Keywords

1.1 Introduction

Aquaculture is one of the world’s fastest growing food sectors (Willer and Aldridge 2019). It is necessary to meet the global seafood demand, which is being accountable for 50% of the world’s seafood consumption (Gómez et al. 2019). However, sustainable development of aquaculture industry is constantly defeated by the outbreak of diseases, which is considered as main obstacles to the economical profitability of the industry (FAO 2020). The outbreak of diseases is also linked to application of antibiotics, posing a significant danger to the public health (World Health Organization 2014). Thus, new and natural alternatives that prohibit the incidence of diseases and improve human and animal health are urgently needed. The use of probiotics, “live organisms that can give a health benefit to the host when administered in the appropriate amounts,” is a potential alternative to boost the global health (FAO/WHO 2001). The scientific community has been searching for the environmentally friendly solutions to prevent aquacultural disease, where probiotics emerged as crucial alternative to antibiotics due to advert effects of antibiotics, such as the modulation of microbiota in the aquaculture systems and the development of resistance bacteria (Kuebutornye et al. 2019; Resende et al. 2012; Ringø 2020; Wang et al. 2019a). Consequently, wide range of probiotics, such as Bacillus, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Pediococcus, Enterobacter, Vibrio, Pseudomonas, Rhodopseudomonas, Roseobacter, and Shewanella, have been found and applied to improve growth performance, immune response, and disease resistance of farmed fish and shellfish (Abd El-Rhman et al. 2009; Adel et al. 2017; Feng et al. 2019; Kuebutornye et al. 2019; Li et al. 2006; Li et al. 2020; Ringø 2020; Yang et al. 2019). In aquaculture, probiotics have been applied as functional feed additives to boost host’s health and well-being via increasing growth, supplying nutrient, modulation gut microbiota, enhancing immunity, improving feed efficiency, increasing digestive enzyme activities and digestibility, and controlling diseases (Kuebutornye et al. 2019; Ringø 2020; Selim and Reda 2015).

Bacillus species are one of the most commonly used probiotics in the aquaculture industry because of their ability to form endospores, which is a benefit for industrial applications without losing their characteristics (Hong et al. 2005; Kuebutornye et al. 2019; Cutting 2011; Hai 2015). In addition, Bacillus is known to generate natural antimicrobial compounds, which are able to prohibit the proliferation of harmful bacteria in the aquaculture systems and host’s intestines (Abriouel et al. 2011; Caulier et al. 2019; Sumi et al. 2015). Similarly, Bacillus species are known to stimulate the digestive enzymes, antioxidant enzymes, relative immune gene expression, and stress-related genes, which in turn improve disease resistance of the host against pathogenic bacteria (Elshaghabee et al. 2017; Nayak 2010; Soltani et al. 2019). Bacillus species also increase the use of feed in fish, contributing to better growth rates (Mukherjee et al. 2019; Nair et al. 2020; Xia et al. 2020). Therefore, these chapters gather recent data on the role of Bacillus species in promoting growth performance, disease resistance, and immune response in aquaculture.

1.2 Mode of Action of Probiotics in Aquaculture

Probiotics can affect the host’s immune responses, as well as the interrelationship between these responses and their gastrointestinal microflora (Hemarajata and Versalovic 2013; La Fata et al. 2018; Yan and Polk 2011). Over the past decades, extensive researches on probiotics have provided insight into the significance of probiotics and their modes of action and numerous mechanisms have been suggested (Santacroce et al. 2019; Shi et al. 2016).

  1. 1.

    Probiotics improve feed efficiency and growth rate of farmed fish and shellfish (Ringø 2020; Romano 2021). They also enhance the host’s appetite and feed digestion via decomposition of indigestible components, enhance vitamin productions, and detox diet’s substances (Ashaolu 2020; Cencic and Chingwaru 2010; Hoseinifar et al. 2018).

  2. 2.

    Probiotics could compete the exclusion of gastrointestinal harmful bacteria via the secretion of peroxide, bacteriocin, siderophore, and lysozyme enzymes (Vieco-Saiz et al. 2019; Yang et al. 2014). The physiological and immunological effects are considered as one of the most essential modes of action of probiotics (Klaenhammer et al. 2012; Plaza-Diaz et al. 2019; Vieco-Saiz et al. 2019).

  3. 3.

    Probiotics could enhance aquaculture animal’s disease resistance to stress caused by various environmental threats during aquaculture activities (Hlordzi et al. 2020; Mohapatra et al. 2013; Reverter et al. 2020).

These mechanisms display the favorable impacts of probiotics in farmed fish and shellfish. Future studies, however, on the relationship between probiotics and hosts, including metagenomics and proteomic studies, is important to clarify mode of action of probiotics.

1.3 Bacillus Applications in Aquaculture

1.3.1 Improve Growth Performance

The utmost target of aquaculture practice is to acquire the rapidest growth and lowest production cost. To achieve this goal, several means have been established to boost growth rate and feed consumption by adding functional feed additives and growth natural growth promoters (Hernández et al. 2016; Katya et al. 2014). Probiotics are potential tools to maintain the normal growth, health, and well-being of farmed fish and shellfish because they serve as nutrients source, vitamins, and digestive enzymes. These substances for their part will contribute significantly on feed consumption, nutrients uptake, and host’s growth rate (Lauriano et al. 2016; Nath et al. 2019). Probiotics consumption have been speculated to improve the host’s appetite or boost organisms’ digestibility (Irianto and Austin 2002). Probiotics can improve feed efficiency of fish and shellfish by stimulating the excretion of digestive enzymes and maintaining the balance of intestinal microbes, which lead to the improvement of nutrients absorption and utilization, as well as the survival and growth of the host (Ibrahem 2015; Irianto and Austin 2002). Studies on diets containing probiotics revealed the possible involvement of these probiotics on the improvement of intestinal microflora balance and the production of extracellular enzymes to elevate the feed efficiency and growth of cultured species as growth promoters (Giri et al. 2013; Ringø et al. 2018). Most of the studies using Bacillus in aquaculture focus on growth performance and survival rate (Table 1.1).

