Gut microbiota changes in insect-fed monogastric species: state-of-the-art and future perspectives

Abstract

Insects exert their influence on gut microbiota of monogastric animals by increasing microbiota alpha diversity, selecting bacteria able to produce short-chain fatty acids, reducing potential pathogens, and decreasing the nutrient digestibility. Gut microbiota of fish and pigs seems to respond better to the administration of insect-based diets, while that of poultry generally displays a more favorable outcome when fed low inclusion levels of insect meals. As most of research has only dealt with the characterization of gut microbiota of insect-fed animals, the “-omics” technologies appear to be fundamental to investigate the functional relevance of microbiota changes. The intestinal microbiome can be defined as “the genes and genomes of the gut microbiota, as well as their products and the host environment” (Berg et al., 2020). Therefore, three major microbiome components can easily be unearthed, as the term “microbiome” does not only comprise the “assemblage of living microorganisms present in the intestinal environment” (the so-called “microbiota”), but also its “theatre of activity”, which is the “collection of their genomes and genes” (the so-called “metagenome”) and the “whole spectrum of molecules produced by them, including their structural elements, metabolites, and molecules produced by coexisting hosts and structured by the surrounding gut conditions” (the so-called “metabolome”) (Berg et al., 2020). One of the main factors affecting the intestinal microbiome in either humans or animals is the diet, as feed nature and characteristics exert a significant influence on nutrient specificity and availability for microbiome members, thus, in turn, selecting/excluding taxa that are adept at/deficient in processing the available biomolecules. In particular, the relationship between diet and gut microbiome seems to have a key role in the animal production systems, as they represent two of the main components involved in the establishment and maintenance of a proper health status of the intestine, which is of vital importance to animal health and growth performance (Biasato et al., 2018). Nowadays, when choosing a diet for monogastric species, insects (especially Hermetia illucens [HI] and Tenebrio molitor [TM]) cannot be taken out of the picture, not only for their excellent nutritional profile, but also for their interesting nutraceutical components (i.e., chitin, antimicrobial peptides [AMPs], and lauric acid), which have recently been suggested to exert a primary influence on animal gut microbiota (Biasato et al., 2022). So far, most of the research dealing with the binomial “insects-animal gut” focused on the intestinal microbiota (on the bacterial composition only), with very few studies had started exploring the metagenome potential and metabolome as well. Microbiome study (applying various -omics technologies, such as metataxonomics, metagenomics, metaproteomics, metabolomics, and metatranscriptomics) can be used for an in-depth characterization of the complexity of the microbial ecosystems to highlight any shift related to dietary modifications. Indeed, the gut microbiome has an enormous functional potential for the host, as changes in dietary nutrients play a fundamental role in shaping the structure of the gut microbiome and, in turn, determining the inter-relationship between the latter and host. The present review aims to critically summarize—for the first time—the current knowledge about the intestinal microbiota (and microbiome, when available) of monogastric species intended for production purposes (poultry, fish, pigs, and rabbits) fed diets including or supplemented with insect-based products (meals, fats, and live larvae). In particular, a focus on the mode of action of insect-based products and the different species-specific response is herein provided, with final remarks about the future challenges and perspectives as well. The insect-based products (mainly insect meals) seem to exert their influence on animal gut microbiota in four different ways (Figure 1): 1) Increase in microbiota alpha diversity, which can be attributable to the chitin fermentation (as recently suggested in humans [Refael et al., 2022]); 2) Selection of short-chain fatty acids (SCFAs)-producing bacteria, as a consequence of their ability to degrade the chitin (Borrelli et al., 2017; Rangel et al., 2022); 3) Reduction in pathogens, which can be related to the antimicrobial