Sustainable Ornamental Fish Aquaculture: The Implication of Microbial Feed Additives

Simple Summary In recent decades, trade of ornamental fish has significantly increased. A rise in demand was observed, especially from top importing countries that contributed majorly to the growth of the market. The destructive fishing methods caused a severe impairment of natural and environmental resources. Thus, there is a need to improve ornamental fish aquaculture, increasing the number of cultured species and limiting wild fish handling and transport stress losses. In this light, the use of microbial feed additives such as probiotics, prebiotics, and synbiotics, could help in improving the immune system and growth as well as increasing reproductive performance in captivity-bred species. Abstract Ornamental fish trade represents an important economic sector with an export turnover that reached approximately 5 billion US dollars in 2018. Despite its high economic importance, this sector does not receive much attention. Ornamental fish husbandry still faces many challenges and losses caused by transport stress and handling and outbreak of diseases are still to be improved. This review will provide insights on ornamental fish diseases along with the measures used to avoid or limit their onset. Moreover, this review will discuss the role of different natural and sustainable microbial feed additives, particularly probiotics, prebiotics, and synbiotics on the health, reduction in transport stress, growth, and reproduction of farmed ornamental fish. Most importantly, this review aims to fill the informational gaps existing in advanced and sustainable practices in the ornamental fish production.


Introduction
Ornamental fishes, due to their different and brilliant colors, shapes and behavior, are often referred to as living jewels and are kept in aquaria or garden pools for their beauty as well as entertainment. Ornamental fishes thank to their different and brilliant colors, shape and behavior, are often referred as living jewels and are kept in aquaria or garden pools for entertaining and fancy thus resulting, together with photography, among the

Economic Aspects of Ornamental Fish Trade
The ornamental fish market was valued at USD 5.4 billion in 2021 and is anticipated to expand at a compound annual growth rate (CAGR) of 8.5% from 2022 to 2030. The tropical freshwater fish segment dominates the market and accounted for the largest revenue share of 51.7% in 2021. Among most popular tropical freshwater fish for beginners are guppy (Poecilia reticulata), molly (Poecilia sphenops), and zebrafish (Danio rerio); most of them are quite inexpensive and usually priced between USD 1 and 6. Nearly 15% of the total traded fish consists of marine ornamental species, with their attractive colors and attention-grabbing behavior [11], largely derived from wild catches [12].
Approximately 99% of the market is kept by hobbyists and less than 1% by public aquaria and research institutes. It has been estimated that more than 1.5 million people are involved in this sector and there are over 3.5 million hobbyists worldwide involved in this trade. The sector has provided employment opportunities, alleviated poverty, and contributed to national income by enhancing foreign exchange earnings [13].
World exports of ornamental fish increased steadily from USD 177.7 million to a peak of USD 364.9 million in 2011, before declining slightly to USD 347.5 million in 2014 [14].
In 2014, Asian countries accounted for more than 50% of the trade, and export was valued at USD 130 million. Europe accounted for 27.6% of the total market (valued at USD 95.8 million) followed by South America (7.5%), North America (3.98%), Africa (2.2%), Oceania (1.4%) and the Middle East (0.5%) (Figure 1). Singapore was the top ornamental fish exporter (20% of the total) and the turnover was valued at USD 69.32 million [14] ( Figure 2). Japan was the leader in koi carp production, thus the second most important exporting country (USD 41.34 million). Starting from 2000, for many years, the USA had the world's largest ornamental fish market, but imports declined from USD 60 million in 2000 to USD 42.9 million in 2014 [14]. In Europe, the UK remains the largest importer of ornamental fish (USD 29.5 million) [15]. of USD 364.9 million in 2011, before declining slightly to USD 347.5 million in 2014 [14].
In 2014, Asian countries accounted for more than 50% of the trade, and export wa valued at USD 130 million. Europe accounted for 27.6% of the total market (valued at USD 95.8 million) followed by South America (7.5%), North America (3.98%), Africa (2.2% Oceania (1.4%) and the Middle East (0.5%) (Figure 1). Singapore was the top ornamenta fish exporter (20% of the total) and the turnover was valued at USD 69.32 million [14 (Figure 2). Japan was the leader in koi carp production, thus the second most importan exporting country (USD 41.34 million). Starting from 2000, for many years, the USA ha the world's largest ornamental fish market, but imports declined from USD 60 million i 2000 to USD 42.9 million in 2014 [14]. In Europe, the UK remains the largest importer o ornamental fish (USD 29.5 million) [15].

