Characterization of intestinal bacteria in wild, domesticated adult black tiger shrimp - P1

The black tiger shrimp 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. This work by Wanilada Rungrassame et al, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand, 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.

Introduction

Characterization of microbiota in animal intestines has been central in advancing understanding of the relationship between host and microorganism. Microbial communities in humans and mice are estimated to comprise >1,000 taxa. While some microbes can be pathogenic to their hosts, other microbial symbionts are beneficial to the development and physiology of their host, playing roles in nutrient absorption, immune response, and epithelial development. Maintaining a balance in the population of intestinal bacteria is crucial to the health of the host. In turn, several host factors, such as diet, developmental stage and physiological condition have been identified to affect intestinal bacterial composition. The host, by its own gut immune system and gut environment (e.g. pH and bile acids), also shapes the composition of intestinal bacteria to maintain a relatively stable level of diversity.

In addition to understanding the host-microbiota relationship, characterization of intestinal bacteria will help identify bacteria with potential to become probiotics, which are increasingly used as an alternative means for preventive medicine in humans and animals. In aquatic animal farming, the concept of replacing antibiotic use with probiotics will help prevent environmental contamination and adverse health effects for consumers. Indeed, several studies have developed probiotic applications for aquaculture. This is no exception for Penaeus monodon (black tiger shrimp), which has been a commercially important marine crustacean for the past few decades in many Asian countries and Australia. With increasingly high shrimp consumption, the decline of wild harvests has forced domestication to become the major source of shrimp production. However, the farming industry has been deteriorating due to several factors, in particular the outbreak of disease.

As alternative means to address the disease outbreak problem, disease control using probiotics or maintaining healthy intestinal bacterial balance requires comprehensive understanding of bacterial diversity in the intestines of commonly farmed aquatic animals. Although there have been several studies on bacterial diversity in commonly cultured aquatic species animals from the wild such as P. merguiensis (banana shrimp), Salmo salar L (Atlantic salmon), Gadus morhua L (Atlantic cod), and Danio rerio (zebrafish), there are limited findings on the intestinal bacteria of P. monodon. It was only recently that studies of bacterial diversities in the intestines of P. monodon post-larval and juvenile stages were reported, yet there has been no report on the intestinal bacteria of wild-caught P. monodon, which feed on slow-moving benthic animals such as small crabs, shrimp, mollusks, and polychaetes. In contrast, domesticated shrimp in rearing facilities are fed with commercial feed pellets and reared in monoculture with higher stocking density. Comparing the differences in intestinal bacteria of wild-caught and domesticated shrimp can unveil changes in intestinal bacteria as shaped by aquaculture. These changes have implications for shrimp health and consequently, application of this knowledge may improve survival rates of P. monodon under domestication.

In this study, we examined bacterial populations associated with the intestines of wild-caught and domesticated adult black tiger shrimp using high-throughput next-generation pyrosequencing analysis in parallel with denaturing gradient gel electrophoresis (DGGE). Similarities and differences in intestinal bacteria between wild and domesticated shrimp provide initial evidence for a core bacterial community as well as probiotic candidates for P. monodon.

Results

Overview of 16S rRNA Pyrosequencing Analysis

Barcode pyrosequencing of 16S rRNA sequences was employed to determine bacterial populations in intestines of six individual adult shrimp from wild (WC, n = 3) and domesticated (DB, n = 3) habitats (Fig. 1). A total of 61,499 reads were obtained from pyrosequencing of the V3-4 regions of 16S rRNA genes (Table 1). The barcodes assigned the sequences to distinct libraries: WC1 (5,029 sequences), WC2 (9,969 reads), WC3 (11,532 reads), DB1 (2,961 reads), DB2 (3,190 reads) and DB3 (1,009 reads). Sequences were clustered into operational taxonomic units (OTUs) at 0.03 dissimilarity levels, in which each OTU represented a unique phylotype. The total numbers of OTUs ranged from 138 to 806, where the WC group exhibited higher variation in the number of OTUs. The OTUs were further classified to genus level using the RDP II Classifier. All six bacterial communities contained five phyla: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria. DB1 from the DB group had the lowest number of bacteria genera (21, 43 and 28 genera for DB1, DB2 and DB3, respectively), whereas the highest number of bacteria genera was detected in WC2 from the WC group (39, 89, and 52 genera for WC1, WC2 and WC3, respectively).

Table 1. 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) P. monodon.

To estimate and compare bacterial diversity in each individual shrimp, bacterial diversity indices were calculated from OTUs of each library. Good’s coverage indices, used to estimate the percentage of total bacterial OTUs represented in a sample, were in the range of 0.90–0.97, suggesting the 16S rRNA results from each library represented the majority of bacteria in the shrimp intestines. Bacterial diversity estimated by the Shannon index varied from 2.45 to 3.21 in the WC group, and 2.61–3.58 in the DB group, suggesting a similar range of diversity between the two environments. Chao1 analysis for bacterial richness estimated a range of 597 to 2,454 phylotypes, higher than the actual observed OTUs in each library, indicating that the true bacterial richness in P. monodon intestines was underestimated. Additionally, rarefraction analysis was carried out to determine whether OTUs from each library had been adequately obtained from pyrosequencing analysis (Fig. 2). Consistent with the Chao1 analysis, rarefraction curves for the WC (Fig. 2A) and DB groups (Fig. 2B) did not plateau, suggesting that bacterial richness of adult P. monodon intestines were not yet determined. Additional sequence sampling will still be required to capture the true intestinal bacterial diversity of adult P. monodon. Even with the help from the next-generation pyrosequencing technique, a microbial ecology study to reveal a complete picture of bacterial communities is still very challenging. For instance, in soil bacteria community analyses, rarefraction curves have not been saturated, even when a range of 5,000 to 10,000 pyrosequencing reads were obtained.

Operational taxonomic units (OTUs) were clustered based on 97 per cent 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.