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By: Ann Chang Cheng, Shinn Pyng Yeh, Shao Yang Hu, Hsueh Li Lin, Chun Hung Liu*
The use of probiotics in shrimp farming is known to bring benefits such as improved nutrient absorption and feed conversion efficiency, promoting growth, immunity, and disease resistance; as well as the exclusion of undesirable bacteria in the digestive tract of animals. This study performed by researchers of the Aquaculture department of Pingtung National University of Science and Technology, Taiwan, and associated research institutions was conducted with the objective of profiling the microbiota structure in the intestines of the white shrimp, Litopenaeus vannamei, after being fed for 60 days with diets that present these alternative features and a control diet.
Shrimp aquaculture has been prompted due to increasing demands and decreasing fishery catches. The production of white shrimp, Litopenaeus vannamei, is the highest penaeid culture in the world. The shrimp aquaculture industry has been impacted by increases in feed costs, serious diseases and environmental deterioration.
Feed costs have the highest share in the total cost of shrimp aquaculture, and shrimp diseases have profound negative impacts on their production. The development of feed additives and novel feed ingredient substitutes to reduce feed costs and prevent or reduce disease is important.
In aquaculture, probiotics are known to have benefits, such as improving nutrient absorption and the feed conversion efficiency, thereby enhancing growth performance, immunity and disease resistance, and excluding undesirable forms of bacteria in the gut. Probiotics are also used to improve the nutritional value and utilization of plant ingredients through fermentation.
Fermented soybean meal (FSBM) contains biologically active peptides that exert physiological effects on microbes in the gut, including antimicrobial activity. In a previous study by the same authors, a biofunctional ingredient, Bacillus subtilis E20-FSBM, was shown to have antimicrobial peptides (AMPs) that inhibited two important shrimp pathogens: Vibrio alginolyticus and V. parahemolyticus.
In addition, white shrimp fed a diet containing B. subtilis E20-FSBM (FS-BMD) and a diet containing an AMP isolated from B. subtilis E20-FSBM (AMPD) had better disease resistance after an oral challenge with V. parahemolyticus, suggesting that FSBMDs or AMPDs might alter the composition of microorganisms in the intestine of shrimp and reduce the risk of disease.
Materials and methods
Preparation of the experimental diets
Bacillus subtilis E20-FSBM and AMP derived from B. subtilis E20-FSBM were prepared according to procedures described previously. Three experimental diets were used to feed shrimp in this study, including a control diet, and diets containing FSBM (called the FSBMD), and AMP (control diet containing AMP at 62.5 μg/g, designated the AMPD).
Diets were prepared based on the protein (37%) and lipid (7%) requirements of white shrimp.
Shrimp and the experimental design
Shrimp in the intermolt stage with a mean weight of 0.76 ± 0.02g were used for this study. After collection, shrimp were acclimatized in a cement tank (6 × 2 × 1 m) with aerated brackish water (25‰) and fed the control diet twice daily until the experiment was initiated.
Three groups were tested: shrimp fed the control diet, the FSBMD, and the AMPD. Each feeding group, comprised of triplicate with 20 shrimp per replicate, was reared in cement tanks containing 0.8 tons of 25‰ aerated brackish water. Shrimp were fed the experimental diets twice daily at a rate of 5% of their biomass.
Water temperature, dissolved oxygen and pH were maintained at 28 ± 1°C, >5 mg/L and 8.01–8.13 respectively. NH3-N and NO2-N were at optimal ranges of 0.01–0.11 mg/L and 0.01–0.06 mg/L, respectively. After the 60-day feeding period, shrimp were harvested and weighted, and then, nine shrimp from each group were selected for DNA isolation.
16S metagenomic sequencing and taxonomic classification
DNA of whole intestine of shrimp was isolated in triplicate using a FavorPrepTM Tissue Genomic DNA extraction Mini Kit. The V3-V4 region of the 16S rRNA gene was amplified using SureCycler 8,800 with bacterium-specific primers, including the forward primer (S17): 5´-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3´ and reverse primer (A21): 5´-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3´ in the preparation solution (Nextera XT Index Kit).