Effects of Microbial Feed Additives on Ornamental Fish Health
Disease outbreak is the major problem in ornamental fish production, causing a sub stantial economic loss of approximately 400 million US dollars [16]. Diseases can be eithe of parasitic, bacterial, viral or fungal origins and common symptoms include dropsy of USD 364.9 million in 2011, before declining slightly to USD 347.5 million in 2014 [14].
In 2014, Asian countries accounted for more than 50% of the trade, and export wa valued at USD 130 million. Europe accounted for 27.6% of the total market (valued at US 95.8 million) followed by South America (7.5%), North America (3.98%), Africa (2.2% Oceania (1.4%) and the Middle East (0.5%) (Figure 1). Singapore was the top ornament fish exporter (20% of the total) and the turnover was valued at USD 69.32 million [1 ( Figure 2). Japan was the leader in koi carp production, thus the second most importan exporting country (USD 41.34 million). Starting from 2000, for many years, the USA ha the world's largest ornamental fish market, but imports declined from USD 60 million i 2000 to USD 42.9 million in 2014 [14]. In Europe, the UK remains the largest importer o ornamental fish (USD 29.5 million) [15].

Effects of Microbial Feed Additives on Ornamental Fish Health
Disease outbreak is the major problem in ornamental fish production, causing a sub stantial economic loss of approximately 400 million US dollars [16]. Diseases can be eithe of parasitic, bacterial, viral or fungal origins and common symptoms include drops

Effects of Microbial Feed Additives on Ornamental Fish Health
Disease outbreak is the major problem in ornamental fish production, causing a substantial economic loss of approximately 400 million US dollars [16]. Diseases can be either of parasitic, bacterial, viral or fungal origins and common symptoms include dropsy, ulcers, fin and tail rot, constipation, swim bladder inflammation, clamped fins, pop eyes, flip over disease, skin flukes, and cloudy eye. The resulting losses negatively affect the financial and socioeconomic status of the ornamental fish farming community. Tables 1-4 list the most common parasitic, bacterial, viral, and fungal diseases identified so far.   Commercial aquaculture practices commonly used to prevent or heal damage in fish intended for human consumption, can represent a good starting point to reduce losses The aquaculture industry still represent a major area of antibiotic misuse [17] and so far, oxytetracycline hydrochloride and kanamycin have positively resolved a wide spectrum of fish bacterial diseases, including furunculosis, aeromonosis, pseudomonosis, lactococcosis, and vibriosis, being administered via feed, bath treatment or injection [18]. Excessive and unregulated use of antibiotics in aquaculture, as well as in the ornamental fish industry, has led to the development of gut antibiotic-resistant bacteria [19] and antimicrobial-resistant pathogens [20], subsequently affecting the immune system of fish.
Starting from the regulation of antibiotic use, researchers are now focused on considering and discussing valid alternatives and promising functional feed additives (vaccination, bacteriophages, quorum quenching, probiotics and prebiotics, chicken egg yolk antibody and medicinal plant derivative) that could also be successfully applied in ornamental fish culture [21].
Lilly and Stillwell [22] first proposed the term probiotics "to be used for substances that favors the growth of microorganisms". Since then, several definitions have been proposed and the most common is "live microorganisms that when administrated in adequate amounts, confer a health benefit to the host". proposed by the World Health Organization (WHO). Kozasa [23] was the first researcher who used probiotics in aquaculture and the very first review article on probiotics was published in 1998 [24]; since then, several reviews have been published [25][26][27][28][29][30][31][32][33]. Live bacteria as well as inactivated bacteria and spore formers have been used as probiotics in aquaculture [34]. Among microbial fish additives, lactic acid bacteria (LAB) and Bacillus probiotics are the most used; however, Aeromonas, Alteromonas, Arthrobacter, Bifidobacterium, Clostridium, Paenibacillus, Phaeobacter, Pseudoalteromonas, Pseudomonas, Rhodosporidium, Roseobacter, Streptomyces, Vibrio, microalgae (Tetraselmis), and yeast (Debaryomyces, Phaffia, and Saccharomyces) are also beneficial [33]. Probiotics can be administered via feed supplementation (single or in mixture) or dissolved in water.

