QUEEN BEE LARVAE AND FISH HEALTH: AN EX VIVO APPROACH FOR TESTING THE INFLAMMATORY RESPONSE IN THE ANTERIOR INTESTINE OF EUROPEAN SEA BASS (Dicentrarchus labrax)

Introduction Nowadays there is a growing demand for new sustainable ingredients adhering to circular economy principles and improving animal performance and health. Within these, research has focused on exploring functional ingredients (FI) that act preventively against potential diseases or dysfunctions, thus fostering fish health status (Ruiz-Cano et al., 2022). Insects, such as honeybees (Apis mellifera), are gaining popularity as a possible FI. The queen bee larvae (QBL) are usually discarded during royal jelly production and are typically considered waste. However, recent studies have shown that QBLs are rich in protein, fatty acids, and essential amino acids, enhancing their nutritional value for use in food and feed (Addeo et al., 2021; Zhao et al., 2022). For evaluating new ingredients, animal nutrition research mainly relies on conventional in vivo feeding trials, which are time-consuming, expensive, and ethically challenging. The ethical concerns surrounding animal experimentation have prompted the adoption of the 3Rs principle to minimize the use of animals and reduce their suffering (Mukherjee et al., 2022). The ex vivo model is ethically advantageous as a first approach to evaluating novel ingredients, as it uses tissue slices obtained from an organism and thus substantially reduces the number of animals used in experimentation. The slices (explants) are kept under optimal conditions, mimicking the natural environment, maintaining intercellular connections and interactions, and ensuring that physiological processes more closely represent the in vivo situation (Wang et al., 2015). The purpose of this study was to apply an ex vivo approach to evaluate the viability and inflammatory response of anterior intestine explants of European sea bass juveniles after 4 hours of incubation with QBL immersed in royal jelly (raw ingredient, RAW), its extract (RAWex), and freeze-dried product (FD) and its extract (FDex) and after a lipopolysaccharide (LPS) challenge. Materials & Methods QBL extracts were prepared from the raw ingredient and freeze-dried using a sequential extraction with methanol (50/50) and acetone (70/30) solutions. Six European sea bass (Dicentrarchus labrax) juveniles with 106.4±15.7 g were euthanized with excess anesthesia (2-phenoxyethanol); thereafter, the anterior intestine was sampled and slices of 5 mm diameter were cut using a biopsy scalpel. Three slices were placed in each well of 24 well plates and incubated with 1 mL of pre-treatment medium (Penstrep 500 U, 5.5 mM glucose, 2 mM glutamine, and 10% FBS) for 1 hour. Then, the pre-treatment medium was removed, and 1 mL of treatment medium containing Penstrep 100 U, 5.5 mM glucose, 2 mM glutamine, and 10% FBS and, respectively, 0 (Control), 1, 2, 4, 8, and 10% of the RAW, RAWex, FD, and FDex were added to each well and incubated at 22 °C, 100 rpm, for 4 hours. Each treatment was tested in triplicate. At the end of the incubation time, one slice per well was collected and used for a viability test and another slice was stored in Trizol at -80 oC until gene expression analysis. The remaining slice was challenged with 10 or 100 ng of LPS for 4 hours and then stored at -80 oC until gene expression analysis. The slices' viability was assessed based on cellular metabolic activity using MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). The expression of pro- inflammatory genes, namely tumor necrosis factor α (TNF-α), cyclooxygenase 2 (COX2), and interleukin β (ILβ), was evaluated to assess the immune response. Results After 4-hour incubation, the viability of the anterior intestine explants was not affected by the ingredients, independently of the type and concentration tested. However, compared to the Control, the inclusion of FD at 10% led to an increase in TNFα expression. After the challenge with 10 or 100 ng of LPS, no differences in the expression of immune genes tested were observed. Nevertheless, when exposed to a 10 LPS challenge, there was an augmentation in ILβ expression within 4% FD as opposed to the unchallenged 4% FD. Similarly, the application of 100 ng LPS led to a rise in ILβ expression within 10% FDEx compared to the unchallenged 10% FDEx. Conclusions This preliminary study aimed to assess the potential of the ex vivo approach as a preliminary screening of FIs for use in aquafeeds, using QBL as a potential functional ingredient. According to the present results, tissue viability was not affected by QBL, either as raw or as an extract, independently of the concentration tested. The explants responded to the treatments by increasing the pro-inflammatory cytokinin TNFα expression but only in the FD treatment, whilst differences were observed in 4% FD and 10% FDEx for the ILβ expression after the two LPS-challenging intensities. Overall, results suggest that QBL does not seem to enhance the immune response of European sea bass, but further studies are required to confirm these results and the potential of ex vivo assays as pre-screening tests of the tissue immune responses. Acknowledgments Maria Vittoria Tignani was supported by PON-Ricerca & Innovazione 2014-2020 Ph.D. fellowship “InGreen-Insects meals and oils as green ingredients to increase resilience and sustainability in animal husbandry and aquaculture” (CUP: B11B21004830007) funded by MUR. Nicole Martins was supported by a research contract (2023_029_IS_MB4AQUA) in the R&D&I project “MB4Aqua: Macroalgae biorefinery: a novel approach to produce sustainable feedstuffs and functional additives towards low carbon footprint aquafeeds” reference 2022.06587.PTDC. Filipa Fontinha was supported by an FCT grant (2020.07212.BD). References Addeo et al. (2021). Potential use of a queen bee larvae meal (Apis mellifera ligustica) in animal nutrition: a nutritional and chemical-toxicological evaluation. Journal of Insects as Food and Feed, 7(2), 173-186. Mukherjee et al. (2022). Role of animal models in biomedical research: a review. Laboratory Animal Research, 38(1), 18. Ruiz-Cano et al. (2022). Essential oils and melatonin as functional ingredients in dogs. Animals, 12(16), 2089. Wang et al. (2015). Advances and challenges for neural regeneration research. Neural Regeneration, 3-17. Zhao et al. (2022). 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