Characterization of intestinal bacteria in wild, domesticated adult black tiger shrimp - P3

Discussion

The influence of intestinal bacteria on their hosts has been mostly elucidated in vertebrate hosts, with profound effects on host genes involved in nutrient absorption, mucosal modification and immune response. Some intestinal bacteria have been linked to the health status of their hosts. Due to the influence of intestinal microbiota on animal fitness, characterization of gut microbiota would be essential for farm animals. Here, we characterized bacterial populations associated with the intestines of P. monodon, an economically important shrimp species in Asia. To identify a core bacterial population, intestinal bacterial communities were determined in P. monodon obtained from wild and domesticated environments using a high-throughput pyrosequencing approach in parallel with DGGE analysis.

In this study, pyrosequencing analysis revealed some OTUs shared among P. monodon intestines from wild and domesticated environments. Although these OTUs represent only a small percentage of the total intestinal bacterial population, the dominant OTUs in each shrimp were commonly found in all six shrimp libraries. The closest relatives were classified to six genera from Proteobacteria (Vibrio, Photobacterium, Pseudomonas, Sphingomonas, Novosphingobiumcommon and Undibacterium), two genera from Firmicutes (Fusibacter and Lactobacillus), and one genus from Bacteroidetes (Cloacibacterium). Among the shared bacterial genera in this study, Vibrio, Photobacterium, Pseudomonas, Sphingomonas and Novosphingobium were found to be highly prevalent in the intestines collected from P. monodon post-larva and juveniles in our previous study. In particular, Vibrio and Photobacterium are commonly associated with marine habitats and many marine organisms such as crustaceans, oyster, and fish. Although the bacteria in these genera have been characterized as free-living or commensal in marine animal digestive tracts, the consistent detection of Vibrio, Photobacterium, Pseudomonas, Sphingomonas and Novosphingobium genera in this work and previous studies could imply that they are an indigenous bacterial population in P. monodon intestines. Some have been reported as pathogens in aquaculture, such as Vibrio harveyi, an opportunistic pathogen that causes infection in shrimp under conditions of high nutrient concentrations and high animal density in rearing environments. Fusibacter were commonly found in shrimp from both groups. Thus far, bacteria isolates belonging to the genus Fusibacter have been found to be strictly anaerobic and halotolerant bacteria. Interestingly, Lactobacillus was also found to be associated with P. monodon intestines. Members of Lactobacillus are indeed employed as probiotics in many animals, including some aquatic organisms. For instance, the group of Litopenaeus vannamei (Pacific white shrimp) fed with Lactobacillus plantarum supplemented diet shows a higher survival rate under pathogen exposure than the control diet group. The detection of Lactobacillus in P. monodon intestines in this report as well as our previous work also suggests that the bacterium could withstand P. monodon gut and aquatic environments. Hence, application of Lactobacillus as probiotics is a promising mean to enhance disease resistance in the P. monodon farming. Despite diverse intestinal bacteria members associated in P. monodon adults, the dominant bacteria detected in intestines of WC and DB P. monodon were Proteobacteria, followed by Firmicutes and Bacteroidetes. This observation is consistent with previous reports on intestinal bacteria in P. monodon post-larvae and juveniles. The prevalence of these phyla in animal intestines has been reported in aquatic organisms such as Fenneropenaeus chinensis (Chinese shrimp), P. merguiensis (banana prawn), Nephrops norvegicus (Norway lobster) and Danio rerio (zebrafish).

The similarity of bacterial communities among the wild and domesticated shrimp suggested the establishment of specific bacteria by selective pressures from the environment within the host gut. For instance, a germ-free mice model study reveals that mucosal environment influences selection and establishment of bacteria in the gastrointestinal tract. The mucosal immune system in a host’s digestive tract has been demonstrated to play important roles on maintaining commensal bacteria. Although understanding of P. monodon gut physiology remains elusive, expression of P. monodon immune-related genes upon pathogenic bacteria challenge provides evidence of a shrimp gut immune system. Furthermore, the observation of shared bacterial members is congruent with previous comparative studies of other wild and domesticated model animals. For instance, comparison of bacteria in intestines of D. Rerio (zebrafish) from wild and lab-reared environments showed similar bacterial members. Analyses of bacterial communities in wild and domesticated Mus musculus (mice), Drosophila melanogaster (fruit flies) or Hydra species (Hydra) reveal shared bacterial members regardless of individual variation, and similar bacteria compositions were observed at the phylum or class level in both wild and domesticated hosts. Finally, the similarities of intestinal bacteria in P. monodon with different life histories also imply that there has been long-term co-evolution between intestinal bacteria and host shrimp.

