# Characterization of expression response in post-prandial situation of food sensing system in rainbow trout (Oncorhynchus mykiss) fed a plant-based diet: Focus on free fatty acid receptors and their signaling pathways

Elodie Baranek, Karine Dias, Cécile Heraud, Anne Surget, Laurence Larroquet, Sandrine Skiba-Cassy, Jérôme Roy

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Elodie Baranek, Karine Dias, Cécile Heraud, Anne Surget, Laurence Larroquet, et al.. Characterization of expression response in post-prandial situation of food sensing system in rainbow trout (Oncorhynchus mykiss) fed a plant-based diet: Focus on free fatty acid receptors and their signaling pathways. Aquaculture, 2024, 581, pp.740362. 10.1016/j.aquaculture.2023.740362. hal-04311147

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https://hal.inrae.fr/hal-04311147v1

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Type of article: Research Article

Characterization of expression response in post-prandial situation of food sensing system in rainbow trout (*Oncorhynchus mykiss*) fed a plant-based diet: focus on free fatty acid receptors and their signaling pathways

Elodie Baranek, Karine Dias, Cécile Heraud, Anne Surget, Laurence Larroquet,

Sandrine Skiba-Cassy and Jérôme Roy

INRAE, University of Pau and Pays de l'Adour, E2S UPPA, UMR1419 Nutrition Metabolism and Aquaculture, Aquapole, F-64310, Saint-Pee-sur-Nivelle, France.

Corresponding author:

Jérôme Roy

Phone: +33 5 59 51 59 65

Email: jerome.roy@inrae.fr
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ABSTRACT

Recently identified and characterized in rainbow trout (Oncorhynchus mikyss), the main gustatory receptors for fatty acid, known as free fatty acids receptor (FFAR) 1 and 2, may play a crucial role in nutrient detection and regulation of feeding behavior. However, our understanding of their post-prandial roles remains limited. This study investigates the impact of a single meal (acute effect) with an alternative plant-based diet (vegetal diet, V diet devoid of fish meal and fish oil) on the food detection mechanisms in trout. After 30 days of feeding with commercial-like diet (C diet made up of a mix of fish meal, fish oil and plant ingredients) followed 5 days of fasting, we conducted a post-prandial kinetic experiment after single meal with C or V diet. We assessed the expression pattern of *ffar* genes, their associated signaling pathways (calcium and indolamine pathways) in the tongue along with the expression pattern of appetite-regulating neuropeptides in the hypothalamus. Additionally, we evaluated central activity through brain activity markers in the telencephalon of trout at various post-meal time points, including 20 minutes, 2, 6, 10 and 24 hours. Our results revealed that the diet nature (C vs V) influences the expression of *ffar* genes, while some paralogs of *ffar2* (*ffar2a2* and *ffar2b1.2*) were early up-regulated by V diet, *ffar2b1.1* and *ffar1* were latter up-regulated by C diet. Differential regulations in the tongues of V diet fish were also observed on calcium signaling and an early alteration in serotonin turnover. The expression increase of neuropeptide *npya* from 6 to 10 hours after meal, could suggest a greater sensation of hunger in fish fed the C diet. Finally, our findings suggest that rainbow trout exhibit a keen ability to detect a novel feed after a single meal with V diet at the brain level, particularly in areas associated with decision-making and behavior control. Altogether, results not only show that the integrative feeding response appears to be dysregulated by V diet, but also highlight the noteworthy sensitivity of nutrient detection mechanisms in rainbow trout to new food sources. FFARs may play a role in finely discerning diets with varying levels of ω-3 LC-PUFAs in rainbow trout. This heightened detection sensitivity holds the potential to significantly influence the regulation of feeding behavior and food detection in farmed fish.

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KEYWORDS: single meal, farmed fish, GPCR taste receptor, gustation, post-prandial kinetic.

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# 1. INTRODUCTION

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Resource conservation and sustainability have become paramount concerns in various industries, and aquaculture is no exception. Traditionally, aquafeeds have predominantly relied on fishmeal (FM) and fish oil (FO) as key components. These ingredients are sourced from wild fish stocks with finite limits, making their use unsustainable. Consequently, for both economic and ecological reasons, it has become imperative to substitute these ingredients with renewable, environmentally friendly, and cost-effective alternatives (Naylor et al., 2009). In recent years, due to the steadily rising demand from the aquaculture sector, efforts to replace these traditional ingredients have been actively pursued, with a notable emphasis on incorporating plant-based alternatives (*The State of World Fisheries and Aquaculture 2020*, 2020).

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In the context of rainbow trout (RT, *Oncorhynchus mykiss*) production, a species widely farmed in Europe, the complete replacement of FM and FO in their diet yields adverse outcomes. This includes early deterioration of survival rates and a decline in growth, in contrast to RT fed with commercial diets (Lazzarotto et al., 2018). These effects are primarily ascribed to alterations in feeding behavior (Turchini et al., 2009). It is well-established that alterations in feeding behavior exert a profound impact on fish throughout their entire life cycle which specifically exhibit often reduced food intake (Torstensen et al., 2008). Moreover, an increase in abnormal feeding behavior was also observed (Benítez-Santana et al., 2014). Therefore, it is essential to carefully consider the dietary composition to ensure the optimal growth and development of RT. Certainly, the composition of nutrients in the diet is recognized to have a significant impact on the feeding and growth of fish (Bertucci et al., 2019). Several studies have demonstrated that the nutrient content of the diet can influence feeding behavior of farmed fish (Comesaña et al., 2020; Conde-Sieira et al., 2015a; Librán-Pérez et al., 2014, 2015a; Soengas, 2014; Velasco et al., 2016), which is mediated by neuropeptide pathways, leading to changes in the expression of anorexigenic and orexigenic neuropeptides (Comesaña et al., 2020; Conde-Sieira et al., 2015b; Librán-Pérez et al., 2015b, 2014; Velasco et al., 2016). Among nutrients, these changes in feeding behavior can be attributed to differences in the fatty acid (FA) profiles between plant-based

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ingredients and FM/FO, underscoring the importance of careful dietary composition considerations to achieve a better feed intake.

Undoubtedly, FM and FO serve as valuable natural sources of ω-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential nutrients for all vertebrates. These ω-3 LC-PUFAs play crucial roles in the regulation of feeding behavior and associated mechanisms in fish, as supported by recent research (Roy et al., 2020a, 2020c, 2022). Although plants do contain α-linolenic acid (ALA), a precursor to ω-3 LC-PUFAs, the absence of EPA and DHA in their composition can potentially impact feeding behavior and influence the regulation of feed intake mechanisms in RT (Roy et al., 2020b). Moreover, given the prevailing economic circumstances and the imperative to reduce the reliance on FM and FO in aquafeed formulations, aquaculture producers are actively pursuing cost reduction measures. The cost of feed represents the largest portion of production expenses for farmers, often accounting for more than 50% of total production costs. Consequently, optimizing food consumption becomes paramount to ensure favorable growth, robust health, and enhanced survival of fish, particularly in farm-raised environments.

Because of their textural properties, it is well accepted that dietary lipids such as ω-3 LC-PUFA can modulate post-meal feeding behavior through mechanisms related to craving, hedonic perception, or pleasure. Consequently, the absence of some of them in the composition of the alternative plant ingredients may significantly influence the regulation of fish feed intake, primarily through appetite control. One of the initial mechanisms involved in this feeding response is taste detection via gustatory system which is crucial for food acceptance. This sensory system, among the first to detect nutrients, plays a key role in regulating feeding behavior and energy balance (Chaudhari and Roper, 2010). Some stimuli trigger the acceptance of a feed (Dressler and Smith, 2013), while others, conversely, lead to its rejection (Drewnowski et al., 1999).

In the literature, it has been demonstrated that oral fat activates the gustatory system (Besnard et al., 2016; Running et al., 2015). In mammals, FAs are detected by G protein-coupled receptors (GPCR) known as “free FA receptors” (FFARs). FFAR1 and FFAR4 are responsible for detecting medium and long-chain FAs, while FFAR 2 and 3 detect the short chain FAs (Kimura et al., 2020). These various compounds
108 interact with taste receptors located in the taste bud cells (TBC) eliciting intracellular calcium signals. This
109 results in calcium accumulation within the cell leading to membrane depolarization, which, in turn, triggers
110 the release of serotonin. Serotonin plays a central role in the regulation of food intake and whole-body energy
111 homeostasis (Galen et al., 2021) by facilitating the transmission of the gustatory signal from the tongue to
112 the brain, thereby inducing cerebral activity, and an integrated response at the central level
113 (Shanmugamprema et al., 2020). Recently, we highlited the presence of these key taste receptors, FFAR, in
114 the RT genome, with their regulation varying depending on the composition of the aquafeed (e.g., a diet rich
115 in FM/FO vs 100% plant-based diet) (Roy et al., 2023) suggesting an involvement of these receptors in the
116 modulation of fatty acid sensing pathways.

117 In the current context, where minimizing the environmental impact of aquaculture is a top priority,
118 it becomes crucial to gain insights into the regulation of the mechanism involved in food detection. This
119 understanding is pivotal for enhancing the composition and effectiveness of plant-based diets compared to
120 those containing FM and FO. However, to the best of our knowledge, the characterization of food detection
121 mechanisms and its regulations dietary composition, especially in ω-3 LC-PUFAs, within the 24 hours
122 following a meal, remains unreported in RT.

123 Recently, we made an important discovery by showing that the absence of FM/FO (and ω-3 LC-
124 PUFAs) in the diet for eight months, starting from the first meal, leads to significant changes in nutrient
125 detection (Baranek et al., 2023). However, it is crucial to note that the observed results reflect the chronic
126 effects (8 months of diet) of this regimen on the food detection system, rather than the acute effects of a single
127 meal. Therefore, this approach does not accurately assess the specific impact, without habituation, of a food
128 lacking FM/FO on the nutrient detection mechanism. In this way, the aim of the present study was to evaluate
129 the acute effect of a single meal with a plant-based diet (referred as V diet), in comparison to a commercially
130 formulated diet (containing FM, FO and alternative plant ingredient, referred as C diet) with different levels
131 of ω-3 LC-PUFA, on RT feed detection mechanism. To this end, we conducted a post-prandial kinetic
132 experiment to investigate the expression patterns of *ffar* and their associated signaling pathways, including
133 the calcium and indolamine pathways, in RT tongue following a single meal. The experiment covered several
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time points, encompassing measurements before the meal (after a 5-day fasting period), as well as 20
minutes, 2, 6, 10 and 24 hours after the meal. Additionally, we characterized the expression patterns of genes
encoding neuropeptides that regulate appetite in the hypothalamus. Moreover, we assessed the expression
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patterns of genes and proteins associated with brain activity markers to assess central activity triggered by
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the meal. This comprehensive analysis, conducted in the telencephalon of RT, provided valuable insights
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into the regulatory mechanisms governing appetite and brain activity in response to dietary intake.

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## 2. MATERIALS AND METHODS

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### 2.1. Ethics statement

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The trial was conducted according to the guidelines of the National Legislation on Animal Care of the
French Ministry of Research (decree no. 2013-118, 1 February 2013) and in accordance with European
Union legal frameworks relating to the protection of animals used for scientific purposes (i.e. Directive
2010/63/EU). The trial was conducted at the INRAE's NuMeA facilities
(https://doi.org/10.15454/GPYD-AM38) and approved by the ethical committee (C2EA-73) of INRAE
"Comité d'éthique Aquitaine poissons oiseaux" (INRAE agreement no. 21699, 19 December 2019).
All efforts were made to minimize the number of fish used and their suffering. The scientists in charge
of the trial received training and personal authorization.

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### 2.2. Experimental diets design

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Diets were manufactured at INRAE's experimental facilities at Donzacq (permit no. A40-228.1, Landes, France) using a twinscrew extruder (BC 45, Clextral, Firminy, France) after formulated using Allix® software (A-Systems, City, France). The pellets had a diameter of 4 mm and a length of 4 mm.
Details about the ingredients and composition of the experimental diets are given in Table 1 and the proportions of the main fatty acid in the diets in Table 2. The experiment was conducted with two different experimental diets: a commercial-like diet (C diet) containing a mix of FM (19%), FO (8%) and plant ingredients, and a plant-based diet (V diet), completely free from FM and FO, which were

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replaced by a blend of plant ingredients 18% of soybean meal and 15.2% of corn gluten to replace FM
and 8% of rapeseed oil, 6% of linseed oil and 3.6% palm oil to replace FO). This vegetal oil blend in V
diet was chosen in order to achieve a similar proportion of fatty acid classes as found in C diet. For V
diet, DHA and EPA (present in FO for C diet) was replaced to the benefit of ALA by adding linseed oil
(6%).

Diets were isoenergetic (c. 24.49 and 24.33 kJg⁻¹ for C and V diet respectively) and were formulated
to cover the nutritional requirements of the RT (“The National Research Council [NRC] (2011).
Nutrient Requirements of Fish and Shrimp. Washington, DC: The National Academies Press.
doi: 10.1146/annurev. nutr.17.1.405,” n.d.). Diets were also isoproteic (46.11% ± 1.18% dry
matter) and close to be isolipidic (21.98% ± 2.3% dry matter). The ω-3 PUFAs were present in
comparable proportions in both diets. C diet contained 19.47% while the V diet had 20.13% ω-3
PUFAs, expressed as a percentage of total fatty acids. In terms of the overall composition of the diets,
these percentages corresponded to 4.59% and 4.09% of the total diet, respectively, but consisted mainly
of EPA, ALA and DHA in the C diet and ALA in the V diet. This amount of ω-3 FA class was chosen
in order to be close to the proportions of ω-3 FA classes found in traditional diet of RT rich in FM/FO
(more than 80% of aquafeed composition) (Lazzarotto et al., 2018). In order to avoid exceeding anti-
nutrient threshold levels, we used a blend of wheat gluten, soybean meal and whole wheat, corn gluten
meal, soy protein and fatabean as protein sources (c. 46.11% of total diet). L-lysine, L-methionine,
dicalciumphosphate and soy-lecithin were added to all diets to correct the deficiency in essential amino
acids, phosphorous and phospholipids. Mineral and vitamin premix were also added to each diet.

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## 2.3. Proximate Composition

Nutrient compositions of the diets, crude protein and lipids, gross energy, ash and starch content
and fatty acid profiles were analyzed as previously described (Baranek et al., 2023) and following
Official Methods of Analysis of AOAC INTERNATIONAL (Latimer and Latimer, 2023).
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Briefly, the nutrient composition of the diets was analyzed after 24 h of drying at 105°C. Gross energy was determined using an adiabatic bomb calorimeter (IKA, Heitersheim Grißheimer, Germany). Ash content was measured by combustion in a muffle furnace (550°C for 8 h). Crude protein was quantified using the Kjeldahl method (Kjeldahl, 1883) and crude lipids using the Soxhlet method. Total lipid content was extracted, purified, and measured gravimetrically according to the Folch method (Folch et al., 1957) using dichloromethane.

