Marine probiotics to positively balance shrimp microbiota and promote better growth and survival

Carine Le Ker, Emilie Giudicelli, Fanny Giudicelli, Marine Akwa

The microbiota is known to interact with its host and contribute to a number of key host processes including nutrition, development, immunity and behavior. It is therefore involved in the regulation of shrimp health and disease. Its composition changes with different factors: the stage of development, the feed quality, environmental conditions and the presence of pathogens. Microbiota of farm-raised shrimp is less diverse than in wild shrimp due to lower microbial diversity in pond farms and industrial feed lacking live microorganisms.

An alternative to the imbalance of farmed shrimp’s microbiota is the introduction of beneficial bacteria to restore bacterial diversity. Not all bacteria can be successfully introduced in shrimp microbiota or its water environment. Shrimp production is commonly performed in salted water, so probiotics (i.e. live microorganisms) used in shrimp production must be active in a salinity range from 2-35‰. Most of the probiotics used on the market are of terrestrial origin and, therefore, not fully adapted to the aquatic living environment or the host itself. Stressed terrestrial bacteria slow down or stop their metabolism and reproduction, and consequently stop playing their probiotic function. An illustration is the use of a terrestrial Pediococcus isolated from natural-pasture Gramineae in salmon feed supplementation. The terrestrial probiotic did not persist in salmon microbiota following the transfer from freshwater to seawater. However, benefits have been observed over time (Jaramillo-Torres et al., 2019).

Figure 1. Experimental design of trials from nursery to pre-grow out.

Table 1. Growth parameters at the end of the experiment in control and treated shrimp with marine probiotics consortium encapsulated in algae.

T test, p< 0,05

Marine probiotics encapsulated in algae

Probiotics have to resist the salted environment and be able to enter shrimp or fish mucus and compete for space and nutrients in the animal’s gut. For that purpose, the use of “host-associated probiotics” from fish and shrimp has gained attention (Van Doan et al., 2020).

Our team strategy was to focus on sessile organisms with holobiont free of aquaculture pathogens genus (more specifically Vibrio). When facing a predator or a pathogen, sessile organisms cannot run and have developed an arsenal of bioactive molecules to fight. Many of them are produced thanks to the symbiotic bacteria they harbor.

From our screening and characterization of marine bacteria studies, we developed two formulas dedicated to fish and shrimp, named AKWABIOTIC, consisting in marine Bacillus probiotics consortium encapsulated in algae providing synergic and beneficial effects to shrimp and fish health and growth.

The marine probiotics consortium encapsulated in algae was evaluated in triplicate on shrimp from nursery (3,500 PLs/tank) to pre-grow out (350 PLs/tank) in two experimental stations in China and Peru (Fig. 1).

Growth performance and pathogen challenges

Marine probiotics encapsulated in algae significantly increased the final body weight by 11%, weight gain rate by 27%, specific growth rate by 6% and decreased the feed coefficient rate by 15% after the 80 days of feed supplementation (Table 1). Under normal controlled conditions, marine probiotic solutions increase the

Figure 2. Survival with (B) and without (A) salinity stress of control and treated of PL30 shrimp with marine consortium probiotics encapsulated in algae (n=125 shrimps/tank).

Figure 3. Shrimp survival during the challenge against Vibrio parahaemolyticus (carrying the phage responsible for the Early Mortality Syndrome), at PL30 (A) and after an 80-day experiment (B), treated or not with the marine probiotics consortium encapsulated in algae (n=125 shrimps/tank). Vibrio colonies counted from shrimp’s hepatopancreas on TCBS (C).

Figure 4. Relative abundance of hepatopancreas microbiota at phylum level in control or treated shrimp with marine probiotics consortium encapsulated in algae (n=10 HP). survival rate by 6% (Fig. 2). During salinity stress, where salinity was reduced to 1‰, the probiotic solution also improved the shrimp resistance showed 15% more survivors.

