Phyllanthus amarus enriched Artemia nauplii enhanced survival, growth and nutritional quality of early post-larvae of the prawn Macrobrachium rosenbergii

The presence of primary and secondary phytochemical compounds in petroleum ether, acetone and ethanol extracts of Phyllanthus amarus was detected qualitatively as well as quantitatively. Alkaloids, terpenoids, tannins, polyphenols and quinines were present in the petroleum ether extract of P. amarus. Terpenoids, flavonoids, tannins, saponins, cardiac glycosides and quinines were present in the acetone extract, whereas alkaloids and terpenoids were absent in ethanol extracts of P. amarus. From the different solvent extracts of P. amarus, overall, 20 secondary phytochemicals were identified. Among these, presence of six bioactive compounds {Dodecanoic acid; Propanoic acid, 2-(tricyclo [3.3.1.13, 7] dec-2-ylidene)-; Tetradecanoic acid; Neophytadiene; Hexadecanoic acid, ethyl ester, and 9, 12, 15-Octadecatrienoic acid, (Z, Z, Z)-} have been identified through literature. Among the three solvents, ethanol extract of P. amarus enriched Artemia nauplii was found to produce the best survival, growth, nutritional indices (weight gain and specific growth rate) and concentrations of basic biochemical constituents (total protein, amino acid, carbohydrate, and lipid) in M. rosenbergii early post larvae. This suggests that the general health of the prawn was improved. This study indicates the fact that the primary phytochemical quality and secondary bioactive compounds of P. amarus have the potency to sustainably enhance the survival, growth and nutritional quality of M. rosenbergii. Thus, this herb is recommended as a feed additive. *Correspondence to: Saravana Bhavan P. Department of Zoology, Bharathiar University, Coimbatore 641046, Tamil Nadu, India; E-mail: bhavan@buc.edu.in


Introduction
Medicinal plants provide an inexhaustible resource of raw materials for the pharmaceutical, cosmetics, food industries and more recently in agriculture for pest control [1]. The herbal extract could be defined as the compounds and /or mixture of compounds obtained from fresh or dried plants, or parts of plants, such as leaves, flowers, seeds, roots and barks. The carry me seed plant, Phyllanthus amarus is a common pantropical weed that grows well in moist, shady and sunny places. It is widely spread throughout tropics and subtropics [2]. It has been used in Ayurvedic medicine for over 2,000 years and has a wide number of traditional uses. It has been reported to be used in treating jaundice by blocking DNA polymerase of hepatitis B virus [3][4][5]. There are also reports that it is used to treat diabetes, otitis, diarrhoea, gonorrhoea, and frequent menstruation, galactogogue, leucorrhoea, menorrhagia, mammary abscess, skin ulcer, hypertension, pain, inflammation, cough, asthma bronchitis, gastrointestinal disturbances, helminth infestation, nociceptive pain, mutation and cancer. It has a urolithic property, dissolving renal calculi [6]. P. amarus contains moderate amount of protein, rich in carbohydrate and low in fat, ash, crude fiber. It also contains Mg, Ca, K, PO and ascorbic acid, Fe; Zn, thiamine, niacin and riboflavin [7]. It has been reported that P. amarus contains many valuable compounds, such as lignans, flavonoids, hydrolysable tannins (ellagitannins), polyphenols, triterpenes, sterols and alkaloids [8]. The extracts and the compounds isolated from P. amarus reported to have a wide spectrum of pharmacological properties including anti-viral and anti-fungal, anti-bacterial, anti-plasmodial and anti-inflammatory, anti-tumor, anticancer, anti-diabetic, anti-oxidative, anti-hyperglycemic, hypolipidemic, immuno-stimulatory, hepatoprotective, nephroprotective and diuretic [7,[9][10][11][12][13][14][15][16][17][18].
The research in the use of plant extracts for aquatic animals is increasing with the demand for eco-friendly and sustainable aquaculture [19]. The medicinal plants have phytochemicals, the bioactive substances which are responsible for the biological activities they exhibit. For this reason, herbal extracts were introduced in the pharmacopoeias [20]. The herbal biomedicine active principles in the aquaculture have the characteristics of growth and survival promoting ability, tonic to improve the immune system, anti-microbial capability, anti-stress characteristics and stimulating appetite due to presence of alkaloids, flavonoids, pigments, phenolics, terpenoids, starch, steroids and essential oils. Recent studies showed that the incorporation of medicinal plants (raw material, extracts and phytocompounds) as additives of fish/prawn feeds stimulates the growth and immune responses [21][22][23][24][25][26][27][28][29][30][31][32][33][34].
The freshwater prawn, Macrobrachium rosenbergii is the most popular one for the commercial farming in many countries. It gained momentum after the setback in shrimp farming due to disease outbreaks and other factors. Newly-hatched M. rosenbergii larvae are around 2 to 2.2 mm in length. They normally start feeding about 1 day after hatching and initially require live zooplankton. Their prey should be of a suitable size for ingestion; the optimal size is estimated at 300 to 500 µm.