Species Probiotic Strain Effects References
Marbled hatchetfish (C. strigata) Commercial mixture (B. subtilis, B. licheniformes, L. acidophilus and S. cerevisiae) Improved water quality by reducing metabolic waste and stress response [75] Cardinal tetra (P. axelrodi) P. acidilactici Significant increase in all non-specific immune system biomarkers (lysozyme activity, total immunoglobulin and alternative complement activity) [76] Green terror (A. rivulatus) P. acidilactici Improved stress response (modulation of lysozyme activity, immunoglobulin and protease levels) faecium Improved fish viability [78] E. cloacae Improved resistance against P.shigelloides challenge (increased blood cell counts and respiratory activity) [79] Kenyi cichlid (M. lombardoi) B. infantis Alteration of gut microbiota composition [57] Rosy barb (P. conchonius) Gold black molly (P. sphenops) S. cerevisiae Improvement of reproduction, stress response and resistance against pathogens [81] Sailfish molly (P. latipinna) S. cerevisiae Improvement of growth performance, feed utilization and disease resistance [82] Orange clownfish (A. percula) LAB, a variety of Gram-positive bacteria, are the main microorganisms that ferment plants, vegetables, meats, fish, and dairy products in the intestine [83,84]. LAB are also commonly used to produce various compounds, such as small organic acids, vitamins, and biological peptides [85][86][87]. Within LAB, L. acidophilus is among the most industrially utilized strain in the manufacture of dairy products and dietary supplements [88,89]. Given that LAB may supply several organic molecules via various metabolic processes, these microbes could be utilized as valuable and specific sources of a wide range of enzymes with novel properties [90,91].
Production of Siamese fighting fish (Betta splendens) represents a great example of good practice using LAB. This species provides a great income among exported ornamental fish in Thailand. A study reported that a diet supplemented with L. plantarum (KKU CRIT5) exerted no significant improvement of digestive enzymes compared to control, with decreased protein depositions in body and muscle, suggesting that additional trials could be set up in this species in order to verify the effects of this probiotic focusing on dose and time of administration [47]. In Xiphophorus helleri, a dietary supplementation of L. acidophilus positively modulated mucosal immune parameters, e.g., skin mucus protein and alkaline phosphatase levels. In addition, the response to salinity stress was improved, clearly indicating a better healthy condition of the fish, as also supported by the modulation of intestinal microbiota towards beneficial LAB [48]. Surprisingly, in the same species, the administration of a commercial probiotic formulation containing two Lactobacillus species, two Bacillus species, Streptococcus faecium, and S. cereviasiae did not confer better tolerance against bacterial challenges [49]. Similar results were observed in goldfish, Carassius auratus, where the administration of the same commercial probiotic or a mixture of Lactobacillus sp. and Bacillus sp. had no significant effect on resistance against Pseudomonas fluorescens infection [49]. Additionally, a study on goldfish reported that when a 40% protein diet was supplemented with different concentrations of three commercial Lactobacillus probiotic-based formulations, an increase in protein and a decrease in fat content were observed. This suggested that these probiotics could improve the nutritional properties of the fillet [54], which is an important factor, especially for species intended for human consumption. Porthole livebearer (Poecilopsis gracilis), when fed with Artemia nauplii enriched with L. casei, showed increased production of skin mucus and a faster recovery after the air-dive/stress test, suggesting that this probiotic could be helpful in obtaining healthier organism for the ornamental fish market [56].
B. coagulans and B. mesentericus, used to enrich Thermocyclops decipiens cultures and administered via diet to Puntius conchonius post larvae, significantly changed gut microflora composition. Microbiota analysis further revealed that B. coagulans poorly adheres to the gut with respect to B. mesentericus [57].