Table 1.1 Weight gain (WG), specific growth rate (SGR), food conversion efficiency (FCE), food conversion ratio (FCR), protein efficiency ratio (PER), survival rate (SR), digestive enzyme, and disease resistance of fish and shellfish fed Bacillus probiotics. ➝ no change, ↑ increase, ↓ decrease
Full size table

1.3.1.1 Tilapias

During past decades, Bacillus spp. have been intensively applied in Nile tilapia aquaculture. Han et al. (2015) indicated that 10 weeks feeding trial with B. licheniformis significantly enhanced growth performance. However, there were no significant discrepancies in survival rate and feed conversion ratio (FCR) and in villi length and muscular layer thickness of anterior intestine among the treatments. In contrast, Iwashita et al. (2015) reveal that administration of the probiotic had no significant effect on the growth rates of Nile tilapias, although the fish fed probiotics had better feed conversion. Likewise, no significant difference in growth performance and FCR was observed in Nile tilapia fed Bacillus amyloliquefaciens (Silva et al. 2015). This can be explained due to the low temperatures during experimental period. Marcusso et al. (2015) reported that the homeostasis of Nile tilapia rearing at temperatures below 24 °C could be affected, enhancing the susceptibility to bacterial infections and impairing the growth performance. No effects were observed on the growth performance of Nile tilapia fed Bacillus subtilis (Aqua NZ and AP193) and Bacillus subtilis strains (Addo et al. 2017a, 2017b). These results are not unexpected given the short duration of this trial. This statement agrees with Apún-Molina et al. (2009) who observed a tendency toward improved growth in Nile tilapia fry (0.14 g) only after 75 d of feeding with diets composed of Bacillus or Lactobacillus probiotics. On the contrary, dietary inclusion of Bacillus subtilis significantly improved body weight, percent weight gain, specific growth rate, and feed conversion ratio (Liu et al. 2017). It is well documented that Bacillus exoenzymes are very efficient at metabolizing a large variety of carbohydrate, lipids, and proteins (Liu et al. 2009). The exoenzymatic activity of Bacillus spp. is one of the main reasons for its ability to improve digestive enzyme activities (Han et al. 2015). Higher enzyme activities in the digestive tract enhance digestive capability and growth performance of the host. It is widely accepted that the level of digestive enzyme activity is a useful comparative indicator of food utilization rate, digestive capacity, and growth performance of the host (Suzer et al. 2008; Ueberschär 1995). Liu et al. (2017) also reported that 4-week B. subtilis HAINUP40 diet supplementation significantly increased protease and amylase activities of tilapia. This is because B. subtilis HAINUP40 could secrete exoenzymes; the improvement of indigestive tract enzyme activities may be partially due to enzymes synthesized by the bacteria. However, the proportion of enzymes contributed by bacteria cannot be assessed since the probiotic may also stimulate the production of endogenous enzymes in the fish (Dawood et al. 2016; Suzer et al. 2008; Wu et al. 2012; Ziaei-Nejad et al. 2006). In the same trend, supplementation of B. subtilis and B. licheniformis or B. subtilis and Bacillus licheniformis (BS) combined with traditional Chinese medicine (TCM) significantly enhanced weight gain and specific growth rate of Nile tilapia and Mozambique tilapia (Abarike et al. 2018b; Abarike et al. 2018a; Gobi et al. 2018). It is known that an increase in the body weight gain in fish fed with probiotic supplemented diets, could contribute to the increase in digestive enzyme activity, increase in appetite, increase in the production of vitamin, breakdown of indigestible components, as well as possible improvement of intestine morphology (Irianto and Austin 2002). In addition, Bacillus spp. could secret several digestive enzymes like protease, amylase, and lipase (Cai et al. 2019; Caulier et al. 2019).

1.3.1.2 Shrimps

Shrimp is a commercially important aquatic species with high economic value and good flavor, which has been widely farmed in the world, particularly in some Asia countries (Chen et al. 2020a). However, the shrimp industry has suffered severe economic losses because of the frequent outbreaks of diseases such as early mortality syndrome (EMS) and white spot syndrome virus (WSSV) (Alavandi et al. 2019; Castex et al. 2009; Chang et al. 2012). Chemotherapeutant and antibiotics are usually applied to settle this problem. Unfortunately, prolonged use of chemotherapeutant and antibiotics could lead to severe outcomes such as resistant bacteria, drug residues, and toxins, which pose a substantial threat to human beings and environment (Dash et al. 2015). Therefore, to seek an alternative way to solve this threat has caused increasing concern (Huynh et al. 2018). Probiotics have been widely applied in shrimp aquaculture. Jamali et al. (2015) revealed that dietary enrichment with B. licheniformis and B. subtilis significantly enhanced growth performance and survival rate of Pacific white shrimp, Litopenaeus vannamei. Elevation of growth performance has been demonstrated as the Bacillus could colonize shrimp digestive tract. In P. monodon, Bacillus, when used as a probiotic, was able to colonize both the culture water and the shrimp digestive tract; the Bacillus also was able to replace Vibrio spp. in the gut of the shrimp, thereby increasing shrimp survival (Rengpipat et al. 1998), via out-competing other bacteria for nutrients and space by producing antibiotics (Moriarty 1998; Verschuere et al. 2000). Similarly, significant improvement in growth performance of L. vannamei supplemented with Bacillus spp. has been reported in previous studies (Sadat Hoseini Madani et al. 2018; Sánchez-Ortiz et al. 2016; Swapna et al. 2015). Also, Amoah et al. (2019) indicated that dietary inclusion of B. coagulans significantly improved growth performance and feed utilization of Pacific white shrimp. The nutritive values as reported by Vijayavel and Balasubramanian (2006) is highly dependent on their biochemical constituents such as crude protein, crude lipid, ash content, and moisture, which also is noted to be an indication of improved meat quality. In addition, higher inclusion levels of probiotic BC at 1 × 108 CFU g−1 feed in diets could modulate gut microflora of L. vannamei (Amoah et al. 2019), which play an ardent role in the digestive enzyme activities and the intestinal health. It has been well documented that digestive enzymes are known to break down food and absorb nutrients (Gobi et al. 2018). The digestive enzymes including amylase, lipase, and trypsin (Rawlings and Barrett 1994; Svendsen 2000) in this study significantly increased in the treated group compared to the untreated. Similar results of improved digestive enzyme activities in Litopenaeus vannamei (Zokaeifar et al. 2012) and Fenneropenaeus indicus (Ziaei-Nejad et al. 2006) have been established. Verschuere et al. (2000) in their work also noted that, Bacillus genus secrets a wide range of exoenzymes which aid in the nutritional enhancement of the host. More recently, significant increase in growth performance, villus number, villus height, thicker submucosa, and propionic acid content has been reported in L. vannamei fed different Bacillus species (B. licheniformis, B. cereus, and B. subtilis) singularly or combined with other probiotics (Pediococcus acidilactici, P. pentosaceus, and Lactococcus lactis) (Chen et al. 2020a; Chen et al. 2020b; Khademzade et al. 2020; Won et al. 2020a).

Dietary supplementation of Bacillus coagulans on growth and feed utilization of freshwater prawn Macrobrachium rosenbergii showed that growth performance and feed utilization were found to be significantly higher (P < 0.05) in prawn fed 109 cfu g−1 diet. In addition, the specific activities of protease, amylase, and lipase digestive enzymes were significantly higher (P < 0.05) for 109 cfu g−1 diet (Gupta et al. 2016).

In Marron (Cherax cainii), Ambas et al. (2017) found that synbiotic use of B. mycoides and organic selenium (OS) significantly improved some immune parameters of marron, particularly the glutathione peroxidase, and to some extent total hemocyte counts. However, the synbiotic feed did not synergistically improve marron growth; in fact, the use of B. mycoides-supplemented diet alone demonstrated significantly higher growth in marron compared with the growth of marron fed on other test diets. A study conducted by Ock Kim et al. (2020), it was indicated that strain Bacillus subtilis isolated from the gut of Penaeus indicus and added at 2 × 102 CFU 100 g−1 as probiotics in feed, resulted in weight gain of the juvenile shrimp (16.8 ± 0.11 g) after 40 days. The weight gain was 16.8 ± 0.11 CFU 100 g−1 at 10 × 102 CFU 100 g−1 probiotic concentration.

