Analysis of bacterial community and identification of pathogenic bacteria in the diseased clam larvae of Meretrix petechialis

The clam Meretrix petechialis is an important commercial bivalve mollusk widely distributed in China, Japan and Korea. The development of clam culture depends on the plenty of seed supply. However, mass mortalities of M. petechialis larvae often occur during hatchery rearing. Outbreak of disease is the main bottleneck for successful spat production. So, the characterization of pathogenic bacterial strains causing epizootics in larval culture is essential for developing efficient management strategies to prevent and control disease outbreaks in clam hatchery. In present study, the moribund umbo larvae of clam sampled from hatchery were analyzed by constructing bacterial 16S rDNA clone library. The results showed that the bacterial community diversity of moribund larvae was low, and the Vibrio mediterranei clones accounted for more than 75% in the library, indicating they may be the causative bacteria of clam larvae mortality. Furthermore, the dominant strain was isolated from the moribund larvae, and identified as V. mediterranei by 16S rDNA sequencing and phylogenetic analysis. The virulence of the obtained strain US2-01 was determined by artificial infection test, a dose effect was observed for the pathogenicity of US2-01 on clam larvae. The onset time of US2-01 infection was related to its concentration, and the onset time was delayed in the low concentration group (1.0 × 10⁴ CFU/mL). The cumulative mortality rate of infected clam larvae was 84% when they were immersed in final concentration 1.0 × 10⁶ CFU/mL US2-01 for 96 hours. The clinical signs were observed during the artificial infection experiment, the treated larvae gradually lost their swim ability, showing symptoms such as the shedding of veliger cilia, partial tissue dissociation, and the empty stomach, which were consistent with the symptoms of the naturally diseased larvae in hatchery. Finally, the drug sensitivity test was performed and the results showed that V. mediterranei strain US2-01 was resistant to commonly used antibiotics such as penicillin, ampicillin and erythromycin, moderately sensitive to tetracycline and doxycycline, and highly sensitive to the other seven antibiotics such as ceftazidime, gentamicin and kanamycin. Taken together, this is the first report that V. mediterranei is the potential pathogen of M. petechialis larvae in hatchery. The results could provide a scientific basis for the disease research and control for larvae breeding in the clam hatchery.