Single and Multi-Strain Probiotics Supplementation in Commercially Prominent Finfish Aquaculture: Review of the Current Knowledge

The Nile tilapia Oreochromis niloticus, Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, olive flounder Paralichthys olivaceus, common carp Cyprinus carpio, grass carp Ctenopharyngodon idella and rohu carp Labeo rohita are farmed commercially worldwide. Production of these important finfishes is rapidly expanding, and intensive culture practices can lead to stress in fish, often reducing resistance to infectious diseases. Antibiotics and other drugs are routinely used for the treatment of diseases and sometimes applied preventatively to combat microbial pathogens. This strategy is responsible for the emergence and spread of antimicrobial resistance, mass killing of environmental/beneficial bacteria, and residual effects in humans. As an alternative, the administration of probiotics has gained acceptance for disease control in aquaculture. Probiotics have been found to improve growth, feed utilization, immunological status, disease resistance, and to promote transcriptomic profiles and internal microbial balance of host organisms. The present review discusses the effects of single and multi-strain probiotics on growth, immunity, heamato-biochemical parameters, and disease resistance of the above-mentioned finfishes. The application and outcome of probiotics in the field or open pond system, gaps in existing knowledge, and issues worthy of further research are also highlighted.


Introduction
Aquaculture, the farming of aquatic organisms under controlled environments, is a diverse food producing activity growing annually by 4.5%, accounting for a value of USD 243.26 billion [1]. Data suggest that fish, macroalgae, molluscs and crustaceans are meeting the protein demand of an increasing global population, contributing 49.1, 27.3, 15.6, and 7.9%, respectively, to total aquaculture production, equalling ~20% of total global animal protein supplies [2]. Aquaculture practices have shifted from extensive to super-intensive to elevate production at the expense of huge amounts of artificial feeds and degradation of aquatic environments. Poor culture management causes immunosuppression, stress and creates suitable conditions for the proliferation of opportunistic microbial pathogens (bacteria, viruses, and parasites) leading to infectious disease outbreaks. Hemorrhage, exophthalmia, meningoencephalitis, anorexia, and disruption of the nervous system are common impacts of infectious diseases responsible for high mortality of in cultured fishes. To control diseases, antibiotics, drugs and vaccines are routinely used in aquaculture [3].
Vaccination is a well-established strategy for long-term disease protection, but it has been associated with

Probiotics Functionality Linked to Growth, and Cellular and Humoral Immunity
Probiotics not only reduce intestinal pathogens but also detoxify harmful compounds in feed through hydrolytic enzymes and the production of vitamins, biotin, and vitamin B12 [24,25]. Growth elevation with improved feed utilization can ensure improved yields at lower production costs which are of value to aquaculture. Several authors have characterized responses to probiotics including alterations of digestive enzyme synthesis and release [26] and of intestinal structure to increase nutrient digestion and absorption [27], influencing the final body weight gain and production scale.
In aquaculture, probiotics can be supplemented through feed or water to manipulate the microbial balance in host and culture environment. Different nutrient specific digestive enzymes (e.g., amylase, protease, chitinase and lipase) can be increased by probiotics in the intestine [28], improving digestion and absorption while removing toxicity. Jang et al. [29] mentioned that probiotics improved digestive process by enhancing beneficial bacterial population, microbial enzymatic activity, and microbial balance, ultimately improving digestibility and absorption of nutrients toward increased growth rates and diet digestibility. It is unknown whether digestive enzymes are produced directly by probiotics or by modulating synthesis and secretion of enzymes by intestinal cells, or possibly by both mechanisms. However, probiotics reportedly demonstrate in vitro extracellular amylase, cellulase, lipase and protease production and could potentially modulate those enzymatic functions in vivo [30].
