THERMAL HISTORY DIFFERENTIALLY SHAPES THE GUT MICROBIOME RESPONSE OF GILTHEAD SEABREAM Sparus aurata JUVENILES UNDER WARMING CONDITIONS

Introduction

Aquatic organisms closely interact with their environment and associated microorganisms, driving growing interest in microbiota–host–environment interactions. Farmed fish are particularly sensitive to rearing conditions, as ectotherms whose growth, welfare and disease resistance are strongly affected by water temperature. This is especially relevant in Mediterranean aquaculture, where the semi-enclosed Mediterranean Sea is highly vulnerable to global warming and gilthead seabream (Sparus aurata) reared in sea cages are directly exposed to seasonal fluctuations and extreme warming events. Recent evidence showed that extreme summer heat episodes can induce marked intestinal microbiota shifts in this species (Domingo-Bretón et al., 2025). However, responses to thermal stress may depend not only on the maximum temperature reached, but also on the previous thermal trajectory experienced by the fish. Thus, this study aimed to evaluate whether differential pre-acclimatation can modulate the intestinal microbiome response of gilthead seabream exposed to a subsequent acute temperature increase.

Materials and methods

Gilthead seabream juveniles were acclimated from April to May 2025 in an open-flow system under natural photoperiod and temperature at the IATS facilities (40°5′N, 0°10′E). Then, 220 fish were randomly distributed into ten 90 L tanks connected to two RAS systems (22 fish per tank). After 10 days at 20 °C, baseline intestinal microbiome sampling was performed before temperature rise (t0). Tanks were assigned to two thermal regimes, each with five replicate tanks: Warming Programmed (WPr) and Warming Abrupt (WAb) (Figure 1A).

Fig. 1. (A) Representation of both thermal trajectories performed (B) Alpha diversity (Shannon index) between sampling points (t0-t2) for both thermal regimes.

In WPr, temperature was progressively increased from 20 °C to 24 °C, whereas WAb remained at 20 °C. After 12 days, intermediate sampling was performed (t1). Temperature was then increased to 30 °C in both treatments and maintained for three weeks until final sampling (t2). Growth parameters were recorded at each sampling point, and anterior intestinal samples were collected for DNA extraction from 10 fish per group/sampling point. Full-length 16S ribosomal RNA gene were sequenced in a PromethION P2 of Oxford Nanopore platform. Reads were basecalled with SUP model of Dorado v1.4, and taxonomic assignment was performed with minimap2 against SILVA v138.2.

Results and discussion

Specific growth rate (SGR) did not differ between groups during the initial period, indicating comparable starting conditions before temperature differentiation. During the second period, WPr showed higher SGR than WAb, consistent with its rearing in warmer water during programmed acclimatation. Notably, during the final period, when both groups were maintained at 30 °C, SGR remained higher in WPr, suggesting an effect of the previous thermal trajectory and a reduced growth response after abrupt warming in WAb.

Regarding the gut microbiome, alpha diversity showed a temperature-associated decrease (Figure 1B). In WPr, significant differences were observed among all sampling points, indicating gradual restructuring during programmed warming. In WAb, no differences were detected between t0 and t1, consistent with both points being maintained at 20 °C, but diversity declined significantly after the abrupt increase to 30 °C. At the final sampling point, diversity was significantly lower in WAb than in WPr, suggesting that programmed warming mitigated the loss of microbial diversity after exposure to 30 °C. At the phylum level, Pseudomonadota dominated the anterior intestinal microbiome, followed by Bacillota and Actinomycetota. In WPr, phylum-level composition remained comparatively stable, with a transient increase in Actinomycetota at t1. In WAb, no significant differences were detected between t0 and t1, whereas t2 showed a strong taxonomic shift, with Pseudomonadota increasing to 95.2% and displacing Bacillota, Actinomycetota and Bacteroidota phyla. Bray-Curtis-based PERMANOVA revealed significant shifts in microbial community structure in both thermal regimes. In WPr, all pairwise comparisons were significant, with moderate and similar effect sizes, supporting gradual microbiome differentiation. In WAb, the strongest changes occurred after the abrupt increase to 30 °C, with higher effect sizes in the t1 to t2 transition than in WPr. At genus level, the final reduction in diversity was associated with Pseudomonadota-affiliated taxa in both systems, but in WAb this shift was mainly driven by Enterovibrio, which dominated the final microbiome and represented more than 80% of the community. This finding agrees with previous evidence in greater amberjack (Seriola dumerili), where Enterovibrio was associated with high-temperature microbiome shifts under marine heatwave conditions (Sánchez-Cueto et al., 2023). Overall, these results suggest that acute warming reduces intestinal microbial diversity in gilthead seabream juveniles, while the magnitude and structure of the microbiome response depend on previous thermal background.

Funding

BreamHOLOBIONT (PID2023-146990OB-I00); CSIC-MOMENTUM (MMT24-IATS-01-01); AICO 2025 (CIAICO/2024/281) and APOSTD 2024 (CIAPOS/2024/092).

References

Domingo-Bretón et al. (2025) Aquac. Rep. 40:102566; Sánchez-Cueto et al. (2023) ISME Commun. 3:3.