The commercial mixture containing B. subtilis, B. licheniformes, L. acidophilus, and S. cerevisiae was beneficial in fish facing extreme conditions including handling and transport stress. Based on these results, these probiotic strains have been largely used in the trade of Cardinal tetra, Paracheirodon axelrodi [75] and the marbled hatchet fish, Carnegiella strigata [74], which showed a decrease in stress levels and related metabolite secretion. In these trials, a sensible improvement of water quality was also described.
Probiotics (Bacillus sp., Lactobacillus sp., and their mixtures) were used in the giant gourami, Osphronemus goramy, to produce higher-quality fish and to reduce the risk of diseases outbreak. Fish exposed to a mixture of Bacillus sp. and Lactobacillus sp. via water presented a higher survival rate. These species also improved the quality of the rearing water [58].
L. fermentum (KT183369) and B. subtilis sp. inaquasporium (KR816099) isolated from coconut were used in a feeding trial with the black molly, Poecilia sphenops. At the end, their adhesive properties towards the host cells were found, which led to the speculation that both strains could be used against Vibrio parahaemolyticus. The ability to fight V. parahaemolyticus was demonstrated by a challenge test. In addition, L. fermentum displayed a higher capacity to colonize the gut, suggesting that could be an excellent feed additive for ornamental aquaculture species [59,60]. An additional challenge test against V. anguillarum was set up using B. pumilus RI06-95Sm and results showed its ability to colonize the molly's gut, reverse the negative impacts of antibiotic treatment and decrease the mortality rate [61].
Three different probiotic strains-L. rhamnosus, B. coagulans, and B. mesentericus-were administered separately to zebrafish, D. rerio, through enrichment of artemia and compared with control fish fed with unenriched artemia. The positive effect of probiotic administration was demonstrated by the decrease of the number of gut pathogenic bacteria in all experimental groups compared to control fish. One week after the end of the trial, gut microbiota was significantly colonized by L. rhamnosus, suggesting artemia as an effective means for probiotic delivery [62]. L. rhamnosus administration via water to zebrafish larvae significantly accelerated skeletogenesis acting on lipid and vitamin D metabolism [63] and positively modulated the expression of genes responsible for bone formation [38]. A positive modulation of signal involved in lipid and vitamin D metabolism was also observed in clownfish, Amphiprion ocellaris [71]. Positive outcomes were also observed using caudal fin regeneration as a process to investigate the effects of a mix of B. subtilis, B. licheniformis, B. coagulans, and L. acidophilus plus the yeast S. cerevisiae. In this study, evidence regarding the treatmentability to promote osteoblast differentiation, suppress osteoclast activity and modulate phosphate homeostasis was provided, strongly promoting its use to support bone homeostasis and reduce deformity [92].
Zebrafish also resulted an excellent experimental model to demonstrate the positive role of L. rhamnosus IMC 501 on fish welfare: an increase of genes involved in immunity was recorded in intestinal tissue, while in the liver, a downregulation of stress and apoptoticrelated biomarkers was documented, suggesting that administration of probiotics could help against pathogens [40]. In zebrafish, B. amyloliquefaciens R8 administration induced a significant increase of gut xylanase activity in respect to fish fed a control diet. At the hepatic level, mRNA expression of glycolysis-related and anti-apoptotic genes was regulated; this occurred concomitantly to an increase of 3-hydroxyacyl-coenzyme A dehydrogenase and citrate synthase enzyme activities, responsible for fatty acid β-oxidation and mitochondrial integrity [64]. In addition, treating zebrafish with probiotics significantly increased disease resistance against A. hydrophila and S. agalactiae. This evidence strongly supports the idea that the administration of xylanase-expressing B. amyloliquefaciens R8 can improve stress response, increasing immunity and disease resistance against pathogen challenges [64]. In X. hellerii, diet supplementation with B. subtilis significantly improved growth by increasing feed assimilation and improving metabolism [52]. Inclusion of B. subtilis in P. latipinna diet, significantly improved growth and disease resistance against A. hydrophila, and led to a higher survival rate than in control or antibiotic-treated groups [72]. The administration of a commercial probiotic, containing B. subtilis and B. licheniformis to goldfish, C. auratus, significantly improved the survival rate, food digestibility, stress resistance, and immune response [55]. These two probiotic strains enhanced immune parameters, increased total protein, albumin, total globulin, lysozyme and hemolytic complement activity levels compared to control when sprayed on a dry diet and administered to tinfoil barb, Barbonymus schwanenfeldii. In addition, the increase in peroxidase and trypsin levels suggested a possible activation of leucocyte phagocyte activity as well as an increased resistance to pathogens and strongly advises their use against bacterial challenges [73]. The probiotic strain P. acidilactici was co-administered with fish oil to green terror, A. rivulatus, and the effects on the innate immune parameters were measured. Compared to a control diet, the experimental fish displayed a significant increase in non-specific immune system biomarkers, suggesting the positive effects of this probiotic administration [76]. A preliminary study on P. scalare also revealed that E.faecium could be used to improve the viability of this species [78]. Dietary administration of E. cloacae with a 2% mannan oligosaccharide (MOS), significantly increased blood cell counts and respiratory activity in the Kenyi cichlid, Maylandia lombardoi, against Plesiomonas shigelloides, suggesting the use of this probiotic strain to prevent this infection in ornamental fish aquaculture [79].
