Probiotics, Prebiotics, and Synbiotics Utilization in Crayfish Aquaculture and Factors Affecting Gut Microbiota

Aquaculture is affected by numerous factors that may cause various health threats that have to be controlled by the most environmentally friendly approaches. In this context, prebiotics, probiotics, and synbiotics are frequently incorporated into organisms’ feeding rations to ameliorate the health status of the host’s intestine, enhancing its functionality and physiological performance, and to confront increasing antimicrobial resistance. The first step in this direction is the understanding of the complex microbiome system of the organism in order to administer the optimal supplement, in the best concentration, and in the correct way. In the present review, pre-, pro-, and synbiotics as aquaculture additives, together with the factors affecting gut microbiome in crayfish, are discussed, combined with their future prospective outcomes. Probiotics constitute non-pathogenic bacteria, mainly focused on organisms’ energy production and efficient immune response; prebiotics constitute fiber indigestible by the host organism, which promote the preferred gastrointestinal tract microorganisms’ growth and activity towards the optimum balance between the gastrointestinal and immune system’s microbiota; whereas synbiotics constitute their combination as a blend. Among pro-, pre-, and synbiotics’ multiple benefits are boosted immunity, increased resistance towards pathogens, and overall welfare promotion. Furthermore, we reviewed the intestinal microbiota abundance and composition, which are found to be influenced by a plethora of factors, including the organism’s developmental stage, infection by pathogens, diet, environmental conditions, culture methods, and exposure to toxins. Intestinal microbial communities in crayfish exhibit high plasticity, with infections leading to reduced diversity and abundance. The addition of synbiotic supplementation seems to provide better results than probiotics and prebiotics separately; however, there are still conflicting results regarding the optimal concentration.


Introduction
The growing global demand for animal protein due to the world population's rapid growth has led to the prevalence of aquaculture in fish and shellfish production (~50% of global production) [1]. The aquaculture industry provides consumers with animal products of high quality that possess an increased protein percentage while simultaneously contributing to global food safety, and its production is estimated to increase further in tandem with the increasing demand [2]. In total, during the last 70 years, the amount and synbiotics seem to play crucial roles, affecting various health and production factors. However, prior knowledge of microbial composition and interaction is needed in order to develop the most suitable supplement in each case. Previous review studies discuss the effect of these supplements in fish, shellfish, and shrimp aquaculture; however, a comprehensive review focusing on crayfish aquaculture is missing. Although there are occasionally contradictory results regarding the administration of pre-, pro-, and synbiotics in crayfish culture, there is no such comprehensive review combining all this information.
Therefore, the scope of the present study is to review the utilization of pre-, pro-, and synbiotics in crayfish aquaculture, their effects on crayfish farming, and proposed benefits and their mechanism of action, as well as to present some future perspectives. Furthermore, the microbiome of crayfish, apart from being a dynamic and complex biological system, exhibits a key role in many physiological processes of the organism [27]. Thus, in the present study, the factors affecting crayfish microbiota (including growth, diseases, and farming type) are reviewed and discussed.

Pro-, Pre-, and Synbiotics
Shellfish aquaculture's intensification relies on the world's strong appetite for shellfish; specifically, their rich valuable protein content and healthy profile of unsaturated fats [28], which, when substituting saturated fats (SFAs) and trans fats, decrease the risk of cardiovascular disease (CVD) [29,30]. However, the intensification of aquaculture practices exerts versatile stresses on cultured aquatic organisms [31], primarily due to infectious pathogens, which trigger their immune defense system responses and pose serious threats to the aquaculture industry's growth and sustainability [32]. Since crustaceans' productivity is highly dependent on their habitat, and therefore different environmental changes may cause viral and bacterial diseases, their stock's conservation demands intensive and rigorous management [33]. Since sustainable cultivation is an integral and economically viable component of the aquaculture sector, shellfish aquaculture has largely relied on cutting-edge technologies, such as recirculating aquaculture systems (RAS), to effectively address threats posed by pathogens to shellfishes [34][35][36]. Additionally, aquaculture practices regarding the treatment of pathogens commonly and widely depend on antibiotics, which are administered in the diets of cultured aquatic species [32]. However, disease prevention treatments through antibiotics may increase opportunistic pathogens' infestation [37] and favor antibiotic-resistant pathogens. The latter can pose serious negative health effects due to their ability to be transmitted toward terrestrial animals and enter the human food chain [38,39]. Moreover, indiscrete antibiotic application impedes the beneficial activity of gut microbiota. This can have serious effects on the physiological processes of cultured aquatic organisms, such as altered microbial systems, disrupted nutrition, and immunological competence [40,41].
Because antimicrobial agents' health threats and adverse side effects have become uncomfortably apparent to both producers and consumers, alternative approaches are urgently needed to address the threat of pathogens in aquaculture [42,43]. Probiotics have been acknowledged as significant replacement agents for those stressors and for their detrimental effects, serving as immune modulators and boosting resistance to different microbial infections [42][43][44]. In this context, feed additives, such as prebiotics, probiotics, and synbiotics, are frequently incorporated into cultured aquatic organisms' diets [5]. The aim of such practices is to considerably ameliorate the microbial and morphological health status of the host's intestine [45,46], enhancing its functionality [47]. Prebiotics (indigestible fiber) increase the preferred gastrointestinal tract microorganisms' growth and activity, and provide a clear balance between the gastrointestinal and immune system's microbiota, thus benefiting the host's immunity and health [48][49][50]. Probiotics (non-pathogenic bacteria-based products) are mainly focused on organisms' energy production and efficient immune response [51], increasing resistance against various pathogens [42][43][44]. However, mounting evidence enhances their role in increased nutrients' absorption, stress resistance, and fertility of the host species [52], thus shaping them as positive promoters of aquatic · no significant impact on growth and survival [77]         (1 × 10 7 CFU/g), LS2 (1 × 10 9 CFU/g), PE1 (5 g/kg), PE2 (10 g/kg), LS1PE1 (1 × 10 7 CFU/g + 5 g/kg); LS2PE2 (1 × 10 9 CFU/g +10 g/kg) for 18

