "Agar well diffusion assay" (Das et al., 2005) against V. harveyi. Cells of V. harveyi (50 μL/10 8 CFU/mL cells (18 hrs, old) were inoculated into Petri plates. The LB agar (35 mL) was dispensed into plates and solidified for 1 hr at room temperature. Two wells (6.0 mm) were made on the LB agar plates using a sterile steel borer. The wells were sealed at the bottom with 10 μL of 1.0% soft agar and filled with 200 μL of crude leaves extract. The plates were then incubated at 37°C for 48 hrs and zones of inhibitions on V. harveyi around the well were measured excluding the well. The antimicrobial activity of the R. mucronata extract was determined by dissolving it in 30% of DMSO in various concentrations. DMSO was used as a negative control. Similarly, the extract obtained through "cold extraction" was also tested. Each test was performed in triplicate and values were expressed as average with standard deviation.

Estimation of minimum inhibitory and minimum bactericidal concentrations (MIC & MBC)
The minimum inhibitory concentrations (MIC) for the extract were evaluated as described by Islam et al. (2008). Dilution methods were used to determine the MIC. In dilution tests, V. harveyi was tested for their ability to produce visible growth against the extracts on a series of LB agar plates against the extracts. Several concentrations of extracts (5.0 to 50 μg) were tested and the lowest concentration which inhibited the visible growth of V. harveyi was observed as the MIC. The plates were incubated at 37°C for 24 hrs and 20 μL of V. harveyi (1.8 OD or 2.19×10 7 CFU/mL) was confirmed for the MIC on the LB agar medium. The MBC was evaluated as the lowest concentration of a crude plant extract required to kill 99.9% of 20 μL of V. harveyi (1.8 OD or 2.19×10 7 CFU/mL).

Effects of R. mucronata extract on the growth and virulences of V. harveyi
The leaves of extract at 300 µg/mL was added to 100 mL of LB medium. Five hundred μL (24 hrs) of V. harveyi (1.8 OD) cells were inoculated into LB broth and incubated under shaker incubator at 37°C for 100 rpm in 5 days. Three mL of V. harveyi inoculum was estimated for their growth for 30 days at OD 600 nm. The growth and virulence factors such as proteolytic, lipolytic, phospholipase, thermonuclease, crude bacteriocin, exopolysaccharide (EPS), and proteases produced were estimated. Salt aggregation test (SAT) was carried out for cell surface hydrophobicity and cell adhesion was examined through bacterial adhesion to hydrocarbons test (BATH) (Soto-Rodriguez et al., 2012). Each test was performed in triplicate and values were expressed on average with standard deviation.

Cell lysate preparation, estimation of luciferase, and luminescence
The luciferase enzyme produced by V. harveyi was tested by the luciferase kit (LUC1, Technical Bulletin MB-260, and Sigma, USA) and read by a luminometer (Victor TM X3, Perkin Elmer, USA). V. harveyi cells were harvested by centrifugation at 10,000 rpm for 5 min. The pellet was re-suspended in 333 μL of 1X cell lysis buffer per mL of V. harveyi and incubated at 25°С for 10 min. Thereafter the suspension was centrifuged at 12,000 rpm for 1 min at 4°С. The supernatant was stored in ice. Luciferase substrate (lyophilized, suspended in luciferase assay buffer) as cell lysate was equilibrated to 25°С before use. Cell lysate (20 μL) was added to 100 μL of the luciferase substrate and mixed well. Readings were recorded in 10 sec for light emission by the luminometer and expressed as counts per second (CPS, i.e., photons). The light intensity was closely constant for 20 sec. The LB broth medium and 1X lysis buffer were used as a negative control for luciferase assay. For the estimation of luminescence, the V. harveyi cells, harvested by centrifugation at 10,000 rpm for 5 min, and its spent culture medium, were estimated for luminescence by a luminometer .

Fourier transform infrared spectroscopy (FT-IR) analysis
The shade-dried powder (1.0 mg) was mixed thoroughly with 2.5 mg of dry potassium bromide (KBr) by a pestle and mortar. The powder was filled in a micro-cup (2.0 mm internal diameter) to obtain the diffuse reflectance infrared (IR) spectrum for replicate samples. All the IR spectra were recorded at 37°C in the mid-infrared range (4000-400 cm -1 ) using Fourier Transform Infrared Spectrometer (FT-IR) Bruker IFS 66, Shimadzu. Normally, 20 scans were signal-averaged for a single spectrum. Each spectrum is displayed in terms of absorbance as calculated from the reflectance-absorbance spectrum using the Hyper-IR software (D' Souza et al., 2008).