When adult zebrafish were fed a diet supplemented with C. aquaticum, a "potentialcandidate" probiotic isolated from lake water samples, a set of protease and xylanase and a bacteriocin-like substance, able to counteract the negative effects of diverse pathogens, including aquatic, foodborne and plant pathogens, were produced. In fish receiving the probiotic, carbohydrate and immune-related gene expression were upregulated. Additionally, dietary probiotics increased disease resistance against A. hydrophila and S. iniae. These results clearly showed that C. aquaticum, as a probiotic, not only improved nutrient metabolism but also increased innate immune parameters as well as resistance against pathogens [66]. In the same fish species, administration of Actinobacteria phylum, B. infantis and B. longum decreased the number of gut pathogenic species. Despite this positive evidence, one week after the end of the trial, gut microflora was not significantly colonized by these two species, suggesting that these probiotics should be administered longer or should be supplied with other beneficial species for a more stable result [62].  [65]. A decrease in the number of apoptotic cells was observed in the gut, suggesting the capacity of these bacteria to control immune response and inflammation [65]. In the black molly, P. sphenops, when challenged with V. anguillarum, the proteobacteria probiotic strain Phaeobacter inhibens S4Sm colonized the gut, causing changes in the fish microbiome able to contrast the negative impact of antibiotic treatment, suggesting that its use in aquaculture could protect fish from external pathogens [61].
The administration of Thermocyclops decipiens enriched with B. infantis significantly changed gut microflora composition in P. conchonius post-larvae [57]. Dietary protein supplement with a multispecies bacteria formulation made of several probiotic strains B. bacterium, S. silivarius, E. faecium, A. oryzae, and Candida pintolopesii exerted positive effects on hematological factors of the Oscar, A. ocellatus fingerlings, suggesting positive outcomes against pathogens in both commercial and ornamental fish trade and opens new possibility for further research [80].
Bacteria can also be used as a source of proteins. In a trial with X. hellerii, a set of artificial diets were prepared by substituting fish meal with different proportions of microbial single-cell proteins (SCPs) extracted from the gut. SCPs could be bacterial cells, either Micrococcus or Bacillus, Azobacter or Streptomyces-aand -b-enriched diets, that induced a significant improvement of food conversion efficiency and conversion rate and a higher protein content in X. hellerii, thus representing a promising area of research [50]. Results presented are summarized in Table 5.