Probiotics Administration
All information regarding the administration of probiotic supplements in crayfish aquaculture is summarized in Table 1. Singe probiotics such as Bacillus subtilis and Bacillus licheniformis exhibit positive effects on the immunity and survival rate of P. leptodactylus [79] and P. clarkii [74] when administered as dietary and water additives, respectively. After the administration of Lactobacillus plantarum on P. leptodactylus [63] and C. cainii [60,70], positive results in immunity parameters were observed in both, while in C. cainii, the diversity of intestinal microbiota increased. However, severe histopathological effects in both the guts and hepatopancreas were observed when non-industrial effective microorganisms were added to the diet of P. leptodactylus, while no effect was observed on their growth rate [76]. Furthermore, no positive effect on the growth and survival of stage II P. leptodactylus juveniles was observed when lactic acid bacteria and Hafnia alvei were applied both as dietary and water additives [77]. From another study on P. clarkii, a probiotic strain A23 Bacillus amyloliquefaciens, isolated from healthy individuals and added to the diet, demonstrated promising results, providing multiple benefits for crayfish cultivation. More specifically, it was found to enhance intestinal digestive enzyme activities, innate immune genes expression, and enzyme activities, as well as white-spot syndrome virus (WSSV) resistance [64,69]. Further, B. amyloliquefaciens supplements the decreased apoptosis of hemocytes [69]. The above results were further confirmed in a recent study investigating two other fish-derived probiotics, namely Bacillus coagulans (SCC-19) and Lactococcus lactis (Z-2), where increased activities of immune-related enzymes and mRNA expression of two AMP genes, better integrity, and a thicker mucosal layer, together with higher density granules in epithelial cells and increased phagocytosis rate of hemocytes and pathogen resistance, were observed [65]. Finally, intestinal microbiota diversity was found to be elevated [65]. Dietary Limosilactobacillus fermentum GR-3 revealed positive effects on P. clarkii gut microbiota, as it was observed that the dysbiosis incurred from Arsenic (As) reduced and further field application led to a significant increase in production [66]. In addition, dietary supplementation of Saccharomyces cerevisiae [73] resulted in increased weight gain, SGR, expression of LYZ, prophenoloxydase (proPO), and resistance towards Citrobacter freundii. Positive effects were also observed on the health status of C. cainii by the means of immune indices and microbial composition of the midgut after the dietary inclusion of Bacillus [67,80], Clostridium butyricum [78], and Lactobacillus acidophilus [60]. More specifically, Holdemania and Vibrio were identified as the most abundant bacteria in the groups fed the probiotic and in the control group, respectively [60], while Lactobacillus abundance was associated with the up-regulation of immune genes expression after the probiotic inclusion [70]. In Cherax tenuimanus, improved resistance towards Vibrio mimicus was also observed after dietary supplementation of probiotic bacteria (Bacillus sp.) (A10 (Bacillus mycoides), A12 (Shewanella sp.), PM3 (B. subtilis), and PM4 (Bacillus sp.)), whereas they were found to positively affect the physiological condition of crayfish with no impact on intermoult period, growth, and survival [68]. However, contradictory results were observed on C. quadricarinatus, where commercial probiotics [62], including Bacillus, Acinetobacter, and Chryseobacterium genera, could not control A. hydrophila in the system, while (Ecoterra ® ) [72] supplementation only led to an increase in some hemolymph parameters. When Cambarellus montezumae was studied, the dietary probiotic Spomune© inclusion resulted in increased survival and growth rate, as well as weight gain [71]. Furthermore, in the same species, Lactobacillus inclusion also resulted in increased final weight and improved overall welfare [75] (Table 1).

