Engineering the fish gut microbiome: could it serve as future-proof strategy for sustainable aquaculture?

Abstract

As the global population rises the need for sustainable production of nutritious food intensifies. Recent estimates show that 70% more food needs to be produced to feed the projected human population of 9.1 billion by 2050. Aquaculture plays a critical role in meeting these demands through sustainable practices. The significance of the gut microbiome in maintaining the health of aquatic animals is increasingly recognized particularly within sustainable aquaculture. Modification of the gut microbiome is an emerging field that enhances the growth and health of fish and other aquatic species. Probiotics prebiotics and synbiotics are pivotal for gut microbiota modification. Additionally, biotechnological techniques such as fecal microbiota transplantation (FMT) CRISPR-Cas bacteriophage-mediated methods and synthetic biology are advancing these efforts. Looking ahead the integration of current techniques with advanced engineering approaches is expected to create optimally modified gut microbiota for aquatic species. Achieving this goal requires a comprehensive understanding of the basic composition of the intestinal microbiota and the available methods for modification. In this review, we mainly focus on the basic characteristics and significance of the gut microbiome in aquatic animal health while exploring mechanisms techniques and applications of microbiome engineering. Emerging trends challenges and limitations are also examined alongside ethical and safety considerations that are vital for progress in this field.

Introduction

The relationship between the gut microbiota and host health has gained tremendous focus in recent years. Gut microbiota comprises a complex and diverse community of microorganisms that inhabit the gastrointestinal (GI) tract of animals. It includes bacteria, fungi, viruses and other microbes. The majority of these consist of bacteria, and they can be aerobic, facultatively anaerobic, or obligately anaerobic, depending on the specific microenvironment within the GI tract [190]. It plays a significant role in various physiological functions, such as digestion, nutrient absorption, and immune system regulation. Hence, a diversified and balanced gut microbiota is always essential to the overall well-being of the host [263].

The fish gut microbiota composition is influenced by host preferences, feeding habitats, diets, environmental factors, and developmental stages. The development of the mucosal system, epithelial proliferation, and angiogenesis are all influenced by gut microbiota, which helps digestion and nutrient absorption more effectively [228]. It is important for maintaining the immune system, feeding behaviour, energy balance and disease resistance of the host (fish). By offering nutritional competition, limiting direct interaction with the gut, and providing resistance to colonization, gut microbes prevent pathogens from flourishing. Also, it is very crucial in maintaining the average health of the fish [81, 295].

The majority of research on gut microbial populations have been conducted on mammals. Studies on the fish gut microbiome have been relatively limited in comparison. However, there has been a surge in attention to understanding fish gut microbiomes in recent years due to their ecological and economic importance [93, 134, 171]. The study of gut microbiota composition and relative abundance also extends towards a wide range of animals such as birds [72], reptiles [307], amphibians, and various invertebrates [46].

Fish have a very different bacterial ecology than other animals. The human gut microbiome is dominated by bacteria and firmicutes, while the amphibian gut microbiome is represented by Firmicutes, Bacteroidetes, and Proteobacteria. In contrast, Proteobacteria is the main phylum in fish microbiota, with Fusobacteria constituting another common abundant group [37, 268, 274]. Identifying the microbial communities within the fish gut is crucial. It is equally important to understand the complex dynamics of their interactions with the host organism and also to discern the consequential impacts on the physiological processes and overall health of the host [28, 171]. Deeper insights into the functional roles of these microbiomes can provide valuable insights into their impact on digestion, immunity, metabolism and other physiological traits of the fish. This knowledge can then be leveraged to develop strategies for engineering the microbiome to enhance fish health and productivity [276].

A significant portion of our understanding of the complex interaction between the gut microbiota and fish hosts is derived from terrestrial vertebrates, with additional insights coming from studies involving gnotobiotic zebrafish (Danio rerio). Gnotobiotic animal models represent a highly significant in vivo experimental approach for studying the functions of the gut microbiota [55, 291]. Initially, research on fish gut microflora relied on culture-dependent techniques. However, the advent of metagenomics and next-generation sequencing (NGS) has revolutionized fish gut microbiome research in recent years [128]. These techniques enable direct analysis of microbial genomes from samples (gut or other environment), creating a deeper understanding of the relation between microorganisms and their hosts. The principal NGS platforms employed in fish gut microbiome research encompass the Illumina system, the Roche 454 system, and the Ion Torrent Personal Genome Machine (PGM). Notably, the Illumina system stands out as the most prevalent choice [55, 263].

Microbiome engineering holds a crucial role in achieving improvements in host health by leveraging the manipulation of microbial communities to achieve specific functions. Several approaches, including engineered probiotics, microbial transplantation, chemical-based approaches, and genetic engineering, play a vital role in the rapidly growing field of microbiome engineering [245]. Current practices in gut microbiota engineering mainly focus on humans, with less attention given to fish. However, applying microbiome approaches could significantly enhance fish health as well [171].

Aquaculture emerged as essential in meeting the increasing demand for high-quality animal protein [94] while alleviating pressure on wild fish stocks and marine ecosystems. Estimates indicated that 70% more food would be required to feed the projected human population of 9.1 billion by 2050, with aquaculture expected to play a key role in meeting this demand sustainably [40]. The anticipated doubling of aquaculture production by 2050 highlights its critical role in future global food security [171]. With the increasing frequency of disease outbreaks in aquaculture, farmers have turned more often to antibiotics, resulting in antimicrobial-resistant bacteria in both fish and the environment due to antibiotic residues. This practice also reduces microbiome diversity, which negatively impacts fish nutrition and health [205]. Implementing beneficial microbiome communities in the fish gut via probiotics or prebiotics could provide promising strategies for enhancing growth rates, disease resistance, and overall well-being in aquaculture settings. This comprehensive approach to fish health management offers significant potential for sustainable aquaculture practices and safeguarding the health and welfare of farmed fish populations [275]. Apart from this, engineering gut microbiome is gaining interest due to its potential to increase resistance to pathogens, enhance growth, boost metabolism, and improve immunity, thereby enhancing the overall gut physiology [178].

This review highlights the importance of gut microbiome engineering in fish health and addresses the challenges of its application in aquaculture, emphasizing the need for microbiome engineering. It also discusses various mechanisms, techniques, and applications of gut microbiome engineering while evaluating its effects on disease resistance, growth, and other physiological traits. Furthermore, this study explores emerging trends and innovations in the field, addressing the challenges and limitations for the broader adoption of gut microbiome engineering while considering ethical, regulatory, and safety considerations.

Fish gut microbiome: composition and function

Overview of the fish gut microbiome

The developing field of research concerning the gut microbiome of fish reflects a notable surge in interest. The majority of fish gut microbiomes comprise bacteria, and they play a significant role in various physiological functions, such as digestion, nutrient absorption, and immune system regulation. They have a significant impact on the overall health and physiological balance of the host, which is similar to that of humans and other animal species [263]. Hence, a long-standing, intimate, intricate relationship exists between animal hosts and their gut flora. Research conducted in humans has demonstrated the involvement of microorganisms within the gastrointestinal tract (GIT) in the gut-brain axis. The gut-brain axis is a complex bidirectional communication system that involves the interaction between the central nervous system (CNS), the enteric nervous system (ENS) of the gut, the endocrine system, and the immune system. The main component that regulates the gut-brain axis is the gut microbiome [28].

The composition of gut microbiota can fluctuate over time and can either be transient or persistent. The transient (allochthonous) microbiota refers to the temporal inhabitants of the gut, and a significant portion of these microorganisms typically enter the gastrointestinal system through diet. Even though they may not permanently colonize the gut, they can still play a role in maintaining the physiological processes of the host. In contrast, the persistent (autochthonous) microbiota establishes a more stable and long-term symbiotic association with the host’s gut. They contribute to overall well-being and functioning of the gut [188, 303]. Autochthonous microbes appear to be attached to the intestinal epithelium of the gut, whereas allochthonous microbes associate with the lumen [188, 225].

The development of the gut microbiota in fish is a complex process. The first microbes that the fish eggs come into contact with are mainly determined by the surrounding water, which acts as a repository of microorganisms [268, 313]. Hence, the eggs get a chance to be colonized by these microbes. Upon hatching, the larvae come into contact with these microbes and get colonized in their GIT. This early colonization stage acts as a basic platform for the development of the gut microbiota in fish [73, 282]. Research across various fish species suggests that the diversity of the gut microbiome varies as fish progress from early larval to juvenile stages [152, 169, 257]. Although the composition of the gut microbiota appears to be significantly affected by the introduction of external food sources, a prevailing gut microbial community existed even before that [64, 83], In a study conducted on Nile tilapia, the composition of the gut microbiota seems to vary in biofloc system. However, despite these fluctuations, a stable core community of prokaryotes persists within the gut of fish. This indicates that certain microbial populations maintain a consistent presence and functionality within the gut ecosystem, even as the overall composition varies due to external factors. New microbes interact with this core community, coexisting and forming a dynamic microbiome composition within the gut. This evolving microbial community potentially contributing to host health and homeostasis [63, 295].

Roles and functions of gut microbiota in fish health and performance

The gut microbiota forms a complex ecosystem within the fish GIT, actively contributing to various aspects of host physiology, metabolism, and immune function (Fig. 1). Gut microbiota aid in the breakdown of complex dietary components, such as carbohydrates, proteins, and lipids, into simpler forms that can be absorbed by the fish. They produce enzymes that the fish host may lack, facilitating digestion. Many bacterial strains of the fish gut can secrete enzymes that aid in digestion [222]. This enhances nutrient absorption and ensures efficient utilization of nutrients from the diet. Apart from this, intestinal microbiota is also involved in epithelial proliferation and maturation which aids in the efficient absorption of nutrients from the gut [190]. Intestinal microbiota in humans is known to facilitate protein/amino acids (AAs) digestion and absorption by decomposing complex subunits, which are easy to absorb and consequently modify metabolic pathways in the host cell [12].