So far, several yeast species have been used in ornamental fish culture and the positive effects of their use have been demonstrated. Molly, P. latipinna, fed on artemia enriched with S. cerevisiae cell wall, showed an improvement of reproduction, stress response, and resistance against A. hydrophila [81]. Similarly, a feeding trial was carried out to study the effects of S. cerevisiae in juvenile A. percula and the results showed that the treatment increased hematological and serum values (markers of the non-specific immune parameters) improving defense against Streptococcus sp. [82]. Diet supplementation with RNA extracted from Kluyveromyces fragilis significantly modulated the stress response during zebrafish early larval development, suggesting an anti-inflammatory action of RNA yeast extract [69]. Two non-Saccharomyces species, Yarrowia lipolytica 242 (Yl242) and Debaryomyces hansenii 97 (Dh97), significantly improved the immune system of zebrafish larvae and defense against V. anguillarum when administered with the diet. Treated fish showed a downregulation of immune-related genes (IL-1β, TNF-α, IL-10, C3, MPx), suggesting that this yeast could be used against pathogens [70]. Since a healthier status was already described in several aquaculture species including catfish, Clarias gariepinus [93], salmon, Salmo salar, [94], and trout, Oncorhynchus mykiss [95], the results strongly suggest the use of yeast also in ornamental aquaculture.

Effects of Microbial Feed Additives on Ornamental Fish Growth
The main target of aquaculture practice is to acquire the most rapid growth and the lowest production cost. To achieve this goal, several means have been established to boost the growth rate and feed consumption by adding functional feed additives [96,97]. Probiotics are among those functional feed additives showing strong effects on growth, health, and well-being. In aquaculture, investigations on probiotic-containing diets demonstrated the role of these favorable bacteria in improving gut microflora balance and in the production of extracellular enzymes able to enhance feed utilization and increase growth performance [98,99]. Probiotics can increase the uptake of nutrients, the assimilation capacity, the feed conversion ratio and improve digestibility [99][100][101]. In addition, probiotics have been proven to promote the absorption of feed through the production of extracellular digestive enzymes, i.e., amylases, proteases and lipases or intestinal alterations, resulting in a better growth [25,[102][103][104][105].
Ahmadifard et al. [72] indicated that dietary inclusion of B. subtilis in diets of P. latipinna significantly improved growth and reproductive performance. It has been further reported that probiotics colonized in the gastrointestinal tract could stimulate broodstock and larvae nutrition by synthesizing the necessary nutrients and enzymes, such as proteins, vital fatty acids, and amylase, as well as protease and lipase [106]. In addition, probiotics in fish gut promoted enzyme excretion in the host by inducing maturation of the gut secretory cells [99,101]. These enzymes improved the digestion efficiency of complicated proteins and lipids included in the diet that can per se affect the assimilation rate. Similarly, He et al. [107] disclosed that the inclusion of B. subtilis in the diet significantly improved Koi carp, C. carpio, weight gain and feed conversion ratio. However, dietary probiotics had no effect on fish body skin coloration.
The supplementation of Lactobacillus in black swordtail, X. hellerii, resulted in the promotion of growth and survival rates [48]. Significantly improved growth performance can be due to increased digestive enzymes and appetite, vitamin production, degradation of undigested elements and potential enhancement in intestinal morphology [108] ( Table 6). However, dietary administration of L. acidophilus did not have any significant effect in goldfish, C. auratus gibelio, growth performance and feed utilization [109]. The inconsistent nature of these findings can be attributable to a species-specificity, stage of life, dosage, and trial condition and suggests the need for different probiotics to be assessed on desired species [110]. P. acidilactici has also been used in green terror, A. rivulatus [76], Oscar, A. ocellatus [111], and convict cichlid, Amatitlania nigrofasciata [112]. The results suggested that its dietary administration significantly improved growth efficiency and survival rate. Dietary incorporation of two Bacillus probiotics (B. subtilis and B. licheniformis) significantly increased growth performance and feed utilization in goldfish, C. auratus [55]. Bacillus sp. are dominant in the gastrointestinal tract of fish and shellfish [105], which are able to produce several amino acids [28] and vitamins (K and B12) [113] to boost the host's growth performance. Similarly, the combination of L. plantarum, L. delbrueckii, L. acidophilus, L. rhamnosus, B. bifidum, S. silivarius, E. faecium, A. oryzae, and C. pintolopesii significantly improved the growth rate, but decreased food conversion rate (FCR) in Oscar, A. ocellatus [80]. Dhanaraj et al. [114] found that dietary administration of L. acidophilus and S. cervisiae showed a higher growth rate and FCR in koi carp, C. carpio, compared to the control. In addition, β-glucan, found in the yeast cell wall, was capable of improving immunity, growth performance, and resistance against pathogens [115]. It is likely that dietary Lactobacillus sp., Azotobacter sp., Clostridia, sp., Enterbacter sp., Agrobacterium sp., Erwinia sp., and Pseudomonas sp. significantly improved goldfish growth performance [116]. This mixture could increase the fermentation rate of feeds in fish intestine, and increase the absorption rate of nutrition in the digestive system [33]. In contrast, Abraham and collaborators [49] indicated that dietary inclusion of L. sporogenes, L. acidophilus, B. subtilis, B. licheniformis, S. faecium, and S. cerevisiae displayed no effect on the growth performance and feed utilization of C. auratus and X. hellerii. Similarly, dietary inclusion of B. licheni-formis, B. latrospore, and S. cerevisiae did not make a difference in weight gain, the specific growth rate, and the feed conversion ratio in guppy, P. reticulata [117]. Table 6. Effect of microbial feed additive supplementation on ornamental fish growth performance and survival rate.