1.3.1.3 Catfish

To the best of our knowledge, there were few studies regarding the use of Bacillus spp. on this fish. Afrilasari and Meryandini (2016) reported that Bacillus megaterium PTB 1.4 increased the activity of digestive enzymes and the growth of catfish. It is known that isolate PTB 1.4 is B. megaterium, where Bacillus spp. group is known to have ability to produce extracellular enzymes (Moriarty 1998). Probiotic bacteria are capable of producing digestive enzymes that help fish use feed nutrients and digest (Bairagi et al. 2002). Generally, endogenous enzyme can be produced by fish, but the presence of probiotics can improve digestive enzyme. Probiotics improve digestive enzyme activity by stimulating the synthesis of endogenous enzyme in the digestive tract (Mohapatra et al. 2012). Similarly, combination of B. subtilis, B. amyloliquefaciens, B. cereus, and a commercial B. amyloliquefaciens significantly improved growth performance of C. gariepinus (Reda et al. 2018). This improvement could be attributed to the production of amylase and protease by the same strain (Selim et al. 2019). In addition, Bacillus sp. are capable to detoxify the harmful substance in feed, produce essential vitamins such as vitamin B12 and biotin, and increase the intestinal villus heights (Ramirez and Dixon 2003; Reda and Selim 2015; Sugita et al. 1992).

In hybrid catfish (C. macrocephalus × C. gariepinus), Meidong et al. (2017) indicated that Bacillus siamensis strain B44v, selectively isolated from Thai pickled vegetables (Phak-dong), displayed a high potential as a probiotic in catfish culture. Fish fed diet containing strain B44v (107 CFU g−1 feed) displayed not only no mortality but also growth improvement. The potential probiotic B. siamensis strain B44v could produce cellulase and protease, whereas the Bacillus sp. strain B51f produced protease and amylase enzymes. Ability to produce some hydrolytic enzymes is beneficial to the host. Enzymes increase the digestion of macromolecules in animal feed and improve feed intake by reducing digesta viscosity and increasing nutrient absorption in host animals (Ray et al. 2012).

For striped catfish, Pangasianodon hypophthalmus, the mixture of probiotics (B. amyloliquefaciens 54A and B. pumilus 47B) isolated from striped catfish at concentrations of 1 × 108, 3 × 108, and 5 × 108 CFU g−1 was added to the fish feed and conducted for 90 days. Truong Thy et al. (2017) reported that AWG (476.6 ± 7.81 g fish−1) of fish fed probiotics at 5 × 108 CFU g−1 was significantly higher than the control (390 ± 25.7 g fish−1) after 90 days of feeding, but there was no significant (P > 0.05) effect of probiotics on FCR and SGR. However, in basa fish, Pangasius bocourti (Meidong et al. 2018) reported that the administration of strain B81e isolated from the fish’s gut (1 × 107 CFU g−1) for 60 days had significant effects (p < 0.05) on weight gain, specific growth rate, and feed utilization efficiency of P. bocourti. This growth improvement might be related to the capability of the putative probiotics in producing extracellular protease and lipase within fish gut and thus exert beneficial effects to the digestive processes of the host fish as bacterial enzymes can help degrade the proteinaceous and lipid substrates (Ramesh et al. 2015; Ray et al. 2012). The significant reduction in FCR indicated that the fish utilized dietary nutrients more efficiently when feed was supplemented with strain B81e.

1.3.1.4 Japanese eel (Anguilla japonica)

Bacillus spp. supplementations have been recently applied in Japanese eel. Lee et al. (2017) indicated that dietary supplementation of Bacillus subtilis WB60 at 108 CFU g−1 in diet of Japanese eel (Anguilla japonica) resulted in better weight gain, feed efficiency, and protein efficiency ratio compared to the control and Lactobacillus plantarum diets. Similar results were observed in Japanese eel fed Bacillus subtilis WB60 and mannanoligosaccharide (MOS), as well as (Bacillus subtilis or licheniformis) and (mannan or fructooligosaccharide) (Lee et al. 2018; Park et al. 2020). There is growing evidence that gastrointestinal bacteria facilitate the decomposition of nutrients in the host organism and provide physiologically active materials, such as enzymes, amino acids, and vitamins (Cencic and Chingwaru 2010; Morowitz et al. 2011; Wang et al. 2020a). These materials can positively influence the digestive tract and improve feed digestion and utilization (Bairagi et al. 2004; Dawood et al. 2019; Ramirez and Dixon 2003; Wang et al. 2020b).

1.3.1.5 Sea Cucumber (Apostichopus japonicus)

Supplementation of Bacillus cereus EN25 at 0 (control), 105, 107, and 109 CFU g−1 for 30 days showed no significant effects on growth of sea cucumbers A. japonicus (Zhao et al. 2016). Growth performance of sea cucumbers was one of the important indices to evaluate the effects of potential Bacillus spp. on culturing of sea cucumbers. Previous studies had proved that dietary Bacillus spp., such as indigenous B. subtilis T13 (Zhao et al. 2012), indigenous B. cereus (Yang et al. 2015), and commercial B. subtilis (Zhang et al. 2010), could improve the growth performance of sea cucumbers at suitable doses. This difference could be attributed to the differences in Bacillus strains, sizes of sea cucumbers, sources of sea cucumbers, experimental period, and experimental conditions. The present study was conducted with the same source of sea cucumbers at the same experimental period and conditions with Zhao et al. (2012), except Bacillus strain and initial sizes of sea cucumbers. Recently, Liu et al. (2020) indicated that dietary supplementation of B. baekryungensis MS1 at 107 cfu g−1 for a total of 60 days significantly improved the growth performance of the sea cucumber cultured under low temperature. This is related to the mode of action of probiotics, including the production of digestive enzymes, the production of antibacterial substances, immune stimulation, and interference of quorum sensing, all of which depend on the long-term growth and reproduction of probiotics. Studies have also shown that probiotics work by managing community assembly of the water and gut microbiota (Selim and Reda 2015; Wang et al. 2017a).