Fig. 1

Schematic representation of gut microbiome engineering methods and their impacts on fish. The figure illustrates tools and techniques for gut microbiome engineering in aquaculture species, including probiotic supplementation, prebiotic application, synbiotic approaches, microbial transplantation, and genetic engineering. Key impacts include enhanced growth, disease resistance, nutrient absorption, and gut health, emphasizing their role in sustainable aquaculture

In the investigations using the zebrafish (D.rerio) model, researchers delved into the intricate interplay where the diet influences the microbial composition, inturns effects the metabolism, immunity and overall physiological processes. Semova et al. [237] demonstrated that the presence of microbiota within the zebrafish gut amplifies both the number and size of lipid droplets within the intestinal epithelium, highlighting the pivotal role of microbiota in modulating lipid absorption and energy equilibrium. Cheesman et al. [43] found that the resident microbiota and Wnt signalling jointly stimulate epithelial cell proliferation in the developing zebrafish intestine. Specifically, they revealed that gut microbiota support cell proliferation by enhancing the stability of β-catenin in intestinal epithelial cells, thus shedding light on a mechanism by which microbiota influence intestinal development.

Zhang et al. [307] explored the impact of dietary supplementation with L. delbrueckii on C. carpio, uncovering a notable enhancement in intestinal enzyme activities alongside improved growth performance. Likewise, Xia et al. [293] found that the bile acids can also improve the gut barriers of fish through both direct and indirect effects mediated by the gut microbiota. In addition, intestinal microorganisms within fish have been shown to enhance carbohydrate digestion through the secretion of a variety of enzymes and by boosting the activities of host digestive enzymes. These processes facilitate the breakdown of carbohydrates into decomposition intermediates and fermentation products. These products can subsequently serve as substrates for secondary utilizers, influencing the intestinal environment accordingly [306]. In addition, gut microbiota exerts influence over the feeding behaviour of fish through the modulation of appetite and satiety signalling pathways. Through the production of metabolites, they impact host metabolism, facilitate energy extraction from the diet, and influence energy storage mechanisms. This dynamic interplay between gut microbiota and host metabolism significantly contributes to the maintenance of energy balance and overall metabolic well-being in fish [124, 170]. Also, a variety of factors have been implicated in appetite control, including gut microbiota, which develops complex interactions to manipulate metabolic requirements and hedonic feelings. Gut microbial metabolites and components act as appetite-related signalling molecules to regulate appetite-related hormone secretion and the immune system, or act directly on hypothalamic neurons. To date, numerous studies support the notion that gut microbiomes exert a profound influence on eating behaviour in humans and other animals [78, 106].

Gut microbiota also plays a crucial role in promoting health through the development and regulation of the mucosal innate and adaptive immune systems [114]. The mutual interactions between a host and its intestinal microbiota are pivotal in moulding immune development. The immune system of the host co-evolves with its native microorganisms, which are essential for numerous physiological functions [130]. By actively monitoring the composition and changes in intestinal microorganisms, the host's immune system regulates the balance of the gut microbiota, ensuring the health of the intestinal lining. This delicate balance is crucial for maintaining optimal GI function and overall physiological health [16, 170, 306]. The mucosal immune system of fish encompasses both innate and adaptive immunity, with all four mucosal organs harbouring mucosa-associated lymphoid tissues (MALT), with particular significance attributed to the gut-associated lymphoid tissue (GALT). Goblet cells, found throughout these mucosal organs, secrete mucus, forming a protective barrier rich in mucins (glycoproteins), proteases, antimicrobial peptides (AMPs), esterase, and lysozymes. Notably, mucosal epithelial cells are equipped with pathogen recognition receptors (PRRs) that engage with microbial entities. Furthermore, fish mucosal tissues host various immune cells including macrophages, neutrophils, natural killer cells, mast cells, eosinophils, and basophils. Immunoglobulin M (IgM) predominates in the plasma of teleost, serving as a key mediator in systemic immune responses [97]. The colonization of intestinal mucosal surfaces by indigenous microbiota exerts a favourable influence on the regulatory mechanisms of the gut immune system. Within this context, the mucosal immune system employs PRRs to distinguish between pathogenic and commensal bacterial entities. These PRRs serve as pivotal mediators in the identification of bacterial antigens, triggering intricate signalling cascades that promote immune responses within the intestinal environment [206]. The microbiota plays a crucial role in shielding the host against colonization and the proliferation of diverse pathogens through the mechanism of colonization resistance [146]. Furthermore, the microbiota exerts its protective effects via several intricate pathways, including competitive nutrient utilization, constraining direct gut interaction, and destroying the resistance mechanisms. This results in the establishment and propagation of pathogens which enables them to flourish within the host environment [81, 192, 295]. Moreover, gut bacterial species actively secrete AMPs as they compete with pathogens for niche occupancy. Consequently, a delicate equilibrium is established among the gut microbiota, pathogens, and gut mucosa. Disruption of this equilibrium not only predisposes the host to infections but also initiates the activation of the GALT. The gut microbiota serves as a pivotal factor in the progression and refinement of the GALT by playing a fundamental role in its development and maturation [28].

Dong et al. [69] investigated the effect of infectious hematopoietic necrosis virus (IHNV) infection in the digestive tract of rainbow trout (O. mykiss). They focused on assessing the immune response and microbial communities across various segments of the digestive tract, ranging from the buccal mucosa to the hindgut post-infection. Their observations unveiled that immune responses induced by IHNV within the mucosal linings disrupt the micro-ecological balance. This disruption facilitates the infiltration of opportunistic pathogens, leading to subsequent secondary infections. It is evident that well-established gut microbiota mitigate infections and sustain overall fish health [69]. A well-established diverse gut microbiota in fish can offer protection against infections thereby lowering the chances of infection and improving overall health. Any changes to this equilibrium of microbes inside the fish can weaken their immune response, potentially leading to disease [177, 183].

Furthermore, the intestinal microbiota exerts a significant influence on various physiological processes, extending beyond digestion and immunity. It plays a crucial role in modulating stress responses, impacting reproductive functions, and contributing to developmental processes [140]. The administration of L. rhamnosus probiotics to zebrafish (D. rerio) elicited a significant enhancement in ovarian function, notably impacting oocyte maturation and ovulation. This effect was achieved through the downregulation of genes that typically impede these reproductive processes. Additionally, L. rhamnosus induced substantial modifications in the zebrafish gut microbiota, leading to larval development. This is characterized by improved growth dynamics and more efficient sex differentiation within the zebrafish population [36]. The coordination between gut microorganisms and reproductive endocrine regulation is mainly based on their ability to directly modulate hormonal profiles [109]. An experiment involving the dietary incorporation of probiotics in goldfish revealed notable enhancements across multiple reproductive parameters. Specifically, supplementation of feed with probiotics led to increased gonadosomatic index (GSI), heightened fecundity, and elevated fry production among spawning females. Additionally, this intervention correlated with augmented length and weight metrics in the resultant fry population [179].

The relationship between the composition of the gut microbiome and neuroendocrine system emerges as a crucial determinant in the modulation of crowding stress and growth regulation. This symbiotic relationship influences stress response mechanisms and behavioural patterns, especially impacting anxiety-like behaviours and locomotor activities. These behavioural alterations, in turn, affects the feeding behaviours and the maintenance of energy homeostasis [28]. In zebrafish, enriching the microbiota has been shown to mitigate anxiety-related behaviours and alleviate stress responses. This effect is achieved through the downregulation of corticotropin-releasing hormone (CRH) expression and reduction in cortisol levels. Conversely, periods of feed deprivation in zebrafish have been found to adversely affect their behaviour and stress physiology, potentially increasing their susceptibility to disease outbreaks [87]. Alterations in the gut microbiota can impair fish foraging behaviour and inhibit feeding activity by elevating stress hormone levels. Changes in hormonal levels disrupt central and peripheral mechanisms involved in nutrient sensing which is crucial for metabolic appetite control. Stress-induced inhibition of these mechanisms underlies the decreased appetite observed in fish under stress conditions [53].

Factors influencing the composition of fish gut microbiome

The fish gut microbiome composition is influenced by a diverse array of factors, including host genetics, developmental stage, feeding habits, diet, stress, disease, and environmental conditions [73, 263, 295, 312]. Environmental factors viz. season, temperature, pH, water and sediment quality play a significant role in shaping microbial communities (Fig. 2). Understanding these interconnections is essential for maintaining fish health and optimizing aquaculture practices [66, 202]. Environmental parameters, including water and sediment quality, exert substantial influence on the composition and diversity of the gut microbiota of aquatic animals. Water acts as a conduit for microbial transportation, facilitating the ingestion of microbes by fish through drinking or water intake. Subsequently, a fraction of these ingested microbes can colonize the fish gut microbiota, thereby contributing to its complexity and functionality [122, 303, 315].

