Microbiome Dynamics in a Shrimp Grow-out Pond with Possible Outbreak of Acute Hepatopancreatic Necrosis Disease

Abstract

Acute hepatopancreatic necrosis disease (AHPND) (formerly, early mortality syndrome) is a high-mortality-rate shrimp disease prevalent in shrimp farming areas. Although AHPND is known to be caused by pathogenic Vibrio parahaemolyticus hosting the plasmid-related PirAB^vp toxin gene, the effects of disturbances in microbiome have not yet been studied. We took 62 samples from a grow-out pond during an AHPND developing period from Days 23 to 37 after stocking white postlarvae shrimp and sequenced the 16S rRNA genes with Illumina sequencing technology. The microbiomes of pond seawater and shrimp stomachs underwent varied dynamic succession during the period. Despite copies of PirAB^vp, principal co-ordinates analysis revealed two distinctive stages of change in stomach microbiomes associated with AHPND. AHPND markedly changed the bacterial diversity in the stomachs; it decreased the Shannon index by 53.6% within approximately 7 days, shifted the microbiome with Vibrio and Candidatus Bacilloplasma as predominant populations, and altered the species-to-species connectivity and complexity of the interaction network. The AHPND-causing Vibrio species were predicted to develop a co-occurrence pattern with several resident and transit members within Candidatus Bacilloplasma and Cyanobacteria. This study's insights into microbiome dynamics during AHPND infection can be valuable for minimising this disease in shrimp farming ponds.

Figure 1

(A) Quantitative profiles of 16S rRNA genes of aquatic bacterial and archaeal populations and PirAB^vp gene (Toxin 1) in the shrimp cultivation pond. Histograms represent the relative abundances of Bacteria and Archaea in pond seawater samples at individual sampling points. (B) Numbers of stomach samples with (AHPND(+)) and without (AHPND(−)) PirAB^vp gene (Toxin 1) tested in this study. The samples with sufficient 16S rRNA gene amplicons obtained were then sequenced using the Miseq platform.

Figure 2

Distribution of Shannon diversity index values for samples of AHPND(−) shrimp, AHPND(+) shrimp, and pond seawater during shrimp cultivation.

Figure 3

Principal co-ordinates analysis of bacterial community structures of the samples collected from the pond seawater (Group PS), healthy shrimp stomach (Group C), and shrimp stomach affected by AHPND (Groups D1 and D2).

Figure 4

Distribution of sequence read abundance of core bacterial populations (sample occupancy > 85%) at the order level in each sample of pond seawater (Group PS), healthy shrimp stomach (Group C), and shrimp stomach affected by AHPND (Groups D1 and D2). The bacterial taxa and their clustering dendrograms are displayed on the right and left sides of the figure, respectively. Only the taxa with sequence read abundance > 4% in the samples are shown. Bubble size represents the relative abundance.

Figure 5

Relative abundance of abundant bacterial populations (82 OTUs) distributed in the samples of pond seawater (PS), healthy shrimp stomach (C), and AHPND-affected shrimp stomach (D). The results of Tukey multiple-comparison test of preferential existence on each OTU in the pond seawater (PS type), stomach (S type), and both environments (M type) are shown on the right side of the figure with different letters to indicate significantly different values (P < 0.05). The phylogeny of each OTU was coloured and illustrated on the left side of the figure.

Figure 6

Vibrio-related consensus interactions in the AHPND(−) and AHPND(+) stomach communities. The interaction strength was ranked among the top 1% of the entire network. The solid (or dashed) arrow represents the activation (or repression). The hub OTUs are underlined.