Each forward and reverse primer for V3-V4 amplification of samples was 5´-barcode-tagged with an 8-bp specific DNA sequence as shown in Table 1. Sizing of the amplified product was checked using a 4,200 TapeStation. After library construction, sequences of 2 × 300-bp paired-end reads were amplified.
The Illumina paired-end reads were aligned to long reference sequences using Bowtie 2. Sequences were filtered, and potential chimeric sequence filtering was performed using Mothur. The effective reads and corresponding clean reads were in a range of 95.12% ~ 97.7%.
In order to group sequences into operational taxonomic units (OTUs), the UPARSE algorithm was used, and sequences from each OTU that agreed were created. The Ribosomal Database Project (RDP) classifier was employed for classifying OTU sequences into taxa after an analysis of the bacterial community.
Taxonomic levels from phylum to genus were analyzed, and the clustering results were visualized by observing a principal component analysis (PCA) plot.
Abundances and biodiversity of gut microbiota
The alpha_diversity.py script in the website of QIIME (http://qiime.org/scrip ts/alpha_diver sity.html) was used to calculate genera, Pielou’s evenness (J), Shannon diversity index and Margalef ’s species richness.
Results
Growth performance
Growth of shrimp fed experimental diets for 60 days is shown in Table 2. Shrimp survival was 91.38%–96.22%. Final weight, percentage of weight gain and feed efficiency of shrimp fed FSBMD and AMPD were not significantly different from control.
Characteristics of the shrimp gut microbiome
Totals of 579,196, 555,204 and 503,798 non-redundant sequences were generated for the control, FSBMD and AMPD groups respectively.
Sequences in the control, FSBMD and AMPD treatments were clustered, respectively, into 1,443, 1,374 and 2,169 OTUs, at an identity cut-off of 97%.
The obtained OTUs were then assigned to eight phyla, 20 classes, 40 orders, 68 families and 96 genera with taxonomic names (Figure 1).
Similarities and differences in intestinal microbiota
Figure 2 shows similarities and differences in observed genera among the control and treatment groups. Shared OTUs (Figure 2a), FSBMD replicates (Figure 2b), AMPD replicates (Figure 2c), OTUs shared as core microbiota among the control and treatment groups (Figure 2d), taxonomic classifications of unique OTUs at the generic level can be seen.
Two representative genera in the control group were Lachnoclostridium and Maribacter, while Kiloniella, Bilophila, Enterococcus, Acidaminococcus, Rhizobium, Anderseniella, Tyzzerella, Incertae, Sedis, Pseudospirillum, Winogradskyella and Desulfovibrio were unique to both the FSBMD and AMPD groups (i.e. not present in the control group).
Microbiome structure and diversity
Feeding of AMPD or FSBMD had a strong influence on the bacterial profiles of the shrimp. According to PCA data, the intestinal microbiota of shrimp was dependent on dietary supplementation and clustered into three distinct groups.
Contributions of principal component 1 (PC1) and PC2 were 36.4% and 18.3%, respectively, and together, they explained 54.7% of the variation in the dataset.
“At the phylum level, the microbial taxonomic composition showed the Proteobacteria to have the highest abundance in all groups, but Proteobacteria abundance was lower (82.94%) in shrimp fed the AMPD.”
Relative abundances of the Bacteroidetes (1.21% in the control) and Actinobacteria (0.56% in the control) were slightly higher in the FSBMD group (1.64% and 3.36%, respectively) and the AMPD group (1.35% and 4.24%, respectively).
Across all of the data, the predominant genera (with the highest relative abundances) in shrimp intestines were Vibrio, Sphingomonas, Pseudoalteromonas, Shewanella, Shimia and Ruegeria. However, the relative abundances of the predominant genera varied among the various groups.
In shrimp fed the control diet and FSBMD, Vibrio, Sphingomonaas, Shewanella, Shimia and Mesorhizobium were predominant, whereas Vibrio, Pseudoalteromonas, Sphingomonas, Ruegeria, Marinicella, Pseudoruegeria and Planctomyces were predominant genera in the AMPD group.
The analysis by PCA eigenvector plots showed that shrimp in the control group had higher accumulation of Vibrio in the intestinal tract compared to that of shrimp in the FSBMD and AMPD groups.
