Common problems in aquaculture occur by naturally present opportunistic bacteria that may become pathogenic when the host immune system is weakened by environmental stress. The selective establishment of beneficial microbiota in the gut is crucial for the stable production of healthy fish larvae. 

The microbial colonization of the fish gut is mainly influenced by rearing water and feed, apart from the selective pressure from the fish host itself.

Recent studies showed that the gut microbiota of fish larvae, such as zebrafish, is more similar to the surrounding environment than adult fish, which indicates the great importance of the early-life rearing environment.

For example, Nile tilapia (Oreochromis niloticus) larvae reared in recirculating aquaculture system (RAS) and active suspensions tanks showed distinct gut microbiota composition.

Traditionally, flow-through systems (FTS) are used for fish larvae culture. In FTS, the nutrient load and microbial density are continuously diluted due to water exchange, which has been reported to select for fast-growing bacteria, known as rstrategists.

On the other hand, RAS allows maintaining a stable microbial community composition in the water selective to the growth of slow-growing bacteria, known as K- rstrategists Opportunistic bacteria are characterized as r-strategists, which often can affect negatively fish health, while Kstrategists can deal better with perturbations in nutrient availability and are considered harmless for fish survival.

The use of probiotics, which are beneficial microbes that can modulate the microbial community of its host, improve feed utilization and reduce disease susceptibility, has been proposed as a strategy for sustainable aquaculture. It has been demonstrated that probiotics increase the survival of marine fish larvae.

“Considering the differences in the rearing environment between RAS and FTS, as described above, we hypothesize that tilapia larvae rea red in RAS will develop a different gut microbiota and show better survival and growth than those reared in FTS.”

In this study, two rearing systems, namely FTS and RAS, were tested for Nile tilapia larvae culture. To test the impact of B. subtilis as dietary probiotic in RAS treatment, a control diet and a control + B. subtilis coated diet (RASB) were applied.

The effect of the three treatments (FTS, RAS and RASB) on survival, growth performance and gut microbiota were evaluated in Nile tilapia larvae, starting from first feeding, for 26 days.

Water quality maintenance and fish survival

The hatching rate of Nile tilapia eggs were 82%, 73% and 78% in the incubator for the FTS, RAS and RASB treatment, respectively. The three rearing systems shared the same source for water supplementation, and no significant differences in pH, TAN and NO2-N was observed between the systems.

The repeated measure ANOVA showed that the cumulative mortality was significantly higher in FTS (P = 0.002), while RAS and RASB had similar cumulative mortality over time.

Gut microbial community composition

Numerically, there was trend that FTS < RAS < RASB in bacterial richness in the gut of tilapia fry at 26 dof.

Our analysis showed that one genus, namely Plesiomonas, was shared among all three treatments, while Escherichia and Shingella showed high prevalence in RAS, and Gemmobater and Bacillus were prevalent in the RASB treatment.

No core genus was solely identified in FTS treatment. Looking at the phylum level, Proteobacteria, Actinobacteriota and Planctomycetota were the dominating phyla in the gut of tilapia fry from the three treatments, which accounted for 87% of the total population.

At last, the genera significantly enriched in each treatment were selected by LDA. A total of 39 genera were detected significantly enriched in the gut from each of the three treatments. 

In detail, FTS was enriched with Shinella (RA = 8.3%) and Hyphomicrobium (RA = 1.6%). RAS was enriched with Paracoccus (RA = 8.7%), Mycobacterium (RA = 8.7%) and Cetobacterium (RA = 5.0%). RASB was enriched with Gemmobacter (RA = 5.7%) and Bacillus (RA = 4.0%).

Discussion

Environmental rearing conditions during early life and diet determine the microbial community composition and structure in the fish intestine. The assembly of gut microbiota urther influences fish larvae’ immunological and histological development, which plays a crucial rule in fish health and growth.

Our study demonstrated the feasibility of modulating the bacterial community in the fish gut by creating different rearing systems or by dietary probiotic supplementation during early life, which could influence survival and lead to a healthy gut microbiota composition.

Rearing system affected mortality and gut microbiota of tilapia fry

The water quality in the three rearing systems was optimal for the growth of Nile tilapia. Supplementation of Bacillus spp. in the feed or water was shown to enhance the water quality by reducing the ammonia and nitrate concentrations in the systems.

A trend for a lower nitrate concentration in RASB than RAS was observed in our experiment during the later experimental period. However, this difference could also be due to the numerically higher water exchange in RASB when compared with RAS.

Our study showed that the rearing system (FTS vs RAS) had no significant effect on the growth of tilapia larvae. At the same time, RAS significantly improved the survival rate of tilapia larvae. Compared with FTS, RAS has a more stable and diverse microbial community composition in the tank water than FTS, which is typically dominated by potentially pathogenic r-strategists in the water.

