Abstract
Bacillus aquimaris SH6 spores produce carotenoids that are beneficial to white-leg shrimp (Litopenaeus vannamei) health. However, the optimal dose and mechanisms behind these effects are not well understood. We investigated the fate of SH6 spores in the gut of L. vannamei. Shrimp were divided into six groups administrated with either feed only (negative control) or SH6 spores at 5 × 106 CFU/g pellet (high dose, SH6 spore-H group), 1 × 106 CFU/g pellet (medium dose, SH6 spore-M group), 2 × 105 CFU/g pellet (low dose, SH6 spore-L group), astaxanthin at 0.5 mg/g pellet (Carophyll group), or carotenoids from SH6 vegetative cells at 5 μg/g pellet (SH6 carotenoid group). The growth rate was highest in SH6 spore-H (3.38%/day), followed by SH6 spore-M (2.84%/day) and SH6 spore-L (2.25%/day), which was significantly higher than the control (1.45%/day), Carophyll (1.53%/day) or SH6 carotenoid (1.57%/day) groups. The astaxanthin levels (1.9–2.0 μg/g shrimp) and red-colour scores (21–22) in SH6 spore-H/M were higher than the control (astaxanthin: 1.2 μg/g shrimp; red score: 20) or SH6 spore-L, but lower than the Carophyll and SH6 carotenoids. Feeding with medium and high doses of SH6 spores after 28 days resulted in respective 1.3-2-fold increases in phenol oxidase activity and 8–9 fold increases in Rho mRNA expression compared to the control and low dose group. The live-counts of SH6 in the gut gradually increased during the 28-day feeding period with SH6 spores at different concentrations, starting from 4.1, 8.2, and 5.4 × 104 CFU/g gut at day 1 and reaching 5.3, 5.1, and 4.4 × 105 CFU/g gut in the SH6-H/M/L groups, respectively, at day 28. Gut microbiota became more diversified, resulting in a 2-8-fold increase in total bacterial live-counts compared to the controls. SH6 spore germination was detected by measuring the mRNA expression of a specific sequence coding for SH6 amylase at 4 h, reaching saturation at 24 h. Our results confirm that SH6 spores colonize and germinate in the gut to improve the microbial diversity and boost the immune system of shrimp, exhibiting beneficial effects at >1 × 106 CFU/g pellet.
Highlights
For sustainable shrimp aquaculture, the most useful and safe method consists of adjusting the diet of shrimp by feeding with probiotic-supplemented food [1,2,3,4]
Based on the weight at day 0 and day 28, we calculated the growth rate (GR) of shrimp administrated with SH6 spores and other groups as described in the Methods
To determine whether the increase in SH6 colonies in the shrimp gut is effective in improving the live counts and diversity of the gut bacteria, we identified the colonies at the species level and counted other non-pigmented bacteria colonies in the shrimp gut of the SH6 spore H/M/L groups during feeding time
Summary
The most useful and safe method consists of adjusting the diet of shrimp by feeding with probiotic-supplemented food [1,2,3,4]. Probiotic supplements can improve the production yield in shrimp aquaculture by adjusting and strengthening the microbiota in the gut. They promote bio-competition by creating adverse conditions for disease causing microbes through the modulation of gut microbiota, as well as adjusting the immune response of the host [5,6,7,8,9]. The first and second factors, Rho and Ran, belong to the GTPase protein group and are known to be involved in shrimp phagocytosis [10], where Rho is involved in superoxide formation and opsonisation, two indispensable mechanisms of phagocytosis in shrimp [11,12]. The third and fourth factors, PO and SOD activity, represent the level of immune response in shrimp. Conversion of pro-phenol oxidase (Pro-PO) to active form phenol oxidase (PO) is highly correlated with the formation of melanin as well as the innate immune system [14]
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