Introduction
Yellow mealworm (Tenebrio molitor) is a prominent sustainable alternative to fishmeal in aquafeeds. However, replacing fishmeal can compromise thermal stress resistance if formulation lack essential nutrients critical for antioxidant defence and membrane integrity [1]. This is particularly relevant for insect meals, which often lack long-chain n-3 polyunsaturated fatty acids [2]. In contrast, mealworm-derived immunostimulants and bioactive compounds can potentially modulate fish immune and oxidative status [3].
This study aimed to characterize the comparative effects of full-fat (ffTM) and defatted (dTM) mealworm meal on the physiological, immunological and inflammatory status of rainbow trout (Oncorhynchus mykiss) subjected to a 15-day progressive thermal stress challenge
Materials and methods
A single batch of mealworm larvae was either subjected or not subjected to a thermos-mechanical defatting process, resulting in two distinct mealworm meals (TM): a full-fat mealworm meal (ffTM, crude protein: 51.9%, crude lipids: 32.8%) and a defatted mealworm meal (dTM, crude protein: 66.7%, crude lipids: 12.9%). Three isoproteic (45%) and isolipidic (22%) experimental diets were formulated for juvenile rainbow trout. A control diet (CTRL) was formulated to mimic the composition of a conventional commercial trout diet. Two other diets were formulated based on the CTRL diet, but incorporated 25% of the full-fat and defatted MMs, totally replacing fishmeal (FFMM and DMM diets). All diets included 11.5% of fish oil (dry matter basis), and rapeseed oil was adjusted to maintain consistent lipid levels across all formulations. The diets were tested in triplicate during an initial 8-week growth trial under optimal rearing conditions. Following this, nine homogeneous groups of 7 fish each (159 ± 15 g) were assigned to 120L fiberglass tanks within a recirculating aquaculture system. The thermal challenge was initiated by raising the water temperature from 17 ��C to 23 ��C over 7 days. During the subsequent 15-day challenge period . Fish were fed 2% of the initial biomass, twice daily, over a. In this 15-day period the temperature was raised in 5-days intervals until reaching 25 ��C. At the end of the challenge blood was sampled to isolate the plasma for the innate immune parameters analyses, livers were sampled for oxidative stress enzymatic and non-enzymatic indicators analyses and head-kidneys were sampled for inflammatory status markers expression.
Results
No mortalities or signs of disease were recorded during the thermal trial, and fish showed a mean 30% weight increase. The haemoglobin (Hb), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV) and red blood cell count showed no significant differences between experimental diets. In contrast, haematocrit levels were significantly higher in the CTRL and FFMM groups relatively to the DMM groups. Furthermore, fish fed the FFMM diet also showed higher white blood cell counts (WBC) than fish fed the DMM diet. No significant difference between experimental treatments was observed on glucose and lactate levels, nor on the lysozyme, protease and anti-protease activities. However, fish fed the DMM diet showed a significantly higher peroxidase activity than fish fed the FFMM diet. No significant dietary impact was observed on the hepatic activities of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) total glutathione (TG) and lipid peroxidation (LPO) levels. In contrast, the fish groups fed the DMM diet showed significantly higher reduced glutathione (GSH) levels than fish groups fed the CTRL diet, and significantly lower oxidized glutathione (GSSG) levels than fish groups fed the FFMM diet. Ultimately leading to the DMM groups showing a hepatic reduced - oxidised glutathione ratio (GSH/GSSG ratio) significantly higher than the CTRL and FFMM groups The expressions of Hsp70, tnf-α, il-8, tgf-β, and Casp3 was not affected by dietary composition. In contrast, il-1β was significantly upregulated in DMM group relative to CTRL.
Conclusion
Under a 15-day thermal stress challenge, rainbow trout fed defatted mealworm meal (DMM) showed higher plasma peroxidase activity, hepatic GSH levels and redox status ((GSH/GSSG). However, the upregulation of pro-inflammatory cytokines and reduced WBC in the DMM group suggest a complex physiological response. Both ffTM and dTM support high survivability and similar hepatic oxidative protection compared to fishmeal. While defatted mealworm meal (dTM) may offer specific antioxidant advantages, full-fat mealworm meal (ffTM) remains a viable alternative that reduces the requirement for supplementary vegetable oils without compromising the ability of rainbow trout to withstand thermal stress .
Acknowledgements
This work was funded by the SAFE project (SmartAqua4FuturE) within the European Union's Horizon Europe programme under grant agreement no. 101084549. This research was also supported by national funds through FCT (Foundation for Science and Technology), within the scope of UIDB/04423/2020, UIDP/04423/2020 and D. Amaral doctoral grant (2023.03476.BD).
References
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