The growing demand for seafood, as a significant supply of high-quality proteins for human consumption (Stankus 2021), has sustained the rise of the aquaculture sector for the last decades (FAO, 2022). Inclusion of insect meals in aquafeeds has gained interest due to their nutritional qualities, production efficiency and sustainability (Bruni et al. 2018; Henry et al. 2015; van Huis and De Prins 2013). Among the eight insect species authorized for feed production by the EU (Regulation (EU) 2021/1925), H. illucens has been reported as one of the most promising ones to be used in finfish diets (Barry 2004; Alfiko et al. 2022). Links between H. illucens meal in aquafeeds and its effects on fish growth, microbiome and health have been explored over the last years (Terova et al. 2019, 2021; Rimoldi et al. 2019, 2021, 2024; Biasato et al. 2022; Busti et al. 2024; Rangel et al. 2022; Antonopoulou et al. 2019; Rimoldi et al. 2019). In gilthead sea bream, the inclusion of HI has been found to modulate fish microbiome diversity and composition when used to replace 5 to 30% FM in the diet (Panteli et al. 2021; Rimoldi et al. 2024; Busti et al. 2024). In the present study, fish growth, gut microbiota and liver histology were assessed in gilthead seabream fed on diets in which up to 50% of FM was replaced with HI. At the end of the feeding trial, no drastic differences in zootechnical performances were highlighted within the different experimental groups, with the exception of the condition factor (K) which was lower in fish fed HI25 diet. Also, fish from this group displayed a higher HSI and a lower VSI compared to the other groups. These results may indicate a more marked effect of low inclusion of HI compared to higher ones, in this case, affecting liver size particularly.
As regards microbiome analyses, a high homogeneity within the microbiomes extracted from tissues and those extracted from contents and scraping was observed, suggesting that despite the efforts to distinguish them during the sampling, it remains challenging to analyze the transient microbiome separately from the resident microbiome (Tarnecki et al. 2017). The transient microbial community is primarily affected by environmental conditions and the host's feeding habits, since it is composed of free-living microorganisms that enter the host's body along with the water or feed and are soon expelled (Moschos et al. 2022).
At the end of the experiment, all the test diets induced a higher alpha diversity compared to the beginning (T0), with higher diversity values in HI35 and HI50 compared to HI0 and HI25 groups. A higher microbial richness should always be considered a positive effect, since it may potentially provide further metabolic capabilities to the host, thus improving its general health conditions (Borrelli et al. 2017). Busti and colleagues (2024) reported a decrease in alpha diversity in gut microbiome from juvenile gilthead sea bream fed with 5 to 15% HI dietary inclusion, compared to a control FM-based diet, while no differences were reported in response to 30% and 35% FM substitution with HI (Panteli et al. 2021; Rimoldi et al. 2024). Our results indicate a possible role of HI percentage in increasing alpha diversity in the gut microbiome of gilthead seabream, although other studies performed in different conditions (replacement percentage, duration and size) have shown discordant results, highlighting how the microbial composition is certainly influenced by the combination of several factors (Rimoldi et al. 2024, Quero et al. 2023, Busti et al. 2024).
According to recent researches on sea bream (Busti et al. 2024; Panteli et al. 2021), Proteobacteria, Actinobacteria, and Firmicutes are the predominant taxa populating gut microbiome, with Firmicutes exhibiting the largest proportions, regardless of the diet used (Panteli et al. 2021). Both Panteli et al. (2021) and Rimoldi et al. (2024) reported lower relative abundances of Firmicutes in sea bream fed on HI-based diets, compared to those fed a control diet in which FM was used as the only protein source. Remarkably, Firmicutes were not among the most prevalent phyla in our study, and their abundance decreased as HI meal percentage increased in the diet. A decrease in the relative abundance of Firmicutes was already evidenced as a consequence of FM replacement by HI in the diet in this fish species (Estruch et al. 2015) and may be related to the different fatty acid profile of the two ingredients.