Fig. 2

Factors influencing the fish gut microbiome. The figure outlines key factors shaping the fish gut microbiome, including environmental conditions, diet composition, host genetics, health status, and microbial interactions, highlighting their combined effects on microbiome structure and function

Geographic location

Various geographical regions exhibit divergent environmental parameters encompassing temperature, salinity levels, seasonal patterns, water chemistry, and microbial constituents. These environmental variables exert a direct influence on the diversity and abundance of microorganisms within aquatic ecosystems, subsequently impacting the composition of the gut microbiota in fish [122, 203]. Liu et al. [162, 164, 166, 167] utilized high-throughput 16S rRNA gene sequencing to investigate how genetic variation and environmental differences affect the gut microbiome of three geographic populations of the black Amur bream (Megalobrama terminalis) from Pearl river, Moyang river and Wanquan river. Their study suggested that prolonged geographic isolation leads to diverse environmental conditions and genetic variations, which together influence the diversity of the gut microbiome in wild M. terminalis. Additionally, these findings offer valuable insights for studying ecological adaptation in fish populations [162, 164, 166, 167]. In contrast, Nikouli et al. [193] studied adult Sparus aurata and Dicentrarchus labrax individuals and found no significant geographic impact on gut microbial species. They observed a strong similarity in gut microbial communities between the two species, with only minor differences noted in the luminal gut communities. When Hanson-Regan [108] compared the gut microbiomes of both native and introduced species of the freshwater fish invader Cyprinella lutrensis, they observed a significant change in gut microbiota between host sites. Additionally, they noted a lesser extent of variation among host species. Their findings emphasize that beyond geographical factors, the collective influence of host genetics, diet, and geography holds substantial importance in shaping the intestinal microbiota. However, in S. salar, a dramatic difference between environmental and gut bacterial communities was observed. Moreover, community composition was not significantly impacted by geography [168]. Gut microbial composition shift was also found in humans according to geography [238].

Season

Dulski et al. [71] performed high-throughput sequencing of 16S rRNA gene amplicons to investigate the gut microbial populations within Tench (Tinca tinca ). They examined two distinct populations, wild Tench (lake) and Tench in semi-intensive farming (pond), during both the summer and autumn seasons. They observed a notable disparity in the abundance of Candidatus xiphinematobacter between lake and pond fish during the summer, but not in the autumn. Additionally, in pond Tench, the abundance of Methylobacterium exhibited a significant reduction during the summer compared to the autumn. Consequently, they propose that seasonal variations can exert an influence on the composition of the gut microbiome [71]. Similarly, in Nile tilapia (O. niloticus), conspicuous differentiations in microbial diversity were observed across diverse sampling periods and among populations inhabiting both natural habitats and captivity. These observations underline the pronounced impact exerted by environmental variables in modulating the intricate composition of the intestinal microbiota [20].

Seasonal fluctuations were noted in the gut microbiota of Atlantic salmon (S. salar L.). During the yearly cycle, lactic acid bacteria (LAB) consistently make up a substantial portion of the intestinal microbiota. However, bacterial species like Vibrio spp. were only detected at specific sampling points. The indigenous (autochthonous) microbial population appeared relatively stable across seasons, whereas the non-indigenous (allochthonous) population may change in response to different seasons. This suggests that while certain microbial groups remain consistent in the salmon gut throughout the year, others may exhibit seasonal shifts, possibly influenced by environmental factors or host physiology [120].

Temperature

Temperature is a key factor in regulating animal physiology and function. Response to temperature by each host species will be unique. Especially in the case of microbes, some may have the capacity to proliferate in elevated temperatures, while some of them get suppressed or may not have any effect. Likewise, certain gut bacteria like Firmicutes and Proteobacteria show consistent changes according to the variation in temperature [239] Hassenrück et al. [113] demonstrated that the temperature variation of 26 to 33 °C in juvenile milkfish induced changes in gut microbiota composition which in turn helped the host to acclimatise to temperature variation. Similarly, temperature variation from 8, 12, 16 and 20 °C in Chinook salmon (O. tshawytscha) resulted in the replacement of Vibrionaceae (Photobacterium phosphoreum and/or P.pisciola) by the Fusobacteriaceae (C.somerae) and Brevenemataceae (Brevinema) as the dominant population [256]. In addition, variation in water temperature from 8 to 18 °C in Chum Salmon (O. keta) showed the dominancy of Flavobacterium with an increasing level of Tenacibaculum at 8 °C and the most diverse population of other microbes with an increasing level of vibro at 18 °C. This indicates that temperature variation is very important in regulating dissolved oxygen, salinity, and pH levels in water, which is crucial for aquatic animal health [95].

In addition, most opportunistic pathogens get activated at elevated temperatures and can also cause gut dysbiosis in fish [95, 162, 164, 166, 167, 260]. In mussel larvae, (M. coruscus) a shift in gut microbiome composition was reported according to the temperature variation from 21 to 27 °C. As the temperature increases, the composition of several bacteria including Delftia, Neptunomonas, Pseudoalteromonadaceae, Rhodococcus, and Stenotrophomonas, while the composition of Tenacibaculum genera elevated significantly. Tenabacibaculum is an opportunistic pathogen that can cause the death of the larvae [320]. Elevated temperature of 28 °C was also known to impair the intestinal health of crabs [10]. Similarly, acute heat stress of 24.5 °C in Rainbow trout altered the normal gut morphology, permeability, and antioxidant capacity altogether harming metabolism (Zhou et al., 2020). Heat stress is also reported to damage the intestines, their microbiota, and overall physiology in sturgeons [162, 164, 166, 167]. However, low temperatures (14 °C) also showed a negative effect on gut microbial composition and thereby altered the normal physiological condition of largemouth bass (Micropterus salmoides) [172]. Cold stress is also known to affect intestinal health by reducing the intestinal microbiota and enhancing the proliferation of pathogenic Pleisomonas bacteria [121]. Moreover, many studies documented that temperature is a key factor in regulating gut microbiome health and composition [162, 164, 166, 167],).

Salinity

Salinity is regarded as another important factor which maintains the composition of intestinal microbiota. Apart from this, salinity is a significant factor that helps to maintain the osmotic pressure and, thereby, regulates respiration, metabolism, digestion, growth, and even the survival rate [98]. The effect of salinity variation among freshwater, brackish water, and marine water species may vary. Many studies have reported the change in gut microbiome in different species in different salinity ranges [133, 135, 163, 165]. Since Atlantic salmon is an anadromous fish, variation in salinity could affect its gut microbial diversity. Changes in the composition of the gut community have been reported during the transfer from seawater to freshwater. Proteobacteria were found as a dominant community in all groups, yet the presence of Escherichia coli or Shigella occupied a dominant population in the seawater [185]. There was a dominant community shift from Actinobacteria to Proteobacteria reported after acute other communities like phages did not show any response [98]. However, abundance of proteobacteria and lactobacillus in farmed Chinook Salmon remained unaffected by salinity shifts. Hence, some gut bacteria populations in fish can remain stable regardless of salinity [314].

Salinity stress also enhances the chances of infections among aquatic species. Low salinity stress in Pacific white shrimp made the species susceptible to pathogenic V. parahaemolyticus by reducing the gut microbiome richness and promoting the proliferation of pathogenic species [41]. High salinity exposure in grass carp resulted in boosting the growth of harmful bacteria with the suppression of beneficial bacterial growth. A shift in gut microbiome composition according to the salinity shift caused a deleterious effect on the overall health of the host [160, 161]. Both the low and high salinity levels induce changes in the intestinal microbiota diversity, increase the risk of susceptibility towards various diseases, and altogether disrupt the normal physiological functions of the host [56].

pH

pH level in water also has a significant role in balancing intestinal microbiota. Aquatic animals always come up with varying pH levels due to anthropogenic interventions. A change in pH level beyond the optimum level will disrupt gut-microbiota balance and also promote the enhancement of pathogenic microbes [261]. Low pH levels resulted in dysbiosis in marine sea bream, reducing the abundance of LAB in the intestine and finally promoting the growth of pathogenic strains [85]. Ocean acidification modified the intestinal community structure in Crassostera gigas [137] and edible oysters [57]. The relative abundance of Firmicutes declined and boosted the growth of mycoplasma in the case of C. gigas at low pH [137]. Elevated pH also possesses deleterious effects on gut microbiota. High alkaline conditions enhanced the growth of Bacteroides, ammonium-producing Providencia sp., and Vibrio pathogenic bacteria in common carp and disrupted the overall balance of intestinal microbiota [242]. Niu et al. investigated the changes in the gut microbiota of C. elegans in different acidic conditions and suggested that there exists a deep relationship between the environmental pH and microbial ecology. Extreme pH also resulted in gut dysbiosis in C. elegans [194]. Similarly, a pH drop caused an imbalance of intestinal microbiota in Colossoma macropomum (Sylvain et al., 2016). Hence, a change in pH level can cause dysbiosis of gut microbiota and can favour the growth of pathogenic microbes in aquatic animals [137].

Sediment and water quality

In aquatic systems, water and sediment are primary components that determine the composition of intestinal microbiota [124]. The microbial community present in the water and sediment represents the dominant community in the gut of the species in that habitat [124]. In the study conducted on shrimp L. vannamei, the bacterial community structure was different in the water, sediment, and intestine. Even though the bacterial communities were different, they shared a similar dominant bacterial profile. This indicates the deep relationship between the gut microbiota and the microbiota in sediment and water [122]. Similar findings were also observed in P. japonicus. The shrimp intestinal microflora closely mirrored the surrounding water microbiota and influenced the health of the host by regulating the immune activity [254]. Another study conducted by Fan et al. also demonstrates that bacterial composition is almost similar in the intestine and sediment in the L. vannamei, irrespective of its relative abundance [79]. Several studies documented that water and sediment are the main key factors in shaping the gut microbiome of aquatic animals [70, 117].

