The effect of Lactobacillus plantarum administration on the intestinal microbiota of whiteleg shrimp Penaeus vannamei

Highlights

• Four different preparation of L. plantarum groups increased the abundance of Acidobacteria.

• The potential beneficial bacteria were enriched in the cell-free extract of L. plantarum group.

• The composition and diversity of shrimp intestinal microbiota varied in different preparations of L. plantarum

Abstract

Along with the intensification of culture systems to meet the increasing global demands, there is an elevated risk of disease outbreak and substantial loss for shrimp farmers. Probiotics, e.g., Lactobacillus plantarum, are among the most promising feed supplements for controlling or treating microbial diseases in shrimp aquaculture. The maintenance of live and viable probiotic bacterial culture during storage is, however, quite challenging. This study investigated the paraprobiotic potential of L. plantarum on whiteleg shrimp (Penaeus vannamei), specifically on the intestinal microbiota. Four different experimental diet preparations of L. plantarum (fermentation supernatant (FS), live bacteria (LB), dead bacteria (DB), and cell-free extract (CE)) were used in the 15-day feeding trial. Subsequently, 16S rDNA gene sequencing technology was applied to analyze and characterize the whiteleg shrimp intestinal microbiota. Results showed that Proteobacteria was the most prevalent phylum in whiteleg shrimp gut microbiota, independent of the diet. Other phyla, including Bacteroidetes, Verrucomicrobia, Tenericutes, and Euryarchaeota were also recorded and independent of the diet. The microbiota of the whiteleg shrimp fed with the L. plantarum CE diet showed the highest abundance of Acidobacteria, with a 70-fold increase compared to that of the animals on the control diet. The abundance of Verrucomicrobia was the highest in the whiteleg shrimp fed with L. plantarum DB diet. Moreover, the abundance of Cyanobacteria, Crenarcheota, and Euryarchaeota in the microbiota of animals fed with L. plantarum FS diet were more than 10-fold higher, compared to that of the whiteleg shrimp fed with the control diet. An increased number of the potential beneficial bacteria from the genera Demequina, Rubritalea, Tenacibaculum, Marinicella and Phaeobacter versus a significantly decreased amount of the potential pathogenic genus Synechococcus were encountered in the microbiota of the CE diet-fed animals. There were 382 operational taxonomic units (OTUs) shared among all gut samples. The microbiota of the shrimps on the CE diet had the most unique OTUs, followed by those included in the FS and DB diet groups. These results demonstrated that P. vannamei fed with L. plantarum CE and FS diets had a significantly altered gastrointestinal microbiota. In conclusion, L. plantarum CE and FS diets have the potential to be used as paraprobiotics, due to the capacity to positively modulate the gastrointestinal microbiota in P. vannamei.

Introduction

Whiteleg shrimp, Penaeus vannamei, is one of the most important farmed shrimp species in the world (FAO, 2018). Due to an increase in global demand, the pressure for intensification and expansion of shrimp aquaculture systems has rendered most aquaculture business fragile. At intensive and land-based high-density shrimp culture, the degraded aquatic environment becomes stressful and ideal for disease outbreaks leading to the devastating economic impacts (Kalaimani et al., 2013). In the past decade, the bacterial disease associated with production loss in shrimp aquaculture has become increasingly important. Particularly in Asia and South America, acute hepatopancreatic necrosis disease (AHPND), has resulted in collective losses exceeding an estimated US $43 billion (Kumar et al., 2018; Roy et al., 2019). The conventional approach so far applied in the mitigation or cure of bacterial diseases, such as antibiotics and disinfectants, had limited success and is more often associated with the development of multiple antibiotic resistance phenotypes (Shah et al., 2014). Additionally, the use of antimicrobial agents can create an imbalance in the intestinal microbiota. The latter may lead to a negatively impacted immune response and growth performance of the hosts (Shah et al., 2014; Gao et al., 2017; Yang et al., 2017). Therefore, an alternative approach that focuses on the prevention of aquatic animal diseases will have the highest chance of decreasing the risk of pathogenic bacterial infection in shrimp aquaculture.

The use of probiotics has become an alternative in the aquaculture industry and is considered as a new tool for disease control and water quality improvement (Chauhan and Singh, 2019; Ringø, 2020). Among several probiotic bacterial species, numerous reports have been published on the beneficial role of Lactobacillus plantarum and its use as probiotics in aquaculture (Piccolo et al., 2014; Wang et al., 2016; Van Nguyen et al., 2019). Lactobacillus plantarum is a Gram-positive, rod-shaped, catalase-negative, non-spore forming facultative anaerobic bacterium (Son et al., 2009; Dash et al., 2014, Dash et al., 2015). Previous work from our laboratory indicated that four different treatments of L. plantarum (live bacteria (LB), dead bacteria (DB), cell-free extract (CE) and fermentation supernatant (FS)) improved the growth performance of whiteleg shrimp (Zheng et al., 2018). Another study showed that L. plantarum cell-free extract diet enhanced the ability of whiteleg shrimp to overcome acute low salinity (from 30 to 32 ppt dropped to 5 ppt within 10 s) (Zheng et al., 2017). There is, however, no information regarding the beneficial effect that L. plantarum might have on the microbiota in P. vannamei.

The micro-ecosystem of gut microbiota is a symbol of the health status and therefore, it should be highlighted when studying the physiology and nutrition of shrimp (Suo et al., 2017). Balancing the intestinal microbiota would confer important health benefits, for instance, stimulating the host digestive function (Duan et al., 2019), inhibiting the colonization of pathogens in the gastrointestinal tract (Buffie and Pamer, 2013), and improving the integrity of the intestinal epithelium (Perales-Puchalt et al., 2018). The most effective strategy to maintain the equilibrium of the intestinal microbiota is via the administration of probiotics (De Almada et al., 2016). Live probiotics are most commonly used in shrimp aquaculture, however, the need to maintain the viable probiotic bacterial culture in the substrates is often considered as a major challenge. Paraprobiotics are non-viable bacterial products or metabolic byproducts from probiotic microorganisms (De Almada et al., 2016). Concerning probiotics, the application of paraprobiotics in aquaculture can offer certain advantages, such as (i) less or no interaction with other components in the aquatic environment; (ii) greater processing ease as paraprobiotics can be added before thermal processes; (iii) storage and transport simplicity (Chuang et al., 2007; Ishikawa et al., 2010). Hence, in this study, the aim is to determine the effect of different L. plantarum preparations as potential paraprobiotics (non-viable probiotics) in the intestinal microbiota of whiteleg shrimp (P. vannamei) using 16S rDNA gene sequencing. This is the first study in its kind to evaluate whether L. plantarum preparations positively modulates the gastrointestinal microbiota in whiteleg shrimp.