Among the most abundant taxa identified in our results, Alphaproteobacteria, Actinobacteriota, and Cyanobacteria were prevailing.Alphaproteobacteria, were predominantly composed of amplicon sequence variants (ASVs) from the family Rhizobiaceae, which were notably enriched in the HI0 and HI25 treatments. Rhizobiaceae are primarily known for containing anaerobic nitrogen-fixing bacteria (Lindstrom and Mousavi, 2020) and for being present in recirculation system waters, but members of the Rhizobiaceae family were abundant in the intestinal microbiota of fish fed with the antibiotic florfenicol (FF) (Gupta et al. 2019). Also, in marine environment, phylogenetic groups from Alphaproteobacteria contributes to the uptake of low molecular compounds such as amino acids and protein playing, and a possible role in assimilating nutrients from feed cannot be ruled out (Cottrell and Kirchman, 2000; Yokokawa and Nagata, 2010).
On the other hand, microbiome from HI35-HI50 groups was markedly characterized by the Chloroflexi phylum, primarily represented by the classes Anaerolineae and Dehalococcoidia. Chloroflexi is a widespread and metabolically diverse phylum of bacteria, common in biofloc and other wastewater management systems, where it has been reported to be involved in organic matter degradation processes (Guan et al. 2015). Anaerolineae is typically observed in water recirculation systems as a precursor to biofilm formation on filters (Almeida et al. 2021, Chen et al. 2024), suggesting that, while their presence in the gut microbiome might be imputable to the transient microbiota, a medium-high inclusion of HI in the diet may have played an important role in adhesion and proliferation of these classes of bacteria.
Remarkably, this phylum has been recently found as part of microbiota in gut of different cultured fish species, including gilthead seabream (Liu et al. 2021; Nikouli et al. 2021; Ruiz et al. 2023a,b) and other captive bred fish. As an example, Chloroflexi were found in domesticated zebrafish raised in indoor laboratory systems, but not in wild type (Pham et al. 2008), and also in wild caught mullets (Chelon ramada) (Le et al. 2020; Floris et al. 2024). However, the impact of this phylum on fish physiology, still remains largely unknown. In gilthead seabream increased Chloroflexi abundance was previously correlated with increased gut mucous production, which, in turn, may have a further role in favoring gut colonization by these specific taxa (Naya-Català et al. 2021). Interestingly, in a previous study, an increase in mucous cell number and in mucosal folds width was observed in the intestine of gilthead seabream fed the same HI35 diet used in the present study, indicating an improved lubrication together with better absorptive mucosa condition in this group (Di Rosa et al. 2023). Thus, Chloroflexi may have had a pivotal role in improving gut condition in seabreams fed on medium-high HI percentage in the diets. Moreover, HI meal is known to act by selecting bacterial communities able to produce short chain fatty acids (mainly butyrate) induced by chitin fermentation (Biasato et al. 2022; Rimoldi et al. 2019, 2021). Thus, a possible increase in Chloroflexi could be induced also by this factor, given that Choloflexi are also known as butyrate-oxidizing bacteria (Yi et al. 2020). Also, Chloroflexi, as well as Actinobacteria, were related to improved gut bacterial metabolic potentials involved in energy metabolism, carbohydrate metabolism, amino acid metabolism, environmental information processing, and cellular processes in crucian carp (Carassis auratus) (Li et al. 2023). The abundance of the phylums Chloroflexi and Actinobacteria, was also positively correlated with improved growth in hybrid fish derived from herbivorous Megalobrama amblycephala (♀) × carnivorous Culter alburnus (♂) (Li et al. 2023). In our study, Chloroflexi were remarkably represented in all the experimental groups, with a significant increase in HI35. As previously reported, the fatty acid profile of the HI35 diet used in the present study was dominated by Lauric acid (C12:0) and Palmitic acid (C16:0) (Oteri et al. 2021). Lauric Acid (C12:0), particularly, is a short-medium chain FA, highly abundant in H. illucens meal and its role in exhibiting anti-inflammatory properties at intestinal level, and antimicrobial activity against Gram-positive bacteria has been widely demonstrated (Skrivanova et al. 2006; Spranghers et al. 2018; Vargas et al. 2019; Randazzo et al. 2023). Chloroflexi are mainly Gram-negative bacteria (Sutcliffe et al. 2010), and the role of certain SCFAs could play a role in selecting microbiota communities, differently to what happens in marine wild fish populations, which feed on a varied diet, rich in long chain polyunsaturated fatty acids (LCPUFAs). Even though the role of Chloroflexi in fish gut is still unclear (Bovio et al. 2019), even a potential role in boosting fish detoxicant defense should be worthy of further investigations, since Chloroflexi have been shown to increase, in gut microbiome of fish treated with different xenobiotics, such as microplastics (Zhang et al. 2024) and aromatic compounds (styrene and fluorobenzoate) (García-Márquez et al. 2022).
