Dietary Effect of Padina boergesenii on Growth, Immune Response, and Disease Resistance Against Pseudomonas aeruginosa in Cirrhinus mrigala

In the aquatic environment, seaweeds have the potential to be renewable resources. The current study was designed to assess the impact of seaweed Padina boergesenii incorporated into a basal diet at various concentrations. The phytoconstituents of the seaweeds were characterised by gas chromatography-mass spectrometry. Diets were designed to include elevated levels of 0.5%, 2.5%, 4.5%, and 6.5% of seaweed meal. Significant differences in Cirrhinus mrigala fed with P. boergesenii incorporated into the basal diet for 45 days. The growth parameters (weight gain, specific growth rate), biochemical constituents, and immunological assays were observed. The extract fraction demonstrated effective inhibitory activity against Pseudomonas aeruginosa. As a result, this research suggests that extracts of the seaweed P. boergesenii contain potential bioactive compounds with significant antibiotic activity.


Introduction
Aquaculture is an antiquated form of husbandry. Aquaculture is the cultivating aquatic organisms in marine, brackish water, and freshwater zones, and they involve interventions in the rearing process to augment the production [1]. Aquaculture cultivation has expanded significantly, particularly in the last few epochs, resulting in intensive culture performances. In recent years, aquaculture has grown in importance as a component of the global economy. Aquaculture now accounts for half of global aqua food production, with 179 million tonnes of fish caught per year in 2018. Aquaculture will continue to be the primary factor behind worldwide fish output increase, extending a decade-long trend [2]. Nutrition and protein are provided through aquaculture and fisheries. Aqua food, fish, and by-products provide about 3 billion people globally with 20% of their daily animal protein [3].
For many years, aquaculture of fish or other aquatic organisms with any illness or pathogen infection has been treated with commercially available antibiotics. The emergence of antibiotic-antagonistic bacteria and other organisms associated with fish diseases is a global threat to aquaculture that has received widespread attention in recent years and is increasing the number of organisms resistant to antibiotics. Infectious diseases are major problems in the field. They cause direct losses in biomass and productivity, as well as indirect effects on trade restrictions. The aquaculture animals are incapacitated by various pathogenic microorganisms and hosts [4]. These trends show that in recent years, there has been an increase in the use of aquatic organisms as a potential and promising source of pharmaceutical agents [5].
Macroscopic marine algae have been closely associated with human life since ancient times and have been extensively used as a source of food, feed, fertiliser, and medicine in a variety of ways. Seaweeds are non-blooming plants with no root, stalk, or leaves. Seaweed is abundant in shallow marine water and in estuaries. "Holdfasts" are typically used to attach marine macroalgae to the bottom or other solid structures. Algae generate several valuable natural metabolites that can be used in the pharmaceutical and food industries. Algae are currently used to identify biologically active complexes like omega-6 fatty acids, stearic acid, omega-9 fatty acids, fucoxanthin, fucoidyl, loloride, and dechlorophysterol [6].
Seaweeds contain more than 60 trace elements, compared to terrestrial plants [7]. Oil fatty acids, peptides, lipids, brominates, aromatics, nitrogen-heterocyclic, nitrosulphuric heterocyclic, dibutanoids, proteins, alkaloids, flavonoids, terpenoids, and sulfated polycarbohydrates are among the many bioactive compounds isolated from marine macroalgae, These compounds have the ability to promote growth and body biochemical constituents in fish, as well as improve immunity, antibacterial activity, and anti-stress [8]. Seaweeds contain a variety of immunologically active substances, primarily polysaccharides, which can alter the activity of certain immune system components and increase protection against certain diseases.
Plant-based proteins have a comparatively low price and are easily available resources. Anti-nourishment factors (e.g. phytoestrogens, protease inhibitors, lectins, and phytic acid) and scantling or asymmetry in their vital amino acid melange may compromise the growth and health of farmed aquatic species [9]. Recent studies have revealed that seaweeds have stimulatory effects on the non-specific immune systems of humans and animals, attracting the attention of many researchers who want to use seaweeds as immunostimulants to improve animal health and prevent disease outbreaks, thereby reducing the use of antibiotics and chemotherapeutics. Immunostimulants are used to boost the immune system and are effective against a variety of opportunistic and secondary pathogens [10]. Despite the fact that seaweeds have the potential to be used in a wide range of commercial and therapeutic applications, many people are unaware of their enormous impact. In light of the foregoing, the current study was designed to assess the phytocompounds and biological activity of selected seaweed.
Pseudomonads are among the most dangerous fish pathogens, causing ulcerative syndrome and haemorrhagic septicemia. P. aeruginosa is an aerobic Gram-negative motile rod with catalase and oxidase activity. Furthermore, this bacterium has many virulence-related determinants, including cell-mediated and secreted virulence. Pseudomonas species have demonstrated varying resistance patterns to varied antimicrobial agents. P. aeruginosa, especially, could develop innate and/or acquired. Nowadays, serious efforts to detect P. 1 3 aeruginosa are being made not just for economic reasons, but also for public health reasons [11]. Pseudomonas infection, most ordinarily caused by P. aeruginosa, maybe a public health concern that causes many healthcare-associated illnesses in consumers. Pseudomonas species are presently catalogued as a food-borne illness that affects humans by consuming spoiled foods and ready-to-eat products, as well as manipulating contaminated seafood. Although few studies have claimed possible routes of disease transmission, people with weakened immune systems or those who work with infected fish are usually in danger. In this context, the present study focused on determining the immunostimulatory effect of P. boergesenii on Cirrhinus mrigala against P. aeruginosa [12].