Effects of Microbial Feed Additives on Ornamental Fish Reproduction
The beneficial effect of probiotics in reproduction was demonstrated, thanks to their ability to produce vitamin B and certain unknown stimulants [118], which in turn could play a vital role in increasing the reproduction rate of the host [126]. One example is represented by B. subtilis, which is able to synthesize vitamins B1 and B12, responsible for the reduction of the number of abnormal and dead larvae [51]. The effects of one year of B. subtilis dietary supplementation were evaluated in five ornamental fish species-Cirrhinus mrigala, P. reticulata, P. sphenops, X. helleri and X. maculatus-and the results obtained highlighted better reproductive performances, as witnessed by the increase in the gonadosomatic index (GSI), fecundity and fertility rate in all species analyzed. In addition, fries presented higher survival rates as well as decreases of deformities [52]. Similarly, an Artemia diet enriched with B. subtilis significantly improved the reproductive performance of P. latipinna, in terms of fry production and survival [72]. The administration of L. rhamnosus strongly improved zebrafish reproductive performance, acting on both fertility and fecundity [26,42]. Probiotics acted at the gonadal level by inducing follicle maturation [43,67]. A similar effect of L. rhamnosus was also evidenced in killifish (Fundulus heteroclitus) [127].
The positive effects of probiotics on male reproductive performance were first described in zebrafish by Valcarce, et al. [128], who reported an improvement of sperm quality and reproductive behavior in fish treated with L. rhamnosus and B. longum [128]. In this last model, similar results were reported by other authors, using two different Lactobacillus strains, L. rhamnosus CIC 6141 and L. casei BL23 [68]. Dietary administration of P. acidilactici (0.2%) and nucleotide (0.5%) positively affected sperm quality, motility and density in goldfish, C. auratus [129].
A novel aspect in this field is represented by the ability of probiotics to contrast endocrine disruptor reproductive toxicity, as observed in zebrafish exposed to bisphenol A. Probiotic co-administration, indeed, mitigates bisphenol A reproductive toxicity in zebrafish [130].
These results are summarized in Table 5.

Role of Microbial Feed Additives in Maintaining Good Water Quality of Ornamental Fish Holding Systems
Administration of probiotics in culture water can offer an advantage at any point in the species life cycle. This is of high importance, especially during larval stages, when their use can improve health conditions [131]. Probiotics in water can proliferate using available substrates and competitively exclude the pathogenic bacteria [132].
It has been suggested that water probiotics (B. acidophilus, B. Subtilis, B. licheniformis, Nitrobacter sp., Aerobacter sp., and S. cerevisiae) beneficially affect water quality through enhancing organic matter decomposition of the undesirable organic substances [133], increasing the population of food organisms, reducing pathogenic bacteria [134] and nitrogen and phosphorus concentrations and controlling ammonia, nitrite, hydrogen methane, etc., levels [135][136][137][138]. Considering that fish feed and waste are two significant parameters of the aquaculture ecological footprint, it can be argued that probiotics can contribute to reduce the environmental impact of aquaculture [139].

