Doing More with Less: A Comparison of 16S Hypervariable Regions in Search of Defining the Shrimp Microbiota

Abstract

The shrimp has become the most valuable traded marine product in the world, and its microbiota plays an essential role in its development and overall health status. Massive high-throughput sequencing techniques using several hypervariable regions of the 16S rRNA gene are broadly applied in shrimp microbiota studies. However, it is essential to consider that the use of different hypervariable regions can influence the obtained data and the interpretation of the results. The present study compares the shrimp microbiota structure and composition obtained by three types of amplicons: one spanning both the V3 and V4 hypervariable regions (V3V4), one for the V3 region only (V3), and one for the V4 region only (V4) using the same experimental and bioinformatics protocols. Twenty-four samples from hepatopancreas and intestine were sequenced and evaluated using the GreenGenes and silva reference databases for clustering and taxonomic classification. In general, the V3V4 regions resulted in higher richness and diversity, followed by V3 and V4. All three regions establish an apparent clustering effect that discriminates between the two analyzed organs and describe a higher richness for the intestine and a higher diversity for the hepatopancreas samples. Proteobacteria was the most abundant phyla overall, and Cyanobacteria was more common in the intestine, whereas Firmicutes and Actinobacteria were more prevalent in hepatopancreas samples. Also, the genus Vibrio was significantly abundant in the intestine, as well as Acinetobacter and Pseudomonas in the hepatopancreas suggesting these taxa as markers for their respective organs independently of the sequenced region. The use of a single hypervariable region such as V3 may be a low-cost alternative that enables an adequate description of the shrimp microbiota, allowing for the development of strategies to continually monitor the microbial communities and detect changes that could indicate susceptibility to pathogens under real aquaculture conditions while the use of the full V3V4 regions can contribute to a more in-depth characterization of the microbial composition.

Bacillus subtilis delivery route: effect on growth performance, intestinal morphology, cecal short-chain fatty acid concentration, and cecal microbiota in broiler chickens

“…The resolution of the V4 region of the 16S rRNA gene sequenced in this study could have influenced this outcome. García-López et al. (2020) have recently shown that both the V3 and V3V4 hypervariable regions capture a broader spectrum of microbiota diversity than the V4 region.…”

“…(2020) have recently shown that both the V3 and V3V4 hypervariable regions capture a broader spectrum of microbiota diversity than the V4 region. Although the V3 region offers the advantage of faster sequencing time and lower cost, the V3V4 region offers a higher taxonomic resolution at an increased cost ( García-López et al., 2020 ). Increased abundance of F irmicutes has been associated with increased nutrient absorption and energy harvest from diets ( Jumpertz et al., 2011 ).…”

OTUs and ASVs Produce Comparable Taxonomic and Diversity from Shrimp Microbiota 16S Profiles Using Tailored Abundance Filters

“…The taxonomic variations in the microbiota have been mainly explored using 16S rDNA profiling from environmental and aquaculture samples [ 4 , 6 , 7 ], frequently selecting up to two consecutive hypervariable regions [ 9 , 11 ]. A previous study determined that the hypervariable region V3 is a cost-effective alternative to determine the microbiota diversity in the shrimp hepatopancreas and the intestine [ 12 ]. Although amplicons spanning both the V3 and V4 regions bore a higher taxonomic resolution and diversity, the V3 region showed optimal family-level resolution and better performance than V4 at the genus level using current sequencing technologies [ 12 ].…”

Microbiomes of Clownfish and Their Symbiotic Host Anemone Converge Before Their First Physical Contact.

“…We observed a better taxonomic accuracy with the forward reads covering the V3 region of the 16S rRNA gene. Recent studies have shown that the V3 represents the best choice for the pro ling of complex microbial communities [25], for increased and more accurate estimates of community richness and diversity [26]. Thus, 270 bp reads covering V3 were used for downstream analyses, as in [25,26,27].…”

“…Recent studies have shown that the V3 represents the best choice for the pro ling of complex microbial communities [25], for increased and more accurate estimates of community richness and diversity [26]. Thus, 270 bp reads covering V3 were used for downstream analyses, as in [25,26,27]. The standard dada2 pipeline was used, with a maximum of 2 expected errors (maxEE) and with default parameters as per version 1.14.1, except for the "dada" step where all samples were pooled for ASV inference [24].…”