Experimental Animals
The freshwater fish, Cirrhinus mrigala (Hamilton 1982), was obtained from the Tamil Nadu Fisheries Development Corporation, Bhavani Sagar Dam-Erode, Tamil Nadu. For 15 days before start of the experiments, 500 fish were kept in a cement tank (1000 L) filled with water in the laboratory. Fish were fed rice bran during the acclimatisation period. To maintain a healthy environment, 80% of the water is renewed every day.

Collection and Identification of Seaweeds
Algae were collected in the coastal areas of Mandapam and Rameshwaram on the southeast coast of India in June 2019. After gathering, it was thoroughly washed and cleaned with seawater, freshwater, and distilled water to remove all holdfasts, epiphytes, calcareous sand, and other adhering detritus material. The water was drained from the thallus, which was then put on blotting paper to absorb any remaining moisture. The seaweeds were identified as Padina boergesenii with the help of the Botanical Survey of India. The collected samples were air-dried and ground into a fine powder [13,14].

Preparation of Extract and GC-MS Analysis
Methanol was used to extract the powdered material that had been subjected to the Soxhlet apparatus. A Perkin Elmer Clarus Series gas chromatographic system with a capillary column was used to analyse the phytochemical constituents of the seaweed sample. The products were identified by comparing retention time and fragmentation pattern with known reference complexes as well as mass spectra in the library search results kept in the software (Turbo. Mass. ver 5.42). The GC-MS results were compared with the standard compounds in the library.

Preliminary Phytochemical Tests
The extracted samples were analysed to determine the presence of different phytocompounds [15].

Collection of Bacterial Strain
The P. aeruginosa bacterial strain was obtained from PSG (IMSR), Coimbatore, Tamil Nadu. A subculture of this bacterium was made in Pseudomonas broth and maintained at 37 °C for 24 h.

Antibacterial Susceptibility Testing by Kirby-Bauer Method
The disc diffusion method was used to test antimicrobial susceptibility using a Muller Hinton agar plate (HI Media Laboratories, Mumbai, India, MV1084), as recommended by the Clinical and Laboratory Standards Institute. Commercially available antibiotic discs such as gentamicin (10 g/disc), amikacin (30 g/disc), ciprofloxacin (5 g/disc), neomycin (30 g/disc), tobramycin (10 g/disc), ceftazidime (30 g/disc), oxacillin (30 g/disc), meropenem (10 g/disc), imipenem (/disc), and vancomycin (30 g/disc) were used to examine the multidrug resistant (MDR). The zone of inhibition was measured using the HI media antibiotic scale and compared with the zone interpretative chart [16].

Antibacterial Assays-Well Diffusion Method
The antibacterial activity of Padina boergesenii was assessed by the well diffusion method using standard methods [17].

