The localization, abundance and community structure of bacteria associated with Artemia nauplii was analyzed using a combination of culture-based, microscopy-based and molecular methods for both culturable and not readily culturable populations.Artemia cysts were hatched using a standard protocol and 4 nauplii sample types were generated - Newly Hatched, Microalgae enriched, Super Selco® enriched, and Protein Selco® enriched (DC Super Selco® or DHA Protein Selco®; INVE Aquaculture, Dendermonde, Belgium). Newly hatched Artemia nauplii (instar I) were harvested for analysis 19 h after addition of cysts to the hatching tank. The remaining nauplii were enriched for 24 h.The antimicrobial treatment of Artemia nauplii included an initial treatment with formalin (1500 ppm) for 5 min, followed by a mixture of Virkon S® (30 ppm), and the antibiotics oxytetracycline (25 ppm), erythromycin (25 ppm) and streptomycin (10 ppm) for 4 h.For the culture based enumeration and isolation, triplicate samples of 20 Artemia nauplii were collected and colony forming units (CFUs) enumerated after 24 h and 48 h.In the analysis by scanning electron microscopy, the bacterial density on the surfaces of Artemia nauplii was determined for each treatment by counting the number of bacteria per field of view(f.o.v.) and counting a minimumof 6 f.o.v. for at least 24 animals per treatment.Fluorescence in situ hybridization (FISH) and visualization was performed to visualize bacterial cells and to detect Vibrio.For direct extraction of DNA from Artemia nauplii, triplicate samples of 50¿100 nauplii were collected. Isolated bacterial strains were grown over night in Luria Bertani (LB) broth, Miller (Difco Laboratories) and DNA was extracted using the Promega Wizard Genomic DNA Purification Kit (Promega, Madison,WI, USA).For bacterial clone library construction, primers 63f and 1387r were used to amplify bacterial 16S rRNA genes from DNA extracted from Artemia nauplii using a modified PCR protocol. PCR amplification of bacterial 16S rRNA gene fragments for denaturing gradient gel electrophoresis (DGGE) analysis was performed with primers 1055F and 1392R-GC. Phylogenetic affiliation was made of bacterial strains isolated from newly hatched and enriched Artemia nauplii.
To determine and compare the localization, abundance, and community structure of bacteria associated with newly hatched and enriched Artemia nauplii.The effect on the bacterial community of treating nauplii with a mixture of antibiotics and the disinfectants formalin and Virkon S® was also investigated, with special emphasis on the potentially pathogenic Vibrio population.
The use of live feeds presents a biosecurity risk for aquaculture due to the potential for inadvertent introduction of bacterial pathogens. Reared Artemia nauplii constitute the most commonly used live feed for larval aquaculture species including fish, crustaceans, and molluscs.Closest characterized cultured relative (accession number): Brevundimonas sp. V4.BO.05 (AJ244704), Pseudomonas azelaica (AM088475), Pseudomonas sp. AU2510 (AY486376), Rhizobium giardinii CCBAU 85040 (EU256415), Rhizobium sp. 6-1C1 (DQ395341), Rhizobium sp. RPA12 (EF201804), several Micrococcus sp. (e.g. AM913979), several Pseudomonas sp. (e.g. AB276372), Sphingomonas dokdonensis (DQ178975), Vibrio alginolyticus (AY332566)a, Vibrio coralliilyticus (AJ316167), Vibrio proteolyticus (DQ995521)
