Eﬀects of Probiotic Bacillus on Fish Growth Performance, Immune Response and Disease Resistance in Aquaculture

Aquaculture is one of the fastest-growing animal food-producing agricultural industries in the world and proper performance of fish in morphological, physiological and immunological aspects is important for fish production and sustainable expansion of aquaculture. But several inhibitors like disease, pathogen, and adverse environment can overpower these performances. At present, antibiotics in preventing these inhibitors have been seen as becoming favorable to those inhibitors. So, Bacillus , an important group of probiotic bacteria can be an alternative to these antibiotics in aquaculture. Bacillus has been seen used in different experiments, mainly as a supplement in feed at various concentrations. Bacillus showed effective results like improved growth with minimum cost, improvement in reproduction, hematology, improved immune response and disease, and stress resistance as well as better proximate composition in different fish species. Application of Bacillus strains has proven efficient in improving water quality by reducing ammonia and nitrite toxicity, harmful algal blooms and utilization of H + ion. Larger application of probiotic Bacillus instead of the hazardous synthetic chemicals would promote eco-friendly low-input sustainable aquaculture for food and nutritional security of the increasing world population. This study reviewed the effectiveness of Bacillus sp. as probiotic on fish growth and reproduction performances, immune response, hematological improvement, and stress resistance and water quality improvement in aquaculture.

circulans, Bacillus aerius and Bacillus polymyxa [28,29,30]. This study reviewed the efficiency of Bacillus spp. as a probiotic, and the major beneficial roles of probiotic Bacillus for promoting the sustainable aquaculture considering the antagonistic, health-promoting, immunity-inducing, bioremediation and other beneficial properties for the immense welfare of fish and fish production.