1.3.1.6 Tambaqui (Colossoma macropomum)

Dietary inclusion of Bacillus subtilis (109 UFC g−1) and Saccharomyces cerevisiae (109 UFC g−1) showed that no differences were found for the growth parameters between the treatments with probiotics (da Paixão et al. 2017). Although probiotics are supposed to be beneficial, the literature mentions possible synergistic effects. The total replacement of indigenous populations with probiotics may not be desirable to improve growth performance (Merrifield et al. 2010). The control of the endogenous balance between pathogenic and beneficial bacteria is still the target of many studies. According to Merrifield et al. (2010), the lack of improvements regarding growth and feed use may be explained by the level of gastrointestinal colonization that could be too high and any possible synergistic effect with the normal gut microbiota was negated. Thus, it is expected that the beneficial effects of probiotics for tambaquis are not on its performance but on its health and welfare. However, in another study with tambaqui, Dias et al. (2018) indicated that the use of the autochthonous bacteria B. cereus improves the growth performance, productivity, hematological profile, and survival of tambaqui juveniles. This enhanced growth performance of fish supplemented with probiotics is probably due to an improvement in digestion as well as an increase in the synthesis and absorption of nutrients (Hoseinifar et al. 2017). Similar results were obtained by El-Haroun et al. (2006) reporting increased growth performance and feed efficiency in tilapia fed the probiotics Bacillus licheniformis and Bacillus subtilis. According to these authors, the added probiotics improved digestibility, dietary protein, and energy utilization. These positive effects can be attributed to the capacity of the probiotics to promote an increase in the gut absorbent surface area, and stimulate and/or produce several enzymes on the intestinal tract, which improve digestibility and nutrient retention, leading to higher growth rates (El-Haroun et al. 2006; Ibrahem 2015).

1.3.1.7 Carp Species

Dietary administration of BioPlus 2B, a probiotic containing Bacillus licheniformis and B. subtilis, and Ferroin solution indicate that the combination of probiotic and Ferroin solution represents an effective dietary supplement for improving carcass quality, growth performance, and hematological parameters in kutum fry (Azarin et al. 2015). In mrigal fingerlings, Cirrhinus mrigala (avg.wt. 2.5 ± 0.20 g) were fed with three different doses (2 × 104, 2 × 105, and 2 × 106 CFU) of Bacillus sp. PP9 admixed with 100 g feed for a period of 60 days. It was found that the feed with Bacillus concentration of 2 × 104 CFU exhibited significantly higher growth and lower food conversion ratio compared to the control and other supplemented diets (Bandyopadhyay et al. 2015). More recently, Qin et al. (2020) found that dietary inclusion of B. licheniformis at the low-dose 1 × 105 cfu g−1 and the high-dose (HD) group with 1 × 106 cfu g−1 led to significantly (p < 0.05) improved percent weight gain (PWG) and specific growth rate (SGR) parameters. The improvement of growth performance parameters such as PWG and SGR with increasing concentrations of supplemented B. licheniformis FA6 observed in this study is in agreement with Han et al. (2015) observed a significant increase in the growth performance of tilapia fed with B. licheniformis. The increase in the growth performance of grass carp may due to the secretion of digestive enzymes by B. licheniformis, which improves feed digestibility (Kuebutornye et al. 2019).

In Pengze crucian carp, Carassius auratus, dietary supplementation with prebiotics β-glucan (BG group) and probiotics Bacillus subtilis (BS group) resulted in better growth performance than other groups whereas feed efficiency was unaffected by dietary treatments. The textures of muscle in terms of hardness, springiness, cohesiveness, gumminess, chewiness, and resilience were higher in BG and BS groups than the control group. Supplementation of β-glucan and B. subtilis acted as a hypolipidemic in terms of decreasing the total cholesterol, high-density lipoprotein, and low-density lipoprotein, whereas increased the immune responses in serum measured by acid phosphatase, alkaline phosphatase, and catalase activities. Dietary supplementation of β-glucan and B. subtilis significantly improved the fold height and microvillus height in contrast to basal diet. Moreover, β-glucan could significantly increase digestive capacity observed in terms of an increase in amylase and trypsase activities, and B. subtilis significantly increased amylase and lipase activities in intestine (Cao et al. 2019).

1.3.1.8 Trout

A commercial probiotic (4.2 × 109 CFU g−1 of additive) was supplemented to the experimental diets at 0% (control), 0.03% (P1; 6 × 103 CFU g−1of diet), or 0.06% (P2; 1.5 × 106 CFU g−1 of diet) and fed to brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) for 9 and 20 weeks, respectively. Rainbow trout showed significantly better growth performance than brown trout, regardless of the dietary treatment. No effect of dietary probiotic supplementation was detected on growth performance and body composition (Ramos et al. 2017). However, in Caspian Brown Trout (Salmo trutta caspius)Aftabgard et al. (2019) found that the combined effects of IMOS, a prebiotic, and BetaPlus®, a probiotic containing B. subtilis and B. licheniformis, demonstrated a better performance of select growth indices, including BWI and FCR, than fish that were fed the control diet; these results were probably due to improved nutrition and digestive processes (Cerezuela et al. 2011).

1.3.1.9 Other Aquacultured Species

Two probiotics (Virgibacillus proomii and Bacillus mojavensis) were used to study their effects on the digestive enzyme activity, survival, and growth of sea bass, Dicentrarchus labrax at various ontogenetic stages in three separate experiments (Hamza et al. 2016). The results indicated that the two probiotics V. proomii and B. mojavensis were adequate for improved growth performance and survival and for healthy gut microenvironment of the host (Hamza et al. 2016).

In the study of Hauville et al. (2016) Florida pompano (Trachinotus carolinus) larvae were fed either live feed enriched with Algamac 3050 (Control), Algamac 3050, and probiotics (PB), or the previous diet combined with a daily addition of probiotics to the tank water (PB+). The results indicated that a mix of Bacillus sp. can promote growth through an early maturation of the digestive system during the early larval stages of pompano and snook.

In grouper Epinephelus coioides (Yan et al. 2016, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 108 CFU g−1 live (T1) and heat-inactivated B. pumilus SE5 (T2). The results indicated that the heat-inactivated probiotic significantly improved the final weight, weight gain (WG), and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). This suggested that live and heat-inactivated B. pumilus could promote the efficient utilization of dietary nutrients. Interestingly, significant increased growth was only observed in fish fed the heat-inactivated B. pumilus containing diet for 60 days, but not in fish fed the live B. pumilus containing diet. Likewise, Hoseinifar et al. (2011) observed that dietary supplementation of 20 g kg−1 inactive brewer’s yeast Saccharomyces cerevisiae var. ellipsoideus significantly improved the growth performance in juvenile beluga sturgeon (Huso huso). In rock bream, Oplegnathus fasciatus, Kim et al. (2017), revealed that supplementation of B. amyloliquefaciens spores at a concentration of 1.4 × 106 colony-forming units per gram (CFU g−1) of feed for 90 days resulted in significant improvements in body weight (BW), weight gain (WG), specific growth rate (SGR), and food conversion ratio (FCR) when compared with control group fish.