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## 2.4. Experimental design

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The female (genetically all female) juvenile RT came from the same parental stock (INRAE Fish Farm of Lees-Athas, permit no. A64.104.1, Aspe valley, France). The feeding trial was conducted in a raceway system at the INRAE's facilities of Donzacq, France. The trial involved juvenile female RT with an average weight of 140 grams. Water flow was set to ensure a dissolved oxygen concentration greater than 90% of saturation. Fish were exposed to natural photoperiod condition and the water temperature was set at 17 ± 1°C. During the trial, the water conditions were as follows: dissolved oxygen was measured at 9 mg L⁻¹, ammonia levels were below 0.01 mg L⁻¹, nitrite levels below 0.04 mg L⁻¹ and nitrate concentration was approximately 17 ppm. The flow rate in each tank was 0.3 l/s, resulting in a complete water renewal of each tank 6 times per hour. During 30 days, all fish were fed by hand twice a day with an interval 8 hours with C diet, until apparent satiety. After this, fish were starved during 5 days to obtain control condition (exclude feeding effect) and observe the post-prandial regulation of feed intake by a meal. Afterwards, the trout were fed once either the C diet or the V diet (Figure 1).
For tissue sampling, fish were first anesthetized by immersing them in a benzocaine solution (30 mg/l) and euthanized in a bath containing 60 mg/l of benzocaine. Tongue and brain (telencephalon, hypothalamus) were sampled before the single meal (T0) as well as 20 minutes (T20'), 2 hours (T2), 6 hours (T6), 10 hours (T10), and 24h after the single meal (T24). A total of 240 fish were included in the sampling, with an equal distribution of 120 fish per diet. To minimize stress on the fish during sampling, we employed three tanks per diet (48 fish per tank), thus avoiding the need to repeatedly sample the same tank six times for each timing point. Out of the total sample, 72 fish were collected for qPCR analysis, covering the tongue, hypothalamus, telencephalon, and myelencephalon (6 fish per sampling point and per diet). Additionally, 72 supplementary samples consisting of tongue, myelencephalon, and telencephalon were dissected for western blotting analysis (6 fish per sampling point and per diet). Moreover, 96 myelencephalon and tongues samples per treatment were dissected for ultra-high-performance liquid chromatography/Fluorescence (UHPLC-FL) analysis (8 fish per sampling point and per diet).

<figure><img src="image_3.png" /><figcaption>Figure 1. Scheme of experimental design.</figcaption></figure>

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## 2.5. Total RNA isolation and real-time quantitative PCR

Total RNA was isolated from the tongue, myelencephalon, hypothalamus and telencephalon (n=6 per dietary treatment) using the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) with Precellys® 24 (Bertin Technologies, Montigny-le-Bretonneux, France) in accordance with the instructions of the manufacturer, as previously described (Roy et al., 2023). Total RNAs (2 µg) were reverse-transcribed into cDNA using the Super-Script III RNase H-Reverse transcriptase kit (Invitrogen) and random

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primers (Promega, Chartonniéres-les-Bains, France) according to the manufacturer's recommendations.

The quantities of extracted RNA were analyzed using a spectrophotometer (ND-1000, Nanodrop, Thermo, Vantaa, Finland), and samples with an OD 260 nm:280 nm ratio > 1.8 were used for analysis.

High throughput real-time quantitative PCR (RT-qPCR) was performed using the BioMark microfluidic system from Fluidigm (Fluidigm, San Francisco, CA, USA), in which each gene combination was quantified using a 96.96 Dynamic Array™ IFCs (BMK-M-96.96, Fluidigm) as previously described (Cardona et al., 2022). Pre-amplification of the samples, chip loading, and RT-qPCR were performed according to the protocol of the manufacturer. Firstly, 5 ng of each cDNA were initially pre-amplified (10 min at 95°C activation and 14 PCR cycles 15 sec at 95°C, 4 min at 60°C) with PreAmp Master Mix (100-5581, Fluidigm) with a pool containing all the primers (0.2 µM). After an exonuclease treatment (M02935, NEB, Biolabs, Ipswich, MA, USA), pre-amplified samples were diluted to 1/5. In order to prepare samples for loading into the IFC, a mix was prepared consisting of 440 µL 2× TaqMan® Gene Expression Master Mix (Applied Biosystems 1369510, Thermofischer Scientific, Maryland, USA), 44 µL 20× DNA Binding Dye Sample Loading Reagent (100-0388, Fluidigm), 44 µL 20× EvaGreen (BI1790, Biotium, San Francisco, CA, USA), and 132 µL TE buffer.

In each well of a 96-well assay plate, 6 µL of the sample pre-mix and 2 µL of diluted cDNA sample were dispensed and the plate was briefly vortexed and centrifuged. A primer pre-mix was also prepared consisting of 440 µL 2X Assay Loading Reagent (85000736, Fluidigm) and low EDTA TE. In each well of a 96-well assay plate, 6 µL of the primer pre-mix and 2 µL of primer were dispensed and the plate was briefly vortexed and centrifuged.

For the assays, 5 µL of each primer mix were dispensed to each Assay inlet of the BioMark chip, and 5 µL of sample mix were dispensed to each Sample inlet of the BioMark chip. After loading the assays and samples into the BioMark, PCR was performed using the following thermal protocol: thermal mix of 50 °C, 2 min; 70°C, 30 min; 25 °C, 10 min; hot start at 50 °C, 2 min; 95 °C, 10 min, 30 cycles of (95 °C, 15 s; 60 °C, 60 s), and melting analysis (60 °C, 30 s; 95 °C, 1 °C/3 s). The Fluidigm
PCR analysis software v.4.1.3, was used to analyse the results. Data were extrapolated from standard curves and normalized to the housekeeping elongation factor 1α gene (eef1α) for data from myelencephalon, hypothalamus and telencephalon and keratin 8b gene (krt8b) for data from tongue. Relative expression of the target genes was determined using the ΔΔCT method (Pfaffl, 2001). The values for each group were expressed in fold changes relative to the T0 and presented as means ± 1 standard error of the mean (SEM) for all genes. Primer sequences used to amplify all paralog genes were previously validated and their accession numbers are presented in Table 3.

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## 2.6. Indolamine release measurement using UHPLC-FL

Indolamine in tongue (n=8) from standard mixtures and polar metabolite extracts were separated and detected using UHPLC (ACQUITY H-Class PLUS System, Waters, Milford, MA, USA), equipped with a thermostatted autosampler supported by a fluorescence detector (ACQUITY Multi-λ, Waters). Waters® Empower™ Pro software was used to obtain and quantify results.

All reagents and standards were purchased from Merck KGaA (Darmstadt, Germany). All organic solvents used were gradient MS grade (ADL & Prochilab, Lormont, France). Each tongue was homogenized using Precellys® 24, a tissue homogenizer, in 20 mM phosphate and 1 mM EDTA buffer (pH = 6.5 ± 0.05). After first centrifugation (14,000× g, 20 min, 4 °C), deproteinization of the supernatant was performed with a volume-to-volume 10% metaphosphoric acid solution. After second centrifugation (14,000× g, 5 min, 4 °C), the supernatant was filtered with a 0.22 µm poly-vinylidene di-fluoride unit through a third and last centrifugation (14,000× g, 5 min, 4 °C). Metabolite mixtures were stored at -20°C (for no longer than one week) until analysis. Chromatographic separation was performed on a HPLC column (150 mm × 4.6 mm, i.d. 3 µm) at 30°C (Luna PFP(2), Phenomenex, Torrance, CA, USA). The injection volume was 10 µL, and the flow rate was set at 0.4 mL/min. A quaternary solvent system was used, which consisted of (A) a 10 mM

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phosphate buffer (pH 4.3 ± 0.05), (B) methanol, (C) ultrapure water, and (D) acetonitrile. The mobile phase was filtered through in-line 0.2 µm membrane filters. The following linear gradient elution was used: 0–11 min: 80% A, 20% B; 16 min: 50% C, 50% D; 16–26 min: 50% C, 50% D; 27 min: 80% A, 20% B; and 27–35 min (column equilibration): 80% A, 20% B. The eluate was monitored using fluorescence detection set at 285 nm (excitation) end 355 nm (emission) for serotonin (5-hydroxytryptamine; 5-HT) and 5-hydroxyindolacetic acid (5-HIAA) respectively. Metabolites of interest were identified by comparing retention times to standards. Quantification was based on integrating peak areas and comparing them to standard calibration curves (R² > 0.999) of each metabolite of interest. Calibration curves were linear from 0.01–3 pmol per injection for 5-HT and 5-HIAA.

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## 2.7. Protein extraction and western blotting

Tongues were ground with a pestle and mortar under liquid nitrogen, 200 mg of powder was collected, and then homogenized in 1ml extraction reagent (#78510, Thermo Fisher Scientific, Waltham, MA, USA) with a tissue homogenizer (Precellys® 24) at 4°C.

The myelencephalon and telencephalon were homogenized in 0.5 ml reagent. The supernatants were collected after two successive centrifugations (1,000× g, 15 min, 4°C and 20,000× g, 30 min, 4°C). Protein concentrations were determined using the bicinchoninic acid assay method (#BCA1-1KT, Sigma-Aldrich, Saint Louis, MO, USA). Proteins were separated by SDS-PAGE gel electrophoresis (20 µg total protein loaded/sample) and transferred to a 0.45 µm PVDF membrane (#IPFL00010, Merk Millipore) via electrophoretic transfer. Membranes were incubated at room temperature for 1 h with an Intercept® phosphate buffered saline (PBS) blocking buffer and then incubated overnight at 4°C with the appropriate primary antibody already validated (Baranek et al., 2023): ORAI1 (calcium release-activated calcium modulator) rabbit polyclonal IgG (PA5-26378, Invitrogen) (1:2000), STIM1 (Stromal interaction molecule) rabbit monoclonal IgG (D88E10#5668, Cell
300 Signaling, Danvers, MA, USA) (1:1000), ERK1/2 (extracellular signal-regulated kinases) rabbit
301 monoclonal IgG (137F5#4695, Cell Signaling, Danvers, MA, USA) (1:1000), p-ERK1/2
302 (Thr202/Tyr204) rabbit monoclonal IgG (D13.14.4E#4370, Cell Signaling, Danvers, MA,
303 USA) (1:1000), TPH2 (tryptophane hydroxylase) rabbit polyclonal IgG (PA5-37803,
304 Invitrogen) (1:1000), VMAT2 (vesicular monoamine transporter 2 of 5-HT) mouse monoclonal
305 IgG (Sc-374079, Santa Cruz Biotechnology, Dallas, TX, USA) (1:1000), BDNF (brain-derived
306 neurotrophic factor) rabbit monoclonal IgG (ab108319, Cambridge, UK) (1:1000) or cFOS
307 (proto-oncogen factor) rabbit polyclonal IgG (ab190289, Cambridge, UK) (1:1,000). Membranes
308 were washed with PBS containing 0.1% TWEEN® 20 (Sigma-Aldrich) three times before 1 h
309 incubation at room temperature, with the corresponding secondary antibodies anti-rabbit IgG or anti-
310 mouse IgG, diluted 1:10,000 in PBS with 0.1% Tween 20. Membranes were then washed with PBS
311 containing 0.1% TWEEN® 20 three times and twice with PBS. Finally, membranes were exposed to
312 enhanced chemiluminescence reagents, Super Signal™ West Pico PLUS Chemiluminescent Substrate
313 (#34580, Thermo Fisher Scientific) for 5 min at room temperature before revelation using an imager
314 (iBright FL1500, Thermo Fisher Scientific). The cFOS protein signals were measured by fluorescence
315 using a fluorescent secondary antibody (A32735, Invitrogen) and the same imager. Two types of
316 normalization were required due to the weight of the protein and the protein-loading control. ORAI1,
317 VMAT2 and BDNF were normalized using β-tubulin (2146, Cell signaling), while STIM1, TPH2,
318 cFOS and p-ERK1/2 were normalized with total protein staining, using No-Stain™ Protein Labelling
319 Reagent (#44717, Thermo Fisher Scientific). The protein abundance of housekeeping protein and No-
320 Stain are presented in the supplemental information 1.

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2.8. Statistical analysis

323 All statistical analyses were performed using R software (version 3.6.1) (R Core Team (2019) ("R: The
324 R Project for Statistical Computing. https://www.r-project.org/ (accessed January 2023)," n.d.).
Individual fish were the experimental unit for data on gene expression, indolamine release and protein abundance. The results were calculated as means ± 1 standard error of the mean (SEM). Analyses were carried out on untransformed data as criteria for normality of distribution and homeogeneity of variances were fulfilled (Shapiro-Wilk and Levene's test, respectively). Firstly, a one-way analysis of variance (ANOVA) was conducted to assess the individual effects of each diet after feeding for each timing (supplemental 2-8). Secondly, the interaction between the two factors, diets and time, were assessed using a two-way analysis of variance (ANOVA). When a significant interaction was observed, a one-way analysis of variance (ANOVA) was conducted to assess the individual effects of time after feeding between the two diets. This allowed us to specifically examine the impact of each factor in isolation and determine their respective contributions to the observed results. When appropriate, analysis was followed by Tukey's post-hoc test. Differences were considered significant at p < 0.05.

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337 **3. RESULTS**

338 **3.1. Kinetic influence of the V diet on changes in *ffar* gene expression in RT**

339 The mRNA levels of *ffar1* and *ffar2* in the tongue of RT before meal, 20 minutes, 2, 6, 10 and 24 hours after meal varied (Figure 2) with timing effect (higher) for all transcript by diets. For each diet, the mRNA levels of *ffar1* and *ffar2* were also up-regulated after meal (timing effect, confirmed by one-way anova for each diet) for all transcripts except for *ffar2b2a* for V diet (supplemental information 2).

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343 The mRNA levels of *ffar1* were up-regulated 20 minutes after meal in fish fed the C and V diet, and at 2 and 6 hours and at 10 and 24 hours in fish fed the V and C diet respectively (supplemental information 2). When comparing the two diets, transcript level of *ffar1* was up-regulated in fish fed the C diet (diet effect) with a significant interaction (diet.time) showing an up-regulation at 10 and 24 hours after feeding in fish fed the C diet (Figure 2).

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The mRNA levels of *ffar2a1a* were only up-regulated 6 hours after meal in fish fed the C and V diet (supplemental information 2). When comparing the two diets, the mRNA levels of the *ffar2a1a* were not differed by the diet proposed (Figure 2).

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The mRNA levels of *ffar2a2* were increase 2, 10 and 24 hours after meal in fish fed the C diet, and at 20 minutes and 10 hours in fish fed the V diet (supplemental information 2). When comparing the two diets, the transcript level of *ffar2a2* gene was not differed by the diet proposed but revealed a significant interaction (diet.time) showing an up-regulation at 20 minutes and at 24 hours after feeding in fish fed the V and C diet respectively (Figure 2).

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The mRNA levels of *ffar2b1.1* were up-regulated only 10 hours after meal in fish fed the C diet, and at 20 minutes and 2 hours in fish fed the V diet (supplemental information 2). When comparing the two diets, transcript level of *ffar2b1.1* gene was up-regulated in fish fed the C diet (diet effect) with a significant interaction (diet.time) showing an up-regulation at 10 and 24 hours after feeding in fish fed the C diet (Figure 2).

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The mRNA levels of *ffar2b1.2* were up-regulated 10 and 24 hours after meal in fish fed the C diet, and at 20 minutes, 2 hours and 6 hours in fish fed the V diet (supplemental information 2). When comparing the two diets, the transcript level of *ffar2b1.2* gene was not differed by the diet proposed but revealed a significant interaction (diet.time) showing an up-regulation at 20 minutes and 10 hours after feeding in fish fed the V and C diet respectively (Figure 2).

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The mRNA levels of *ffar2b2a* were up-regulated 6 and 24 hours after meal in fish fed the C diet, (supplemental information 2). When comparing the two diets, the mRNA levels of the *ffar2b2a* was not differed by the diet proposed (Figure 2).

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The mRNA levels of *ffar2b2b1* were up-regulated only at 10 hours after meal in fish fed the C diet, and at 20 minutes in fish fed the V diet (supplemental information 2). When comparing the two diets, the mRNA levels of the *ffar2b2b1* was not differed by the diet proposed (Figure 2).

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The mRNA levels of *ffar2b2b2* were up-regulated 20 minutes, 10 and 24 hours after meal in fish fed the C diet, and at 20 minutes and 6 hours in fish fed the V diet (supplemental information 2).

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When comparing the two diets, the mRNA levels of the *ffar2b2b2* was not differed by the diet proposed (Figure 2).