Probiotics in Aquaculture
Fish is the richest source of animal protein and is the fastest food-producing sector in the world.
Worldwide, people obtain about 25% of their animal protein from fish and shellfish and consumer's demand for fish continues to climb [31]. The production can be maximized through intensification with the addition of commercial diets, growth promoters, antibiotics, and several other additives. Application of these products leads to high production beyond any doubt, but the most worrisome factor is that the routine use of these measures causes severe complications and even a stage has come where its sustainability is in the stake [32]. In aquaculture practices, probiotics are used for a long time however within the most recent couple of years probiotics turned into a basic part of the aquaculture practices for improving growth and disease resistance. This strategy offers plentiful advantages to overcome the limitations and side effects of antibiotics and other drugs and also leads to high production through enhanced growth and disease prevention [33][34][35][36].
The Greek word probiotic means "for life", was introduced by Parker [34]. Probiotics are living organisms and substances, which contribute to intestinal and microbial parity. A more extensive meaning of the term probiotic is characterized as a live microbial aide which beneficially affects the host by modifying the host-associated microbial community and the related microbial network, by ensuring improved use of the feed or upgrading its dietary benefit, by enhancing the host reaction towards disease, or by improving the condition of its surrounding environment [36][37][38]. According to the currently adopted definition by FAO/WHO, probiotics are live microorganisms, which when administered in sufficient amounts confer a health advantage on the host animal [39].
A probiotic organism must have resistance to the acidic stomach environment, bile and pancreatic enzymes, accession to the cells of the intestinal mucosa, capable of colonization for a long period of time they can stay alive so that they can colonize the host efficiently, capable of producing antimicrobial substance against the pathogenic bacteria, and the absence of translocation and should be non-pathogenic and non-toxic [40]. Probiotic bacteria have become the fore of current research in fish culture because of their excellent effects in improving fish growth and health while ensuring environmental safety [41]. Several works [42,43,44] have reported growth enhancement following probiotic application in fish culture. In addition, increased of the activities of lysozyme, peroxidase, superoxide dismutase (SOD), catalase (CAT), protease, and anti-protease and immunoglobulin M (IgM) levels have been reported in serum and mucosal surfaces which are important defense molecules against many infectious pathogens in mass-cultured fish [45,46,47].
Probiotics are microorganisms that are claimed to supply health benefits when consumed [48]; the term became more common after 1980 and therefore being widely-used from 1989. The definition of the probiotics may be a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance [49]. The introduction of the concept (but not the term) is mostly attributed to laureate bacteriologist Elie Metchnikoff who postulated that yogurt-consuming Bulgarian peasants lived longer lives [50]. The dependence of the intestinal microbes on the food made it possible to adopt measures to change the microbiota in our bodies and replace the harmful microbes by using useful microorganisms [51].
Probiotics are initially defined as organisms or substances that contribute to intestinal microbial balance [37]. The term probiotic has emerged from the Greek words "Pro" and "bios" meaning "for life" and is commonly recognized as a support for living organisms that helps naturally to enhance the health condition of the host animal. According to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), probiotics are live microorganisms that, when administered in an appropriate amount provides health benefits to the host [36].
Probiotics are often defined as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance [49]. It can even be defined as a viable micro-organism which when ingested in a sufficient quantity confers a beneficial effect on the host due to an improvement of the intestinal microbial balance [54]. A decent definition for probiotics in aquaculture is, "live, dead or component of a microbial cell that when administered via the feed or to the rearing water benefits the host by improving either disease resistance, health condition, growth performance, feed utilization, stress response or general vigor, which is achieved a minimum in partially via improving the hosts microbial balance or the microbial balance of ambient environment" [55]. Another definition of probiotics is employed in aquaculture as "live microbial cultures added to feed or environment (water) to extend viability (survival) of the host" [56].
Nevertheless, aquaculture has been an expansion undertaken by FAO/WHO to incorporate a wide range of Gram-positive and Gram-negative bacteria, bacteriophages, microalgae and yeast with the appliance through the aqueous/water route, additionally as by supplying the feed. The essence of the definition of FAO/WHO is that probiotics are living organisms, which are administered orally having some tangible health benefits, and are being widely used for the control of diseases in aquaculture, especially in developing countries [11,53,57,58,59]. In practice, only limited evidence is there that the viability of probiotic cultures checked as soon as they are added to feed, and therefore the health benefits may be inaccurately described. Of course, it's going to be a control on the viability of the processing of diet, namely pelleting/granulation and extrusion. For example, the administration of the probiotic extruded diet led to an improvement in nonspecific immunity of Nile tilapia compared with granulated/pelleted diet [44,60].

Mechanism of Action of Probiotics
Probiotic selection depends on their colonization, antagonism to pathogens and therefore the production of beneficial compounds like vitamins, fatty acids and digestive enzymes [61]. For the successful application of probiotic strains as microbial ingredients in fish, other characteristics seem to be essential, like high viability during processing, storage and after gastro-intestinal transit [62]. A probiotic dosage is often bringing positive and negative results to different receivers, whose responses to different dietary probiotic levels are observed [63,64]. Major Probiotic mechanisms of action include enhancement of the epithelial barrier, increased adhesion to the intestinal mucosa, competitive exclusion of pathogenic microorganisms and production of anti-microorganism substances is as follow [60]: i.
Enhancement of the epithelial barrier: The intestinal barrier is a major defense mechanism used to maintain epithelial integrity and to protect the organism from the environment.
ii. Increased adhesion to intestinal mucosa: Adhesion to intestinal mucosa is regarded as a prerequisite for colonization and is important for the interaction between probiotic strains and the host.

