Introduction
Early microbial exposure is increasingly recognized as an important factor shaping host development and immune function in marine organisms. In bivalve hatcheries, however, larvae are typically reared in UV-treated seawater, resulting in reduced microbial diversity during early life stages. Whether this matters in the long run is still an open question for Ruditapes philippinarum, a species whose aquaculture is already under severe pressure from Perkinsus infections and climate-driven heatwaves. Here we present the preliminary results from WP2 of SHIELD, a Marie Sklodowska-Curie Actions Postdoctoral Fellowship project, testing whether exposing hatchery larvae to microbiota-enriched (ME) seawater, conditioned by healthy donor clams, produces juveniles with better growth performance and, ultimately, greater resilience to environmental stressors.
Materials and Methods
Broodstock from a Perkinsus-free population were induced to spawn at Naturedulis (Goro, Italy). Resulting larvae were divided into two groups from day 1: an ME group, exposed for 10 days to seawater conditioned by donor clams from a natural, pathogen-free environment placed upstream of the larval tanks, and a control group maintained in standard UV-treated seawater. Shell length was measured at day 8 and day 13 post-fertilisation. Larval volume was recorded at the transition from conical tanks to downwelling baskets, after which larvae were distributed into three replicate baskets per treatment for the nursery phase. Once juveniles reach approximately 4 months of age, they are transferred to a Perkinsus-endemic site (Chioggia, Venice Lagoon Italy) for a natural field challenge throughout the summer. A separate cohort kept in hatchery conditions is later exposed to a simulated heatwave under controlled laboratory conditions. Microbiota composition (16S rRNA gene sequencing), gene expression (RNA-seq), and epigenetic modifications (ATAC-seq) will be characterised at key time points across both challenges.
Results
At day 8, ME larvae were slightly smaller on average than controls (184.2 +/- 14.0 um vs. 190.1 +/- 36.9 um) but showed considerably more uniform size distribution (CV 7.6% vs. 19.4%). By day 13, ME larvae had overtaken controls in mean shell length (231.0 +/- 10.5 um vs. 218.4 +/- 14.2 um), corresponding to growth rates of 23.25 um day-1 and 14.21 um day-1 respectively. At the time of transfer to downwelling baskets, larval volume was approximately 14 ml in the ME group compared to 5.5 ml in controls, roughly 2.5 times more larvae available for the nursery stage. Juveniles from both groups are currently growing in replicate tanks at Naturedulis (Goro, Italy); heatwave and field exposure experiments are planned for the coming months.
Discussion
Even at this early stage, the WP2 data suggest a noticeable benefit of ME exposure: faster and more uniform larval growth, and substantially higher larval yield at the nursery transition. For hatchery operators, a 2.5-fold increase in larval volume at transfer represents a meaningful gain.
These results indicate that early microbial exposure can enhance larval growth performance, improve batch uniformity, and substantially increase production yield at the hatchery stage. Importantly, these benefits are achieved without changes to infrastructure, making this approach directly applicable in aquaculture settings. Ongoing experiments will determine whether these early advantages persist under heat stress and pathogen exposure.
Acknowledgment
This project has received funding from the European Union's Horizon Europe research and innovation programme under the Marie Sklodowska-Curie Actions grant agreement No. 101207800 (SHIELD).
References
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Milan M., et al. (2019). Shifts in the microbiome of the Manila clam. Molecular Ecology, 28(19), 4486-4499.
Fernandez-Boo S., et al. (2023). Perkinsus infections and gonadal development in bivalves. Parasitology, 150(4), 321-328.