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

Abstract

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.

Figure 1. Frequency distribution of phylogenetic groups 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).

(A) Percent distribution of bacterial phylum by pyrosequencing analysis and (B) relative abundance of the six bacterial phylogenetic groups estimated by real-time PCR.

Figure 2. Frequency distribution of selected genera with high abundance in Gammaproteobacteria from pyrosequencing analysis.

PL15 denotes 15-day-old post-larva (PL15) whereas J1, J2 and J3 denote 1-, 2- and 3-month-old juveniles, respectively.

Figure 3. Comparison of the bacterial compositions in shrimp intestines of four growth stages: 15-day-old post-larva (PL15) and 1-, 2- and 3-month-old juveniles (J1, J2 and J3, respectively).

Pie charts of the hierarchical tree reflect relative abundance for each genus from each library (red represents PL15, blue represents J1, green represents J2 and yellow represents J3).

Figure 4. Top five most abundant bacterial genera in shrimp intestines of four growth stages: A) 15-day-old post-larva (PL15), B) 1-month-old juveniles (J1), C) 2-month-old juveniles (J2) and D) 3-month-old juveniles (J3).

Figure 5. PCR-DGGE analysis of the predominant bacterial population in intestines of black tiger shrimp from different growth stages: 15-day-old post-larva (PL15), 1-, 2- and 3-month-old juveniles (J1, J2 and J3, respectively) and S is in-house standard marker.

(A)Dendogram analysis of DGGE profile and (B) bacterial profiles and species similarity of selected DGGE bands.

Figure 6. 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).

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.