Prebiotics Administration
The administration of prebiotic supplements in crayfish aquaculture is summarized in Table 2. In P. leptodactylus, 75% dietary fishmeal substitution with Chlorella vulgaris showed the highest values of final weight, SGR, protein efficiency ratio (PER), protein productive value (PPV), in vivo apparent digestibility coefficients of organic matter (ADC OM ), and in vivo apparent digestibility coefficients of crude protein (ADC CP ), while the lowest FCR was observed. Additionally, with the Chlorella inclusion, activities of alkaline protease, lipase, amylase, PO, SOD, LYZ, and NOS were stimulated [82]. When Mannanoligosaccharide (MOS) and fructooligosaccharide (FOS) were added to the diet of narrow-clawed crayfish, a positive impact on crayfish immunological responses to air and bacterial exposure challenges, feed utilization, and growth performance was observed [83]. Additionally, in the same species, it was revealed that dietary galactooligosaccharide (GOS) exhibits advantageous effects on innate immunity, stress resistance, intestinal microbiota, and digestive enzyme activity, while no significant improvement in growth performance and survival was observed [84]. Concerning P. clarkii, dietary Haematococcus pluvialis administration was found to increase WGR, SGR, and hemolymph immune-related enzyme activities while leading to a malondialdehyde (MDA) content decrease [81]. Additionally, activities of alkaline protease, lipase, amylase, PO, SOD, LYZ, and NOS were promoted [81]. Further, P. clarkii fed with sulfated β-glucan revealed improved overall growth performance together with antioxidant capacity and immunity. Additionally, the intestinal flora improved as abundances of beneficial probiotics increased, while those of maleficent decreased [87]. On red claw crayfish C. quadricarinatus, an injection of 3-HB with a monomer of poly-β-hydroxybutyrate (PHB) caused improved phagocytosis, suppressed the growth of pathogenic bacteria, and increased the expression of microtubule-related genes. Hence, this prebiotic helped the crayfish individuals to be more resistant to pathogens [85] overall. Similarly, in the same genus, C. tenuimanus (Smith, 1912), the prebiotic MOS' inclusion in the diet led to survival, health status, and immunity improvement, especially under certain circumstances such as bacterial infection and stress conditions incurred by exposure to NH 3 and air [86]. Dietary supplementation with Bio-Mos©, which has S. cerevisiae as a main ingredient [88], resulted in better WGR and SGR while also exhibiting positive results on the health status, intestinal microbiota composition, immune parameters, and disease resistance of Cherax distructor individuals (Table 2).

Synbiotics Administration
All information regarding the administration of synbiotic supplements in crayfish aquaculture is summarized in Table 3. Experiments conducted on P. leptodactylus individuals sought to evaluate the effects of prebiotics (galactooligosaccharide (GOS, MOS, and xylooligosaccharide (XOS)), probiotics (Enterococcus faecalis and Pediococcus acidilactici), and synbiotics on different physiological markers. Results indicate that crayfish fed with the GOS+ Enterococcus [89] and XOS + E. faecalis [90] diet revealed the highest activities of PO, SOD, LYZ, alkaline phosphatase (ALK), and NOS. Furthermore, after implementing the aforementioned diets, the survival of A. hydrophila exposure had increased [89,90]. These results highlight that crayfish fed with synbiotic-enriched diets had a better effect than a single administration with probiotics and/or prebiotics [89]. The above is in line with a previous study [59], where synbiotics Lactobacillus salivarius and pectin (PE) inclusion in the diet exhibited better results on growth performance, immunocompetence, and disease resistance in comparison to the single inclusion of prebiotics and probiotics separately in the diet. Experimental diets containing Biogen as probiotics, Allium sativum (garlic), Cynodon dactylon as immunostimulant, and sodium alginate as prebiotics revealed improvement in the growth and immune response of P. clarkii juveniles [91]. Lactobacillus sp. dietary administration, together with coconut pulp, operating as a prebiotic for crayfish individuals, including in the Cherax genus, led to an increased growth rate but had no effect on survival [93] (Table 3). In addition, higher survival towards V. mimicus was observed in C. cainii fed poultry by-product meal, fermented by Lactobacillus casei and S. cerevisiae. From the same study, it was concluded that these dietary inclusions were beneficial to crayfish specifically related to microbial community and immune-related cytokines [92]. (Table 3).