Preparation of extracts
The powdered whole plant sample of P. amarus (50 g) was taken and packed in Whatmann No. 1 filter paper and put into soxhlet apparatus. The extracts were successively soaked with 300 ml (1:6 w/v) of petroleum ether (99.98% purity, SRL Pvt. Ltd. India), acetone (99.5% purity, SD Fine Chemicals, India) and ethanol (99.9% purity, Changshu Yangyuan Chemicals, China) individually and sequentially extracted for 6-9 h each (30 to 36 cycle) based on their polarity (non-polar to polar). Repeated extraction was done until a clear colorless solution was obtained. The extracts were filtered by using double layer muslin cloth and concentrated at 40-50 °C using rotary vacuum evaporator (ROTAVAP). The extracts obtained were vacuum-dried under 40 °C and used for further investigation. The extracts obtained were appeared as dark green, gummy solid.

Qualitative analysis of primary phytochemical substances
The extracts were subjected to detection of the presence of primary phytochemical bio-molecules, such as alkaloids, terpenoids, flavonoids, tannins, polyphenols, saponins, and cardiac glycosides using the standard qualitative procedures of Trease & Evans [40].

Gas Chromatography-Mass Spectrum (GC-MS) analysis for secondary phytochemical compounds
Different solvent extracts of P. amarus were subjected to GC-MS analysis (Thermo GC-trace ultra ver-5.0; Thermo MS-DSQ-II; ZB 5-MS capillary standard non-polar column (30 mts, 0.25 mm id, 0.25 µm film) for identification of different phytochemical compounds (South India Textile Research Association (SITRA), Coimbatore, Tamil Nadu, India). The working condition of GC-MS was as follows: Carrier Gas, He; Flow, 1.0ml/min; Temperature Programme, oven temperature raised from 70 °C to 260 °C at 6 °C/min; Injection Volume, 1 µl; Detector, mass selective detector MS-DSQ-II with XCALIBUR software; Injector temperature, 250 °C; Ion source temperature, 200 °C; Interface temperature, 250 °C; Total running time, 40 min; Relative percentage constituents was expressed as percentage with peak area normalization.
Peaks resolved with relative abundance of 0-100 were considered as major compounds. To show the minor peaks, the chromatogram was magnified. Identification of various components present in each extract was confirmed based on the peak area, retention time and molecular formula. Identification of various components present in these extracts were assigned by the comparison of their retention indices and mass spectra fragmentation patterns with those stored on the computer library and also with published literatures. National Institute Standard and Technology (NIST4) and WILEY9 [41], on-line library source was also used for matching the identified components.