Normal light microscopic observation on fish gut histology depicts an intact epithelium barrier, goblet cells, and well organised villi and microvilli. Supplemented probiotics metabolize the carbohydrates (oligosaccharides/ prebiotics) of diet for their growth and survival in the intestine. A combined application of probiotics and prebiotics, termed synbiotics, produced significantly higher effects compared to each individual component [11,31]. Fermentation of prebiotics by probiotics is reported to produce short chain fatty acids, which can be utilized as an energy source by intestinal epithelial cells [32], and cell proliferation ultimately increases goblet cell intensity, villus and micro villus height, width and density [33]. Alteration of these morphometric structures also can be found in intestinal thickness, fold of change, and mucus layer, contributing to the elevation of nutrient absorption. Higher nutrient absorption through increased absorption area in the intestine in response to probiotics supplementation showed positive changes in apparent digestibility coefficient [19] that correlated with fish growth and utilization of diets.
Probiotics mainly upregulate the fish innate immune parameters through antigen presenting dendritic cells (DCs) to maintain the linkage between innate and adaptive immunity [34]. Mucus on body surface, scale, skin, and gills serve as physical or epithelial barriers as a first line of defence in fish innate immune system. The role of probiotics to improve epithelial barrier is not well defined, but immunoglobulins (Ig), antibacterial peptides and complement proteins containing skin mucus lysozyme activity were enhanced after probiotics supplementation [35,36]. Macrophage and phagocytic cells (natural killer cell, neutrophil, monocyte, lymphocyte, and cytotoxic cells) provide cellular immune defence in fish [37]. Interaction between microbial-associated molecular patterns (MAMPs) and pattern recognition receptors (PRRs) on DCs can stimulate and activate the phagocytic cells or increased production of cytokines [38]. Lactic acid bacteria (LAB), for examples Bacillus and bifidobacteria are commonly used gram-positive probiotics, and their cell wall components like peptidoglycans, lipopolysaccharides, flagella, and microbial nucleic acids are collectively known as MAMPs. MAMPs are attracted by PRRs on DCs and after interaction/binding of MAMPS with PRRs, DCs stimulate and activate phagocytic cells [39] to locate and engulf invading pathogens. Stimulation of plasma cells by DCs produces antibodies which can pass through the enterocyte and neutralize the pathogens [8]. Moreover, binding of MAMPs with Toll-like receptors (TLR) and stimulation of T cells by DCs produce mostly pro-inflammatory cytokines [31]. Among different pro-inflammatory cytokines, tumor necrosis factor (TNF)-α stimulate neutrophil base immunity [40], interleukin (IL)-1β proliferate lymphocyte and macrophage [41], interferon (IFN)-γ stimulate resting macrophages to secrete IL-6 and IL-1β, and multi-functional IL-6 are responsible for B-lymphocyte differentiation and maturation [42] to elevate cellular innate immunity.
Humoral immunity refers to activities of bacteriolytic or haemolytic enzymes like myeloperoxidase or peroxidase, serum/skin mucus lysozyme, serum antiprotease, superoxide dismutase, serum bactericidal activity, alternative complement activity, transferrin and Ig in body or tissue fluids [10,39,43], which contribute to the elimination of pathogens. Serum proteomic study after Bacillus sp. JB-1 supplementation increased transferrin protein quantity in rainbow trout [44]. In human, oral administration of probiotics interacts with intestinal epithelial cells to enhance the production of macrophage chemoattractant protein-1, to send signals to other immune cells characterised by an increment of IgA + cells in the intestine, bronchus, and mammary glands [45]. Lactobacillus acidophilus La1 can persist in the gastrointestinal tract and act as an adjuvant to increase serum IgA titre to Salmonella typhi Ty21 in human [46]. Lactococcus lactis increased the expression of complement receptors and adjuvant potential of Lactobacillus GG to elevate the humoral immunity [47]. Lysozyme, defensins, cathelicidins, and phospholipase are antimicrobial peptides secreted by Paneth cells located in the bottom of intestinal crypts [48]. A transmission electron microscopic study depicted Lactobacillus casei CRL 431 and Lactobacillus paracasei CNCM I-1518 increased Paneth cells activity, apparently increasing the secretion of antimicrobial peptides in intestines [49].