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## 3.2. Calcium-signaling pathway gene expression in tongue tissue

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The mRNA levels of calcium-signaling pathway markers in the tongue of RT before meal, 20 minutes, 2, 6, 10 and 24 hours after meal varied (Figure 3) with timing effect (higher) for all transcript by diets (except for *plcb2*). For each diet, the mRNA levels of calcium-signaling pathway markers after meal varied (supplemental information 3).

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The mRNA level of the three isoforms of phospholipase C β were differentially expressed depending to diet proposed. While the mRNA level of the isoform *plcb2* and *plcb4* were not different by the diet proposed with no timing effect (supplemental information 3), the mRNA levels of the isoform *plcb3* were up-regulated 2 hours after meal in fish fed the C diet, and at 24 hours in fish fed the V diet (supplemental information 3). When comparing the two diets, transcript level of *plcb3* gene was up-regulated in fish fed the C diet (diet effect) with significant interaction (diet.time) showing an up-regulation at 2 hours after feeding in fish fed the C diet (Figure 3).

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For the two isoforms of *orail*, the mRNA levels of *oraila* and *b* were up-regulated 2 hours after meal in fish fed the C diet, and at 10 and 24 hours in fish fed the V diet for *oraila* but no difference for fish fed the V diet for *orailb* (supplemental information 3). When comparing the two diets, transcript level of *orailb* gene was up-regulated in fish fed the C diet (diet effect) not for *oraila* but with significant interaction (diet.time) for *oraila* and *orailb* showing an up-regulation at 2 hours after feeding in fish fed the C diet (Figure 3).

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For the two isoforms of *stim1*, the mRNA levels of *stim1a* and *b* were up-regulated 2 hours after meal and 24h only for *stim1b* in fish fed the C diet, and only 24 hours in fish fed the V diet for

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397 *stim1b* but no difference for fish fed the V diet for *stim1a* (supplemental information 3). When comparing the two diets, transcript level of *stim1b* gene was up-regulated in fish fed the C diet (diet effect) not for *stim1a*, but with significant interaction (diet.time) for *stim1a* and *stim1b* showing an up-regulation at 2 hours after feeding in fish fed the C diet (Figure 3).

401 For the two isoforms of *serca3*, the mRNA level of *serca3a* was up-regulated 2 hours after meal in fish fed the C diet, and 10 hours in fish fed the V diet for *stim1b* but no difference for fish fed the C and V diet for *serca3b* and *serca3a* respectively (supplemental information 3). When comparing the two diets, as for *oralb* and *stim1b*, the transcript level of *serca3b* gene was up-regulated in fish fed the C diet (diet effect) not for *serca3a*, but with significant interaction (diet.time) for *serca3b* and *serca3a* showing an up-regulation at 2 hours after feeding in fish fed the C diet (Figure 3).

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### 3.3. Calcium-signaling pathway protein abundance in tongue tissue

409 The protein levels of calcium-signaling pathway markers in the tongue of RT before meal, 20 minutes, 2, 6, 10 and 24 hours after meal varied (Figure 4) with timing effect (higher) for ORAI1 and p-ERK1/2 by diets (Figure 4) and for each diet (supplemental information 4) but not for STIM1.

412 The ORAI abundance was increased 6 hours after meal in fish fed the C and V diet (supplemental information 4). By comparing the two diets, ORAI1 was not differed by the diet proposed (figure 4).

415 Abundance of STIM1 was not modified over the 24 hours following the meal and showed no difference between diets (Figure 4) and for each diet (supplemental information 4).

417 The phosphorylation of ERK1/2 was increased 2 and 6 hours after the meal in fish fed the C diet, and 20 minutes and 2 hours in fish fed the V diet (supplemental information 4). When comparing the two diets (figure 4), the phosphorylation of ERK1/2 was higher in fish fed the C diet (diet effect)

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421 **3.4. Serotonin-signaling pathway in tongue tissue and myelencephalon tissue**
422 The metabolites levels of indolamine turnover in the tongue of RT before meal, 20 minutes,
423 2, 6, 10 and 24 hours after meal varied (Figure 5).

424 The 5-HT was increased early and only 20 minutes after meal in fish fed the C and V diet
425 (supplemental information 5). When comparing the two diets, fish fed the V diet had less 5-HT than
426 fish fed the C diet from 20 min until 24 hours (Figure 5A). For 5-HIAA, the direct degradation
427 products of 5-HT, the metabolite level did not differ between diets (Figure 5A) but was
428 increased for fish fed the V diet at 2 hours compared to 10 hours (supplemental information 5).
429 The ratio 5-HIAA/5-HT ratio was lower for fish fed the C diet with a peak 20 minutes after
430 meal and higher for fish fed the V diet with a peak 2 hours after meal (supplemental information
431 5). Fish fed the V diet had a higher 5-HIAA/5-HT ratio than fish fed the C diet (Figure 5A)
432 from 20 minutes until 24 hours.

433 For the mRNA level of the two tryptophan hydroxylase (isozyme of 5-HT synthesis
434 from tryptophan), while expression of *tph2* was affected by neither time nor diet, for *tph1b*, the
435 mRNA level was higher in myelencephalon for fish fed C diet (Figure 5b). For each diet, the mRNA
436 levels of the two tryptophan hydroxylase were no differed (supplemental information 5).

437 The mRNA level of *sert* (serotonin transporter) was down-regulated after meal in fish fed the
438 C diet, and *mao* (an enzyme responsible for degradation of serotonin) at 10 hours compared to 2 hours
439 in fish fed the V diet (supplemental information 5). When comparing the two diets, significant
440 interactions were detected for mRNA levels of *sert* and *vmat*. These genes were up-regulated
441 for *sert* at 2 hours after feeding in fish fed the V diet and for *vmat2* at 2 hours in fish fed the C
442 diet (Figure 5b).

443 The protein abundance of TPH2 (tryptophan hydroxylase, isozyme of 5-HT synthesis
444 from tryptophan) was decreased 24 hours after meal in fish fed the C and V diet (supplemental
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information 5). When comparing the two diets, TPH2 was not differed by the diet proposed but decrease
after meal (timing effect) (figure 5B).

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The protein abundance of VMAT2 (vesicular monoamine transporter 2 of 5-HT) was
increased at 2 hours, 6 hours and 24 hours after meal in fish fed the C and V diet (supplemental
information 5). When comparing the two diets, VMAT2 was not differed by the diet proposed but
increase after meal (timing effect) (Figure 5B).

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**3.5. Neuropeptide gene expression profile in hypothalamus tissue**

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The mRNA levels of neuropeptide gene in the hypothalamus of RT before meal, 20 minutes, 2,
6, 10 and 24 hours after meal varied (Figure 6) with timing effect (higher) for *npya*, *npyb*, *agrp2* and
*pomcb* and for *cartpt* (lower) by diets. For each diet, the mRNA levels of neuropeptide varied
(supplemental information 6).

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For orexigenic markers, the mRNA level of *npya* was up-regulated 20 minutes and 2 hours
after meal in fish fed the V diet (compared to 6 hours), and only at 2 hours for *npyb* (supplemental
information 6). When comparing the two diets, the transcript level of *npya* gene was not differed by the
diet proposed but revealed a significant interaction (diet.time) showing an up-regulation at 6 hours
and 24 hours after feeding in fish fed the C diet (Figure 6). The mRNA level of *agrp2* was up-regulated
24 hours after meal in fish fed the C diet (supplemental information 6). When comparing the two diets,
the transcript levels of *agrp1* and *agpr2* genes were not differed by the diet proposed (Figure 6).

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For anorexigenic markers, the mRNA levels of *pomcb* were up-regulated 20 minutes after
meal in fish fed the C and V diet (supplemental information 6). When comparing the two diets, the
transcript levels of *pomca* and *pomcb* genes were not differed by the diet proposed (Figure 6). The
mRNA level of *cartpt* was down-regulated 2 hours and 6 hours after meal in fish fed the C and V diet
respectively (supplemental information 6). When comparing the two diets, the transcript level of *cartpt*
gene was not differed by the diet proposed (Figure 6).

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### 3.6. Gene expression of brain activity in telencephalon tissue

The mRNA levels of the brain activity genes in telencephalon of RT before meal, 20 minutes, 2, 6, 10 and 24 hours after meal varied (Figure 7) with timing effect (higher) for all transcripts by diets. For each diet, the mRNA levels of the brain activity genes varied in telencephalon (supplemental information 7).

The mRNA level of *ntkr2b* (receptor activated by BDNF) was up-regulated 2, 6 and 24 hours after meal in fish fed the V diet (supplemental information 7). When comparing the two diets, the transcript level of *ntkr2b* gene was not differed by the diet proposed but revealed a significant interaction (diet.time) showing an up-regulation at 20 minutes and 10 hours after feeding in fish fed the C diet (Figure 7).

The mRNA level of *bdnf* was up-regulated 2, 6 and 10 hours after meal in fish fed the C diet and 2 hours in fish fed the V diet (supplemental information 7). When comparing the two diets, the transcript level of *bdnf* gene was not differed by the diet proposed but revealed a significant interaction (diet.time) showing an up-regulation at 20 minutes after feeding in fish fed the C diet (Figure 7).

The mRNA level of *delta-fosb* was up-regulated 2 hours after meal in fish fed the C diet and from 2 hours until 24 hours in fish fed the V diet (supplemental information 7). When comparing the two diets, the transcript level of *delta-fosb* gene was not differed by the diet proposed but revealed a significant interaction (diet.time) showing an up-regulation at 20 minutes and 24 hours after feeding in fish fed the C and V diet respectively (Figure 7).

The mRNA level of *cfos* was up-regulated 20 minutes and 2 hours after meal in fish fed the C diet and 2 hours and 24 hours in fish fed the V diet (supplemental information 7). When comparing the two diets, the transcript level of *cfos* gene was not differed by the diet proposed (Figure 7).

The mRNA level of *neurabin* was up-regulated 20 minutes, 2 and 10 hours after meal in fish fed the C diet and 2 and 6 hours in fish fed the V diet (supplemental information 7). When comparing
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the two diets, the transcript level of *neurabin* gene was higher in C diet with a significant interaction (diet.time) showing an up-regulation at 20 minutes and 10 hours after feeding in fish fed the C diet (Figure 7).

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The mRNA level of *ncam* was up-regulated after meal in fish fed the C diet and 24 hours in fish fed the V diet (supplemental information 7). When comparing the two diets, the transcript level of *ncam* gene was higher in C diet with a significant interaction (diet.time) showing an up-regulation at 20 minutes after feeding in fish fed the C diet (Figure 7).

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### 3.7. Protein abundance of brain activity in telencephalon tissue

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The protein levels of brain activity markers in the telencephalon of RT before meal, 20 minutes, 2, 6, 10 and 24 hours after meal varied (Figure 8) with timing effect (higher) for BDNF, cFOS and p-ERK1/2 by diets (Figure 9) and for each diet (supplemental information 8) but not for cFOS.

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The BDNF abundance was increase 20 minutes and 2 hours after meal in fish fed the C diet and only 2 hours in fish fed the V diet (supplemental information 8). By comparing the two diets, BDNF was not differed by the diet proposed (Figure 8).

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Abundance of cFOS was not modified over the 24 hours following the meal and showed no difference between diets (Figure 8) and for each diet (supplemental information 8).

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The phosphorylation of ERK1/2 was decreased 6 hours after the meal in fish fed the C diet, and decreased 10 and 24 hours in fish fed the V diet (supplemental information 8). When comparing the two diets (Figure 8), the phosphorylation of ERK1/2 was not differed by the diet proposed but revealed a significant interaction (diet.time) showing an increase at 2 hours and 6 hours after feeding in fish fed the C and V diet respectively.

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# 4. DISCUSSION

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The orogustatory sensing system plays a crucial role in enabling individuals to distinguish between different foodstuffs, aiding in the selection of nutritious feeds and maintaining energy homeostasis. While various factors such as density, hierarchy, temperature, and the species under study can impact diet selection, the instantaneous attraction sparked by the food taste serves as the primary driving force behind food preference in both mammals (Sellayah et al., 2014) and fishe (Lall and Tibbetts, 2009). This study aimed to unravel the signaling pathways involved in the orosensorial detection of nutrients by understanding their activation kinetics, from taste detection to the integrated response at the brain level in RT that were refed with once with a novel food source (V diet). The originality of this study lies in the fact that the V diet had never been consumed or detected in fish orosensory system before. Previous researches has suggested that RT exhibit a dietary preference for diets enriched in DHA (Roy et al., 2020b) and that their sensory system for nutrient detection is less receptive to the V diet compared to C diet (Heraud et al., 2022; Roy et al., 2022). Moreover, whilst the mounting evidence highlights the relationship between fat sensing and its impact on food intake, not only in mammals (Abdoul-Azize et al., 2014; Behrens and Lang, 2022; Besnard et al., 2016; Narukawa M1., n.d.) but also in fish (Kasumyan, 2019; Librán-Pérez et al., 2015a; Luo et al., 2020; Roy et al., 2020a; Velasco et al., 2020, 2017), it is likely that not only the amount of fatty acids in diets modulates fat detection in RT but also the nature of these fatty acids. However, we have recently demonstrated that nutrient detection, especially regarding fatty acids, is modulated after eight months of feeding, starting from the first meal, when trout were fed diets without FM/FO. These groundbreaking results, however, do not allow for an in-depth study of nutrient detection since we observed a long-term chronic effect of the diet (Baranek et al., 2023). This leads to habituation to this diet, thereby altering nutrient detection throughout this period. Our study here is, to our knowledge, the first to investigate the effect of a single meal on nutrient detection in fish, revealing the acute impact on this detection, particularly concerning ω-3 LC-PUFAs.

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Firstly, our results indicated that the expression of all *ffar* genes (*ffar1* and 7 paralogs of *ffar2*), was upregulated after the meal, regardless of the diet provided. These findings align with previous studies in
rodents (Hichami et al., 2022) and humans (Ozdener et al., 2014), which demonstrated increased expression of the *ffar4* gene just after meal. This suggests a potential role for this receptor family in the postprandial fat detection mechanisms. Another study in mice (*Mus booduga*) taste bud cells (TBC) revealed a rapid increase in *ffar4* gene expression following a meal, paralleled by changes at the protein level (Shanmugamprema et al., 2022). Consequently, the authors concluded that this receptor mediates the orogustatory perception of dietary lipids. Regarding the dietary impact, our results unveiled differential upregulation kinetics of *ffar* gene expression. Two paralogs of *ffar2* (*ffar2a2* and *ffar2b1.2*) exhibited early upregulation (at 20 minutes) in fish fed the V diet after the meal compared to those fed the C diet. This effect was even more pronounced when comparing the two diets, with upregulation observed at 20 minutes compared to before the meal for *ffar2a2* and *ffar2b1.2*, as well as for *ffar2b1.1*, *ffar2b2b1*, *ffar2b2b2* and *ffar1*. This effect persisted for up to 2h for *ffar2b1.1*, and up to 6h for *ffar1* and *ffar2b1.2* in fish fed the V diet. In contrast, for C diet, an early upregulation at 20 minutes compared to before the meal was only observed for *ffar1* and *ffar2b2b2* but was not maintained at 2 hours. Additionally, our results indicated delayed regulation of *ffar* gene expression. Paralogs *ffar2b1.1* and *ffar1* displayed more pronounced upregulation in fish fed the C diet from 10 to 24 hours after meal and did not return to basal levels (pre-feeding/fasting period) as observed in fish fed the V diet. When comparing the two diets, this effect was even more prominent with delayed upregulation observed compared to before the meal for all *ffar1* and *ffar2* transcripts at 10, 24 hours or both in fish fed the C diet, and only for *ffar2a1a* and *ffar2a2* at 10 hours in fish fed the V diet.