properties (i.e., chitin, AMPs, and lauric acid) of insects (Dabbou et al., 2021; Biasato et al., 2022); 4) Decrease in nutrient digestibility (especially crude protein [CP]), as a consequence of the chitin presence (Biasato et al., 2020a) or the use of full-fat meals (Basto et al., 2021). Graphical summary of the different modes of action of insects on the gastrointestinal tract of monogastric animals. A) Insects and their derivates (chitin, lauric acid, proteins, and oils) increase microbiota richness and diversity. B) Chitin selects beneficial bacteria, and it is used as the main fermentation component to increase short chain fatty acids (SCFAs) in the gut. C) Antimicrobial compounds present in insects reduce the incidence of pathogenic bacteria in the gut. D) Insects decrease nutrient digestibility. Furthermore, a different species-specific response can be highlighted, as fish and pigs overall respond better to the administration of insect-based diets, while poultry generally displays a more favorable outcome when fed low inclusion levels of insect meals (i.e., 5–10%). Differently, data about gut microbiota changes in insect-fed rabbits are still too limited to include them in such scenario. The following sections and subsections will provide a rationale for all these aspects. Tables 1–4 will also summarize the main gut microbiota (and microbiome, when available) findings in the different monogastric species. Main intestinal microbiota and microbiome findings in insect-fed poultry Main intestinal microbiota and microbiome findings in insect-fed poultry Main intestinal microbiota and microbiome findings in insect-fed pigs Main intestinal microbiota and microbiome findings in insect-fed pigs Main intestinal microbiota and microbiome findings in insect-fed fish Main intestinal microbiota and microbiome findings in insect-fed fish Main intestinal microbiota and microbiome findings in insect-fed monogastric rabbits Main intestinal microbiota and microbiome findings in insect-fed monogastric rabbits When comparing intestinal microbiotas, the quantification of the existing differences among groups can be performed at two levels: the alpha (within-sample) and beta (between-sample) diversity. Alpha diversity indices (such as phylogenetic diversity [PD], observed number of amplicon sequence variants, Chao1, Simpson, and Shannon) summarize the structure of a microbial community with respect to its richness (number of taxonomic groups) and/or evenness (distribution of abundances of the groups) (Kers and Saccenti, 2022). Including different levels of TM (5–15% [poultry], 18–25% [fish], and 5–10% [pigs]) and HI (5–17% [poultry], 8–60% [fish], and 5–30% [pigs]) meals—as well as oils (0.16–0.29% [poultry, HI], 1.5% [rabbits, TM and HI], and 6.24% [fish, HI])—in diets for monogastric species led to unaffected (Biasato et al., 2019, 2020a, 2020c; Yu et al., 2019; Meyer et al., 2020; Colombino et al., 2021; Fabrikov et al., 2021; Håkenåsen et al., 2021; Jin et al., 2021; Terova et al., 2021; Rangel et al., 2022; Dabbou et al., 2020, 2021) or increased alpha diversity (in terms of higher Shannon [Borrelli et al., 2017; Biasato et al., 2018; Terova et al., 2019; Kar et al., 2021], Chao1 [Kawasaki et al., 2019; Terova et al., 2019; Biasato et al., 2022], and Simpson [Rimoldi et al., 2019; Terova et al., 2019] indices, and PD [Terova et al., 2019; Weththasinghe et al., 2022] and number of observed species [Borrelli et al., 2017; Terova et al., 2019; Rimoldi et al., 2021]) than that of animals fed the control diet. Even if limited microbial diversity may be desirable as not all microbes are beneficial, high α-diversity of gut microbiota is generally favorable for the overall health and productivity of production animals, as it helps in maintaining the stability of intestinal microbiota and determining the colonization resistance against invading pathogens (Biasato et al., 2018). Refael et al. (2022) recently performed an in vitro oral, gastric and intestinal digestion of powders from crickets (Acheta domesticus), silkworm pupae (Bombyx mori) or isolated chitin and their subsequent fermentation in anaerobic bioreactors inoculated with human feces. Interestingly, they demonstrated that chitin alone supported an increase in Shannon index more than the whole insect powders, thus potentially suggesting its active role in the modulation of gut alpha diversity. However, considering that the abovementioned studies mainly characterized the gut microbiota rather than microbiome, further