Proximate composition of P. amarus
The proximate composition of P. amarus whole plant powder, such as contents of moisture, crude protein, crude fibre, crude fat and ash were estimated following AOAC methodology [42], and total nitrogen free extract (carbohydrate) content was calculated by subtracting all the above contents adopting Castell & Tiews method [43]. P. amarus is a small erect annual herb (10-60 cm tall) with numerous small oblong-elliptic or squarish leaves and glabrous (6-12 mm long). The main stem is simple or branched, terrete smooth or scabridulous in younger parts [39]. Flowers are very small, yellow in colour and hang down in beautiful array hidden below the leaves. The flowers produce very small (2 mm) fruits that burst open and the seeds are hurled away. When the plants are picked, the feathery leaves fold in, completely closing themselves. P. amarus is a common pantropical weed that grows well in moist, shady and sunny places. It is widely spread throughout tropics and subtropics [2].

Artemia cyst hatching and its enrichment
The brine shrimp, Artemia franciscana cysts were purchased from Aqua world, Paris Corner, Chennai, India. The cysts (2 g/ 20 L and 15 g kg-1 body biomass of the prawns for feeding trial) were taken and hydrated in 1 L-1 of purified artificial saltwater (prepared from artificial sea salt powder 35.0 g L-1, pH of 6.5). After 12-15 h, the cysts burst, and the embryo surround by the hatching membrane become visible. After a few hours, the brownish orange colored nauplii came out. The 48-hr old Artemia nauplii were collected on a sieve and enriched with 1% of petroleum ether, acetone and ethanol extracts each of P. amarus separately for 1 h at the rate of 1 g in 100 ml.

Procurement and acclimatization of experimental animal
The early post larvae (PL-10) of the freshwater prawn, M. rosenbergii were procured from Sri Durgai Hatcheries, Chengalpattu, Tamil Nadu, India. They were transported to the laboratory in polythene bags filled with oxygenated water. The prawns were acclimatized to the ambient laboratory condition with ground water in cement tanks (6 × 3 × 3 feet) for a week (temperature, 26.5±1.3 °C; pH, 7.01±0.12; TDS, 0.80±0.05 g/l; DO, 7.30±0.49 mg/l; BOD, 29.0±1.24 mg/l; COD, 124.0±3.2 mg/l; ammonia, 0.026±0.007 mg/l). During acclimatization the PL were fed with un-enriched Artemia nauplii. About half of the tank water was renewed each day and adequately aerated to maintain a healthy environment. This ensures an environment devoid of accumulated metabolic wastes and sufficient oxygen supply to the prawns. The unfed feeds, faeces, moult and dead prawns were removed by siphoning.

Feeding trails with extracts of P. amarus enriched Artemia nauplii
M. rosenbergii early PL ranging from 0.8±0.06 cm in length and 0.06 ± 0.02 g in weight were used in a triplicate experimental set up. The prawns were divided into four groups. One group served as control and fed with un-enriched Artemia nauplii and the other three groups were fed with 1% of petroleum ether, acetone, and ethanol extracts of P. amarus enriched Artemia nauplii at the rate of three times per day (8.00 a.m., 2.00 pm and 10.00 pm) for 30 days. At the end of the feeding trial the final length and weight were measured for calculating nutritional indices and estimating basic biochemical constituents. During the feeding trial the water medium was renewed daily by siphoning method without severe disturbance to the prawn and was aerated adequately.

Estimations of basic biochemical constituents
The initial and final concentrations of basic biochemical constituents, such as total protein, amino acid and carbohydrate were estimated in test prawns adopting the methodology of Lowry et al. [45], Moore & Stein [46], and Roe [47], respectively, and the total lipid was extracted following the method of Folch et al. [48] and estimated gravimetrically following the method of Barnes & Blackstock [49].

Statistical analysis
Data between control versus experiments and between experiments were subjected to statistical analysis through one-way ANOVA and subsequent post hoc multiple comparison with DMRT by adopting SPSS (v20). All the details of statistical analyses were given in respective tables. The P values less than 0.05 were considered statistically (95%) significant.