Although shared OTUs were observed among intestines of the six adult shrimps, there were still a vast number of unique OTUs, which reflect the high degree of bacterial variation among individual P. monodon, especially in wild-caught shrimp. In this study, bacterial profiles from the DGGE analysis were congruent with pyrosequencing results in that there was higher individual variation in intestinal bacterial populations among the wild-caught than the domesticated shrimp. As intestinal bacteria can be influenced by bacteria present in the surrounding aquatic habitats. Domesticated shrimp reared in the same water tank would therefore experience less fluctuation in bacterial composition in the surrounding water compared to wild habitats. Transiently persistent bacteria and those associated with ingested food could also inflate the observed individual bacterial variation. As wild P. monodon fed on smaller live animals, unlike domesticated shrimp fed mainly with dried feed pellets, the small live animals ingested by wild P. monodon might influence the number of intestinal bacteria genera, resulting in higher numbers of genera detected in the wild-caught P. monodon than the domesticated group. To further determine the effect of diets on intestinal bacteria, the characterization of bacterial compositions in P. monodon intestines is underway to compare differences between P. monodon feeding on live food (e.g. polychaete worms) to those fed with commercial feed pellets.

Most intestinal bacterial population analyses in penaeid shrimp have been characterized by both culture-dependent and culture-independent techniques based on Sanger sequencing of 16S rRNA genes and a recent study has applied the high-throughput 454 pyrosequencing platform to the analysis of intestinal bacterial diversity in P. monodon. The introduction of pyrosequencing technology has enabled a greater depth to bacterial population analysis in various environments; for instance, bacterial richness in the deep sea environment has been reported to be one or two orders of magnitude higher than those obtained under the traditional Sanger based approaches. Despite its advantages, even pyrosequencing may not uncover the entire range of bacterial diversity. Although the reading length obtained from pyrosequencing technology has been continuously improving, the full length of 16S rRNA (~1.5 kb) has not yet been achieved with current 454 pyrosequencing capability. Primer selection and their target regions influence bacterial diversity analyses and indeed the detection of phyla Verrucomicrobia, Planctomycetes and Chlamydiae is highly variable with 16S rRNA primer sets. We chose primers target variable regions 3 and 4 of the 16S rRNA gene, which may not be effective in detecting the presence Verrucomicrobia and Planctomycetes. Nonetheless, these bacteria phyla were not found in high abundance in P. monodon intestines in a previous study using full-length 16S rRNA clone library approach. While pyrosequencing analysis provided more comprehensive view of bacterial diversity in a community, the DGGE analysis in parallel provided an overview of structures of dominant bacterial groups, which can be used for comparison of bacterial patterns.

Our work reports intestinal bacterial composition in wild-caught and domesticated P. monodon adults by using the 454-pyrosequencing approach and bacterial patterns were also compared by DGGE analysis. We provided evidence of common intestinal bacteria in P. monodon intestines, and suggest that the intestine may be viewed as a selective environment that allows only certain bacterial taxa to persist. Characterization of the bacterial composition in P. monodon intestines is fundamental to understanding of the beneficial effects of bacteria and the importance of host-microbe interaction in a non-model organism.

Conclusion

Recent studies have reported the importance of balanced gut microbiota to the health of animal hosts. Hence, the main purposes of this study were to utilize a culture-independent high-throughput pyrosequencing technique to extend our knowledge concerning the breadth of bacterial diversity in intestines of adult P. monodon, and to determine the composition of bacterial communities in P. monodon from the wild and those under domestication. The bacterial profiles showed similar dominant genera in wild-caught and domesticated shrimp, suggesting the occurrence of a resident bacterial population in P. monodon. We identified shared bacterial members associated with adult shrimp intestines from wild and domesticated environments. This work provides evidence that host intestinal conditions exert stronger selective pressure for bacterial community establishment in P. monodon than rearing environments.