58 SEPTEMBER 2024 • WORLD AQUACULTURE • WWW.WAS.ORG and Gammaproteobacteria. Alphaproteobacteria were found in greater numbers in the severely infected Pond A2 at 43 percent with Pond A4 only having 19.68 percent. This class can grow at low levels of nutrients and some induce nitrogen fixation. The top three orders from Alphaproteobacteria, Rhodobacterales, Hyphomicrobiales, and Sphingomonadales, increased in numbers in severely infected Pond A2. The Betaproteobacteria are at about the same level in both ponds with Burkholdleriales being the order represented. This order has a wide range of metabolic activity and includes opportunistic pathogens. Gammaproteobacteria contains nitrogen-fixing organisms, cellulose degraders, and Vibrio. Pond A4 had the highest (8.47 percent) number of Gammaproteobacteria compared to Pond A2 (1.49 percent) with Pond A4 having the most Vibrionales (5.99 percent). Pond A4 had 8 different genera of Vibrio: alginolyticus, fortis, harveyi grp, harveyi, mytii, porteresiae, tasmaniensis, vulnificus, and a high percentage, 5.46 percent, of undefined Vibrio spp. Pond A2 had 5 genera of vibrio spp; fischeri, alginolyticus, harveyi, nigripulchritudo, and tasmaniensis. Additional phyla abundant in both ponds that demonstrated a downshift in the severely infected pond included: Cyanobacteria at 13.73 percent for Pond A4 and 6.49 percent for Pond A2, Bacteroidota at 5.64 percent for Pond A4 and 3.46 percent for Pond A2, 5.64 percent. An upshift of microbes was observed for Planctomycetota in the severely infected Pond A2 at 7.18 percent, compared to 1 percent for Pond A4. Although it has been noted that the gut microbiota is distinct from the rearing water, we were interested in identifying differences and similarities. It has been suggested that the gut microbiota may differ because of environmental filtering and colonization (Holt 2021). A meta-analysis study combined published data from different shrimp microbiome studies and identified the following microbes as dominant in generalized shrimp microbiota: Proteobacteria (65.99 percent), Firmicutes (16.42 percent), Actinobacteria (synonym Actinomycetota) (3.24 percent) and Bacteroidetes (2.17 percent) and Fusobacteria (0.76 percent) (Cornejo-Granados 2018). These Phyla were all represented in our study but at different percentages of the population with Fusobacteria below 1 percent. This meta-analysis study highlighted important facts about the shrimp microbiome including; differences between marine and freshwater, differences in developmental stages of the shrimp, differences within the organs of the shrimp, and differences in microbial diversity in disease. Another meta-analysis studied the gut bacteria of healthy versus diseased shrimp (Yu 2018). The healthy gut of L. vannamei is dominated by members of the Gammaproteobacteria, Alphaproteobacteria, and Bacteroides. Additional Phyla present in the gut included Actinobacteria, Planctomycetes, and Tenericutes. Shrimp disease is closely associated with dysbiosis and the degree of dysbiosis is related to the severity of the disease. In diseased shrimp, across four different diseases, there was a consistent decrease in Actinobacteria and Bacteroidetes but an increase in Gammaproteobacteria. The reduction in Actinobacteria numbers is expected because this species exhibits antibacterial activity found in healthy shrimp. The rise in Gammaproteobacteria numbers would be expected because this group of bacteria contains shrimp pathogens including vibrio spp. In our study, Proteobacteria increased in the severely infected Pond 2 with the Alphaproteobacteria increasing and the Gammaproteobacteria decreasing compared to Pond 4. Because not all vibrio spp. are pathogenic and some are beneficial, the diversity in the vibrio spp. in Pond 4 may be consistent with the stage of the disease. Because WFS is associated with pH<8 and the lowered pH can be from eutrophication, we looked at a study of microbial diversity over a eutrophication gradient. The study identified Actinobacteria, Proteobacteria, and Cyanobacteria as the dominant Phyla. Actinobacteria contributed 20 percent to the microbial population in a highly eutrophic pond and more than 40 percent in a less eutrophic pond. If you compare the eutrophication gradient to disease severity the results are comparable, with 19.95 percent of the population Actinobacteria in the severely infected pond and 45.65 percent of the population Actinobacteria in the less severely infected pond. There was a direct correlation between the eutrophic index, disease severity, and the abundance of Planctomycetes, Verrucomicrobia, Cyanobacteria, and Chloroflexi. The abundance of Actinobacteria and Bacteroides was indirectly correlated to eutrophic index and disease severity. Proteobacteria is a diverse group and did not show a correlation with the eutrophic level. Alphaproteobacteria, a class within Proteobacteria, showed a negative correlation to the eutrophic level but was directly correlated to disease severity (Kiersztyn 2019). Microbial activities, such as the degradation of organic matter, respiration and nitrification, and accumulated dissolved carbon dioxide can alter the hydrogen concentration lowering the pH of the water. The lower pH is associated with WFS and the changes in the microbial diversity of the pond water as the disease progresses were similar to the changes in microbial diversity you would see in pond eutrophication. Reversing the water quality in a WFS-infected pond by adding lime to raise the pH led to less mortality (Alfiansah 2020). White Feces Syndrome occurs after 50 DOC when water quality is declining as indicated by lower pH. At this point, the microbes in the water are responding to the poor conditions, as are the shrimp. Changes in the water quality negatively affect the beneficial bacteria providing an opportunity for the colonization of EHP and pathogenic bacteria in the shrimp. Because this started with the poor water quality maintaining the water quality would mitigate the disease. Probiotics are commonly used in aquaculture environments to improve water quality. Probiotic bacteria have also been documented to improve the immune system, modulate the gut microbiome, improve nutritional status, and decrease disease. Probiotics would increase the microbial diversity in WFS ponds with bacteria that have the functions lost from the Actinomycetes. As the water quality improves the original microbial diversity should be restored in both the water and the shrimp. The improved water quality will lead to healthier shrimp and greater profits. Probiotics are commonly used in aquaculture environments to improve water quality. Probiotic bacteria have also been documented to improve the immune system, modulate the gut microbiome, improve nutritional status, and decrease disease. Probiotics would increase the microbial diversity in WFS ponds with bacteria that have the functions lost from the Actinomycetes.
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