Tilapia
Tilapia alone contributed 10% (5.377 million tonnes) of global aquaculture production in 2016 [1]. Nevertheless, its intensive production is more susceptible to stress-related disease and mass mortalities [50], and probiotics are viewed a promising potential solution [51].
Dietary Clostridium butyricum improved final body weight and feed digestion at 1.5 × 10 8 CFU g -1 [56], but not at 10 5 CFU g -1 [57] and protection against A. hydrophila and S. agalactiae, respectively. This probiotic decreased serum malondialdehyde and diamine oxidase but upregulated immune genes expression levels. Similar parameters were modulated along with increment of immunity, and digestive enzymes were also induced by B. subtilis HAINUP40 [58] and B. licheniformis DAHB1 [59]. Protection against S. agalactiae and A. hydrophila was also reported after feeding with a combination of B. subtilis and B. licheniformis [60] and various strains of Bacillus spp. [61], respectively.
Immunity modulation was also confirmed by B. cereus as a water supplement at 10 4 CFU ml −1 [62]. Combination of Bacillus with Pediococcus sp., Enterococcus sp. and Lactobacillus sp. did not produce any notable enhancement of digestive enzymes or other serum biochemistry parameters [63]. Expression of inflammatory, antimicrobial and T cell response genes after administration of B. subtilis ABP1 is an indication of its oral vaccine property [64]. Paenibacillus ehimensis NPUST1 [65] and Aspergillus oryzae [66] improved growth and immunological competence, but lowered the levels of glucose and cortisol in blood. Similarly, gut probiotic Rummeliibacillus stabekisii modulated the aforementioned parameters with enriched gut microbiota and inhibit A. hydrophila and S. iniae [67]. In addition, supplementation with Psychrobacter maritimus S and P. namhaensis S089 showed similar modulation except lower HSP70 [16,68] and B. amyloliquefaciens increased fat digestibility [69]. B. velezensis TPS3N, B. subtilis TPS4 and B. amyloliquefaciens TPS17 [70]; Bacillus sp. KUAQ1 and KUAQ2 [71] improved tilapia innate immunity. In addition, intestinal lipase activity and morphometric alteration, viz. micro-villus height and width, goblet cells count, and muscle thickness were reported by the former Bacillus probiotics. Administration of B. subtilis WB60 with L. lactis for 8 weeks resulted in better growth, immunity and immune genes expression, villus height, without changing the haematology [72]. Similar modulations were observed after supplementation with L. plantarum L-137 [73] and only gene transcription by L. plantarum [74]. The Bacillus and Lactobacillus probiotics specified above conferred elevated protection against different infectious diseases caused by E. faecalis, A. hydrophila, and S. agalactiae.

Atlantic Salmon
Comparatively less information is available on the effects of probiotics in Atlantic salmon. Available information can be categorized into in vivo and in vitro studies as summarized in Table 2.
Wang et al. [80] assessed the effect of B. velezensis V4 CGMCC 10149 and Rhodotorula mucilaginosa CGMCC 1013 on juvenile salmon raised in a recirculating aquaculture system. After 62 days feeding with different doses of these strains, fish weight gain, immunity as well as disease resistance against A. salmonicida improved significantly. In the same species, administration with P. acidilactici MA18/5M containing commercial probiotic (Bactocell) upregulated TLR3, TNF-α, and IL-1β, enriched gut microbial community and probiotic count in the intestine [81]. Meanwhile, fish growth rate and protection against infectious pancreatic necrosis virus were not affected by this commercial probiotic. In another study, Bactocell reduced intestinal inflammation, maintaining intestinal homeostasis of Atlantic salmon after inducing inflammation with oxazolone [82].