Host genetics, age, developmental stage, and diet

Intestinal microbiota is primarily shaped by genetics and phylogeny of the host, development, and other environmental factors [154]. Host genetics controls the responses toward the colonisation of bacteria in the gut, secretion of various enzymes, and other factors. Hence, both may have an equal role in shaping the intestinal microbial community [189]. Beyond other environmental factors, host genomics is the primary factor that shapes the gut microbiota in stickleback. Smith et al. observed that the more genetically divergent population of stickleback fish consists of the most divergent intestinal community [251]. Despite this in Channel catfish and blue catfish, host genetics played a minimal effect in regulating the gut microbial composition. This study strongly suggests that the same environmental condition and similar life history diminish the effect of host genetics in influencing gut microbial diversity [24].

In addition to the host genetics, the developmental stage is also a key factor in regulating gut microbiota. The environment of the gut will be different at different life stages and also the associated microbes. Microbial colonisation will change over time in each species. It may also be due to feeding at that specific period [313]. Gut microbiota associated with the younger and adult stages, including their relationships, were better studied than the intermittent stages [204]. In gilthead bream, the later age represented the high diversity of gut microbiota. In addition to age or developmental stage, the sex of the host also influences the microbial composition [209]. Basil et al. proved that the intestinal microbial composition mainly depends upon the fish's age more than the sex and sampling site. Age-related community shifts in the Bacteroidia and Desulfobacteria populations were also documented [19]. Similar findings can also be observed in the southern catfish. The gut microbial population underwent sequential changes according to the developmental stage in catfish [313] and also in sturgeon fish [204].

Host diet

Fish possess specific gut traits according to their diet and feeding habits. Herbivorous fish exhibit longer guts compared to the carnivorous species. The gut microbiota associated with the gut also varies according to the nutrition available through the diet [162, 164, 166, 167]. Several studies have been recorded on the modulation of gut microbiota with the application of specific feeds [94, 145, 269, 270]. Villasante et al. demonstrated the effect of a carbohydrate diet on the gut microbiome of Atlantic salmon. They found that the carbohydrate diet caused a low abundance of several bacteria except carbohydrate-dependent bacteria [281]. It was reported that the microbial shift and change in digestive enzyme activity were found in the gut of M. amblycephala during the feed transition. Mainly, the metabolic activity is changed during the feed transition [288]. Fasting periods also have a key role in maintaining the gut microbial balance. Proteobacteria were the representatives during the fasting period, and Firmicutes represented the feeding period in the Leopard coral grouper (P. leopardus). Host diet and fasting are also the main factors that shape the gut microbiome of the host [180].

Challenges in aquaculture and the need for microbiome engineering

Food security is a crucial factor required for acquiring sustainable development worldwide [80]. Aquatic food plays a significant role in global nutrition and food security [196]. However, wild fisheries are decreasing rapidly due to the overexploitation of aquatic wild stock [80]. Aquaculture offers a solution to the overexploitation of wild fisheries in a sustainable way [32, 39]. Aquaculture is regarded as a rapidly growing industry, expanding at a rate of 1.1% each year. It has been the fastest-growing food sector in the world over the past 50 years, with fish food production of more than 80 million metric tons [80].

The global aquaculture sector has thrived over the past 20 years and continues to grow by meeting critical goals of environmental, economic, and social sustainability. Aquaculture plays a significant role in meeting the need for protein sources for millions of people worldwide. Many people, especially those in coastal areas, mainly depend on aquaculture for their livelihoods [26]. Aquaculture contributed about 50% of fish to meet the needs of human beings in 2020. In addition, 62% of the global live-weight volume and 75% of the global edible weight volume will be contributed by inland freshwater aquaculture alone in 2020 [17, 27]. The rise in population has resulted in increased protein requirements, which in turn caused a surge in demand for aquaculture products [182]. About 40 million metric tons of aquatic food will have to be produced to meet the needs of people by 2030 [3, 23].

Sustainable aquaculture practices mainly focus on using low-impact farming methods, including non-fed aquaculture, recirculating aquaculture systems, or integrated agriculture–aquaculture, which helps to reduce waste and water use [175]. Although the aquaculture sector is growing, various challenges pose a serious threat to achieving sustainability [27]. Aquatic pollution, disease outbreaks, genetic degradation of aquaculture species, use of chemicals and drugs, decline in comparative profitability, lack of knowledge on market risks, and financial crises are some of the main challenges faced by aquaculture [155, 265]. Disease outbreaks are the main challenge faced by aquaculture today [243, 265]. Emerging various viral bacterial fungal diseases with changes in weather and climate make them even worse [173, 191].

Good aquaculture practices, including proper diet and maintaining optimal rearing conditions, are now being practised to mitigate disease-causing conditions. Along with this, antimicrobial products have been used to prevent microbial infections in aquaculture systems. However, some pathogens are capable of resisting these antimicrobials. Despite this, another serious issue arising as a result of using antimicrobials is antimicrobial resistance, especially antibiotic resistance [181, 201, 290]. Vaccinations [264] have been used as an alternative to antimicrobials. Limited efficacy, ineffectiveness against juvenile fish, farmed crustaceans, and molluscs, and inefficiency in developing long-term acquired immunity become major limitations in vaccine implementation [208]. Biosecurity approaches and vaccine implementations have been used to mitigate various infectious diseases in aquaculture. Species-specific approaches and the severe contingency and efficacy of diseases still act as a barrier against these approaches [191]. The application of probiotics, prebiotics, synbiotics, and phytobiotics opened a new biological approach to replace the use of antimicrobials and vaccines [208]. Preventive measures against diseases and infections are far better than treating the diseases [5, 14]. Maintaining a healthy gut microbiome is a significant key to good health [73, 217].

Recently, there has been a significant rise in interest in gut microbiome research, as a healthy gut is considered an indication of the well-being of the host. Several studies are now going based on diversity, functions, and factors influencing the gut microbiome. Moreover, advances in high-throughput sequencing, including 16S rRNA sequencing and shotgun sequencing, have greatly contributed to deeper insights into gut microbial studies. Emerging technologies like metatranscriptomics, metabolomics, culturomics, and synthetic biology also open up a more detailed mechanistic approach to gut microbial studies than a simple phylogenetic description [86, 143]. Now, gut microbiome engineering seeks more interest due to its different approach to maintaining the health of individuals [126].

In humans, gut microorganisms have been used as biosensors to treat the dysbiosis of the microbiome against several diseases like cancer, metabolic, infectious, and auto-immune diseases. Intestinal gut microbiome engineering to produce a keystone consortium is an emerging area of research to produce healthy individuals [15]. Several application studies are ongoing in humans and other animals, rather than fish. Techniques like manipulation of the gut microbiome community, application of in vitro gut stimulators, and synthetic microbial communities are now focused mainly on humans. Similar technologies, including biotechnological approaches, can also be executed in fish, which will open a new door to gut microbiome research in fish [171]. Numerous strategies for intestinal microbiota manipulation together with probiotics in a beneficial way to maintain host health will bring solutions for various challenges in aquaculture, including disease outbreaks [93]. Emerging in situ genome engineering approaches to gut microbiome manipulation also boost the microbiome applications in fish hosts [245]. Implementations of these gut microbiome engineering techniques aim to achieve not only the maintenance of a healthy environment for fish but also the sustainable growth of the entire aquaculture sector [65].

Mechanisms and techniques of gut microbiome engineering

Gut microbiome engineering is an emerging field that aims to create a healthy host by manipulating gut microbes [304]. Microbial manipulation is done by altering gut microbiota composition or their metabolic activity to maintain host health [148, 149]. Gut microbiome engineering involves various techniques such as probiotics, prebiotics, synbiotics, phage therapy, genetic engineering, and faecal microbial transplantations [289]. Now, gut engineering studies are mainly concentrated in humans, mice, and rats [86].

Probiotics, prebiotics, and synbiotics: mechanisms and applications

Probiotics

Probiotics, prebiotics, and synbiotics possess significant applications in the aquaculture sector [60, 61]. Probiotics are living microorganisms with beneficial effects that can be administered to individuals in proper amounts. These microorganisms usually proliferate and colonize the gut of the host and help to maintain the good health of the host [116]. Probiotics are used to promote the growth of intestinal microbes and improve digestion, absorption, immune responses, and disease resistance in the host [300]. In addition, it can also remove other pathogenic microorganisms by providing a competitive environment [136]. Feed probiotics and water probiotics have been used to maintain microbial balance in the host.

Bacterial or fungal strains can be isolated and administered by mixing with feed as a probiotic. Whereas, these microbes can be simply served in the water as water probiotics. Water probiotics improve water quality by removing organic matter and other contaminants [59]. However, most of the probiotics are administered by mixing with feed. Several bacterial strains have been used in probiotics as live or dead strains [42]. Probiotic strains are selected based on several criteria. The strain should be free from antibiotic genes and could not affect host genetics. Microorganisms should be able to be administered to the host, and they should have the ability to survive the drastic conditions in the digestive tract and to proliferate in the host gut. These microbes should have antimicrobial properties and must affect promoting the health of the host [184]. In addition, these microbial communities should be native to the aquatic environment and compatible with the host. Mainly, probiotic communities must be safe for application, both for the host and the environment and free from any antibiotic-resistant gene. [59].