Current Knowledge Regarding Prebiotic and Synbiotic Use in Ornamental Fish
Prebiotics, as promising immunostimulants, have been introduced to aquaculture as a preventive action [140]. They mainly consist of oligosaccharides such as fructooligosaccharide (FOS), galactooligosaccharide (GOS), mannan-oligosaccharide (MOS), and xylooligosaccharide (XOS), which have been proven to promote beneficial bacterial growth within the gastrointestinal tract [141,142]. Many prebiotic compounds such as FOS, GOS, MOS, inulin or β -glucan have been used in the ornamental fish culture industry (Table 7) and extensively employed as immunostimulants to improve growth performance, modulate microbial activities of the digestive tract, stimulate the immune system and enhance stress resistance. In the literature, data about the effect of prebiotic administration on growth performance in ornamental fish are reported. GOS administration was effective in gibel carp, C. auratus gibelio [110], and zebrafish, D. rerio [143]. MOS administration did not affect Siamese fighting fish, B. splendens [144], and clownfish, A. ocellaris [145] survival and growth, but positively acted on zebrafish, D. rerio. [146] and regal peacock cichlid, A. stuartgranti [147]. Focusing on XOS, it was effective on Oscar, A. ocellatus [148], and crucian carp, C. auratus gibelio [149] (Tables 7 and 8).   The prebiotic type, dose, duration of treatment as well as host gut microbiota are the main reasons for observing contradictory results.
Nutritional supplements combining probiotics and prebiotics in the form of synergism is known as symbiotic, resulting in a more beneficial effect when compared with individual administration [162]. In a trial, angelfish, P. scalare, were fed with three different diets-a control diet, a diet enriched with P. acidilactici, and a diet enriched with P. acidilactici and fructooligosaccharide (FOS)-and were then exposed to environmental stress (temperature and salinity stress). Results demonstrated that fish fed P. acidilactici and FOS presented higher levels of lysozyme activity, immunoglobulin, and protease measured in skin mucus, in respect to P. acidilactici alone, suggesting the beneficial and synergic role of FOS to potentiate the probiotic effects [77].
In addition, in koi, C. carpio koi, under dietary COS combined with B. coagulans [140], or in rockfish, Sebastes schlegeli, under dietary GOS combined with P. acidilactici [163] or in Japanese flounder, P. olivaceus, dietary MOS combined with B. clausii [160], a significant improvement of growth performance and immune response was observed. Since synbiotic treatment has been suggested as a suitable alternative technique for pathogen prevention, their effectiveness in terms of defense against infectious diseases could be evaluated by a challenge test. Challenge tests using A. veronii and Edwardsiella tarda as pathogens were conducted in koi, C. carpio koi [140], and rockfish, Sebastes schlegeli [163], following a dietary synbiotic administration.Post-challenge mortality of fish was significantly higher in the control group than in the synbiotic group, which showed COS + B. coagulans and GOS + P. acidilactici to have a synergistic effect ( Table 8).

Research Gaps and Future Perspectives
The use of probiotics, prebiotics and synbiotics offers viable alternatives for a new generation of a higher-quality live products in terms of size, health, safety, and production time. Several studies on probiotics suggested the many advantages and benefits of their use in terms of growth performance, water quality and immune system. Despite the extensive research attempts to increase disease resistance of cultured fish by using dietary probiotics, prebiotics, and synbiotics, a limited number of studies were so far conducted on ornamental fish. In the near future, the application of prebiotics and synbiotics will be pivotalfor the development of a sustainable ornamental fish production. However, there is still a need to understand the mechanisms of action of pre-, pro-and synbiotics on both gastrointestinal health and animal welfare. Further studies must be undertaken to determine the composition of microbial communities and their administration as live feed additives. They are, indeed, temperature sensitive and can be killed by the high temperature during pelleting procedures, thus great efforts have been made to fill this gap, resulting their microencapsulation, freezing and inclusion in protective matrices, promising strategies to increase microbe viability and survival in feed [164]. Additionally, detailed investigations about the time, type, frequency and dose of live feed additive application must be conducted. Considering the promising immunomodulatory effects of these functional feed additives, their application can also be taken into account in ornamental fish larviculture.