The observed pattern of clustered expression of these receptor classes is highly intriguing; indicating that both the meal itself and the nature of the meal exert influence over *ffar* expression, whether in the rapid postprandial phase or over a longer term. Specifically, the V diet induced earlier modulation within 6 hours followed by dowregulation, whereas the C diet appeared to trigger this modulation later, starting around 10 hours after the meal. In a related study, Choo and colleagues reported that increased *ffar* expression following a meal was associated with a downregulation of other fat-sensing receptors, such as *cd36* (Choo et al., 2020). Notably, their study also found higher *ffar4* expression after 20 min, coinciding with decreased *cd36* expression, a pattern similar to the kinetics we observed. Furthermore, while some fatty acid receptor,
572 like CD36 in mammals are associated with oral recognition of fatty acids and "taste detection", FFAR4 might
573 particularly play a role in signal amplification for the "sustained sensing" of food. Considering the FFAR
574 expression kinetics in our experiment, it is plausible that *ffar1* and *ffar2b1.1* may be involved in "sustained
575 sensing" along with *ffar2a2* and *ffar2b1.2*, which also appear to have a role in fatty acids recognition in RT.
576 Regarding the diets, the V diet induced a more rapid but less sustained expression of these *ffar* compared to
577 C diet.

578 Our findings regarding the gene expression of FFAR align with our data obtained from the long-
579 term effect (8 months of feeding) of a diet without FM/FO. Specifically, we observed that the gene expression
580 of *ffar1*, *ffar2a1a*, *ffar2b1.1*, and *ffar2b1.2* were significantly upregulated 20 minutes after the meal in fish
581 fed with diet V compared to C diet (Baranek et al., 2023). This dual result is particularly interesting because
582 it appears to demonstrate that both chronic (8 months of feeding) and acute effects (a single meal)
583 significantly impact fat detection in trout, without inducing a chronic effect on the nutrient detection systems
584 in this animal.

585 Furthermore, beyond the impact of a single meal or different diets, the distinct profiles and levels of
586 ω-3 LC-PUFAs could explain the clustered expression patterns of *ffar*. The literature suggests that activation
587 of *ffar1* and *ffar4* may result in anorectic effect, leading to reduced food intake. For instance, Gorski et al.,
588 demonstrated that FFAR1 activation (by specific agonist) in mice led to early reductions in food intake and
589 growth (Gorski et al., 2017). Given that multiple studies have shown upregulation of *ffars* shortly after meal,
590 along with associated protein level changes, FFAR1, 2 and 4 could play critical roles in various physiological
591 homeostasis mechanisms, especially in the regulation of appetite, eating disorders, or food preferences
592 (Ichimura et al., 2012; Kimura et al., 2020; Sclafani et al., 2013; Sleeth et al., 2010). Additionally, Ozdener
593 et al., demonstrated that incubation of TBC with LA (ω-6) induced upregulation of gene expression (Ozdener
594 et al., 2014) in human and mice, suggesting a role for this fatty acid in *ffar4* regulation.

595 Considering the demonstrated anorectic role of these receptors in numerous mammalian studies, we
596 can formulate some hypotheses in the context of our study. Firstly, we observe that the expression of certain
597 *ffar* is influenced by the proposed diet, particularly by its composition. This could have implications for the
abundance of these receptors at the lingual level and, ultimately for feed intake, given their known involvement in regulating food intake in other species. Indeed, in our diet composition, among the major fatty acid present in V diet (more than 10%), ALA stands out as the primary fatty acid with the most substantial difference in content (amounting to 4.96% in the C diet and 18.75% in the V diet, measured as a percentage of fatty acids). Therefore, while ALA could potentially exert rapid modulation on some *ffar2* paralogs, in the C diet, DHA/EPA might influence *ffar1* and certain *ffar2* paralogs, potentially playing a role in signal amplification for the “sustained sensing” of food. It is important to acknowledge that further evidence particularly at the protein level, is required to confirm these observations. While lingual lipases in the oral cavity hydrolyze dietary fatty acids (Kawai and Fushiki, 2003), enabling their release and interaction with taste receptors, we did not use free fatty acids in our study. Therefore, it will be essential to investigate the role of isolated free fatty acids on the activity of trout FFARs determine the impact of free fatty acids on FFAR receptor activation and the regulation of food intake in trout. However, we can speculate that ALA, as in mammals, may modulate *ffar*, leading to a rapid anorectic effect observed in both mammals and RT fed the V diet as demonstrated in one of our previous study (Heraud et al., 2022). On the other hand, EPA and DHA may support food craving until the next meal (between 6 and 10 hours in a breeding situation) in fish fed the C diet.

Additionally, several other studies have underscored the role of various nutrients, such as amino acids, carbohydrates, and vitamins, in modulating fat perception and regulating feeding behavior (Comesaña et al., 2020; Jones, 1990; Kasumyan and Døving, 2003; Kohbara and Caprio, 2001). Additionally, the choice of raw materials, particularly those providing fatty acids, has been shown to be influential (Geurden et al., 2005). These findings collectively suggest that RT are sensitive to a wide range of compounds beyond just fatty acids. Investigating these aspects will be crucial, especially in the context of alternative plant-based products. Finally, our effects observed at later time points (10 and 24 hours) could result from the effects of fatty acids on metabolism, potentially leading to the effects observed at the cerebral level. Therefore, it would be interesting to investigate the mechanisms of the gut-brain axis, the kinetics of intestinal hormone activity, as well as the activity of FFARs, as these receptors have demonstrated their strong expression in these
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metabolic organs in trout (Roy et al., 2022), along with their well-documented roles in mammals (Hara et al., 2014; Itoh et al., 2003; Kimura et al., 2020).

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As anticipated, our results revealed a general upregulation in the expression of numerous genes markers (8 out of 9 genes) and an increase at protein level within the calcium signaling pathways following a meal. Notably, when comparing the dietary effects, a clear trend of upregulation 2 hours after the meal was observed for the C diet compared to V diet as evidenced by most of the markers tested within the calcium signaling pathways at the gene expression level. At the protein level, this trend was confirmed for the phosphorylation of ERK1/2 at 2 hours after the meal with a higher level observed for the C diet. In mice and humans, ERK1/2-MAPK cascade is known to be activated by Ca²⁺ and regulated by FAs. The impact of ERK signaling on fat taste was validated in knockout mice maintained on high-fat diet, which a reduced preference for dietary fatty acids (Khan et al., 2017). Moreover, ablation of CALHM1, which decreases the amplification of calcium elevation after meal in TBC, impairs preference for dietary fat, underscoring the significance of this cascade in orogustatory lipid detection (Subramaniam et al., 2016). These findings suggest that the phosphorylation of ERK1 and ERK2 is a critical step in the fat taste transduction pathway, necessary for dietary fat preference in mammals. Additionally, Murtaza et al., demonstrated that the phosphorylation of ERK1/2 is regulated by FFAR activation, using FFAR4 agonists that increased the phosphorylation of ERK1/ERK2 compared to control conditions in mouse and human TBC (Murtaza et al., 2020). This result implies that ERK1/2 could play a role in fat sensing. While results indicate the involvement of ERK1/2 in nutrient detection 2 hours after a meal, it appears that this involvement occurs later for ORAI1 (6 hours). Although our findings were not confirmed at protein level for STIM and ORAI (only at the gene expression level), the higher phosphorylation of ERK1/2 suggests that at 2 hours after the meal, the intensity of Ca²⁺ signaling is higher in fish fed the C diet compared to the V diet. The activation of FFAR receptor is closely linked to Ca²⁺ signaling which is one of the earliest key signals in gustatory perception, facilitating the transmission of taste information from the tongue to the brain (Hichami et al., 2022). Knowing that, it is crucial to explore the interplay between FFAR, other nutrient receptors, the calcium-signaling pathway and the regulation of food intake particularly in the context of diets with distinct profiles of ω-3 LC-PUFAs.

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Many taste receptors, including those for fatty acids, amino acid or for sweet, bitter, acid and umami tastes, belong to the class of GPCRs (Ahmad and Dalziel, 2020) and their activation are associated with an increase in calcium signaling (Medler, 2015). Therefore, it is imperative to investigate these receptors, as they could be modulated with similar intensity by nutrients in both diets resulting herein in a significant activation of the Ca²⁺ pathway and showing no significant differences between the diets in terms of STIM and ORAI protein level. In particular, the free amino acids present in FM, which are absent from our vegetarian diet, could play a crucial role as stimuli. It would therefore be relevant to explore T1R-type amino acid receptors in future studies and their involvement in the regulation of food intake in fish. Following the intracellular calcium elevation in TBC in the tongue, the gustatory signal is transmitted to the brain through the release of neurotransmitters, among which serotonin plays a crucial role. Our findings reveal a substantial increase in 5-HT levels in the tongue tissue, 20 minutes after the meal in fish fed the C diet, with notably higher levels compared to fish fed the V diet. Given that serotonin is known to play a role in the homeostatic and hedonic regulation of food intake (Tavares et al., 2020; van Galen et al., 2021), our findings suggest that the C diet induces a sense of well-being in fish during the meal, thereby promoting its consumption (Galen et al., 2021). Serotonin level gradually decline after the meal but remains higher than baseline (T0) up to 24 hours, potentially reflecting a sustained sense of well-being that stimulates the desire to eat at the next meal. A similar pattern is observed in fish fed the V diet but the initial increase at 20 minutes is only half as high and is followed by a rapid decrease, resulting in a lower serotonin level than baseline (T0) at 24 h. Importantly, in several species including drosophila, rodents, mammals and crayfish, serotonin is a key neurotransmitter known to play a crucial role in the modulation of cognitive and behavioral functions especially in depressive disorders, motivation, stress or anxiety-like behavior when low levels are detected (Bacqué-Cazenave et al., 2020). In line with findings in mammals (Pepino et al., 2012), the lower release of serotonin observed in the tongues of fish fed the V diet may contribute to reduced oral sensitivity to fatty acid. Consequently, this lower level of serotonin level in fish fed the V diet could lead to a decrease in food intake, as previously observed (Heraud et al., 2022). These results are further supported by the 5-HIAA/Serotonin ratio which reflects serotonin turnover (Bacqué-Cazenave et al., 2020). In fish fed the V diet, this ratio is approximately 2 fold
higher (and higher after the meal). This difference could explain the anorexigenic effect observed in previous studies on RT (Gesto et al., 2018; Johnston and Glanville, 1992). Although this effect occurs more rapidly than alterations in calcium signaling, it aligns with findings in mammals, suggesting an association between calcium signaling pathways, ERK phosphorylation and serotonin release in mouse and human TBC (Subramaniam et al., 2016).

Just as with the FFAR receptors, the data on serotonin turnover were identical to those observed in our previous long-term study. After 8 months of feeding, we observed lower serotonin levels 20 minutes post-meal and a significantly higher 5-HIAA/5-HT ratio after the meal in trout fed with V diet compared to the C diet (Baranek et al., 2023). Once again, this illustrates that both acute and chronic effects of the V diet induce similar impacts on the nutrient detection system, suggesting that nutrient detection and integration systems were rapidly and consistently regulated, without being altered by chronic effects over time.

Furthermore, we observed a similar pattern of serotonin metabolite levels after the meal (increased by the C diet vs. V diet) in the brain regions responsible for serotonin synthesis (myelencephalon; datas not shown). However, previous studies have indicated a higher quantity of serotonin in the fasting period in the telencephalon, a key decision-making center known to receive inputs from serotonin neurons in fish (Rink and Wullimann, 2004; Wullimann and Mueller, 2004), potentially stimulating the desire to eat. In summary, although further confirmation is needed, these findings suggest a reduced food detection at the brain level in fish fed the V diet, which could impact feeding behavior as previously observed from the first meal (Heraud et al., 2022).

Furthermore, most of the neuropeptides implicated in the regulation of satiety showed no significant differences in expression in the hypothalamus between the two diets except for *npya*, an orexigenic neuropeptide. However, although this requires further investigation, the higher expression of *npya* in fish fed the C diet from 6 hours onwards, corresponding to the typical interval until the next meal in normal feeding conditions (such as in fish farming), suggests that fish on the C diet may experience a greater sensation of hunger.
In addition, our findings regarding the serotonin release in mice TBC, which transmits the fat taste response to the brain *via* afferent nerve fibers (El-Yassimi et al., 2008) are relevant to fish. Learning and memory processes are highly sensitive in fish when it comes to changes in feeding habits, and the markers of plasticity and brain activity allow us to study rapid responses related to feeding habit transformation in fish (Dou et al., 2018; Shi et al., 2021). For all the markers of brain activity and plasticity except for *cfos*, we observed lower expression in fish fed the V diet compared to the C diet, even though there was no effect at the protein level for immediate early genes (BDNF and cFOS). While this still requires confirmation, these results suggest that the V diet may disturb feeding habits and feeding behavior in RT. Thus, it confirms that dietary changes can affect feeding habits, as shown in a previous study with different food properties and palatability (Liu, H.; Li, S.; Bai, J., 2015). Therefore, early environmental change, such as introducing a novel food source (V diet here) could transform the feeding habits (after consuming the C diet for 30 days) of fish by influencing the detection of feeding cues in the brain. Our results indicate an early and rapid feeding integrative response in telencephalon of RT, with the maximum peak occurring 2 hours after the meal for gene markers of synaptic plasticity. These genes are known to be markers of memory retention of feeding habits in the telencephalon and were downregulated in fish fed the V diet for the first time with a single meal. In contrast, for mandarin fish (*Siniperca chuatsi*), the acquisition of a novel feed (dead prey fish) is closely associated with an upregulation of these genes in parallel with the acceptance of the new diet (He et al., 2013). Therefore, we can speculate that the downregulation observed in RT fed the V diet is linked to the detection of a novel food source and may potentially signify a programming effect to remember this new food when consumed in the future. These results suggested that RT can early detect a novel food source (V diet) at the brain level in the area of memory and learning, even if the intensity of the feeding integrative response appears to be decreased. As observed in other fish (He et al., 2013), it would be interesting to study the dietary response and the response of these genes during a subsequent meal with this new diet to determine if RT have the capacity to overwrite the original memory of natural food preference for C diet to enhance the acceptance of the new diet (V diet).

