Introduction
Manila clam (Ruditapes philippinarum) is one of the most economically important farmed aquatic species in Italy (EUROSTAT, 2023). Since its introduction in 1983, this species became one of the most exploited resources of the lagoons of the Northern Adriatic Sea, forming large natural benches that satisfied the request of juveniles by local farmers (Brusà et al., 2013).
In the last few years, the natural recruitment of Manila clam juveniles decreases (Ponti et al., 2017), so the hatchery-reared seed emerged as a source of juveniles to support the clam industry.
Infectious diseases represent one of the bottlenecks for the mass production of seed in hatchery, due to mortality peaks caused by Vibrio bacteria (Dubert, 2017).
Currently, the prevention of diseases outbreaks is preferred to treatments with antibiotics, in order to avoid the emergence of antibiotic resistant bacterial strains, so the administration of probiotic microorganism represents a beneficial practice to fight vibriosis in shellfish hatchery (Prado et al., 2010).
The aim of this preliminary study is to verify the beneficial effects also on R. philippinarum larvae, starting from a test on survival of larvae treated with Phaeobacter inhibens DSM17395. Phaeobacter inhibens has been reported to produce antibacterial compounds and it has been verified to exerts a probiotic activity in other mollusk and fish larvae (Sohn et al., 2016; D’Alvise et al., 2012).
Materials and methods
The marine bacteria P. inhibens DSM17395, identified as potential probiotics (Ruiz-Ponte et al., 1998) was grown in marine broth, at 25°C, with gentle rocking.
Once fecundation was carried out, larvae were maintained in flasks containing 200 ml SSW (Sterile Sea Water) at 24±1°C, 28 psu and daily fed with microalgae of the species Isochrysis galbana (var. T-ISO) and Chaetoceros calcitrans, dosed with a total concentration of 100.000 cells/ml.
Selected concentrations of the probiotic were 106, 105 and 104 CFU, referring to the most cited in similar work (Karim et al., 2013; Prado et al., 2009). Bacterial density was determined by measuring OD 600 through spectrophotometer. For each concentration, experiment was run in triplets, and in addition, a group of larvae were treated without the addition of probiotic, as control group.
SSW was periodically changed, together with the count of survived individuals, carried out through Sedgwick rafter cell counter. Then, survival rate has been calculated through the following formula (Prado et al., 2009):
In the treated groups, the probiotic was dosed at the beginning of the test and in correspondence of water changes.
Results
Preliminary results of the study are summarized in Fig. 1. Survival of larvae has been monitored from T0 (the day right after the emission) to T4 (ten days after the emission), when the metamorphosis event begins.
Among the three different treatments tested, the one involving the lower concentration of the probiotic, 104 CFU, is the one showing result similar to the control. In addition, at T3 and T4, 104 treatment results show a better survival rate.
Despite the decline in number of larvae through the time, this study shows that the survival rate of the treated groups is similar or improved when compared to the control ones (that should reflect the standard condition at which this species is farmed in the hatcheries).
Further results will be available for the oral presentation and will be focused on the effectiveness of the above-mentioned treatment.
References
Brusà, B. R., Cacciatore, F., Ponis, E., Molin, E., Delaney, E., (2013) Clam culture in the Venice lagoon: stock assessment of Manila clam (Venerupis philippinarum) populations at a nursery site and management proposals to increase clam farming sustainability. Aquat. Living Resour. 26 (1) 1-10.
D’Alvise, P. W., Lillebø, S., Prol-Garcia, M. J., Wergeland, H.I., Nielsen, K. F., et al. (2012) Phaeobacter gallaeciensis Reduces Vibrio anguillarum in Cultures of Microalgae and Rotifers, and Prevents Vibriosis in Cod Larvae. PLOS ONE 7(8): e43996.
Dubert, J., Barja, J. L., Romalde, J. L., (2017) New Insights into Pathogenic Vibrios Affecting Bivalves in Hatcheries: Present and Future Prospects. Frontiers in Microbiology, 8.
EUROSTAT. Production from aquaculture excluding hatcheries and nurseries (from 2008 onwards). Available online: https://ec.europa.eu/eurostat/databrowser/view/fish_aq2a/default/table?lang=en (accessed on 24 April 2023).
Karim, M., Zhao, W., Rowley, D., Nelson, D., & Gomez-Chiarri, M. (2013). Probiotic strains for shellfish aquaculture: protection of eastern oyster, Crassostrea virginica, larvae and juveniles againsl bacterial challenge. Journal of Shellfish Research, 32(2), 401-408.
Ponti, M., Castellini, A., Ragazzoni, A., Gamba, E., Ceccherelli, V. U., & Abbiati, M. (2017). Decline of the Manila clams stock in the northern Adriatic lagoons: a survey on ecological and socio-economic aspects. Acta Adriatica: International Journal of Marine Sciences, 58(1), 89-103.