Synopsis of Pro-, Pre-, and Synbiotics Administration and Limitations
Administration of probiotics, prebiotics, and synbiotics in crayfish aquaculture has increasingly gained attention. First, there is evidence that these supplements improve growth performance and feed utilization (Tables 1-3). Many parameters linked to growth and feed utilization have been examined (SGR, WG, FCR, PER, LER). These effects could be attributed to the provision of necessary nutrients and increased activity of digestive enzymes, which will further increase the digestibility of feed. More specifically, synbiotics referred to increased fat decomposition, which led to beneficial effects on growth parameters. Furthermore, in some cases, the intestinal morphology improved, leading to more efficient gut functions.
Apart from digestive enzymes, the above supplements were found to enhance antioxidant enzymes (CAT, SOD, GRx). These enzymes operate as barriers towards oxidative stress, reducing the harmful effects of reactive oxygen species (ROS) and protecting the host against susceptibility to pathogens. More specifically, SOD led to decomposition of reactive O 2 − to H 2 O 2 , while CAT turned H 2 O 2 into O 2 and H 2 O [94]. GRx is an enzyme that protects the integrity of the cells by catalyzing the reduction between reduced glutathione and H 2 O 2 [95].
Further, the administered substances exhibit immunostimulatory effects, as they were found to increase LYS, PO, proPO, and NOS activity. Additionally, increases were observed in other physiological parameters that depict immune modulation, such as THC, TVC, TPP, LGC, and SGC. However, no significant effects or adverse effects were obtained. More specifically, a severe pathological finding in both the guts and hepatopancreas was observed, combined with reduced survival. Still, the existing knowledge is insufficient with core information still missing. Hence, the administration of these supplements is not a simple process. With the exception of increased cost, attention is needed for their optimal application, as contradictory information exists regarding the optimal doses. Furthermore, the injected substances are not very practical due to the number of cultured individuals and the elevated stress caused to them. Thus, further research is needed for the clarification of the optimal doses, substances, and method of administration. Following this direction, the development of the optimal supplements in order to avoid the administration of substances operating as a threat to public health (i.e., antibiotics) requires prior knowledge of the organism's microbiome. In comparison to shrimp, the crayfish microbiome is less studied [96], so further investigation is needed due to its high plasticity, and many factors influence its abundance and composition. The knowledge and understanding of the crayfish microbiota complex system have the potential to provide solutions for crayfish aquaculture.

Main Factors Affecting Crayfish Microbiota Abundances and Composition
The intestinal microbiome of crayfish and of all aquatic organisms in general is a dynamic and complex biological system that plays a key role in physiological functions. Additionally, the microbiota of aquatic organisms are closely related to environmental factors, with water ranking among the most important [27]. Most studies investigating crayfish microbiota alterations and compositions have been conducted primarily on four species, P. clarkii, C. quadricarinatus, C. cainii, and P. leniusculus. The main results of these studies concerning the main phylum and genera abundances are summarized in Figures 1-5. Considering the broad range of crayfish species in comparison with its wide distribution, a clear conclusion cannot be drawn so far. However, investigating microbiota alterations, diversity, and composition is the first step towards an enhanced understanding of the interactions between the host, environment, and microbes.      [110][111][112]. C. cainii and C. quadricarinatus photos retrieved from Refs. [113,114], respectively.

Figure 3.
Main factors affecting microbiota of crayfish from Cherax genus: (A) Impact of DIV1 infection on abundance of main phyla and genera of microbiome of C. quadricarinatus; (B) Impact of starvation on main phyla abundance and composition of C. cainii's microbiome; (C) Most significant changes of main phyla of C. cainii gut microbiome when cultured in water with different biological filters. Analyzed data obtained from Refs. [110][111][112]. C. cainii and C. quadricarinatus photos retrieved from Refs. [113,114], respectively.   Figures A and B obtained from Ref. [115]. C. cainii and C. quadricarinatus photos retrieved from Refs. [113,114], respectively. level of gut microbiota of C. quadricarinatus individuals after exposure to different nanoplastic concentrations. Data for Figures A and B obtained from Ref. [115]. C. cainii and C. quadricarinatus photos retrieved from Refs. [113,114], respectively. Analyzed data obtained from Refs. [116,117]. P. leniusculus personal photo from Greece.

P. Clarkii
As P. clarkii represents one of the most extensively cultured crayfish species [118], many factors have been addressed in order to assess their influence on microbiota (Figures 1 and 2). When P. clarkii's intestine microbiome from ponds and from rice co-culture fields was studied, no significant differentiation was found between the different breeding models [97]. However, conflicting results obtained from other studies that examined the same culture methods revealed a significantly different relative abundance of bacterial and archaeal communities in the gut of red swamp crayfish [119]. The most dominant phyla were Proteobacteria, Actinobacteria, Tenericutes, Firmicutes, Bacteroidetes [98], Cyanobacteria, Chloroflexi, Acidobacteria, RsaHF231 and Nitrospirae [97]. In fungal and viral communities, no significant differences were observed [119]. Furthermore, the abundance of intestinal microbiota in autumn was found higher than in the summer in Figure 5. Main factors affecting microbiota of P. leniusculus individuals: (A) Differences in abundance and composition of microbiota in main phyla of P. leniusculus individuals in four different tissues (exoskeleton, intestine, hepatopancreas, and hemolymph); (B) Differences in abundance and composition of gut microbiota in phylum level of P. leniusculus individuals after exposure to different antibiotics concentrations; (C) Differences in abundance and composition of gut microbiota in genus level of P. leniusculus individuals after exposure to different antibiotics concentrations. Analyzed data obtained from Refs. [116,117]. P. leniusculus personal photo from Greece.