Primary phytochemicals of P. amarus
The petroleum ether extract of P. amarus showed presence of 5 primary compounds, such as alkaloids, terpenoids, tannins, polyphenols and quinines. Of which, tannins were luxuriantly present, alkaloids and terpenoids were moderately present. The other compound, such as polyphenols and quinones were poorly present (Table 1). In acetone extract of P. amarus, the presence of 6 compounds, such as terpenoids, flavonoids, tannins, saponins, cardiac gylcosides and quinines were detected. Of which, tannins and cardiac gylcosides were luxuriantly present, flavonoids and saponins were moderately present. The other compounds, such as terpenoids and quinones were poorly present (Table 1). Similarly, the ethanol extract of P. amarus contains 6 primary compounds, such as, flavonoids, tannins, polyphenols, saponins, cardiac gylcosides and quinines. Of which flavonoids and tannins were luxuriantly present, polyphenols and quinones were moderately present, and other compounds, such as saponins and cardiac gylcosides were poorly present (Table 1). Therefore, overall, P. amarus contains 8 primary phytochemical compounds. In this study, ethanol has served as the best solvent for extraction of flavonoids. For the extraction of tannins, all three solvents can be used. Ethanol can be used for the extraction of saponins, while, acetone can be used for the extraction of cardiac glycosides. For extractions of alkaloids and terpenoids, petroleum ether can be used. Ethanol can also be used for extractions of polyphenols and quinines.
Kalaiselvi VC (2018) Phyllanthus amarus enriched Artemia nauplii enhanced survival, growth and nutritional quality of early post-larvae of the prawn Macrobrachium rosenbergii

Nutritional indices and basic biochemical constituents in M. rosenbergii PL fed with P. amarus extracts enriched Artemia nauplii
The morphometric data, length and weight, and nutritional indices, such as SR, WG, and SGR were found to be significantly increased (P<0.05) in petroleum ether, acetone and ethanol extracts of P. amarus enriched Artemia nauplii fed PL groups when compared with control (Table 6). Similarly, the contents of biochemical constituents, such as total protein, total amino acid, total carbohydrate and total lipid were found to be significantly higher (P<0.05) in petroleum ether, acetone and ethanol extracts enriched Artemia nauplii fed PL when compared with control (Table 6). Among these, ethanol extracts enriched Artemia nauplii produced the best results followed by acetone and petroleum ether extracts.

