Introduction
Natural functional feed additives can enhance both fish growth and health (Onomu et al., 2024) through the interplay between bioactive compounds and intestinal mucosa. I nsects and their derivatives are considered a promising feed ingredient and additive to promote animal growth and health enhancement (Veldkamp et al., 2021).This study aimed to describe the effects of two compounds derived from black soldier fly ( Hermetia illucens, BSF ): a polysaccharide (chitin from exuviae, CHI) and a fatty acid (lauric acid, LAU ). A feeding trial was performed with juvenile rainbow trout (Oncorhynchus mykiss ) to assess if and to what extent raw chitin from BSF used alone or in combination with increasing levels of lauric acid can affect liver and intestinal morphology by focusing on goblet cells mucus composition, intestinal cell proliferation and differentiation a s bio-markers.
Materials and methods
Four semi-purified diets were formulated for juvenile rainbow trout to be grossly isoproteic (44%) and isolipidic (14 %). Starting from a control basal diet (CTRL), three other test diets were produced to include a fixed amount of raw chitin from exuviae of BSF (1.5% dietary inclusion) (C-CHI) without lauric acid or with increasing level of it at 0.1% and 0.2% (C-CHI-LAU1 and C-CHI-LAU2; respectively). A total of 144 rainbow trout juveniles (91.3 ± 2.9 g) were randomly distributed into 12 fiberglass tanks (0.2 m3; 3 tanks per dietary treatment) and fed over 1 0 weeks at a daily feeding rate of 2 % body weight. At the end of the trial, growth parameters were measured and intestinal samples from pyloric caeca (PC) and proximal intestine (PI) were collected and immediately fixed in formalin for histological and immuno -histochemical analyses to assess the gut mucosae status. In particular, hematoxylin-eosin-stained sections were used to evaluate the general morphological aspects, PAS combined with Alcian Blue pH 2.5 staining to analyze the overall complex carbohydrates, and immunostaining for proliferating cell nuclear antigen (PCNA) and peptide transporter 1 (PepT1) to assess cell proliferation and peptides absorption respectively. Finally, the histochemical detection of alkaline phosphatase (ALP) was used to detect fully differentiated enterocytes.
Results
There were no significant differences in the growth performance and body indices between the fish fed the control and the experimental diets. There were no histomorphological changes in the liver of the fish fed the different diets. However, there was a significant reduction in villi length (p<0.05 ) and changes in the mucus composition of goblet cells in the PC and PI (p<0.05). Lauric acid (0.2%) in the diet increased the number of mixed mucins in the PC, while chitin (1.5%) significantly increased the acid mucins alongside a significant decrease in mixed mucins in the PI (p<0.05). The signal intensity of peptide transporter 1 (PepT1) was lower in fish fed with the C-CHI diet (Fig 1) . T he PCNA and ALP expressions in PI did not differ significantly among dietary treatments (p>0.05).
Discussion and Conclusion
This study showed that inc luding BSF exuviae and LAU into trout diets resulted in no differences in growth performances and biometric indices, similarly to what was observed in the same species by Rimoldi et al. (2023) with an inclusion of 1.6% , but unlike what was reported in sea bream fed diet including 0.02% LAU (Ullah et al., 2025) .
Raw chitin alone caused a reduction in the folds length, and it affected goblet cell mucin production in rainbow trout fed diet with BSF meal (Elia et al. , 2018). Furthermore, raw chitin decreased PepT1 signal intensity , indicating reduced intestinal peptide absorption, similar to what was observed when trout were fed a plant-protein based diet (Verdile et al. (2023). None of the diets affected PCNA and ALP expression, indicating that both raw chitin or LAU, did not induce proliferative state of intestinal tissue and cellular damage in the rainbow trout intestine (Kilemade et al. 2002).
Overall, these findings indicate that 1.5% BSF exuviae supplementation can negatively impact intestinal morphology, mucin production, and PepT1 expression, whereas a combination with 0.1% LAU in the diet seems to ameliorate the adverse effects of chitin.
References
Elia et al. (2018). Aquaculture 496, 50–57. https://doi.org/10.1016/j.aquaculture.2018.07.009
Kilemade et al. (2002). Aquatic Toxicology 60, 43–59. https://doi.org/10.1016/S0166-445X(01)00269-7
Onomu and Okuthe (2024). Fishes 9, 167. https://doi.org/10.3390/fishes9050167
Rimoldi et al. (2023). Aquaculture 567, 739256. https://doi.org/10.1016/J.AQUACULTURE.2023.739256
Ullah et al (2025). Fish Physiol Biochem 51, 1–15. https://doi.org/10.1007/S1069501457-3/FIGURES/1
Veldkamp et al. (2022). JIFF 8(9) : 1027-1040 DOI 10.3920/JIFF2021.0031
Verdile et al. (2023). Aquaculture 564. https://doi.org/10.1016/j.aquaculture.2022.739031
Funding
The present study was supported by Ministry of University and Research within the framework of the PRIN2022 Call for proposals by proceeding down the ranked list.