In challenges against several aquaculture pathogens, marine probiotics at least doubled the number of survivors and shift half-life by 30% (data not published). The challenge with Vibrio parahaemolyticus (carrying the phage responsible for the acute hepatopancreatic necrosis disease) led to an increase of survival by 120% at PL30 (end of nursery) and by 125% at the end of the experiment after the 80-days probiotics supplementation, with mortalities, in control group, being 83% and 72%, respectively (Fig. 3). These high survival rates were associated with a low level of Vibrio in the gut when marine probiotics were administered (Fig. 3).

Microbiota profiles

After the 80-day trial, both hepatopancreas and intestines were isolated and analyzed to build microbiota profiles (only results from hepatopancreas are presented). At the phylum level, most of the

Figure 5. Relative abundance of hepatopancreas microbiota at genus level in control and treated shrimp with marine probiotics consortium encapsulated in algae (n=10 HP).

bacteria population in the control group belonged to Proteobacteria phylum, up to 95% of relative abundance (Fig. 4). The dominance of this phylum is standard with marine animals’ microbiota (Yukgehnaish et al., 2020).

The use of marine probiotics increased the richness and diversity of bacterial flora. The Shannon index for probiotics treated shrimps was 2,913 points higher than the control. An enrichment of phyla containing beneficial bacteria with probiotics activities like Firmicutes and Actinobacteria (producing antibacterials), and Bacterioides (digesting vegetal carbohydrates) was observed in hepatopancreas microbiota of treated shrimp with marine probiotics.

At genus level, Vibrio spp. dominate, with up to 65% (Fig. 5) in the control group. The use of marine probiotics decreased the content of Vibrio by 63%. Looking at the specific pathogen species, probiotics

Table 2. Relative abundance of probiotics and Vibrio in hepatopancreas microbiota of control and treated shrimps with marine probiotics encapsulated in algae.

solution decreased by a factor of 3 the relative abundance of Vibrio harveyi and V. parahaemolyticus (Table 2). This decrease was associated with an increase of probiotics genus abundance, here Bacillus, and perfectly illustrates the mechanism of exclusive competition between probiotics and pathogens in the animal gut (Knipe et al., 2021).

In this study, Bacillus probiotics colonized microbiota at a relative abundance of 4%. It is important to remember that both introduced probiotics and beneficial commensal bacteria don’t need to be abundant to play a crucial function and act positively on animal physiology (Wang et al., 2020). Probiotic purpose is not to replace microbiota. Above all, probiotics modulate microbiota composition in favor of beneficial bacteria presence, including probiotic ones.

At genus level, we also observed the emergence of beneficial bacteria in shrimp treated with marine probiotics encapsulated in algae. Bacterioides are known to be the major producer of B12 vitamin and powerful vegetal fiber digesters (Nathan & Eduardo, 2017). They also are associated with resistance to low salinity in shrimps’ microbiota (Landsman et al., 2019). Dietzia and Bacillus genera produce antibacterial, antifungal, anti-parasite and antiviral substances (Elshaghabee et al., 2017; Modolon et al., 2020). On the other hand, Nautella, an indicator of bad health, disappeared from microbiota when shrimp were treated with marine probiotics (Zheng et al., 2017).

Conclusion

The evaluated AKWABIOTIC shrimp formula made of a marine Bacillus probiotics consortium encapsulated in algae allowed the controlled release of the right bacteria at the right place for a colonization and action of adapted marine Bacillus in the shrimp gut. Marine probiotics and algae showed antagonist effects against Vibrio populations.

In addition, marine probiotics and algae positively modulated shrimp microbiota to provide a balanced good/bad bacteria ratio. Induced microbiota modulations improve shrimp health and growth and marine probiotics also improve survival under normal and both biotic- and abiotic-challenged conditions.

The preventative use of marine probiotics solutions strengthens the bacterial shield which is the microbiota and decreases the need for antibiotics or other chemical inputs during shrimp production. When used after an event, for example, environmental stress, the appearance of pathogens, the use of antibiotics or disinfectants, the marine probiotic solution allows to correct the caused dysbiosis. Finally, the synergy between algae and marine probiotics significantly improves growth and feed efficiency for better productivity.

References available on request.

More information: Carine Le Ker

R&D Manager Marine Akwa E: carine@marineakwa.com

Fanny Giudicelli

Sales Manager Marine Akwa E: fanny@marineakwa.com