Environmental drivers of virulence in Vibrio europaeus: Temperature-dependent transcriptional reprogramming is associated with mass mortality in Manila Clam (Ruditapes philippinarum)

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Vibrio europaeus is an emerging bacterial pathogen responsible for recurrent mass mortality outbreaks in shellfish aquaculture, yet the environmental mechanisms regulating its virulence remain poorly understood. Here, we investigated the combined effects of temperature and salinity on the virulence and global transcriptional response of V. europaeus CECT8136 T by integrating outcomes from experimental infections in Manila clam ( Ruditapes philippinarum ) juveniles with RNA-seq of bacteria grown in vitro under corresponding controlled conditions, phenotypic characterization, and species-wide pangenome analyses. Infection experiments demonstrated that temperature was the primary determinant of virulence: at 25 °C, V. europaeus caused high mortality across a broad salinity range, whereas mortality was markedly reduced at 15 °C in seawater and completely abolished under brackish conditions. Potential transcriptional mechanisms associated with these virulence phenotypes were inferred from in vitro RNA-seq, which revealed extensive temperature-dependent transcriptional reprogramming, whereas salinity acts as secondary modulator. Warm-temperature conditions promoted a highly virulent phenotype, while the corresponding in vitro transcriptional response was characterized by enhanced bacterial growth, energy production, nutrient acquisition, motility, adhesion, and coordinated expression of virulence-associated functions. In contrast, cold-temperature conditions were associated with reduced virulence, while in vitro RNA-seq revealed slower bacterial growth, enhanced biofilm formation, iron acquisition, and metabolic strategies consistent with environmental persistence. Most genes associated with virulence or persistence belonged to the core genome, indicating that these genetic determinants are widely conserved across the species. These findings suggest a strong association between environmental conditions, transcriptional plasticity, and virulence, improving our understanding of opportunistic vibriosis and informing disease prediction and management in shellfish aquaculture.