Gas chromatography and mass spectrometry analysis
Gas chromatography-mass spectrometry (GC-MS) analysis was performed by using Agilent GC-MS-5975C with the Triple-Axis Detector equipped with an auto sampler. The GC column used was fused with a silica capillary column (length 30 m × diameter 0.25 mm × film thickness 0.25 mm) with 1.51 mL helium for 1 min as a carrier gas. The mass spectrometer was operated in the electron impact (EI) mode at 70 eV in the scan range of 40-700 m/z. The split ratio was adjusted to 1:10 and injection volume was 1.0 μL. The injector temperature was kept at 250°C, and the oven temperature was 70°C for 3 min, which was later increased to 250°C at 14°C/min (total run time 34 min). The temperature of the transfer line and the ion source was set to a value of 230°C and the interface temperature at 240°C, the full mass data being recorded between 50-400 Daltons and scan speed 2000. Mass start time was set at 5 min and end time at 35 min. Peak of the crude R. mucronata extract was identified by comparison with retention times of standards and the mass spectra obtained was compared with those available in the NIST libraries (NIST 11-Mass Spectral Library 2011 version) with an acceptance criterion of a match above a critical factor of 80% (Musharraf et al., 2012).

Effect of R. mucronata extract against V. harveyi during P. monodon larviculture
Plastic tubs were washed with 10 mg/mL of KMnO 4 solution (w/v) for 10 min and filled with 20 L of saline water at 20 practical salinity unit (PSU). Postlarvae (PL 10) of P. monodon from a private shrimp hatchery, were tested by PCR to be disease-free, following Ananda Raja et al. (2017), for the OIE 1 listed diseases, and were acclimatized at 20 PSU for 5 days with aeration under laboratory conditions. About 1000 PL, in the weight range of 17 to 18 ± 0.2 mg were stocked per tub. The first tub was inoculated with V. harveyi (10 mL of 1.8 OD) alone as a control. The second tub was considered as treatment and inoculated with V. harveyi (2.0g for 10 L of the extract). The third tub was considered as control, where the crude extract was added at 200 μg/mL with PL. The fourth tub was a control for PL, where neither V. harveyi nor extract was added. Aeration was provided for each container and the PLs were fed twice a day at 15% of their body weight. All the experimental tubs were covered on the top with a plastic lid to avoid external contamination. The water temperature, salinity, and pH were recorded once in 5 days. Experiments were carried out in triplicate. The mortality of PL was recorded daily. No water exchange was made in the containers for 30 days. The total heterotrophic and V. harveyi counts were enumerated (Traifalgar et al., 2009) using LB agar and V. harveyi selective agar medium under the spread plate method (Biswas et al., 2012).

Statistical analysis
The data were analyzed and expressed as means along with the standard deviation. Analysis of variance (SPSS, 1 The former Office International des Epizooties (OIE) inter-governmental organization coordinating, supporting and promoting animal disease control, is now called World Organisation for Animal Health ver. 16.0) was carried out to find the significance (p<0.05) difference, if any. The cumulative percent mortality (CPM) was calculated as a Cumulative frequency/total number of observations (n) ×100.

Effects of R. mucronata extract on growth and virulences of V. harveyi
The crude extract showed the maximum active antagonistic efficiency to control the growth of V. harveyi (OD) from the 5 th day onwards. The highest OD difference was observed on the 20 th day (0.37) compared to the control and the lowest on the 10 th day (p<0.05). The bioluminescence production was decreased from 1100 to 2200 CPS against the R. mucronata extract for 30 days as compared to control (Table 1). The maximum control on bioluminescence was detected on the 15 th day (2230 CPS) and the minimum was on the 25 th day (1090 CPS) (p<0.05). The production of intracellular luciferase by V. harveyi was decreased with the addition of extract from 20 to a maximum of 76 CPS in 30 days (Fig. 1). Table  1 shows that the maximum decrease in bacteriocin production (OD) by the extract was observed between the 5 th and 15 th days (0.39 and 0.27 respectively, p<0.05). The maximum decrease in protease was 0.15 OD against the extract on the 20 th day. Treatment with the extract showed a weak level (+) of phospholipase, produced by V. harveyi as compared to very high phospholipase (++++) in the control for all the 30 days. In SAT, V. harveyi revealed strong hydrophobic activity from the 5 th to 30 th day in the control whereas, the treatment showed moderate to weak hydrophobic activities.

FT-IR analysis of R. mucronata extract
The FT-IR spectrum of dried leaves powder reveals various functional groups of compounds that were compared with the FTIR standard library data. The FT-IR spectrum showed the presence of significant functional groups such as aromatics, alkanes, alcohol, carboxylic acids, esters, ethers, aliphatic amines, and alkyl halides ( Table 2).