Trout intestinal Lactobacillus (RII and RIII) was administered at a dose of ~10 8 CFU g -1 , where RIII enhanced species richness and phylogenetic bacterial community diversity in distal intestine [83]. Moreover, Lab. fermentum and Lab. plantarum modulated gill epithelium through upraising the number of mucous cells, surface area and lamina propria [84]. Lactobacillus was reported to be the dominant genus in the intestine of fish fed probiotics ( Table 2). In vitro incubation of salmon foregut with 1 ml Lab. delbrueckii subsp. lactis (1.6 × 10 5 CFU ml -1 ) exhibited that probionts adhere to the proximal intestinal mucus and did not cause any damage to morphology and integrity of the intestine [85]. Pre-incubation with the same probiotic offered protection against A. salmonicida infection. Similarly, Kristiansen et al. [86] showed that Carnobacterium divergens adhered to the proximal intestine following in vitro exposure at a concentration of 10 8 CFU ml -1 for 1 h and inhibited the growth of pathogenic Y. ruckeri. In another study, in vitro incubation of C. divergens prevented A. salmonicida-and V. anguillarum-induced damage in the foregut of Atlantic salmon to some extent [87].

Rainbow Trout
Rainbow trout contributed 2% of total global finfish production in 2016 [1]. Numerous benefits of probiotics have been reported in rainbow trout aquaculture, for example, both L. plantarum and its strain KC426951 promoted growth and immunity with some exceptions in heamato-biochemical parameters [88,89] (Table 3). The former probiotic increased alkaline phosphatase (ALP), IgM, and hemoglobin to a significant level, whereas the latter one only increased white blood cells with no alteration of hemoglobin, mean corpuscular volume, and red blood cells concentration. In contrast to the results reported by Soltani et al. [89], some haematological parameters like ALP, triglyceride and total cholesterol depicted improved modulation after Lab. rhamnosus treatment [90].
Dietary supplementation with LABs L. buchneri, L. fermentum, and the yeast S. cerevisiae together for 130 days improved innate immunity and immune genes transcription without alteration of heamato-biochemical parameters [91]. Modulation of these parameters was also observed after feeding with various strains of LAB [92,93]. Administration with L. plantarum, Lac. lactis, and Leuconostoc mesenteroides upregulated IL-8 & 10, TNF-α, and IgT and decreased IL-1β, TNF-α and TLR5 transcription before and after Lac. garvieae infection, respectively directly in contrast to results reported by Pérez-Sánchez et al. [94] (Table 3).
Combination of Bacillus sp. with Lactobacillus sp. reportedly elevated humoral immunity, intestinal structure and decreased plasma phenoloxidase [95]. The dietary administration of Bacillus sp. and B. licheniformis individually or in combination showed improved [96], unchanged [63], and decreased [97] results in weight gain and specific growth rate. Humoral immunity was elevated with B. subtilis + B. licheniformis, but this combination had no influence on serum biochemical parameters apart from total leukocytes. Trout gut probiotics Kocuria SM1 and Rhodococcus SM2 failed to induce changes in growth, digestive enzymes, and haematology, with the exceptions of lowered trypsin and increased hemoglobin levels [30]. In another study, single administration of Kocuria SM1 improved immunity and serum biochemistry [98]. Besides these, Kocuria SM1-fed rainbow trout fingerlings decreased mortality by 10-28% from 73-92% in response to V. anguillarum challenge. Dietary administration of Enterobacter cloacae and B. mojavensis showed improved protection from Y. ruckeri, where survival rate increased to 99.2% compared to 35% in the control group [99]. Probiotics like E. casseliflavus positively modulated serum biochemistry by decreasing malondialdehyde and lymphocytes without changing alanine and aspirate aminotransferase [100]. Supplementation of Bifidobacterium animalis PTCC-1631 and Bif. lactis PTCC-1736 at the lowest dose (10 7 CFU g -1 ) improved fish body weight and Gut Morphology Goblet cells, Symbol →, no change; ↑, increase; ↓, decrease versus controls.   digestibility compared to higher doses and to the control group [101] (Table 3). Treatment with a commercial probiotic, Proviotic containing L. bulgaricus and Aqualase (mixture of S. cerevisiae and S. elipsoedas) demonstrated better growth and diet digestion [102,103]. Similarly, PrimaLac composed of L. acidophilus, L. casei, E. faecium and Bif. bifidium elevated these two parameters along with protease, amylase and lipase activity in the intestine [15].

