Introduction
Fish meal (FM) has long been considered the ideal protein source for carnivorous fish production, due to its well-balanced nutritional profile, high digestibility, and palatability. However, due to its limited global availability and environmental concerns, current research is focused on reducing the reliance on FM in aquafeeds by substituting it with more available, eco-friendly, and cost-efficient protein alternatives. Significant progress has been made in utilizing alternative proteins derived from terrestrial plants. However, traditional plant ingredients used as substitutes often contain several anti-nutritional factors and could present conflicts, as some these resources are intended for human consumption. In recent years, after the European Commission approved the use of specific insect meals as feed materials for aquafeeds. Black soldier fly (Hermetia illucens) larvae meal (BSF) has gained significant attention from the scientific community as a sustainable and novel protein source
Materials and Methods
Black soldier fly (Hermetia illucens, BSF) larvae reared on plant-based residual substrates were experimentally produced and incorporated into aquafeed formulations, replacing fishmeal (30% incorporation in the control group - CTRL) at inclusion levels of up to 60%. A three-month feeding trial was carried out at the experimental facilities of the Hellenic Centre for Marine Research (HCMR), using European seabass (Dicentrarchus labrax, initial mean weight ~30 g). Fish were fed six experimental diets in which fishmeal was progressively replaced by black soldier fly larvae meal at 0 (CTRL), 20, 30, 40, 50, and 60% substitution levels. At the end of the trial, all fish were starved for 48 h to evacuate the digestive tract, anesthetized with phenoxyethanol: ethanol (1:1), and individually weighed to assess growth performance. A total of 19 fish were randomly selected from each tank and pooled. Four fish were euthanized by anesthetic overdose, and samples of skeletal muscle, perivisceral fat, the intestinal tract, and liver were collected to calculate the somatometric indices. Histological samples were preserved in 10% buffered formalin (with sodium phosphate and sodium hydroxide; pH 7.4) until analysis. Additionally, fillet samples were collected from the same fish and stored at ���20 °C. Another five fish from each tank were used for whole-body composition analyses. Additionally, 108 tissue samples (liver and anterior intestine) were collected for molecular analyses. Gene expression profiling was performed using customized qPCR arrays targeting seabass markers that had been previously optimized at the IATS-ANA infrastructure. The data normality and homogeneity of variances were checked through the Shapiro-Wilk and Levene's tests, respectively. In a normality and equal variance scenario, One-way ANOVA followed by a Tukey post hoc test was used to assess statistical differences (p < 0.05). Whenever data normality or homogeneity was not achieved, data transformation (i.e., logarithmic) or nonparametric statistical analysis (i.e., Kruskal-Wallis for gene expression, Spearman for correlation; p < 0.05) were employed. All statistical analyses were performed using SigmaPlot (version 15). To further explore tissue-specific gene expression patterns, partial least-squares discriminant analysis (PLS-DA) was performed using EZinfo v3.0 (Umetrics, Sweden), which enabled the visualization of multivariate clustering and discrimination among dietary groups.
Results and Discussion
At the end of the feeding trial, fish growth performance and feed utilization indices were not significantly affected by the FM substitution up to 60%. Additionally, the whole-body proximate composition showed minimal variation among treatments. Significant differences were observed in the fillet fatty acid composition, showing increased n-6 polyunsaturated fatty acids and reduced long-chain n-3 PUFA (EPA and DHA) associated with higher BSF content in the diets. While the histological assessment showed no alterations in intestinal morphology and no signs of liver inflammation. Interestingly, the largest lipid deposits were observed in the Control and BSF 60% groups, while the smallest deposits were noted in the BSF 20% group. The remaining dietary treatments had intermediate values (p < 0.05). A multi-tissue partial least-squares discriminant analysis (PLS-DA) of gene expression revealed that at 60% inclusion of defatted black soldier fly (BSF) meal, there was notable immune modulation. This was characterized by increased pro-inflammatory markers and decreased regulatory cytokines in the intestine. No statistically significant differences in the expression of individual genes were initially detected among dietary groups in either tissue (p > 0.05). To further investigate potential dose-dependent effects across tissues, a partial least-squares discriminant analysis (PLS-DA) was performed by grouping the samples into CTRL–20–30–40 and 50–60. The PLS-DA model effectively discriminated between the two groups and identified 15 genes with a variable importance in projection (VIP) score > 1 as the primary contributors to class separation.
Acknowledgment
The feeding trial was funded through the Greek national project "Insects4Aqua" (Grant Agreement 5029335, Ministry of Rural Development and Food). The access of Efstratios Roussos to the IATS-CSIC facilities for gene expression analyses was supported by the TNA program (PID36931) within the H2020 AQUAEXCEL3.0 project (871108). This publication reflects only the authors' viewpoints, and the European Union cannot be held responsible for any use that may be made of the information contained herein.