The microbial matured water has improved marine larval survival in the early life stage, which also applies to freshwater fish species like tilapia in the RAS and RASB treatment of this study.

The difference in the microbial composition and bacterial loading of tank water between FTS and RAS might explain the differences in the fish gut microbiota. The fish sampled from different tanks within the same RAS showed similar gut microbiota composition in this study, in line with our previous study.

“However, fish sampled from different tanks within FTS showed different gut microbiota compositions, according to the large number of unique ASVs detected in fish from FTS.”

The high variability between individuals in FTS is also a characteristic of rstrategists, potentially explaining the significantly higher variability in the water microbial community between parallel tanks in FTS than RAS.

Our study further demonstrated that RAS as a water microbial maturation strategy in larvae culture delivered a more stable and reproducible gut microbial community in tilapia guthan FTS.

Dietary probiotic supplementation altered fry gut microbiota but not growth

The growth-promoting effect of Bacillus spp. on tilapia is dose-dependent. Although B. subtilis can improve the growth and survival of juvenile or adult tilapia in some studies, dietary supplementation of Bacillus spores to 2 g tilapia fry for 8 weeks did not affect their growth.

In this study, dietary supplementation of B. subtilis at the dosage of 108 CFU/g did not significantly influence the growth of Nile tilapia larvae, which might be due to the restricted feeding masking the probiotic effect on fish growth. Bacillus spp. were reported to increase the disease resistance of fish.

Microbial functionality influenced by rearing system and probiotic supplementation

Both the rearing system and the probiotic supplementation  in the current study modulated the microbial composition in the gut of tilapia. In the present study, FTS treatment group was significantly enriched with Shinella and Hyphomicrobium.

Both were previously reported to be present in high abundance in RAS, however, the role of these genera in the fish gut is still not clear. Besides, the fish gut microbiota from RAS and RASB treatments were dominant with Ceto bacterium, while it was detected in low abundance in FTS (RA = 0.02%).

“C. somerae is an anaerobic microbe which produces vitamin B12 in the freshwater fish intestine and is related to fermentative metabolism of peptides and amino acids. C. somerae was commonly detected as core species in freshwater fish species, including tilapia.”

Moreover, a decrease in abundance of Cetobacterium in zebrafish gut by antibiotic treatment was shown to increase the susceptibility of fish to pathogen infection, in our study, the high mortality of fish larvae in FTS could be related to the low occurrence of C. somerae in the fish gut.

In addition, RAS was enriched with Mycobacterium (RA = 8.7%).

Some species belonging to Mycobacterium genus, such as M. marinum, were reported as pathogens and cause mycobacteriosis in fishes. However, whether Mycobacterium causes pathology depends on the species and the host’s susceptibility.

Dietary supplementation of B. subtilis spores enriched the Bacillus spp. in the gut of RABS treatment (RA = 4.0%), which implied its colonization in the tilapia gut. Besides, dietary probiotic supplementation increased the abundance of Gemmobacter in our study.

“Gemmobacter was shown to be a dominant genus in the gut of zebrafish larvae, confirming the presence of these taxa in freshwater fish gut.”

To summarize, recirculating system and probiotic administration may benefit the gut microbial colonization of tilapia larvae as evidenced by the observed positive correlation between the gut microbiota distribution and the standard body length as well as survival in RAS and RASB treatments.

Conclusions

This study demonstrated the feasibility of modulating the gut microbiota of tilapia larvae through different rearing systems (i.e. FTS and RAS) and dietary probiotic supplementation (RASB). Though FTS had similar or even superior water quality compared to RAS, RAS showed better survival of larvae than FTS.

This result could be partly explained by the alterations in the gut bacterial colonization, for instance, the absence of Cetobacterium in FTS. Dietary B. subtilis supplementation in RAS increased the abundance of potentially beneficial Bacillus and Gemmobacter in the fish gut.

Our study indicated that RAS is superior to FTS for fish larvae culture concerning survival, while dietary probiotic supplementation may further improve gut health with potential implications during later life stages.

This is a summarized version developed by the editorial team of Aquaculture Magazine based on the review article titled “EFFECT OF REARING SYSTEMS AND DIETARY PROBIOTIC SUPPLEMENTATION ON THE GROWTH AND GUT MICROBIOTA OF NILE TILAPIA (OREOCHROMIS NILOTICUS) LARVAE” developed by: YALE DENG – Wageningen University, MARC C.J. VERDEGEM – Wageningen University, EP EDING – Wageningen University, FOTINI KOKOU – Wageningen University.
The original article was published on AUGUST 2021 through ELSEVIER under the use of a creative commons open access license.
The full version can be accessed freely online through this link: https://doi.org/10.1016/j.aquaculture.2021.737297

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