726
727

5. CONCLUSION

728

729

730

731

732

733

734

735

In conclusion, our present study has unveiled the high sensitivity of RT to detect a novel food source after a single meal. This novel food source triggers distinct regulatory responses, affecting genomic expression of fat receptors, calcium signaling and inducing early alteration in serotonin turnover. These regulatory changes likely contribute to the detection and subsequent integration of brain responses following a meal. Therefore, environmental changes, particularly the introduction of a novel food source with significant nutrient variations, such as ω-3 LC-PUFAs in the diet, have the potential to disrupt the feeding habits of RT. These disruptions can influence food detection in the brain to such an extent that they affect the fish's feeding behavior and, consequently, their acceptance of the new diet.
736 ABBREVIATIONS

<table><tr><td>737</td><td>5-HIAA</td><td>5-hydroxyindolacetic acid</td></tr><tr><td>738</td><td>5-HT</td><td>5-hydroxytryptamine (serotonin)</td></tr><tr><td>739</td><td>ω-3 LC-PUFA</td><td>omega-3 long chain polyunsaturated fatty acid</td></tr><tr><td>740</td><td>ALA</td><td>α-linolenic acid</td></tr><tr><td>741</td><td>BDNF</td><td>brain-derived neurotrophic factor</td></tr><tr><td>742</td><td>C diet</td><td>commercial-like diet</td></tr><tr><td>743</td><td>DHA</td><td>docosahexaenoic acid</td></tr><tr><td>744</td><td>EPA</td><td>eicosapentaenoic acid</td></tr><tr><td>745</td><td>ERK</td><td>extracellular signal-regulated kinases</td></tr><tr><td>746</td><td>FFAR</td><td>free fatty acid receptor</td></tr><tr><td>747</td><td>FM</td><td>fish meal</td></tr><tr><td>748</td><td>FO</td><td>fish oil</td></tr><tr><td>749</td><td>GPCRs</td><td>G protein-coupled receptors</td></tr><tr><td>750</td><td>ORAI</td><td>calcium release-activated calcium modulator</td></tr><tr><td>751</td><td>PBS</td><td>phosphate buffered saline</td></tr><tr><td>752</td><td>PVDF</td><td>polyvinylidene fluoride</td></tr><tr><td>753</td><td>RT-PCR</td><td>real-time quantitative PCR</td></tr><tr><td>754</td><td>SEM</td><td>standard error of the mean</td></tr><tr><td>755</td><td>STIM</td><td>stromal interaction molecule</td></tr><tr><td>756</td><td>TBC</td><td>taste bud cells</td></tr><tr><td>757</td><td>TPH</td><td>tryptophan hydroxylase</td></tr><tr><td>758</td><td>V diet</td><td>vegetal diet</td></tr><tr><td>759</td><td>VMAT</td><td>vesicular monoamine transporter 2 of serotonin</td></tr><tr><td>760</td><td>RT</td><td>rainbow trout</td></tr><tr><td>761</td><td>UHPLC</td><td>ultra-high-pressure liquid chromatography</td></tr><tr><td>762</td><td></td><td></td></tr></table>

763 AUTHOR'S CONTRIBUTIONS

<table><tr><td>764</td><td>E.B.: methodology, software, data curation, writing-original draft preparation; K.D.: data curation, software, analysis of protein abundance in brain tissues C.H.: data curation, software, analysis of indolamine brain concentration; L.L.: data curation, analyzed lipid content; A.S.: data curation, analysis of diet composition; S.SC.: conceptualization, methodology, writing-review and editing. All authors have read and agreed to the published version of the manuscript; J.R.: conceptualization, methodology, software, data curation, writing-original draft preparation, funding acquisition. All authors have read and agreed to the published version of the manuscript.</td></tr><tr><td>765</td><td></td></tr><tr><td>766</td><td></td></tr><tr><td>767</td><td></td></tr><tr><td>768</td><td></td></tr><tr><td>769</td><td></td></tr><tr><td>770</td><td></td></tr><tr><td>771</td><td></td></tr></table>

772 ACKNOWLEDGMENTS

<table><tr><td>773</td><td>We dedicate this study to Frank Sandres, who cared for the fish during the feeding trial and sadly passed away. We thank Anthony Lanuque and Frederic Terrier for the preparation of the diets, monitoring the feeding experiment and in their help for the sampling for the kinetic experiment.</td></tr><tr><td>774</td><td></td></tr><tr><td>775</td><td></td></tr></table>
776 For the purpose of Open Access, a CC-BY public copyright licence has been applied by the
777 authors to the present document and will be applied to all subsequent versions up to the Author
778 Accepted Manuscript arising from this submission.

779

FUNDING

781 This research was funded by the French National Research Agency (grant no. ANR-21-CE20-0009-01
782 "FEEDOMEGA").

783

784 AVAILABILITY OF DATA AND MATERIALS

785 All data generated or analyzed during this study are available from the corresponding author on reasonable
786 request.

787

788 DECLARATIONS

789

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

790

790 Experiments were conducted at the INRAE facilities of Donzacq, France (authorisation number A40-
791 228.1, Landes). All fish were reared and handled in strict accordance with the French (Decree No 2013-
792 118, 1 February 2013) and European (i.e. Directive 2010/63/EU) animal welfare policies, followed
793 ARRIVE guidelines.

794

795 CONSENT FOR PUBLICATION

796

797 Not applicable.

COMPETING INTERESTS

798

799 The authors declare that they have no competing interests.

AUTHOR DETAILS

800

801 INRAE, University of Pau and Pays de l'Adour, E2S UPPA, UMR1419 Nutrition Metabolism and
802 Aquaculture, Aquapole, F-64310, Saint-Pee-sur-Nivelle, France.

803

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    <td>Librán-Pérez, M., Velasco, C., Otero-Rodiño, C., López-Patiño, M.A., Míguez, J.M., Soengas, J.L., 2015b. Metabolic response in liver and Brockmann bodies of rainbow trout to inhibition of lipolysis; possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis. J. Comp. Physiol. [B] 185, 413-423. https://doi.org/10.1007/s00360-015-0894-8</td>
  </tr>
  <tr>
    <td>904</td>
    <td>Liu, H.; Li, S.; Bai, J., 2015. Research advances in gene regulation and genetic improvement of fish feeding. Agric. Sci. Technol.</td>
  </tr>
  <tr>
    <td>905</td>
    <td>Luo, H., Liang, X.-F., Li, J., Zhang, Y., Xiao, Q., Peng, B., Zhang, Z., 2020. Effect of long-chain saturated and unsaturated fatty acids on hypothalamic fatty acid sensing in Chinese perch (Siniperca chuatsi). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 241, 110395. https://doi.org/10.1016/j.cbpb.2019.110395</td>
  </tr>
  <tr>
    <td>906</td>
    <td>Medler, K.F., 2015. Calcium signaling in taste cells. Biochim. Biophys. Acta 1853, 2025-2032. https://doi.org/10.1016/j.bbamcr.2014.11.013</td>
  </tr>
  <tr>
    <td>907</td>
    <td>Murtaza, B., Hichami, A., Khan, A.S., Shimpukade, B., Ulven, T., Ozdener, M.H., Khan, N.A., 2020. Novel GPR120 agonist TUG891 modulates fat taste perception and preference and activates tongue-brain-gut axis in mice. J. Lipid Res. 61, 133-142. https://doi.org/10.1194/jlr.RA119000142</td>
  </tr>
  <tr>
    <td>908</td>
    <td>Narukawa M1., n.d. Physiological responses to taste signals of functional food components. Biosci Biotechnol Biochem. https://doi.org/2018 Feb;82(2):200-206. doi: 10.1080/09168451.2017.1422385. Epub 2018 Jan 12.</td>
  </tr>
  <tr>
    <td>909</td>
    <td>Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin, D.M., Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era of finite resources. Proc. Natl. Acad. Sci. 106, 15103-15110. https://doi.org/10.1073/pnas.0905235106</td>
  </tr>
  <tr>
    <td>910</td>
    <td>Ozdener, M.H., Subramaniam, S., Sundaresan, S., Sery, O., Hashimoto, T., Asakawa, Y., Besnard, P., Abumrad, N.A., Khan, N.A., 2014. CD36- and GPR120-mediated Ca2+ Signaling in Human Taste Bud Cells Mediates Differential Responses to Fatty Acids and is Altered in Obese Mice. Gastroenterology 146, 995-1005.e5. https://doi.org/10.1053/j.gastro.2014.01.006</td>
  </tr>
  <tr>
    <td>911</td>
    <td>Pepino, M.Y., Love-Gregory, L., Klein, S., Abumrad, N.A., 2012. The fatty acid translocase gene CD36 and lingual lipase influence oral sensitivity to fat in obese subjects. J. Lipid Res. 53, 561-566. https://doi.org/10.1194/jlr.M021873</td>
  </tr>
  <tr>
    <td>912</td>
    <td>Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2003-2007. https://doi.org/10.1093/nar/29.9.e45</td>
  </tr>
  <tr>
    <td>913</td>
    <td>R: The R Project for Statistical Computing. https://www.r-project.org/ (accessed January 2023), n.d.</td>
  </tr>
  <tr>
    <td>914</td>
    <td>Rink, E., Wullimann, M.F., 2004. Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio). Brain Res. 1011, 206-220. https://doi.org/10.1016/j.brainres.2004.03.027</td>
  </tr>
  <tr>
    <td>915</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>916</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>917</td>
    <td>Librán-Pérez, M., Geurden, I., Dias, K., Corraze, G., Panserat, S., Soengas, J.L., 2015a. Feeding rainbow trout with a lipid-enriched diet: effects on fatty acid sensing, regulation of food intake and cellular signaling pathways. J. Exp. Biol. 218, 2610-2619. https://doi.org/10.1242/jeb.123802</td>
  </tr>
  <tr>
    <td>918</td>
    <td>Librán-Pérez, M., Otero-Rodiño, C., López-Patiño, M.A., Míguez, J.M., Soengas, J.L., 2014. Central administration of oleate or octanoate activates hypothalamic fatty acid sensing and inhibits food intake in rainbow trout. Physiol. Behav. 129, 272-279. https://doi.org/10.1016/j.physbeh.2014.02.061</td>
  </tr>
  <tr>
    <td>919</td>
    <td>Librán-Pérez, M., Velasco, C., Otero-Rodiño, C., López-Patiño, M.A., Míguez, J.M., Soengas, J.L., 2015b. Metabolic response in liver and Brockmann bodies of rainbow trout to inhibition of lipolysis; possible involvement of the hypothalamus-pituitary-interrenal (HPI) axis. J. Comp. Physiol. [B] 185, 413-423. https://doi.org/10.1007/s00360-015-0894-8</td>
  </tr>
  <tr>
    <td>920</td>
    <td>Liu, H.; Li, S.; Bai, J., 2015. Research advances in gene regulation and genetic improvement of fish feeding. Agric. Sci. Technol.</td>
  </tr>
  <tr>
    <td>921</td>
    <td>Luo, H., Liang, X.-F., Li, J., Zhang, Y., Xiao, Q., Peng, B., Zhang, Z., 2020. Effect of long-chain saturated and unsaturated fatty acids on hypothalamic fatty acid sensing in Chinese perch (Siniperca chuatsi). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 241, 110395. https://doi.org/10.1016/j.cbpb.2019.110395</td>
  </tr>
  <tr>
    <td>922</td>
    <td>Medler, K.F., 2015. Calcium signaling in taste cells. Biochim. Biophys. Acta 1853, 2025-2032. https://doi.org/10.1016/j.bbamcr.2014.11.013</td>
  </tr>
  <tr>
    <td>923</td>
    <td>Murtaza, B., Hichami, A., Khan, A.S., Shimpukade, B., Ulven, T., Ozdener, M.H., Khan, N.A., 2020. Novel GPR120 agonist TUG891 modulates fat taste perception and preference and activates tongue-brain-gut axis in mice. J. Lipid Res. 61, 133-142. https://doi.org/10.1194/jlr.RA119000142</td>
  </tr>
  <tr>
    <td>924</td>
    <td>Narukawa M1., n.d. Physiological responses to taste signals of functional food components. Biosci Biotechnol Biochem. https://doi.org/2018 Feb;82(2):200-206. doi: 10.1080/09168451.2017.1422385. Epub 2018 Jan 12.</td>
  </tr>
  <tr>
    <td>925</td>
    <td>Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin, D.M., Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era of finite resources. Proc. Natl. Acad. Sci. 106, 15103-15110. https://doi.org/10.1073/pnas.0905235106</td>
  </tr>
  <tr>
    <td>926</td>
    <td>Ozdener, M.H., Subramaniam, S., Sundaresan, S., Sery, O., Hashimoto, T., Asakawa, Y., Besnard, P., Abumrad, N.A., Khan, N.A., 2014. CD36- and GPR120-mediated Ca2+ Signaling in Human Taste Bud Cells Mediates Differential Responses to Fatty Acids and is Altered in Obese Mice. Gastroenterology 146, 995-1005.e5. https://doi.org/10.1053/j.gastro.2014.01.006</td>
  </tr>
  <tr>
    <td>927</td>
    <td>Pepino, M.Y., Love-Gregory, L., Klein, S., Abumrad, N.A., 2012. The fatty acid translocase gene CD36 and lingual lipase influence oral sensitivity to fat in obese subjects. J. Lipid Res. 53, 561-566. https://doi.org/10.1194/jlr.M021873</td>
  </tr>
  <tr>
    <td>928</td>
    <td>Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2003-2007. https://doi.org/10.1093/nar/29.9.e45</td>
  </tr>
  <tr>
    <td>929</td>
    <td>R: The R Project for Statistical Computing. https://www.r-project.org/ (accessed January 2023), n.d.</td>
  </tr>
  <tr>
    <td>930</td>
    <td>Rink, E., Wullimann, M.F., 2004. Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio). Brain Res. 1011, 206-220. https://doi.org/10.1016/j.brainres.2004.03.027</td>
  </tr>
  <tr>
    <td>931</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>932</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>933</td>
    <td>Medler, K.F., 2015. Calcium signaling in taste cells. Biochim. Biophys. Acta 1853, 2025-2032. https://doi.org/10.1016/j.bbamcr.2014.11.013</td>
  </tr>
  <tr>
    <td>934</td>
    <td>Murtaza, B., Hichami, A., Khan, A.S., Shimpukade, B., Ulven, T., Ozdener, M.H., Khan, N.A., 2020. Novel GPR120 agonist TUG891 modulates fat taste perception and preference and activates tongue-brain-gut axis in mice. J. Lipid Res. 61, 133-142. https://doi.org/10.1194/jlr.RA119000142</td>
  </tr>
  <tr>
    <td>935</td>
    <td>Narukawa M1., n.d. Physiological responses to taste signals of functional food components. Biosci Biotechnol Biochem. https://doi.org/2018 Feb;82(2):200-206. doi: 10.1080/09168451.2017.1422385. Epub 2018 Jan 12.</td>
  </tr>
  <tr>
    <td>936</td>
    <td>Naylor, R.L., Hardy, R.W., Bureau, D.P., Chiu, A., Elliott, M., Farrell, A.P., Forster, I., Gatlin, D.M., Goldburg, R.J., Hua, K., Nichols, P.D., 2009. Feeding aquaculture in an era of finite resources. Proc. Natl. Acad. Sci. 106, 15103-15110. https://doi.org/10.1073/pnas.0905235106</td>
  </tr>
  <tr>
    <td>937</td>
    <td>Ozdener, M.H., Subramaniam, S., Sundaresan, S., Sery, O., Hashimoto, T., Asakawa, Y., Besnard, P., Abumrad, N.A., Khan, N.A., 2014. CD36- and GPR120-mediated Ca2+ Signaling in Human Taste Bud Cells Mediates Differential Responses to Fatty Acids and is Altered in Obese Mice. Gastroenterology 146, 995-1005.e5. https://doi.org/10.1053/j.gastro.2014.01.006</td>
  </tr>
  <tr>
    <td>938</td>
    <td>Pepino, M.Y., Love-Gregory, L., Klein, S., Abumrad, N.A., 2012. The fatty acid translocase gene CD36 and lingual lipase influence oral sensitivity to fat in obese subjects. J. Lipid Res. 53, 561-566. https://doi.org/10.1194/jlr.M021873</td>
  </tr>
  <tr>
    <td>939</td>
    <td>Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2003-2007. https://doi.org/10.1093/nar/29.9.e45</td>
  </tr>
  <tr>
    <td>940</td>
    <td>R: The R Project for Statistical Computing. https://www.r-project.org/ (accessed January 2023), n.d.</td>
  </tr>
  <tr>
    <td>941</td>
    <td>Rink, E., Wullimann, M.F., 2004. Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio). Brain Res. 1011, 206-220. https://doi.org/10.1016/j.brainres.2004.03.027</td>
  </tr>
  <tr>
    <td>942</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>943</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>944</td>
    <td>Pepino, M.Y., Love-Gregory, L., Klein, S., Abumrad, N.A., 2012. The fatty acid translocase gene CD36 and lingual lipase influence oral sensitivity to fat in obese subjects. J. Lipid Res. 53, 561-566. https://doi.org/10.1194/jlr.M021873</td>
  </tr>
  <tr>
    <td>945</td>
    <td>Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2003-2007. https://doi.org/10.1093/nar/29.9.e45</td>
  </tr>
  <tr>
    <td>946</td>
    <td>R: The R Project for Statistical Computing. https://www.r-project.org/ (accessed January 2023), n.d.</td>
  </tr>
  <tr>
    <td>947</td>
    <td>Rink, E., Wullimann, M.F., 2004. Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio). Brain Res. 1011, 206-220. https://doi.org/10.1016/j.brainres.2004.03.027</td>
  </tr>
  <tr>
    <td>948</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>949</td>
    <td>R: The R Project for Statistical Computing. https://www.r-project.org/ (accessed January 2023), n.d.</td>
  </tr>
  <tr>
    <td>950</td>
    <td>Rink, E., Wullimann, M.F., 2004. Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio). Brain Res. 1011, 206-220. https://doi.org/10.1016/j.brainres.2004.03.027</td>
  </tr>
  <tr>
    <td>951</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>952</td>
    <td>Roy, J., Baranek, E., Marandel, L., 2023. Characterization of free fatty acid receptor family in rainbow trout (Oncorhynchus mykiss): towards a better understanding of their involvement in fatty acid signalisation. BMC Genomics 24, 130. https://doi.org/10.1186/s12864-023-09181-z</td>
  </tr>
  <tr>
    <td>953</td>
    <td>Pepino, M.Y., Love-Gregory, L., Klein, S., Abumrad, N.A., 2012. The fatty acid translocase gene CD36 and lingual lipase influence oral sensitivity to fat in obese subjects. J. Lipid Res. 53, 561-566. https://doi.org/10.1194/jlr.M021873</td>
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    <td>954</td>
    <td>Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, 2003-2007. https://doi.org/10.1093/nar/29.9.e45</td>
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958

959 Roy, J., Larroquet, L., Surget, A., Lanuque, A., Sandres, F., Terrier, F., Corraze, G., Chung-Yung Lee, J., Skiba-Cassy, S.,

960 2020a. Impact on cerebral function in rainbow trout fed with plant based omega-3 long chain polyunsaturated fatty acids enriched with DHA and EPA. Fish Shellfish Immunol. 103, 409–420. https://doi.org/10.1016/j.fsi.2020.05.044

961

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963 S., 2020b. Rainbow trout prefer diets rich in omega-3 long chain polyunsaturated fatty acids DHA and EPA. Physiol.