research is recommended to confirm this hypothesis in monogastric species as well. High Throughput 16S amplicon target sequencing (or metataxonomics) allows for identifying a potential microbial signature associated with the use of a specific diet or ingredient. Beta diversity metrics (such as Bray-Curtis dissimilarity, Jaccard, unweighted UniFrac, and weighted UniFrac) summarize the differences among intestinal microbiotas by considering sequence abundances or considering only the presence–absence of sequences (Kers and Saccenti, 2022). Significant changes in β-diversity and relative abundances of phyla have commonly been highlighted in monogastric animals after administering insect-based diets. At the highest taxonomic level, insect-fed birds may display higher Firmicutes and/or lower Bacteroidetes and higher Firmicutes to Bacteroidetes ratios when compared to the control groups (Biasato et al., 2018). The identification of increased Firmicutes (Panteli et al., 2021; Rimoldi et al., 2021; Rangel et al., 2022; Weththasinghe et al., 2022) and decreased Proteobacteria (Rimoldi et al., 2019, 2021; Terova et al., 2019; 2021; Weththasinghe et al., 2022)—sometimes resulting in higher Firmicutes to Bacteroidetes and lower Proteobacteria to Bacteroidetes ratios (Panteli et al., 2021)—seem to be characteristics of fish species fed diets containing insect-based products, while Actinobacteria and Bacteroidetes phyla are, instead, less constant in their changes (Terova et al., 2019; Panteli et al., 2021; Biasato et al., 2022). Lastly, dietary insect meal inclusion in pigs usually leads to higher Actinobacteria (Meyer et al., 2020; Kar et al., 2021) and lower Firmicutes (Kar et al., 2021), as well as heterogeneous Bacteroidetes variations (Meyer et al., 2020; Håkenåsen et al., 2021). As an interesting aspect to consider, independently of the monogastric species, these phyla profiles are mainly driven by the selection of specific genera that are able to SCFAs, such as Clostridium, Ruminococcus, Lactobacillus, Oscillospira, Coprococcus, Alistipes, Faecalibacterium, Blautia, Roseburia, Eubacterium, and Bifidobacterium in poultry (Biasato et al., 2018, 2019, 2020b; Colombino et al., 2021), Actinomyces, Bacillus, Enterococcus, Lactobacillus, Staphylococcus, Mycoplasma, Pediococcus, and Carnobacterium in fish (Rimoldi et al., 2019, 2021; Terova et al., 2019; Biasato et al., 2022; Li et al., 2022; Rangel et al., 2022; Weththasinghe et al., 2022), Bifidobacterium, Roseburia, Lactobacillus, Pseudobutyrivibrio, Clostridium, Faecalibacterium, Blautia, Coprococcus, Eubacterium, Prevotella, Ruminococcus and Staphylococcus in pigs (Yu et al., 2019; Biasato et al., 2020a; Meyer et al., 2020; Jin et al., 2021; Kar et al., 2021), and Akkermansia and Ruminococcus in rabbits (Dabbou et al., 2020). Chitin seems to be the preferred source of microbial fermentations, since it acts as a prebiotic and promotes, in turn, the selection of beneficial taxa in the gut microbiome. Indeed, at the lowest taxonomic level, Alkaliphilus transvaalensis, Christensenella minuta, and Flavonifractor plautii have previously been identified in the intestinal microbiome of HI-fed laying hens in correlation with the highest levels of KEGG genes responsible for the chitin degradation (β-N-acetylhexosaminidases [K01207] and N-acetylglucosamine 6-phosphate deacetylase [K01443]), which boosted the production of butyrate and propionate (Borrelli et al., 2017). Similarly, the intestinal microbiome of insect-fed European seabass has recently displayed a Paenibacillus-related increase in chitinase ChiA-encoding genes (Rangel et al., 2022), while increased SCFAs production (mainly butyrate, isobutyrate, valeric, and isovaleric) has also been reported in pigs fed insect-based diets (Yu et al., 2019; Meyer et al., 2020). The production of SCFAs—especially butyrate—is considered beneficial for the gut, as they can enhance the intestinal epithelial cell barrier function by acting as energy source for the enterocytes and stimulating goblet cell differentiation and mucus production, as well as reducing the enteric pathogens because of their antimicrobial properties (Biasato et al., 2018). The administration of insect-based diets to monogastric species may also cause a reduction in pathogens in their gut microbiota, even if less research studies have reported this outcome when compared to the identification of the SCFAs-producing bacteria. Such a positive effect seems to be exclusively related to the use of HI (either the meal or the fat), as