In hybrid sturgeon, Acipenser schrenckii ♂ and Acipenser baerii ♀, fish were fed with Bacillus amyloliquefaciens (GB-9) and Yarrowia lipolytica lipase2 (YLL2): Diet 1 (0-control), Diet 2 (5.0 g kg−1 GB-9), Diet 3 (4.0 g kg−1 YLL2), and Diet 4 (5.0 g kg−1 GB-9 + 4.0 g kg−1 YLL2), respectively (Fei et al. 2018). The results indicated that supplementations of GB-9 + YLL2 resulted in a significant increase in final weight, Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) concentration, compared with that of control (p < 0.05). This might be because the DHA and EPA hydrolyzed by YYL2 improved the poor establishment of the GB-9 in the gastrointestinal tract of hybrid sturgeon and might have promoted the growth of GB-9 (Menni et al. 2017). Similarly, combination of B. licheniformis and B. amyloliquefaciens indicated up to 2.5 times higher survival with probiotic addition, as well as 20% higher survival 7 days following a transport event. These benefits could not be explained by faster larval growth. In fact, CONT larvae were significantly longer than probiotic-treated larvae, likely due to decreased competition for food in CONT tanks which exhibited significantly lower survival. The other differing morphometric in this study was oil globule volume which was lowest in CONT larvae, suggesting that CONT larvae were consuming their endogenous reserves more quickly than probiotic-treated larvae. Retention of oil globules allows for a longer transition time to exogenous feeding, and studies indicate larvae that retain their endogenous reserves longer demonstrate increased survival (Avila and Juario 1987; Berkeley et al. 2004). The probiotic may alter development of the digestive tract and thus the start of exogenous feeding, as has been demonstrated in previous studies involving Bacillus probiotics and common snook (Hauville et al. 2016).

Dietary supplementation of B. subtilis has been reported to improve the growth performance, feed utilization, amylase, protease, and lipase enzymes of parrotfish (Oplegnathus fasciatus) and red sea bream (Pagrus major) (Liu et al. 2018; Zaineldin et al. 2018). The observed improvement in growth performance might be ascribed to the enhanced intestinal digestive enzyme activity and beneficial intestinal microbiota (Dawood et al. 2014; Liu et al. 2009; Sun et al. 2010). Bacillus sp. can produce certain essential micronutrients to promote better growth and feed utilization of hosts (Sanders et al. 2003). Further, Bacillus species may participate in digestion processes to break down nutrients such as carbohydrates, proteins, and lipids by producing extracellular enzymes (Liu et al. 2009; Sun et al. 2010). In abalone, Haliotis discus hannai, Gao et al. (2018) indicated that the food containing 105 cfu mL−1 Bacillus licheniformis promoted food intake and growth of abalones. Bacillus licheniformis is an aerobic nonpathogenic bacterium that inhabits the intestinal microbial community in the form of spores, which can reduce intestinal pH, reduce ammonia concentration, and promote decomposition of starch and cellulose. Thus, it is generally considered to be a relatively stable probiotic (Hong et al. 2005; Vine et al. 2006).

1.3.2 Increase Disease Resistance

Probiotics have been proven as an effective tool for disease prevention in aquaculture (Hoseinifar et al. 2018). Probiotics can interact with or antagonize other enteric bacteria by resisting colonization or by directly inhibiting and reducing the incidence of opportunistic pathogens (Chiu et al. 2017). They can also improve host’s health and well-being via physiological or immune modulation (Butt and Volkoff 2019). Probiotics can produce effective molecules that have bactericidal activity on intestinal pathogenic bacteria of the host, providing a barrier against the proliferation of opportunistic pathogens (Martínez Cruz et al. 2012; Seghouani et al. 2017). The functional molecules produced during the bactericidal activity are antibiotics, bacteriocins, siderophores, enzymes and/or hydrogen peroxide as well as the alteration of the intestinal pH due to the generation of organic acids (Verschuere et al. 2000). The inhibition of intestinal related diseases has been reported in several cultured species by probiotic incorporation in aquafeeds (Ringø et al. 2018; Serra et al. 2019; Wanka et al. 2018). Thus, it can be confirmed that the ability of aquatic animals to avoid the infectious diseases mainly depends on the immunomodulatory effect that happened due to the administration of beneficial bacterial cells.

1.3.2.1 Tilapias

Dietary inclusion of B. licheniformis at 0%, 0.02%, 0.04%, 0.06%, 0.08%, and 0.1% containing live germ 2 × 1010 CFU/g for 10 weeks significantly increased disease resistance of Nile tilapia, Oreochromis niloticus against Streptococcus iniae (Han et al. 2015). Bacillus strains supplementation in diet could increase disease resistance in fish through the stimulation of both the cellular and humoral immune function, such as phagocytic activity, lysozyme activity, and complement activity (Arena et al. 2006; Queiroz and Boyd 1998; Sookchaiyaporn et al. 2020; Zhou et al. 2010). It was reported that Bacillus bacteria are able to outcompete other bacteria for nutrients and space and can exclude other bacteria through the production of antibiotics, and as usually lead to the enhanced immunity of fish (Cha et al. 2013). Similarly, dietary inclusion of B. licheniformis Dahb1 at 107 cfu g−1 could improve disease resistance of Mozambique tilapia (Oreochromis mossambicus) against A. hydrophila (Gobi et al. 2018). In terms of Bacillus subtilis HAINUP40, H. Liu et al. (2017) reported that dietary supplement of B. subtilis HAINUP40 at 108 cfu g−1 can effectively enhance disease resistance of Nile tilapia against Streptococcus agalactiae. In addition, combination of B. subtilis with S. cerevisiae and A. oryzae; Bacillus subtilis with Aqua NZ and AP193; Bacillus subtilis strains SB3086, SB3295, SB3615 with AP193; B. subtilis and B. licheniformis, and Bacillus subtilis and Bacillus licheniformis (BS) combined with traditional Chinese medicine (TCM) A. hydrophila and S. iniae. Higher intestinal Bacillus spp. counts can regulate the gut microbiota of fish, selectively stimulate other beneficial probiotic bacteria, and depress some potential harmful bacteria (Yang et al. 2012).