Discussion Primary phytochemicals of P. amarus
Similar to that of the present study, the presence of alkaloids, tannins, terpenoids, saponins, phenolics, flavonoids and steroids have been reported in the hexane extract of P. amarus [55]. Alkaloids in ethyl acetate extracts of leaf and root, saponins in aqueous and methanol extracts of the stem, and flavonoids in petroleum ether extract of all parts of P. amarus have been reported [56]. Phenols and flavonoids from the root, stem and leaf of P. amarus have also been reported using different solvents, ethyl acetate, dimethylformamide, chloroform, dichloromethane and n-Hexane [57]. Dimethylformamide was reported to be the best solvent for extraction of phenols and flavonoids from the root, stem and leaf of P. amarus [58]. Qualitative phytochemical studies have also been reported in different species of Phyllanthus [59,60].
It has been reported that alkaloids, such as morphine, atropine and quinine are biologically and therapeutically active [61]. In the present study, alkaloids are moderately present in petroleum ether extract of P. amarus.
Terpenoids are known to possess antimicrobial, antifungal, anti-parasitic, anti-viral, antiallergenic, anti-spasmodic, antihyperglycaemic, anti-inflammatory, immunomodulatory and anticancer properties, and they have inhibition of cholesterol synthesizing property as well [62,63]. In the present study, terpenoids are moderately present in petroleum ether extract of P. amarus.
The flavonoids and other polyphenols are potentially beneficial to human health. They are known to contain a broad spectrum of chemical and biological activities including antioxidant, free radical scavenging, anti-ageing, anti-allergic, anti-inflammatory, anti-microbial, anti-    leukemic, vasodilator, anticancer and antibacterial properties and are reported to be useful for improving blood circulation in brain of Alzheimeric patients [64 -68]. The flavonoids, anthrax quinines and terpenes stimulate glucose uptake in cells [69]. Certain flavonoids exhibited hypoglycemic activity [70] and also beta cell regeneration in pancreas [69]. The leaf was reported to have the highest activity of phenols and flavonoids compared to root and stem. The antioxidant activity of flavonoids is efficient in trapping superoxide anion (O2), hydroxyl (OH), peroxyl (ROO-) and alcohoxyl (RO) radicals [71].
In the present study, ethanol extract of P. amarus contains luxuriant presence of flavonoids.
Tannins are used as the astringent substance in treatment of burns. They precipitate the proteins of exposed tissues to form a protective covering. They are used as mild antiseptics in treatment of diarrhoea, and to check small haemorrhages [72]. They are also used as healing agents in inflammation, leucorrhoea, gonorrhoea and piles. Tannins have been found to have antiviral, antibacterial, anti-parasitic, antiinflammatory, anti-ulcer and antioxidant properties [73]. In the present study, all the three solvent extracts of P. amarus contain luxuriant presence of tannins.
Phenols are structural and allelopathic components which are associated with diverse functions, like nutrient uptake, protein synthesis, enzyme activity and photosynthesis [74]. Phenolic compounds have biological and pharmacological properties, such as anti-microbial, anti-viral, anti-inflammatory, cytotoxic, anti-mutagenic and anticarcinogenic activities [67,75]. In the present study, moderate presence of polyphenols was detected in the ethanol extract of P. amarus.
The cardiac glycosides are one of the most naturally occurring plant phytoconstituents and are basically steroid drug with an inherent ability to afford a very specific and powerful action mainly on the cardiac muscle when administered through injection. Cardiac glycosides and catecholamines are agent of choice in treatment of congestive cardiac failure [78]. In the present study, luxuriant presence of cardiac glycosides was detected in the acetone extract of P. amarus.
Quinonines have anti-inflammatory, anti-bacterial and immunomodulatory potentials, and are very much used in the treatment of malaria and more recently of tumors [79,80]. In the present study, moderate presence of quinones was detected in the ethanol extract of P. amarus. The primary phytochemicals serve as health tonic in aquaculture nutrition [31,81,82].

