Hermetia illucens defatted larvae meal as partial dietary protein source for rainbow trout (Oncorhynchus mykiss): Intestinal microbial communities and hepatic oxidative stress

Scientific effort is being done on replacing fishmeal (FM) with more sustainable dietary protein sources in fish feeds. Insect meals have been reported to have a good nutritional composition and to be a promising sustainable alternative protein source. Moreover, insects grow quickly on low-quality organic waste, do not require arable land and present a high feed conversion efficiency. Recent studies have been demonstrating the impact of diet composition on intestinal bacterial communities, which, in turn, may modulate health and well-being status of fish. Therefore, the purpose of this study was to investigate the effects of dietary inclusion of Hermetia illucens (Diptera: Stratiomydae) defatted larvae meal (Hi) as partial replacer of FM on rainbow trout (Oncorhynchus mykiss) performance, hepatic oxidative stress and intestinal mucosa- (MAB) and digesta-associated bacterial communities (DAB). Three isoproteic (about 45% crude protein), isolipidic (about 15% crude lipids) and isoenergetic (about 22MJ/kg d.m.) experimental diets were formulated with FM as main protein source (Hi0) or substituting 25% or 50% of FM with Hi (Hi25 and Hi50, respectively). The Hi was purchased from Hermetia Baruth GmbH (An der Birkenpfuhlheide Baruth/Mark, Germany). Rainbow trout (initial average body weight: 178.9 ± 9.81 g, mean ± SEM) were randomly allocated to four replicate tanks per dietary group and fed each experimental diet (daily feeding rate corresponding to 1.5% of body weight), for 11 weeks. At the end of the growth trial, fillet yield was recorded for 30 fish per dietary group, while organosomatic parameters were determined in six fish per dietary group. The antioxidant enzymes involved in the redox potential of fish, catalase, glutathione peroxidase, glutathione reductase, superoxide dismutase and glucose 6-phosphate dehydrogenase, as well as lipid peroxidation, were also determined in 10 livers per group. To study the MAB and the DAB, denaturing gradient gel electrophoresis (DGGE) was performed on six intestine tissues and six intestine contents per dietary group, respectively. To get an overview of the complex communities, richness and biodiversity parameters were calculated on the DGGE banding patterns and ANOSIM was performed to determine the extent of the differences between the three dietary groups in the MAB and in the DAB. To identify dominant bacterial groups, as well as suggesting to which functions the sequenced strains could have on fish, selected bands were excised from the DGGE gel, sequenced and deposited in the GenBank database. The Web-based BLASTN tool was used to find closely related nucleotide sequences; to increase the accuracy of the assignments, different sequence similarity thresholds were used for different taxonomic levels: a similarity ≥97% for species level identification and 95%, 90%, 85%, 80% and 75% for assignment at the genus, family, order, class and phylum level, respectively. Growth performances, fillet yield and organosomatic parameters were similar among the dietary groups. Similarly, specific activity of antioxidant enzymes in rainbow trout liver was not affected by the dietary treatment, while hepatic lipid peroxidation level decreased with the increase of dietary H. illucens level in the diet (p<0.01). Dietary inclusion of Hi affected the structure and the composition of the both MAB and DAB communities. The biodiversity of the MAB Hi25 group was significantly higher than that of the control group (p<0.05) and the Hi50 group stayed in an intermediate position between the control and Hi25 groups. As showed in table 1, the results of the pairwise comparisons showed that differences between all the groups were significant (p<0.01) in the DAB, while in the MAB only the pair Hi0-Hi25 displayed a significant difference (p<0.05). Irrespective of the diet, the sequencing of the excised bands highlighted a clear prevalence of γ-Proteobacteria, though α-, β-Proteobacteria and Actinobacteria were also present in the MAB of fish fed Hi-based diets; the DAB of the fish fed Hi-based diets presented a clear increase in bacteria belonging to the Firmicutes phylum. The global analysis of our preliminary results suggests that H. illucens defatted larvae meal is a valid alternative protein source to FM for rainbow trout feeding. Up to 50% of FM may be replaced by Hi without affecting growth performance, hepatic liver oxidative status, while liver peroxidation is inclusively reduced. The results of the microbiological assays revealed that also the intestinal bacterial communities were sensitive to dietary changes, having shown an increased community biodiversity in the Hi-fed groups. Several OTUs retrieved could be accountable for effects on fish physiology, going from probiotic bacteria, to bacteria able to produce eicosapentaenoic acid, to those able to hydrolyse carbohydrates. Some bacteria may protect fish from pathogens as well as enhance digestion mechanisms, physiological functions and welfare in general. The outcome of this study may benefit future research into both the microbial community of Salmonids and into the interaction between host, guest and diet. To delve into the biological consequences of the host-guest interaction, we encourage to combine the description of the bacterial community with functional analyses, such as metagenomics, metabolomics, challenge tests and alike.