Several species of Lactobacillus, Bifidobacterium, and Streptococcus were commonly used in probiotics. They include L. acidophilus, L. casei, L. fermentum, L. gasseri, L. plantarum, L. reuteri, L. helveticus, L. bugaricus, L. salivarius, L. rhamnosus, L. johnsonii, L. paracasei, B.bifidum, B. breve, B. lactis, B. longum, Saccharromyces sp., S. boulardii, S. thermophiles, and S. cremoris [42, 88, 272]. B. subtilis is regarded as one of the most potent probiotic microbes used for various applications. B. subtilis is used in O. niloticus, Labeo rohita, and L. vannamei for the secretion of digestive enzymes; in M. rosenbergii and O. niloticus for improving water quality. It is also used in aquaculture to improve water quality, maintain reproductive health, minimize stress, and promote antioxidant responses [96]. Streptomyces, another potent probiotic microorganism, is widely used due to its ability to secrete chemical compounds like streptomycin and oxytetracycline. Streptomyces provide disease-resistant capacity, antimicrobial activity, and enzyme modulation activity [29]. Several species of Lactobacillus were also widely used in aquaculture in therapeutic and prophylactic feed formulations [248].

Prebiotics

Prebiotics are non-digestible feed additives that can promote the growth and activity of bacteria in the gut [226]. Prebiotics are non – digestible carbohydrates that can be classified into monosaccharides, oligosaccharides, and polysaccharides based on the degree of polymerization [34]. These feed additives can promote growth, immunity, disease resistance, and stress reduction in aquatic animals. Prebiotics are mainly derived from plants and naturally occur in vegetables, fruits, beans, seaweeds, microalgae, edible mushrooms, and animal milk [285]. The beneficial effect of prebiotics is due to the by-products generated during fermentation by gut bacteria such as Lactobacillus and Bifidobacterium and gut enzymes [34, 225]. Short-chain fatty acids produced during this fermentation cause a pH drop in the gut and, in turn, affect the gut environment in a positive way that enables these prebiotics to be absorbed and utilized by the host [102].

Ideal prebiotics are selected based on several criteria. The prebiotic food additive should have the ability to overcome the enzyme degradation in the upper gastrointestinal tract. It should be utilized by the beneficial gut bacteria as an energy source and have a positive effect on the gut environment. It must be able to stimulate the immune responses in the gut environment [195]. Prebiotics help to minimize the chances of infection by promoting the growth of beneficial gut bacteria that produce inhibitory compounds, act as competent for adhesion sites, inhibit the quorum sensing in the gut and stimulate immune responses. [102]. These in-digestible carbohydrates directly interact with pattern recognition receptors (PRR) and activate the innate immune system. They can also stimulate the microbial-associated molecular patterns (MAMPs) activity [255].

Mannan-oligosaccharides (MOS), Arabinoxylan-oligosaccharides (AXOS), fructo-oligosacchrides (FOS), galacto-oligosccharides (GOS), Isomaltooligosaccharides (IMO), and trans oligosaccharides are the main oligosaccharides used as prebiotics in aquaculture. Inulin, β-glucans, stachyose, lactylol, lactosucrose, lactulose, and oligofructose constitute the other important prebiotics [8]. MOS has been used as an immunostimulant against A. hydrophyla infection in Nile tilapia [219]. Inulin is used to promote growth, immunization responses [301], and stress reduction [318] in Nile tilapia. In addition, XOS and GOS were known to enhance antioxidation and detoxification in Nile tilapia [297]. Fructo-oligosaccharides possess positive effects on growth performance, phagocytic activity, and resistance against A. hydrophila infection in C. macropomum (De et al., 2019). Growth performance, intestinal enzyme activity, and metabolism in M. amblycephala were stimulated by the use of XOS as a probiotic [1].

Synbiotics

Combinations of probiotics and prebiotics were denoted as synbiotics. Synbiotics gained much importance due to the application of live microbes into the gut along with dietary supplements [6]. Synbiotics promote the proliferation of good bacteria in the gut, which boosts intestinal enzyme activity and digestion. It also promotes the nutrient absorption ability, thereby ensuring efficient feed utilization by the host [227]. Moreover, synbiotics stimulate the intestinal epithelial cells to secrete immune functional cells and promote innate immune responses [125]. Synbiotics, as a combination of probiotics and prebiotics, offer not only growth performance and immune stimulation but also disease resistance capacity and gastrointestinal microbial balance. It also acts as a biological control agent in water and, consequently, supports the overall well-being of the animal [198].

The synbiotic composition is determined based on the prebiotic component. Prebiotics with high degrees of polymerization are highly recommended for use in synbiotics, as they can provide primary and secondary metabolites by fermentation and can be utilized by the host. In addition, more survival and action of probiotics were observed in combination with the prebiotics [30]. Synbiotics must be biocompatible and biodegradable, and they should ensure host and environmental safety [212]. Synbiotics have been applied to several aquaculture species to promote their growth and well-being [227].

Feed additives with microencapsulated Bacillus and oat β-glucan were known to improve the growth performance and feed utilization in Nile tilapia [67]. A combination of MOS with B. sp., L. acidophilus, and E. faecium in feed was regarded as a prophylactic alternative for fish farming [38]. Probiotic L. casei with S. ferox extract (SFE) in catfish (C. gariepinus var.) promoted growth, feed efficiency, and resistance against bacterial infections [110]. Feed supplementation of the synbiotic culture of B. amyloliquefacien promoted the growth rate and lyzozymal activity in Nile tilapia [92].

The concentration of prebiotics, probiotics, and synbiotics is a crucial factor to be determined before supplementation. These feed supplements beyond the optimal level have a negative effect on species and vice versa. Moreover, compatibility between the probiotics and prebiotics is also very important. These feed additives should be formulated based on the environmental conditions of the implementing species. Consequently, a long-term study is required for the formulation of probiotics, prebiotics, and synbiotics for each species [7].

Phage therapy and bacteriophage-based approaches

Bacteriophages are viruses that can infect and kill specific bacteria. They play a vital role in controlling the bacterial population and are capable of eliminating half of the bacterial population in each 24 h [214, 215]. The tail end of the virus can specifically identify and bind to the receptors on the surface of the bacteria, which makes them host-specific. The specificity can extend up to genus level, species level or to specific strains [266]. The phage life cycle comprises lytic and lysogenic cycles. Phages replicate and lyse the bacterial cell in the lytic cycle whereas in the lysogenic cycle, phage DNA gets incorporated into the host genome and transferred to the subsequent generations [218]. Phage-based microbiome engineering is a different approach that specifically reduces the targeted bacterial population by utilizing the phage life cycle as they lysis the bacterial cell [266]. Several studies on phage application to control or prevent pathogenic bacteria are well documented [141, 142, 211, 230, 249].

Phages can be isolated by using various methods including double layer agar method, concentration methods and filtration or adsorption-based separation methods from soil, water and sediment. An ideal phage must be strictly lytic, and free of antibiotic resistance and virulence gene. Moreover, they should be polyvalent, environmentally tolerant and able to co-exist with other phages. The ability to destroy the bacterial biofilm is also considered a priority in phage selection [162, 164, 166, 167]. Engineered phages through various techniques viz. Chemical mutagenesis, CRISPR/Cas and Caspe technique Cell-free transcription techniques and Bacteriophage recombineering with electroporated DNA (BRED) also can be used to improve phage specificity [127]. Broad-spectrum phages, also known as phage cocktails, are mixtures of multiple phages that can infect multiple hosts that belong to distinct species or genera [62].

Feed pellets fixed with phage are considered an effective method for delivering phages to the fish through oral administration. Application of phage therapy together with dietary supplements and anti-microbials could enhance its efficacy (Donati et al., 2022). Combination of phages with probiotics has been practised in various studies. Phages that can overcome the dry conditions in the environment are only chosen as a coating for the fish feed [214, 215]. Biopolymers like edible whey protein isolates (WPI) coating on fish feed incorporated with phages were known to improve the loading efficiency and storage stability and act as a barrier against gastrointestinal digestion. Hence, the phage activity can be limited during the storage phase and can ensure effective activation in the target site. WPI coating also helps to overcome phage dissociation in saltwater. Consequently, WPI remains a cost-effective method for treating bacterial infections in aquaculture [123]. Oral application of phages is regarded as the most convenient method of phage administration rather than the method of injection and direct application to the surrounding water [214, 215].

Phage therapy remains an excellent approach to eradicating pathogenic bacteria compared to antibiotics. Antibiotics can only avoid the infection for a particular time, but phage therapy can maintain the overall gut microbiota composition irrespective of the infection [68]. Phage therapy also provides a solution for multidrug resistance in bacteria [82]. Bacteriophage-based approaches helped to eradicate multi-antibiotic-resistant bacteria like Aeromonas  subsp. Masoucida in aquaculture [298]. Apart from this, phage Pzl-Ah152 was known to be well effective against A. hydrophila, used to treat enteritis of crucian carps. Pzl-Ah152 boosted the gut microbial diversity without disrupting its balance [82]. MJG Phage are also used to treat the A. hydrophila infection in Rainbow trout [33] and Phage D6 in fish pellets [216]. Cocktail of Phages VB_VaC_TDDLMA (phage TDD) and VB_VaC SRILMA (phage SRI) can control the V. alginolyticus infection in brine shrimp culture (A. franciscana) [262]. Motile Aeromonas Septicemia (MAS) caused by Aeromonas spp. in striped cat fish (P.hypophthalmus) is effectively treated by the A. hydrophila-phage 2 and A. hydrophila-phage 5 [147]. Early or simultaneous administration of phage could have more effect against bacterial infections in aquaculture [141].