P. Clarkii
As P. clarkii represents one of the most extensively cultured crayfish species [118], many factors have been addressed in order to assess their influence on microbiota (Figures 1 and 2). When P. clarkii's intestine microbiome from ponds and from rice co-culture fields was studied, no significant differentiation was found between the different breeding models [97]. However, conflicting results obtained from other studies that examined the same culture methods revealed a significantly different relative abundance of bacterial and archaeal communities in the gut of red swamp crayfish [119]. The most dominant phyla were Proteobacteria, Actinobacteria, Tenericutes, Firmicutes, Bacteroidetes [98], Cyanobacteria, Chloroflexi, Acidobacteria, RsaHF231 and Nitrospirae [97]. In fungal and viral communities, no significant differences were observed [119]. Furthermore, the abundance of intestinal microbiota in autumn was found higher than in the summer in both culture methods [81]. When ditchless rice-crayfish co-culture was compared with traditional rice-crayfish culture, it was revealed that it has a superior bacterial system, which led to a lower abundance of pathogen colonization in the crayfish's intestine [120]. Bacterial communities of the environment and from the intestinal microbiota of P. clarkii as a host have been proposed to interact with each other [121].
Further, the bacterial communities in the hepatopancreas of P. clarkii at different health statuses, including healthy, anorexic, moribund, and whitish muscle statuses, were investigated, and distinct differences were found in the structure, composition, and predicted function of the hepatopancreatic microbiota between the healthy and sick crayfish. More specifically, the LEfSe analysis revealed that the synbiotic bacterial species that were significantly enriched were Proteus penneri, Citrobacter sensu stricto, and Lactococcus garvieae, and the potential probiotics, such as Weissella cibaria and Lactobacillus murinus in the healthy crayfish in comparison to sick crayfish, while the opportunistic pathogens, including C. freundii, Plesiomonas shigelloides, Citrobacter sensu stricto 7, and Terrisporobacter, in the hepatopancreas of sick crayfish were significantly more enriched than those of healthy crayfish. In addition, compared with that of healthy crayfish, the hepatopancreas of moribund crayfish had significantly enriched bacterial genera, such as Dubosiella, Candidatus, Bacilloplasma, and Phreatobacter, whereas the hepatopancreas of crayfish with whitish muscle disease was observed with a significant enrichment of some opportunistic pathogens, including Morganella morganii, Providencia alcalifaciens, Vagococcus fluvialis, Clostridium lundense, and Bacteroides [122]. Furthermore, the intestinal microbiota of P. clarkii individuals at different health statuses after WSSV infection (healthy crayfish (HC), WSSV-infected active crayfish (IAC), and WSSV-infected diseased crayfish (IDC)) demonstrated that the relative abundances of certain phyla changed significantly in WSSV-infected crayfish, as indicated by a decrease in Tenericutes, Firmicutes and an increase in Proteobacteria and Bacteroidetes in WSSV-infected groups [99,123]. The IAC group exhibited the highest species diversity [123], while the overabundance of Aeromonas and Citrobacter and the decrease in Acinetobacter and Kurthia were associated with severe WSSV disease [99,123]. In addition, significant differences were indicated in the composition of the gut microbiome after infection with C. freundii, which pose a threat to crayfish farming and can also cause human infection through consumption [124].
A key factor that seems to enact a crucial role in the intestinal microbiota of P. clarkii is thermal stress. More specifically, increased abundance of Proteobacteria and decreased abundance of Bacteroidetes and Firmicutes was observed as the temperature elevated. However, some adaptive mechanisms were also observed as the abundance of phyla Bacteroidetes and Firmicutes, and pathogenic genera Shewanella and Acinetobacter gradually decreased while the abundance of beneficial Tenericutes and Rhodobacter gradually increased [125]. Furthermore, the effects of cadmium (Cd) at different concentrations were investigated, and from the results, it was indicated that Cd exposure could induce intestinal histological damage and affect intestinal microbiota composition and functions [126]. A possible solution to Cd increased concentration could be the inoculation of probiotic B. subtilis, which was found to mineralize Cd and attenuate Cd accumulation in crayfish [105]. Except for Cd polystyrene and polyethylene nanoplastic, nitrite, and sulfide, mercury and Hepatotoxin microcystion-LR exposure seem to also have an adverse effect on intestinal microbiota [66,100,101,106,107]. The relative abundance of lactic acid bacteria, Citrobacter, and other probiotics decreased, while the relative abundance of some intestinal pathogens and some other genera such as Shewanella and Acinetobacter increased [66,106].
Additionally, the diversity of gut microbiota was found to decline during development stages, while a specific pattern was associated with each stage [108,127]. Except for developmental stage, diet seemed to affect relative abundance in the intestine microbiota of crayfish. More specifically, the main phyla identified in groups fed pelleted feed and extruded feed were Proteobacteria, Tenericutes, and Firmicutes. The composition of Proteobacteria in the intestine of the pelleted feed group was significantly lower in comparison with the extruded feed group [102]. A relative abundance of Bacteroidetes was also found to be higher in P. clarkii gut microbiota when fed with fermented feed [127]. Additionally, environmental conditions and sampling site are suggested to shape carapace microbiota, while gut microbiotas seem to be more stable and associated with the factors linked to the host [109].

