Application of indigenous Rhodobacteraceae probiotic strains and microbiota characterization in the rearing of greater amberjack (Seriola dumerili) larvae
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Date
2025-07-08
Authors
Παραλίκα, Βασιλική
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Abstract
This study aimed to isolate putative indigenous antagonistic bacteria from the greater amberjack (Seriola dumerili) rearing system—including larvae, live feed, rearing water, and tank wall biofilm—and evaluate their impact on the microbial assemblage within the rearing environment (larvae, water, and biofilm). Two main experiments were carried out at a commercial marine hatchery.
In the first experiment, four 3.000 L cylindroconical tanks were each stocked with approximately 150.000 greater amberjack larvae. From 3 to 17 days after hatching (dah), larvae in two of the tanks received a combination of copepod (Acartia tonsa) nauplii and rotifers (Brachionus sp.), referred to as the "Copepods group." The remaining two tanks, the Control group, were fed rotifers only during the same period. All tanks followed a common feeding protocol that included rotifers (3–27 dah), Artemia nauplii (10–23 dah), enriched Artemia metanauplii (17–the end of the trial), and a formulated diet (23–the end of the trial).
Larval samples were collected at 9, 16, 23, and 31 DAH for the isolation and identification of antagonistic bacterial strains. Additional samples were taken from the live feed, rearing water, and tank wall biofilm. Bacterial strains were isolated primarily from greater amberjack larvae and Artemia and identified via 16S rRNA gene sequencing. These strains belonged to Vibrionaceae, Pseudoalteromonadaceae, Alteromonadaceae, and Rhodobacteraceae. All isolates were screened in vitro for antagonistic activity based on their ability to inhibit the growth of marine fish pathogenic bacteria (three Vibrio strains and one Aeromonas strain). Several Vibrionaceae isolates demonstrated inhibitory effects, suggesting intra-genus competition. Additionally, strains from the Rhodobacteraceae family—Phaeobacter sp., Phaeobacter gallaeciensis JL 2886, and Ruegeria sp. InS-296—isolated from greater amberjack larvae, also exhibited antagonistic activity. This inhibition is attributed to the production of tropodithietic acid (TDA), a well-known antimicrobial secondary metabolite with physiological role which inhibits Vibrio growth. Also, in an in vivo preliminary test the three indigenous probiotic strains improved the hatching rate of Pagrus major eggs and enhanced the survival of newly hatched larvae, indicating their non-pathogenic nature. These findings highlight the larval microbiota of greater amberjack as a promising source of putative probiotic candidates.
The host-associated microbiota of these larvae—whether fed with copepods in addition to conventional live feed or not—was characterized and found to be more strongly influenced by age rather than diet. Rhodobacteraceae was the dominant bacterial family across all larval samples, followed by Vibrionaceae. In the control group, the relative abundance of Vibrionaceae increased over time, while Rhodobacteraceae declined. In contrast, larvae fed with copepods showed an age-related increase in Rhodobacteraceae, reaching 69% at 31 days after hatching, while Vibrionaceae remained consistently low. These findings suggest that copepod supplementation may positively influence the composition of the larval microbiota.
The second greater amberjack larval rearing experiment was conducted under the same conditions and rearing parameters as the first. In this experiment, the indigenous putative probiotic bacteria (Phaeobacter sp., Phaeobacter gallaeciensis JL 2886, and Ruegeria sp. InS-296) were immobilized on ceramic cylinders and used as biofilters in the water of two rearing tanks—"Probiotics group”. Two additional tanks, which did not receive probiotics, served as the "Control group." Both groups followed the same feeding protocol, which included rotifers, Artemia, and dry feed. The impact of probiotics on the microbial assemblage of the greater amberjack rearing system (larvae, tank water, and biofilm) was assessed. Sample type and time were the main factors shaping microbial communities, with probiotics having a limited effect on microbial richness. Probiotics significantly influenced the larvae's microbiota only at 23 dah, while both probiotics and time had a significant effect on the microbiota of all sample types. Seven core bacterial families were shared across all sample types: Vibrionaceae, Rhodobacteraceae, Pseudoalteromonadaceae, Nitrincolaceae, Alteromonadaceae, Saprospiraceae, and Flavobacteraceae, though their relative abundances varied by sample. Key unique taxa included NS10 marine group (in water), Saprospiraceae (in biofilm), and Cutibacterium (in larvae). Vibrionaceae increased in water as larvae began feeding on live feed. Dominant families were Vibrionaceae in larvae, Rhodobacteraceae in water, and both in biofilm. Ruegeria, notably, became more abundant in larvae from 23 dah.
Overall, while indigenous probiotics influence microbial dynamics in the rearing of greater amberjack larvae to some extent, practical zootechnical and managerial issues must be resolved before using indigenous probiotics as biofilters at a commercial scale.
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Keywords
Marine fish larvae, Microbiota, Greater amberjack (seriola dumerili)