a consequence of the synergic activity of its three nutraceutical components—lauric acid, chitin, and AMPs—, which all concur with its antimicrobial properties (Biasato et al., 2022). In particular, a decrease in potentially pathogenic bacteria (such as Corynebacterium, Streptococcus, Sarcina, Treponema, Aeromonas, Deefgea, Vagococcus, and Lactococcus) can be highlighted in broiler chickens (Dabbou et al., 2021), pigs (Yu et al., 2019; Meyer et al., 2020; Jin et al., 2021; Kar et al., 2021) and rainbow trout (Rimoldi et al., 2019, 2021; Fabrikov et al., 2021; Terova et al., 2021; Biasato et al., 2022). Interestingly, a reduction in selected foodborne pathogens (such as Listeria and Campylobacter) has also recently been observed in HI-fed rainbow trout (Biasato et al., 2022). However, further studies performing bacterial or parasitic challenges on monogastric species-fed insect-based products are mandatory to confirm this hypothesis. Even if the use of insect-based products in monogastric species is mainly associated with positive outcomes in terms of intestinal microbiota modulation, some “side effects” can be pointed out as well, especially in poultry. Indeed, reduced alpha diversity, increased Proteobacteria, decreased Firmicutes and Firmicutes to Bacteroidetes ratios, selection of Helicobacter, and decreased SCFAs-producing bacteria (such as Clostridium, Coprococcus, and Ruminococcus) have been reported in broiler chickens fed HI- and TM-based diets (Biasato et al., 2019, 2020b, 2020c), with a reduction in Lactobacillus and Bifidobacterium being also observed in HI-fed laying hens (Kawasaki et al., 2019). On the one hand, these negative outcomes can reasonably be attributed to the chitin-related reduction in CP digestibility (Biasato et al., 2020b); on the other, the use of full-fat meals rather than defatted ones (mainly HI) may determine itself a reduction in CP digestibility (75.8% vs. 87.2% [Basto et al., 2021]). In both situations, the nondigested protein increases at the ileal level, thus leading to hindgut protein fermentation and, in turn, formation of toxic compounds potentially capable of creating a non-healthy gut environment (Biasato et al., 2020b). Even if the different insect species seem to exert a similar influence on the gut microbiota of monogastric species, it is possible to underline a different response of fish, pigs, and poultry to the administration of the insect-based products. As already mentioned before, while fish and pigs usually respond well to both low (5–10%) and high (≥15%) inclusion levels of insect meals—with only very few minor negative effects being highlighted—, poultry species are less predisposed to eat diets containing more than 15% of insect meals. Indeed, chitin- or fat-related reduction in CP digestibility does not only negatively affect the bird intestinal microbiota (as previously discussed in “Decrease in nutrient digestibility” section), but also other gut health parameters, such as morphology (in terms of low villus height, high crypt depth, and reduced villus height to crypt depth ratio) and mucin dynamics (in terms of reduced mucin staining intensity) (Biasato et al., 2020b, 2020c). Interestingly, the negative modulation of the health status of the intestine seems to have direct repercussions on bird growth performance, as worsening in feed efficiency is also commonly observed (Biasato et al., 2020b, 2020c). Differently, gut microbiota of selected fish species (sea bream, sea bass, rainbow trout, and Atlantic salmon) and pigs may occasionally display a reduction in selected alpha diversity metrics (Chao1 [Panteli et al., 2021] and Shannon [Rimoldi et al., 2019; Biasato et al., 2022; Li et al., 2022; Weththasinghe et al., 2022] indices, and PD [Li et al., 2022]) and SCFAs-producing bacteria (Lactobacillus [Håkenåsen et al., 2021] and Coprococcus [Meyer et al., 2020]), and selection of potential pathogens such as Chlamydia (Biasato et al., 2020a). However, these negative findings have always been observed along with predominant, positive outcomes, as well as unaffected gut morphology and mucin dynamics, and preserved overall health status and animal growth performance (Biasato et al., 2020a, 2022). Despite the highest chitinase activity among the omnivorous monogastric species having previously been reported in chicken stomachs (Tabata et al., 2018), such dichotomy between fish-pigs and poultry can potentially be explained by the remarkable genetic selection broiler chickens and laying hens have faced in the last