Microbial transplantation and other strategies

Faecal Microbial Transplantation (FMT) or simply microbial transplantation is the engraftment of a microbial community from the stool of a healthy donor to the recipient to restore or modify the normal gut microbiota [25, 132]. The FMT concept was first described in humans during the fourth century in China [310]. Microbial Transplantation also called Faecal bacteriotherapy opens a new way to treat dysbiosis in the gastrointestinal tract [49]. Apart from the faecal transplantation, the Bacterial consortium is utilized as the most convenient method of FMT in aquaculture. Bacterial transplantation is implemented through probiotics, with feed or by simply increasing their concentration in surrounding water [279]. Understanding and observing the current status of the recipient, donor selection and selection of method of implementation are the main factors taken care of during FMT. In addition, compatibility between the species and environment also should be considered [22, 279]. Though the FMT have good beneficial effects, side effects like diarrhoea and abdominal pain are reported in humans [4]. Similarly, the side effects of FMT in fish also need to be evaluated prior to the application.

FMT can restore the gut microbial diversity after antibiotic treatment or dysbiosis. Han et. al. demonstrated the restoration of gut microbiota after florfenicol-perturbation to the intestine. The microbiome diversity lost as a result of florfenicol application was successfully restored by the FMT than that of natural recovery. FMT also promotes intestinal damage repair and stabilizes intestinal metabolites [105, 107]. FMT in large yellow croaker (L. crocea) larvae were observed to promote intestinal health by enhancing intestinal microbial diversity. The larvae gut after FMT were shown to increase in α-amylase and trypsin, leucine-aminopeptidase activity and a well-established gut environment than normal (Zhang et al., 2023). FMT implementation in the early life stage of Rainbow trout resulted in improved muscle yield and fast growth through gut microbiota modulation [223]. Three Lactobacillus strains isolated from Patagonian fish were colonised and established in the gut of zebrafish and modified their intestinal microbiota composition [240].

Inter-species Intestinal microbial transplantation (IMT) was carried out between donor Atlantic salmon (S.salar) to recipient gilthead seabream (S. aurata), after disrupting the normal gut microbial community of seabream with antibiotics. IMT was observed to be maintained and enriches the growth of novel bacteria over time. This study suggests that IMT can be done within the species or between different species and could be used to improve the digestive mechanisms and thereby maintain the fish's health [231]. Reciprocal FMT has been carried out from the Zebrafish and Mice to the germ-free recipients showing the development of a new microbial community in the gut. A newly introduced microbial community will proliferate and shape the intestinal microbiota composition, where the host acts as a habitat or basic niche [221]. Similar reciprocal microbial transfer was also applied in Zebra fish from humans and some of the bacterial communities were known to be colonised successfully [11, 273, 278]. Lactic acid bacteria isolated from the Atlantic Salmon (S. salar) [9], Salmonids [280], and freshwater fish [100] can be used as probiotic candidates in aquaculture.

Application of phytogenic, immune and metabolic modulators are the other strategies used to modify the gut microbiota. Herbal seed powder supplementation consisting of fennel, fenugreek and anise seeds improved intestinal microbiota health and the overall health of European sea bass [13]. Allium-derived compounds also modulated the intestinal microbial component in Gilthead Seabream (S. aurata). Many studies documented the effect of phytobiont effect in shaping the gut microbiota [44, 269, 270, 294]. RNase 1 was used as a gut microbiota and metabolome modulator in Blunt snout after the Aeromonas infection. RNase of M.amblycephala with antimicrobial activity and digestive activity played a significant role in reducing inflammation of the gut, reducing pathogen invasion and improving intestinal function [90]. Similarly, other roles of RNase can also be evaluated for enhancing gut microbiota [45, 259].

Genetic engineering and biotechnological interventions

Advancement in biotechnology and genomic techniques enables researchers to modify microbial strains and improve gut microbiome diversity. CRISPR/Cas-based tools, synthetic biology techniques, and metabolic engineering are the main engineering techniques applied in gut microbial studies [321]. Synthetic biology approaches in the gut microbiome are now mainly practised in humans, where it engineers bacteria to improve their functions by modifying their natural biological components. Synthetic biology approaches mainly focus on designing the cells and enzymes thereby effectively improving cell communication [139]. Gene editing tools like CRISPR/Cas technique, Transcription-activator effector nucleases (TALEN) and Zinc finger nucleases (ZFNs) unlocked new entities of synthetic biology [139].

CRISPR-based genome editing techniques are widely used to modify the functional genes of lactic acid bacteria (LAB). CRISPR/Cas systems can induce double-strand break in the target site of nucleic acid by the action of Cas9 nuclease and repairs, which result in genome editing [160, 161]. Genome engineering is an important synthetic biology tool that can be applied to probiotics. The inducible plasmid self-destruction (IPSD) technique is one of the genome engineering or editing techniques that delivers homologous DNA into the bacteria and can result in gene knock-out or knock-in. IPSD was effectively implemented in lactobacilli and bifidobacteria to modify the probiotics [321].

Metagenomic Alteration of the Gut microbiome by In situ Conjugation (MAGIC), is a tool of synthetic biology in which genetic modification is carried out with the help of their natural DNA exchanging capability. In MAGIC, a donor strain can be modified and developed, so that they can able to transfer the genetic pyloads to other bacterial strains by normal gene transfer methods like conjugation [150, 229]. Reproducible pGT vectors also can be transferred between the bacterial strains with the help of the MAGIC tool [139]. General methods of gene transfers, including transformation, transduction, and conjugation, can be applied to insert required DNA into the bacterial cells to modify their functions to attain a modified gut microbiota. Chemical transformation and electroporation are commonly used transformation tools, whereas suicidal plasmid vectors, conjugative transposons and shuttle plasmid vectors belong to the conjugating techniques [244] [150]. Apart from this, synthetic biology utilises several tools like, molecular docking, artificial intelligence techniques, nanotechnology and post-translational modification to engineer the microbial enzyme secretions [247].

Applications

Successful applications of gut microbiome engineering in aquaculture and its impact on fish health and performance

Application of probiotics in aquaculture

Previous research shows that probiotics play a significant role in fish health (Table 1). Probiotics possess various applications in aquaculture to improve growth, immunity, and the overall health of the animal. Various probiotic strains exhibit different effects on each aquaculture species. Several species of Bacillus, Streptococcus, Pseudomonas, and Enterococcus are commonly used probiotic strains in aquaculture. A combination of several bacterial strains or individual strains can be formulated as probiotics [31], demonstrated that the application of Lactococcus lactis enhances the immunity in O. niloticus by excluding the pathogenic bacteria through competition. It also boosts the nutritional value of its food [31]. A similar effect in improving growth, disease resistance, and intestinal health was observed in O.niloticus with the combined application of S. agalactiae, B. cereus NY5, and B. subtilis [138]. A mixture of E. xiangfangensis (GFB-1), P. stutzeri (GFB-2), B. subtilis, Citrobacter freundii, and P. aeruginosa was known to improve growth, reproductive performance, and gut health in B. gonionotus [233].

Table 1 Applications of probiotics in aquaculture

B. cereus and G. candidum have been associated with the growth enhancement and stimulation of digestive enzyme activities in L. rohita [94]. Similarly, the combination of B. amyloliquefaciens and B. subtilis was reported to improve immune responses and growth in L.rohita and Heteropneustes fossilis [235, 252]. B. amyloliquefaciens is shown to improve the immunity growth and the lipid metabolism in O. niloticus [246], whereas B.subtilis and B. licheniformis together are linked to influencing reproductive performance in Red tilapia (O. niloticus x O. mossambicus) [74]. Lactiplantibacillus plantarum enhances nutrient utilisation and intestinal enzyme activities in Larimichthys crocea [163, 165]. All these studies highlight the different applications of probiotics in aquaculture and their potential to enhance immunity, growth, disease resistance, and feed utilisation efficacy in various aquaculture species.

Applications of prebiotics in aquaculture

Prebiotics also share similar applications with probiotics, as they enhance gut health by improving immunity, growth, disease resistance, and survival rate (Table 2). Fructooligosaccharides (FOS), galactooligosaccharides (GOS), mannanoligosaccharides (MOS), and inulin are the main prebiotics widely used in aquaculture. A combination of FOS, GOS, MOS and inulin showed a positive impact on the growth and health of C. gariepinus [89]. Improved stress resistance and survival rates were reported by the application of FOS, xylooligosaccharides (XOS), chitosan, β-glucan, and MOS in hybrid grouper (Epinephelus lanceolatus x E. fuscoguttatus) [319]. Similarly, in O.niloticus, MOS with vitamin E was found to enhance its reproductive performance [253]. Chito-oligosaccharides also showed improved growth performance, innate immunity, and intestinal health in O. niloticus [197], whereas it was found to improve gut health and resistance against A. hydrophila in hybrid tilapia (O. niloticus x O. aureus) [213]. Chitosan was given to Oreochromis niloticus to promote innate immunity, antioxidant capacity, and overall intestinal morphology [159].

Table 2 Application of prebiotics in aquaculture

Guerreiro et al., demonstrated that FOS, XOS and GOS could enhance immunity in Diplodus sargus [101]. A combination of Inulin, GOS with D-sorbitol is known to boost growth performance, serum complement and cytokine levels, lysozyme activity, intestinal antioxidant capacity and gut microbial diversity in Nibea coibor [158]. Inulin has also been reported to improve the growth, serum immunity and immune responses in O. mykiss [91] and promote growth and immunohematological indices in O. niloticus [301]. Moreover, inulin together with Jerusalem artichoke tuber enhanced the growth activities of O. niloticus [271]. FOS enhanced growth, survival rate, digestive capacity, intestinal barrier function, digestive enzyme activities gut morphology in Atractosteus tropicus [207, 239].