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966 Oger, C., Galano, J.-M., Corraze, G., Durand, T., 2020c. Characterization and modulation of brain lipids content of rainbow trout fed with 100% plant based diet rich in omega-3 long chain polyunsaturated fatty acids DHA and EPA.

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978 Shanmugamprema, D., Muthuswamy, K., Subramanian, G., Ponnusamy, V., Krishnan, V., Subramaniam, S., 2020. Fat taste signal transduction and its possible negative modulator components. Prog. Lipid Res. 79, 101035.

979 Shanmugamprema, D., Muthuswamy, K., Subramanian, G., Ponnusamy, V., Krishnan, V., Subramaniam, S., 2020. Fat taste signal transduction and its possible negative modulator components. Prog. Lipid Res. 79, 101035.

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986 Sensing: a proposed role for fibre, fermentable carbohydrates and short-chain fatty acids in appetite regulation. Nutr. Res. Rev. 23, 135–145. https://doi.org/10.1017/S0954422410000089

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1009 Velasco, C., Conde-Sieira, M., Comesaña, S., Chivite, M., Díaz-Rúa, A., Míguez, J.M., Soengas, J.L., 2020. The long-chain fatty acid receptors FFA1 and FFA4 are involved in food intake regulation in fish brain. J. Exp. Biol. 223, jeb227330.

1010 https://doi.org/10.1242/jeb.227330

1011 Velasco, C., Conde-Sieira, M., Comesaña, S., Chivite, M., Díaz-Rúa, A., Míguez, J.M., Soengas, J.L., 2020. The long-chain fatty acid receptors FFA1 and FFA4 are involved in food intake regulation in fish brain. J. Exp. Biol. 223, jeb227330. https://doi.org/10.1242/jeb.227330

1012 Velasco, C., Conde-Sieira, M., Comesaña, S., Chivite, M., Díaz-Rúa, A., Míguez, J.M., Soengas, J.L., 2020. The long-chain fatty acid receptors FFA1 and FFA4 are involved in food intake regulation in fish brain. J. Exp. Biol. 223, jeb227330. https://doi.org/10.1242/jeb.227330

1013

1014 Velasco, C., Librán-Pérez, M., Otero-Rodiño, C., López-Patiño, M.A., Míguez, J.M., Soengas, J.L., 2016. Ceramides are involved in the regulation of food intake in rainbow trout (Oncorhynchus mykiss). Am. J. Physiol. Regul. Integr. Comp. Physiol. 311, R658–R668. https://doi.org/10.1152/ajpregu.00201.2016

1015 Velasco, C., Librán-Pérez, M., Otero-Rodiño, C., López-Patiño, M.A., Míguez, J.M., Soengas, J.L., 2016. Ceramides are involved in the regulation of food intake in rainbow trout (Oncorhynchus mykiss). Am. J. Physiol. Regul. Integr. Comp. Physiol. 311, R658–R668. https://doi.org/10.1152/ajpregu.00201.2016

1016

1017 Wullimann, M.F., Mueller, T., 2004. Teleostean and mammalian forebrains contrasted: Evidence from genes to behavior. J. Comp. Neurol. 475, 143–162. https://doi.org/10.1002/cne.20183

1018

1019

1020
1022 TABLE.

1023 Table 1. Ingredients and composition of the experimental diets.

<table><thead><tr><th rowspan="2">Ingredient (%)</th><th colspan="2">DIET</th></tr><tr><th>Commercial-like diet</th><th>Plant-based diet</th></tr></thead><tbody><tr><td>Fish meal</td><td>19.0</td><td>0.0</td></tr><tr><td>Soybean meal</td><td>11.0</td><td>18.0</td></tr><tr><td>Extruded whole wheat</td><td>13.5</td><td>11.5</td></tr><tr><td>Corn gluten</td><td>6.0</td><td>15.2</td></tr><tr><td>Wheat gluten</td><td>0.0</td><td>8.0</td></tr><tr><td>Soy protein concentrate</td><td>18.0</td><td>6.8</td></tr><tr><td>Fababean protein concentrate</td><td>9.5</td><td>8.0</td></tr><tr><td>Soy lecithin</td><td>2.5</td><td>2.5</td></tr><tr><td>L-Lysine</td><td>0.6</td><td>7.16</td></tr><tr><td>L-methionine</td><td>0.6</td><td>0.6</td></tr><tr><td>CaHPO4.2H2O</td><td>0.1</td><td>1.44</td></tr><tr><td>Mineral premixa</td><td>1.4</td><td>2.0</td></tr><tr><td>Vitamin premixb</td><td>1.3</td><td>1.2</td></tr><tr><td>Fish oil</td><td>8.0</td><td>0.0</td></tr><tr><td>Rapeseed oil</td><td>8.5</td><td>8.0</td></tr><tr><td>Palm oil</td><td>0.0</td><td>3.6</td></tr><tr><td>Linseed oil</td><td>0.0</td><td>6.0</td></tr></tbody></table>

<table><thead><tr><th colspan="3">Composition (% of dry matter)</th></tr></thead><tbody><tr><td>Dry matter (in % of diet)</td><td>97.60</td><td>94.83</td></tr><tr><td>Crude protein</td><td>45.28</td><td>46.95</td></tr><tr><td>Crude lipid</td><td>23.6</td><td>20.35</td></tr><tr><td>Starch</td><td>17.66</td><td>11.37</td></tr><tr><td>Ash</td><td>4.10</td><td>7.58</td></tr><tr><td>Energy (kJg-1 DM)</td><td>24.49</td><td>24.33</td></tr></tbody></table>

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

1035

1036

1037

1038

1039

1040

<footnote>a Mineral premix: (g or mg kg⁻¹ diet): calcium carbonate (40% Ca), 2.15 g; magnesium oxide (60% Mg), 1.24 g; ferric citrate, 0.2 g; potassium iodide (75% I), 0.4 mg; zinc sulphate (36% Zn), 0.4 g; copper sulphate (25% Cu), 0.3 g; manganese sulphate (33% Mn), 0.3 g; dibasic calcium phosphate (20% Ca, 18% P), 5 g; cobalt sulphate, 2 mg; sodium selenite (30% Se), 3 mg; KCl, 0.9 g; NaCl, 0.4 g (UPAE, INRAE). b Vitamin premix: (IU or mg kg⁻¹ diet): DL-a tocopherol acetate, 60 IU; sodium menadione bisulphate, 5 mg; retinyl acetate, 15,000 IU; DL-cholecalciferol, 3000 IU; thiamin, 15 mg; riboflavin, 30 mg; pyridoxine, 15 mg; B12, 0.05 mg; nicotinic acid, 175 mg; folic acid, 500 mg; inositol, 1000 mg; biotin, 2.5 mg; calcium pantothenate, 50 mg; choline chloride, 2000 mg (UPAE, INRAE).</footnote>
<table><caption>Table 2. Fatty acid composition of experimental diets (% of total fatty acids).</caption><thead><tr><th rowspan="2"></th><th colspan="2">Diet</th></tr><tr><th>Commercial-like diet</th><th>Plant-based diet</th></tr></thead><tbody><tr><td>C12:0</td><td>0.08</td><td>0.22</td></tr><tr><td>C14:0</td><td>3.76</td><td>0.65</td></tr><tr><td>C15:0</td><td>0.23</td><td>0.05</td></tr><tr><td>C16:0</td><td>12.78</td><td>13.65</td></tr><tr><td>C17:0</td><td>0.23</td><td>0.09</td></tr><tr><td>C18:0</td><td>2.76</td><td>2.81</td></tr><tr><td>C20:0</td><td>0.35</td><td>0.34</td></tr><tr><td>C22:0</td><td>0.22</td><td>0.19</td></tr><tr><td>C24:0</td><td>0.04</td><td>0.09</td></tr><tr><td>Sum of saturated fatty acids</td><td><strong>20.45</strong></td><td><strong>18.09</strong></td></tr><tr><td>C14:1 ω-7</td><td>0.09</td><td>0.0</td></tr><tr><td>C16:1 ω-7</td><td>3.76</td><td>0.55</td></tr><tr><td>C17:1 ω-7</td><td>0.1</td><td>0.04</td></tr><tr><td>C18:1 ω-9</td><td>33.06</td><td>38.55</td></tr><tr><td>C20:1 ω-9</td><td>1.3</td><td>0.61</td></tr><tr><td>C22:1 ω-9</td><td>0.9</td><td>0.12</td></tr><tr><td>Sum of MUFAs</td><td><strong>39.20</strong></td><td><strong>39.87</strong></td></tr><tr><td>C16:2 ω-4</td><td>0.76</td><td>0.02</td></tr><tr><td>C16:3 ω-4</td><td>0.83</td><td>0.12</td></tr><tr><td>C18:2 ω-4</td><td>0.19</td><td>0.02</td></tr><tr><td>C18:3 ω-4</td><td>0.1</td><td>0.08</td></tr><tr><td>Sum of ω-4 PUFAs</td><td><strong>1.88</strong></td><td><strong>0.24</strong></td></tr><tr><td>C18:2 ω-6 (LA)</td><td>15.86</td><td>21.23</td></tr><tr><td>C18:3 ω-6</td><td>0.14</td><td>0.0</td></tr><tr><td>C20:2 ω-6</td><td>0.10</td><td>0.05</td></tr><tr><td>C20:3 ω-6</td><td>0.06</td><td>0.0</td></tr><tr><td>C20:4 ω-6 (ARA)</td><td>0.48</td><td>0.0</td></tr><tr><td>C22:2 ω-6</td><td>0.0</td><td>0.08</td></tr><tr><td>C22:4 ω-6</td><td>0.0</td><td>0.0</td></tr><tr><td>C22:5 ω-6</td><td>0.12</td><td>0.0</td></tr><tr><td>Sum of ω-6 LC-PUFAs</td><td><strong>16.76</strong></td><td><strong>21.36</strong></td></tr><tr><td>C16:4 ω-3</td><td>0.0</td><td>0.0</td></tr><tr><td>C18:3 ω-3 (ALA)</td><td>4.96</td><td>18.75</td></tr><tr><td>C18:4 ω-3</td><td>0.93</td><td>0.09</td></tr><tr><td>C20:3 ω-3</td><td>0.0</td><td>0.0</td></tr><tr><td>C20:4 ω-3</td><td>0.3</td><td>0.0</td></tr><tr><td>C20:5 ω-3 (EPA)</td><td>7.94</td><td>0.77</td></tr><tr><td>C21:5 ω-3</td><td>0.32</td><td>0.05</td></tr><tr><td>C22:5 ω-3</td><td>0.96</td><td>0.08</td></tr><tr><td>C22:6 ω-3 (DHA)</td><td>4.06</td><td>0.39</td></tr><tr><td>Sum of ω-3 LC-PUFAs</td><td><strong>19.47</strong></td><td><strong>20.13</strong></td></tr><tr><td>Sum of ω-3 (EPA + DHA)</td><td><strong>12.0</strong></td><td><strong>1.16</strong></td></tr><tr><td>ω-3 (DHA + EPA) / ω-6</td><td><strong>0.72</strong></td><td><strong>0.05</strong></td></tr></tbody></table>
<table><caption>Table 3. Nucleotide sequences of the PCR primers used to evaluate mRNA abundance of transcripts by real-time quantitative PCR.</caption><thead><tr><th>Transcript</th><th>Forward Primer</th><th>Reverse Primer</th><th>Accession Number</th></tr></thead><tbody><tr><td colspan="4"><strong>Reference</strong></td></tr><tr><td>eef1a1</td><td>TCCTCTTGGTCGTTTCGCTG</td><td>ACCCGAGGGACATCCTGTG</td><td>ENSOMYG000000038328</td></tr><tr><td>krt8b</td><td>TGGCTACTCCAGTGGTTTCG</td><td>CCGCTACCGGAGCTGTAGTT</td><td>ENSOMYG000000057091</td></tr><tr><td colspan="4"><strong>ffar receptor</strong></td></tr><tr><td>ffar1</td><td>ACTGTTGCACCTGAGTCTGG</td><td>GCTGGTCCTGGGTGAAGTTC</td><td>ENSOMYG000000041396</td></tr><tr><td>ffar2a1a</td><td>CCGAGTTCCTCTGCTCCATC</td><td>TAGGTGATGGGGAAGGCAAC</td><td>ENSOMYG00000004986</td></tr><tr><td>ffar2a2</td><td>GACAACTTCACCCAGGAGCA</td><td>AGCAGAACACACAGGCCAG</td><td>ENSOMYG000000030315</td></tr><tr><td>ffar2b1.1</td><td>TTTTCCACACACAGTTGGCC</td><td>AGGTAGTGTTGTCGGCATCT</td><td>ENSOMYG000000041393</td></tr><tr><td>ffar2b1.2</td><td>GTGTGGCCTTCCCTATCAGA</td><td>GCAGGGCACAATGTACACAA</td><td>ENSOMYG000000041387</td></tr><tr><td>ffar2b2a</td><td>CCCATCCAACACTCGCTGAA</td><td>TGATGACGACGATGCTCAGG</td><td>ENSOMYG000000030493</td></tr><tr><td>ffar2b2b1</td><td>TGACCGCAATCAGTGTCGAA</td><td>CCCAGAAGAAGACGCTAGCC</td><td>ENSOMYG000000030500</td></tr><tr><td>ffar2b2b2</td><td>GTCCAGTACCATCAACGCCA</td><td>CTGCACACTCTCCAACAGGGT</td><td>ENSOMYG000000005604</td></tr><tr><td colspan="4"><strong>ffar signaling pathways</strong></td></tr><tr><td>plcb2</td><td>TTATCACCCACGGCTTCACC</td><td>GGAGAGGATGACGGGGTACT</td><td>ENSOMYG000000026272</td></tr><tr><td>plcb3</td><td>ATAGTGGACGGCATCGTAGC</td><td>TGTGTCAGCAGGAAGTCCAA</td><td>ENSOMYG000000038153</td></tr><tr><td>plcb4</td><td>CCGAGGCGTCTGAGGATAAC</td><td>GTAGGGGTGGATGTTGGTGG</td><td>ENSOMYG000000018058</td></tr><tr><td>stim1a</td><td>GAACCTTGACCCCAGCATCA</td><td>GGAGATTGCAGAGTACCGGG</td><td>ENSOMYG000000038250</td></tr><tr><td>stim1b</td><td>CGGACGTGTACAACTGGACA</td><td>CCTTCCGTCCAGATTGTGCT</td><td>ENSOMYG000000011142</td></tr><tr><td>orai1a</td><td>CTATGGTGGAGGTGCAGCTG</td><td>TCATCAGGGCGAAAAGGTGC</td><td>ENSOMYG000000005009</td></tr><tr><td>orai1b</td><td>TGAAGGCTTCCAGTCGAACG</td><td>GGCACTGAAGGCAATGAGGA</td><td>ENSOMYT00000069145</td></tr><tr><td>atp2a3a (SERCA3a)</td><td>GCTGTTATCACCACCTGCCT</td><td>AGATGACAGAAGTCAGCCC</td><td>ENSOMYT00000017316</td></tr><tr><td>atp2a3b (SERCA3b)</td><td>CCTGCGTCTCCTTTGTGCTA</td><td>CAGACTCCGATCACAGCGTT</td><td>ENSOMYG000000012673</td></tr><tr><td colspan="4"><strong>Brain activity</strong></td></tr><tr><td>ntkr2b</td><td>ACTATCCTGGAGCTGCTGGA</td><td>CTGTTCGTGGAGCTCTTGGT</td><td>ENSOMYG000000021208</td></tr><tr><td>bdnf</td><td>GCTGCCGTGGAATAGACAAG</td><td>TCCTTATAAACCGCCAGCCA</td><td>ENSOMYG000000036728</td></tr><tr><td>delta fosb</td><td>TGCAGCCAACTCTCATCTCG</td><td>GAGGAATAACTGGGCCCTGG</td><td>ENSOMYG000000009639</td></tr><tr><td>cfos</td><td>AACAGACTCTCCATGGCAGT</td><td>TGCTGATGTGATGACGGTGG</td><td>ENSOMYT00000070275.1</td></tr><tr><td>neurabin</td><td>AGGAGAGAGAGGAGACAGCA</td><td>TTCCTCCGCCTGTTTCTCAT</td><td>ENSOMYP00000130463</td></tr><tr><td>ncam</td><td>GCTAACGTCACCAAAGCCAA</td><td>GGCAGCAGTACAGTTGTAGC</td><td>ENSOMYP00000054444</td></tr><tr><td colspan="4"><strong>Neuropeptide</strong></td></tr><tr><td>npya</td><td>AAGGCAGAGGTGAGTGCTGT</td><td>AGCCTGTGGCTCACTAATC AA</td><td>ENSOMYG000000006572</td></tr><tr><td>ncyb</td><td>CGTATTTGTTAGCGCCTTTC</td><td>TCATTCGATTTCTTCACTT CCA</td><td>ENSOMYG000000013012</td></tr><tr><td>agrp1</td><td>ACCAGCAGTCCTGTCTGGGTAA</td><td>AGTAGCAGATGGAGCCGAACA</td><td>ENSOMYG000000068541</td></tr><tr><td>agrp2</td><td>CCAGGAGACGGATTTTGCCA</td><td>GAGGGGCACAGCTATCCATC</td><td>ENSOMYG000000066626</td></tr><tr><td>pomca</td><td>ACAAGATGAACCACTTCCGCT</td><td>TGAACAACGTGAGCAGTGGT</td><td>ENSOMYG000000014502</td></tr><tr><td>pomcb</td><td>AGAGCTCCCACCATCTCGT</td><td>GCCGCCCCATCAAAGTCTAT</td><td>ENSOMYG00000002756</td></tr><tr><td>cartpt</td><td>ACCATGGAGAGCTCCAG</td><td>GCGCACTGCTCTCCAA</td><td>ENSOMYG000000036163</td></tr><tr><td colspan="4"><strong>Indolamine metabolism</strong></td></tr><tr><td>tph1b</td><td>AGCGTCCGTTTACAGTGAGG</td><td>GCCCACGATGTCCAGTTCAT</td><td>ENSOMYG000000000567</td></tr><tr><td>tph2</td><td>AGCACCTCAAAGACCACGTC</td><td>ACTGGTCGAGCTCTGCAATC</td><td>ENSOMYG000000010750</td></tr><tr><td>slc6a4a (sert)</td><td>CCTGCTGCCCTACATGTTGA</td><td>GGGGCAGATGTGTTTCCAGA</td><td>ENSOMYG000000028988</td></tr><tr><td>slc18a2 (vmat2)</td><td>TCGCCTTGCTGCTGGATAAT</td><td>GAGGCCGTGGTGTGATTCTT</td><td>ENSOMYG000000010158</td></tr><tr><td>mao</td><td>GGTACGTACAAGCAGTGTGGA</td><td>GCTCCTTGGCCATACACTCA</td><td>ENSOMYT000000086290.1</td></tr></tbody></table>