GC-MS analysis of the R. mucronata extract
To document preliminary data on bioactive components, ethyl acetate extract was subjected to GC-MS analysis. The crude extract was found to have a mixture of secondary and volatile compounds. Fatty acid methyl esters were investigated quantitatively by GC-MS in multiple reactions monitoring the mode and thus allowing a better signal resolution without a preliminary fractionation of the extract. A total of 21 peaks were observed with retention times as presented in Fig. 2. The main phytoconstituent reported was tetramethyl-6,7,8,8a-tetrahydro-5H-naphthalene-1-one   (38.63% peak area), followed by squalene (31.19% peak area), α-amyrin, (7.07%) and β-amyrin (8.75%). These bioactive compounds could be involved in the antagonistic activity (Table 3) against V. harveyi along with other compounds. Chemical constituents were identified using spectrum database NIST 11 software installed in GC-MS.

Effects of the extract of R. mucronata against V. harveyi during P. monodon larviculture
During the larviculture of P. monodon, the mortality varied from 0 to 50.81% till the 30 th day after treatment with the extract. The CPM increased in the control from 4% on the 5 th day to 66.5% on the 30 th day respectively.  Table 3. GC-MS profile of R. mucronata leaves extract.
The difference in the overall decrease of CPM among the treatments was 12 to 16% as compared to control from 20 th to 30 th days. On the 30 th day, a maximum reduction of 15.70% was found in the treatment, compared to control. The growth of postlarvae uniformly increased from 4-7 mg on the 10 th day. The growth of V. harveyi when treating with the extract treatment was from 5.2×10 4 to 3.2×10 3 CFU/mL on the 30 th day as compared to control (9×10 5 to 1.0×10 3 CFU/mL). In the treatment, the total heterotrophic bacterial (THB) count decreased from 4.3×10 4 CFU/ mL to 1.2×10 3 CFU/mL, compared to control (6.9×10 4 to 7.2×10 3 CFU/mL) ( Table 4). Significant differences were found in CPM (p<0.033) and V. harveyi counts (p<0.049) between the leaves extract of R. mucronata treated with V. harveyi and control.