Role of Bacillus in aquaculture
The beneficial roles of Bacillus as probiotic microorganism have been broadly studied during the most recent decade. Bacillus can contribute a lot to the inward physiological, morphological, hematological, and immunological status of fish by being available in the fish through the administration at an optimum level and improves water quality in aquaculture. The findings of the roles of Bacillus in fish as a probiotic are being illustrated below:

Bacillus in growth and reproductive performance
Probiotic microbes are able to colonize in the gastrointestinal tract and adhere to the intestinal mucosa of fish and exert their multiple benefits [52]. Dietary supplementation of probiotics in aquaculture offers an eco-friendly prophylactic measure in fish growth performance and health.
Gobi et al. [61] reported that the dietary supplementation of probiotic Bacillus licheniformis to Oreochromis mossambicus significantly improved the growth performance, immune parameters, enzyme activities and resistance against Aeromonas hydrophila. Analysis of growth parameters shows that the final weight, Specific Growth Rate (SGR) and Feed Conversion Ratio (FCR) of Oreochromis mossambicus increased significantly [61]. Weight gain and specific growth rate of fish fed with 10 7 CFUg -1 (D2) were significantly higher than those fed with 10 5 CFUg -1 (D1) and control diet. Fish fed with a control diet showed the lowest weight gain and specific growth rate ( Figure 2). There was no significant difference in FCR for fish fed diets, between D2 and D1. Adorian et al. [62] reported that the dietary supplementation with probiotic Bacillus (Bacillus licheniformis and Bacillus subtilis) in Asian sea bass Lates calcarifer showed significantly better growth than those fed the basal diet. The treatments were supplemented with 10 3 , 10 6 , and 10 9 CFUg −1 probiotics B3, B6, and B9 respectively. Highest SGR was found in B9 treatment while increasing the dose resulted in growth depression. Lowest FCR value was found in B9 treatment rather than others (Table 1). Based on this experiment, the relationship between different diets and SGR can be graphically illustrated in Figure 3. The addition of Bacillus sp. in the Guppy (Poecilia reticulata) increased fry production and reduced the fry mortality during spawning [63].

Figure 3 Relationship between dietary probiotics supplement level and specific growth rate in juvenile Lates calcarifer
The probiotic test feeds were prepared by gently delicately showering the necessary measure of bacterial suspension on the control diet and blending it part by part in a drum blender to obtain a prominent concentration of 5×10 8 cellsg 1 and 5×10 7 cellsg 1 control feed respectively.
Significant differences were found in average fecundity per female, the average number of fry survived per female, average weight and length of fry per female ( Table 2). The average fecundity/female, the average number of fry survived/female, average weight and length of fry/female were high in Bacillus sp. inoculated group than in control. The percentage survival of fry was more in Bacillus inoculated group than the control group thereby reflecting its positive influence on the reproductive performance of Poecilia reticulates (Figure 4 and 5).   Saputra et al. [66] reported that the dietary supplementation of B. amyloliquefaciens significantly improved the growth performance of Nile tilapia (Oreochromis niloticus) and immunity against Aeromonas hydrophila. Growth and feeding parameters including final weight (FW), specific growth rate (SGR) and feed conversion ratio (FCR) were significantly influenced by all probiotics with better growth performance and survival [74]. In another study, it has been found that the dietary administration of Bacillus aerius strain B81e significantly influenced weight gain, specific growth rate and feed utilization efficiency of Pangasius bocourti [76]. Ghosh et al. [79] reported that B. circulans significantly improve growth, reduce feed conversion ratio, and increase protein efficiency ratio in Labeo rohita when fed at a rate of 1.5 × 10 5 CFU100g −1 feed at 3% of the bodyweight of fish. Elsabagh et al. [83] reported that the growth performance, feed conversion ratio, and blood profiles in tilapia fed on Bacillus treated diets were notably increased. John et al. [84] demonstrated that the weight of Macrobrachium malcolmsonii fed on the Bacillus subtilis coated diet increased 3.5 times more compared to the control diet.