1.3.2.2 Shrimps

The efficiency of these isolates in controlling pathogens, which is a key factor in selecting appropriate bacteria as probiotics, was evaluated (Kesarcodi-Watson et al. 2008). Based on in vitro laboratory results, B4, B6, and B12 inhibited V. parahaemolyticus; however, only B. subtilis AQHPS001 (B12) showed the highest antagonistic property against VPAHPND strains. However, among the VPAHPND strains, there were different sizes of the inhibitory clear zone, and VPAHPNDAQH3.2 was the only strain that resisted B12. This suggests that there are varieties of VPAHPND and that each strain may employ different mechanisms in response to the target B12 (Kewcharoen and Srisapoome 2019). Previous reports found that Bacillus spp. could produce many kinds of bacteriocins, such as subtilin, subtilosin, coagulin, megacin, bacillin, bacillomycin, mycosubtilin, toximycin, and xanthobacidin, which could reduce pathogen colonization by directly inhibiting pathogens while having no resulting effects on the virulence resistance genes of pathogenic bacteria (Desriac et al. 2010; Hammami et al. 2012; Joseph et al. 2013). Zhao et al. (2015) also reported that Bacillus spp. could secrete quorum-quenching enzymes, which are expected to be quorum-sensing blockers to reduce disease infection. These results suggest that B. subtilis AQAHBS001 possesses more effective characteristics that are important for controlling the various harmful VPAHPND strains than other candidates. For these reasons, it was further chosen to study its application on a laboratory scale. Similarly, dietary inclusion of Bacillus subtilis WB60, Pediococcus pentosaceus, and Lactococcus lactis at 108 CFU g−1 could improve disease resistance of whiteleg shrimp Litopenaeus vannamei against Vibrio parahaemolyticus (Won et al. 2020b). Generally, administration of probiotics in the shrimp diet was shown to decrease mortality rates compared to the CON diet (Balcázar et al. 2007; Sapcharoen and Rengpipat 2013; Zhang et al. 2009). Previous studies demonstrated that probiotic supplementation can be used for modulating fish health and disease resistance (Wang et al. 2018; Zuo et al. 2019). Indeed, probiotics can beneficially influence the disease resistance of fish to pathogen bacteria by producing antimicrobial substances and competing with pathogens for physical occupation of space (Lim et al. 2020). As a result, the enhanced survival and cumulative survival rates could be due to probiotic supplementation. Chen et al. (2020a) recently indicated that dietary MOS and/or B. licheniformis supplementation could positively increase ammonia resistance of Litopenaeus vannamei. According to Chen et al. (2012), immune parameters decrease after ammonia stress, yet these parameters recover faster when they were initially stimulated by a probiotic. Faster recovery of immune parameters might have contributed to the increased survival after ammonia stress for the Rps. palustris fed shrimp.

1.3.2.3 Catfish

Meidong et al. (2017) revealed that Bacillus siamensis strain B44v and Bacillus sp. strain B51f, derived from indigenous fermented foods, displayed strongly antagonistic activity against the bacterial fish pathogens, A. hydrophila and S. agalactiae. Both strains effectively inhibited Gram-positive and Gram-negative bacteria, indicating their broad spectrum as a useful antagonistic property as the two most striking bacterial fish pathogens in aquaculture in Thailand belong to the genera the Aeromonas and Streptococcus (Maisak et al. 2013). Besides fish pathogens, the bacteriocin-like substance from B. siamensis strain B44v inhibited several foodborne pathogens suggesting potential applications in human foods (Sivamaruthi et al. 2018). Likewise, Reda et al. (2018) showed that supplementation of three autochthonous Bacillus strains (B. subtilis, B. amyloliquefaciens, and B. cereus) and a commercial B. amyloliquefaciens at a dose of 1 × 1010 CFU kg−1 significantly increased disease resistance of African catfish against Aeromonas sobria. This may be returned to the ability of Bacillus spore to resist gastrointestinal conditions, survive and transit cross gastrointestinal tract, germinate and vegetate with heterologous antigen expression before being excreted (Duc et al. 2003). In striped catfish, Truong Thy et al. (2017) indicated that the mixed probiotics of Bacillus amyloliquefaciens 54A and B. pumilus 47B isolated from striped catfish (Pangasianodon hypophthalmus) intestine significantly enhanced disease resistance of the fish against Edwardsiella ictaluri and ammonia tolerance. Antimicrobial activity of probiotics has been demonstrated on many in vitro and in vivo studies in animals. The study of Corr et al. (2007) reported trial mice received protection from Lactobacillus salivarius against Listeria monocytogenes involved bacteriocin produced by L. salivariusUCC118. Additionally, antimicrobial activities of probiotics against pathogens include secretion of hydrogen peroxide (Pridmore et al. 2008), lactic acid (Fayol-Messaoudi et al. 2005), competitive exclusion (Lee et al. 2003), and stimulation of immune system (Ryan et al. 2009). The positive effect on barrier function of probiotics is to protect the host intestine by prevention of pathogen attachment to epithelial cells on gut surface (Mennigen et al. 2009). In basa fish, Pangasius bocourti, Meidong et al. (2018) found that B. aerius B81e has beneficial effects on growth performance, innate immunity, and disease resistance of P. bocourti against Aeromonas hydrophila and Streptococcus agalactiae. Bacterial co-aggregation has considerable significance in the host gut as co-aggregation ability of bacterial probiotics might interfere with the ability of pathogenic bacteria to infect the host and can prevent colonization of the pathogens (Spencer and Chesson 1994). In addition, B. aerius B81e has an absence of hemolysin and is susceptible to most of the common antibiotics tested which demonstrated that it is likely a nonpathogen and has an inability to transfer antibiotic-resistant genes to recipient bacteria in the host gut, thus preventing the development of antibiotic-resistant pathogens (Meidong et al. 2018).

1.3.2.4 Japanese eel (Anguilla japonica)

The combination of Bacillus subtilis WB60 and Lactobacillus plantarum KCTC3928 or Bacillus subtilis WB60 and mannanoligosaccharide (MOS) significantly improved disease resistance of Japanese eel against V. anguillarum (Lee et al. 2017, 2018). Similarly, Park et al. (2020) reported that dietary inclusion of B. subtilis with FOS (BSF) and B. licheniformis significantly increased disease resistance against Aeromonas hydrophila. Significant increase in disease resistance in these works may be attributable to the stimulation of cellular and humoral immune function.

1.3.3 Sea Cucumber (Apostichopus japonicus)

Zhao et al. (2016) indicated that the cumulative mortality after V. splendidus challenge decreased significantly in sea cucumbers fed with EN25 at 107 CFU g−1 (P < 0.05). The present study confirmed dietary B. cereus EN25 at 107 CFU g−1 could significantly improve disease resistance in juvenile A. japonicus. Recently, Liu et al. (2020) showed that B. baekryungensis MS1 significantly reduced the mortality of sea cucumbers infected with Vibrio splendidus. By regulating the expression of immune-related genes and signaling pathways, B. baekryungensis MS1 improved the immunity of sea cucumber in winter and effectively controlled the infection of pathogenic bacteria such as V. splendidus.

1.3.4 Tambaqui (Colossoma macropomum)

da Paixão et al. (2017) indicated that supplementation of two probiotics Bacillus subtilis and Saccharomyces cerevisiae at 109 UFC g−1 significantly increased disease resistance of tambaqui, Colossoma macropomum, against Streptococcus agalactiae. Similarly, Dias et al. (2018) reported that B. cereus (4.2 × 104, 3.9 × 106 and 3.3 × 108 CFU g−1) supplemented as probiotics to C. macropomum for 120 days significantly increased disease resistance against Aeromonas hydrophila. The probiotic promoted a nonspecific response against bacterial infection, increasing fish survival after challenge with A. hydrophila.

