Introduction
Immunonutrition strategies are increasingly recognized as effective tools for enhancing disease resistance and post-stress resilience in farmed fish . One of those strategies involves the use of alternative ingredient, either protein- or lipid-based, that include bioactive compounds to improve fish health and robustness. Among the different alternative ingredients with bioactive compounds, single-cells proteins (SCPs ) cover the different characteristic, as they combine readily digestible protein with bioactive compounds that support health , making it a nutrient-dense addition to aquafeeds. Among the different SCPs available, PEKILO® is a single-cell protein produced by cultivating the filamentous fungus Paecilomyces variotii on agricultural or forestry by-products. Animals fed PEKILO®- based diets exhibit better feed conversion efficiency, and they demonstrate higher survival rates under typical farming conditions. Moreover, the β-glucans and nucleotides in PEKILO® act as immunostimulants, enhancing the expression of pro-inflammatory cytokines and chemoattractant molecules and thereby strengthening the innate immune response of fish, including Atlantic salmon ( Salmo salar ) (Hooft et al., 2024; Mesah et al., 2025). However, the effect of this ingredient in marine warmwater fish, particularly during the early-development stages , when rapid growth coincides with immune system development—remain poorly understood . Therefore, the present study aimed to evaluate the inclusion of PEKILO® in weaning diets for gilthead sea bream larvae, with the goal of improving both growth performance and health status during this critical life stage.
Material & Methods
For that purpose , four different weaning diets were formulated diets with increasing levels of PEKILO® ( 0, 5, 10, and 15% inclus ion) and the effect on larval growth performance, survival, stress resista nce, and disease resistance against Vibrio anguillarum was studied. The experiment wa s conducted with 28 days post hatching (dph ) larvae fed over 18 days in triplicates. Larvae were assigned to 12 fiberglass cylinder tanks with conical bottoms (170l ), at 890 larvae/100L. Tanks were supplied with filtered seawater at an increasing debit from 1 L/min at the beginning of the experiment to 2 L/min at the end of the experiment and continuous aeration during the entire experiment. Water temperature was 22.2 ± 0.8 ºC and the dissolved oxygen was 6.57 ± 0.1 g/L . Larvae were hand-fed every 45 min from 8:00 am to 8:00 pm for 18 days until 47 dph . Photoperiod was 12:12 L:D and larvae mortality was daily registered by individually counting the dead larvae. At the end of the feeding period, two different tests were conducted with groups of thirty larvae from each tank (triplicates for each experimental diet) : i ) a stress test ( 2 min of air exposure) ii) a challenge test against infection with a sublethal dose of Vibrio anguillarum (105 CFU/ml). Mortality was recorded for 48 h, and the relative percentage of survival (RPS) was calculated at the end of the challenge.
At the beginning and at the end of the experiment, samples of whole larvae were taken to analyze proximal composition (crude protein, crude lipid, ash, moisture) and whole-body fatty acid and amino acid profiles. Whole larvae samples were also taken for histological studies. At the end of the feeding period and after challenge test, samples of whole larvae were also taken for analysis of relative expression of immune-related genes through real-time quantitative PCR (RT-qPCR) analysis . The analyzed genes included interleukin-1β (il1b), interleukin-10 (il10 ), tumor necrosis factor-α (tnf-a ), nuclear factor kappa β subunit 2 (nfkβ2 ) caspase 3 (casp3) , cluster of differentiation 4 (cd4), cluster of differentiation 8 (cd8) and major histocompatibility complex II (mhcII ) and using b -actin as housekeeping . Relative expression was calculated by the 2–ΔΔCt , where ΔCt was determined by subtracting the housekeeping from the Ct value of the target gene described by Livak and Schmittgen (2001).
Results
Results indicated a dose/dependent effect of P. variotii and could effectively use up to 10% of PEKILO® in diet without negatively affecting growth performance and survival , the inclusion of 15% of PEKILO® significantly (p<0.05) affecting larvae growth both in terms of weight and length. Larvae fed with higher levels of PEKILO® (15%) showed a significant (p<0.05) decrease resistance to pathogen in terms of survival, but had no effect on survival after stress test. Increasing levels of P. variotii did not affect whole-larvae proximate composition but did affect their fatty acid content, by increasing the n-6 fatty acids in whole larvae, and specifically the linoleic (18:2n-6) content in whole larvae . T he inclusion of PEKILO® in seabream larval weaning diets improved the response of immune-related genes after 48h of infection with V. anguillarum , by increasing the relative expression of genes implied in the immune response against pathogens, such as il1b , il10, tnf-a , nfkβ2 or mhcII . However , t he inclusion of 15% of P. variotii meal i n larval diets failed to induce a proper response to the infection with V. anguillarum (Fig. 1).
This study demonstrates the potential of Paecilomyces variotii fungal meal as a sustainable and functional ingredient in weaning diets for gilthead sea bream . Levels of inclusion up to 10%. improve larval growth and the response of larvae to infection with Vibrio anguillarum, but higher level of inclusion (15%) induced negative effects on larval survival after challenge test against pathogen , denoting a dose-dependent effect.
Bibliography
Hooft, J.M., et al. (2024) Paecilomyces variotii (PEKILO®) in novel feeds for Atlantic salmon: effects on pellet quality, growth performance, gut health, and nutrient digestibility and utilization, Aquaculture 740905, https://doi.org/10.1016/j.aquaculture.2024.740905.
Mensah, D.D., et al. 2025. Paecilomyces variotii improves growth performance and modulates immunological biomarkers and gut microbiota in vaccinated Atlantic salmon pre-smolts . Fish & Shellfish Immunology 160,110223 . https://doi.org/10.1016/j.fsi.2025.110223
Acknowledgments
The project leading to these results has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 871108 (AQUAEXCEL3.0). The work was
performed at ULPGC-FITU (Feed Ingredients and Additives Testing Unit) research infrastructure, under grant agreement PID is 28649 (TNA programme). Silvia Torrecillas is financed by a Ramón y Cajal fellowship (RYC2021-031414-I) funded by MICIU/ AEI/10.13039/501100011033 and, as appropriate, by “ESF Investing in your future”, by “ESF+” or by “European Union NextGeneration EU/PRTR.