Feed Preparation
Fishmeal and soybean meal were used as protein sources in the diets, which were prepared using commercially available feed ingredients. Carbohydrate sources included rice bran, groundnut oil cake, wheat flour, and tapioca flour. Cod liver oil was used as a lipid source, and egg albumin was used as a binding agent. Vitamins and minerals were also added.
The dough mixer was prepared and cooked in a closed aluminium container at 105 °C for 20 min followed by cooling at room temperature. Subsequently, the 0.5%, 2.5%, 4.5%, and 6.5% seaweed powder were added individually.
The egg albumin, cod liver oil, vitamins, and minerals were mixed until a stiff dough was obtained. The dough was pelletised and cut into 3.0 ± 0.97 mm pieces using an indigenous hand pelletiser with a mesh size of 0.1 mm diameter (pigeon manufactures, Kolkata, India). The pellets were dried at room temperature until they reached a constant weight. The prepared feed was stored individually in airtight plastic containers at − 20 °C until used for the feeding trials (Table 6).

Experiment Trial
After acclimatisation, the fish were divided into triplicates of five groups. Each group contains 20 fish. The T 1 to T 4 experimental diets were fed with P. boergesenii extract mixture at varying concentrations (0.5%, 2.5%, 4.5%, and 6.5%), with the basal diet, and the control group (T) was devoid of the seaweed extract [18][19][20].

Growth Parameters and Survival Rate
The following growth parameters were evaluated: The following formula [21,22] is used to calculate specific growth rate (SGR), initial weight (IW), final weight (FW), weight gain (WG), initial length (IL), final length (FL), survival rate, feed conversion ratio (FCR), and protein efficiency ratio (PER).
Survival rate = No of fish treated (Initial)-No of fish remaining (final) × 100 Feed conversion ratio (FCR) = Total feed given (g) / Weight gain (g) Protein efficiency ratio (PER) = Weight gain by fish (g) / Protein intake (g) × 100 Weight gain (WG) = Final body weight of fish (g) -Initial body weight of fish (g) Specific Growth Rate (SGR) = Final mean body weight (g) -Initial mean body weight (g) / Time interval (days) × 100

Analysis of Muscle Biochemical Constituents
At the end of 45 days, the fish analysed for total proteins was determined by the method of [23]. Total carbohydrate was determined by using the method of [24]. Total lipid content was determined by the [25] method.

Immunological Assays
Blood samples were taken from each experimental group of fish. The blood was drawn from the caudal vein with a syringe that had been rinsed with an anticoagulant solution to prevent clotting. The collected blood samples were mixed with EDTA solution for analysis of RBC, WBC, Hb, packed cell volume, MCV, MCH, MCHC, and lysozyme activity [25][26][27].

Statistical Analysis
Triplicate samples were used in all experimental groups. Results for each parameter were measured and expressed as the mean ± (SD). The data was analysed using one-way ANOVA in SPSS version 16, and mean values were compared using the Duncan multiple range test. Differences were considered statistically significant when p < 0.05.

Phytochemical Analysis
Results of phytochemical analysis Padina boergesenii yielded favourable results for a wide range of substances, including flavonoids, alkaloids, saponins, phenolic compounds, terpenoids, amino acids, and carbohydrates (Table 1). Glycosides and phlobatannins are absent in Padina boergesenii.

Gas Chromatography-Mass Spectroscopy Analysis
The bioactive compounds from the methanolic extract of seaweed P. boergesenii were analysed for the presence of secondary metabolites and chemical compounds with respective peak area percentages. GC-MS analysis of the methanol extract of P. boergesenii showed fourteen peaks, which indicate the presence of phytochemical constituents (Graph 1). These compounds were identified through mass spectra compared with the NIST library. Extracted seaweed samples contained fourteen phytochemical constituents that were characterised and identified. They are chloroxylenol, hexadecanoic acid, arachidonic acid, stearic acid, eicosatetraenoic acid, cis-vaccenic acid, pyrano, octadecenoic acid, phytol, butanoic acid, 13-heptadecyn-1-ol, glycidyl palmitate, glycidyl oleate, and glucobrassicin, and mass spectra chromatogram was presented. Other chemical compounds and their functions based on reports were tabulated ( Table 2).