Secondary phytochemicals of P. amarus
In the present study, dodecanoic acid (C 12 H 24 O 2 ), propanoic acid, 2-(tricyclo[3.3.1.13,7]dec-2-ylidene)-(C 13 H 18 O 2 ), tetradecanoic acid (C 14 H 28 O 2 ), neophytadiene (C 20 H 38 ), hexadecanoic acid, ethyl ester (C 18 H 36 O 2 ) and 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-were the active principle compounds of secondary phytochemicals/ bioactive substances identified from P. amarus. The dodecanoic acid (lauric acid: N-dodecanoic acid) is a common saturated fatty acid with a 12-carbon atom chain. Lauric acid is found in coconut and palm kernel oils. It is also found in human breast milk, cow's milk and goat's milk [83,84]. It is used for treating viral infections including influenza (the flu), swine flu, avian flu, the common cold, fever blisters, cold sores, genital herpes caused by herpes simplex virus (HSV), genital warts caused by human papilloma virus (HPV) and HIV/AIDS. It is also used for preventing the transmission of HIV from mothers to children. Further, it is also used in treatment of bronchitis, gonorrhoea, yeast infections, chlamydia, intestinal infections caused by a parasite called Giardia lamblia, and ringworm. Some research suggests lauric acid might be a safer fat than trans-fats in food preparations. It is safe for pregnant and breast-feeding women in food amounts (Lauric acid Wikipedia).
The propanoic acid is a naturally occurring carboxylic, short chain fatty acid. Its anion CH 3 CH 2 CO 2 as well as the salts and esters are known as propionates/ propanoates. Propionic acid inhibits the growth of mould and some bacteria, as a result, used as a food preservative. It is also used to make pesticides and pharmaceuticals. The esters of propionic acid are used as solvents or artificial flavorings [85]. In biogas plants, propionic acid is a common intermediate product, which is formed by fermentation with propionic acid bacteria. Its degradation in anaerobic environments requires the activity of complex microbial communities [86]. In propionic acidemia, a rare inherited genetic disorder, propionate acts as a metabolic toxin in liver cells by accumulating in mitochondria as propionyl-CoA (the initial metabolic product of propanoic acid) and its derivative, methylcitrate, two tricarboxylic acid cycle inhibitors [87,88]. In a study, Al-Lahham et al. [89] demonstrated that propionic acid lowers fatty acids content in liver and plasma, reduces food intake, exerts immunosuppressive actions and probably improves tissue insulin sensitivity. Thus, increased propionic acid in the body might be considered beneficial in the context of prevention of obesity and diabetes.
The tetradecanoic acid (myristic acid: 1-tetradecanoic acid) is also a common saturated fatty acid. Its salts and esters are commonly referred to as myristates. It was first isolated from nutmeg (Myristica fragrans) by Playfair Lyon [90]. Both lauric acid and myristic acid have positive effects on HDL (good cholesterol) level [91][92][93]. They have high hydrophobicity and act as lipid anchors in bio membranes of eukaryotic cells.
Terpenes and terpenoids are the primary constituents of the essential oils of many types of medicinal plants and flowers. The neophytadiene is a naturally occurring diterpenoid branched hydrocarbon belongs to the group of compounds known as phytanes. It is a member of the class of compounds known as sesquiterpenoids, which are terpenes with three consecutive isoprene unit makes neophytadiene a potential biomarker for the consumption of plant food product. Its presence can be estimated in the subcutaneous fat of animals [94]. These hydrocarbons are derived mainly from plant origin, being important components of vegetable wax [95]. They often have a strong odour and may protect the plants that produce them by deterring herbivores and by attracting predators and parasites of herbivores [96,97]. Terpenes have desirable properties for use in food, cosmetics and pharmaceutical products [98,99]. They are usually active ingredients of natural agricultural pesticides [100].
The hexadecanoic acid (palmitic acid) is the most common saturated fatty acid found in animals, plants and microorganisms [101]. Palmitates are the salts and esters of palmitic acid. The palmitate anion is the observed form of palmitic acid at physiologic pH (7.4). The excess carbohydrates in the body of human and animals are converted to palmitic acid. It is the first fatty acid produced during fatty acid synthesis and is the precursor to longer fatty acids. Palmitate negatively feeds back on acetyl-CoA carboxylase, which is responsible for converting acetyl-CoA to malonyl-CoA, which in turn is used to add to the growing acyl chain, thus preventing further palmitate generation. According to the WHO excess consumption of palmitic acid increases the risk of developing cardiovascular disease due to increase in LDL levels in the blood [102]. Among all fatty acids, palmitic acid has the strongest effect in boosting the metastatic potential of CD36+ metastasis-initiating cells in mouse models [103].  [104]. It is associated with cardiovascular (by helping to maintain normal heart rhythm, heart pumping and reduces blood clots) and neuropathologic diseases, and type 2 diabetes [105]. It is a potential nutraceutical to protect the brain from stroke, characterized by its pleiotropic effects in neuroprotection, vasodilation of brain arteries, and neuroplasticity [106]. It is also used to reduce high cholesterol, high blood pressure, asthma (decrease inflammation and improve lung function). ALA is used to treat rheumatoid arthritis, multiple sclerosis, lupus, diabetes, renal disease, ulcerative colitis, and Crohn's disease. It is also used to prevent pneumonia, chronic obstructive pulmonary disease, migraine headache, skin cancer, depression, and allergic and inflammatory conditions such as psoriasis and eczema. Ironically, ALA may raise some men's risk of getting prostate cancer. Its isomer is γ-linolenic acid (GLA, 18:3, n-6).

