Structure and co-occurrence patterns of bacterial communities associated with white faeces disease outbreaks in Pacific white-leg shrimp Penaeus vannamei aquaculture

Abstract

Bacterial diseases cause production failures in shrimp aquacultures. To understand environmental conditions and bacterial community dynamics contributing to white faeces disease (WFD) events, we analysed water quality and compared bacterial communities in water as well as in intestines and faeces of healthy and diseased shrimps, respectively, via 16S rRNA gene sequencing and qPCR of transmembrane regulatory protein (toxR), thermolabile haemolysin (tlh), and thermostable direct haemolysin genes of pathogenic Vibrio parahaemolyticus as a proxy for virulence. WFD occurred when pH decreased to 7.71-7.84, and Alteromonas, Pseudoalteromonas and Vibrio dominated the aquatic bacterial communities. The disease severity further correlated with increased proportions of Alteromonas, Photobacterium, Pseudoalteromonas and Vibrio in shrimp faeces. These opportunistic pathogenic bacteria constituted up to 60% and 80% of the sequences in samples from the early and advances stages of the disease outbreak, respectively, and exhibited a high degree of co-occurrence. Furthermore, toxR and tlh were detected in water at the disease event only. Notably, bacterial community resilience in water occurred when pH was adjusted to 8. Then WFD ceased without a mortality event. In conclusion, pH was a reliable indicator of the WFD outbreak risk. Dissolved oxygen and compositions of water and intestinal bacteria may also serve as indicators for better prevention of WFD events.

Figure 1

Principal component analysis (PCA) of observed environmental parameters. Point labels indicate the pond without white faeces disease (non-WFD pond P1) and ponds with disease events (P2, P3, and P4). Water parameters presented in the PCA were taken at day 60, 53, 63, and 67 for P1, P2, P3, and P4, respectively. SPM: suspended particulate matter; suc(+) TPPV: sucrose-fermenting colonies of total presumptive pathogenic Vibrio (yellow colonies of presumptive pathogenic Vibrio); suc(−) TPPV: non sucrose-fermenting colonies of presumptive pathogenic Vibrio (green colonies); DO dissolved oxygen; Chl a chlorophyll a concentration; NO₂^-: nitrite; NO₃⁻: nitrate; NH₄⁺: ammonium; r-Silicate: reactive silicate; PO₄³⁻: phosphate.

Figure 2

Contribution of the most dominant bacterial genera in pond water communities (A) and shrimp intestines and faeces (B). Samples were collected from a pond with healthy shrimps (P1) and ponds with diseased shrimps (P2, P3, and P4). Intestine (P1) for intestinal bacteria (IB) and faecal strings (P2, P3, P4) for faecal string bacteria (FSB) were sampled at rearing day 60, 53, 63, and 67 for P1, P2, P3, and P4, respectively. FL free-living fraction, PA particle-associated fraction, bWFD before WFD event, WFD during WFD event, aWFD after WFD event.

Figure 3

Bray–Curtis dissimilarity values of WB in the free-living (A) and particle-associated (B) fractions compared to the intestinal (IB) or faecal string bacteria (FSB) for samples from P1 and P2-P4, respectively. bWFD: before WFD event, WFD: during WFD event, aWFD: after WFD event. P1: pond with healthy shrimps; P2, P3 and P4: ponds with diseased shrimps.

Figure 4

Bacterial co-occurrence network generated by SPIEC-EASI. Node size corresponds to the average sequence proportion of operational taxonomic units in intestinal and faecal samples. Network modules detected by Louvain clustering are shown in different colours, grouped by the samples they predominantly occurred in: healthy, diseased, both (general). Network modules identified as characteristic for healthy and diseased shrimps by random forest models are indicated by (−) and (+), respectively. Edge width corresponds to the strength of the association between OTUs.

Figure 5

Sequence proportion of the members of the most dominant and most distinguishing network modules between healthy (−) and diseased shrimps (+), as well as their contribution to the particle-associated fraction from the respective ponds and sampling times. Their taxonomic affiliation is provided on genus level. Water samples were not used for the network construction.