Common Carp
Common carp is among the most important commercially cultured fish in the world contributing 8% of total global finfish aquaculture production in 2016 [1]. Probiotics reportedly affect growth, immunological parameters, and disease protection in common carp [7]. Administration of L. delbrueckii for 8 weeks showed improved production, feed intake and various digestive enzymes including protease, amylase, lipase, Na+/K+-ATPase, creatine kinase and γGT activity [104,105]. Moreover, similar to L. delbrueckii, Lactobacillus sp. and  Bacillus spp. modulated immune-gene transcription when used as water supplements [106]. Comparable modulation of parameters was noted after 60 days administration of L. plantarum 44ª, although the probiotic had no effects on lipase and haematological parameters [107]. In another study, same probiotic supplementation unveiled better serum biochemistry, phagocytic and λ-globulin activity in head kidney and protection against A. hydrophila, whereas phagocytic activity in spleen remains unchanged [108] (Table 4). Supplementation with B. coagulans, B. licheniformis and P. polymyxa (MTCC 122) at 10 9 CFU g -1 influenced growth and feed utilizations, innate immunity [109], survival percentage, protection from A. hydrophila and V. harveyi [110]. Combined applications of Lactobacillus sp. with Bacillus sp. at 0.75, 1.5 and 2.25 g kg -1 improved protease activity in the intestine and modulated water quality parameters like pH, temperature and lowered dissolved oxygen concentration [111]. Administration of P. acidilactici [112] and P. pentosaceus [113] for 60 and 45 days, respectively, exhibited contrasting results. P. acidilactici improved feed conversion ratio, but decreased final body weight and daily weight gain, protease activity and immune gene expression. On the other hand, P. pentosaceus improved growth and digestive enzymes activity. However, both probiotics increased innate immunity and serum biochemistry ( Table 4). Commercial probiotic mixture viz. Primalac ® elicited a similar pattern of results, but only level of white blood cells and serum total protein increased among all haematobiochemical parameters [114,115].

Japanese/Olive Flounder
Olive flounder is among increasingly commercially important cultured marine species in Northeast Asian countries like China, Japan, Korean peninsula and some other countries [11]. This fish is widely accepted all over the world due to fast growth, taste, palatability, wide range of environmental adaptation and disease resistance. In 2018, its production was 37,239 metric ton equaling ~52% of total fisheries production in the Republic of Korea [116].
Extensive studies have focused on the use of single L. lactis BFE920 (LLBF) [17,117], LLBF and L. plantarum FGL0001 (LPFG) [17] as well as single and combination of LLBF and LPFG [35] in olive flounder. LLBF and LPFG were isolated from bean sprouts and the hindgut of olive flounder respectively, and their probiotic properties were confirmed in vitro before dietary supplementation. LLBF was also administered orally as vaccine after cloning with E. tarda membrane protein and S. iniae antigen termed as OmpA, FlgD, and OmpA-FlgD [118] and SiMA [117] respectively (Table 5). These oral vaccines increased transcription levels of cluster of differentiation (CD) 4-1, CD4-2, CD8-α, T-bet, INF-γ, weight (20-21%) and feed digestion. Both vaccines also elevated antigen specific antibodies in fish tissues, as well as protection against E. tarda (5 × 10 5 CFU ml -1 ; LD 80 ) and S. iniae (10 5 CFU ml -1 ) producing 82% higher rates of survival relative to controls. Individual supplementation of probiotics LLBF and LPFG at 10 7 CFU g -1 increased expression levels of regulatory and pro-inflammatory genes, respectively [17] ( Table 5). In addition, gut permeability was increased by LPFG compared to control and LLBF, and immune tone was stable for 30 days. Similar concentration level with combination of LLBF and LPFG increased levels of immunity and immune genes transcription and conferred higher protection against S. iniae challenge [35].