Bacillus in hematological and lysozyme contribution
Several studies have reported significant stimulations of serum lysozyme and phagocytic activities in Oreochromis niloticus [15,18,19 (Table 4). Diets containing 10 6 and 10 9 CFUg −1 probiotics led to a higher count of red blood cells when compared to other treatments.
For white blood cells and hematocrit independent of level probiotic supplementation, increased cell count and hematocrit percentage were compared to the control. A significantly higher percentage of hemoglobin was observed in the blood of a fish fed diet supplemented with 10 6 CFUg −1 probiotics. Lysozyme activity of plasma differs significantly between the treatments tested. Lysozyme activity was higher in animals fed the diet containing 10 6 CFUg −1 probiotics ( Figure 5). The treatments were supplemented with 10 3 , 10 6 , and 10 9 CFUg −1 probiotics B3, B6, and B9 respectively. Paralichthys olivaceus individuals treated with a commercial probiotic demonstrated significantly higher plasma LYS activity [48]. Abarike et al. [68] reported that the dietary supplementation of commercial probiotic Bacillus subtilis and Bacillus licheniformis significantly improved the protease, lysozyme, anti-protease activities, catalase activities and immunoglobulin in Nile tilapia, Oreochromis niloticus. Another study reported that the probiotic-treated L. vannamei postlarvae showed enhancement of antibacterial activity with a considerable increment of LYS [70]. A recent study reported that the isolated gut probiotic B.
subtilis with formulated diet insignificantly increased the hematological parameter values of the hemopoietic stimulation in freshwater fish Labeo rohita [77].

Bacillus in innate immunity and disease resistance
Probiotics in aquaculture enhance the immunity and disease resistance against bacterial pathogens. Using probiotics in aquaculture to control and prevent diseases has gained interest increasingly [64]. There have been reports of using probiotic bacteria to protect fish from bacterial infections [17,65,66]. The administration of probiotics can improve immunity and protect against several pathogens in many fishes such as Labeo rohita, Oreochromis niloticus, Epinephelus bruneus, Oncorhynchus mykiss, and Cyprinus carpio. Das et al. [16] remarked that the B. amyloliquefaciens can be utilized in aquaculture to improve health status and disease resistance in Catla (Catla catla) with an optimal dietary supplementation of 109 CFUg −1 . Beck et al. [40] supplemented the mixed probiotics of Lactococcus lactis BFE920 and Lactobacillus plantarum FGL0001 to olive flounder, Paralichthys olivaceus and reported that the growth performance, innate immunity, and disease resistance were significantly improved. Mixed probiotic showed better performance than the single probiotic agent in improving lysozyme activity and phagocytic activity of innate immune cells. In addition, neutrophil activity was also improved significantly compared to the control group.
Probiotics can be also used as immunostimulants in aquaculture. Selim and Reda [19] demonstrated that the dietary supplementation of Bacillus amyloliquefaciens improves innate immunity and disease resistance in Oreochromis niloticus with significant increases in IL-1 and TNF α mRNA levels in the kidneys. Fish that were fed Bacillus amyloliquefaciens exhibited better relative survival percentages when challenged by Yersinia ruckeri or Clostridium perfringens also appeared to enhance in vitro serum bactericidal activities against Aeromonas hydrophila.