Fucoidan has been linked with the growth, digestive tract maturation and maintenance of an ideal intestinal microbial composition in L. crocea [308, 309]. The combined action of FOS, GOS, MOS, inulin, β-glucan, isomaltooligosaccharides (IMO) and XOS resulted in pathogen exclusion, improved digestion, nutrient absorption and modification of the gut microbiota in L. vannamei [317]. MOS application enhanced growth and digestive enzyme activity in Atractosteus tropicus [176]. Similarly, growth enhancement and improvement in digestion and immune activities were observed in L. japonicus by the action of FOS [283]. Moreover, MOS and inulin were also known to evoke growth promotion in this species [277]. All these findings emphasize the significance of probiotics in the aquaculture sector.

Synbiotics applications in aquaculture

Synbiotics, which combine probiotics and prebiotics, exhibit a significant role in aquaculture by enhancing the growth, immunity, disease resistance, and gut health of various species (Table 3). Effects of probiotics and prebiotics will vary in the same species itself based on their combinations and proportions. Bifidobacterium sp., L. acidophilus, and E. faecium, along with MOS and chitosan, have been reported to improve the immunity and resistance against the A. hydrophila infection in O. niloticus [38]. The combination of A. oryzae and β-glucan also promotes the immune response in the same species [60, 61]. In A.japonica, the administration of B. subtilis with MOS and E. faecium with FOS was found to improve its disease resistance [200]. Enhanced growth, feed utilisation, intestinal health, and non-specific immunity were the notable improvements observed in L. vannamei with the administration of B. subtilis and S. cerevisiae with β-glucan and MOS [299]. Whereas L.plantarum L20 and S. polycystum enhanced the growth, disease resistance, and protection of gastrointestinal structure by modulating the gut microbiota in P. monodon [47]. Similarly, in M.rosenbergii, the combination of L. rhamnosus and Yu-Ping-Feng polysaccharide improved lipase activity and maintained a balanced environment [156].

Table 3 Applications of Synbiotics in aquaculture

The inclusion of B. subtilis and L. plantarum with xylooligosaccharides was found to promote the immunity and overall performance of O. niloticus [284]. Acinetobacter KU011TH and chitosan in hybrid catfish (C. gariepinus x C. macrocephalus) influenced growth, immunity, and disease resistance against A. hydrophila [236]. A combination of galactooligosaccharides (GOS) and P. acidilactici was found to increase innate immune responses and skin mucus parameters in O. mykiss [115]. Moreover, L. delbrueckii subsp. bulgaricus, along with A. officinalis initiated improvements in digestive enzyme activity and overall metabolic efficiency in the same species [302]. Reproductive performance and growth of D. rerio were also found to be influenced by the combined action of Lactobacillus acidophilus and L. delbrueckii subsp. bulgaricus [305]. Overall, the combined action of prebiotics and probiotics as synbiotics brings more significant effects in aquaculture in terms of their immunity, growth, and overall development and thereby supports the sustainability of aquaculture.

Application of bacteriophage-mediated vector transfer

The applications of bacteriophage-mediated vector transfer in aquaculture also offer a promising approach to modifying gut microbial communities and enhancing the overall health of the aquaculture species (Table 4). In the same way as prebiotics, probiotics, and synbiotics, bacteriophage application also basically influences the growth, immunity, and disease resistance in aquaculture species. For instance, the application of FpV4 and FPSV-D22 targeting F. psychrophilum was reported to positively alter the gut microbiota of O. mykiss, independent of the target bacterium. This study also underlines that the intervention of phage in the gut affected their interaction with other bacteria [68]. Treatment of A.hydrophila with phage PZL-Ah152 positively influenced the gut microbiota in C.carassius [82]. Vb AsM ZHF phage targeted S.maximus, promoting immunity and disease resistance against A. salmonicida subsp. and reducing the mortality rate [298]. Introduction of FCL-2 phage against F. columnare increased the survival rate of Salmo salar by reducing the relative amounts of the pathogen in water. Similarly, phage MJG phage targeting A. hydrophila resulted in restoration of liver tissue damage and elimination of the clinical signs of infections in O. mykiss [33]. All these reports strongly denote the significance of phage-mediated vector transfer in fish gut microbiome engineering as a way forward.

Table 4 Application of bacteriophage mediated vector transfer in aquaculture

Lessons learned and challenges encountered in real-world implementations

Gut microbiome engineering is widely applied in humans, and its implementation in aquaculture is more challenging. However, research on these gut microbiome techniques and their applications in aquaculture is focussing more now. Now, the use of probiotics, prebiotics, and synbiotics has become more common in aquaculture, where biotechnological approaches remain rare and need to be focused more [171]. Probiotics, prebiotics, and synbiotics can be applied in aquaculture to improve growth, digestion, immunity, disease resistance, nutrient absorption, and water quality [31, 74, 77, 284, 319].

Probiotic strain identification and isolation, including determination of its proportion of use and evaluation of its safety and effectiveness on required species, is the main challenge faced during the formulation of a probiotic, especially in aquatic species. Beyond the probiotic formulation, its application at the farm level in agriculture is another challenge. Knowledge of the benefits of probiotics and their administration of use among farmers is to be boosted to achieve the effective use of probiotics in aquaculture [258]. Evaluation of safety and efficacy is very important to selecting a bacterial strain as a probiotic. Temperature is a significant factor that affects the viability of the probiotic strain, especially during storage. Storage of probiotics at 4°C can maintain viability and efficiency for a long period [50]. In addition, water quality, feed utilisation, species, and other environmental factors are also affecting the viability of probiotic strains [7]. Genetic exchange between the probiotic strains changes the function of the strains, making the formulation less effective in the host. Moreover, they can also be emulated as opportunistic pathogens and can infect the host [75]. Along with this, prebiotics and synbiotics applications are also facing similar limitations in modulating gut microbiota. Formulation of prebiotics, probiotics, and synbiotics in different aged and sized fish and their application level at each species, together with the evaluation of effectiveness and safety in each administration method, are the main challenges faced in this sector [186].

Likewise, formulation and administration techniques of safe phages are the main challenge in phage-mediated vector transfer in aquaculture. Jun et al. demonstrated that phage pVp-1 has bacteriolytic activity against three representative AHPND-V. Parahaemolyticus strains in L. vannamei. Phage therapy resulted in no such resistance against the AHPND as its proliferation is so rapid. Hence, the relationship between the period of phage division and the proliferation is an important factor to be considered in phage therapy [129]. Sometimes, single-phage-type applications will not be enough to control the infection due to the rapid growth of the pathogen. Advanced methods like phage cocktail combined with toxin binder can be applied in these situations [99]. Another main challenge faced in this technique is the safety issue due to the virulence genes associated with phages. Virulence genes associated with the bacteriophages, especially in the case of temperate phages, should be observed properly and ensure the safety of these phages before use [157]. In aquaculture, mainly phage therapy is applied to bathing, feeding, injection, and wiping. The effective optimal safety delivery method must be developed before its implementation [54].

FMT is considered a powerful tool to treat dysbiosis and to restore the gut microbiome in animals. The selection of healthy donors and receivers is the important factor considered first and can be achieved by rigorous screening experiments [171]. FMT is mainly applied in humans now, though the studies in advanced use of FMT remain in an unoccupied area [4]. Selection and screening of the desired donor strains from the donor is a very large and time-consuming method. Its application in other individuals belonging to the species or different species must be tested and screened properly to evaluate the effects [94]. Some recipient populations may not be able to accept and develop the new gut microbiome, mainly in stressful conditions. Receptance ability will be directly correlated with the health of the recipient. FMT will be successful mostly in the younger ages than in the older ones [121]. Tang et al. demonstrated the effective transplantation of young faces enhanced metabolic capacity in older ones to restore gut microbiome diversity [267].

Gut microbiome modulation through the CRISPR method is much more expensive. Inappropriate aquatic genome data is another limitation of the CRISPR technique in aquaculture [104]. It is much more difficult to incorporate genes into a group of mixed microbiotas, and it is only possible in the population of E. coli. Moreover, gene insertion can occur multiple times in multiple target sites and can also bring a negative result. Like other engineering techniques, precise standardisation of the safer CASPER editing technique is the most important challenge [244].

Environmental consequences and sustainability considerations.

As the researchers focus more on gut microbiome engineering to improve sustainability in aquaculture, environmental consequences are gaining more concern and attention. The main concern is regarding the biotechnological application in this field [151]. Intensified aquaculture production and practices began to depend on antibiotics to overcome disease outbreaks. Gradually, it led to the development of antibiotic-resistant bacteria and started transferring these resistant genes into the aquatic environment. Intrusion of these antibiotic-resistant genes into the terrestrial environment through the aquatic environment brought adverse effects not only to humans and terrestrial animals but also to aquatic animals [118]. In this circumstance, antibacterial vaccines, probiotics, and bacteriophages gained more attention [52]. Henceforth, biotechnological interventions in the aquaculture field, especially in gut microbiome engineering, opened a new path for the improvement of aquaculture production and sustainability [131, 171].