Except Sporolac, Lactobacil and mixture of probiotics improved immune parameters and resistance against lymphocystis. In other studies, infectious S. parauberis-injected flounder fed mixed probiotics [119] and Uronema marinum infected group fed individual L. plantarum, L. acidophilus, L. sakei, B. subtilis and S. cerevisiae [120] at 0.1% and 2.42 × 10 8 CFU g -1 respectively. In both cases, fish production, haematology and immunity levels were higher in disease infected probiotics treated groups as compared to controls. Among individual probiotics only L. plantarum elevated survival in U. marinum infected fish compared to other 4 probiotics and control. Another commercial LAB probiotic, L. fermentum was supplemented at 0.5% for 8 weeks to compare the effects with yacon, ginger and blueberry at 0.1% [121]. Commercial probiotics showed no effects except higher protection against S. iniae infection.
A probiotic Bacillus SJ-10 (BSJ-10) was isolated from Korean traditional fermented food [122] and complete genome sequence demonstrated similarity with two well-known olive flounder probiotics, B. subtilis and B. licheniformis [123,124]. Dietary supplementation with this probiotic both live [21,116] and heat-killed (HK) [125] forms at 10 8 CFU g -1 for 8 weeks increased rates of diet digestion and growth. Live and HK BSJ-10 and mixture with L. plantarum KCCM 11322 (3:1) protect olive flounder against streptococcosis [21,43,125] and edwardsiellosis [116]. Two different forms of BSJ-10 demonstrated no effect on heamto-biochemistry and intestinal structure, but both increased immunity and cytokines production in different localised organs like liver, kidney, gill and spleen (Table 5). Live BSJ-10 increased probiotics count in the intestine, but had no effect on IL-6, CD4-1 and CD4-2 transcription, and were able to ferment β-glucooligosaccharides and barley β-glucan as a synbiotic disease biocontrol model in olive flounder. Treatment with 3:1 Bacillus sp. SJ-10 and L. plantarum KCCM 11322 at 0.75 + 0.25 × 10 8 CFU g -1 feed resulted in improved growth, immunity and disease resistance in olive flounder [43]. Similarly, feeding with B. subtilis, B. pumilus and B. licheniformis demonstrated identical patterns of response in olive flounder [22]. Among these probiotics, B. subtilis and B. licheniformis improved disease protection compared to control. In addition, only B. subtilis increased fish survival and decreased ammonia concentration (5 days experiment) in controlled culture environments. Jang et al. [29] used vegetative cells of BSJ-10 and noted improved growth and feed utilization, and spore-modulated immune genes transcription, leading to improved richness and diversity of intestinal bacterial population.
A study with individual (BSJ-10 and L. plantarum) [28] and multi-strain probiotics [126] as supplemented with 30% reduced fish meal diet was found to maintain intestinal homeostasis and immune competence in olive flounder. Both BSJ-10 and L. plantarum increased lipase and trypsin, but amylase activity was only improved by BSJ-10. Individual and multi-probiotics also improved concentrations of proteobacteria, actinobacteria, and Immune tone   IRGE   B  S  J  -1  0  t  r  a  n  s  c  r  i  p  t  T  N  F  -α  ,  I  L  -6  ,  I  L  -1  β  ,  &  I  L  -1  acidobacteria, and Lactobacillus, Marinilactibacillus and Globicatella in the intestine, while also elevating cytokines transcription levels. Moreover, multiple and individual probiotics had no effects on production and intestinal structure respectively but improved infectious disease resistance of experimentally-cultured fish.

Grass Carp
Grass carp is predominant among cultured finfishes, comprising 11% of total global aquaculture production in 2016 [1] with production increasing to over 5.7 million tons in 2018; FAO ranked grass carp as the #1 aquacultured species worldwide in 2020 [127]. Several studies have examined the effects of probiotics in grass carp. Dietary B. licheniformis FA6 enhanced production, humoral immunity, upregulated immune gene transcription, intestinal morphology and Aeromonas infection protection in grass carp [128]. Identical results [129] along with modulation of intestinal microbiome [130] were noted after dietary supplementation of B. subtilis Ch9, and both B. methylotrophicus WM-1 [131] and Streptomyces amritsarensis N1-32 [132] contributed in the modulation of immunity and aeromonosis protection ( Table 6).
