WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2024 57 is a finite space in the gut for colonization, microbial and antimicrobial compounds may limit the proliferation of pathogenic species. A highly diverse microbiota can resist potential pathogens but the loss of microbial diversity allows the pathogenic bacteria to colonize the gut and contribute to disease. However, as in the case of WFS, the biological and physical changes elicited by EHP led to a decline in the diversity of the gut microbiome. Dysbiosis is the term used to describe the loss of microbial diversity and microbial balance (Holt 2021). We initiated a long-term study of the microbiomes of shrimp pond water during infection with WFS. The water microbiome evolves with changes in water quality, and the microbial species diversity responds to environmental pressures. Our goal is to collect microbiome data from WFS-infected ponds, to study the microbial diversity throughout the disease, and to determine triggers for dysbiosis from changes in the microbial content in the rearing pond. This knowledge would allow the development of treatment regimens to reinstate microbial diversity in the pond and the shrimp. For the study, we selected two ponds in India from the same farm that share the same borewell water source. The ponds were stocked with Litopenaeus vannamei and both ponds exhibited signs of WFS. Pond A2 was sampled at 60 days of culture (DOC), had a pH of 7.8, and was severely infected with WFS. Pond A4 was sampled at 49 DOC, had a pH of 8.3, and was in the early stages of WFS. We established a method to collect the pond microbiome on Sterivex™-GP filters from 150 ml of pond water. DNA was purified from the filters for the PCR detection of EHP and Vibrio species associated with WFS and for Targeted Amplicon Sequencing. We confirmed the presence of microbes that are part of the pathobiome for WFS through colony isolations and PCR. Enterocytozoon hepatopenaei is unculturable, so no colony isolations can be made but PCR of the isolated DNA can be used to detect its presence or absence. Colony isolations from Pond A2 and Pond A4 included V. parahaemolyticus toxR+ (VPtoxR+) isolates and several unknown toxR- Vibrio spp. In addition, Pond A4 contained V. alginolyticus toxR+ and toxR-. Polymerase chain reaction (PCR) analysis of the DNA from Pond A2 and Pond A4 identified the presence of EHP and toxR+368, which was expected because of their WFS status (Caro 2021). After we confirmed WFS visually and by molecular procedures we were interested in studying the microbiome of the two ponds at different stages of infection and comparing the results to published studies. An increasing number of microbiome studies of the digestive systems of shrimp species and rearing water have been undertaken and reviewed (Palaniappan et al. 2024, Chen et al. 2020, Cornejo-Granados 2018, Yu 2018, Holt 2021). Finding a consensus from these studies is challenging because differences in experimental procedures and bioinformatic tools implemented in different studies can affect outcomes. Geographical location, shrimp species, water quality, shrimp age, and farming techniques can affect microbiota in both shrimp and rearing water. The amount of data generated by metagenomics is vast and the analysis and reporting of the data is cumbersome. DNA isolated from the filters was sent to Charles River Microbial Solution Services for their TAS NGS-based bacterial identification by Targeted Amplicon 16S/ITS Sequencing (NGS-TAS-16S-20), a process used to identify microbial populations. Although they offer a fungal sequencing option we focused on the bacterial microbiome because most studies on shrimp gut and pond water have been on the bacterial populations. Since the samples were taken from ponds that exhibited different stages of infection the data was examined for similarities and possible shifts in microbial diversity throughout disease. The sequencing gave 186,572 reads for Pond A2 and 217,302 reads for Pond A4 with 57 percent classified for both samples. The ponds exhibited a similar background of microbes; Pond A2 had 15 Phyla and Pond A4 had 21 Phyla represented in their bacteria populations. The similarity in microbes is not surprising because the ponds were from the same farm, shared the same water supply, contained the same shrimp species, and had the same farming practices. The Phyla that represented over 1 percent of the microbial population in both ponds were Bacteroidota, Chloroflexota, Cyanobacteriota, Planctomycetota, Pseudomonadota (synonym Proteobacteria), and Actinomycetota. Actinomycetota and Proteobacteria had a high percentage of reads from both ponds. However, Pond A2 with the severe infection had about half the number of Actinomycetota as Pond A4, (21 percent and 45.60 percent respectively) while Pond A2 had almost twice the number of Proteobacteria (45.725 percent and 25.95 percent). Actinomycetota, a diverse Phylum of gram-positive bacteria, are denitrifiers, produce vitamins, and produce bioactive metabolites that break down organic compounds including cellulose, alginates, and various hydrocarbons. Five orders represented over 1 percent of the microbial population in the 2 ponds. Micrococcales have probiotic potential and represented 29.34 percent of the population in Pond A4 and 11.71 percent of the population in Pond A2. The Streptomycetales are microbes that produce bioactive compounds that are antibacterial, antiparasitic, and antifungal. The Streptomycetales represented 3.15 percent of the sample in Pond A4 but were not detected in Pond A2. Frankiales declined from 1.33 percent of the population in Pond A4 to 0.02 percent of the population in Pond A2. Two orders showed an increase in population: Acidimicrobiales was 0.82 percent in Pond A4 and 3.41 percent in Pond A2, and Kitasatosporales was 0.63 percent in Pond A4 and 1.97 percent in Pond A2. Proteobacteria contains diverse gram-negative bacteria that belong to six classes. Three of the six classes were found in the two samples; Alphaproteobacteria, Betaproteobacteria, (CONTINUED ON PAGE 58) We initiated a long-term study of the microbiomes of shrimp pond water during infection with WFS. The water microbiome evolves with changes in water quality, and the microbial species diversity responds to environmental pressures. Our goal is to collect microbiome data from WFS-infected ponds, to study the microbial diversity throughout the disease, and to determine triggers for dysbiosis from changes in the microbial content in the rearing pond. This knowledge would allow the development of treatment regimens to reinstate microbial diversity in the pond and the shrimp.
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