The result obtained using the PICRUSt-predicted metagenomes analysis can unveil functional redundancy across microbial communities, where different taxa perform similar ecological roles through convergent metabolic pathways (Louca et al. 2018). In this study, Cyanobacteria were observed across all the treatments, but in particular, showed a discriminant role for the HI0 and HI25 groups. This is in contrast with findings from Panteli and colleagues (2021), which found an increase in Cyanobacteria related with higher HI meal inclusion levels; unfortunately, is not possible to compare with other studies since Cyanobacteria sequences are often removed from the analysis (Rimoldi et al 2020). The presence and activity of Cyanobacteria were further supported by PICRUST results, which identified genes associated with photosynthesis activity, even if, this function did not showed correlation with any specific treatment, suggesting that photosynthetic activity was not affected by the substitution of FM with HI. Predictive functional profiling of the microbial communities revealed slight differences in several metabolic pathways across the samples. Among all the predicted genes, particular focus was paid towards the pathways associated with fatty acid metabolism and revealed a decrease in fatty acid conversion pathways corresponding to increasing HI levels. This result, coupled with the lack of differences in fatty acid biosynthesis, suggests a possible effect due to the different fatty acid profile in the diets (Oteri et al. 2021), resulting in selection of bacteria with a higher expression of FAs degradation pathway in HI0 and HI25 (with a higher percentage of FM in the diet) compared to HI35 and HI50 groups. Moreover, the HI35 and HI50 groups displayed increased activity in enzymatic pathways related to protein synthesis and translation, including ribosome and tRNA biosynthesis, leading to assume that protein synthesis was higher in gut microbiota from these groups. Chloroflexi, as the most representative phylum found in microbiota from HI35 and HI50 groups, are involved in several metabolic pathways in fish gut (Li et al. 2023) and may be responsible of the results obtained by the PICRUST analyses for these groups. However, further speculations cannot be done, since it is difficult to unambiguously identify bacterial strains responsible for a higher protein synthesis within the ones identified.
Overall, the adaptation of the microbial community to dietary changes point out to the importance of examining functional profiles, as they provide important insights into the metabolic adjustments and resilience of microbiomes to dietary interventions. It is important to note that the functional pathways were predicted using 16S rRNA gene sequencing data, and further functional validation is necessary.
Histological analyses highlighted a significantly higher granulocyte infiltration in liver from HI25 group, compared to the other experimental groups. Since no other significantly appreciable morphological alterations were highlighted in the HI25 group, the high granulocyte influx observed may be ascribable to a hormesis effect, as observed for HSI and VSI in the present study. Hormesis is defined as an adaptive response to low-intensity/dose stimuli (Calabrese et al. 2007). An adaptive hormetic response was observed often in vertebrate models, including fish, subject to dietary and toxicant stimuli and was likely related to a triggering in immune system activity (Rix et al. 2022). On the other hand, significant results emerged by the analyses of hepatocytes number per area, which provide an estimation of hepatocytes number: the less they are for area, the bigger they are. The lower number of hepatocytes per area recorded in group HI50, indicates a larger size of hepatocytes in this group and thus a higher lipid deposition, tending to a steatotic state. Liver plays a pivotal role in lipid metabolism and deposition, and its histological architecture is strictly dependent on lipid composition and profile of the diet. As previously mentioned, HI35 and HI 50 diets were particularly rich in SCFAs compared to the other diets, as a consequence of a high HI meal inclusion (Oteri et al. 2021). A similar high lipid deposition in liver was already reported in gilthead bream fed on a vegetable-based diet in which HI meal was used as protein source (40% of the dietary crude protein), and has been related to the HI lipid profile (Randazzo et al 2021). Compared to freshwater fish species, saltwater ones retain a lower ability in converting short-chain precursors in highly unsaturated FAs through the enzymatic elongation and desaturation pathways (Tocher et al. 2010), which in turn may lead to a higher lipid deposition in liver parenchyma.