Bacterial population in intestines of the black tiger shrimp (Penaeus monodon) under different growth stages.

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities

Rarefaction analysis of shrimp intestines.

<p>Operational taxonomic units (OTUs) were clustered based on 97% sequence similarity for the (A) wild-caught group (WC1, WC2, and WC3) and (B) the domesticated group (DB1, DB2, and DB3). The number of sequences sampled represents the number of pyrosequencing reads.</p

<i>Penaeus monodon</i> from wild and domesticated habitats (A).

<p>Wild-caught <i>P. monodon</i> adults (WC) were captured from the Andaman Sea, whereas 10-month-old domesticated adults (DB) were reared at the Shrimp Genetic Improvement Center, Surat Thani, Thailand (B). The map was adapted from <a href="http://www.mapsofworld.com/thailand/thailand-political-map.html" target="_blank">http://www.mapsofworld.com/thailand/thailand-political-map.html</a>.</p

Characterization of Intestinal Bacteria in Wild and Domesticated Adult Black Tiger Shrimp (<i>Penaeus monodon</i>)

<div><p>The black tiger shrimp (<i>Penaeus monodon</i>) is a marine crustacean of economic importance in the world market. To ensure sustainability of the shrimp industry, production capacity and disease outbreak prevention must be improved. Understanding healthy microbial balance inside the shrimp intestine can provide an initial step toward better farming practice and probiotic applications. In this study, we employed a barcode pyrosequencing analysis of V3-4 regions of 16S rRNA genes to examine intestinal bacteria communities in wild-caught and domesticated <i>P. monodon</i> broodstock. Shrimp faeces were removed from intestines prior to further analysis in attempt to identify mucosal bacterial population. Five phyla, <i>Actinobacteria</i>, <i>Fusobacteria</i>, <i>Proteobacteria</i>, <i>Firmicutes</i> and <i>Bacteroidetes,</i> were found in all shrimp from both wild and domesticated environments. The operational taxonomic unit (OTU) was assigned at 97% sequence identity, and our pyrosequencing results identified 18 OTUs commonly found in both groups. Sequences of the shared OTUs were similar to bacteria in three phyla, namely <i>i) Proteobacteria</i> (<i>Vibrio</i>, <i>Photobacterium</i>, <i>Novosphingobium</i>, <i>Pseudomonas</i>, <i>Sphingomonas</i> and <i>Undibacterium</i>), <i>ii) Firmicutes</i> (<i>Fusibacter</i>), and <i>iii) Bacteroidetes</i> (<i>Cloacibacterium</i>). The shared bacterial members in <i>P. monodon</i> from two different habitats provide evidence that the internal environments within the host shrimp also exerts selective pressure on bacterial members. Intestinal bacterial profiles were compared using denaturing gradient gel electrophoresis (DGGE). The sequences from DGGE bands were similar to those of <i>Vibrio</i> and <i>Photobacterium</i> in all shrimp, consistent with pyrosequencing results. This work provides the first comprehensive report on bacterial populations in the intestine of adult black tiger shrimp and reveals some similar bacterial members between the intestine of wild-caught and domesticated shrimp.</p></div

Summary of pyrosequencing read analysis, bacterial diversity richness (OTUs), sample coverage (Good’s coverage), diversity index (Shannon) and estimated OTU richness (Chao1) for intestinal bacterial diversity analysis of wild-caught (WC, n = 3) and domesticated (DB, n = 3) <i>P. monodon</i>.

<p>Summary of pyrosequencing read analysis, bacterial diversity richness (OTUs), sample coverage (Good’s coverage), diversity index (Shannon) and estimated OTU richness (Chao1) for intestinal bacterial diversity analysis of wild-caught (WC, n = 3) and domesticated (DB, n = 3) <i>P. monodon</i>.</p

Denaturing gradient gel electrophoresis (DGGE) analysis of intestinal bacterial communities in <i>Penaeus monodon</i>.

<p>The bacteria profiles were from intestines of wild-caught shrimp (WC1, WC2, and WC3) and domesticated shrimp (DB1, DB2, and DB3). The selected DGGE bands were sequence to identify bacteria (A) and DGGE profiles were clustered using UPMGA based on Pearson correlation (B).</p

Shared OTU analysis of pyrosequencing reads from <i>Penaeus monodon</i> intestines.

<p>The Venn diagram represents the unique and shared OTUs at 97% similarity level in libraries of (A) wild-caught group (WC1, WC2, and WC3) and (B) the domesticated group (DB1, DB2 and DB3). The shared reads were classified based on BLAST (C) with the following criteria; similarity of ≥97% were classified to the nearest type strain, ≥90% to the nearest genus, and ≤90% to the nearest phylum.</p

Frequency distribution of bacterial phyla in <i>Penaeus monodon</i> intestines from pyrosequencing libraries of the wild-caught group (WC1, WC2 and WC3), and the domesticated group (DB1, DB2 and DB3).

<p>Frequency distribution of bacterial phyla in <i>Penaeus monodon</i> intestines from pyrosequencing libraries of the wild-caught group (WC1, WC2 and WC3), and the domesticated group (DB1, DB2 and DB3).</p

Principal coordinate analysis (PCoA) based on weighted-UniFrac analysis of bacterial populations associated with <i>P. monodon</i> intestines from the wild-caught group (WC1, WC2, and WC3) and the domesticated group (DB1, DB2 and DB3).

<p>Principal coordinate analysis (PCoA) based on weighted-UniFrac analysis of bacterial populations associated with <i>P. monodon</i> intestines from the wild-caught group (WC1, WC2, and WC3) and the domesticated group (DB1, DB2 and DB3).</p

Principal component analysis of bacterial populations in intestines of different growth stages of the black tiger shrimp: 15-day-old post-larva (PL15) and 1-, 2- and 3-month-old juveniles (J1, J2 and J3, respectively).

<p>The principal component analysis (PCA) was performed using R script to compare bacterial community structures among the samples based on relative abundance of various bacteria genera.</p