Environmental concerns regarding genetically modified organisms also came up with the biotechnological applications. The main concerns about genetically engineered microbes were competition, habitat alteration, horizontal gene transfer, and toxicity due to their small size and rapid growth. The persistence of unwanted genes, transfer of modified genes to the wild population, and uncontrollable proliferation are the main issues that gain more attention. A solution for the gene transfer is the implementation of a toxin and antitoxin system in different parts of the genetic repository of the cell, as the plasmid that does not carry the antitoxin will kill the new host [174]. Even though the bacteriophage-mediated vector transfer technique can replace antibiotics to overcome antibiotic resistance, resistance mutation can cause environmental concerns. But it develops ten times slower than antibiotic resistance [162, 164, 166, 167]. Loss and gain of function of genes in engineered microbes were the other concerns because they can change the basic characteristics of the bacteria, which can be a challenge to the stability of the environment [119]. Altogether, the escape of the modified microbes to the environment and the disruption in native genes are the main subjects to be more concerned about and need to ensure safety [174].

Future directions and challenges

Emerging trends and innovations in gut microbiome engineering

Gut microbiome engineering has mainly been applied in humans now. Its applications in agriculture are an emerging area. Only a few studies are documented on the various microbiome engineering techniques. Now, the gut microbiome engineering techniques look forward to widening their study mainly towards therapeutic and preventive applications. In aquaculture, it's mainly targeting the enhancement of fish health, improvement of feed efficiency, immunity, disease resistance, and other sustainable practices. Deep knowledge of the composition, diversity, and factors affecting the gut microbiome is the primary data that is required prior to the application of engineering techniques. For the proper database of the gut microbiome, integrated application of multi-omics, metatranscriptomics, and metabolome single-cell genomics is required. A well-documented database provides a strong basis for gut microbiome engineering [296]. Further improvements in gnotobiotic models to study the host and microbiome interaction can help achieve this. Culturing of fish gut microbiota will promote its application through probiotics, prebiotics, and synbiotics [162, 164, 166, 167].

Biotechnological innovation is regarded as the new opening in gut microbiome engineering. The combined action of different methods together can bring a better result. Rasmussen et al. documented the combined action of vibriophage KVP40 and probiotic P. inhibens, which reduced the mortality of fish pathogenic vibrio thereby helping to maintain fish fry health [220]. In addition, application of probiotics with feed supplements also enhances sustainable outcomes. Ribonucleotide supplementation with probiotic strain B. subtilis improved the health of Asian seabass [232]. Implementation of FMT and probiotics together can bring a huge result in gut microbiome modulation. B. subtilis from the intestine of Microstomus kitt possesses a high probiotic potential [234]. B. velezensis isolated from the M. salmoides acts as a potential feed additive to promote growth disease resistance in aquaculture [292].

The combination of the CRISPR/Cas technique and phage therapy is another emerging area. As the CRISPR/Cas system works against many anti-microbial resistances of pathogens, you can use it to eliminate pathogens from the gut. Applying phage as a delivery system of the CRISPR/Cas system to improve its potential to eliminate pathogens [187]. Engineering a key consortium of single microbes that support mutually is the main emerging trend in this field [15]. CRISPR/Cas involves novel gene editing techniques like, Base editors that work on single base conversions, primer editors rely on base substitutions and precise DNA insertions and deletions; and CRISPR-associated transposases perform recombination-independent, multi-kilobase DNA insertions. These are applied in the formulation of probiotics in humans [287] and can be applied in aquaculture.

Addressing challenges and limitations for wider adoption

Safety issues are the main problems that are faced by all engineering techniques in the aquaculture field. Emergence of antibiotic-resistant pathogens together with their capability to transfer these genes to other microorganisms [272]. Genetically engineered bacteria are a huge goal, and their development is still stuck in its initial phase. Its application is much more concerned and restricted due to its potential pathogenicity and local regulations. The future challenges regarding this engineered bacterium will be its capacity to effectively proliferate and work in its target site, how to localise these bacteria only to the target site, and also its elimination after use. New advancements in technologies could address these safety challenges and can be used for therapeutic applications [287]. When it comes to aquaculture, it is very difficult to apply to water and aquatic animals, development and usage of probiotics and other techniques, their activity in aquaculture, production cost, and storage capacity include another set of challenges [66].

Ethical, regulatory, and safety considerations

Gut microbiome engineering involves various ethical, regulatory, and safety considerations. Mainly, the ethical principle includes individual good/non-maleficence that ensures the animals are not harmed intentionally and carelessly. Other principles involve collective good/beneficence that secures the proper well-being of animals; autonomy relies on individual rights and free will; and lastly, justice assures equality in availing of all benefits and costs for all [111]. The ethical view consists of different aspects, including bioethics, environmental ethics, and food ethics. Bioethics focuses on and supports the overall well-being of animals. Environmental ethics mainly supports biodiversity in terms of minimising the use of chemicals. Food ethics relies on avoiding unhealthy food that is high in fat, sugar, or any other substance that destroys the gut microbiota [144]. Ethical concern tries to avoid the potential hazards of the various techniques that cause hazards or risks towards the animal health and environment before their application. [111]. The safety controls of worldwide regulatory authority regarding genetically engineered microbes (GEM) include the minimisation or controlling of biocontainment to limit the spread and persistence of GEM in the environment. Genetic instability is ensured for the stability of GEM regardless of the loss or gain of function. In addition, uncontrolled growth of GEM and competition with stable and long-term eubiotic communities are also evaluated before the application of GEM [151].

Conclusion

Summary of key findings and implications

Gut microbiome studies in aquaculture are still in their infant stage, where more studies have been documented on its composition and diversity. More studies are required to develop microbiome applications for the sustainable growth of aquaculture species. In this review, we summarise the details and knowledge about the gut microbiome composition, and structure, its importance in aquaculture, and the various engineering techniques for modulating the intestinal microbiota. We also focus on its applications in aquaculture and other ethical and safety concerns regarding these techniques. As we concentrate on the gut microbiome in fishes, Fusobacteria, proteobacteria, Firmicutes, and Bacteroidetes constitute the abundant group. They have a significant influence on host physiology, digestion, feed utilisation, metabolism, immune function, and stress responses. Several abiotic and biotic factors, including host genetics, developmental stage, feeding habits, diet, stress, disease, and environmental conditions consisting of season, temperature, pH, water, and sediment quality, play a significant role in shaping intestinal microbial communities. Hence, factors such as seasonal shifts and other environmental factors that influence the non-indigenous species of the gut microbiome highlight the need for dynamic, environmentally adaptive strategies-based approaches in microbiome engineering. As disease outbreaks become more frequent in aquaculture, farmers increasingly mainly depend on antibiotics and vaccines, leading to antimicrobial-resistant bacteria in fish and the environment becoming a serious issue. Maintaining the proper health of individuals and good aquaculture practices helps to avoid many of these disease conditions. Well-balanced gut microbiota composition and diversity promote the better health of an individual. At this point, modulation of these intestinal microbiotas emerges as a new platform for achieving the sustainable growth of aquaculture.

Gut microbiome engineering is a new approach towards the modulation of intestinal microbial diversity that involves various techniques such as probiotics, prebiotics, synbiotics, phage therapy, genetic engineering, and faecal microbial transplantations. Among these, probiotics, prebiotics, and synbiotics constitute those practices much more explored in the aquaculture sector. Techniques are well utilised to promote the growth of intestinal microbes and improve digestion, absorption, immune responses, and disease resistance in the host. Bacteriophage-mediated vector transfer is another biotechnological approach also utilised in aquaculture. It mainly helped to eradicate antibiotic-resistant bacteria. FMT studies are mainly documented in humans, and only a few studies have been reported in aquaculture. Likewise, CRISPR-CasPER-mediated engineering techniques are also trapped in their initial state; more concentration is required for their advanced application in aquaculture.

The main challenge regarding these techniques is their application in farm conditions. Development of these techniques for each aquatic species in consideration of the safety issues is also time-consuming and more difficult. Combining these different techniques towards the development of key consortiums for each species can be looked forward to in the future. Much more advanced studies and application of each technique in gut microbial studies enhance the sustainability of aquaculture.

Prospects and recommendations for further research and application

To achieve further advancements in gut microbiome studies, improvements from the basic level of experiments are required. Applying advanced techniques with multi-omics approaches provides greater insights into the role, function, and composition of gut microbiota and will help to optimise the intestinal community towards its future applications [202]. Gnotobiotic fish models, including zebrafish and stickleback, can be utilised for the same [311]. Various engineering techniques can be applied to achieve a healthy microbiota or to treat dysbiosis in animals. Yet, deep knowledge of the relationship between the host disease condition and the intestinal fauna is required for its proper implementation [84]. A combination of various engineering techniques and its target implementation, gut microbiome modulation, is the main area kept before performing in the future [187]. Beyond the usual engineering techniques, metabolic engineering strategies can also be included for better outputs from gut microbiome modulation [153]. CRISPR-related tools can expand engineering techniques in a much more precise and efficient way [35]. Next-generation probiotics are another emerging area in aquaculture. The application of synthetic biology with genetic modification of probiotics will boost the personalised enhanced future in next-generation probiotic therapies [2]. Natural bacteria-based drugs in treating different diseases are also an upcoming therapeutic application in humans and can also be applied in aquaculture in the future. More advancement in gut microbiome engineering techniques together with artificial intelligence (AI) and synthetic biology can bring great development in sustainable aquaculture [139, 202]. The development of comprehensive guidelines and well-structured training programs incorporating advanced strategies and addressing safety concerns is essential for the successful real-world application of microbiome engineering. With its potential to enhance aquaculture sustainability through improved productivity, disease resistance, and environmental balance, microbiome engineering emerges as a robust, future-proof strategy for advancing sustainable aquaculture practices.