decades, which has made the current strains reasonably more prone to utilize the commercial feeds and progressively less used to consume insects in their feeding regime. Insects are highly complex organisms and a of beneficial compounds that can animal health and In the present the pointed out insect-based products can affect the gut microbiota composition in monogastric species. However, most of the studies are only on bacterial considering and or the shift in their The of study is to insects with the microbiome and the effects on the host. The of including to the potential of the gut microbiota with insects is still an is also fundamental to more about the functional relevance of specific microbiota changes at a level, and this is that has started to be So far, the “-omics” has identifying an increase in chitin genes (Borrelli et al., 2017; Rangel et al., 2022), different of acid, and degradation genes (Borrelli et al., 2017; Yu et al., 2019; Meyer et al., 2020; Panteli et al., 2021; Rimoldi et al., 2021; Weththasinghe et al., 2022), and an in mucin degradation genes et al., 2022). As a aspect to consider, as an high between the microbiota associated with gut and feed has also previously been observed in insect-based diets et al., 2022; Weththasinghe et al., 2022), following the in the different of the insect-based to the insect-based feed microbiome from the among and may to animal gut microbiome, in to the and to a feed and the of the feed composition alone on the microbiome et al., 2022). Furthermore, since insects potentially be on different of microbial along the chain is still this sequencing can a of potential microbial that can be from the to the the insect-fed animals and, the final associated with the including antimicrobial resistance genes and along the insect-based chain also be as the of the can a for microbes to specific genes or Lastly, considering that specific of microbes may potentially influence growth outcomes in insect-fed animals, it be interesting to specific microbiome at a can be related to an increase or a decrease in the growth In the present review critically the existing about the intestinal microbiota and microbiome in monogastric animals pigs, and rabbits) fed insect-based diets, identifying four different modes of action of insects in the animal gut in microbiota alpha diversity, selection of SCFAs-producing bacteria, reduction in pathogens, and decrease in nutrient as well as a different response in fish and pigs when compared to poultry (as a consequence of the genetic selection of the The herein also three different research to the existing knowledge about the binomial 1) to studies (especially and on the intestinal 2) to the whole of the insect-based feed microbiome on gut microbiome structure and the and/or and 3) to the relationship between microbiome and animal growth performance performing correlation and Biasato is a of the at the of and of the of in and the of insect meal in poultry feeding at the of of the of research activity has been focused on and of fish and dealing with the of products and on the performance, gut health (especially gut health and and of the insect-fed animals. 2020, has also been on the effects of different on the production of Hermetia illucens and Tenebrio molitor is a at the of and of the of at the of in in at the of and a in research on fish, and poultry and their performance and the of on research has been focused on the use of insect meals as in animal and on the of insect to produce In particular, has a with the and the is a of at the of of the of in the focus of research various of poultry especially and has also and energy to out in and in with the with the primary of to with the community in has led to a of and research in and has been and in animal and poultry research activity on the of products on the performance and of the insect-fed poultry species. is an of the and at the of of the of in and in at the of main research are focused on the of gut following the use of dietary antimicrobial or and on and In particular, since research has been focused on the of products on gut health with for including production and is an of the and at the of of the of in and and in and of at the of main research are focused on the of the structure and of the microbiome in different research and of for microbial and of microbial in (mainly and by and characterization of production and its study in vitro and in of the human and animal gut microbiome.

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