Cherax Genus
Among the Cherax genus, C. quadricarinatus and C. cainii are the most popular species involved in crayfish farming. Thus, many factors affecting their microbiota abundance and composition have been studied (Figures 3 and 4). Suspended zeolite, which is known for toxic metals uptake and nitrogenous waste filtering, has been proposed to improve the gut microbial diversity, metabolic functions, and immune response of the organisms [128]. For the same species, the long-term effects of starvation on health indices influence the gut microbiota and innate T immune response, indicating a significant modulation on the microbiome as the bacterial abundance at both genus and species level in post-starved marron, while core microbiota was replaced by Vibrio [110]. Further, significant differences were found in the composition of the gut microbiome after infection with a new-emerging viral pathogen, namely the Decapod iridescent virus 1 (DIV1) [111]. Interestingly, the effects of nanoplastics on C. quadricarinatus led to significant changes in gut microbiota, including a decrease in abundance of Bacteroidetes, Actinobacteria, and Firmicutes [115]. Other necessary studied parameters in assessing their effect on the intestinal microbiota of crayfish are supplementation with trace elements (manganese, silica, and phosphorus and two different biological filters, i.e., Gravel, Bio-Ball). From the results, it was observed that trace element supplementation at higher levels led to a significant increase in abundance of phosphate-solubilizing bacteria [129], while biological filters demonstrated higher microbial diversity in the gut of C. cainii [112].

Other Genera
Generally, in rice-crayfish culture, enriched microbes in crayfish gut from distinct sets are observed, which include Shewanella, Ferroplasma, Leishmania, and Siphoviridae genera [130]. Further, in rice co-culture fields, beneficial bacterial taxa, including Bacillus sp., Streptomyces sp., Lactobacillus sp., Prevotella sp., Rhodobacter sp., Bifidobacterium sp., Akkermansia sp., and Lactococcus sp., have been identified, while opportunistic pathogens, (Citrobacter sp., Aeromonas sp.) have been observed [131]. In P. leptodactylus individuals fed with diets including polyphenols extracted from olive mill wastewaters (OMWW), any pathological changes in the midgut and hindgut were found by histological analysis. In crayfish fed on an OMWW-enriched diet, total intestinal microbiota decreased, except for anaerobes and yeasts [132]. In the gut of P. leniusculus, high heterogenicity of bacterial abundance and composition among individuals has been demonstrated, while no significant alterations in the microbiome were revealed [116] following their exposure to environmentally relevant concentrations of sulfamethoxazole. P. leniusculus represents one of the most successful crayfish invaders in Europe, and as the microbiome plays a crucial role in the overall fitness of the host, it may also affect or be affected by the invasion range. Exoskeletal, hepatopancreatic, and intestinal microbiota exhibited differentiation among invasion core and invasion front populations [117] (Figure 5).