Discussion
Mangroves are a potential source of various compounds with higher biological activity than toxicity (Manilal et al., 2010). The compounds are used as antimicrobial agents and in pharmaceutical preparations. In this study, crude R. mucronata extracts showed inhibition on V. harveyi, but the extract obtained through the Soxhlet method gave a better inhibition than cold extraction. The crude extract obtained from the bark and collar of the Asiatic mangrove showed inhibition on V. harveyi (7.0 mm) and various other fish pathogens (Kesavaraju & Sreeramulu, 2017). The extract also showed anti-fungal activities on Acremonium sp and Aspergillus niger at a concentration of 75 µg/mL (Arunprabu et al., 2016). Kannappan et al. (2018) reported the inhibition on bioluminescence disease-causing V. harveyi (12 mm at 350 µg/mL) by the crude extract of R. apiculata. The mechanism of inhibition on V. harveyi was due to the phytochemicals such as alcohols, phenols, alkanes, carbonyls, unsaturated aldehydes, nitro compounds, aromatics, esters, ethers, alkyl halides & aliphatic amines present in the crude extracts alone and not by DMSO or pH of the extract which was neutralized during the experiment. Ernawati & Hasmila (2016) reported that the secondary metabolites present in the leaf extract of R. mucronata were flavonoids and steroid compounds. The presence of phenol and flavonoid contents of the R. mucronata has been proved for its antioxidant properties (Kaur et al., 2019). Treatment of the crude extract showed a weak level of phospholipase against V. harveyi. It also revealed strong hydrophobic activity on the 30 th day in the control experiment indicating, therefore, that the extract definitely can be used to control V. harveyi. The R. apiculata extract treatment, showed a weak level of phospholipase produced from V. harveyi and showed moderate Values are the average of three determinations with a standard deviation (SD). PL: postlarvae. PSU: practical salinity unit (1 PSU = 1 g/kg). to weak hydrophobic activities (Kannappan et al., 2018). The methanol extract of R. apiculata and R. mucronata (1.0 mg/mL) showed a reduction of the virulence factors like protease, pyocyanin pigments, and biofilm produced by Pseudomonas aeruginosa . Quebrachitol, a bioactive compound extracted from the R. mucronata inhibited biofilm and virulence production in Staphylococcus epidermidis by impairment of initial attachment and intercellular adhesion (Karuppiah & Thirunanasambandham, 2020). The extract of R. mucronata showed a maximum decrease in bioluminescence production on the 15 th day and the minimum decrease was on the 25 th day. The production of intracellular luciferase also decreased on the 30 th day. The methanol extracts of R. apiculata and R. mucronata showed significant inhibition against quorum-sensing dependent virulence factors such as protease, elastase, and production of pyocyanin and biofilm in P. aeruginosa PAO1 (Annapoorani et al., 2013). We found numerous significant biochemical compounds in R. mucronata (Table 3). In our study, the main phytoconstituents detected from R. mucronata were tetramethyl-6,7,8,8a-tetrahydro-5H-naphthalene-1-one, squalene, α-amyrin, and β-amyrin. In particular, the tetramethyl compounds have been reported to exhibit anticancer and antioxidant activity (Mohammed et al., 2016). Also, bioactive components such as alkaloid, tannin, saponin and flavonoid extracted from the ripe fruit of R. mucronata were found to lower the blood glucose level in rats (Hardoko et al., 2015). The squalene detected from the R. mucronata extract is an organic compound normally obtained from shark liver oil for commercial purposes. Adhikari et al. (2018) reported that R. mucronata leaves contained magnesium and squalene contents. R. mucronata-based squalene also may be used in the formulation of a wide variety of cosmetic products. Palaniyandi et al. (2018) found in-vitro anti gastric cancer activity of squalene, isolated from R. mucronata leaves against AGS cell line.
During the P. monodon larviculture, the mortality was reduced significantly when the extract was used to a considerable level. The CPM caused by V. harveyi decreased in postlarvae, (12-16% from 20-30 th days by treating with the extract). Saptiani et al. (2019) reported that the ethanol extract (1500 ppm) of R. mucronata leaves inhibited V. harveyi and protected the tiger shrimp from infection with an improved survival rate and this treatment was akin to one using antibiotics. Dietary administration of crude extract of R. mucronata was found to enhance the growth response and innate immunity of clownfish (Dhayanithi et al., 2020). Fermented mangrove propagules of R. mucronata have been used in fish feed, causing no negative effects on the growth of Nile tilapia fish fry (Andriani et al., 2018). The crude extract of R. apiculata at 200 μg/ mL, when tested against V. harveyi during larviculture of P. monodon, reduced mortality by 10.6% (Kannappan et al., 2018). Nurhidayah & Atmomarsono (2020) reported on the potential of R. mucronata against disease-causing V. harveyi in tiger shrimp, with a MIC ranging between 1.0 and 10,000 mg/L. Similarly, the use of bark extract of R. mucronata extract (64.0 ppm) also resulted in curing V. harveyi infection with an enhanced survival rate (76.66%) in Nile Tilapia fish (Mulyani et al., 2020). Also, the fruit extract of R. stylosa showed anti-vibrio activities as the cause of Vibriosis in mangrove crab larvae (Scylla serrata) (Burhanuddin et al., 2019).
The growth of postlarvae increased when treated with the R. mucronata extract treatment coupled with the decreased total heterotrophic bacterial load. Even, R. apiculata plantation improved the water quality, growth, and health of mud crab (Scylla paramamosain) during the grow-out system, and promoted gut microbiota (Dai et al., 2020). R. mucronata has been used in traditional medicine as an antiseptic, antibacterial, and anti-inflammatory agent (Suganthy & Devi, 2016). The values observed from the bio-assay of R. mucronata extract against the V. harveyi during P. monodon larviculture (p<0.05). Our study revealed significant differences between the extract-treated V. harveyi infection and control. Also exposed the anti-vibrio and anti-virulence potential of R. mucronata extract against bioluminescent V. harveyi and forms the baseline information for further research on other mangrove species.
In summary, our results reveal that the extracts of R. mucronata hold potent phytoconstituents which inhibit the growth and modulate the virulence factors produced by V. harveyi. Further, the extract was found to control the mortality caused by V. harveyi in the P. monodon larviculture and could be used as an alternative agent against aquaculture bacterial pathogens. Moreover, the purification of crude extracts of R. mucronata will provide huge opportunities on the detection of many bioactive compounds that could be utilized for making eco-friendly therapeutics. The chemical constituents inherent in R. mucronata were also presented. With the succeeding research on this plant, it is apparent that there are still unexplored new impending compounds obtainable from this plant. The cost of production of extract would be lower as compared to molecular grade chemicals used in animal experiments. The application of such bio-products would reduce the undesirable effects caused by the use of chemical preservatives in aquaculture with reduced production cost and also be eco-friendly. Furthermore, we intend to assess the effect of R. mucronata extract on the equilibrium between the growth and survival among the P. monodon postlarvae against V. harveyi in follow-up studies.