Bacillus in stress resistance and water quality improvement
Probiotics are also known to play significant role in developing innate immunity in fishes, and thus help them to fight against any pathogenic microbes and also against environmental stressors. Taoka et al. [51] reported that Japanese flounder, Paralichthys olivaceus had greater heat tolerance after being treated with a commercial probiotic supplied in the diet and in the rearing water compared to the control. Abdollahi-Arpanahi et al. [74] studied the efficacy of probiotics Bacillus subtilis and Bacillus licheniformis on growth performance, immunophysiology and resistance response of juvenile white shrimp (Litopenaeus vannamei) and concluded that the shrimps fed with probiotics showed higher resistance to environmental stressors including low and high salinities, formalin, chlorine, high and low water temperatures, and ammonia. However, commercial probiotics fed shrimps demonstrated a better resistance than the individuals fed with the indigenous probiotic group. Such resistance is obviously due to better immunological functions of the animal, as higher levels in immune defensive cells, lysozyme activity, total protein as well all a lower cortisol level were measured in the commercial group compared to indigenous one. Liu et al. [75] reported an improved stress tolerance of Litopenaeus vannamei postlarvae after being treated with the probiotic B. subtilis E20, which may be due to the regulation of physiological response of shrimp by probiotic B.
subtilis to adapt acute environmental stresses. Rollo et al. [96] fed Sparus aurata fry with two probiotics (Lactobacillus fructivorans and Lactobacillus plantarum) using Brachionus plicatilis and subjected the fries to acute pH stress. They reported a significantly lower cortisol level, higher gene expression of heat shock protein 70, and significantly lower cumulative mortality.
The use of probiotics in lentic water bodies could improve fish wellbeing by boosting up many water quality parameters since they modify the bacterial arrangement of the water and dregs.
The water temperature, pH, dissolved oxygen, ammonia, and hydrogen sulfide were accounted for to be of more excellent when Bacillus were included the shrimp hatchlings culture in the green water system (Table 5). Bacillus spp. has advantageous effects in water quality improvement in aquaculture with better efficiency of transforming organic matter than Gramnegative bacteria [58]. Ammonia and nitrite concentration increase as the culture turns into an intensive operation. Ammonia and nitrite toxicity can be removed by the use of nitrifying bacteria into the fish environment [77]. Nitrosomonas spp. can be used in treating them to keep water quality in a suitable range, which may be conceivable in light of different roles played by the probiotic microscopic organisms. A buffering framework to dodge wide pH vacillation is fundamental for aquaculture [78]. Bacillus spp. have known to utilize different nitrogen sources, including both NH3 and NH4 + for catabolism of proteins and subsequently use H + particle [74].
Zink et al. [75] reported that the exchange of HCO3 − as a waste product resulting from respiration diminished the pH, which prompted physiological pressure. Probiotic Bacillus produces substances that hinder the growth of harmful algae by algicidal compound and forestall blooms. Algal bloom can possibly cover the surface of the water and prevent the daylight to enter into the water to provide the vitality to phytoplankton production [75][76].
Probiotic Bacillus also improves the decay of organic matters; diminishes nitrogen and phosphorus availability, and controls ammonia, nitrate, and hydrogen sulfide [65].
In situ bioremediation has also been widely used in aquaculture through bio-augmentation abuse endemic or exogenous probiotics, which modifies water quality. Devaraja et al.

Bacillus in enzyme activity and body composition improvement
According to [71], Cyprinus carpio juveniles were supplemented with different treatment of Bacillus circulans probiotic under laboratory conditions with following dosages: The treatments were CD (control), ED1 (2 x 10 2 CFUg -1 B. circulans), ED2 (2 x 10 4 CFUg -1 B. circulans) and ED3 (2 x 10 6 CFUg -1 B. circulans). In general, the total and specific activity of digestive enzymes (Protease, amylase, and lipase) remained significantly higher in the fish fed diet containing B.
circulans at ED2 diet in comparison with other treatments and control ( Table 6). The body composition of the fish can also be affected by the probiotics concentrations in diets. According to an experiment on Cyprinus carpio [71], a specific amount of probiotics can positively result in the improvement of body composition. The accumulation of carcass protein, lipid and ash were significantly higher in groups fed diets ED2. Carcass moisture contents remained significantly low at ED2 (Table 7).

Conclusions
The Gram-positive probiotic Bacillus is thought to be an important tool for promoting sustainable aquaculture because of they have significant advantageous effects for aquaculture industry. Bacillus exerts beneficial effects via multiple mechanisms including promoting growth performance of aquaculture organisms, suppress irritation and disease infestation by induction of immunity, promote nutrition, and improve aquatic ecosystems for growth and reproduction

Conflicts of Interest:
The author declares there is no conflict of interest.