The Antibacterial Assays-Well Diffusion Method
In the present study, a methanol extract of P. boergesenii shows significant inhibition activity against P. aeruginosa. The minimum inhibitory concentration of P. boergesenii was shown to be 23 mm.

Growth Parameters
Significant increases in survival, growth rate, weight gain, specific growth rate, protein efficiency ratio, feed conversion ratio, and survival rate of C. mrigala fed with Antifungal [17] different concentrations of seaweed extract mixed were observed in the current study. Over the entire experimental period, the growth parameters of the experimental groups were significantly (p < 0.05) different from the control. There is no significant difference between the T 3 and T 4 groups among the various treatment groups. The use of seaweeds in feed resulted in improved survival and growth of C. mrigala. The maximum weight gain was observed in T 3 (4.5% of brown algae, P. boergesenii) compared to control. Feed conversion ratio (FCR) indicates that the seaweed-incorporated feed was effectively utilised for growth in the dietary treatments, with the best value that was obtained in T 3 (4.5% of brown algae-P. boergesenii) compared to other groups and control. Feed intake was improved due to the presence of essential amino acids such as glutamic acid and aspartic acid, which can enhance the flavour and taste of the feed. Dietary supplementation of P. boergesenii significantly enhances the specific growth rate of C. mrigala when compared to the experimental group T. Whereas reduced growth was observed with an increase in the seaweed content in the fish feed, T 4 experiment was 6.5%. We conclude that the optimum level is T 3 (4.5% of brown algae) fed with C. mrigala ( Table 4). The experimental fish were challenged with the P. aeruginosa pathogen. A 94% survival rate was observed in experiment T 3 fed with 4.5% of brown algae compared to control group T 20% (Fig. 1).

Biochemical Constituents
In the present study, biochemical constituents of C. mrigala were significantly different (p < 0.05) in all experimental setups when compared to the control group. Among the various treatment groups, no significant difference existed between T 3 and T 4 . When compared to other experiments and the control group, the experimental group T 3 had a high protein level. Carbohydrate content was high in all treated groups when compared with control. A high level was detected in experiment T 3 (4.5% of P. boergesenii). Total lipid content in all treated groups was not significant compared to control (Table 4). Overall, experiment T 3 with 4.5% of P. boergesenii shows good activity.

Haematological Parameters
The WBC, RBC, Hb, PCV, MCV MCH, and MCHC count was significantly different from control during the experimental period. Among the various treatment groups, no significant difference existed between T 3 and T 4 . The RBC count was high in group T 3 (4.5% brown algae) whereas lower in the control. A high WBC count was observed in T 4 (6.5% brown algae), among other experimental groups, while a lower WBC count was observed in the control group. Other parameters like Hb, PCV, MCV, MCH, and MCHC were compared with control. The T 3 (4.5% brown algae) experiment shows higher activity compared to the control. The PCV level was high in T 3 (4.5% brown algae), whereas low levels were recorded in the control group. The MCHC level was high in T 3 (4.5% P. boergesenii) compared to control (Table 5).
In the post-challenge study, WBC count (Fig. 2) and lysozyme activity (Fig. 3) were increased to significant levels, except for the control group. The reduction of haematological parameters in fish feed has the lowest supplementation of P. boergesenii (Table 6). Survival rate (%) Fig. 1 Survival rate of C. mrigala. Pre before challenging study, post after challenging study Table 5 Haematological parameters of experimental fish Cirrhinus mrigala, mean ± SD (n = 3); mean values within the same row sharing the same superscript are statistical significant level (p < 0.05) The data highlighted in bold has shown good activity comparatively with other experiments