Individual and mixture of Shewanella xiamenensis A-1, S. xiamenensis A-2, and A. veronii A-7 at 10 8 CFU g -1 feed enhanced cellular and humoral immunity, serum biochemistry, gene transcription and offered A. hydrophila protection [133]. Administration of S. xiamenensis A-1, A. veronii A-7 and B. subtilis modulate the gut microbiota either singly or in combination, in which proportions of Cetobacterium, Citrobacter, Vibrio, Enterococcus and Streptococcus were increased, although a decreasing number was noted for Pseudomonas and Flavobacterium [134].
Several studies were carried out to examine the effects of probiotics on water quality parameters and microbiota in the culture environment of grass carp. The addition of P. stutzeri F11 [135] and B. licheniformis BSK-4 [136] in water led to reduction of ammonia, nitrite, total nitrogen and altered microbial populations at 9 and 18 days, respectively (Table 6). In another study, application of individual and mixture of P. stutzeri SC221-M and B. cereus BSC24 in culture water improved the rearing water quality as revealed by lower levels of total dissolved solids, chemical oxygen demand, ammonium, nitrite and total nitrogen compared to the control after 6-days posttreatment [137]. Moreover, these probiotics increased proteobacteria and decreased bacteroidetes and actinobacteria in ambient water.

Rohu Carp
Labeo rohita, commonly known as Indian major carp or rohu, is among the most cultured fish species in South Asia due to its commercial value, fast growth rate, consumer preferences and comparative simplicity in weaning to artificial diets [138,139]. Global production of rohu was 1.843 million tons in 2016, contributing 3% of total global finfish production [1]. Weakened immune system and lack of necessary digestive enzymes in juveniles can lead to higher rates of pathogenic bacterial infection and relatively poor feed utilization [138,140]. Efforts have been made continuously to subdue these problems, including many involving applications of probiotics.
Higher serum IgM levels were recorded after 30 days, and the level decreased after 60 days. Similarly, mixtures of these probiotics with B. subtilis VSG1 modulated and generally improved similar immunological parameters, Table 7. Effects of probiotic supplementation on growth, feed utilizations, immunological and haematobiochemical parameters and disease resistance in rohu fish (Labeo rohita). including disease resistance [141] (Table 7). Feeding of B. subtilis, L. lactis and S. cerevisiae mixture at different culture temperatures (28,31,34, and 37 o C) reduced the degree of apoptosis, augmented the transcription of HSP70, positively modulated haemato-biochemistry and innate immunity compared to the control [142]. Supplementation with B. methylotrophicus, B. amyloliquefaciens and B. licheniformis either singly or in combination elevated digestive enzymes activity, immunity and offered a significant degree of infection protection [143]. Similarly, disease protection and immunity alteration were observed after administration with B. aerophilus KADR3 [20], B. amyloliquefaciens CCF7 [144] and COFCAU_P1 [145] (Table 7). Improved protease, amylase and cellulose resulting on good growth in rohu fingerlings were observed after adding Geothichum candidum QAUGC01 to rearing water [140]. Similarly, G. candidum QAUGC01, B. cereus and G. candidum QAUGC01 + B. cereus at 10 9 CFU g -1 elevated gut microbiome, digestive enzymes and survivability against A. hydrophila infection [138]. Similar parameters modulation with improvements in the growth increment were observed after administration of single E. faecium QAUEF01 and its combination with G. candidum QAUGC01 [146] and S. cerevisiae [147].

Probiotics Application under Farming Condition
Most studies on probiotics were conducted under laboratory conditions, in which water and ambient environmental parameters are subject to rigid control. The role of probiotics in open systems under farming conditions remains minimally studied. While performance of probiotics under uncontrolled field levels is subject to uncertainty, commercial manufacturers of probiotics frequently boast that their products are highly effective for farm level application.