Overview of Factors Affecting Microbial Composition and Diversity and Limitations Existing
It can be drawn from all the studies summarized in Figures 1-5 that the microbiome represents a very complex and dynamic system, with plenty of factors and conditions affecting it. The crayfish microbiome is characterized by high plasticity, as there are no strict patterns of microbial abundance and composition. Investigation of the crayfish intestinal microbiome is of major importance, as it is the first step towards the development of optimal supplements in order to eliminate the administration of substances operating as a threat to global health. Freshwater crayfish species have suffered from mass reduction events in their natural habitats, mainly due to anthropogenic effects (i.e., degradation of the natural environment; translocation of invasive species). Thus, the understanding of microbiome composition and alteration will shed more light onto successful invasions in new habitats, and will help not only towards conservation issues but also in attaining information regarding increased survival rates. Finding new 'host-associated probiotics', namely bacteria that are originally isolated from the rearing water or the GI tract of the host to improve the growth and health of the host [133], may be more effective than probiotics from other origins, but this is a research direction that requires further investigation.
The main phyla existing under any circumstances are Proteobacteria, Firmicutes, and Tenericutes. More specifically, Proteobacteria have been extensively observed in aquatic organisms and environments. This phylum includes a broad range of bacteria exhibited in the gut, with some of them operating as opportunistic pathogens (i.e., Vibrio, Pseudomonas) and causing diseases to crayfish as well [134,135]. Firmicutes represent another major phylum identified in the crayfish microbiome. In general, this genus includes Gram-positive bacteria that are used as probiotic supplements (i.e., Bacillus, Clostridium, and Lactobacillus genera) in crayfish aquaculture [70,78]. The next common phylum was Tenericutes, which included bacteria that have been found in plenty of organisms, including plants, vertebrates, invertebrates, and water, and have been observed as crucial components in intestinal health maintenance [136].