Discussion
In this study, we investigated that the phytocompounds and biological activity of P. boergesenii and C. mrigala fed with dietary supplementation with extracts of P. boergesenii led to an increase in growth performance, chemical body composition, and different immunological parameters. Based on the in vitro examination of antimicrobial, phytochemical, and bioactive compounds, P. boergesenii was selected as an immune stimulant to be incorporated into the diet of C. mrigala. Several studies have shown that seaweed or its extracts have different biological activities, including antitumor [28], antiprotozoal, antiviral antioxidant [29], and cytotoxic activity against human cancer cell lines. Active compounds from seaweed were found to be active against humans and animals, particularly fish and bacterial pathogens. Seaweeds contain many fatty acids and secondary metabolites. They have higher antibacterial activity against gram-positive bacteria and gram-negative bacteria. An antimicrobial susceptibility test of P. aeruginosa showed that it was resistant to different antibiotics. The current results are similar to those reported [30] and show that P. aeruginosa has multifactorial mechanisms and Besides, P. aeruginosa has natural protection from numerous antimicrobials due to the bacterium's external film obstruction, the presence of multidrug efflux carriers, and endogenous antimicrobial inactivation. In this study, P. boergesenii exhibited positive results for flavonoids, saponins, and terpenoids [31]. Fao [3] reported that seaweed is the source of bioactive compounds that can produce a great variety of secondary metabolites characterised by a broad spectrum of biological activities (cytostatic, antiviral, anthelmintic, antifungal, and antibacterial). The existence of secondary metabolites and chemical components in the methanolic extract of P. boergesenii was demonstrated in this study using gas chromatography and mass spectroscopy. Seaweeds contain polysaccharides, fatty acids (omega 3), vitamins, and minerals. These compounds can promote growth and biochemical constituents in fish, as well as help to improve non-specific immunity, antibacterial activity, and anti-stress. Similar findings were also reported in the fish fed with A. paniculata. They fed fish that were responsible for increasing the feed intake and giving resistance to invading pathogens [8].
C. mrigala was fed with dietary seaweed P. boergesenii at elevated levels, and improved growth parameters were observed in a dose-dependent way. In the present study, the survival rate, weight gain, and specific growth rate in three experimental groups were increased compared to the control group, and this research agreed with the similar findings of [32]. The decrease in fish growth and feed utilisation was observed in experimental group T 4 (6.5 percent P. boergesenii) because seaweeds contain more anti-nutritional factors (saponins, tannins, and phytic acid) that may affect fish growth; similar findings have been reported by other researchers [33].
Biochemical constituents are important quality traits of aquatic animals for marketing [34,35]. In the present study, increased protein, carbohydrate, and lipid content in experiment T 3 were compared to control and other experimental groups. It indicates that supplement feed additives can promote the absorption and storage of nutrients in the muscles of C. mrigala; similar findings are reported by [19,36]. Whereas reduced biochemical constituents increased the inclusion of P. boergesenii meal, this is in agreement with the results of [37]. According to this study, C. mrigala had higher levels of RBC, WBC, and HCT. This could be due to the enhanced non-specific immune system of C. mrigala.
The findings from this study are supported by preliminary studies showing that dietary supplementation of seaweed can improve the immunity of fish [38,39]. V. Uthayakumar et al. [40] reported enhancement of non-specific immunity through the application of natural immunostimulants. In a post-challenge study, the administration of methanol extract of 4.5% of brown algae increased SR in Cirrhinus mrigala, compared to control. P. boergesenii meal has improved the non-specific immune parameters and disease resistance against P. aeruginosa. This might be due to the enhancement of the non-specific immune system of the fish by P. boergesenii meal. There is strong experimental evidence for P. boergesenii that feeding can modify the activity of the fish immune system by stimulating the nonspecific immune response and increasing disease resistance in several fishes reported by [41][42][43].
Haematological parameters are useful indicators of health status in fish. In our study, fishes were fed a seaweed mixed diet, and the experiment was carried out for 45 days. The haematological and growth parameters increased from the first day to the last day. It has proved to improve fish health. From this study, we have concluded that seaweed is a good food ingredient, and it enhances the non-specific immunity (WBC count and lysozyme activity) and therefore increases the disease resistance of C. mrigala.

Conclusion
The presence of active antibacterial compounds such as alkaloids, flavonoids, saponins, and terpenoids and fatty acids such as hexadecanoic acid, arachidonic acid, octadecenoic acid, and phytol in the experimental feed containing Padina boergesenii extract enhanced the growth, biochemical content, immune response, and protection against P. aeruginosa. The experiment T 3 with a 4.5% concentration of seaweed incorporated into the diet is optimum to stimulate the immune response. The baseline data will be critical in the fish farming industry. As a result, seaweeds may be beneficial to aquaculture, pharmaceutical, and therapeutic applications in the future.