Tilapia cultured in earthen ponds treated with two doses of B. pumilus and a commercial probiotic for 8 months showed increase growth, immunity and haematological parameters, and A. hydrophila infection protection [79] ( Table 1). Artificially cultured fishes are subjected in the hatchery to water quality and other parameters that can compromise the consistent production of healthy juveniles [148], although probiotic applications among broodstock, embryos and larvae have been studied inadequately. Female rainbow trout brood stock in raceway ponds supplemented with multi-probiotics before spawning season demonstrated production of eggs with higher diameters, increased fecundity, fertilization and hatching, yolk sac absorption, and eye development [14]. These results are consistent with beneficial responses to multiple-species LAB probiotic applications in the zebrafish hatchery, which led to consistent improvements in every measurable parameter of larval performance [149]. Moreover, multi probiotics supplementation in rainbow trout during grow out in floating cages led to improved haemato-immunological responses and operational taxonomic units in the intestine [150] (Table 3).
Common carp (2.75 g) cultured in raceways and fed with B. sublitis for 180 days at 4 × 10 6 & 8 CFU g -1 showed elevated production and feed utilizations as contrasted with controls [151]. Moreover, probiotics increased muscle RNA-DNA ratio along with crude lipid, protein, and ash content in the body (Table 4). Single supplementation of L. lactis BFE920 at different concentrations increased protection against streptococcosis (68-77%) after 2 weeks. Subsequently, 2.5 × 10 7 CFU g -1 was chosen for field experiment for 3 months in 12,000 olive flounder [152]. That experiment revealed that probiotics fed group displayed improved immunity and immune related gene expression, resulting in 65.7% survival relative to 5.7% in control group when challenged with S. iniae (Table 5).
Cage culture of grass carp (~44 g) for 56 days with B. natto NT supplementation improved body weight and feed utilization [153]. Myostatin and myocyte enhancer factor C were downregulated, whereas micro-RNA (miR)-1a, miR-181a, miR-23a, and miR-206 transcription were upregulated compared to control group (Table 6). Rohu carp in earthen ponds displayed improved immunity and digestive enzyme activities along with enhanced tissue HSP70 expression after 11 weeks feeding with encapsulated G. candidum QAUGC01 [154]. Multi-probiotics administration in that species in outdoor tanks resulted in higher survival of hatchlings and fry at 8 and 38 days, respectively but those differences were no longer detectable after 68 days [155] (Table 7).

Research Gaps and Concluding Remarks
Some urgency is attached to the transition from the use and at times excessive use of antibiotics to control pathogens in aquaculture. The effectiveness of antibiotics is counteracted by intense selection favouring resistance, leading to the emergence of dangerously capable pathogens. The hazards of routine antibiotic applications are well-recognized now, and alternatives are critically needed. Probiotics are being marketed to aquaculture professionals, but it is generally true that their effectiveness in any given situation is uncertain.
Probiotics are typically found among microorganisms inhabiting the digestive tracts of healthy aquatic species, and their presence has been found to induce beneficial changes such as improved growth, immunity, and survival. Although the concept of promoting improved health by altering the population structure of the microbiome has gained favour, the mechanisms involved are far from clear. Probiotic organisms decrease the likelihood of the impairment or mortality of culture subjects by pathogens through a poorly understood complex of mechanisms, and more specific understanding of these mechanisms is highly desired. Interactive mechanisms of probiotic species with intestinal cells of host subjects require further investigation and analysis.
Probiotics contribute to the development of an unfavourable environment for the proliferation of pathogens, thereby disabling opportunistic microbes including many pathogens, thereby reducing the prevalence of outbreaks of infectious organisms. This can happen by direct and indirect means, for example by generating metabolites that are inhibitory to pathogens, or that promote activation of the immune system, or sometimes simply by competing for nutrients and environmental niches. As discussed above, activation of growth-and immune-related genes have been observed and may be more symptomatic of beneficial health effects than unknown causative factors. Further study of the mechanisms resulting in beneficial impacts of probiotic organisms may lead to more effective treatments and ultimately to improved environmental management of commercially cultured fishes and invertebrates.