Fishmeal Protein Replacement by Defatted and Full-Fat Black Soldier Fly Larvae Meal in Juvenile Turbot Diet: Effects on the Growth Performance and Intestinal Microbiota

{"Background": "Black soldier fly larvae (Hermetia illucens, BSFL) meal is a promising alternative protein source for aquafeeds, with crude protein content ranging from 30% to 65%. However, the lipid content of BSFL meal varies with processing: defatted BSFL meal typically contains below 30% lipid, whereas full-fat BSFL meal contains above 30%. Published results on the effects of full-fat BSFL meal on fish growth are conflicting, with reports of negative, neutral, or positive outcomes depending on species and inclusion level. Because intestinal microbiota strongly influence host development, metabolism, and immunity, dietary shifts in BSFL meal formulation may have downstream effects on microbial communities and gut health. Turbot (Scophthalmus maximus L.) is a commercially important carnivorous marine species with a high dietary fishmeal requirement, making it a relevant model for evaluating alternative proteins.", "Methods": "Three isonitrogenous (~52% protein) and isolipidic (~12% lipid) diets were formulated: a fishmeal-based control (FM), a diet in which 14% of fishmeal protein was replaced by defatted BSFL meal (DF; crude protein 43.45%, crude lipid 23.77%), and a diet with the same fishmeal protein replacement using full-fat BSFL meal (FF; crude protein 31.37%, crude lipid 43.21%). Juvenile turbot (initial body weight 12.64 g) were randomly assigned to nine 200-L fiberglass tanks (40 fish per tank) in a flow-through seawater system and fed to apparent satiation twice daily for 12 weeks. Water conditions were: dissolved oxygen >7 mg/L, temperature 16\u201321\u00b0C, pH 7.59\u20137.84, salinity 28\u201330. At the end of the trial, distal intestine samples were collected for histology (perimeter ratio, PR = internal perimeter/external perimeter), real-time qPCR of barrier (Tricellulin, Claudin-3) and cytokine (TGF-\u03b2, IL-1\u03b2, TNF-\u03b1) genes normalized to \u03b2-actin, and 16S rRNA V4 region Illumina NovaSeq sequencing analyzed with UPARSE, Mothur, QIIME, and Metastats.", "Key Results": "Final body weight, specific growth rate (SGR), weight gain (WG), and feed efficiency (FE) were significantly lower in the FF group than in the FM group (P < 0.05), with no significant difference between FM and DF (P > 0.05). Histology showed no obvious morphological changes across groups, but the distal intestine perimeter ratio was lowest in turbot fed the FF diet. Sequencing yielded 1,292,666 tags across 18 samples clustered into 23,608 OTUs (\u226597% similarity). Proteobacteria and Firmicutes were the dominant phyla in all groups. Dominant genera in FM were Vibrio (12.56%), Mycoplasma (9.24%), Ralstonia (11.33%), Pseudoalteromonas (10.67%), and Pseudomonas (9.33%); in DF, Vibrio (31.84%), Mycoplasma (12.31%), and Lysinibacillus (9.08%); in FF, Vibrio (29.70%), Ralstonia (18.78%), Pseudoalteromonas (7.59%), and Pseudomonas (13.83%). Alpha diversity indices did not differ significantly among treatments (P > 0.05). PCoA and UPGMA analyses showed that DF microbiota clustered more closely with FM than with FF. Metastats analysis revealed that DF significantly increased the relative abundance of Bacillus compared with both FM and FF (P < 0.05) and significantly decreased Candidatus_Arthromitus, Chryseobacterium, and [Eubacterium]_fissicatena_group compared with FM (P < 0.05). FF significantly decreased the abundance of Lactobacillus, Dubosiella, and Bifidobacterium relative to FM (P < 0.05), and significantly increased Chryseobacterium. Compared with DF, FF significantly increased the relative abundance of Lachnoclostridium, Romboutsia, Aeromonas, and Chryseobacterium (P < 0.05). For mucosal barrier markers, both DF and FF significantly upregulated Tricellulin expression versus FM (P < 0.05), and Claudin-3 expression was significantly higher in DF than FF (P < 0.05). FF significantly downregulated TGF-\u03b2 expression (P < 0.05), while IL-1\u03b2 and TNF-\u03b1 expression did not differ among groups (P > 0.05).", "Clinical Implications": "In juvenile turbot diets, defatted BSFL meal (lipid content 23.77%) can replace 14% of fishmeal protein without compromising growth performance, intestinal morphology, or mucosal barrier function, and may offer additional benefits through enrichment of the probiotic genus Bacillus. Full-fat BSFL meal (lipid content 43.21%) at the same protein replacement level should be avoided in turbot because it suppresses growth, reduces intestinal absorptive area (lower PR), downregulates the anti-inflammatory cytokine TGF-\u03b2, decreases beneficial genera (Lactobacillus, Dubosiella, Bifidobacterium), and enriches potential pathogens (Chryseobacterium, Lachnoclostridium, Romboutsia, Aeromonas). The reduced growth in the FF group may stem from a combination of higher dietary chitin (note: calculated chitin content was about 1.16% in DF versus 0.59% in FF, attributable to differing inclusion levels of BSFL meal), absence of supplemental fish oil leading to lower DHA and EPA, and unfavorable microbial shifts. These findings support prioritizing defatted over full-fat BSFL meal in carnivorous marine fish feeds and emphasize that lipid content and resulting fatty acid composition must be carefully controlled when formulating insect-based aquafeeds. The authors note that larger sample sizes would strengthen future microbiota studies."}