Based on data of alpha diversity shown in Table 3, 96 genera of bacteria were detected in shrimp intestines. Shrimp fed the experimental FSBMD and AMPD diets both contained 84 genera. A slightly lower number of genera (78) were detected in the intestines of control shrimp.
Discussion
Fermentation is an effective method for processing shrimp food to prolong the duration of preservation and enhance the food’s flavor and nutritional value. Some health benefits of the FSBM diet to animals may be attributable to AMPs or other compounds that alter the animal’s gut microflora.
In our previous study, white shrimp fed the diets with maximal replacement levels of fish meal with SBM and B. subtilis E20-FSBM at 37.42% and 61.67% had no significant difference in growth performance as compared to fish meal basal diet, which explain the normal growth of shrimp fed the diet containing fish meal replaced by SBM or B. subtilis E20-FSBM at the levels of ~10% in this study.
“In general, white shrimp might get higher weight gain in outdoor culture system as compared to that of shrimp in this study because many natural food, like algae, aquatic insect, zooplankton and other organic matter, in outdoor culture system can support shrimp growth.”
However, shrimp in the indoor culture system reflect directly the effects of the experimental diet. In addition, an AMP from B. subtilis E20-FSBM was demonstrated to strongly inhibit the growth of the shrimp pathogens, V. alginolyticus and V. parahaemolyticus.
The AMP and B. subtilis E20-FSBM were individually incorporated into shrimp diets, and results showed that the shrimp’s resistance to V. parahaemolyticus significantly improved. The positive effects of the AMP and FSBM on the disease resistance of shrimp might be due to changes in the intestinal microbiota, and perhaps more specifically decreases in the abundances of potential pathogens, that is, Vibrio spp., as observed in this study.
Venn diagrams used for the analysis of differences in detected bacteria at the generic level showed that nine unique OTUs were recorded in the FSBMD group, whereas there were just two unique OTUs in both the control and the AMPD groups. Some genera, such as Gordonia and Rhodococcus were only found in the FSBMD and AMPD groups during this study.
“These genera are also known to be potential probiotics. Beneficial bacteria were more abundant in the FSBMD group. FSBM may be able to alter the structure of the intestinal microflora and support the colonization of potential probiotics.”
Vibriosis is one of the most important diseases in shrimp aquaculture and causes serious mortality. Vibrio spp. are widely distributed in marine environments worldwide and are typically among the most abundant flora in shrimp digestive systems.
Endemic pathogens, Photobacterium and Vibrio, are predominant during the post-larval and juvenile stage (89.1% ~ 94.2%).
Similarly, during this study, Vibrio spp. were predominant in shrimp intestines, accounting for 42.6% ~ 70% of the microbiota. In this study, shrimp in the FSBMD and AMPD groups had lower abundances of Vibrio in the intestines compared to that of shrimp in the control.
The reduction in the abundance of Vibrio was due to the AMP supplemented in the diet, in addition to the AMP originally in B. subtilis E20-FSBM. Flavobacterium, a potential fish pathogen, was also reduced in the intestines of shrimp fed the FSBMD or AMPD.
Changes in the intestinal microbiota of the intestines of shrimp receiving the FSBMD and AMPD were beneficial.
Conclusions
The B. subtilis E20-FSBM and an AMP derived from B. subtilis E20-FSBM enhanced the bacterial diversity, species richness and evenness in the shrimp gut. In addition, alterations in the intestinal microbiota of shrimp consuming the FSBMD and AMPD included an increase in colonization of beneficial bacteria and decreases in the potentially harmful pathogens, Vibrio and Flavobacterium. A decrease in potentially harmful pathogens can result a decrease in risk of disease.
This is a summarized version developed by Ph.D. Carlos Rangel Dávalos, researcher and professor at the University of Baja California Sur México.
The original article on which is based is titled: “Intestinal microbiota of white shrimp, Litopenaeus vannamei, fed diets containing Bacillus subtilis E20‐fermented soybean meal (FSBM) or an antimicrobial peptide derived from B. subtilis E20‐FSBM” by: Ann Chang Cheng, Shinn Pyng Yeh, Shao Yang Hu, Hsueh Li Lin y Chun Hung Liu.
The article was originally published on 2020 through the Aquaculture Research Journal of Wiley.
The full version of the article can be accessed at: https ://doi.org/10.1111/are.14345