agrp (1 and 2), agouti-related-peptide; *atp2a3* (a, b), sarcoendoplasmic reticulum calcium transport
ATPase (SERCA); *bdnf*, brain-derived neurotrophic factor; *cartpt*, amphetamine-related transcript; *delta fosb*,
*cfos*, proto-oncogen factor; *efla*, elongation factor 1α; *ffar*, (1, 21a1, 2a2, 2b1.1, 2b1.2, 2b2a, 2b2b1, 2b2b2), free
fatty acid; *krt8b*, keratin 8b; *mao*, monoamine oxydase; *ncam*, neural cell adhesion molecule; *neurabin*, F-actin-
binding protein; *npy* (a, b), neuropeptide Y; *ntkr2b*, neurotrophic tyrosine kinase receptor; *orai* (1a, 1b), calcium
1051 release-activated calcium modulator; *plcb* (2, 3, 4), phospholipase C-β; stim (1a, 1b), (stromal interaction molecule);
1052 *pomc* (a, b), proopiomelanocortin; *slc6a4a* (sert), serotonin transporter; *slc18a2* (vmat2), vesicular monoamine
1053 transporter 2; *tph* (1b, 2) tryptophan hydroxylase.
1054

1054

FIGURE LEGENDS

1055 **Figure 2. Relative expression of mRNA of *ffar* after meal in tongue tissue**

1056 Relative gene expression measured by RT-PCR of *ffar* genes (*ffar1*, *ffar2a1a*, *ffar2a2*, *ffar2b1.1*, *ffar2b1.2*, *ffar2b2a*, *ffar2b2b1*, *ffar2b2b2*) in tongue tissue of RT fed the C or V diet 20 mns, 2, 6, 10 or 24 hours after meal. Datas are expressed as group mean ± SEM; fold change 2<sup>-ΔΔCt</sup> vs before meal (T0) for all genes. Two-way ANOVA following by one-way ANOVA when the interaction is significant, Tukey's post-hoc test when appropriate; an asterik indicate a significant difference (*p* < 0.05) between diet (n = 6).

1063

1064 **Figure 3. Relative expression of mRNA encoding *ffar*-signaling pathways of genes after meal in**
1065 **tongue tissue.**

1066 Relative gene expression measured by RT-PCR of *plcb2*, *plcb3*, *plcb4*, *stim1a*, *stim1b*, *oraila*, *orailb*, *serca3a* and *serca3b*, in tongue tissue of RT fed the C or V diet 20 mns, 2, 6, 10 or 24 hours after meal.
1067 Relative gene expression was measured by RT-qPCR. Datas are shown as means ± SEM; fold change 2<sup>-ΔΔCt</sup> vs. before meal (T0) for all genes. Two-way ANOVA following by one-way ANOVA when the interaction is significant, Tukey's post-hoc test when appropriate; an asterik indicate a significant difference (*p* < 0.05) between diet (n = 6).
1068
1069
1070
1071
1072
1073
1074
1075
1076
1077
1078
1079
1080
1081

1072

**Figure 4. Protein abundance encoding *ffar*-signaling pathways after meal in tongue tissue.**

1082 Protein abundance of ORAI1, STIM1 and p-ERK1/2 were measured by western blotting in tongue tissue.
1083 Data is expressed as the ratio between total amount of the tubulin protein for ORAI1, and expressed as the
1084 ratio of target protein to amount of total protein for STIM1 and p-ERK1/2 (after ratio to total ERK1/2),
1085 relative to the control group (T0). Datas are shown as means ± SEM; fold change 2<sup>-ΔΔCt</sup> vs. before meal
1086 (T0). Two-way ANOVA following by one-way ANOVA when the interaction is significant, Tukey's
1087 post-hoc test when appropriate; an asterik indicate a significant difference (*p* < 0.05) between diet (n =
1088 6).
1089
1090
1091
1092
1093

1081

1082 **Figure 5. Influence of C and V diets after feeding on serotonin-signaling pathways in tongue and**
1083 **myelencephalon tissue.**

1084 (A) Tongue concentration of indolamines (5-HT, 5-HIAA and turnover ratio 5-HT/5HIAA (n = 8).
1085 (B) Relative gene expression was measured by RT-PCR and protein abundance of metabolic pathways of
1086 indolamines in myelencephalon (*tph1b*, *tph2* and *TPH2* for protein abundance), and tongue tissue (*sert*,
1087 *mao*, *vmat2* and *VMAT2* for protein abundance) of RT fed the C or V diet 20 mns, 2, 6, 10 or 24 hours
1088 after meal. For RT-PCR, Datas are shown as means ± SEM; fold change 2<sup>-ΔΔCt</sup> vs. before meal (T0).
1089 Two-way ANOVA following by one-way ANOVA when the interaction is significant, Tukey post hoc;
1090 letters indicate a significant difference (*p* < 0.05) between diet and time (n = 6).
1091 For protein abundance, Datas are expressed as the ratio between total amount of the tubulin protein for
1092 VMAT2, and expressed as the ratio of target protein to amount of total protein for TPH2. Datas are shown
1093 as means ± SEM; fold change compared before meal (T0). Two-way ANOVA following by one-way
ANOVA when the interaction is significant, Tukey's post-hoc test when appropriate; an asterisk indicate a significant difference (p < 0.05) between diet (n = 6).

**Figure 6. Influence of C and V diets after feeding on FI markers in the hypothalamus**

Relative gene expression measured by RT-PCR of *npya*, *npyb*, *agpr1*, *agrp2*, *pomca*, *pomcb*, *cartpt* in hypothalamus tissue of RT fed the C or V diet 20 mns, 2, 6, 10 or 24 hours after meal. Data are shown as means ± SEM; fold change 2<sup>-ΔΔCt</sup> vs. before meal (T0). Two-way ANOVA following by one-way ANOVA when the interaction is significant, Tukey's post-hoc test when appropriate; an asterisk indicate a significant difference (p < 0.05) between diet (n = 6).

**Figure 7. Relative expression of mRNA encoding brain activity of genes after meal in telencephalon tissue.**

Relative gene expression measured by RT-PCR of *ntkr2b*, *bdnf*, *delta fosb*, *cfos*, *neurabin* and *ncam* in telencephalon tissue of RT fed the C or V diet 20 mns, 2, 6, 10 or 24 hours after meal. Relative gene expression was measured by RT-qPCR. Datas are shown as means ± SEM; fold change 2<sup>-ΔΔCt</sup> vs. before meal (T0) for all genes. Two-way ANOVA following by one-way ANOVA when the interaction is significant, Tukey's post-hoc test when appropriate; an asterisk indicate a significant difference (p < 0.05) between diet (n = 6).

**Figure 8. Protein abundance encoding brain activity after meal in telencephalon tissue.**

Protein abundance of BDNF, cFOS and p-ERK1/2 were measured by western blotting in telencephalon tissue. Data is expressed as the ratio between total amount of the tubulin protein for BDNF, and expressed as the ratio of target protein to amount of total protein for cFOS and p-ERK1/2 (after ratio to total ERK1/2), relative to the control group (T0). Datas are shown as means ± SEM; fold change 2<sup>-ΔΔCt</sup> vs. before meal (T0). Two-way ANOVA following by one-way ANOVA when the interaction is significant, Tukey's post-hoc test when appropriate; an asterisk indicate a significant difference (p < 0.05) between diet (n = 5).
<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /><figcaption>1153 **Figure 4.**</figcaption></figure>

1154

1155

1156

1157

1158

1159

1160

1161
<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /></figure>

Commercial-like (C) diet

<figure><img src="image_9.png" /></figure>

Plant-based (V) diet

npya

<figure><img src="image_10.png" /></figure>

diet effect > 0.05

time effect: 0.00

diet.time > 0.05

npyb

<figure><img src="image_11.png" /></figure>

diet effect > 0.05

time effect: 0.00

diet.time > 0.05

agrp1

<figure><img src="image_12.png" /></figure>

diet effect > 0.05

time effect > 0.05

diet.time > 0.05

agrp2

<figure><img src="image_13.png" /></figure>

diet effect > 0.05

time effect: 0.00

diet.time > 0.05

pomca

<figure><img src="image_14.png" /></figure>

diet effect > 0.05

time effect > 0.05

diet.time > 0.05

pomcb

<figure><img src="image_15.png" /></figure>

diet effect > 0.05

time effect: 0.00

diet.time > 0.05

cartpt

<figure><img src="image_16.png" /></figure>

diet effect > 0.05

time effect: 0.00

diet.time > 0.05
<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /></figure>

1177

1178

1179

1180

1181

1182

1183

1184
1185 Supplemental Information

1186 Supplemental information 1. Protein abundance of housekeeping protein; β-tubulin for Orai1 and
1187 Vmat2 and No-Stain for Stim1 and Tph2.

1188 Stim1, Orai1, Tph2 and Vmat2 measured by western blotting in tongue tissue (A and B) and
1189 myelencephalon (C and D) of RT fed with C and V diets for eight months, 4 hours after feeding and their
1190 housekeeping protein, β-tubulin for ORAI1 and VMAT2 for tongue tissue, BDNF for telencephalon tissue
1191 and No-Stain for STIM1 and p-EKR1/2 for tongue tissue, TPH2 for myelencephalon tissue and cFOS and
1192 p-EKR1/2 for telencephalon tissue.

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>
1206 **Supplemental information 2.** Statistical analysis by one-way analysis of variance (ANOVA) on the influence of C and V diets before and after feeding (20 minutes, 2, 6, 10 and 24 hours) on mRNA level of *ffars* in tongue tissue of RT. Different letters indicate a significant difference (*p* < 0.05) between timing (n = 6).