1.3.5 Other Species

In rock bream, Oplegnathus fasciatus, Kim et al. (2017) demonstrated the benefit of incorporation of B. amyloliquefaciens as a feed supplement to improve the health status of Oplegnathus fasciatus challenged with Streptococcus iniae. The enhancement of the innate immune response with a B. amyloliquefaciens enriched probiotic diet and decreased mortality rate, thereby protecting the fish against S. iniae. Similarly, dietary inclusion of B. subtilis at 108 CFU kg−1 significantly increased disease resistance of parrotfish, Oplegnathus fasciatus, against Vibrio alginolyticus (Liu et al. 2018). The growth performance and health status improvement of aquatic animal might be involved with the gut microbiota change after probiotic administration. The previous study has also demonstrated the positive effects of B. subtilis E20 in terms of intestinal presence and subsequent health benefits for L. vannamei (Liu et al. 2009; Tseng et al. 2009) and E. coioides (Liu et al. 2010). In the same trend, dietary inclusion of Bacillus licheniformis significantly improved disease resistance of abalone, Haliotis discus hannai Ino., against V. parahaemolyticus and grass carp, Ctenopharyngodon idella, against A. hydrophila (Gao et al. 2018; Qin et al. 2020).

1.4 Immune Effects of Bacillus

Enhancement of host immunity is one important benefit of probiotic diet supplementation (Kuebutornye et al. 2019). As stated by Verschuere et al. (2000), probiotics can modulate innate immunity through the modulation of humoral immune responses and expression of immune-related genes. Effects of Bacillus on immune response of different fish and shellfish are displayed in Table 1.2.

Table 1.2 Immune responses of fish and shellfish fed different Bacillus probiotics. → no change, ↑ increase, ↓ decrease
Full size table

1.4.1 Tilapias

Selim and Reda (2015) found that Bacillus amyloliquefaciens spores supplementation at concentrations of 1 × 106 (G3) and 1 × 104 (G2) colony-forming units per gram (CFU g−1) of feed significantly enhanced serum killing, serum nitric oxide, serum lysozyme activities, as well as IL-1 and TNF α mRNA levels in the kidneys of Nile tilapia, O. niloticus. The cell wall components of both Gram-positive and Gram-negative bacteria are able to stimulate cytokine production (Henderson et al. 1999). Probiotic bacteria colonize in the gut and are involved with the gut-associated lymphoid tissue to stimulate systemic signals that end with cytokine production (Kesarcodi-Watson et al. 2008; Rangavajhyala et al. 1997; Rescigno et al. 2001; Ringø 2011). Similarly, dietary inclusion of B. subtilis singularly or B. subtilis combined with S. cerevisiae and A. oryzae; B. subtilis with B. licheniformis; B. subtilis and Bacillus licheniformis (BS) combined with traditional Chinese medicine (TCM), and B. subtilis with Aqua NZ and AP193 significantly enhanced innate immune response, growth, relative immune, and antioxidant gene expressions of Nile tilapia (Abarike et al. 2018a; Abarike et al. 2018b; Addo et al. 2017a, 2017b; Iwashita et al. 2015; Liu et al. 2017; Wang et al. 2020a). Dietary inclusion of B. licheniformis has been found to increase alkaline phosphatase, myeloperoxidase, lysozyme, reactive oxygen species, reactive nitrogen species, superoxide dismutase, and glutathione peroxidase of Mozambique tilapia (Oreochromis mossambicus) (Gobi et al. 2018). Also, supplementation of Bacillus licheniformis HGA8B significantly improved lysozyme activity and content of complement C3 (Han et al. 2015). It is well documented that, the immune system can be nonspecifically modulated by probiotics (Hoseinifar et al. 2015; Lazado and Caipang 2014; Nayak 2010). Moreover, colony formation and adhesion of probiotics in the intestine of fish are necessary to enhance the immune responses (Ausubel 2005). Interaction between probiotic cells and immune system are through microbe associated molecular patterns (MAMPs) consisting of specific cell wall polysaccharides, peptidoglycan, lipoprotein anchors, and lipoteichoic acids (Hosoi et al. 2003). Cells or components of immune system can interact with MAMPs by pattern recognition receptor such as toll-like receptors, C-type receptor, and nucleotide oligomerization domain-like receptors (Bron et al. 2012; Kleerebezem et al. 2010). This fact may indicate that, addition of fresh culture of B. licheniformis to the diet maintains a high level of probiotics in the diet and improve the immune responses in fish. Similar results have been reported in Nile tilapia fed B. cereus and B. pumilus (Srisapoome and Areechon 2017; Wang et al. 2017b).

1.4.2 Shrimps

In shrimp, B. licheniformis has been intensively applied in Pacific white shrimp (Litopenaeus vannamei). Amoah et al. (2019) indicated that dietary inclusion of 1 × 108 CFU g−1 feed significantly enhanced activity of lysozyme (LYZ), acid phosphatase (ACP), superoxide dismutase (SOD), total protein (TP), albumin (ALB) in serum, glutathione peroxidase (GSH-Px) in serum and liver of Nile tilapia. Similarly, dietary administration of B. licheniformis significantly upregulated the expression of catalase, glutathione peroxidase, superoxide dismutase (SOD), penaeidin-3a (Pen-3a), and heat shock protein (Hsp-70) genes of Pacific white shrimp, Litopenaeus vannamei (Chen et al. 2020a, 2020b). In addition, the combination of B. licheniformis with B. subtilis significantly enhanced lysozyme and hemocyte cell count and upregulated the expression of proPO, LvToll1 and SOD, Hsp70, and TGase genes (Sadat Hoseini Madani et al. 2018; Sánchez-Ortiz et al. 2016). Likewise, dietary inclusion of B. subtilis E20 singularly or combined with other probiotics significantly innate immune response and related immune gene expression of Pacific white shrimp, Litopenaeus vannamei (Chien et al. 2020; Won et al. 2020a). Also, Khademzade et al. (2020) reported that dietary inclusion of Bacillus cereus and Pediococcus acidilactici significantly enhanced total hemocyte count, total protein, and lysozyme activities of L. vannamei. Similar results were found in tiger shrimp and freshwater pawn fed Bacillus sp. and Bacillus coagulant where significant increase in total heterotrophic count, amylolytic, cellulolytic, and proteolytic bacterial counts, phagocytic, lysozyme, and respiratory burst activities was recorded (De et al. 2018; Gupta et al. 2016). At molecular levels, Sánchez-Ortiz et al. (2016) indicated that dietary supplementation of Bacillus spp. resulted in upregulation of proPO, LvToll1, SOD genes, except the TGase gene expression. Similarly, Tepaamorndech et al. (2019) revealed that dietary inclusion of Bacillus aryabhattai TBRC8450 significantly upregulated C-type lectin, penaeidin-3, and heat shock protein 60 genes, as well as enhanced thioredoxin, ferritin, phenoloxidase, and total antioxidant activities of Pacific white shrimp, Litopenaeus vannamei. However, no significant increase in total hemocyte count, and superoxide dismutase were observed (Tepaamorndech et al. 2019).