Conclusions and Future Perspectives
Aquaculture represents the fastest-growing sector of primary production, offering high-quality animal protein products that meet the demand for nutrition and food security. At the same time, an urgent need for alternative supplementation has arisen as the microbial resistance leading to global health threats increases. Thus, finding substances to replace or reduce antibiotics use is of major importance. Previously, many reviews have addressed the use of probiotics, prebiotics, and synbiotics in various aquaculture species [4,33,55,[137][138][139][140][141][142][143]. However, no such study focused on crayfish species so far. Notably, in crayfish aquaculture, many pathogenic and viral diseases have been cataloged during the last few years [144]. The utilization of antibiotics in an effort to eliminate these diseases has led to problems concerning the health of both the animal host and consumers. Additionally, considering the rising global demand for sustainable and healthy products, the use of pro-, pre-, and synbiotics is of high importance as natural dietary supplements. These supplements were revealed to act in many beneficial ways, including boosting the immune system, increasing the resistance against pathogens, and improving the growth performance and overall wellbeing of the organisms ( Figure 6). However, there are still many blur points, such as the selection of the appropriate probiotic strain and prebiotic type, as well as the appropriate combination for an optimum synbiotic combination. Further, it remains to be seen if the level of increase in weight gain and other growth parameters following the administration of these supplements can cover the rising demand rates. Most studies investigating the synergistic action of prebiotics and probiotics concluded that synbiotics supplements had better results than probiotics and prebiotics supplements, separately. Apart from a few exceptions [62,76], all the studies indicated that the supplementation with pre-, pro-, and synbiotics in crayfish farming provided positive results. However, many questions still exist regarding the optimal dose of the supplement. Further, in some cases, there are still questions regarding their efficacy as neither positive nor negative influences were observed. In addition to these supplements, other alternative additives can be included, such as paraprobiotics, i.e., non-biological part probiotics, plant extracts, algae, and byproducts with prebiotic properties. Finally, a more detailed investigation into the mechanism behind the beneficial observations and how these supplements affect the crayfish gut microbiome is highly desired. Figure 6. Effect of prebiotics, probiotics, and synbiotics on cultured crayfish. Many probiotics, including genera Hafnia, Bacillus, Swawanella, Clostridium, Acinetobacter, etc., and probiotics, including MOS, GOS, FOS, XOS, PHB, PE, astaxanthin, β-glycan, etc., as well as combination of the aforementioned two as synbiotics were applied in three main ways (through feeding, directly into the culture water or with injection). The administration of these substances resulted in overall improved growth performance and digestion indices. Further, immune parameters and resistance towards some common crayfish pathogens also improved after administration of prebiotics, probiotics, and synbiotics. P. clarkii; C. quadricarinatus; C. cainii; P. leptodactylus; photos retrieved from Refs. [104,113,114,145], respectively. P. leniusculus personal photo.
One of the main mechanisms of action of probiotics towards immunity is by stimulating phagocytosis [146,147] as was found to promote the up-regulation of many defensive parameters (PO, SOD, LYZ, and NOS). In parallel, synbiotics enhance the increase in beneficial bacterial strains in the mucus and by competing for adhesion sites, preventing the growth of pathogenic strains [148]. Additionally, synbiotics facilitate the production of cytotoxic substances (such as cytokines). As far as increased susceptibility towards pathogens is concerned, probiotics produce siderophore substances and antimicrobial agents (antibiotics, antimicrobial peptides) [149]. Further, probiotics eradicate pathogens from the infected GI tract through competitive exclusion for nutrients and adhesion sites [150]. Additionally, from the literature, it occurs that probiotics mainly improve the overall growth by up-regulating the digestive enzymes, improving both feed utilization and digestibility [151], while at the same time, they influence the alteration of beneficial intestinal bacteria, which control the secretion of important digestive enzymes, and as a result, nutrients become more easily available to the organisms [5]. Figure 6. Effect of prebiotics, probiotics, and synbiotics on cultured crayfish. Many probiotics, including genera Hafnia, Bacillus, Swawanella, Clostridium, Acinetobacter, etc., and probiotics, including MOS, GOS, FOS, XOS, PHB, PE, astaxanthin, β-glycan, etc., as well as combination of the aforementioned two as synbiotics were applied in three main ways (through feeding, directly into the culture water or with injection). The administration of these substances resulted in overall improved growth performance and digestion indices. Further, immune parameters and resistance towards some common crayfish pathogens also improved after administration of prebiotics, probiotics, and synbiotics. P. clarkii; C. quadricarinatus; C. cainii; P. leptodactylus; photos retrieved from Refs. [104,113,114,145], respectively. P. leniusculus personal photo.
One of the main mechanisms of action of probiotics towards immunity is by stimulating phagocytosis [146,147] as was found to promote the up-regulation of many defensive parameters (PO, SOD, LYZ, and NOS). In parallel, synbiotics enhance the increase in beneficial bacterial strains in the mucus and by competing for adhesion sites, preventing the growth of pathogenic strains [148]. Additionally, synbiotics facilitate the production of cytotoxic substances (such as cytokines). As far as increased susceptibility towards pathogens is concerned, probiotics produce siderophore substances and antimicrobial agents (antibiotics, antimicrobial peptides) [149]. Further, probiotics eradicate pathogens from the infected GI tract through competitive exclusion for nutrients and adhesion sites [150]. Additionally, from the literature, it occurs that probiotics mainly improve the overall growth by upregulating the digestive enzymes, improving both feed utilization and digestibility [151], while at the same time, they influence the alteration of beneficial intestinal bacteria, which control the secretion of important digestive enzymes, and as a result, nutrients become more easily available to the organisms [5].
The gut microbiome contributes substantially to the development and physiological performance of the host, including the prevention of pathogen growth, immune system modulation, nutrient absorption, metabolic pathways regulation, and vitamin production [152]. Thus, analysis of microbiota is essential in the development of a sustainable aquaculture protocol. It is well established that the gut-associated microbiota of crustaceans are essential for preserving animal health and homeostasis. Therefore, it is of crucial importance to assess the impact of a wide range of factors on these microbial communities, especially in aquatic organisms [116]. However, regarding the complexity and the dynamics of microbial communities in aquatic animals' microbiomes, the investigation of the associated factors is not an easy task. Factors leading to alterations in the abundance and composition of microbiota include diet, culture methods, pathogen infections, developmental stage, and toxin exposure (Figure 7). However, further studies are needed in order to better understand the relationship between microbial species and organisms' health biomarkers, which will enable the mitigation of many diseases. The available molecular tools, such as DNA sequencing and NGS technology, including amplicon and shot-gun approaches, led to microbial communities' identification and shed more light on the investigation of microbiota alterations. Further, the part of the gut studied is not consistent, as in some studies, the midgut, the hindgut, or the complete gut were used, leading to conflicting results as there are different microbial communities in each gut section. Thus, the investigation of the crayfish microbiome, both at abundance and diversity levels, requires consistent standards regarding the tissue type and technical processes in order to produce reliable and comparable results. The gut microbiome contributes substantially to the development and physiological performance of the host, including the prevention of pathogen growth, immune system modulation, nutrient absorption, metabolic pathways regulation, and vitamin production [152]. Thus, analysis of microbiota is essential in the development of a sustainable aquaculture protocol. It is well established that the gut-associated microbiota of crustaceans are essential for preserving animal health and homeostasis. Therefore, it is of crucial importance to assess the impact of a wide range of factors on these microbial communities, especially in aquatic organisms [116]. However, regarding the complexity and the dynamics of microbial communities in aquatic animals' microbiomes, the investigation of the associated factors is not an easy task. Factors leading to alterations in the abundance and composition of microbiota include diet, culture methods, pathogen infections, developmental stage, and toxin exposure (Figure 7). However, further studies are needed in order to better understand the relationship between microbial species and organisms' health biomarkers, which will enable the mitigation of many diseases. The available molecular tools, such as DNA sequencing and NGS technology, including amplicon and shot-gun approaches, led to microbial communities' identification and shed more light on the investigation of microbiota alterations. Further, the part of the gut studied is not consistent, as in some studies, the midgut, the hindgut, or the complete gut were used, leading to conflicting results as there are different microbial communities in each gut section. Thus, the investigation of the crayfish microbiome, both at abundance and diversity levels, requires consistent standards regarding the tissue type and technical processes in order to produce reliable and comparable results.

Conflicts of Interest:
The authors declare no conflict of interest.