50 MARCH 2023 • WORLD AQUACULTURE • WWW.WAS.ORG is now common in any microbial ecology laboratory. The approach is applied by targeting the small subunit rDNA gene (16s subunit), a gene involved in protein production. Comparison of variations from individual gene sequences with other known sequences allow identification of groups of bacteria, including many that were previously unknown. The process allows sequencing of, in some cases, millions of DNA fragments simultaneously. Application of this “deep sequencing” technology to environmental samples has revolutionized microbial ecology and is creating a clearer picture of microbial diversity in the environment and microbial patterns that result in disease. Why is knowing the gut microbial diversity in a fish important to aquaculture? Imbalances in biological equilibrium and of gut microbes bring on disease. This imbalance has been too often treated with antibiotics. Alternative approaches may include probiotics. Benefits for the marine aquaculture industry will accrue if probiotics increase gut microbial balance, decrease stress-related diseases and increase fish health and condition. Once considered as a possible alternative to antibiotics and important to the fish farmer, knowledge of the dosage and frequency of probiotic use will be needed for the most effective results. Although probiotics manufacturers can provide suggestions as to how much probiotics to use, most estimates have not been rigorously tested for commercial fish. Aquaculture and Probiotics Work at UNT-MCAPL For probiotics to be of use in commercial aquaculture, knowledge is needed of the effective doses, viability and particular strains. Moreover, probiotics and gut microbial analysis through deep sequencing will only be valuable if probiotics improves production performance in tandem with improving fish health and condition. Yet, few dose-response relationships or quantified effects on production performance have been established for commercially produced probiotics. In most cases the probiotic manufacturers recommend a specific dose, but this may depend on fish species and/ or environmental conditions. To determine the ‘best’ or optimal dose of probiotics for commercial fish species important to Texas aquaculture, our laboratory — the Marine Conservation and Aquatic Physiology Laboratory (MCAPL) at the University of North Texas (UNT) — has investigated how commercially available strains of probiotics affect health and growth in commercially important aquaculture fish species for over a decade. The work conducted at the UNT-MCAPL employs a probiotic approach to finfish aquaculture. Probiotics — An Alternative to Antimicrobials? The fish intestine is a portal of entry for disease, as has been reported for Aeromonas salmonicida in a variety of fish (Ringø et al. 2004, Jutfelt et al. 2006). Aeromonas infections are among one of the most problematic and devastating diseases in aquaculture. Others evolving rapidly include Streptococcus iniae in tilapia, red drum and sturgeon. The scientific community is racing to learn how to mitigate their effects. Use of antibiotics is undesirable as it leads to an increased prevalence of antimicrobial resistant (AMR) organisms, impacting the ability to treat later infections. This is of particular concern for Aeromonas species, which are resistant to several antibiotics and can be opportunistic pathogens of humans. Additionally, antibiotic usage disrupts the normal microflora in the gut, leading to a breakdown of the functions of the healthy gut microflora. Despite AMR and the ban in the EU of using antibiotics for growth enhancement in 2006, antibiotics use in agriculture and commercial aquaculture are still rising globally. Interest in probiotics as an environmentally friendly alternative to antibiotics is increasing, with the global market for probiotic ingredients, supplements and foods projected to reach over $94.5 billion by 2027. The rising awareness among buyers for preventative health measures, with the increasing demand for natural and safe health-promoting products is thought to be driving global markets. But compared to probiotics use in human and terrestrial livestock nutrition, use of probiotics in aquaculture is still in its infancy. Some beneficial bacteria have been commercialized for use in European aquaculture (Fuller 1992, Rinkinnen et al. 2003). However, commercial use of probiotics in US aquaculture is rare, and there have been few quantitative assessments of their effects on production efficiency, economic viability and production risks, especially in response to increasing environmental stressors. What Is Deep Sequencing? For over 200 years, microbes have been identified by growing them in media on plates. But less than 1 percent of the bacteria in any sample can be grown on plates in media. Often those readily propagated species are studied in the laboratory but may not represent the most dominant members of their environment, leading to a lack of clarity as to which bacteria are the most important, limiting the utility of cultivation-based studies. Over the last 20 years, identification of microbes and microbial community analysis has become molecular in nature. Amplification of DNA by the polymerase chain reaction (PCR) FIGURE 2. Mean Condition Index (CI) for juvenile red drum Sciaenops ocellatus fed probiotics (water-soluble PrimaLac®) vs. control (no probiotics). CIs were based on the external appearance of juvenile red drum, which was evaluated and scored based on physical injuries, pigmentation and scale health. Red bars (control, no probiotics), green bars (probiotics from day 0-28 then discontinued until day 56), blue bars (probiotics fed for 0-56 days). From: Busby (2021).
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