1210

<table><thead><tr><th rowspan="2">gene</th><th rowspan="2">diets</th><th colspan="6">post-prandial effects</th><th rowspan="2">Statistic</th></tr><tr><th>before meal</th><th>20 minutes</th><th>2 hours</th><th>6 hours</th><th>10 hours</th><th>24 hours</th></tr></thead><tbody><tr><td rowspan="2">ffar1</td><td>C diet</td><td>a</td><td>b</td><td>ab</td><td>ab</td><td>b</td><td>b</td><td>4.82E-04</td></tr><tr><td>V diet</td><td>a</td><td>b</td><td>b</td><td>b</td><td>ab</td><td>ab</td><td>3.10E-04</td></tr><tr><td rowspan="2">ffar2a1a</td><td>C diet</td><td>a</td><td>ab</td><td>ab</td><td>ab</td><td>b</td><td>ab</td><td>4.28E-03</td></tr><tr><td>V diet</td><td>a</td><td>ab</td><td>ab</td><td>ab</td><td>b</td><td>ab</td><td>2.02E-02</td></tr><tr><td rowspan="2">ffar2a2</td><td>C diet</td><td>a</td><td>ab</td><td>bc</td><td>ac</td><td>c</td><td>bc</td><td>1.12E-03</td></tr><tr><td>V diet</td><td>a</td><td>b</td><td>ab</td><td>ab</td><td>b</td><td>ab</td><td>1.21E-02</td></tr><tr><td rowspan="2">ffar2b1.1</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>b</td><td>a</td><td>7.84E-05</td></tr><tr><td>V diet</td><td>a</td><td>b</td><td>b</td><td>ab</td><td>ab</td><td>ab</td><td>5.20E-03</td></tr><tr><td rowspan="2">ffar2b1.2</td><td>C diet</td><td>a</td><td>ab</td><td>ab</td><td>ab</td><td>b</td><td>b</td><td>2.97E-03</td></tr><tr><td>V diet</td><td>a</td><td>c</td><td>bc</td><td>bc</td><td>ab</td><td>ac</td><td>2.53E-04</td></tr><tr><td rowspan="2">ffar2b2a</td><td>C diet</td><td>ab</td><td>ab</td><td>ab</td><td>a</td><td>ab</td><td>b</td><td>1.26E-02</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.32</td></tr><tr><td rowspan="2">ffar2b2b1</td><td>C diet</td><td>a</td><td>ab</td><td>ab</td><td>a</td><td>b</td><td>ab</td><td>3.17E-02</td></tr><tr><td>V diet</td><td>a</td><td>b</td><td>ab</td><td>ab</td><td>ab</td><td>ab</td><td>1.65E-02</td></tr><tr><td rowspan="2">ffar2b2b2</td><td>C diet</td><td>a</td><td>bc</td><td>ab</td><td>ac</td><td>bc</td><td>c</td><td>8.69E-04</td></tr><tr><td>V diet</td><td>a</td><td>b</td><td>ab</td><td>b</td><td>ab</td><td>ab</td><td>8.47E-03</td></tr></tbody></table>

1211

1212

1213 **Supplemental information 3.** Statistical analysis by one-way analysis of variance (ANOVA) on the influence of C and V diets before and after feeding (20 minutes, 2, 6, 10 and 24 hours) on mRNA level of *ffar*-signaling pathways in tongue tissue of RT. Different letters indicate a significant difference (*p* < 0.05) between timing (n = 6).

1217

<table><thead><tr><th rowspan="2">gene</th><th rowspan="2">diets</th><th colspan="6">post-prandial effects</th><th rowspan="2">Statistic</th></tr><tr><th>before meal</th><th>20 minutes</th><th>2 hours</th><th>6 hours</th><th>10 hours</th><th>24 hours</th></tr></thead><tbody><tr><td rowspan="2">plcb2</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.514</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.3</td></tr><tr><td rowspan="2">plcb3</td><td>C diet</td><td>a</td><td>a</td><td>b</td><td>a</td><td>ab</td><td>ab</td><td>7.77E-04</td></tr><tr><td>V diet</td><td>ab</td><td>ab</td><td>a</td><td>ab</td><td>ab</td><td>b</td><td>4.58E-02</td></tr><tr><td rowspan="2">plcb4</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.0667</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.0678</td></tr><tr><td rowspan="2">orai1a</td><td>C diet</td><td>ab</td><td>ab</td><td>b</td><td>a</td><td>ab</td><td>ab</td><td>9.57E-03</td></tr><tr><td>V diet</td><td>ab</td><td>ab</td><td>a</td><td>ab</td><td>b</td><td>b</td><td>1.01E-02</td></tr><tr><td rowspan="2">orai1b</td><td>C diet</td><td>a</td><td>ab</td><td>b</td><td>a</td><td>ab</td><td>a</td><td>1.94E-03</td></tr><tr><td>V diet</td><td>a</td><td>ab</td><td>a</td><td>a</td><td>ab</td><td>a</td><td>1.18E-04</td></tr><tr><td rowspan="2">stim1a</td><td>C diet</td><td>ab</td><td>ac</td><td>c</td><td>a</td><td>bc</td><td>bc</td><td>4.78E-05</td></tr><tr><td>V diet</td><td>ab</td><td>ab</td><td>a</td><td>ab</td><td>bc</td><td>c</td><td>4.96E-05</td></tr><tr><td rowspan="2">stim1b</td><td>C diet</td><td>ab</td><td>ab</td><td>b</td><td>a</td><td>ab</td><td>b</td><td>2.70E-03</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.236</td></tr><tr><td rowspan="2">serca3a</td><td>C diet</td><td>a</td><td>a</td><td>b</td><td>ab</td><td>ab</td><td>ab</td><td>4.15E-02</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.088</td></tr><tr><td rowspan="2">serca3b</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.335</td></tr><tr><td>V diet</td><td>ab</td><td>ab</td><td>a</td><td>ab</td><td>b</td><td>ab</td><td>7.28E-02</td></tr></tbody></table>

1218
**Supplemental information 4.** Statistical analysis by one-way analysis of variance (ANOVA) on the influence of C and V diets before and after feeding (20 minutes, 2, 6, 10 and 24 hours) on protein abundance of ffar-signaling pathways in tongue tissue of RT. Different letters indicate a significant difference (*p* < 0.05) between timing (n = 6).

<table><thead><tr><th rowspan="2">protein</th><th rowspan="2">diets</th><th colspan="6">post-prandial effects</th><th rowspan="2">Statistic</th></tr><tr><th>before meal</th><th>20 minutes</th><th>2 hours</th><th>6 hours</th><th>10 hours</th><th>24 hours</th></tr></thead><tbody><tr><td rowspan="2">ORAI1</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>b</td><td>a</td><td>a</td><td><strong>2.74E-03</strong></td></tr><tr><td>V diet</td><td>ab</td><td>a</td><td>a</td><td>b</td><td>a</td><td>a</td><td><strong>4.72E-04</strong></td></tr><tr><td rowspan="2">STIM1</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.377</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.737</td></tr><tr><td rowspan="2">pERK1/2</td><td>C diet</td><td>a</td><td>a</td><td>c</td><td>b</td><td>a</td><td>a</td><td><strong>1.29E-13</strong></td></tr><tr><td>V diet</td><td>ab</td><td>b</td><td>c</td><td>ab</td><td>ab</td><td>a</td><td><strong>4.57E-14</strong></td></tr></tbody></table>

**Supplemental information 5.** Statistical analysis by one-way analysis of variance (ANOVA) on the influence of C and V diets before and after feeding (20 minutes, 2, 6, 10 and 24 hours) on serotonin-signaling pathways in tongue and myelencephalon tissue of RT. Different letters indicate a significant difference (*p* < 0.05) between timing (n = 6).

<table><thead><tr><th rowspan="2">Metabolites/enzymes</th><th rowspan="2"></th><th rowspan="2"></th><th colspan="6">post-prandial effects</th><th rowspan="2">Statistic</th></tr><tr><th>before meal</th><th>20 minutes</th><th>2 hours</th><th>6 hours</th><th>10 hours</th><th>24 hours</th></tr></thead><tbody><tr><td rowspan="2">5-HT</td><td>C diet</td><td>a</td><td>b</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td><strong>1.24E-06</strong></td></tr><tr><td>V diet</td><td>a</td><td>b</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td><strong>1.42E-12</strong></td></tr><tr><td rowspan="2">5-HIAA</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.908</td></tr><tr><td>V diet</td><td>ab</td><td>ab</td><td>b</td><td>ab</td><td>a</td><td>ab</td><td>a</td><td><strong>6.68E-03</strong></td></tr><tr><td rowspan="2">TPH2</td><td>C diet</td><td>b</td><td>b</td><td>b</td><td>b</td><td>b</td><td>a</td><td>a</td><td><strong>3.91E-04</strong></td></tr><tr><td>V diet</td><td>b</td><td>ab</td><td>ab</td><td>b</td><td>ab</td><td>a</td><td>a</td><td><strong>6.00E-03</strong></td></tr><tr><td rowspan="2">VMAT2</td><td>C diet</td><td>a</td><td>ab</td><td>c</td><td>bc</td><td>ab</td><td>c</td><td>c</td><td><strong>1.46E-06</strong></td></tr><tr><td>V diet</td><td>a</td><td>ab</td><td>b</td><td>b</td><td>ab</td><td>c</td><td>c</td><td><strong>1.08E-07</strong></td></tr><tr><td colspan="2">ratio</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">5-HIAA/5-HT</td><td>C diet</td><td>b</td><td>a</td><td>b</td><td>b</td><td>b</td><td>b</td><td>b</td><td><strong>1.19E-05</strong></td></tr><tr><td>V diet</td><td>b</td><td>a</td><td>d</td><td>bd</td><td>bc</td><td>cd</td><td>cd</td><td><strong>4.29E-11</strong></td></tr><tr><td colspan="2">gene</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">tph1b</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.26</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.953</td></tr><tr><td rowspan="2">tph2</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.062</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.836</td></tr><tr><td rowspan="2">sert</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td><strong>4.29E-02</strong></td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.635</td></tr><tr><td rowspan="2">mao</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.719</td></tr><tr><td>V diet</td><td>ab</td><td>ab</td><td>a</td><td>ab</td><td>b</td><td>ab</td><td>ab</td><td><strong>4.34E-02</strong></td></tr><tr><td rowspan="2">vmat2</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.278</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.108</td></tr></tbody></table>
**Supplemental information 6.** Statistical analysis by one-way analysis of variance (ANOVA) on the influence of C and V diets before and after feeding (20 minutes, 2, 6, 10 and 24 hours) on FI markers in the hypothalamus of RT. Different letters indicate a significant difference (*p* < 0.05) between timing (n = 6).

<table><thead><tr><th rowspan="2">gene</th><th rowspan="2">diets</th><th colspan="6">post-prandial effects</th><th rowspan="2">Statistic</th></tr><tr><th>before meal</th><th>20 minutes</th><th>2 hours</th><th>6 hours</th><th>10 hours</th><th>24 hours</th></tr></thead><tbody><tr><td rowspan="2">npya</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.4086</td></tr><tr><td>V diet</td><td>ac</td><td>bc</td><td>c</td><td>a</td><td>ab</td><td>a</td><td><strong>1.64E-04</strong></td></tr><tr><td rowspan="2">npyb</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.0576</td></tr><tr><td>V diet</td><td>ab</td><td>ab</td><td>b</td><td>a</td><td>ab</td><td>a</td><td><strong>7.66E-03</strong></td></tr><tr><td rowspan="2">agrp1</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.315</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.817</td></tr><tr><td rowspan="2">agpr2</td><td>C diet</td><td>ab</td><td>a</td><td>a</td><td>a</td><td>ab</td><td>b</td><td><strong>1.49E-02</strong></td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.0778</td></tr><tr><td rowspan="2">pomca</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.385</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.0807</td></tr><tr><td rowspan="2">pomcb</td><td>C diet</td><td>ac</td><td>c</td><td>bc</td><td>ac</td><td>ab</td><td>a</td><td><strong>1.15E-03</strong></td></tr><tr><td>V diet</td><td>a</td><td>b</td><td>ab</td><td>a</td><td>a</td><td>a</td><td><strong>3.02E-03</strong></td></tr><tr><td rowspan="2">cartpt</td><td>C diet</td><td>b</td><td>ab</td><td>a</td><td>ab</td><td>ab</td><td>ab</td><td><strong>3.99E-02</strong></td></tr><tr><td>V diet</td><td>b</td><td>ab</td><td>ab</td><td>a</td><td>ab</td><td>ab</td><td><strong>1.17E-02</strong></td></tr></tbody></table>

**Supplemental information 7.** Statistical analysis by one-way analysis of variance (ANOVA) on the influence of C and V diets before and after feeding (20 minutes, 2, 6, 10 and 24 hours) on mRNA level of brain activity genes in telencephalon tissue of RT. Different letters indicate a significant difference (*p* < 0.05) between timing (n = 6).

<table><thead><tr><th rowspan="2">gene</th><th rowspan="2">diets</th><th colspan="6">post-prandial effects</th><th rowspan="2">Statistic</th></tr><tr><th>before meal</th><th>20 minutes</th><th>2 hours</th><th>6 hours</th><th>10 hours</th><th>24 hours</th></tr></thead><tbody><tr><td rowspan="2">ntkr2b</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.0868</td></tr><tr><td>V diet</td><td>ab</td><td>a</td><td>b</td><td>b</td><td>ab</td><td>b</td><td><strong>2.07E-03</strong></td></tr><tr><td rowspan="2">bdnf</td><td>C diet</td><td>a</td><td>ab</td><td>b</td><td>b</td><td>bc</td><td>ab</td><td><strong>8.18E-03</strong></td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>b</td><td>ab</td><td>ab</td><td>ab</td><td><strong>5.67E-04</strong></td></tr><tr><td rowspan="2">delta-fosb</td><td>C diet</td><td>a</td><td>a</td><td>b</td><td>a</td><td>a</td><td>a</td><td><strong>9.47E-06</strong></td></tr><tr><td>V diet</td><td>a</td><td>ab</td><td>c</td><td>bc</td><td>bc</td><td>c</td><td><strong>4.38E-05</strong></td></tr><tr><td rowspan="2">cfos</td><td>C diet</td><td>a</td><td>b</td><td>c</td><td>ab</td><td>ab</td><td>ab</td><td><strong>6.55E-06</strong></td></tr><tr><td>V diet</td><td>a</td><td>ab</td><td>c</td><td>ab</td><td>ab</td><td>bc</td><td><strong>8.89E-06</strong></td></tr><tr><td rowspan="2">neurabin</td><td>C diet</td><td>a</td><td>c</td><td>bc</td><td>ac</td><td>bc</td><td>ab</td><td><strong>2.77E-03</strong></td></tr><tr><td>V diet</td><td>ab</td><td>a</td><td>c</td><td>bc</td><td>ac</td><td>bc</td><td><strong>2.43E-04</strong></td></tr><tr><td rowspan="2">ncam</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td><strong>3.47E-02</strong></td></tr><tr><td>V diet</td><td>ab</td><td>a</td><td>ab</td><td>ab</td><td>ab</td><td>b</td><td><strong>2.52E-02</strong></td></tr></tbody></table>

**Supplemental information 8.** Statistical analysis by one-way analysis of variance (ANOVA) on the influence of C and V diets before and after feeding (20 minutes, 2, 6, 10 and 24 hours) on protein
1251 abundance encoding brain activity in telencephalon tissue of RT. Different letters indicate a significant
difference (*p* < 0.05) between timing (n = 6).

1253

<table><thead><tr><th rowspan="2">protein</th><th rowspan="2">diets</th><th colspan="6">post-prandial effects</th><th rowspan="2">Statistic</th></tr><tr><th>before meal</th><th>20 minutes</th><th>2 hours</th><th>6 hours</th><th>10 hours</th><th>24 hours</th></tr></thead><tbody><tr><td rowspan="2">BDNF</td><td>C diet</td><td>ab</td><td>bc</td><td>c</td><td>ab</td><td>ab</td><td>a</td><td>7.83E-05</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>b</td><td>a</td><td>a</td><td>a</td><td>3.29E-02</td></tr><tr><td rowspan="2">cFOS</td><td>C diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.087</td></tr><tr><td>V diet</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>a</td><td>0.0567</td></tr><tr><td rowspan="2">pERK1/2</td><td>C diet</td><td>ab</td><td>ab</td><td>ab</td><td>b</td><td>a</td><td>a</td><td>9.62E-02</td></tr><tr><td>V diet</td><td>b</td><td>ab</td><td>ab</td><td>ab</td><td>a</td><td>a</td><td>7.49E-04</td></tr></tbody></table>

1254