Different colonization and residence time of Listonella anguillarum and Vibrio splendidus in the rotifer Brachionus plicatilis determined by real-time PCR and DGGE

Abstract

Listonella anguillarum 90-11-287 and Vibrio splendidus DMC-1 were incorporated in the rotifer Brachionus plicatilis, which was subsequently maintained under larval rearing conditions to determine the residence time of both pathogens in rotifers. Real-time PCR was applied to specifically detect and quantify both pathogens. L. anguillarum colonized rotifers more efficiently than V. splendidus and both pathogenic strains were released from rotifers to seawater, after infected rotifers were transferred to rearing tanks. V. splendidus grew and became predominant in the seawater of tanks. Both pathogens remained in rotifer or seawater enough time to infect fish larvae, but their different behaviour could determine different infection patterns, preferentially by ingestion of prey or by active intake or contact with surrounding seawater. The effect of L. anguillarumand V. splendidus on the bacterial community associated with rotifers and seawater of rearing tanks was analysed by DGGE of PCR-amplified 16S rDNA fragments. The bacterial community of rotifers did not present a marked species dominance. The incorporation of L. anguillarum or V. splendidus did not reduce bacterial diversity and shifts could be explained by bacterial exchange between rotifers and seawater. Main bacterial groups were identified by sequencing the DNA extracted from Marine Agar (MA) bacterial isolates and DGGE excised bands. Only 2 DGGE bands corresponded to bacteria isolated from MA plates, suggesting that bacterial groups present in rotifers may not be easily cultivable. The bacterial community of rotifers was composed by Gram negative bacteria belonging to α-Proteobacteria (Ruegeria spp), γ-Proteobacteria (Alteromonas alvinellae, Marinobacter sp, an Oceanospirillaceae bacterium and Pseudoalteromonas sp), Cytophaga–Flexibacter–Bacteroides group (Polaribacter dokdonensis, Roseivirga spongicola and Tenacibaculum soleae) and a Gram positive bacterium (Microbacterium sp).