1.4.3 Carps

In mrigal, Cirrhinus mrigala, Bandyopadhyay et al. (2015) indicated that dietary inclusion of Bacillus sp. PP9 significantly improved hemoglobin percentage, total erythrocyte count, total leukocyte count, corpuscular hemoglobin, total serum protein, albumin globulin ratio, and serum bactericidal activity. Similarly, dietary supplementation of B. subtilis singularly or combined with other Bacillus sp. and prebiotics significantly stimulated hematological, antioxidant, and immunological parameters of Labeo rohita (Ramesh and Souissi 2018); Labeo catla, Catla catla (Sangma and Kamilya 2015); common carp, Cyprinus carpio (Wang et al. 2017a); grass carp, Ctenopharyngodon idellus (Zhao et al. 2020), and Pengze crucian carp, Carassius auratus var. Pengze (Cao et al. 2019). At gene level, Yin et al. (2018) found that supplementation of B. subtilis resulted in higher protective effects against lead toxicity, superoxide dismutase, catalase and glutathione, lysozyme and IgM levels, as well as immune-related genes of gibel carp, Carassius auratus gibelio. Likewise, dietary inclusion of B. amyloliquefaciens significantly stimulated innate immune response, antioxidant, and relative immune gene expressions of roho labeo, Labeo rohita (Nandi et al. 2018); Indian major carp, Catla catla Singh et al. (2017), and grass carp, Ctenopharyngodon idella (Qin et al. 2020).

1.4.4 Sea Cucumber (Apostichopus japonicus)

Supplementation of B. cereus singularly or combined with B. subtilis significantly enhanced total coelomocytes count, acid phosphatase, phagocytosis, respiratory burst, total nitric oxide synthase, catalase, phenoloxidase, and superoxide dismutase activities (Li et al. 2015). Recently, Liu et al. (2020) indicated that dietary administration of B. baekryungensis significantly enhanced superoxide dismutase, catalase, alkaline phosphatase, acid phosphatase, nitric oxide synthetase, phagocytosis, respiratory burst activities, and ubiquitin-mediated proteolysis pathway. Ubiquitin-mediated proteolysis plays an important role in the dynamic regulation of host defense against pathogen infection. It has been reported that a number of key joint molecules in the natural immune and antiviral signaling pathways can be modified by ubiquitination to regulate the antiviral immune response of the body (Chuang and Ulevitch 2004; Liu and Chen 2011). Ubiquitination plays an important role in the Toll-like receptor (TLR) signaling pathway. The activation of this pathway leads to the upregulated expression of Toll-like receptors and enhances nonspecific immunity (Bhoj and Chen 2009). The upregulation of TLR in this study is consistent with the above theory. In the immune system, mTOR signaling plays an important role in maintaining immune homeostasis, for example, the survival and migration of natural immune cells and the secretion of inflammatory factors (Katholnig et al. 2013; Weichhart et al. 2008). Studies have found that the mTOR signaling pathway negatively regulates nonspecific immune responses (Weichhart et al. 2008). Therefore, the downregulation of the mTOR pathway in sea cucumber is beneficial to improve sea cucumber immunity.

1.4.5 Catfish

In striped catfish, Pangasianodon hypophthalmus, Truong Thy et al. (2017) reported that dietary inclusion of B. amyloliquefaciens and B. pumilus significantly enhanced phagocytic, respiratory bursts, and lysozyme activities. Similar results were observed in basa fish, Pangasius bocourti fed B. aerius (Meidong et al. 2018). Likewise, combination of B. subtilis, B. amyloliquefaciens, B. cereus, and B. amyloliquefaciens (Reda et al. 2018).

1.4.6 Japanese eel

Dietary inclusion of B. subtilis and Lactobacillus plantarum significantly enhanced lysozyme, superoxide dismutase (SOD), myeloperoxidase (MPO), level of intestine glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 70, 90, and immunoglobulin (IgM). Similarly, dietary inclusion of B. subtilis and mannanoligosaccharide (MOS) significantly improved nonspecific enzymatic activities, heat shock protein 70 mRNA levels, and immunoglobulin M expressions (Lee et al. 2018). More recently, Park et al. (2020) indicated that dietary inclusion of B. subtilis or B. licheniformis and mannan or fructo oligosaccharide upregulated heat shock protein 70 and immunoglobulin M genes.

1.4.7 Other Species

Dietary inclusion of B. subtilis singularly or combined with B. licheniformis, Bacillus cereus toyoi, and isomaltooligosaccharides significantly stimulated hematological, innate immune response, antioxidant, and gene expression of parrotfish, Oplegnathus fasciatus (Liu et al. 2018); red sea bream, Pagrus major (Zaineldin et al. 2018); turbots, Scophthalmus maximus (Fuchs et al. 2017); rainbow trout, Oncorhynchus mykiss and brown trout, Salmo trutta (Ramos et al. 2017), and Caspian brown trout, Salmo trutta caspicus (Aftabgard et al. 2019). Regarding B. amyloliquefaciens, dietary inclusion of B. amyloliquefaciens singularly or combined with Yarrowia lipolytica lipase 2 (YLL2), B. licheniformis significantly enhanced innate immune response, antioxidant, and gene expression of rock bream, Oplegnathus fasciatus (Kim et al. 2017); hybrid sturgeon, Acipenser schrenckii ♂and Acipenser baerii ♀ (Fei et al. 2018), and zebrafish, Danio rerio (Lin et al. 2019).

In juvenile Atlantic salmon (Salmo salar L.), Wang et al. (2019a) reported that B. velezensis V4 and Rhodotorula mucilaginosa compound led to an increase in acid phosphatase, IgM, nitric oxide, glutamic pyruvic transaminase, glutamic oxalacetic transaminase, lysozyme, total superoxide dismutase malondialdehyde, glutathione, glutathione peroxide, total antioxidant capacity, and malondialdehyde. Similarly, dietary inclusion of Bacillus licheniformis significantly enhanced hematological, innate immune response, and Mn-SOD gene expression (Gao et al. 2018). Also, significant increase in innate immune response and relative immune gene expressions were observed in grouper, Epinephelus coioides, fed Bacillus pumilus (Yan et al. 2016) and in olive flounder, Paralichthys olivaceus, fed Bacillus sp. SJ-10 plus β-glucooligosaccharides (Hasan et al. 2018).

1.5 Conclusion

This chapter addressed the role of Bacillus probiotics in sustainable aquaculture. Although a wide range of researches have indicated beneficial effects of Bacillus species on grow rate, immunity, and disease resistance of farmed fish and shellfish, the investigated effects were species specific. In order to evaluate in vivo adherence and colonization of Bacillus bacteria within the complex microbial ecosystem of the intestine, detection of green fluorescence protein (GFP) tagged strains or fluorescence in situ hybridization (FISH) targeting 16S rRNA to identify the probiotics on the mucus surface must be carried out. Furthermore, mucus-associated (autochthonous) microbiome must be investigated by next-generation sequencing (NGS), transcriptomic, metagenomics or proteomic profiling, and not the allochthonous microbiome; mostly investigated per sc. In addition, we recommend that gnotobiotic approaches are used in future studies, as the gnotobiotic approaches have been reported to have important roles to understand the function of gut microbiota on numerous biological processes of the host. Moreover, data is needed to understand the mechanisms by which the immune system of the intestinal mucosa discriminates between pathogenic, probiotics, and commensal microorganisms.