Survival, growth and biochemical constituents of M. rosenbergii PL fed with P. amarus extracts enriched Artemia nauplii
In the present study, ethanol extract of P. amarus enriched Artemia nauplii produced the best SR, WG and SGR (96.6%, 1.01g, and 4.1%, respectively against the control 82%, 0.6g and 3.9%, respectively) in M. rosenbergii PL than that of other two solvent extracts (Table 6). This is because of the bioactive principle compounds like dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid), hexadecanoic acid (palmitic acid) and 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-(linolenic acid) present in P. amarus might have been taken up by Artemia nauplii and transferred to M. rosenbergii PL. In addition to polyunsaturated fatty acid (PUFA), the saturated fatty acids (SFA) also required for better survival and growth of prawns. This in turn reflected on significant (P<0.05) elevations of total protein, amino acid carbohydrate and lipid levels (112, 55, 31 and 18 mg/g wet tissue, respectively against the control 62, 22, 11 and 7 mg/g wet tissue, respectively) in M. rosenbergii PL fed with ethanol extract of P. amarus enriched Artemia nauplii than that of other two solvent extracts ( Table  6). The antimicrobial, immunomodulatory and protective effects of other bioactive compounds, like propionic acid and neophytadiene present in P. amarus might have helped in maintenance of general health of M. rosenbergii PL. Artemia nauplii itself a very good live feed, it serves digestive enzymes and PUFA for larval growth of fishes and prawns. Herbal enriched Artemia has additional advantage that it carries bioactive substances.
The herbal products are eco-friendly, and have the characteristics ability of growth promoting, immune boosting, and appetizing effects in aquaculture animals. They increase consumption, induce maturation, and have antimicrobial as well as anti-stress capacities in aquaculture of shrimps and fin fishes [109]. It has been reported that in P. monodon PL fed with A. franciscana nauplii enriched with diets prepared by using Hygrophila spinosa, Withania somnifera, Zingiber officinalis, Solanum trilobatum, Andrographis paniculata, Psoralea corylifolia and cod-liver oil has significantly been improved the survival, growth and resistance to environmental stresses due to salinity [110]. In fish, Heros severus larvae, canola oil enriched A. nauplii produced better growth and stress resistance to temperature and oxygen deficiency and has converted n-3 fatty acids to EPA and DHA [111]. In rainbow trout, Oncorhynchus mykiss, the Artemia urmiana enriched with fish and plant oils (sunflower and canola oils) have produced significantly higher survival rate, specific growth rate and lower food conversion ratio. Further, they have improved the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels in Artemia nauplii, and the fish fed with canola oil enriched Artemia showed significantly higher stress resistance against thermal, salinity and hypoxia [112]. In Penaeus monodon, the methanol extract of Mucuna pruriens enriched Artemia had improved larval survival, whereas methanol extract of Withania somnifera showed improved total protein and lipid levels [113]. The survival, growth, and disease tolerance have been reported to increase in shrimp, P. indicus fed with individual herbal extract (Ricinus communis, Phyllanthus niruri, Leucus aspera, and Manihot esculenta) and seaweed (Ulva lactuca, and Sargassum wightii) enriched Artemia [114]. The enriched Artemia nauplii with sunflower oil, Spirulina and cod liver oil have been produced better survival, growth and concentrations of biochemical constituents in M. rosenbergii PL also [25,115,116]. No report available so far for P. amarus in Macrobrachium.

Conclusion
In the present study, petroleum ether extract, acetone extract and particularly ethanol extract of P. amarus enriched Artemia nauplii produced significant elevations in survival, growth, and concentrations of total protein, amino acid, carbohydrate and lipid levels in M. rosenbergii PL when compared with un-enriched Artemia nauplii fed PL. This clearly indicates the fact that the primary phytochemical constituents and bioactive substances present in P. amarus should have been taken to prawn through Artemia. Thus this herb sustainably exerted growth promotion in M. rosenbergii. Hence, P. amarus can be incorporated or taken as a feed additive in low cost on-farm feed formulations. This study can be extended further for isolation, purification and characterization of different bioactive substances of P. amarus for various biological applications.